Enhancing oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in surgically induced osteoarthritis

Enhancing oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in surgically induced osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Enhancing oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in surgically induced osteoarthritis Seungwoo HAN, Jin Han, Yoon Hee Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3947364/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted 9 You are reading this latest preprint version Abstract Chondrocytes can shift their metabolism to oxidative phosphorylation (OxPhos) in early stages of osteoarthritis (OA), but as the disease progresses, this metabolic adaptation becomes limited and eventually fails, leading to mitochondrial dysfunction and oxidative stress. This study investigated whether enhancing OxPhos through pyruvate dehydrogenase kinase (PDK) 2 affects the metabolic flexibility of chondrocytes and cartilage degeneration in surgical model of OA. Among the PDK isoforms, PDK2 expression was increased by IL-1β in vitro, and in articular cartilage of the DMM model in vivo, accompanied by an increase in phosphorylated PDH. Mice lacking PDK2 showed significant resistance to cartilage damage and reduced pain behaviors in DMM model. PDK2 deficiency partially restored OxPhos in IL-1β-treated chondrocytes, leading to an increased APT and NAD+/NADH ratio. These metabolic changes were accompanied by a decrease of reactive oxygen species (ROS) and senescence of chondrocytes, as well as the expression of MMP-13 and IL-6 following IL-1β-treatment. At the signaling level, PDK2 deficiency reduced p38 signaling and maintained AMPK activation, without affecting JNK, mTOR, AKT and NF-kB pathways. Among them, p38 MAPK signaling was critically involved in ROS production under glycolysis-dominant condition in chondrocytes. Our study provides the proof-of-concept for PDK2-mediated metabolic reprogramming towards OxPhos as a new therapeutic strategy for OA. Health sciences/Medical research/Translational research Health sciences/Anatomy/Musculoskeletal system/Cartilage Chondrocyte Metabolism Pyruvate dehydrogenase kinase Oxidative phosphorylation Figures Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Osteoarthritis (OA) is the most common form of degenerative joint disease characterized by apoptosis of chondrocyte and degradation of cartilage extracellular matrix (ECM), which ultimately leads to joint failure ( 1 ). Chondrocytes, the only cell type found in cartilage tissues, produce cartilage specific ECM proteins such as type 2 collagen and aggrecan, and they become trapped within ECM proteins they produce, resembling a state of hibernation ( 2 ). As cartilage is an avascular tissue, chondrocytes exist in hypoxic conditions despite the diffusion of oxygen from synovial fluid or subchondral bone ( 3 ). In such hypoxic milieu, chondrocytes rely predominantly on glycolysis rather than oxidative phosphorylation (OxPhos) which comprises only less than 10% of total cellular ATP production ( 4 , 5 ). However, chondrocytes also exhibit a significant level of metabolic flexibility in the catabolic state of early OA, where they can alter their metabolic machinery towards OxPhos ( 5 , 6 ). This allows them to adapt to the catabolic conditions, hence enhancing their survival and function. As OA progresses and mitochondrial dysfunction occurs ( 7 , 8 ), however, the metabolic adaptation of the OA chondrocytes reaches its limit, leading to a metabolic shift towards the glycolytic pathway ( 8 ). Actually, chondrocytes in early-stage OA of Kellgren and Lawrence (K-L) grade 1 exhibit a metabolic phenotype characterized by increased OxPhos and decreased glycolysis, compared to chondrocytes in advanced-stage OA of K-L grade 4 ( 9 ). This metabolic shift not only makes it increasingly difficult for the chondrocytes to meet their energy demands, but also amplifies oxidative stress in lactate dehydrogenase-mediated manner ( 8 ). The increase of oxidative stress, in turn, worsen mitochondrial dysfunction, including mitochondrial DNA damage and increased membrane permeability, which eventually contributes to chondrocyte apoptosis and senescence ( 10 , 11 ). In this way, the glycolysis-prone metabolism of chondrocyte interacts with and amplifies both mitochondrial dysfunction and oxidative stress during OA progression ( 12 ). Despite understanding that metabolic imbalances in chondrocytes play a crucial role in the pathogenesis of OA, we still lack conclusive evidence on whether redirecting chondrocyte metabolism towards OxPhos could indeed impact its progression. Glucose that enters chondrocytes through mainly glucose transporter (GLUT) 1 and GLUT3 is metabolized into pyruvate, the end product of glucose catabolism ( 13 ). This pyruvate moves to the mitochondria, where it is converted to acetyl-Coenzyme A (acetyl-CoA) by pyruvate dehydrogenase (PDH), the enzyme that links the cytoplasmic glycolysis pathway to the mitochondrial tricarboxylic acid (TCA) cycle. As a canonical input to the TCA cycle, an increase of acetyl-CoA levels leads to an enhancement in the rate of the TCA cycle and consequently OxPhos ( 14 ). Therefore, the activity of PDH, which converts pyruvate into acetyl-CoA, is the overall rate-limiting and gatekeeping factor in pyruvate-driven OxPhos ( 14 ). The activity of PDH is regulated by PDH kinase (PDK), a Serine/ Threonine kinase, which phosphorylates the α subunit of PDH, thereby inactivating it ( 15 ). The activity of PDK, in turn, is controlled by pyruvate, NAD+, ATP, acetyl-CoA, and nuclear transcription factors, such as FoxO, PPAR, and PGC1α ( 15 ). From the perspective of the metabolic shift towards glycolysis in OA progression, a potential therapeutic strategy might be derived from the metabolism reprogramming to enhance OxPhos through the inhibition of PDK ( 16 ). PDK has four isoforms, PDK1 through PDK4. Among them, PDK2 is expressed ubiquitously, while the remaining PDK isoforms show tissue-specific distribution: PDK1 is primarily found in heart tissue, the pancreatic islet, and skeletal muscle. PDK3 has a relatively limited tissue distribution in the testis, kidney, and brain, and PDK4 in the heart, skeletal muscle, liver, kidney, and pancreatic islets ( 17 , 18 ). However, the expression of PDKs in chondrocytes and their functional role in OA have yet to be investigated. The objective of this study was to determine whether the inhibition of PDK affects the metabolic flexibility of chondrocytes, and contributes to cartilage degeneration in a surgical model of OA. First, we verified the expression of PDK isoforms in both in vitro catabolic condition and in vivo OA cartilage, which revealed an increase of PDK2 under catabolic conditions. The role of PDK2 in OA progression was investigated using a surgical OA model in Pdk2 -deficient mice, and we subsequently focused on its metabolic phenotype, expression of anabolic and catabolic factors, ROS production, and cellular senescence. From a mechanistic perspective, we identified the signaling pathways affected by Pdk2 -deficiency and elucidated their role on the production of ROS and cellular senescence. MATERIALS and METHODS Ethics and isolation of primary chondrocytes This study was conducted in accordance with the guidelines of National Research Council (US) Committee for the Care and Use of Laboratory Animals ( 19 ) and was approved by the Institutional Review Board of the Kyungpook National University School of Medicine (Daegu, Korea), under the approval number KNU 2022 − 0203. Mice genetically deficient in the PDK2 gene (PDK2 KO) on the C57BL/6J background were kindly provided by Dr. In-Kyu Lee (Kyungpook National University, Daegu, Korea). Primary chondrocytes were isolated from the articular cartilage of femur and tibia of 5-day-old C57BL/6J mice. Isolated cartilage pieces were minced into small fragments, underwent a 15-minute incubation in 0.25% trypsin-EDTA at 37°C with gentle shaking, and digested in a solution containing 0.2% collagenase type II, prepared in DMEM, at 37°C with gentle agitation for 4 hours. After digestion, the cell suspension was filtered through a 40 µm cell strainer and washed twice with PBS. The isolated chondrocytes were resuspended in complete 3:2 F12:DMEM-based culture medium supplemented with 0.25% L-glutamine, and 0.25% penicillin/streptomycin, then seeded in 6-well plates at a concentration of 1 x 10^5 cells per well, and cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. To reduce de-differentiation risks, only chondrocytes at passage 0 were used in experiments. RNA and protein expression analysis To induce a catabolic condition on primary chondrocytes, cells were treated with 10 ng/ml of IL-1β, and incubated for 6 hrs for RNA isolation and 24 hrs for protein isolation. For signaling analysis, cells were starved overnight and then stimulated with 20 ng/ml of IL-1β for the indicated time. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA), followed by the generation of the first strand cDNA with Superscript III reverse transcriptase (Invitrogen). A ViiA™ 7 Real-Time PCR System (Applied Biosystems, CA) and SYBR® Green Master Mix (Applied Biosystems) were employed for real-time qPCR. Primers for real-time qPCR can be found in Supplementary Table 1. Target gene expression levels were calculated using the 2 − ΔΔCT method and normalized to the geometric mean of Gapdh. All qRT-PCR analysis were carried out in triplicate, repeated three to five times, and the average results for each were presented. Total proteins were extracted using 300 µL RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN). Total cell lysates containing 10–20 µg protein were loaded to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore Corporation, Billerica, MA). Membranes were blocked using 5% skimmed milk in PBS with 0.25% Tween-20 (PBST) and incubated with the primary antibodies against PDK1 (Abcam, Cambridge, UK, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDH-E1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, Santa Cruz, CA, #sc-377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, Beverly, MA, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), p-AMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272), and β-actin (Sigma-Aldrich, Burlington, MA, #A1978) overnight at 4°C. After washing with PBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 hours, and then blots were developed using enhanced chemiluminescence western blotting detection reagent (Thermo Fisher Scientific) and examined with the MicroChemi system (DNR Bio-imaging Systems). All analysis was biologically triplicated, and the western blot band intensities were quantified using ImageJ software (version 1.8.0, NIH). Band intensities of target proteins were normalized to the β-actin band intensity of respective lanes. Surgical OA induction OA was surgically induced in 12-week-old male C57BL/6J mice, including 7 PDK2 KO mice and 7 of their littermates, through destabilization of the medial meniscus (DMM) surgery under general anesthesia. The sample size of each group was calculated based on the assumptions of a type I error (α) of 0.1, a standard deviation (σ) of 10, an effect size (δ) of 15, and a power of 0.8. Ligament of medial meniscus was dissected in the right knee to induce OA, and the sham operation was conducted on left knee as a control. After surgery, mice were housed under controlled conditions with a 23°C ± 1°C temperature, 50% humidity, and a 12-hour light/dark cycle. They were kept under specific pathogen-free conditions in the animal facilities of Kyungpook National University Chilgok Hospital. Eight weeks after surgery, the mice were euthanized via cervical dislocation, and knee joints were collected for histological examination, and all specimens were used for analysis. This study was conducted in accordance with ARRIVE guidelines 2.0, and its checklist has also been offered in the Supplementary sections. Pain behavior analysis Two weeks post-surgery, pain behavior was assessed weekly using the spontaneous weight-bearing asymmetry test (Incapacitance test), the hot plate test, and the threshold punctate mechanical stimulation (von Frey test). To assess spontaneous weight bearing on the hind limbs, the incapacitance meter (SangChung commercial, Seoul, Korea) was used to measure the downward force applied by each hind limb. Mice were briefly placed in a restrainer, with hind limbs resting on two weight averaging platform pads. Measurements of the paw pressure of each hind limb were taken for 10 seconds, approximately 10 times, and results were averaged. Data were expressed as the percentage of weight distributed on the ipsilateral hind limb. Next, thermal pain sensitivity was assessed with the hot plate test ( 20 ). Mice were placed on a heated plate (plantar test IR emitter, Ugo Basile, Gemonio, Italy), and the time until they displayed pain behaviors such as licking or shaking their paws was recorded. Finally, tactile allodynia was assessed using the von Frey test. Calibrated monofilaments (von Frey hairs; Stoelting, Wood Dale, IL) were applied to the plantar surface of both ipsilateral and contralateral hind paws, placed on an elevated maze in an acrylic cage. A paw withdrawal was considered a positive response. The 50% withdrawal threshold was determined upon six repeated applications of varying force with the von Frey filament, using the up-down method ( 21 ). Safranin-O and immunofluorescent staining Mouse knee joints were fixed in 4% paraformaldehyde for 24 hours, decalcified in 10% EDTA for three weeks, and then embedded in paraffin. The embedded blocks were sectioned at a thickness of 6 µm. After deparaffinization and rehydration, sections were stained in 0.1% Safranin-O solution for 5 minutes and counterstained with fast green solution for 1 minute. Cartilage destruction was scored in all four quadrants of the joint (grade 0–24) and on the medial tibial plateau (grade 0–6) by two observers under blinded conditions, using the OARSI scoring system ( 22 ). For immunofluorescent staining, rehydrated sections were antigen-retrieved in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). The sections were then blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour, followed by overnight incubation at 4 ℃ with primary antibodies against PDK1 (#ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDH-E1α (Ser232, #AP1063), PDH-E1 (#sc-377092), MMP13 (#ab51072), 8-oxo-dG (#sc-66036), or normal rabbit IgG in 1% BSA. For immunofluorescence, the sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 hours, then counterstained with DAPI. Finally, the sections were mounted with anti-fade mounting solution (Vector Labs) and imaged and quantified using a KI-3000F fluorescence microscope. Seahorse real-time cell metabolic analysis Chondrocyte metabolism was analyzed using an XF96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA). Primary chondrocytes from PDK2 KO mice and their littermates were plated in Seahorse XF96 plates at a density of 50,000 cells per well. Confluent chondrocytes were incubated with IL-1β (10 ng/mL) or without it for 24 hours. For the mitochondrial stress test, cells were equilibrated for 1 hour in serum-free Seahorse XF Base Medium. Basal cellular respiration was initially measured, and mitochondrial respiration inhibitors, including 1 µM oligomycin, 1 µM FCCP, and a 1:1 mixture of 2 µM antimycin A with 1 µM rotenone, were sequentially injected into the assay wells. Mitochondrial ATP production was expressed as the oxygen consumption rate (OCR; pmol of O 2 /min). In the glycolysis stress test, cells were starved for 1 hour in glucose-free Seahorse XF DMEM, followed by sequential treatment with 20 mM glucose, 1 µM oligomycin, and 100 mM 2-DG. Real-time measurements of proton accumulation in the media were taken and quantified as the extracellular acidification rate (ECAR; mpH/min) ( 23 ). Following completion of either the mitochondrial or glycolysis stress test, cell nuclei were counted following in situ DAPI staining. Analysis data were then normalized based on the cell count per well, using a normalization unit of 5,000 cells. ATP and NAD+/NADH measures Chondrocytes from PDK2 KO and their littermates were seeded at 5,000 cells per well in 96-well plates for ATP measure, and at 20,000 cells per well in six-well plate for NAD+/NADH measure, and treated with 10 ng/ml of IL-1β or without it for 24 hrs. ATP concentration was measured using a commercial ATP detection kit (Abcam #ab83355), following the manufacturer's protocol. Briefly, chondrocytes were homogenized in 15 µL of ATP assay lysis buffer, and aliquots of the cell lysates were incubated with the ATP Reaction Mix for 30 minutes in the dark. The absorbance of the mixtures was quantified using a spectrophotometer at a wavelength of 570 nm. ATP levels were calculated from a standard curve, which was plotted using the serial dilution of authentic ATP. The NAD+/NADH ratio was determined using a commercial NAD+/NADH Assay Kit (Abcam, ab65348) following the manufacturer's instruction. Briefly, chondrocytes were collected by cell scraping, washed three times with pre-cooled PBS, and lysed with the extraction buffer solution. After centrifugation at 12,000 rpm for 5 min at 4°C, the supernatant was collected. Protein concentration was determined using a BCA kit (Beyotime Biotechnology, China). Samples were then heated at 60°C for 30 min to completely decompose NAD + in the sample. Then, 50 µl of each sample was mixed with 100 µl of Reaction Mix and incubated at room temperature for 5 min. Subsequently, 10 µl of NADH Developer was added to each well and incubated at room temperature for 2 hr. The absorbance of the mixtures was quantified using a spectrophotometer at a wavelength of 450 nm. Assess oxidative stress: detection of intracellular ROS and oxidative DNA damage Intracellular ROS and mitochondrial ROS was assessed with dihydroethidium (DHE) and Mitosox Red (ThermoFisher Scientific, #M36008) fluorescent dye, respectively. Primary chondrocytes were fixed with 4% paraformaldehyde for 10 min, permeabilized using 0.25% Triton X-100, and then rinsed three times in PBS. To assess intracellular ROS, chondrocytes were incubated with 5 µM DHE for 30 min at 37°C, washed with PBS, and then counter-stained with DAPI. Images were captured using a KI-3000F fluorescence microscope (Korealabtech, South Korea). DNA damage caused by oxidative stress was evaluated by immunostaining using 8-oxo-dG antibody. Fixed and permeabilized cells were incubated with the primary 8-oxo-dG antibody overnight at 4°C. Then cells were incubated with a secondary antibody for 2 hours, counter-stained with DAPI, and imaged using a fluorescence microscope. Fluorescence-positive cells were quantified using ImageJ software. Senescence β-galactosidase staining β-galactosidase activity was detected using β-galactosidase staining kit (Cell Signaling Technology, #9860). Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and then incubated for 2 hrs in the staining solution containing 1 mg/mL X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl₂ in pH 7.4 PBS. Then, cells were washed with PBS and visualized under a light microscope, and the percentage of blue colored cells was calculated by counting at least five random fields per sample. Statistical analysis All data are presented herein as mean ± standard error of the mean (SEM). Statistical analysis to compare the mean values of two groups was performed using Mann–Whitney U test, a nonparametric test as the sample size is small and it cannot be assumed as normal distribution. P -values ≤ 0.05 were considered statistically significant. Statistical analyses were performed with Prism software version 8.0 (GraphPad Software, La Jolla, CA). RESULTS Among PDK isoforms, PDK2 increases under IL-1β-induced catabolic condition and in the cartilage of surgically induced OA To comprehend the PDK-mediated metabolic modulation in OA chondrocytes, we first investigated the expression of PDK isoforms under IL-1β-mediated catabolic conditions. Treatment with IL-1β increased the mRNA levels of PDK2 and PDK4 , but not PDK1 and PDK3 , in primary chondrocytes (Fig. 1 a). At the protein level, however, only PDK2 showed an increase following IL-1β treatment. Simultaneously, the phosphorylated, inactive form of PDH (p-S 293 -PDH) increased, suggesting that PDK2 may be involved in the phosphorylation of PDH under the IL-1β-mediated catabolic conditions (Fig. 1 b). To confirm this increase in PDK2, we examined the expression of PDK isoforms in the articular cartilage over time following DMM surgery in mice. Phosphorylated PDH, which indicates the inactivation of PDH, increased in the early phase of OA 2 weeks post-DMM surgery. This increase was accompanied by a rise in PDK2, which continued to rise over time. PDK4 showed a trend of increase around 8 weeks, but it was not statistically significant, while PDK1 and PDK3 did not show any increase in the DMM-induced model of OA (Fig. 1 c). PDK2 deficiency attenuates the severity of OA, oxidative stress, and pain-related behaviors in DMM-induced murine OA model To assess the impact of chondrocyte metabolic reprogramming on OA progression, we compared the phenotype of DMM-induced OA in WT and Pdk2 KO mice. Compared to WT mice, genetic deletion of PDK2 significantly lessened the progression of DMM-induced OA. This was evidenced by the decrease in cartilage degradation, as quantified by the OARSI score, and reduced osteophyte maturation and subchondral bone thickness (Fig. 2 a, b). This was further supported by the immunofluorescent staining which showed fewer MMP13-positive and 8-oxo-dG-positive chondrocytes in Pdk2 KO mice, indicating a decrease of ECM degrading protease and oxidative stress-induced DNA damage, respectively (Fig. 2 a, c). This was accompanied by decreased pain-related behaviors in Pdk2 KO mice compared to WT from 6 weeks post-DMM surgery, as observed in tests such as static weight bearing over the hind limbs (incapitance test), paw withdrawal time on a hot plate, and Von Frey test (Fig. 2 d). PDK2 deficiency partially restores the IL-1β-mediated metabolic shift toward glycolysis in chondrocytes To determine whether PDK2 deficiency impacts chondrocyte metabolism, we assessed OxPhos and glycolysis under IL-1β–induced catabolic conditions using the Seahorse XF-96 Extracellular Flux Analyzer. IL-1β treatment for 24 h markedly suppressed OxPhos, as reflected by a decrease in OCR of cultured primary chondrocytes, while it enhanced glycolysis, as indicated by an increased ECAR. PDK2 deficiency partially restored the OCR by enhancing basal respiration and ATP production in chondrocytes under IL-1β-treated conditions (Fig. 3 a). On the other hand, the IL-1β-mediated increase in ECAR was significantly reduced in Pdk2- deficient chondrocytes, resulting from decreased glycolysis and glycolytic capacity (Fig. 3 b). This PDK2-mediated metabolic reprogramming also led to a significant increase in ATP and NAD+/NADH ratio in the culture supernatant from Pdk2- deficient chondrocytes (Fig. 3 c, d). These findings suggest that the inhibition of PDK2 enhances OxPhos, potentially restoring the balance of energy homeostasis in chondrocytes under catabolic conditions like OA. PDK2 deficiency enhances PDH activity and anabolic effects, which lead to reduced oxidative stress and cellular senescence in chondrocytes under IL-1β-treated conditions To investigate the impact of PDK2 deficiency on chondrocyte homeostasis under the catabolic condition, we then examined the mRNA expression of chondrogenic markers ( Col2 , aggrecan ), catabolic proteases ( MMP13 , Adamts5 ), and senescence-associated secretory phenotype (SASP)-related genes ( IL-6 , Vegf ). In IL-1β-treated conditions, PDK2 deficiency increased Col2 and Aggrecan expression, while it decreased MMP13 and IL-6 expression, but had no effect on Adamts5 and Vegf (Fig. 4 a). PDH enzymatic activity was enhanced in Pdk2 -deficient chondrocytes, as indicated by lower levels of phosphorylated PDH (p-S 293 -PDH) under IL-1β-treated conditions. Similarly to mRNA expression, the protein levels of Col2 increased in PDK2 deficient chondrocytes compared to WT under IL-1β-treated conditions, while those of MMP13 decreased. However, the levels of SirT1 remained unaffected (Fig. 4 b). Next, we assessed the role of PDK2 in the oxidative stress under IL-1β-treated catabolic conditions. IL-1β treatment (10 ng/ml) significantly increased the production of ROS as well as 8-oxo-dG, a DNA damage product that occurs as a result of oxidative stress. In this condition, Pdk2 -deficient chondrocytes exhibited a significant decrease in ROS and 8-oxo-dG production, suggesting that PDK2 can reduce oxidative stress in IL-1β-mediated catabolic conditions (Fig. 4 c). Consistent with these results, the expressions of Hmox and Fth , target genes of oxidative stress, were found to decrease in Pdk2 -deficient chondrocytes (Fig. 4 d). We further investigated the effect of PDK2 deficiency on the senescence of chondrocytes, as mitochondrial dysfunction and ROS production is closely related to cellular senescence ( 24 ). After 48 hr of treatment with IL-1β, a marked decrease in the expression of SA-β-gal, a senescence marker, was observed in the chondrocytes from PDK2 KO mice compared to WT (Fig. 4 e). Our results collectively indicate that PDK2 loss-of-function leads to anabolic effects, and attenuates ROS levels as well as the senescence of chondrocytes under catabolic conditions. PDK2 deficiency reduces p38 signaling avtivity and sustains AMPK activation in response to IL-1β stimulation We further examined the signaling mechanisms involved in the anabolic effects on PDK2-deficient chondrocytes under catabolic conditions, particulary focusing on metabolism and oxidative stress-related pathways such as AMPK, mTOR, FoxO3a, AKT, NFkB and MAPKs. Among these signalings, the phosphorylation of p38 MAPK (Thr180/Tyr182) was most prominently suppressed in Pdk2 -deficient chondrocytes compared to WT. The phosphorylation of AMPK (Thr172), which typically gets down-regulated by IL-1β stimulation, remained in an activated state in Pdk2 -null chondrocytes. PDK2 deficiency led to a non-continuous increase in FoxO3a phosphorylation (Ser253) at 5 and 60 min after IL-1β stimulation, which suppresses its activity through cytoplasmic export and proteosomal degradation ( 25 ). The activation of mTOR, JNK, p65, and AKT signalings were not affected by PDK2 deficiency (Fig. 5 a, b). p38 MAPK is essential for generating ROS in chondrocytes with mitochondrial dysfunction, and mitochondrial ROS, in turn, activate p38 MAPK under catabolic conditions To determine whether the decrease in p38 MAPK phosphorylation is simply a result of reduced ROS or if it directly involves the reduction of ROS in the PDK2-deficient situation, we treated WT and Pdk2 -deficient chondrocytes with the chemical p38 inhibitor, SB203580, and the specific scavenger of mitochondrial superoxide, MitoTempo. When we compared ROS production and cellular senescence, the inhibition of p38 significantly suppressed ROS production and senescence in WT chondrocytes, but this effect was not observed in Pdk2 -deficient chondrocytes, where ROS generation and senescence remained unchanged despite p38 MAPK inhibition (Fig. 6 a). This implies that in a metabolic environment where OxPhos is increased due to PDK2 deficiency, p38 MAPK does not play a significant role in ROS production and subsequent cellular senescence. Then, we investigated how the inhibition of mitochondrial ROS affects the activation of p38 MAPK. PDK2 deficiency led to a significant reduction in the phosphorylation of p38 MAPK compared to chondrocytes from WT littermates under IL-1β stimulation, and treatment with SB203580 substantially delayed the activation of p38 MAPK in Pdk2 -deficient chondrocytes. As expected, scavenging mitochondrial superoxide using MitoTempo significantly reduced the phosphorylation of p38 MAPK at 15 minutes, compared to that of both WT and Pdk2 -deficient controls, indicating that mitochondrial ROS are crucial for the activation of p38 (Fig. 6 b). Collectively, these results suggest the presence of a positive feedback loop between ROS and p38 MAPK and highlight the significant role of PDK-mediated glycolytic metabolic shift in p38 MAPK-mediated ROS generation. DISCUSSION Increasing evidence suggests that alterations in chondrocyte metabolism toward glycolysis, associated with mitochondrial dysfunction, are critically linked to OA pathogenesis ( 8 , 9 ). In this context, PDK-dependent inhibition of PDH activity may be a pivotal mechanism responsible for the glycolytic metabolic shift in catabolic chondrocytes ( 26 ). Here, we identified that PDK2 is specifically upregulated in OA chondrocytes and its loss-of-function led to an increase of PDH activity to restore the IL-1β-mediated metabolic shift toward glycolysis in chondrocytes. In addition, PDK2 deficiency showed a protective phenotype in surgically induced OA model, which was accompanied by reduced oxidative stress and cellular senescence. Mechanistically, PDK2 deficiency led to decreased activation of p38 MAPK, along with a sustained activation of AMPK signaling under IL-1β-treated conditions (Fig. 7 ). Taken together, our data sheds light on the potential of metabolic reprogramming towards OxPhos as a novel therapeutic approach for OA. Several lines of evidence indicate a critical involvement of mitochondrial dysfunction in the pathogenesis of OA ( 9 , 27 ). Specifically, chondrocytes from patients with advanced OA exhibit a decrease in respiring mitochondria, as evidenced by decreased rhodamine123 staining ( 9 ). Morphologically, these mitochondria are characterized by increased length relative to width, coupled with an overall reduction in count and disrupted morphology ( 9 ). This elongation is particularly noteworthy, as it implies heightened mitochondrial fusion, a phenomenon often observed under conditions such as nutrient withdrawal or increased OxPhos ( 28 , 29 ). Actually, chondrocytes from relatively preserved articular cartilage demonstrate significantly higher mitochondrial respiration capacity compared to those from severely damaged lesions ( 9 ). As OA progresses, however, these metabolic adaptations begin to fail. This is evidenced by a diminished capacity of the respiratory chain and by a decrease in the number of mitochondria coupled with an increase in mitochondrial fission, leading to mitochondrial dysfunction ( 9 , 27 ). Consequently, impaired mitochondrial function can disrupt ATP production and increase oxidative stress in chondrocytes, both of which are key contributors to the pathogenesis of OA ( 30 ). To date, the molecular mechanism behind the metabolic shift towards glycolysis in OA chondrocytes has remained largely unclear. In this study, we demonstrated a significant increase in PDK2 among PDK isoforms under IL-1β-mediated catabolic condition and in OA chondrocytes (Fig. 1 ). Moreover, PDK2 deficiency led to a decrease in the phosphorylation of PDH under IL-1β-treated catabolic conditions, indicating the inactivation of PDH complex that converts pyruvate to acetyl-CoA (Fig. 4 b). These findings suggest that PDK2 may be a key regulator of chondrocyte metabolism under catabolic conditions. Indeed, our data confirmed that PDK2 deficiency, at least partially, enhanced OxPhos in IL-1β-treated chondrocytes, while reducing glycolysis (Fig. 3 ). PDK, serving as a negative feedback mechanism, is activated by the products of the PDH reaction and TCA cycle, such as NADH, high energy charge, and acetyl-CoA ( 17 ). This activation leads to an inactivation of PDH. On the other hand, a decreasing energy charge and increasing pyruvate concentrations inhibit PDK activity, thereby leading to increased PDH activation ( 31 ). Although several mechanisms, including lactate dehydrogenase-A (LDH-A), hypoxia-inducible factor 1A (HIF1A), the AKT-mTOR signaling pathway, and pyruvate kinase M2, are known to control the glycolytic shift in chondrocytes ( 8 , 32 – 34 ), PDK is known to mediate the Warburg effect—characterized by enhanced aerobic glycolysis ( 35 ). It may directly lead to the glycolytic shift observed in OA. Given this, inhibiting PDK2 could be a promising approach for the metabolic reprogramming of chondrocytes. The expression of PDK isoforms in chondrocytes has not been extensively characterized. Our data revealed an increase in PDK2, whereas other PDK isoforms, such as PDK1, PDK3, and PDK4, showed a decrease in IL-1β-treated catabolic chondrocytes and in vivo OA cartilage (Fig. 1 ). Consistent with our findings, a recent study reported a significant downregulation of PDK1 mRNA and protein expressions in OA articular cartilage, although it did not specify the expression of other PDK isoforms ( 36 ). The lack of any noticeable phenotype in endochondral bone formation in Pdk2 -deficient mice also suggests that PDK2 plays a limited role in the physiological maturation of chondrocytes (data not shown). This evidence of PDK2 being specifically expressed in catabolic chondrocytes suggests that targeting PDK2 could minimally affect normal cartilage physiology, while effectively addressing OA conditions, thereby offering a potential advantage in the development of OA drugs targeting PDK2. Our data revealed that IL-1β-mediated p38 MAPK phosphorylation was significantly reduced in Pdk2 -deficient chondrocytes (Fig. 5 ). Apoptosis signal-regulating kinase 1 (ASK1), which is positioned upstream of p38 MAPK, is a well-known redox-sensitive kinase ( 37 , 38 ), implying that lower ROS levels in Pdk2 -deficient chondrocytes may lead to reduced phosphorylation of p38 MAPK. Furthermore, p38 MAPK signaling itself can induce oxidative stress via MAP kinase-activated protein kinase 2 (MK2), potentially creating a positive feedback loop between p38 MAPK and oxidative stress in catabolic condition ( 39 ). Taking one step further, our data showed that p38 inhibitor significantly suppressed ROS generation in WT chondrocytes, whereas this inhibition of ROS was not observed in PDK2 KO chondrocytes (Fig. 6 a). This implies that p38 MAPK does not influence ROS generation in conditions prone to OxPhos due to PDK2 deficiency. In other words, p38 MAPK may primarily contribute to an increase in ROS in situations of mitochondrial dysfunction, characterized by reduced OxPhos. Although OA is primarily a degenerative disease, omics data from OA articular cartilage have revealed a sustained increase in inflammatory signatures ( 40 , 41 ). Our findings indicate that p38 MAPK could be an essential intermediary, linking metabolic alterations to inflammatory gene signature in OA cartilage. Another significant observation regarding signaling changes associated with PDK2 deficiency is the more gradual reduction in AMPK phosphorylation (Thr172) by IL-1β stimulation. As implied by its name 'AMP-activated protein kinase', AMPK is activated by AMP, which typically increases under metabolic stress conditions ( 42 ). Conversely, when metabolic balance is restored and ATP levels rise, this leads to the kinase’s inactivation ( 43 ). Beyond metabolic conditions, the regulation of AMPK also involves several upstream kinases; LKB1, CaMKKβ, and TAK1 are key activators, while PKC, AKT, PKA, and PP2A contribute to its inactivation ( 44 ). With regards to oxidative stress, although AMPK activation helps its suppression, oxidative stress can, in return, lead to the inactivation of AMPK ( 45 ). Thus, the reduced levels of ROS observed in PDK2 deficiency could slow down the inactivation of AMPK, potentially aiding in the maintenance of metabolic homeostasis of chondrocytes under catabolic condition. In conclusion, the loss-of-function of PDK2, which is upregulated under catabolic conditions of chondrocytes, leads to a metabolic shift towards OxPhos. This shift is associated with a reduction in oxidative stress and cellular senescence, and is protective in the progression of OA. Our findings suggest that metabolic modulation towards OxPhos deserves particular attention as a potential target for OA treatment. DECLARATIONS Acknowledgments We thank Prof. In-Kyu Lee (Endocrinology, Kyungpook National University) for valuable advice on this project and for assisting with animal experiments. We thank Dr. Ho-Yeol Lee (Kyungpook National University) for helping with the extracellular flux analysis. Ethics approval and consent to participate All experiments were conducted in accordance with approved animal protocols and guidelines established by the Animal Care Committee of Kyungpook National University (Approval No. KNU-2018-62/54). Competing interests The authors declare that they have no conflicts of interest with the contents of this article. Funding This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (grant numbers NRF-2020R1A2C1004517). Author Contributions Study conception and design: J.H., Y.K., S.H., Acquisition of data: J.H., Y.K., Analysis and/or interpretation of data: J.H., S.H., Wrote the paper: J.H., S.H. Critical revision: J.H., Y.K., S.H., All authors read and approved the final manuscript. Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. REFERENCES Thomas CM, Fuller CJ, Whittles CE, Sharif M. Chondrocyte death by apoptosis is associated with cartilage matrix degradation. Osteoarthritis Cartilage. 2007;15(1):27-34. Poole CA, Flint MH, Beaumont BW. Morphological and functional interrelationships of articular cartilage matrices. J Anat. 1984;138 ( Pt 1)(Pt 1):113-38. Wang Y, Wei L, Zeng L, He D, Wei X. Nutrition and degeneration of articular cartilage. Knee Surg Sports Traumatol Arthrosc. 2013;21(8):1751-62. Otte P. Basic cell metabolism of articular cartilage. Manometric studies. Z Rheumatol. 1991;50(5):304-12. Gavriilidis C, Miwa S, von Zglinicki T, Taylor RW, Young DA. Mitochondrial dysfunction in osteoarthritis is associated with down-regulation of superoxide dismutase 2. Arthritis Rheum. 2013;65(2):378-87. Heywood HK, Knight MM, Lee DA. 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Additional Declarations (Not answered) Supplementary Files PDK.Supplementarydata.pdf Supplementary data Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2025 Read the published version in Experimental & Molecular Medicine → Version 1 posted Editorial decision: revise 22 Mar, 2024 Review # 2 received at journal 20 Mar, 2024 Review # 1 received at journal 14 Mar, 2024 Reviewer # 2 agreed at journal 05 Mar, 2024 Reviewer # 1 agreed at journal 28 Feb, 2024 Reviewers invited by journal 25 Feb, 2024 Submission checks completed at journal 12 Feb, 2024 First submitted to journal 10 Feb, 2024 Editor assigned by journal 10 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3947364","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":274760634,"identity":"c4cfa08b-53fb-41f7-b347-03391f29fdb0","order_by":0,"name":"Seungwoo HAN","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACCWYGhg+M/2rkQJwDD4jUwjiDge2YMVhLAlFaGMBamBMbQDyitEi2Mx9s+MDDlj4/7PBDoC12croNBLRIM7MlNs6QkMndeDvNAKgl2djsAAEtcsw85o95DNhyN85OAGk5kLiNsBb+j81/EpjTDWenfyBOizQzD2MzwwHmBHnpHCJtkWxmM2zsbThmuEE6p+BAggERfpE4f/hhw8+GGnn52embP3yosJMjqAUODMAqDYhVDgLyDaSoHgWjYBSMghEFAIt+Q5GAu69gAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1614-7635","institution":"Kyungpook University university","correspondingAuthor":true,"prefix":"","firstName":"Seungwoo","middleName":"","lastName":"HAN","suffix":""},{"id":274760635,"identity":"b965650e-e34b-425d-a849-6c1c27d61038","order_by":1,"name":"Jin Han","email":"","orcid":"","institution":"Kyungpook national univerisity Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Han","suffix":""},{"id":274760636,"identity":"c3769b15-69e0-4d5f-9877-b1c931434a92","order_by":2,"name":"Yoon Hee Kim","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"Hee","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-02-11 03:00:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3947364/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3947364/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s12276-025-01400-9","type":"published","date":"2025-02-03T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51776098,"identity":"0dbb6b47-a18d-449b-8e84-1fd346e62a88","added_by":"auto","created_at":"2024-02-28 20:46:57","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9689040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDK2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeficiency reduces the severity of cartilage degradation, oxidative stress-related DNA damage, and pain-related behaviors in DMM-induced OA mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Safranin-O staining and immunostaining for MMP13 and 8-oxo-dG were performed on WT and \u003cem\u003ePdk2\u003c/em\u003e KO mice 8 wk after DMM and Sham surgery, and representative figures were displayed. Scale bars indicate 100 μm. (b) Osteoarthritis Research Society International (OARSI) grade, subchondral bone plate thickness, and osteophyte size were quantified, and expressed as mean ± SEM. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Mann–Whitney U test. n = 7. (c) Quantification of the percentage of MMP13– and 8-oxo-dG–positive chondrocytes above the tide mark. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Mean ± SEM; Mann–Whitney U test; n = 7. (d) Pain behavior tests of were conducted once a week in DMM and sham-operated mice. The weight bearing on the hind paw was assessed by incapacitance test, which represent the ratio of the weight bearing between the ipsilateral and contralateral hind paw; thus any percentage less than 100% indicates hind limb unweighting. Thermal and mechanical pain sensation was assessed with Hargreaves test using hot plate and von Frey test, respectively. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, compared between WT and \u003cem\u003ePdk2 \u003c/em\u003eKO mice in DMM group, Mann–Whitney U test. n = 7.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/843230f26e1c0e74ef72d894.jpg"},{"id":51776091,"identity":"fded03ed-25a8-4f0b-9ab6-46160dcd6fbc","added_by":"auto","created_at":"2024-02-28 20:46:56","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4871830,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDK2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeficiency partially restores IL-1β-induced metabolic alterations, leading to an increase in ATP and NAD+/NADH levels in primary chondrocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a, b) Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured in primary chondrocytes isolated from WT and \u003cem\u003ePdk2\u003c/em\u003e KO mice using XF96 Seahorse analyzer. The basal respiration and ATP production in OCR analysis and the glycolysis and glycolytic capacity in ECAR analysis from one time point was quantified and displayed as mean ± SEM.*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Mann–Whitney U test. n = 4. (c, d) The level of ATP and NAD+/NADH ratio in the culture media were quantified by colorimetric assay at 570 nm and 450 nm, respectively; mean ± SEM, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Mann–Whitney U test. n = 3.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/de4046773e1a6ef48d063ee6.jpg"},{"id":51776096,"identity":"f3eb795f-ebb6-4e09-9a0d-6830279a6353","added_by":"auto","created_at":"2024-02-28 20:46:57","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":12726548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDK2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeficiency not only increases the active form of PDH and has anabolic effects in IL-1β-treated chondrocytes, but also decreases oxidative stress and cellular senescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The expression of chondrogenic marker genes, such as \u003cem\u003etype 2 collagen\u003c/em\u003e (\u003cem\u003eCol2\u003c/em\u003e) and \u003cem\u003eaggrecan\u003c/em\u003e, catabolic proteases such as \u003cem\u003eMMP13\u003c/em\u003e and \u003cem\u003eAdamts5\u003c/em\u003e, and SASP-related biomarkers like \u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eVEGF\u003c/em\u003e, was assessed by real-time RT-PCR. Results for mRNA expression are displayed as the fold increase of gene expression normalized to \u003cem\u003eGapdh\u003c/em\u003e. mean ± SEM, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Mann–Whitney U test. n = 4. (b) The phosphorylated-PDH (p-S\u003csup\u003e293\u003c/sup\u003e-PDH), PDH-E1, Col2, Sirt1, and MMP13 protein levels in the WT and \u003cem\u003ePdk2\u003c/em\u003e KO chondrocytes after 24 hr of IL-1β were assessed by Western blot analysis. Western blot band quantification for p-S\u003csup\u003e293\u003c/sup\u003e-PDH protein levels was normalized to PDH-E1, and the levels of Col2, Sirt1, and MMP13 were normalized to β-actin. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Mann–Whitney U test. n = 3. (c) Staining of dihydroethidium (DHE; red), a fluorescent probe for ROS, and 8-oxo-dG (green), a marker for oxidative DNA damage, was conducted in primary chondrocytes from WT and \u003cem\u003ePdk2\u003c/em\u003e KO mice treated with IL-1β (10 ng/mL) for 24 hr. Nuclei of cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 100 μm. DHE and 8-oxo-dG fluorescence-positive cells were quantified and expressed as the ratio to DAPI-positive cells. *p \u0026lt; 0.05. Mann–Whitney U test. n = 4. (d) The relative expression of oxidative stress marker genes such as \u003cem\u003eHmox\u003c/em\u003e and \u003cem\u003eFth\u003c/em\u003e was evaluated with by real-time RT-PCR. mRNA expression results are displayed as the fold increase of gene expression normalized to \u003cem\u003eGapdh\u003c/em\u003e. *p \u0026lt; 0.05. Mann–Whitney U test. n = 4. (e) Senescence-associated β-galactosidase (SA-β-gal) staining was conducted in the primary chondrocytes treated with IL-1β (10 ng/mL) for indicated times. Scale bar indicates 100 μm. β-gal-positive cells were counted from six different fields from 3 biological replicates, and the percentages of positive cells were determined. *p \u0026lt; 0.05. Mann–Whitney U test. n = 3.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/5b0dc63f6855a661d71c11e7.jpg"},{"id":51776094,"identity":"4ac180b9-d2f4-4c48-bdee-5abc4ec68a8d","added_by":"auto","created_at":"2024-02-28 20:46:57","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4777378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDK2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeficiency enhances FoxO3a signaling and prevents the downregulation of AMPK signaling, while it suppresses p38 MAPK activity in the IL-1β-induced catabolic condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The phosphorylation of FoxO3a, AMPKα, p38, JNK, mTOR, p65, and Akt was assessed in primary chondrocytes from WT and Pdk2 KO mice, stimulated by IL-1β (20 ng/mL) for the indicated treatment durations. Experiments were performed in triplicate and representative blot was displayed. (b) Western blot band quantification for phosphorylated proteins was based on normalization to the corresponding non-phosphorylayed total protein. *p \u0026lt; 0.05. Mann–Whitney U test. n = 3.\u003c/p\u003e","description":"","filename":"Figure51.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/bbe32c98efa9c025d9cb1374.jpg"},{"id":51776097,"identity":"4620c4d2-4a2f-4d76-a231-4d79bdc89b0d","added_by":"auto","created_at":"2024-02-28 20:46:57","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":10876087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDK2 is involved in a positive feedback loop between p38 MAPK and mitochondrial ROS production \u003cbr\u003e\n\u003c/strong\u003e(a) p38 MAPK inhibition led to a decrease of ROS production and cellular senescence in WT chondrocytes, but not in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes. The chondrocytes from WT and \u003cem\u003ePdk2 \u003c/em\u003eKO were pretreated with p38 MAPK inhibitor, SB203580, and mitochondrial ROS inhibitor, MitoTempo for 30 min, and then cells were treated with IL-1β (10 ng/mL) for 24 hr, and stained with DHE (red) for whole cellular ROS, 8-oxo-dG (green), and MitoSox (red) for mitochondrial ROS and their nuclei were counterstained with DAPI (blue). Cellular senescence were assessed with SA-β-gal staining. Scale bar indicates 100 μm. DHE, 8-oxo-dG, and MitoSox fluorescence-positive cells were quantified and expressed as the ratio to DAPI-positive cells. The area of SA-β-gal expression was quantified using densitometry with ImageJ® software. *p \u0026lt; 0.05, #p \u0026lt; 0.05. Mann–Whitney U test. n = 4. (b) The phosphorylation of p38 MAPK was assessed in the presence of p38 inhibitor (SB203580) and mitochondrial ROS inhibitor (MitoTempo). The chondrocytes from WT and \u003cem\u003ePdk2\u003c/em\u003e KO mice were stimulated with IL-1β (20 ng/mL) for indicated time, and subjected to Western blot analysis to assess the phosphorylated and total protein levels of p38 MAPK. *p \u0026lt; 0.05 compared to the fold value of p-p38/p38 in WT PBS controls at 15 min post-stimulation. # p \u0026lt; 0.05 compared to that of p-p38/p38 of PDK2 KO PBS controls at 15 min after stimulation. Mann–Whitney U test. n = 4.\u003c/p\u003e","description":"","filename":"Figure61.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/b39804b79cd934ed9d6b2a26.jpg"},{"id":51776099,"identity":"5a3d176f-d54e-4e3f-b8b8-1c759ece5823","added_by":"auto","created_at":"2024-02-28 20:46:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1174871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram depicting the anabolic effects of metabolic reprogramming into oxidative phosphorylation by PDK2-deficiency in catabolic conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong PDK isoforms, PDK2 is primarily expressed in catabolic and in vivo OA condition. The loss of PDK2 function enhances OxPhos and ATP/NAD\u003csup\u003e+\u003c/sup\u003e production, which leads to a reduction in oxidative stress. Mechanistically, PDK2 plays a crucial role in the positive feedback loop between oxidative stress and p38 MAPK under catabolic conditions of chondrocytes. \u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure72.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/31816a255a81eb2e86c4b334.jpg"},{"id":75303670,"identity":"e6636824-c4f2-493e-a03e-e7157bb3bc5a","added_by":"auto","created_at":"2025-02-03 08:05:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45464928,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/6d852de0-4002-4661-8b08-9a2e32304e66.pdf"},{"id":51776635,"identity":"83ab40ec-a777-4e60-978a-ee424e2a97b4","added_by":"auto","created_at":"2024-02-28 20:54:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":613027,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary data\u003c/p\u003e","description":"","filename":"PDK.Supplementarydata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3947364/v1/e5ed37a581503b74f9081701.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Enhancing oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in surgically induced osteoarthritis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOsteoarthritis (OA) is the most common form of degenerative joint disease characterized by apoptosis of chondrocyte and degradation of cartilage extracellular matrix (ECM), which ultimately leads to joint failure (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Chondrocytes, the only cell type found in cartilage tissues, produce cartilage specific ECM proteins such as type 2 collagen and aggrecan, and they become trapped within ECM proteins they produce, resembling a state of hibernation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). As cartilage is an avascular tissue, chondrocytes exist in hypoxic conditions despite the diffusion of oxygen from synovial fluid or subchondral bone (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In such hypoxic milieu, chondrocytes rely predominantly on glycolysis rather than oxidative phosphorylation (OxPhos) which comprises only less than 10% of total cellular ATP production (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, chondrocytes also exhibit a significant level of metabolic flexibility in the catabolic state of early OA, where they can alter their metabolic machinery towards OxPhos (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). This allows them to adapt to the catabolic conditions, hence enhancing their survival and function. As OA progresses and mitochondrial dysfunction occurs (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), however, the metabolic adaptation of the OA chondrocytes reaches its limit, leading to a metabolic shift towards the glycolytic pathway (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Actually, chondrocytes in early-stage OA of Kellgren and Lawrence (K-L) grade 1 exhibit a metabolic phenotype characterized by increased OxPhos and decreased glycolysis, compared to chondrocytes in advanced-stage OA of K-L grade 4 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This metabolic shift not only makes it increasingly difficult for the chondrocytes to meet their energy demands, but also amplifies oxidative stress in lactate dehydrogenase-mediated manner (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The increase of oxidative stress, in turn, worsen mitochondrial dysfunction, including mitochondrial DNA damage and increased membrane permeability, which eventually contributes to chondrocyte apoptosis and senescence (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). In this way, the glycolysis-prone metabolism of chondrocyte interacts with and amplifies both mitochondrial dysfunction and oxidative stress during OA progression (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Despite understanding that metabolic imbalances in chondrocytes play a crucial role in the pathogenesis of OA, we still lack conclusive evidence on whether redirecting chondrocyte metabolism towards OxPhos could indeed impact its progression.\u003c/p\u003e \u003cp\u003eGlucose that enters chondrocytes through mainly glucose transporter (GLUT) 1 and GLUT3 is metabolized into pyruvate, the end product of glucose catabolism (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This pyruvate moves to the mitochondria, where it is converted to acetyl-Coenzyme A (acetyl-CoA) by pyruvate dehydrogenase (PDH), the enzyme that links the cytoplasmic glycolysis pathway to the mitochondrial tricarboxylic acid (TCA) cycle. As a canonical input to the TCA cycle, an increase of acetyl-CoA levels leads to an enhancement in the rate of the TCA cycle and consequently OxPhos (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Therefore, the activity of PDH, which converts pyruvate into acetyl-CoA, is the overall rate-limiting and gatekeeping factor in pyruvate-driven OxPhos (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The activity of PDH is regulated by PDH kinase (PDK), a Serine/ Threonine kinase, which phosphorylates the α subunit of PDH, thereby inactivating it (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The activity of PDK, in turn, is controlled by pyruvate, NAD+, ATP, acetyl-CoA, and nuclear transcription factors, such as FoxO, PPAR, and PGC1α (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). From the perspective of the metabolic shift towards glycolysis in OA progression, a potential therapeutic strategy might be derived from the metabolism reprogramming to enhance OxPhos through the inhibition of PDK (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). PDK has four isoforms, PDK1 through PDK4. Among them, PDK2 is expressed ubiquitously, while the remaining PDK isoforms show tissue-specific distribution: PDK1 is primarily found in heart tissue, the pancreatic islet, and skeletal muscle. PDK3 has a relatively limited tissue distribution in the testis, kidney, and brain, and PDK4 in the heart, skeletal muscle, liver, kidney, and pancreatic islets (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). However, the expression of PDKs in chondrocytes and their functional role in OA have yet to be investigated.\u003c/p\u003e \u003cp\u003eThe objective of this study was to determine whether the inhibition of PDK affects the metabolic flexibility of chondrocytes, and contributes to cartilage degeneration in a surgical model of OA. First, we verified the expression of PDK isoforms in both in vitro catabolic condition and in vivo OA cartilage, which revealed an increase of PDK2 under catabolic conditions. The role of PDK2 in OA progression was investigated using a surgical OA model in \u003cem\u003ePdk2\u003c/em\u003e-deficient mice, and we subsequently focused on its metabolic phenotype, expression of anabolic and catabolic factors, ROS production, and cellular senescence. From a mechanistic perspective, we identified the signaling pathways affected by \u003cem\u003ePdk2\u003c/em\u003e-deficiency and elucidated their role on the production of ROS and cellular senescence.\u003c/p\u003e"},{"header":"MATERIALS and METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics and isolation of primary chondrocytes\u003c/h2\u003e \u003cp\u003eThis study was conducted in accordance with the guidelines of National Research Council (US) Committee for the Care and Use of Laboratory Animals (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and was approved by the Institutional Review Board of the Kyungpook National University School of Medicine (Daegu, Korea), under the approval number KNU 2022\u0026thinsp;\u0026minus;\u0026thinsp;0203. Mice genetically deficient in the PDK2 gene (PDK2 KO) on the C57BL/6J background were kindly provided by Dr. In-Kyu Lee (Kyungpook National University, Daegu, Korea). Primary chondrocytes were isolated from the articular cartilage of femur and tibia of 5-day-old C57BL/6J mice. Isolated cartilage pieces were minced into small fragments, underwent a 15-minute incubation in 0.25% trypsin-EDTA at 37\u0026deg;C with gentle shaking, and digested in a solution containing 0.2% collagenase type II, prepared in DMEM, at 37\u0026deg;C with gentle agitation for 4 hours. After digestion, the cell suspension was filtered through a 40 \u0026micro;m cell strainer and washed twice with PBS. The isolated chondrocytes were resuspended in complete 3:2 F12:DMEM-based culture medium supplemented with 0.25% L-glutamine, and 0.25% penicillin/streptomycin, then seeded in 6-well plates at a concentration of 1 x 10^5 cells per well, and cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO2. To reduce de-differentiation risks, only chondrocytes at passage 0 were used in experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA and protein expression analysis\u003c/h2\u003e \u003cp\u003eTo induce a catabolic condition on primary chondrocytes, cells were treated with 10 ng/ml of IL-1β, and incubated for 6 hrs for RNA isolation and 24 hrs for protein isolation. For signaling analysis, cells were starved overnight and then stimulated with 20 ng/ml of IL-1β for the indicated time. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA), followed by the generation of the first strand cDNA with Superscript III reverse transcriptase (Invitrogen). A ViiA\u0026trade; 7 Real-Time PCR System (Applied Biosystems, CA) and SYBR\u0026reg; Green Master Mix (Applied Biosystems) were employed for real-time qPCR. Primers for real-time qPCR can be found in Supplementary Table\u0026nbsp;1. Target gene expression levels were calculated using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCT method and normalized to the geometric mean of Gapdh. All qRT-PCR analysis were carried out in triplicate, repeated three to five times, and the average results for each were presented.\u003c/p\u003e \u003cp\u003eTotal proteins were extracted using 300 \u0026micro;L RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN). Total cell lysates containing 10\u0026ndash;20 \u0026micro;g protein were loaded to 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore Corporation, Billerica, MA). Membranes were blocked using 5% skimmed milk in PBS with 0.25% Tween-20 (PBST) and incubated with the primary antibodies against PDK1 (Abcam, Cambridge, UK, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDH-E1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, Santa Cruz, CA, #sc-377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, Beverly, MA, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), p-AMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272), and β-actin (Sigma-Aldrich, Burlington, MA, #A1978) overnight at 4\u0026deg;C. After washing with PBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 hours, and then blots were developed using enhanced chemiluminescence western blotting detection reagent (Thermo Fisher Scientific) and examined with the MicroChemi system (DNR Bio-imaging Systems). All analysis was biologically triplicated, and the western blot band intensities were quantified using ImageJ software (version 1.8.0, NIH). Band intensities of target proteins were normalized to the β-actin band intensity of respective lanes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSurgical OA induction\u003c/h2\u003e \u003cp\u003eOA was surgically induced in 12-week-old male C57BL/6J mice, including 7 PDK2 KO mice and 7 of their littermates, through destabilization of the medial meniscus (DMM) surgery under general anesthesia. The sample size of each group was calculated based on the assumptions of a type I error (α) of 0.1, a standard deviation (σ) of 10, an effect size (δ) of 15, and a power of 0.8. Ligament of medial meniscus was dissected in the right knee to induce OA, and the sham operation was conducted on left knee as a control. After surgery, mice were housed under controlled conditions with a 23\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C temperature, 50% humidity, and a 12-hour light/dark cycle. They were kept under specific pathogen-free conditions in the animal facilities of Kyungpook National University Chilgok Hospital.\u003c/p\u003e \u003cp\u003eEight weeks after surgery, the mice were euthanized via cervical dislocation, and knee joints were collected for histological examination, and all specimens were used for analysis. This study was conducted in accordance with ARRIVE guidelines 2.0, and its checklist has also been offered in the Supplementary sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePain behavior analysis\u003c/h2\u003e \u003cp\u003eTwo weeks post-surgery, pain behavior was assessed weekly using the spontaneous weight-bearing asymmetry test (Incapacitance test), the hot plate test, and the threshold punctate mechanical stimulation (von Frey test). To assess spontaneous weight bearing on the hind limbs, the incapacitance meter (SangChung commercial, Seoul, Korea) was used to measure the downward force applied by each hind limb. Mice were briefly placed in a restrainer, with hind limbs resting on two weight averaging platform pads. Measurements of the paw pressure of each hind limb were taken for 10 seconds, approximately 10 times, and results were averaged. Data were expressed as the percentage of weight distributed on the ipsilateral hind limb. Next, thermal pain sensitivity was assessed with the hot plate test (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Mice were placed on a heated plate (plantar test IR emitter, Ugo Basile, Gemonio, Italy), and the time until they displayed pain behaviors such as licking or shaking their paws was recorded. Finally, tactile allodynia was assessed using the von Frey test. Calibrated monofilaments (von Frey hairs; Stoelting, Wood Dale, IL) were applied to the plantar surface of both ipsilateral and contralateral hind paws, placed on an elevated maze in an acrylic cage. A paw withdrawal was considered a positive response. The 50% withdrawal threshold was determined upon six repeated applications of varying force with the von Frey filament, using the up-down method (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSafranin-O and immunofluorescent staining\u003c/h2\u003e \u003cp\u003eMouse knee joints were fixed in 4% paraformaldehyde for 24 hours, decalcified in 10% EDTA for three weeks, and then embedded in paraffin. The embedded blocks were sectioned at a thickness of 6 \u0026micro;m. After deparaffinization and rehydration, sections were stained in 0.1% Safranin-O solution for 5 minutes and counterstained with fast green solution for 1 minute. Cartilage destruction was scored in all four quadrants of the joint (grade 0\u0026ndash;24) and on the medial tibial plateau (grade 0\u0026ndash;6) by two observers under blinded conditions, using the OARSI scoring system (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). For immunofluorescent staining, rehydrated sections were antigen-retrieved in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0). The sections were then blocked with 2% bovine serum albumin (BSA) in PBS for 1 hour, followed by overnight incubation at 4 ℃ with primary antibodies against PDK1 (#ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDH-E1α (Ser232, #AP1063), PDH-E1 (#sc-377092), MMP13 (#ab51072), 8-oxo-dG (#sc-66036), or normal rabbit IgG in 1% BSA. For immunofluorescence, the sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 hours, then counterstained with DAPI. Finally, the sections were mounted with anti-fade mounting solution (Vector Labs) and imaged and quantified using a KI-3000F fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSeahorse real-time cell metabolic analysis\u003c/h2\u003e \u003cp\u003eChondrocyte metabolism was analyzed using an XF96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA). Primary chondrocytes from PDK2 KO mice and their littermates were plated in Seahorse XF96 plates at a density of 50,000 cells per well. Confluent chondrocytes were incubated with IL-1β (10 ng/mL) or without it for 24 hours. For the mitochondrial stress test, cells were equilibrated for 1 hour in serum-free Seahorse XF Base Medium. Basal cellular respiration was initially measured, and mitochondrial respiration inhibitors, including 1 \u0026micro;M oligomycin, 1 \u0026micro;M FCCP, and a 1:1 mixture of 2 \u0026micro;M antimycin A with 1 \u0026micro;M rotenone, were sequentially injected into the assay wells. Mitochondrial ATP production was expressed as the oxygen consumption rate (OCR; pmol of O\u003csub\u003e2\u003c/sub\u003e/min). In the glycolysis stress test, cells were starved for 1 hour in glucose-free Seahorse XF DMEM, followed by sequential treatment with 20 mM glucose, 1 \u0026micro;M oligomycin, and 100 mM 2-DG. Real-time measurements of proton accumulation in the media were taken and quantified as the extracellular acidification rate (ECAR; mpH/min) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Following completion of either the mitochondrial or glycolysis stress test, cell nuclei were counted following in situ DAPI staining. Analysis data were then normalized based on the cell count per well, using a normalization unit of 5,000 cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eATP and NAD+/NADH measures\u003c/h2\u003e \u003cp\u003eChondrocytes from PDK2 KO and their littermates were seeded at 5,000 cells per well in 96-well plates for ATP measure, and at 20,000 cells per well in six-well plate for NAD+/NADH measure, and treated with 10 ng/ml of IL-1β or without it for 24 hrs. ATP concentration was measured using a commercial ATP detection kit (Abcam #ab83355), following the manufacturer's protocol. Briefly, chondrocytes were homogenized in 15 \u0026micro;L of ATP assay lysis buffer, and aliquots of the cell lysates were incubated with the ATP Reaction Mix for 30 minutes in the dark. The absorbance of the mixtures was quantified using a spectrophotometer at a wavelength of 570 nm. ATP levels were calculated from a standard curve, which was plotted using the serial dilution of authentic ATP.\u003c/p\u003e \u003cp\u003eThe NAD+/NADH ratio was determined using a commercial NAD+/NADH Assay Kit (Abcam, ab65348) following the manufacturer's instruction. Briefly, chondrocytes were collected by cell scraping, washed three times with pre-cooled PBS, and lysed with the extraction buffer solution. After centrifugation at 12,000 rpm for 5 min at 4\u0026deg;C, the supernatant was collected. Protein concentration was determined using a BCA kit (Beyotime Biotechnology, China). Samples were then heated at 60\u0026deg;C for 30 min to completely decompose NAD\u0026thinsp;+\u0026thinsp;in the sample. Then, 50 \u0026micro;l of each sample was mixed with 100 \u0026micro;l of Reaction Mix and incubated at room temperature for 5 min. Subsequently, 10 \u0026micro;l of NADH Developer was added to each well and incubated at room temperature for 2 hr. The absorbance of the mixtures was quantified using a spectrophotometer at a wavelength of 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAssess oxidative stress: detection of intracellular ROS and oxidative DNA damage\u003c/h2\u003e \u003cp\u003eIntracellular ROS and mitochondrial ROS was assessed with dihydroethidium (DHE) and Mitosox Red (ThermoFisher Scientific, #M36008) fluorescent dye, respectively. Primary chondrocytes were fixed with 4% paraformaldehyde for 10 min, permeabilized using 0.25% Triton X-100, and then rinsed three times in PBS. To assess intracellular ROS, chondrocytes were incubated with 5 \u0026micro;M DHE for 30 min at 37\u0026deg;C, washed with PBS, and then counter-stained with DAPI. Images were captured using a KI-3000F fluorescence microscope (Korealabtech, South Korea).\u003c/p\u003e \u003cp\u003eDNA damage caused by oxidative stress was evaluated by immunostaining using 8-oxo-dG antibody. Fixed and permeabilized cells were incubated with the primary 8-oxo-dG antibody overnight at 4\u0026deg;C. Then cells were incubated with a secondary antibody for 2 hours, counter-stained with DAPI, and imaged using a fluorescence microscope. Fluorescence-positive cells were quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSenescence β-galactosidase staining\u003c/h2\u003e \u003cp\u003eβ-galactosidase activity was detected using β-galactosidase staining kit (Cell Signaling Technology, #9860). Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, and then incubated for 2 hrs in the staining solution containing 1 mg/mL X-gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl₂ in pH 7.4 PBS. Then, cells were washed with PBS and visualized under a light microscope, and the percentage of blue colored cells was calculated by counting at least five random fields per sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are presented herein as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analysis to compare the mean values of two groups was performed using Mann\u0026ndash;Whitney U test, a nonparametric test as the sample size is small and it cannot be assumed as normal distribution. \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant. Statistical analyses were performed with Prism software version 8.0 (GraphPad Software, La Jolla, CA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eAmong PDK isoforms, PDK2 increases under IL-1\u0026beta;-induced catabolic condition and in the cartilage of surgically induced OA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo comprehend the PDK-mediated metabolic modulation in OA chondrocytes, we first investigated the expression of PDK isoforms under IL-1\u0026beta;-mediated catabolic conditions. Treatment with IL-1\u0026beta; increased the mRNA levels of \u003cem\u003ePDK2\u003c/em\u003e and \u003cem\u003ePDK4\u003c/em\u003e, but not \u003cem\u003ePDK1\u003c/em\u003e and \u003cem\u003ePDK3\u003c/em\u003e, in primary chondrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). At the protein level, however, only PDK2 showed an increase following IL-1\u0026beta; treatment. Simultaneously, the phosphorylated, inactive form of PDH (p-S\u003csup\u003e293\u003c/sup\u003e-PDH) increased, suggesting that PDK2 may be involved in the phosphorylation of PDH under the IL-1\u0026beta;-mediated catabolic conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). To confirm this increase in PDK2, we examined the expression of PDK isoforms in the articular cartilage over time following DMM surgery in mice. Phosphorylated PDH, which indicates the inactivation of PDH, increased in the early phase of OA 2 weeks post-DMM surgery. This increase was accompanied by a rise in PDK2, which continued to rise over time. PDK4 showed a trend of increase around 8 weeks, but it was not statistically significant, while PDK1 and PDK3 did not show any increase in the DMM-induced model of OA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDK2 deficiency attenuates the severity of OA, oxidative stress, and pain-related behaviors in DMM-induced murine OA model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of chondrocyte metabolic reprogramming on OA progression, we compared the phenotype of DMM-induced OA in WT and \u003cem\u003ePdk2\u003c/em\u003e KO mice. Compared to WT mice, genetic deletion of PDK2 significantly lessened the progression of DMM-induced OA. This was evidenced by the decrease in cartilage degradation, as quantified by the OARSI score, and reduced osteophyte maturation and subchondral bone thickness (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). This was further supported by the immunofluorescent staining which showed fewer MMP13-positive and 8-oxo-dG-positive chondrocytes in \u003cem\u003ePdk2\u003c/em\u003e KO mice, indicating a decrease of ECM degrading protease and oxidative stress-induced DNA damage, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, c). This was accompanied by decreased pain-related behaviors in \u003cem\u003ePdk2\u003c/em\u003e KO mice compared to WT from 6 weeks post-DMM surgery, as observed in tests such as static weight bearing over the hind limbs (incapitance test), paw withdrawal time on a hot plate, and Von Frey test (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003ePDK2 deficiency partially restores the IL-1\u0026beta;-mediated metabolic shift toward glycolysis in chondrocytes\u003c/h2\u003e\n\u003cp\u003eTo determine whether PDK2 deficiency impacts chondrocyte metabolism, we assessed OxPhos and glycolysis under IL-1\u0026beta;\u0026ndash;induced catabolic conditions using the Seahorse XF-96 Extracellular Flux Analyzer. IL-1\u0026beta; treatment for 24 h markedly suppressed OxPhos, as reflected by a decrease in OCR of cultured primary chondrocytes, while it enhanced glycolysis, as indicated by an increased ECAR. PDK2 deficiency partially restored the OCR by enhancing basal respiration and ATP production in chondrocytes under IL-1\u0026beta;-treated conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). On the other hand, the IL-1\u0026beta;-mediated increase in ECAR was significantly reduced in \u003cem\u003ePdk2-\u003c/em\u003edeficient chondrocytes, resulting from decreased glycolysis and glycolytic capacity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). This PDK2-mediated metabolic reprogramming also led to a significant increase in ATP and NAD+/NADH ratio in the culture supernatant from \u003cem\u003ePdk2-\u003c/em\u003edeficient chondrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec, d). These findings suggest that the inhibition of PDK2 enhances OxPhos, potentially restoring the balance of energy homeostasis in chondrocytes under catabolic conditions like OA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePDK2 deficiency enhances PDH activity and anabolic effects, which lead to reduced oxidative stress and cellular senescence in chondrocytes under IL-1\u0026beta;-treated conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the impact of PDK2 deficiency on chondrocyte homeostasis under the catabolic condition, we then examined the mRNA expression of chondrogenic markers (\u003cem\u003eCol2\u003c/em\u003e, \u003cem\u003eaggrecan\u003c/em\u003e), catabolic proteases (\u003cem\u003eMMP13\u003c/em\u003e, \u003cem\u003eAdamts5\u003c/em\u003e), and senescence-associated secretory phenotype (SASP)-related genes (\u003cem\u003eIL-6\u003c/em\u003e, \u003cem\u003eVegf\u003c/em\u003e). In IL-1\u0026beta;-treated conditions, PDK2 deficiency increased \u003cem\u003eCol2\u003c/em\u003e and \u003cem\u003eAggrecan\u003c/em\u003e expression, while it decreased \u003cem\u003eMMP13\u003c/em\u003e and \u003cem\u003eIL-6\u003c/em\u003e expression, but had no effect on \u003cem\u003eAdamts5\u003c/em\u003e and \u003cem\u003eVegf\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). PDH enzymatic activity was enhanced in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes, as indicated by lower levels of phosphorylated PDH (p-S\u003csup\u003e293\u003c/sup\u003e-PDH) under IL-1\u0026beta;-treated conditions. Similarly to mRNA expression, the protein levels of Col2 increased in PDK2 deficient chondrocytes compared to WT under IL-1\u0026beta;-treated conditions, while those of MMP13 decreased. However, the levels of SirT1 remained unaffected (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). Next, we assessed the role of PDK2 in the oxidative stress under IL-1\u0026beta;-treated catabolic conditions. IL-1\u0026beta; treatment (10 ng/ml) significantly increased the production of ROS as well as 8-oxo-dG, a DNA damage product that occurs as a result of oxidative stress. In this condition, \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes exhibited a significant decrease in ROS and 8-oxo-dG production, suggesting that PDK2 can reduce oxidative stress in IL-1\u0026beta;-mediated catabolic conditions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). Consistent with these results, the expressions of \u003cem\u003eHmox\u003c/em\u003e and \u003cem\u003eFth\u003c/em\u003e, target genes of oxidative stress, were found to decrease in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). We further investigated the effect of PDK2 deficiency on the senescence of chondrocytes, as mitochondrial dysfunction and ROS production is closely related to cellular senescence (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). After 48 hr of treatment with IL-1\u0026beta;, a marked decrease in the expression of SA-\u0026beta;-gal, a senescence marker, was observed in the chondrocytes from PDK2 KO mice compared to WT (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). Our results collectively indicate that PDK2 loss-of-function leads to anabolic effects, and attenuates ROS levels as well as the senescence of chondrocytes under catabolic conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003ePDK2 deficiency reduces p38 signaling avtivity and sustains AMPK activation in response to IL-1\u0026beta; stimulation\u003c/h2\u003e\n\u003cp\u003eWe further examined the signaling mechanisms involved in the anabolic effects on PDK2-deficient chondrocytes under catabolic conditions, particulary focusing on metabolism and oxidative stress-related pathways such as AMPK, mTOR, FoxO3a, AKT, NFkB and MAPKs. Among these signalings, the phosphorylation of p38 MAPK (Thr180/Tyr182) was most prominently suppressed in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes compared to WT. The phosphorylation of AMPK (Thr172), which typically gets down-regulated by IL-1\u0026beta; stimulation, remained in an activated state in \u003cem\u003ePdk2\u003c/em\u003e-null chondrocytes. PDK2 deficiency led to a non-continuous increase in FoxO3a phosphorylation (Ser253) at 5 and 60 min after IL-1\u0026beta; stimulation, which suppresses its activity through cytoplasmic export and proteosomal degradation (\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e). The activation of mTOR, JNK, p65, and AKT signalings were not affected by PDK2 deficiency (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ep38 MAPK is essential for generating ROS in chondrocytes with mitochondrial dysfunction, and mitochondrial ROS, in turn, activate p38 MAPK under catabolic conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether the decrease in p38 MAPK phosphorylation is simply a result of reduced ROS or if it directly involves the reduction of ROS in the PDK2-deficient situation, we treated WT and \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes with the chemical p38 inhibitor, SB203580, and the specific scavenger of mitochondrial superoxide, MitoTempo. When we compared ROS production and cellular senescence, the inhibition of p38 significantly suppressed ROS production and senescence in WT chondrocytes, but this effect was not observed in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes, where ROS generation and senescence remained unchanged despite p38 MAPK inhibition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). This implies that in a metabolic environment where OxPhos is increased due to PDK2 deficiency, p38 MAPK does not play a significant role in ROS production and subsequent cellular senescence.\u003c/p\u003e\n\u003cp\u003eThen, we investigated how the inhibition of mitochondrial ROS affects the activation of p38 MAPK. PDK2 deficiency led to a significant reduction in the phosphorylation of p38 MAPK compared to chondrocytes from WT littermates under IL-1\u0026beta; stimulation, and treatment with SB203580 substantially delayed the activation of p38 MAPK in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes. As expected, scavenging mitochondrial superoxide using MitoTempo significantly reduced the phosphorylation of p38 MAPK at 15 minutes, compared to that of both WT and \u003cem\u003ePdk2\u003c/em\u003e-deficient controls, indicating that mitochondrial ROS are crucial for the activation of p38 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb). Collectively, these results suggest the presence of a positive feedback loop between ROS and p38 MAPK and highlight the significant role of PDK-mediated glycolytic metabolic shift in p38 MAPK-mediated ROS generation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIncreasing evidence suggests that alterations in chondrocyte metabolism toward glycolysis, associated with mitochondrial dysfunction, are critically linked to OA pathogenesis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). In this context, PDK-dependent inhibition of PDH activity may be a pivotal mechanism responsible for the glycolytic metabolic shift in catabolic chondrocytes (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Here, we identified that PDK2 is specifically upregulated in OA chondrocytes and its loss-of-function led to an increase of PDH activity to restore the IL-1β-mediated metabolic shift toward glycolysis in chondrocytes. In addition, PDK2 deficiency showed a protective phenotype in surgically induced OA model, which was accompanied by reduced oxidative stress and cellular senescence. Mechanistically, PDK2 deficiency led to decreased activation of p38 MAPK, along with a sustained activation of AMPK signaling under IL-1β-treated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Taken together, our data sheds light on the potential of metabolic reprogramming towards OxPhos as a novel therapeutic approach for OA.\u003c/p\u003e \u003cp\u003eSeveral lines of evidence indicate a critical involvement of mitochondrial dysfunction in the pathogenesis of OA (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Specifically, chondrocytes from patients with advanced OA exhibit a decrease in respiring mitochondria, as evidenced by decreased rhodamine123 staining (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Morphologically, these mitochondria are characterized by increased length relative to width, coupled with an overall reduction in count and disrupted morphology (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). This elongation is particularly noteworthy, as it implies heightened mitochondrial fusion, a phenomenon often observed under conditions such as nutrient withdrawal or increased OxPhos (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Actually, chondrocytes from relatively preserved articular cartilage demonstrate significantly higher mitochondrial respiration capacity compared to those from severely damaged lesions (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). As OA progresses, however, these metabolic adaptations begin to fail. This is evidenced by a diminished capacity of the respiratory chain and by a decrease in the number of mitochondria coupled with an increase in mitochondrial fission, leading to mitochondrial dysfunction (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Consequently, impaired mitochondrial function can disrupt ATP production and increase oxidative stress in chondrocytes, both of which are key contributors to the pathogenesis of OA (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date, the molecular mechanism behind the metabolic shift towards glycolysis in OA chondrocytes has remained largely unclear. In this study, we demonstrated a significant increase in PDK2 among PDK isoforms under IL-1β-mediated catabolic condition and in OA chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, PDK2 deficiency led to a decrease in the phosphorylation of PDH under IL-1β-treated catabolic conditions, indicating the inactivation of PDH complex that converts pyruvate to acetyl-CoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). These findings suggest that PDK2 may be a key regulator of chondrocyte metabolism under catabolic conditions. Indeed, our data confirmed that PDK2 deficiency, at least partially, enhanced OxPhos in IL-1β-treated chondrocytes, while reducing glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). PDK, serving as a negative feedback mechanism, is activated by the products of the PDH reaction and TCA cycle, such as NADH, high energy charge, and acetyl-CoA (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). This activation leads to an inactivation of PDH. On the other hand, a decreasing energy charge and increasing pyruvate concentrations inhibit PDK activity, thereby leading to increased PDH activation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Although several mechanisms, including lactate dehydrogenase-A (LDH-A), hypoxia-inducible factor 1A (HIF1A), the AKT-mTOR signaling pathway, and pyruvate kinase M2, are known to control the glycolytic shift in chondrocytes (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), PDK is known to mediate the Warburg effect\u0026mdash;characterized by enhanced aerobic glycolysis (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). It may directly lead to the glycolytic shift observed in OA. Given this, inhibiting PDK2 could be a promising approach for the metabolic reprogramming of chondrocytes.\u003c/p\u003e \u003cp\u003eThe expression of PDK isoforms in chondrocytes has not been extensively characterized. Our data revealed an increase in PDK2, whereas other PDK isoforms, such as PDK1, PDK3, and PDK4, showed a decrease in IL-1β-treated catabolic chondrocytes and in vivo OA cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Consistent with our findings, a recent study reported a significant downregulation of PDK1 mRNA and protein expressions in OA articular cartilage, although it did not specify the expression of other PDK isoforms (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The lack of any noticeable phenotype in endochondral bone formation in \u003cem\u003ePdk2\u003c/em\u003e-deficient mice also suggests that PDK2 plays a limited role in the physiological maturation of chondrocytes (data not shown). This evidence of PDK2 being specifically expressed in catabolic chondrocytes suggests that targeting PDK2 could minimally affect normal cartilage physiology, while effectively addressing OA conditions, thereby offering a potential advantage in the development of OA drugs targeting PDK2.\u003c/p\u003e \u003cp\u003eOur data revealed that IL-1β-mediated p38 MAPK phosphorylation was significantly reduced in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Apoptosis signal-regulating kinase 1 (ASK1), which is positioned upstream of p38 MAPK, is a well-known redox-sensitive kinase (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), implying that lower ROS levels in \u003cem\u003ePdk2\u003c/em\u003e-deficient chondrocytes may lead to reduced phosphorylation of p38 MAPK. Furthermore, p38 MAPK signaling itself can induce oxidative stress via MAP kinase-activated protein kinase 2 (MK2), potentially creating a positive feedback loop between p38 MAPK and oxidative stress in catabolic condition (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Taking one step further, our data showed that p38 inhibitor significantly suppressed ROS generation in WT chondrocytes, whereas this inhibition of ROS was not observed in PDK2 KO chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This implies that p38 MAPK does not influence ROS generation in conditions prone to OxPhos due to PDK2 deficiency. In other words, p38 MAPK may primarily contribute to an increase in ROS in situations of mitochondrial dysfunction, characterized by reduced OxPhos. Although OA is primarily a degenerative disease, omics data from OA articular cartilage have revealed a sustained increase in inflammatory signatures (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Our findings indicate that p38 MAPK could be an essential intermediary, linking metabolic alterations to inflammatory gene signature in OA cartilage.\u003c/p\u003e \u003cp\u003eAnother significant observation regarding signaling changes associated with PDK2 deficiency is the more gradual reduction in AMPK phosphorylation (Thr172) by IL-1β stimulation. As implied by its name 'AMP-activated protein kinase', AMPK is activated by AMP, which typically increases under metabolic stress conditions (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Conversely, when metabolic balance is restored and ATP levels rise, this leads to the kinase\u0026rsquo;s inactivation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Beyond metabolic conditions, the regulation of AMPK also involves several upstream kinases; LKB1, CaMKKβ, and TAK1 are key activators, while PKC, AKT, PKA, and PP2A contribute to its inactivation (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). With regards to oxidative stress, although AMPK activation helps its suppression, oxidative stress can, in return, lead to the inactivation of AMPK (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Thus, the reduced levels of ROS observed in PDK2 deficiency could slow down the inactivation of AMPK, potentially aiding in the maintenance of metabolic homeostasis of chondrocytes under catabolic condition.\u003c/p\u003e \u003cp\u003eIn conclusion, the loss-of-function of PDK2, which is upregulated under catabolic conditions of chondrocytes, leads to a metabolic shift towards OxPhos. This shift is associated with a reduction in oxidative stress and cellular senescence, and is protective in the progression of OA. Our findings suggest that metabolic modulation towards OxPhos deserves particular attention as a potential target for OA treatment.\u003c/p\u003e"},{"header":"DECLARATIONS","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. In-Kyu Lee (Endocrinology, Kyungpook National University) for valuable advice on this project and for assisting with animal experiments. We thank Dr. Ho-Yeol Lee (Kyungpook National University) for helping with the extracellular flux analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in accordance with approved animal protocols and guidelines established by the Animal Care Committee of Kyungpook National University (Approval No. KNU-2018-62/54).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest with the contents of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (grant numbers NRF-2020R1A2C1004517).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy conception and design: J.H., Y.K., S.H., Acquisition of data: J.H., Y.K., Analysis and/or interpretation of data: J.H., S.H., Wrote the paper: J.H., S.H. Critical revision: J.H., Y.K., S.H., All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"REFERENCES","content":"\u003col\u003e\n\u003cli\u003eThomas CM, Fuller CJ, Whittles CE, Sharif M. Chondrocyte death by apoptosis is associated with cartilage matrix degradation. Osteoarthritis Cartilage. 2007;15(1):27-34.\u003c/li\u003e\n\u003cli\u003ePoole CA, Flint MH, Beaumont BW. Morphological and functional interrelationships of articular cartilage matrices. J Anat. 1984;138 ( Pt 1)(Pt 1):113-38.\u003c/li\u003e\n\u003cli\u003eWang Y, Wei L, Zeng L, He D, Wei X. Nutrition and degeneration of articular cartilage. Knee Surg Sports Traumatol Arthrosc. 2013;21(8):1751-62.\u003c/li\u003e\n\u003cli\u003eOtte P. 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Free Radic Biol Med. 2021;166:128-39.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chondrocyte, Metabolism, Pyruvate dehydrogenase kinase, Oxidative phosphorylation","lastPublishedDoi":"10.21203/rs.3.rs-3947364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3947364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChondrocytes can shift their metabolism to oxidative phosphorylation (OxPhos) in early stages of osteoarthritis (OA), but as the disease progresses, this metabolic adaptation becomes limited and eventually fails, leading to mitochondrial dysfunction and oxidative stress. This study investigated whether enhancing OxPhos through pyruvate dehydrogenase kinase (PDK) 2 affects the metabolic flexibility of chondrocytes and cartilage degeneration in surgical model of OA. Among the PDK isoforms, PDK2 expression was increased by IL-1β in vitro, and in articular cartilage of the DMM model in vivo, accompanied by an increase in phosphorylated PDH. Mice lacking PDK2 showed significant resistance to cartilage damage and reduced pain behaviors in DMM model. PDK2 deficiency partially restored OxPhos in IL-1β-treated chondrocytes, leading to an increased APT and NAD+/NADH ratio. These metabolic changes were accompanied by a decrease of reactive oxygen species (ROS) and senescence of chondrocytes, as well as the expression of MMP-13 and IL-6 following IL-1β-treatment. At the signaling level, PDK2 deficiency reduced p38 signaling and maintained AMPK activation, without affecting JNK, mTOR, AKT and NF-kB pathways. Among them, p38 MAPK signaling was critically involved in ROS production under glycolysis-dominant condition in chondrocytes. Our study provides the proof-of-concept for PDK2-mediated metabolic reprogramming towards OxPhos as a new therapeutic strategy for OA.\u003c/p\u003e","manuscriptTitle":"Enhancing oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in surgically induced osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 20:46:51","doi":"10.21203/rs.3.rs-3947364/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-03-22T06:11:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-20T08:13:54+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-03-14T16:18:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-03-05T06:19:54+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-02-28T12:12:52+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-02-25T08:20:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-13T01:39:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2024-02-11T02:59:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-11T02:59:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cd1e6e49-5789-40e3-aa47-1ba0c71371eb","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28955477,"name":"Health sciences/Medical research/Translational research"},{"id":28955478,"name":"Health sciences/Anatomy/Musculoskeletal system/Cartilage"}],"tags":[],"updatedAt":"2025-02-03T08:05:26+00:00","versionOfRecord":{"articleIdentity":"rs-3947364","link":"https://doi.org/10.1038/s12276-025-01400-9","journal":{"identity":"experimental-and-molecular-medicine","isVorOnly":false,"title":"Experimental \u0026 Molecular Medicine"},"publishedOn":"2025-02-03 05:00:00","publishedOnDateReadable":"February 3rd, 2025"},"versionCreatedAt":"2024-02-28 20:46:51","video":"","vorDoi":"10.1038/s12276-025-01400-9","vorDoiUrl":"https://doi.org/10.1038/s12276-025-01400-9","workflowStages":[]},"version":"v1","identity":"rs-3947364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3947364","identity":"rs-3947364","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}