Norepinephrine Modulates IL-1β-induced Catabolic Response of Human Chondrocytes

Norepinephrine Modulates IL-1β-induced Catabolic Response of Human Chondrocytes | 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 Research Article Norepinephrine Modulates IL-1β-induced Catabolic Response of Human Chondrocytes Hyun Sook Hwang, Mi Hyun Lee, Dong Jin Go, Hyun Ah Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-148138/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background The influence of the sympathetic nervous system (SNS) on metabolism of bone and cartilage expressing β-adrenergic receptors (AR) was suggested. Here, we investigated the relation between SNS and interleukin-1beta (IL-1β)-induced cartilage metabolism. Methods Human articular chondrocytes and articular cartilage were collected from patients with osteoarthritis (OA). Chondrocyte monolayer and cartilage explant culture were stimulated with IL-1β. The activity of β-ARs was modulated by an agonist, norepinephrine (NE), and antagonists, including propranolol, atenolol, nebivolol, and nadolol. Results The levels of β 1 -, β 2 -, and β 3 -AR in OA cartilage and IL-1β-treated chondrocytes were lower than normal cartilage and untreated cells. Treatment of chondrocytes with IL-1β and β-blockers, including propranolol, atenolol, nebivolol, and nadolol, for 6 h significantly upregulated IL-1β-induced expression of MMP-1, -3, and -13, compared to chondrocytes treated with IL-1β alone, indicating that antagonism of β-AR confers catabolic signals. On the other hand, NE antagonized IL-1β-induced catabolic response. In addition, NE significantly inhibited IL-1β-induced release of glycosaminoglycan (GAG) from cartilage explant culture. In addition, β-AR activity significantly affected IL-1β-stimulated phosphorylation of JNK and ERK. These results indicate that β-AR signal is associated with cartilage metabolism. Conclusions Our findings showed that β-ARs is a regulator of cartilage catabolism induced with IL-1β. Orthopedics Norepinephrine β-adrenergic receptor β-blocker glycosaminoglycan chondrocyte Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Osteoarthritis (OA) is a prevalent degenerative joint disease, which shows features such as cartilage loss, synovitis, and accompanying joint pain [ 1 ]. Synthesis of extracellular matrix (ECM) in chondrocytes is maintained by balance of anabolic factors and catabolic factors, such as matrix metalloproteinases (MMPs), and are also influenced by a variety of molecules, including pro-inflammatory mediators and cytokines [ 1 , 2 ]. Stimuli from inflammation and mechanical stress are detected by sensory nerve fibers and is transmitted to the central nervous system, which subsequently activates the sympathetic nervous system (SNS). A series of processes, including release of neurotransmitter such as norepinephrine (NE), lymphocyte recruitment, and increase of blood and lymph flow, are induced at the site of inflammation [ 3 ]. In particular, the sympathetic nerve fibers expressing α- and β-adrenergic receptors (AR) are found in various tissues, including synovium, bone, and chondrocytes. Previous studies reported results linking the SNS with the skeletal system. The endogenous catecholamine NE was detected at high concentration in synovial fluid from patients with joint trauma [ 4 ]. NE regulates bone development and chondrocyte metabolism via β-AR [ 5 ]. NE has also been shown to regulate apoptosis and proliferation of several cell types including chondrocytes via β-AR [ 6 – 8 ]. Of note, NE exhibits a distinct impact depending on the receptor it signals through such that inflammatory response of OA chondrocyte signals via β-AR while cell cycle and apoptosis signals via α-AR [ 9 ]. Furthermore, the influence of AR signaling on skeletal tissue homeostasis has been reported in animal and human studies. Intra-cerebroventricular administration of leptin reduced bone formation and bone mass in mice, with leptin acting on the hypothalamus to increase sympathetic outflow, leading to the activation of β-ARs on osteoblasts and resulting in decreases in osteoblast proliferation, differentiation, and bone formation [ 10 , 11 ]. Treatment with the nonselective β-blocker propranolol increased bone mass in ovariectomized as well as ovary-intact female mice [ 11 ]. In addition, a study of postmenopausal women showed that patients treated with β1-AR–selective blockers had decrease of bone resorption marker and increase of bone mineral density than did nonusers [ 12 ]. However, it is little known whether the SNS signaling affects cartilage metabolism in chondrocytes or OA patients. In this study, we investigated whether the regulation of β-AR activity by NE and its antagonists has an influence on interleukin-1β (IL-1β)-induced catabolic and anabolic responses in articular chondrocytes. Methods Materials Recombinant human interleukin-1β (IL-1β) was purchased from R&D Systems (Minneapolis, MN, USA). Propranolol (P), atenolol (A), nebivolol (B), and nadolol (N) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against p-IκBα, p-p38/p38, p-ERK/ERK, and p-JNK/JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primers used in qRT-PCR were obtained from Cosmogenetech (Seoul, Korea). Cartilage collection and chondrocyte isolation from cartilage OA cartilage samples were obtained from the knee joints of OA patients [n = 8, 74.25 ± 4.68 years] at the time of total knee replacement surgery. Patient diagnoses were determined using the criteria set forth by the American College of Rheumatology. Normal cartilage samples were obtained from the femoral head of patients [n = 6, 63.17 ± 12.81 years] with femoral neck fractures and no known history of OA or RA. Sample was obtained only from grossly normal-looking cartilage. The collection and use of human tissue samples was reviewed and approved by the Institutional Review Board of Hallym University Sacred Heart Hospital, Anyang, Korea (approval number 2018-05-040). All patients provided written informed consent. All methods were performed in accordance with the relevant guidelines and regulations of Hallym University and were approved by its ethical committee. Primary chondrocytes were isolated from articular cartilage as previously described [ 13 ]. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) Total RNA was extracted from chondrocytes or cartilage tissues using TRIzol reagent as previously described [ 13 ]. cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed using a StepOnePlus real-time PCR system with the primers in Table S1. Western blot analysis Protein from chondrocytes was prepared with RIPA lysis buffer (Biosesang, Kyunggi, South Korea) and protein concentrations were determined using bicinchoninic acid protein assay (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% (w/v) nonfat milk in TBST and incubated with primary and secondary antibodies. The antibody complex was detected using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). Explant culture For explant culture, articular cartilage was taken from normal donors and incubated in DMEM containing IL-1β with or without NE and antagonists of β-adrenergic receptor for 1, 7, and 14 days. The media was changed every 2 days during the culture period. Media were collected at desired time points. Safranin O staining For measurement of proteoglycan content in the cartilage, Safranin O staining was performed as previously described. Briefly, paraffin-embedded cartilage sections were deparaffinized in xylene, hydrated through gradient 100%, 95%, 90, and 70% ethyl alcohol, and stained in hematoxylin solution for 1 min. Then, the slides were counterstained with 0.05% Fast green solution for 5 min, 1% acetic acid solution, and 0.1% Safranin O staining for 5 min. Measurement of GAG released in culture medium GAG content in sample was measured using Sulfated Glycosaminoglycan kit according to the manufacturer’s instructions. Briefly, Blyscan dye reagent (1.0 ml) was added to the same volume of medium samples and glycosaminoglycan standard solution for a calibration curve followed by mixing by inverting and gentle shaking for 30 min. A sulfated GAG dye complex formed in each reaction tube was obtained by centrifugation at 12,000 rpm for 10 min. The pellets were dissolved in dissociation reagent (0.5 ml) and optical density was measured at 656 nm. GAG contents were calculated from the standard curve. Statistical analysis Data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using a Mann-Whitney test between two groups and Kruskal-Wallis test with post hoc Dunn’s test for multiple comparison (GraphPad Prism 6; GraphPad Software, La Jolla, CA, USA). A value of P < 0.05 was considered statistically significant. Results The level of beta-adrenergic receptors (β-AR) is significantly decreased in OA cartilage and IL-1β-treated chondrocytes To investigate whether the level of subtypes of β-AR was different in normal and OA cartilage, mRNA levels of β-ARs were measured in normal and OA cartilages. The expression level of β 1 , β 2 , and β 3 -AR in normal cartilages varied depending on the donor, however, it was significantly lower in OA cartilage (Fig. 1 A). In addition, IL-1β treatment led to significantly decreased expression of three β-AR subtypes, which lasted for 72 hours in cultured chondrocytes (Fig. 1 B-D). These results demonstrate that the expression of β-ARs is downregulated in chondrocytes in response to a catabolic mediator as well as in OA. β-adrenergic blockers and NE modulate IL-1β-induced expression of catabolic and anabolic factors Sympathetic β-adrenergic blockers positively affect IL-1β-induced bone metabolism [ 12 ]. To examine whether β-adrenergic blockers modulate expressions of anabolic and catabolic factors, chondrocytes were treated for 6 h with IL-1β with or without β-adrenergic blockers (0.1, 1.0, and 10 µg/ml). Non-selective β-adrenergic blocker (propranolol) and β 1 -blocker (atenolol) used in our experiments significantly increased IL-1β-induced expression of MMP-1, -3, and − 13 especially at the highest concentration (Fig. 2 A). In contrast, they reversed IL-1β-induced suppression of anabolic factors aggrecan (ACAN), while only atenolol had such influence on collagen II (Col II) (Fig. 2 B). Our data revealed that blockade of β-adrenergic receptors influenced chondrocyte response to IL-1β, such that it enhances both catabolic and anabolic markers. To next analyze the effect of NE, a β-adrenergic agonist, on cartilage matrix metabolism, chondrocytes were treated with IL-1β with or without various concentrations (0.1, 1.0, and 10 µg/ml) of NE for 6, 24, and 48 h. IL-1β treatment increased MMPs expression in a time-dependent manner (Fig. 3 A). Co-treatment with NE significantly inhibited IL-1β-induced expression of MMPs dose-dependently. Contrary to 6 hour treatment (Fig. 2 ), non-selective β-adrenergic blocker (propranolol and nadolol) and β1-blocker (atenolol and nebivolol) did not increase MMP expression compared to treatment with IL-1β alone after 48 hour culture (Fig. 3 A). NE led to reversal of IL-1β-induced down-regulation of ACAN and Col II after 24 hours, which did not last after 48 hours. β-adrenergic blockers did not affect ACAN and Col II regulation after 48 hours of IL-1β treatment (Fig. 3 B), suggesting that β-adrenergic blockers are effective only for the regulation of short-term response to IL-1β. These results demonstrate that activation of β-AR antagonizes IL-1β-induced catabolism of articular chondrocytes. NE suppresses IL-1β-induced release of glycosaminoglycan (GAG) in cartilage explants culture To investigate the effect of β-adrenergic agonist and blockers on ECM metabolism, GAG release was measured in medium from cartilage explants culture exposed to IL-1β with or without NE or β-adrenergic blockers for 1, 4 and 7 days. Untreated cartilage control showed time-dependent increase in cumulative GAG release (Fig. 3 C). IL-1β treatment further increased GAG release into the medium compared with untreated control. NE significantly suppressed GAG release induced by IL-1β. GAG release in combinational treatment with IL-1β and β-adrenergic blockers (P, A, B, and N) for 4 and 7 days did not increase further compared to IL-1β-treated cartilage (Fig. 3 C). In addition, we analyzed the level of ECM in cartilage tissue 7 days after explants culture using safranin O staining. IL-1β significantly decreased ECM level, whereas NE suppressed the reduction of ECM induced by IL-1β (Fig. 3 D). These results demonstrate that activation of β-AR prevents loss of ECM in cartilage. NE suppresses IL-1β-mediated signaling pathways We investigated the effect of NE and β-adrenergic blockers on IL-1β-activated signaling pathways that contribute to MMPs, Col II, and ACAN expression. Chondrocytes were pre-incubated with NE and β-adrenergic blockers for 2 h and then treated with IL-1β for 15, 30, and 60 min. IL-1β induced phosphorylation of p38, JNK, and ERK MAPKs and Iκ-Bα (Fig. 4 A – B; Fig. S1A-B). β-adrenergic blockers enhanced IL-1β-induced activation of JNK, ERK and Iκ-Bα (Fig. 4 A; Fig. S1A), whereas NE significantly inhibited their activation by IL-1β (Fig. 4 B; Fig. S1B). p38 activation was not affected. These results demonstrate that IL-1β-dependent activation of JNK and ERK MAPK and NF-κB is influenced by NE and β-adrenergic blockers in the opposite way. Discussion β-ARs, target receptors of catecholamine involved in the SNS signaling, were down-regulated in both OA cartilage and IL-1β-treated chondrocytes compared with normal cartilage and untreated chondrocytes, respectively. In the present study we investigated the role of β-AR signaling in cartilage metabolism using β-adrenergic agonist and antagonists. Stimulation of β-AR facilitated overall anabolic responses by suppressing catabolic mediator expression and increasing cartilage matrix proteins in IL-1β-treated chondrocytes accompanied by down-regulation of ERK/JNK MAPK and NF-kB signaling pathway. On the contrary, blocking of β-AR led to short-term increase of IL-1β-mediated catabolic responses only. The decreased effect of β-blockers on IL-1β-induced GAG release in long-term treatment is likely attributed to alteration in signaling pathways associated with IL-1β and β-AR. These results show that SNS signaling via β-AR is closely related with regulation of cartilage metabolism. The autonomic nervous system is composed of SNS and parasympathetic nervous system (PNS) that perform opposite actions. The SNS is responsible for the maintenance of homeostasis in response to harmful events leading to fight-or-flight responses [ 3 ]. During heart failure (HF), long-term increase in endogenous catecholamines, including NE and epinephrine, due to activation of the SNS plays a role in the progression of HF [ 14 ]. This is mediated via β-AR, and β-AR antagonists reduce morbidity and mortality in congestive HF. On the other hand, activation of β-ARs relaxes airway smooth muscle and inhaled β-AR agonists are considered as essential bronchodilator drugs in the treatment of bronchial asthma [ 15 ]. Several clinical and animal model studies have investigated the influence of the SNS on the severity of arthritis. In antigen-induced arthritis mouse model, chemical sympathectomy and pharmacological blockade of AR reduced arthritis severity such as joint inflammation and arthritis score [ 16 ]. Depletion of catecholamines by sympathectomy and elimination of sensory afferents by administration of capsaicin decreased joint injury in arthritic rats [ 17 ]. Patients with RA exhibits milder pain by sympathetic blockade using guanethidine [ 18 ]. In murine chondrocyte monolayer culture, stimulation of β 2 -AR by isoproterenol inhibits Col X and Indian Hedgehog (Ihh) mRNA level through activation of ERK1/2 MAPK [ 19 ]. Sox-6 and Col II expression was inhibited by stimulation of β 2 -AR and this inhibitory effect was suppressed by propranolol, β 2 -AR antagonist [ 20 ], indicating that activation of β-AR signaling has negative effects on matrix protein synthesis in chondrocytes. By contrast, we found that β 1 -, β 2 -, and β 3 -AR expressions were reduced in OA cartilage relative to normal cartilage and pro-inflammatory cytokine IL-1β down-regulated three subtypes of β-AR compared to untreated control. In addition, our data showed that β-AR agonist prevented loss of ECM through decreased expression of pro-catabolic factors and increased expression of anabolic factors. These contradictory results are likely to be due to difference in types and concentrations of AR agonists and antagonists, AR subtypes interacting with them, and subsequent downstream signaling pathways. In line with our results showing the suppression of IL-1β-induced catabolic responses by NE in monolayer and explant culture of human chondrocyte, several reports have demonstrated that catecholamines, including NE and dopamine, a precursor of NE, had anti-inflammatory effects in a variety of cells and in vivo models. NE reversed cartilage catabolism and inflammatory responses stimulated by IL-1β [ 9 ]. Adoptive transfer of tyrosine hydroxylase-positive neuronal cells generated from mesenchymal stem cells which exhibit a typical catecholaminergic phenotype led to markedly reduced severity of collagen induced arthritis in mice [ 21 ]. Dopamine prevented cartilage degradation in a DMM-induced OA mouse model and reduced MMPs expression and elevated Col II expression in IL-1β-treated chondrocytes via NF-κB and JAK2/STAT3 signaling pathway [ 22 ]. In lipopolysaccharide-stimulated microglia cells, dopamine suppressed nitric oxide production [ 23 ]. Bone remodeling is also under sympathetic control, such that β-blocker treatment enhanced bone mass in wild type and ovariectomized mice [ 11 ]. Clenbuterol, a β 2 -AR agonist, suppressed longitudinal growth of bones in young rats together with muscular hypertrophy [ 24 ]. On the contrary, another study showed that clenbuterol relieved sciatic nerve injury-induced loss of bone mineralization [ 25 ]. Regulation of the SNS by leptin via β 2 -AR decreases osteoblast number and increases osteoclast differentiation leading to reduction of bone mass in mice [ 26 , 27 ]. β 2 -AR-deficient mice exhibited greater bone mass in response to mechanical loading compared to wild type, but it was not shown in β 1 -knockout and β 1,2 -AR double knockout mice. In addition, administration of isoproterenol a non-selective β-AR agonist to wildtype mice increased bone resorption, indicating the possibility that β 1 - and β 2 -AR could play opposite role in bone metabolism [ 28 ]. It is also noted that the SNS is closely related with regulation of inflammatory responses, e.g. proinflammatory cytokine production. In a study of postmenopausal women with hypertension, central blockade of the SNS with moxonidine reduced serum TNF-α level [ 29 ]. TNF production is regulated through stimulation of α-AR or β-AR by catecholamines such as NE [ 30 , 31 ]. NF-κB pathway is an inducible transcription factor involved in inflammation and cellular differentiation, and in ECM homeostasis [ 32 ]. In particular, the activation of NF-κB leads to increase in catabolic gene expression such as MMPs, ADAMTS5, and proinflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase. We found that regulation of β-AR activity with NE and four antagonists of AR had opposite influence on activation of JNK and ERK MAPK and NF-κB compared to IL-1β-treated chondrocytes, subsequently leading to alteration in expression of anabolic and catabolic factors. Thus, the SNS modulated pro-catabolic and anti-anabolic responses induced by IL-1β through regulation of JNK and ERK MAPK and NF-κB in chondrocytes. In addition, it is likely that β-ARs induce different biological responses through activating diverse downstream signaling molecules, including adenylate cyclase, depending on types, concentration, and treatment time of agonists and antagonists. Conclusions In conclusion, our findings demonstrate that β-AR agonist NE converted IL-1β-driven catabolic responses to cartilage anabolism in articular chondrocytes. Cartilage homeostasis is likely to be closely related with the SNS level and activity. Whether modulators of the SNS signaling may be beneficial for prevention of OA progression should be studied in further animal experiments. Declarations Ethics approval and consent to participate This study was reviewed and approved by the Institutional Review Board of Hallym University Sacred Heart Hospital, Anyang, Korea (approval number 2018-05-040). Written informed consent was obtained from all participants in this study. All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Availability of data and materials Correspondence and requests for all data required to evaluate the work should be addressed to H.A.K. Competing interests The authors have no conflicting interests. Funding This study was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2017R1A2B2001881 and 2019R1G1A1100461) and in part by Hallym University Research Fund. Authors contributions HSH and MHL were involved in the conception and design of the study and acquisition and analysis of data. DJG and HAK conceived and designed the experiments and wrote the paper. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 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Supplementary Files 20210118BMCsupple.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 22 Mar, 2021 Reviews received at journal 05 Mar, 2021 Reviewers agreed at journal 23 Feb, 2021 Reviewers invited by journal 23 Feb, 2021 Editor assigned by journal 27 Jan, 2021 Editor invited by journal 27 Jan, 2021 Submission checks completed at journal 27 Jan, 2021 First submitted to journal 15 Jan, 2021 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. We do this by developing innovative software and high quality services for the global research community. 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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-148138","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":9085148,"identity":"ae925e44-c2ab-4863-a025-00a2aff798b1","order_by":0,"name":"Hyun Sook Hwang","email":"","orcid":"","institution":"Hallym University Sacred Heart Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Sook","lastName":"Hwang","suffix":""},{"id":9085149,"identity":"cf3bd9de-36f2-4866-bc9f-21de280cb012","order_by":1,"name":"Mi Hyun Lee","email":"","orcid":"","institution":"Hallym University Sacred Heart Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mi","middleName":"Hyun","lastName":"Lee","suffix":""},{"id":9085150,"identity":"5bd7d2e6-ed8e-42e4-8e1f-318c8ae36916","order_by":2,"name":"Dong Jin Go","email":"","orcid":"","institution":"Hallym University Kangnam Heart Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Jin","lastName":"Go","suffix":""},{"id":9085151,"identity":"398eefbf-dba4-4c68-96bc-37371fb70201","order_by":3,"name":"Hyun Ah Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACCWYGNgaGigMMfOwwEeK0nDnAwMZMtBYGoBbGNlK0SLZzpz34Oe+OPBszj+kGhho7BsnZB/BrkWbm3W7Yu+2ZYRszj9kNhmPJDNJ8Cfi1yDHzbpPg3XaYEaKF7QCDHA8Bh4G0SP6dc9geouUfEVqADtsmzdtwOBGsBRQO0oS0SDYDtcgcO5zcxsxWdiOxL5lHsoeAFonzZ7dJvqk5bNvP3rztxodvdnISZwhoQQIcBgwJDAyEnIUC2B+QonoUjIJRMApGEAAAI5Y45BWGNUAAAAAASUVORK5CYII=","orcid":"","institution":"Hallym University Sacred Heart Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Ah","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2021-01-15 07:14:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-148138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-148138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5346710,"identity":"bf894589-4ee5-4c69-80cc-498f0bd161fd","added_by":"auto","created_at":"2021-01-28 18:13:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87706,"visible":true,"origin":"","legend":"The levels of beta-adrenergic receptors are decreased in OA cartilage and IL-1β-treated chondrocytes. (A) The relative expression of β1-, β2-, and β3-adrenergic receptor (AR) in normal and OA cartilage. Data represent the mean ± standard deviation (SD) for duplicate data from different donors. Normal cartilage (n = 6); OA cartilage (n = 8). (B)~(D) Expression of β1-, β2-, and β3-adrenergic receptors in IL-1β-treated chondrocytes. Chondrocytes were stimulated with IL-1β (1 ng/ml) for 6, 24, 48, and 72 h. mRNA levels of β-ARs were quantified using Quanti Fast SYBR Green-based real-time PCR (RT-PCR). The expression ratios of β-adrenergic receptors relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the internal control, are shown. Data represent the mean ± standard deviation (SD) for duplicate data from five different donors. *P \u003c 0.05 and ****P \u003c 0.0001 vs. normal cartilage tissue or untreated chondrocytes by Mann-Whitney test. ","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-148138/v1/d2e4edcdd6bdbf261974424f.png"},{"id":5346709,"identity":"b56bc39f-42e3-4045-8c03-e2d20bc7d908","added_by":"auto","created_at":"2021-01-28 18:13:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79585,"visible":true,"origin":"","legend":"β-AR blockers affect IL-1β-induced expression of catabolic and anabolic factors.\n(A), (B) The effect of β-AR blockers on IL-1β-induced expression of (A) catabolic and (B) anabolic factors. Chondrocytes were pre-incubated with 0.1, 1.0, and 10 ng/ml of propranolol (P; non-selective β-adrenergic blocker) and atenolol (A; β1-adrenergic blocker) for 2 h followed by treatment with IL-1β (1 ng/ml) for 6 h. The mRNA levels of catabolic factors (MMP-1, -3, and -13) and anabolic factors (ACAN and Col II) were measured using RT-PCR. Data represent the mean ± SD of duplicate data from three different donors. *P \u003c 0.05, **P \u003c 0.01, ***P \u003c 0.001, and ****P \u003c 0.0001 vs. IL-1β-treated cells by Kruskal-Wallis test with post hoc Dunn's multiple comparison test.\n","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-148138/v1/0e4289ed5e8e62a9d5060985.png"},{"id":5346805,"identity":"0b20f1dc-bb18-414f-b934-228b5040b54d","added_by":"auto","created_at":"2021-01-28 18:16:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":199438,"visible":true,"origin":"","legend":"Norepinephrine (NE) reverses IL-1β-induced up-regulation of MMPs and down-regulation of matrix proteins and suppresses IL-1β-induced release of glycosaminoglycan (GAG) into the medium in cartilage explants culture. The effect of NE and β-AR blockers on IL-1β-induced expression of (A) MMPs and (B) extracellular matrix proteins. Chondrocytes were preincubated with NE (0.1, 1.0, and 10 ng/ml) or β-AR blockers (propranolol (P), atenolol (A), nebivolol (B), and nadolol (N); 1.0 ng/ml) for 2 h followed by treatment with IL-1β (1 ng/ml) for 6, 24, and 48 h. Data represent the mean ± SD of duplicate data from five different donors. *P \u003c 0.05, **P \u003c 0.01, ***P \u003c 0.001, and ****P \u003c 0.0001 vs. IL-1β-treated cells by Kruskal-Wallis test with post hoc Dunn's multiple comparison test. (C) GAG release in cartilage explants culture treated with IL-1β and NE or β-AR blockers. (Cartilage tissue (40 mg) was pre-incubated with NE (1.0 ng/ml) or β-AR blockers (P, A, B, and N; 1.0 ng/ml) 2 h before treatment with IL-1β (1 ng/ml) for 1, 4, and 7 days. NE or β-AR blockers were contained throughout the experiment. Culture medium was changed every two days for culture period. Concentration of GAG in culture medium was measured using Sulfated Glycosmainoglycan Assay kit. Propranolol (P), atenolol (A), nebivolol (B), and nadolol (N). (C) Data represent the mean ± SD of duplicate data from three different donors. *P \u003c 0.05, **P \u003c 0.01, and ****P \u003c 0.0001vs. IL-1β-treated cartilage tissue by Kruskal-Wallis test followed with post hoc Dunn's multiple comparison test. (D) Safranin O staining of explant culture of human joint cartilage in the presence of IL-1β and NE.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-148138/v1/d1c1c1b4984edc28f25fa0db.png"},{"id":5346707,"identity":"1c8b53a5-2f3b-435b-9fc2-ad750ffa6655","added_by":"auto","created_at":"2021-01-28 18:13:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":233979,"visible":true,"origin":"","legend":"Effect of NE or β-AR blockers on IL-1β-activated signaling pathways in human chondrocytes. Chondrocytes were pretreated with (A) β-AR blockers (P, A, B, and N; 1.0 ng/ml) or (B) NE (1.0 ng/ml) 2 h before stimulation with IL-1β (1 ng/ml) for 15, 30, and 60 min. Phosphorylation of Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 MAPK, and Iκ-Bα was analyzed by Western blot. Western blots shown are representative of four independent experiments. β-actin was used as a loading control. Propranolol (P), atenolol (A), nebivolol (B), and nadolol (N).","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-148138/v1/de6685dccf442af3e02dfd3f.png"},{"id":13652177,"identity":"6f0a06e7-944e-4cf6-ab38-c6963c844b28","added_by":"auto","created_at":"2021-09-17 09:48:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":961079,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-148138/v1/6454e7ea-f6df-4108-a195-93c17ef91f5f.pdf"},{"id":5346706,"identity":"56e5319f-1061-458b-9264-b705ff279fe6","added_by":"auto","created_at":"2021-01-28 18:13:15","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":610344,"visible":true,"origin":"","legend":"","description":"","filename":"20210118BMCsupple.pdf","url":"https://assets-eu.researchsquare.com/files/rs-148138/v1/80692a2d8e072b522f513331.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eNorepinephrine Modulates IL-1β-induced Catabolic Response of Human Chondrocytes\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eOsteoarthritis (OA) is a prevalent degenerative joint disease, which shows features such as cartilage loss, synovitis, and accompanying joint pain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Synthesis of extracellular matrix (ECM) in chondrocytes is maintained by balance of anabolic factors and catabolic factors, such as matrix metalloproteinases (MMPs), and are also influenced by a variety of molecules, including pro-inflammatory mediators and cytokines [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStimuli from inflammation and mechanical stress are detected by sensory nerve fibers and is transmitted to the central nervous system, which subsequently activates the sympathetic nervous system (SNS). A series of processes, including release of neurotransmitter such as norepinephrine (NE), lymphocyte recruitment, and increase of blood and lymph flow, are induced at the site of inflammation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In particular, the sympathetic nerve fibers expressing α- and β-adrenergic receptors (AR) are found in various tissues, including synovium, bone, and chondrocytes. Previous studies reported results linking the SNS with the skeletal system. The endogenous catecholamine NE was detected at high concentration in synovial fluid from patients with joint trauma [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. NE regulates bone development and chondrocyte metabolism via β-AR [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. NE has also been shown to regulate apoptosis and proliferation of several cell types including chondrocytes via β-AR [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Of note, NE exhibits a distinct impact depending on the receptor it signals through such that inflammatory response of OA chondrocyte signals via β-AR while cell cycle and apoptosis signals via α-AR [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the influence of AR signaling on skeletal tissue homeostasis has been reported in animal and human studies. Intra-cerebroventricular administration of leptin reduced bone formation and bone mass in mice, with leptin acting on the hypothalamus to increase sympathetic outflow, leading to the activation of β-ARs on osteoblasts and resulting in decreases in osteoblast proliferation, differentiation, and bone formation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Treatment with the nonselective β-blocker propranolol increased bone mass in ovariectomized as well as ovary-intact female mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, a study of postmenopausal women showed that patients treated with β1-AR\u0026ndash;selective blockers had decrease of bone resorption marker and increase of bone mineral density than did nonusers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, it is little known whether the SNS signaling affects cartilage metabolism in chondrocytes or OA patients.\u003c/p\u003e \u003cp\u003eIn this study, we investigated whether the regulation of β-AR activity by NE and its antagonists has an influence on interleukin-1β (IL-1β)-induced catabolic and anabolic responses in articular chondrocytes.\u003c/p\u003e "},{"header":"Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eRecombinant human interleukin-1β (IL-1β) was purchased from R\u0026amp;D Systems (Minneapolis, MN, USA). Propranolol (P), atenolol (A), nebivolol (B), and nadolol (N) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against p-IκBα, p-p38/p38, p-ERK/ERK, and p-JNK/JNK were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primers used in qRT-PCR were obtained from Cosmogenetech (Seoul, Korea).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCartilage collection and chondrocyte isolation from cartilage\u003c/h2\u003e \u003cp\u003eOA cartilage samples were obtained from the knee joints of OA patients [n\u0026thinsp;=\u0026thinsp;8, 74.25\u0026thinsp;\u0026plusmn;\u0026thinsp;4.68 years] at the time of total knee replacement surgery. Patient diagnoses were determined using the criteria set forth by the American College of Rheumatology. Normal cartilage samples were obtained from the femoral head of patients [n\u0026thinsp;=\u0026thinsp;6, 63.17\u0026thinsp;\u0026plusmn;\u0026thinsp;12.81 years] with femoral neck fractures and no known history of OA or RA. Sample was obtained only from grossly normal-looking cartilage. The collection and use of human tissue samples was reviewed and approved by the Institutional Review Board of Hallym University Sacred Heart Hospital, Anyang, Korea (approval number 2018-05-040). All patients provided written informed consent. All methods were performed in accordance with the relevant guidelines and regulations of Hallym University and were approved by its ethical committee. Primary chondrocytes were isolated from articular cartilage as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from chondrocytes or cartilage tissues using TRIzol reagent as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). qRT-PCR was performed using a StepOnePlus real-time PCR system with the primers in Table S1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eProtein from chondrocytes was prepared with RIPA lysis buffer (Biosesang, Kyunggi, South Korea) and protein concentrations were determined using bicinchoninic acid protein assay (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% (w/v) nonfat milk in TBST and incubated with primary and secondary antibodies. The antibody complex was detected using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExplant culture\u003c/h2\u003e \u003cp\u003eFor explant culture, articular cartilage was taken from normal donors and incubated in DMEM containing IL-1β with or without NE and antagonists of β-adrenergic receptor for 1, 7, and 14 days. The media was changed every 2 days during the culture period. Media were collected at desired time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSafranin O staining\u003c/h2\u003e \u003cp\u003eFor measurement of proteoglycan content in the cartilage, Safranin O staining was performed as previously described. Briefly, paraffin-embedded cartilage sections were deparaffinized in xylene, hydrated through gradient 100%, 95%, 90, and 70% ethyl alcohol, and stained in hematoxylin solution for 1 min. Then, the slides were counterstained with 0.05% Fast green solution for 5 min, 1% acetic acid solution, and 0.1% Safranin O staining for 5 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of GAG released in culture medium\u003c/h2\u003e \u003cp\u003eGAG content in sample was measured using Sulfated Glycosaminoglycan kit according to the manufacturer\u0026rsquo;s instructions. Briefly, Blyscan dye reagent (1.0 ml) was added to the same volume of medium samples and glycosaminoglycan standard solution for a calibration curve followed by mixing by inverting and gentle shaking for 30 min. A sulfated GAG dye complex formed in each reaction tube was obtained by centrifugation at 12,000 rpm for 10 min. The pellets were dissolved in dissociation reagent (0.5 ml) and optical density was measured at 656 nm. GAG contents were calculated from the standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using a Mann-Whitney test between two groups and Kruskal-Wallis test with post hoc Dunn\u0026rsquo;s test for multiple comparison (GraphPad Prism 6; GraphPad Software, La Jolla, CA, USA). A value of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":" \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe level of beta-adrenergic receptors (β-AR) is significantly decreased in OA cartilage and IL-1β-treated chondrocytes\u003c/h2\u003e \u003cp\u003eTo investigate whether the level of subtypes of β-AR was different in normal and OA cartilage, mRNA levels of β-ARs were measured in normal and OA cartilages. The expression level of β\u003csub\u003e1\u003c/sub\u003e, β\u003csub\u003e2\u003c/sub\u003e, and β\u003csub\u003e3\u003c/sub\u003e-AR in normal cartilages varied depending on the donor, however, it was significantly lower in OA cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, IL-1β treatment led to significantly decreased expression of three β-AR subtypes, which lasted for 72 hours in cultured chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). These results demonstrate that the expression of β-ARs is downregulated in chondrocytes in response to a catabolic mediator as well as in OA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eβ-adrenergic blockers and NE modulate IL-1β-induced expression of catabolic and anabolic factors\u003c/h2\u003e \u003cp\u003eSympathetic β-adrenergic blockers positively affect IL-1β-induced bone metabolism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To examine whether β-adrenergic blockers modulate expressions of anabolic and catabolic factors, chondrocytes were treated for 6 h with IL-1β with or without β-adrenergic blockers (0.1, 1.0, and 10 \u0026micro;g/ml). Non-selective β-adrenergic blocker (propranolol) and β\u003csub\u003e1\u003c/sub\u003e-blocker (atenolol) used in our experiments significantly increased IL-1β-induced expression of MMP-1, -3, and \u0026minus;\u0026thinsp;13 especially at the highest concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, they reversed IL-1β-induced suppression of anabolic factors aggrecan (ACAN), while only atenolol had such influence on collagen II (Col II) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Our data revealed that blockade of β-adrenergic receptors influenced chondrocyte response to IL-1β, such that it enhances both catabolic and anabolic markers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo next analyze the effect of NE, a β-adrenergic agonist, on cartilage matrix metabolism, chondrocytes were treated with IL-1β with or without various concentrations (0.1, 1.0, and 10 \u0026micro;g/ml) of NE for 6, 24, and 48 h. IL-1β treatment increased MMPs expression in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Co-treatment with NE significantly inhibited IL-1β-induced expression of MMPs dose-dependently. Contrary to 6 hour treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), non-selective β-adrenergic blocker (propranolol and nadolol) and β1-blocker (atenolol and nebivolol) did not increase MMP expression compared to treatment with IL-1β alone after 48 hour culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). NE led to reversal of IL-1β-induced down-regulation of ACAN and Col II after 24 hours, which did not last after 48 hours. β-adrenergic blockers did not affect ACAN and Col II regulation after 48 hours of IL-1β treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting that β-adrenergic blockers are effective only for the regulation of short-term response to IL-1β. These results demonstrate that activation of β-AR antagonizes IL-1β-induced catabolism of articular chondrocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eNE suppresses IL-1β-induced release of glycosaminoglycan (GAG) in cartilage explants culture\u003c/h2\u003e \u003cp\u003eTo investigate the effect of β-adrenergic agonist and blockers on ECM metabolism, GAG release was measured in medium from cartilage explants culture exposed to IL-1β with or without NE or β-adrenergic blockers for 1, 4 and 7 days. Untreated cartilage control showed time-dependent increase in cumulative GAG release (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). IL-1β treatment further increased GAG release into the medium compared with untreated control. NE significantly suppressed GAG release induced by IL-1β. GAG release in combinational treatment with IL-1β and β-adrenergic blockers (P, A, B, and N) for 4 and 7 days did not increase further compared to IL-1β-treated cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, we analyzed the level of ECM in cartilage tissue 7 days after explants culture using safranin O staining. IL-1β significantly decreased ECM level, whereas NE suppressed the reduction of ECM induced by IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results demonstrate that activation of β-AR prevents loss of ECM in cartilage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNE suppresses IL-1β-mediated signaling pathways\u003c/h2\u003e \u003cp\u003eWe investigated the effect of NE and β-adrenergic blockers on IL-1β-activated signaling pathways that contribute to MMPs, Col II, and ACAN expression. Chondrocytes were pre-incubated with NE and β-adrenergic blockers for 2 h and then treated with IL-1β for 15, 30, and 60 min. IL-1β induced phosphorylation of p38, JNK, and ERK MAPKs and Iκ-Bα (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u0026ndash; B; Fig. S1A-B). β-adrenergic blockers enhanced IL-1β-induced activation of JNK, ERK and Iκ-Bα (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Fig. S1A), whereas NE significantly inhibited their activation by IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Fig. S1B). p38 activation was not affected. These results demonstrate that IL-1β-dependent activation of JNK and ERK MAPK and NF-κB is influenced by NE and β-adrenergic blockers in the opposite way.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Discussion","content":" \u003cp\u003eβ-ARs, target receptors of catecholamine involved in the SNS signaling, were down-regulated in both OA cartilage and IL-1β-treated chondrocytes compared with normal cartilage and untreated chondrocytes, respectively. In the present study we investigated the role of β-AR signaling in cartilage metabolism using β-adrenergic agonist and antagonists. Stimulation of β-AR facilitated overall anabolic responses by suppressing catabolic mediator expression and increasing cartilage matrix proteins in IL-1β-treated chondrocytes accompanied by down-regulation of ERK/JNK MAPK and NF-kB signaling pathway. On the contrary, blocking of β-AR led to short-term increase of IL-1β-mediated catabolic responses only. The decreased effect of β-blockers on IL-1β-induced GAG release in long-term treatment is likely attributed to alteration in signaling pathways associated with IL-1β and β-AR. These results show that SNS signaling via β-AR is closely related with regulation of cartilage metabolism.\u003c/p\u003e \u003cp\u003eThe autonomic nervous system is composed of SNS and parasympathetic nervous system (PNS) that perform opposite actions. The SNS is responsible for the maintenance of homeostasis in response to harmful events leading to fight-or-flight responses [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. During heart failure (HF), long-term increase in endogenous catecholamines, including NE and epinephrine, due to activation of the SNS plays a role in the progression of HF [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This is mediated via β-AR, and β-AR antagonists reduce morbidity and mortality in congestive HF. On the other hand, activation of β-ARs relaxes airway smooth muscle and inhaled β-AR agonists are considered as essential bronchodilator drugs in the treatment of bronchial asthma [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral clinical and animal model studies have investigated the influence of the SNS on the severity of arthritis. In antigen-induced arthritis mouse model, chemical sympathectomy and pharmacological blockade of AR reduced arthritis severity such as joint inflammation and arthritis score [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Depletion of catecholamines by sympathectomy and elimination of sensory afferents by administration of capsaicin decreased joint injury in arthritic rats [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Patients with RA exhibits milder pain by sympathetic blockade using guanethidine [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In murine chondrocyte monolayer culture, stimulation of β\u003csub\u003e2\u003c/sub\u003e-AR by isoproterenol inhibits Col X and Indian Hedgehog (Ihh) mRNA level through activation of ERK1/2 MAPK [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Sox-6 and Col II expression was inhibited by stimulation of β\u003csub\u003e2\u003c/sub\u003e-AR and this inhibitory effect was suppressed by propranolol, β\u003csub\u003e2\u003c/sub\u003e-AR antagonist [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], indicating that activation of β-AR signaling has negative effects on matrix protein synthesis in chondrocytes. By contrast, we found that β\u003csub\u003e1\u003c/sub\u003e-, β\u003csub\u003e2\u003c/sub\u003e-, and β\u003csub\u003e3\u003c/sub\u003e-AR expressions were reduced in OA cartilage relative to normal cartilage and pro-inflammatory cytokine IL-1β down-regulated three subtypes of β-AR compared to untreated control. In addition, our data showed that β-AR agonist prevented loss of ECM through decreased expression of pro-catabolic factors and increased expression of anabolic factors. These contradictory results are likely to be due to difference in types and concentrations of AR agonists and antagonists, AR subtypes interacting with them, and subsequent downstream signaling pathways.\u003c/p\u003e \u003cp\u003eIn line with our results showing the suppression of IL-1β-induced catabolic responses by NE in monolayer and explant culture of human chondrocyte, several reports have demonstrated that catecholamines, including NE and dopamine, a precursor of NE, had anti-inflammatory effects in a variety of cells and \u003cem\u003ein vivo\u003c/em\u003e models. NE reversed cartilage catabolism and inflammatory responses stimulated by IL-1β [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Adoptive transfer of tyrosine hydroxylase-positive neuronal cells generated from mesenchymal stem cells which exhibit a typical catecholaminergic phenotype led to markedly reduced severity of collagen induced arthritis in mice [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Dopamine prevented cartilage degradation in a DMM-induced OA mouse model and reduced MMPs expression and elevated Col II expression in IL-1β-treated chondrocytes via NF-κB and JAK2/STAT3 signaling pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In lipopolysaccharide-stimulated microglia cells, dopamine suppressed nitric oxide production [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBone remodeling is also under sympathetic control, such that β-blocker treatment enhanced bone mass in wild type and ovariectomized mice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Clenbuterol, a β\u003csub\u003e2\u003c/sub\u003e-AR agonist, suppressed longitudinal growth of bones in young rats together with muscular hypertrophy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. On the contrary, another study showed that clenbuterol relieved sciatic nerve injury-induced loss of bone mineralization [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Regulation of the SNS by leptin via β\u003csub\u003e2\u003c/sub\u003e-AR decreases osteoblast number and increases osteoclast differentiation leading to reduction of bone mass in mice [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. β\u003csub\u003e2\u003c/sub\u003e-AR-deficient mice exhibited greater bone mass in response to mechanical loading compared to wild type, but it was not shown in β\u003csub\u003e1\u003c/sub\u003e-knockout and β\u003csub\u003e1,2\u003c/sub\u003e-AR double knockout mice. In addition, administration of isoproterenol a non-selective β-AR agonist to wildtype mice increased bone resorption, indicating the possibility that β\u003csub\u003e1\u003c/sub\u003e- and β\u003csub\u003e2\u003c/sub\u003e-AR could play opposite role in bone metabolism [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIt is also noted that the SNS is closely related with regulation of inflammatory responses, e.g. proinflammatory cytokine production. In a study of postmenopausal women with hypertension, central blockade of the SNS with moxonidine reduced serum TNF-α level [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. TNF production is regulated through stimulation of α-AR or β-AR by catecholamines such as NE [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNF-κB pathway is an inducible transcription factor involved in inflammation and cellular differentiation, and in ECM homeostasis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In particular, the activation of NF-κB leads to increase in catabolic gene expression such as MMPs, ADAMTS5, and proinflammatory mediators, including cyclooxygenase-2 and inducible nitric oxide synthase. We found that regulation of β-AR activity with NE and four antagonists of AR had opposite influence on activation of JNK and ERK MAPK and NF-κB compared to IL-1β-treated chondrocytes, subsequently leading to alteration in expression of anabolic and catabolic factors. Thus, the SNS modulated pro-catabolic and anti-anabolic responses induced by IL-1β through regulation of JNK and ERK MAPK and NF-κB in chondrocytes. In addition, it is likely that β-ARs induce different biological responses through activating diverse downstream signaling molecules, including adenylate cyclase, depending on types, concentration, and treatment time of agonists and antagonists.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eIn conclusion, our findings demonstrate that β-AR agonist NE converted IL-1β-driven catabolic responses to cartilage anabolism in articular chondrocytes. Cartilage homeostasis is likely to be closely related with the SNS level and activity. Whether modulators of the SNS signaling may be beneficial for prevention of OA progression should be studied in further animal experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed and approved by the Institutional Review Board of Hallym University Sacred Heart Hospital, Anyang, Korea (approval number 2018-05-040). Written informed consent was obtained from all participants in this study. All methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for all data required to evaluate the work should be addressed to H.A.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicting interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (2017R1A2B2001881 and 2019R1G1A1100461) and in part by Hallym University Research Fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHSH and MHL were involved in the conception and design of the study and acquisition and analysis of data. DJG and HAK conceived and designed the experiments and wrote the paper. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGoldring MB. The role of the chondrocyte in osteoarthritis. Arthritis Rheum. 2000;43(9):1916\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang HS, Kim HA. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis. Int J Mol Sci. 2015;16(11):26035\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePongratz G, Straub RH. The sympathetic nervous response in inflammation. 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Am J Physiol Endocrinol Metab. 2011;300(4):E633-9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJenei-Lanzl Z, Capellino S, Kees F, Fleck M, Lowin T, Straub RH. Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis. Ann Rheum Dis. 2015;74(2):444\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu W, Ding Z, Liu F, Shan W, Cheng C, Xu J, He W, Huang W, Ma J, Yin Z. Dopamine delays articular cartilage degradation in osteoarthritis by negative regulation of the NF-kappaB and JAK2/STAT3 signaling pathways. Biomed Pharmacother. 2019;119:109419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang B, Chen T, Li G, Jia Y, Wang J, Xue L, Chen Y. Dopamine Alters Lipopolysaccharide-Induced Nitric Oxide Production in Microglial Cells via Activation of D1-Like Receptors. 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Stimulation of alpha-adrenergic receptor augments the production of macrophage-derived tumor necrosis factor. J Immunol. 1990;145(5):1430\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSevern A, Rapson NT, Hunter CA, Liew FY. Regulation of tumor necrosis factor production by adrenaline and beta-adrenergic agonists. J Immunol. 1992;148(11):3441\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi MC, Jo J, Park J, Kang HK, Park Y. NF-kappaB Signaling Pathways in Osteoarthritic Cartilage Destruction. Cells. 2019;8(7).\u003c/span\u003e\u003c/li\u003e\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":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Norepinephrine, β-adrenergic receptor, β-blocker, glycosaminoglycan, chondrocyte","lastPublishedDoi":"10.21203/rs.3.rs-148138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-148138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe influence of the sympathetic nervous system (SNS) on metabolism of bone and cartilage expressing β-adrenergic receptors (AR) was suggested. Here, we investigated the relation between SNS and interleukin-1beta (IL-1β)-induced cartilage metabolism.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eHuman articular chondrocytes and articular cartilage were collected from patients with osteoarthritis (OA). Chondrocyte monolayer and cartilage explant culture were stimulated with IL-1β. The activity of β-ARs was modulated by an agonist, norepinephrine (NE), and antagonists, including propranolol, atenolol, nebivolol, and nadolol.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe levels of β\u003csub\u003e1\u003c/sub\u003e-, β\u003csub\u003e2\u003c/sub\u003e-, and β\u003csub\u003e3\u003c/sub\u003e-AR in OA cartilage and IL-1β-treated chondrocytes were lower than normal cartilage and untreated cells. Treatment of chondrocytes with IL-1β and β-blockers, including propranolol, atenolol, nebivolol, and nadolol, for 6 h significantly upregulated IL-1β-induced expression of MMP-1, -3, and -13, compared to chondrocytes treated with IL-1β alone, indicating that antagonism of β-AR confers catabolic signals. On the other hand, NE antagonized IL-1β-induced catabolic response. In addition, NE significantly inhibited IL-1β-induced release of glycosaminoglycan (GAG) from cartilage explant culture. In addition, β-AR activity significantly affected IL-1β-stimulated phosphorylation of JNK and ERK. These results indicate that β-AR signal is associated with cartilage metabolism.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eOur findings showed that β-ARs is a regulator of cartilage catabolism induced with IL-1β.\u003c/p\u003e","manuscriptTitle":"Norepinephrine Modulates IL-1β-induced Catabolic Response of Human Chondrocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-01-28 18:12:32","doi":"10.21203/rs.3.rs-148138/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-03-22T14:02:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-05T16:14:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"b14650cf-e732-4958-af3c-da516334e8a7","date":"2021-02-23T14:54:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-02-23T11:41:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-01-27T17:15:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-01-27T08:03:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-01-27T05:31:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Musculoskeletal Disorders","date":"2021-01-15T07:07:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e33b7dbe-db8f-4fdb-9ae4-7abb989ef29c","owner":[],"postedDate":"January 28th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2118529,"name":"Orthopedics"}],"tags":[],"updatedAt":"2021-08-03T12:59:05+00:00","versionOfRecord":[],"versionCreatedAt":"2021-01-28 18:12:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-148138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-148138","identity":"rs-148138","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}