Ubiquitination of Smad2 by Smurf1 inhibits endothelial-to-mesenchymal transition in human coronary artery endothelial cells

Ubiquitination of Smad2 by Smurf1 inhibits endothelial-to-mesenchymal transition in human coronary artery endothelial cells | 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 Ubiquitination of Smad2 by Smurf1 inhibits endothelial-to-mesenchymal transition in human coronary artery endothelial cells Panpan Liu, Mingyang Zhang, Shuhui Wang, Zhiheng Liu, Nana Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6878518/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Smad2 is a well-established regulator involved in tissue development and in the pathogenesis of endothelial-to-mesenchymal transition (EndMT) mediated by TGF-β signaling. However, the mechanism underlying the regulation of Smad2 in human coronary artery endothelial cells (HCAECs), particularly the identity of the responsible E3 ubiquitin ligase and its role during EndMT remain unclear. In this study, we identified Smad ubiquitination regulatory factor 1 (Smurf1) as a negative regulator of Smad2 protein levels in HCAECs and demonstrated that the E3 ligase activity of Smurf1 is essential for this function. Mechanistically, Smurf1 interacts with Smad2, promoting its ubiquitination, and subsequent proteasomal degradation. Specifically, Smurf1 catalyzes K48-linked polyubiquitination of Smad2 at lysine residues K156, K383 and K420. Functionally, Smad2 was found to promote EndMT in HCAECs, an effect that was partially attenuated either by co-expression of Smurf1 or by mutation of Smad2 at lysine 420 (Smad2 -K420R), which replaces. Taken together, our findings identify, for the first time, specific lysine residues on Smad2 targeted by Smurf1 for K48-linked ubiquitination, and highlight their crucial regulatory role in modulating EndMT in HCAECs. Biological sciences/Cell biology Health sciences/Cardiology Health sciences/Molecular medicine Health sciences/Pathogenesis Smurf1 Smad2 EndMT HCAEC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endothelial-to-mesenchymal transition (EndMT) is a biological process involved in both normal physiological development and the progression of various diseases, and it is associated with the loss of endothelial barrier integrity [ 1 ] . Initially, EndMT was believed to play a critical role in embryogenesis, particularly during cardiogenesis [ 2 , 3 ] . However, subsequent studies have highlighted its pathological involvement in multiple disease states [ 4 – 6 ] . During EndMT, endothelial cells undergo significant alterations in morphology, function, and gene expression profiles [ 7 ] . This transition is characterized by the upregulation of mesenchymal markers such as vimentin, α-smooth muscle actin (α-SMA), Fn1, n-cadherin, and Snail, and endothelial markers such as von Willebrand factor (vWF), vascular endothelial cadherin (VE-cadherin), and CD31 are decreased) [ 7 ] . These molecular changes lead to enhanced cellular motility and invasiveness [ 8 ] . Morphologically, endothelial cells transition from a tightly-adherent, cobblestone-like monolayer to an elongated, spindle-shaped mesenchymal phenotype with disrupted intercellular junctions [ 9 ] . EndMT is mainly triggered by transforming growth factor β (TGF-β) signaling, which operated through both Smad-dependent and Smad-independent pathways [ 10 , 11 ] . Among the Smad family proteins, Smad2 constitutes a central mechanistic node in mediating TGF-β-induced EndMT [ 12 ] . Given the pathophysiological relevance of EndMT, therapeutic strategies targeting this process—or its reversal, mesenchymal-to-endothelial transition (MET)—may offer promising avenues to restore endothelial function in disease contexts. Ubiquitin-mediated proteolysis is a fundamental post-translational regulatory mechanism that governs a wide array of physiological processes. The ubiquitination cascade involves three sequential enzymatic components: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases [ 13 ] . Among the E3 ligases, the Smad ubiquitination regulatory factor (Smurf) family-comprising evolutionarily conserved HECT-domain proteins-plays a critical role in modulating TGF-β and BMP signaling pathways [ 14 ] . Smurf1, a representative HECT-type ubiquitin ligase, has been shown to regulate multiple signaling pathways, including TGF-β1, BMP, MAPK and TLR pathways [ 15 ] . Within the canonical TGF-β/BMP receptor superfamily, ligand binding leads to the phosphorylation and activation of receptor-regulated Smad proteins (R-Smads). Specifically, Smad1, Smad5, and Smad8 are mainly involved in BMP signaling, whereas Smad2 and Smad3 mediate canonical TGF-β-mediated signaling [ 16 ] . Accumulating evidence indicates that Smurf1 preferentially targets Smad1/5, while Smurf2 predominantly regulates Smad2/3 [ 17 ] . Although our previous study identified Smad2 as a key regulator of EndMT and found its modulation by the deubiquitinating enzyme USP7 [ 18 ] , it remains unclear whether and how Smad2 is regulated by E3 ligase during EndMT process. In this study, we investigated the expression and functional relationship between Smad2 and Smurf1 during EndMT. We found that Smurf1 inhibits EndMT by destabilization Smad2 protein. Mechanistically, Smurf1 interacts with Smad2 and promotes its degradation through enhanced K48-linked polyubiquitination at lysine 420. This post-translation modifications leads to a reduced Smad2 protein levels and subsequent suppression of EndMT in human coronary artery endothelial cells (HCAECs). These findings provide novel insights into the post-translational regulation of Smad2 by Smurf1 and highlight a previously unrecognized role of Smurf1 in modulating TGF-β-induced EndMT. Material and Methods 1.1. Cell culture This study utilized two distinct cell models: human embryonic kidney 293T cells (HEK293T) and HCAECs (Shanghai Biological Cell Bank, Shanghai, China). Both cell types were maintained in Dulbecco's Modified Eagle Medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS, Gibco #10270-106) and 1% penicillin-streptomycin under standard culture conditions (37°C, 5% CO₂, humidified atmosphere). For experimental interventions, cellular stimulation was achieved using recombinant human TGF-β2 (Proteintech #HZ-1092) at specified concentrations and durations as detailed in subsequent sections. 1.2. Cell transfection For Smurf1 knockdown in cells, we employed both small interfering RNAs (siRNAs, si-Smurf1; GenePharma) and a CRISPR/sgRNA plasmid (pLenti-Smurf1-sgRNA, L10610; Beyotime, Shanghai, China). To overexpress target genes, we constructed pcDNA3.1-based expression vectors containing full-length coding sequences of Smurf1, Smad2, ubiquitin (Ub), or K48-linked ubiquitin (K48), with pcDNA3.1 vector serving as negative control. HA-tagged ubiquitin and Myc-tagged wild-type Smurf1 (Dr. Lingqiang Zhang, State Key Laboratory of Proteomics); Flag-tagged wild-type Smurf1 (Dr. Chengjiang Gao, Shandong University); as well as HA-tagged K48 ubiquitinatin (Prof. Zheng Hui, Soochow University). Flag-tagged Smad2, and Myc-tagged Smad2 (KeyGEN, Nanjing, China). We used QuikChange Site-directed Mutation Kit (Agilent Technology) to produce a Smurf1 mutant (C699A) with no catalytic activity. Smad2 mutant plasmids (K63R, K19R, K46R, K420R, K13R, K156R, and K156R/K383R) were created by Dr. Wang Yan in our lab. All transfections were performed following manufacturers' protocols, using jetPRIME (101000046, Polyplus-transfection SA) for siRNA delivery and LongTrans (TF07, UcallM, Wuxi, China) for plasmid transfection. 1.3. RNA Isolation and qRT-PCR Quantification TRIzol reagent (Invitrogen, USA) was used to extract total cellular RNA. Following isolation, RNA samples underwent DNase I treatment (RNase-free) to remove potential genomic DNA contamination. According to the instructions of commercial reverse transcription kit (Takara, Shiga, Japan), DNA (cDNA) complementary to the isolated RNA was synthesized. Gene expression analysis was then conducted through quantitative real-time PCR (qRT-PCR) employing SYBR Green chemistry (Selleck Chemicals, Houston, TX, USA) on a LightCycler 480 system (Roche Diagnostics, Basel, Switzerland). The housekeeping gene GAPDH was used for normalization of all qRT-PCR data. All quantitative PCR reactions were conducted in technical triplicates to ensure experimental reproducibility and data reliability. The following primer sequences were used for amplification: Smurf1(5-CACAAGCCATTTTCTTTGCT-3.&5-ACTTGGCTGCATATCGAAAG-3), Smad2 (5-TGGGGAAGTTTTTGCCGAGT-3.& 5-ACCGTCTGCCTTCGGTATTC-3). 1.4. Immunoblotting (IB) and immunoprecipitation (IP) Cellular proteins were isolated using RIPA buffer (Beyotime, Shanghai, China) or NP-40 lysis buffer (Biosharp, China), both add protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). For ubiquitination studies, lysis buffer was additionally supplemented with 10 mM N-ethylmaleimide (NEM) to preserve ubiquitin conjugates. The biocinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China) was used to determine protein concentration. Extracted proteins were immunoprecipitated with bead-conjugated Flag antibody (Selleck Chemicals, USA), anti-Myc magnetic beads (Selleck Chemicals, USA), or protein A/G beads (Merck Millipore) conjugated with Smad2 antibody in a rotating incubator for 2 h at 4°C. Following immunoprecipitation, protein-bound beads were subjected to four consecutive washes with co-IP lysis buffer. Add Laemmli buffer containing 5% β-mercaptoethanol to the protein complex and boil at 100°C for 10 min. The proteins were then separated by SDS-PAGE using either 7.5% or 10% resolving gels (determined by target protein molecular weight) and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA) using wet transfer conditions. Then, membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature to prevent non-specific binding. Primary antibody incubations were carried out overnight at 4°C with gentle agitation to ensure optimal antigen-antibody interactions. The following primary antibodies were employed for immunoprecipitation and immunoblotting experiments: anti-Smurf1 (Santa Cruz Biotechnology, 4833S; 1:500), anti-Flag (Sigma-Aldrich, F1804; 1:5000), anti-Myc (Abbkine Scientific, A02060; 1:1000), anti-Smad2 (Cell Signaling Technology, 5339; 1:1000), anti-HA (Santa Cruz Biotechnology, sc-7392; 1:2000), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017; 1:1000), anti-GAPDH (Cell Signaling Technology, 188S, 1:5000), β-actin (Proteintech, #78; 1:5000), FNI (Abcam, Cambridge, MA, USA, AB268020, 1:1000), anti-Snail1 (Cell Signaling Technology, 3879S; 1:1000), and anti-N-cadherin (Santa Cruz Biotechnology, sc-393933, 1:500). Membranes were incubated with secondary antibodies for 60 minutes at 25°C with gentle agitation, after primary antibody incubation. Exposure of target proteins using commercially available enhanced chemiluminescence (ECL) substrate systems. 1.5. Immunofluorescence (IF) assay Place sterile glass coverslips in a 12-well plate and seed the cells at 5×10³ cells/well. After relevant transfection of the cells, the medium was discarded, and the cells were gently washed 3 times with PBS, then 1ml of 4% paraformaldehyde was added to fix for 30 min, permeabilized with 0.5% Triton X-100 for 30 min at room temperature, and blocked with 5% BSA solution for 1 h. Subsequently, incubate cells with anti-Smurf1 and Smad2 primary antibodies overnight and then with species-matched fluorescent secondary antibodies (1:500 dilution, 1 h). Counterstain the nuclei with DAPI, and images were using an Olympus microscope (Tokyo, Japan) under standardized exposure conditions to ensure consistency across sample. 1.6. Cell migration assays A scratch wound assay was used to assess the migratory capacity of HCAECs. Inoculate HCAECs using 6-well tissue culture plates and allowed to reach full confluency. After this, change the complete medium to serum-free medium. Using a sterile 200µl pipette tip, a consistent scratch was introduced into each monolayer. Following incubation, Non-adherent cells are removed using a gentle wash with PBS, then replenished with serum-free medium. Wound closure was monitored and photographed at the same predetermined locations at 0, 6, 12, and 24 h post-wounding monitored by phase-contrast microscopy (Olympus, Tokyo, Japan). The relative wound closure rate was calculated by calculating the percentage reduction in the wound area at specified intervals relative to the baseline measurement (0 hour), with ImageJ software used for analysis. 1.7. Cycloheximide chase assay To assess Smad2 degradation kinetics, transfected cells were analyzed under controlled conditions. Plated cells were transfected upon reaching 60% confluency. Following a 48-hour transfection period, cycloheximide (CHX; 10 µg/ml, Sigma) was administered to block new protein synthesis, with subsequent sample collection at predetermined intervals (0, 8, and 16 hours). Cellular proteins were then extracted and subjected to immunoblotting. To validate experimental reliability, three independent biological replicates were conducted for each condition. The protein degradation rate was calculated by quantifying Smad2 band intensity using ImageJ software (National Institutes of Health, USA, https://downloads.micron.ox.ac.uk/fiji_update/mirrors/fiji-latest/fiji-latest-win64-jdk . zip). 1.8. Statistical analysis To ensure reproducibility, three independent biological replicates were performed for all experiments, each containing triplicate technical samples. Semi-quantitative analysis was conducted using ImageJ software, followed by statistical processing with GraphPad Prism 8.0 (GraphPad, USA, https://app.graphpad.com/ ) and SPSS 26.0 software (IBM SPSS Statistics, USA, https://souurl.cn/NhIGed ). Data are presented as mean ± SEM. Intergroup comparisons were analyzed by one-way ANOVA and multiple comparisons use Tukey's post-hoc test. ns, p >0.05, * p < 0.05, ** p < 0.01, *** p < 0.001. 1.9. Statement All experimental protocols were approved by institute of pediatric research, Children’s Hospital of Soochow University, Suzhou, Jiangsu Province, China. We confirm that all methods were performed in accordance with the relevant guidelines and regulations. Results 2.1. Smurf1 represses the expression of Smad2 in protein level Our results demonstrate that overexpression of Smurf1 dose-dependently casued reduced levels of endogenous and exogenous Smad2 protein (Fig. 1 A and 1 B), while knockdown of endogenous Smurf1 by siRNA caused Smad2 protein level increased significantly in HCAECs (Fig. 1 C). Notably, Smurf1 did not alter Smad2 mRNA levels in HCAECs (Fig. 1 D and 1 E). To further confirm that Smurf1 regulates the stability of Smad2, HCAECs were treated with cycloheximide, a protein synthesis inhibitor and the degradation rate was examined. The results showed that transfection of Smurf1 accelerate the degradation rate of Smad2 protein (Fig. 1 F and 1 G). Consistently, depletion of endogenous Smurf1 with siRNA attenuated the degradation rate of Smad2 protein (Fig. 1 H and 1 I). These results together demonstrate that Smurf1 mediates time- and dose-dependent proteasomal degradation of Smad2 proteins, and the expression level of Smurf1 is negatively correlated with the Smad2 protein level and degradation. 2.2. Smurf1 interacts with Smad2 To investigate whether Smurf1 interacts with Smad2, coimmunoprecipitation assays were performed. 293T cells were co-transfected with Flag-Smad2 and Myc-Smurf1 constructs. Subsequent co-immunoprecipitation experiments using anti-Myc antibody successfully pulled down the Smurf1 complex, which demonstrated specific binding to Flag-Smad2 (Fig. 2 A). Reciprocal coimmunoprecipitation experiments with anti-Flag antibody yielded consistent results (Fig. 2 B). To further validate these findings, we examined potential interactions between endogenous Smad2 and Smurf1 proteins, and we found that endogenous Smad2 physiologically interacted with Smurf1 in HCAECs, indicating an interaction between Smad2 and Smurf1 (Fig. 2 C). Furthermore, immunofluorescence staining revealed a co-localization between Smurf1 and Smad2 proteins in HCAECs (Fig. 2 D), demonstrating a colocalization of the two proteins. These findings collectively establish that Smurf1 interacts with Smad2 at the cellular level. 2.3. Smurf1 ubiquitylates Smad2 Given Smurf1 is a HECT-type ubiquitin ligase, we next questioned whether Smurf1 affects the ubiquitination level of Smad2. We overexpressed Flag-Smad2 and HA-ub together with or without Myc-Smurf1 in cells. The data showed that Smurf1 overexpression causes both endogenous and exogenous ubiquitination levels of Smad2 increased in 293T cells (Fig. 3 A&B). This result was further corroborated by the siRNA-mediated knockdown of endogenous Smurf1, which caused a significantly decreased polyubiquitination level of Smad2 (Fig. 3 C). Furthermore, to clarify whether the E3 enzyme activity of Smurf1was required for its role in upregulating the ubiquitination level of Smad2, we generated an enzymatically inactive mutant by replacing 699 cysteine residue with alanine (Smurf1-C699A). The results showed that the catalytically inactive mutant Smurf1-C699A failed to increase the ubiquitination level of Smad2, demonstrating that the ubiquitin ligase activity of Smurf1 was required for down-regulating Smad2 protein level (Fig. 3 D). Together, these results establish Smad2 as a substrate of Smurf1 and demonstrate the essential role of Smurf1's E3 ligase activity in mediating this post-translational modification. 2.4. Smurf1 catalyzes K48-linked ubiquitination of Smad2 Based on the results aforementioned, we next questioned what type of ubiquitin signals of Smad2 is regulated by Smurf1. Because K48-linked polyubiquitination is recognized as the degradation signal, targeting substrates for proteasomal destruction through specific recognition by the 26S proteasome complex. To test this hypothesis, we co-transfected Smad2 and Smurf1 plasmids into 293T cells to examine the endogenous K48 polyubiquitin levels of Smad2. As shown in Fig. 4 A, Smurf1 overexpression significantly increases the endogenous K48 polyubiquitin levels of Smad2. Additionally, we also generated a mutant HA-K48R plasmid by replacing all the lysine residue with arginine except for the lysine 48 and transfected this mutant plasmid with or without Myc-Smurf1 and Flag-Smad2 into 293T cells. The results showed that Smurf1 overexpression also caused exogenous K48 polyubiquitin levels of Smad2 increased significantly (Fig. 4 B). Consistently, Smurf1 knockdown caused a decreased K48-linked polyubiquitination level of Smad2 proteins (Fig. 4 C). To further identify which lysine residue is essential for Smad2 ubiquitination, we generated seven mutants including K13R, K19R, K46R, K56R, K63R, K156R and K383R. To screen the specific lysine residue, we respectively transfected these mutant Smad2 plasmid or wild-type Smad2 plasmid together with Flag-smurf1 plasmid, then we detected the protein level changes of Smad2 proteins in each group. The results showed that Smurf1 overexpression causes a decreased level of Smad2 except for the Smad2 K420R , Smad2 K156R , and Smad2 K156/383R plasmid (Fig. 4 D), suggesting these lysine residues is required for the ubiquitin modification mediated by Smurf1. Next, cells were transfected with the wild-type Smad2 or the mutant including Smad2 K420R , - Smad2 K156R and - Smad2 K156R/K183R , together with or without Smurf1, the results showed that all these mutants had lower levels of ubiquitination compared with the wild-type Smad2 plasmid (Fig. 4 E), suggesting that the three lysine residues, especially Smad2 K420R are the ubiquitin acceptor residues of Smad2. Taken together, our results demonstrate that Smurf1 mediates K48-linked polyubiquitination of Smad2, and lysine 420 is essential for Smad2 ubiquitination. 2.5. Smurf1 antagonizes EndMT by decreasing the expression level of Smad2 protein During EndMT, cells undergo profound functional and transcriptional changes, characterized by the upregulation of mesenchymal marker proteins and a concurrent downregulation of endothelial markers.These alterations enhance the migratory and invasive capacities of the cells [ 8 ] . Based on this rationale, we next investigated whether Smurf1 and Smad2 regulate EndMT in HCAECs. The plasmids of WT Smad2, Smad2 K420R and Flag-Smurf1 or empty vector plasmid were respectively used to transfect HCAECs. And the scratch test showed that the over-expression of WT-Smad2 or the co-expression of Smurf1 and Smad2 k420R could increase the cell migration, while the co-expression of Smurf1 or Smurf1 and WT-Smad2 could reduce the cell migration(Fig. 5 A and 5 B). To examine the molecular shifts underlying the morphological changes, we extracted the proteins and conducted immunoblotting assay. The results showed that over-expression of WT-Smad2 or the co-expression of Smurf1 and Smad2k420R caused an increased expression level of FN1 N-cadherin and Snail. Interestingly, the co-expression of Smurf1 or Smurf1 and WT-Smad2 caused the expression level of FN1 N-cadherin and Snail downregulated, suggesting that Smad2-WT promoted the increase of FN1 and snail expression, and slightly up-regulated N-cadherin, however, when Smad2 and Smurf1 were co-transfected, FN1 N-cadherin and Snail were down-regulated to varying degrees, and this down-regulation trend was partially reversed by Smad2K420R mutants(Fig. 5 C). Discussion Firstly, the Smurf family was identified as key regulators of TGF-β/BMP signal transduction. Early studies established their ability to bind and ubiquitinate receptor-regulated Smads and the TGF-β type I receptor (TβRI), targeting these proteins for proteasomal degradation [ 19 ] . Beyond their canonical role in TGF-β/BMP regulation, subsequent research has revealed that Smurfs participate in diverse cellular processes. Notably, multiple lines of evidence indicate that Smurf1/2 regulate fundamental biological functions including cell cycle progression and migratory behavior through TGF-β/BMP-independent mechanisms [ 20 ] . The Smurf family is a key regulator of protein homeostasis, controlling the stability of diverse substrates to modulate essential cellular processes including migration, proliferation, and programmed cell death. Abnormal cell physiology and imbalances in cell homeostasis, often linked to disruptions in these processes, contribute to the development of various diseases [ 21 ] . Smurf1 exhibits complex regulatory functions in TGF-β/BMP signaling cascades, early studies focused predominantly on its E3 ubiquitin ligase activity targeting Smad1/5 for proteasomal degradation, thereby blocking intracellular BMP signals through ubiquitination and proteasomal degradation [ 22 ] . However, subsequent research has revealed that Smurf1 interactions extend beyond these targets. For instance, Multiple studies have demonstrated that Smurf1 promotes Smad7 ubiquitination and facilitates its cytoplasmic retention. Despite these findings, the regulatory mechanisms of Smurf1 on Smad2 remain unclear and somewhat ambiguous. While Smurf1 significantly affects Smad1 and Smad5 levels, its impact on Smad2 is less pronounced. Specifically, Smurf1 only affects Smad2 protein levels at the highest levels of Smurf1 expression [ 22 ] . Previous studies also have contradictory conclusion. Notably, Smurf1 expression failed to produce any detectable changes in total Smad2/3 abundance or their activated phosphorylated forms (pSmad2/3) in the lens epithelial cell system, further underscoring the selective nature of Smurf1's regulatory role [ 16 ] . Smurf1 significantly decreases endogenous Smad5 protein levels in C2C12 cells, while having no detectable effect on Smad2, Smad3, or Smad7 [ 23 ] . In experimental conditions where Smurf1 expression showed minimal impact on Smad2 and Smad3 protein levels, particularly in estrogen receptors α (ERα)-deficient contexts [ 24 ] . Additionally, in kidney disease, hypoxia can lead to the increased protein levels of TGF-β1,Smurf1, and Smad2/3 [ 25 ] . When Smad2 is co-expressed with Smurf1, higher degrees of Smad4 downregulation are observed [ 26 ] . Smurf1 is a key molecule in TGF-β/BMP signaling and other cellular processes, with its dysfunction linked to critical physiological and pathological outcomes. While its effects on Smad1, Smad5, and Smad7 are well-documented, its regulatory impact on Smad2 remains less clea. Future studies should systematically examine Smurf1-mediated Smad2 regulation and its dual roles in homeostasis and disease pathogenesis. Based on the above research objectives, we further explored its mechanism in HCAEC and found that Smurf1 regulates Smad2 levels in a dose-dependent manner, with overexpression of Smurf1 reducing Smad2 levels and knockdown increasing them. Smurf1 shortened the half-life of Smad2, promoting its degradation, while Smurf1 depletion prolonged Smad2 stability. The physical interaction between Smurf1 and Smad2 was consistently demonstrated through complementary approaches: (1) co-immunoprecipitation assays revealed molecular association in protein complexes, and (2) immunofluorescence microscopy confirmed their spatial co-localization in cellular compartments. Furthermore, Smurf1 catalyzed K48-linked ubiquitination of Smad2, targeting it for proteasomal degradation, with lysine 420 identified as the primary ubiquitination site. The E3 ubiquitin ligase function of Smurf1 proved critical for mediating this biological process, as an enzymatically inactive mutant (Smurf1 C699A) failed to ubiquitinate Smad2. The TGF-β/Smad signaling pathway represents a pleiotropic regulatory system that plays critical roles in both physiological homeostasis and pathological processes within the cardiovascular system. Such as myocardial infarction (AMI), Kawasaki disease (KD), pulmonary hypertension, atherosclerosis have been found to have pathological processes of EndMT [ 27 , 28 ] . For example, Ubiquitin-specific protease 7 (USP7) promotes EndMT in HCAECs through deubiquitination-mediated stabilization of Smad2 and Smad3 proteins [ 18 ] . Apolipoprotein A-I (ApoA-I) exerts a protective effect on endothelial function by suppressing EndMT in HCAECs, decreasing phosphorylated Smad3 and Smad2 [ 29 ] . Consistent with these findings, Ghrelin suppresses TGF-β1-mediated EndMT in HCAECs. This inhibitory effect occurs through the GHSR-1a/AMPK signaling axis, which upregulates Smad7 expression, thereby blocking Smad2/3 phosphorylation [ 30 ] . This investigation presents the initial experimental confirmation that Smurf1 mediates K48-linked ubiquitination of Smad2 at lysine 420s to restrain EndMT in HCAECs, as evidenced by decreased that of EndMT markers (Fn1, N-cadherin, snail). These findings demonstrate that Smurf1 regulates Smad2 stability through ubiquitination and degradation, providing mechanistic insights into its role in TGF-β signaling and EndMT. This study highlights Smurf1 as a potential target for intervention in diseases involving EndMT, such as fibrosis and cardiovascular disorders. Declarations Competing interests The authors declare no competing interests. Funding This work was funded by the National Natural Science Foundation of China (grant number nos. 82171797, 82371806, 82270529, 82470523). Author Contribution Panpan Liu: Writing–original draft, Data curation, Methodology. Mingyang Zhang: Visualization, Methodology, Data curation. Shuhui Wang: Investigation, Formal analysis. Zhiheng Liu: Visualization, Data curation, Methodology. Nana Wang: Resources. Zhiyuan Liu: Software. Yiyi Shen: Investigation, Methodology. Jiaying Zhang: Methodology, Data curation. Jing Li, Investigation, Data curation. Yin Liu, Validation, Data curation. Haitao Lv: Funding acquisition, Writing -Review & Editing, Project administration, Supervision. Guanghui Qian: Project administration, Writing-Review & Editing, Funding acquisition. Data Availability Data supporting the present study are available from the corresponding authors (Haitao Lv, [email protected] ; or Guanghui Qian, [email protected] ) upon reasonable request. References Bischoff, J. Endothelial-to-Mesenchymal Transition[J]. Circ. Res. 124 (8), 1163–1165. 10.1161/circresaha.119.314813 (2019). Arciniegas, E. et al. 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Involvement of USP7 in aggravating Kawasaki disease by promoting TGFβ2 signaling mediated endothelial-mesenchymal transition and coronary artery remodeling[J]. Int. Immunopharmacol. 146. 10.1016/j.intimp.2024.113823 (2025). Zhu, H. et al. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation[J]. Nature 400 (6745), 687–693. 10.1038/23293 (1999). David, D., Nair, S. A. & Pillai, M. R. Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression[J]. Biochim. Biophys. Acta . 1835 (1), 119–128. 10.1016/j.bbcan.2012.11.003 (2013). Wang, D. et al. The role ofSMURFsin non-cancerous diseases[J]. FASEB J. 37 (8). 10.1096/fj.202300598R (2023). Zhu, H. et al. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation[J]. Nature 400 (6745), 687–693. 10.1038/23293 (1999). Ying, S-X., Hussain, Z. J. & Zhang, Y. E. Smurf1 Facilitates Myogenic Differentiation and Antagonizes the Bone Morphogenetic Protein-2-induced Osteoblast Conversion by Targeting Smad5 for Degradation[J]. J. Biol. Chem. 278 (40), 39029–39036. 10.1074/jbc.M301193200 (2003). Ito, I. et al. Estrogen Inhibits Transforming Growth Factor β Signaling by Promoting Smad2/3 Degradation[J]. J. Biol. Chem. 285 (19), 14747–14755. 10.1074/jbc.M109.093039 (2010). Li, L. et al. cMet agonistic antibody prevents acute kidney injury to chronic kidney disease transition by suppressing Smurf1 and activating Smad7[J]. Clin. Sci. 135 (11), 1427–1444. 10.1042/cs20210013 (2021). Morén, A. et al. Degradation of the Tumor Suppressor Smad4 by WW and HECT Domain Ubiquitin Ligases[J]. J. Biol. Chem. 280 (23), 22115–22123. 10.1074/jbc.M414027200 (2005). Xu, X. et al. Snail Is a Direct Target of Hypoxia-inducible Factor 1α (HIF1α) in Hypoxia-induced Endothelial to Mesenchymal Transition of Human Coronary Endothelial Cells[J]. J. Biol. Chem. 290 (27), 16653–16664. 10.1074/jbc.M115.636944 (2015). Wang, J. et al. The miR-214-3p/c-Ski axis modulates endothelial–mesenchymal transition in human coronary artery endothelial cells in vitro and in mice model in vivo[J]. Hum. Cell . 35 (2), 486–497. 10.1007/s13577-021-00653-6 (2022). Feng, J. et al. Apolipoprotein A1 Inhibits the TGF-β1-Induced Endothelial-to-Mesenchymal Transition of Human Coronary Artery Endothelial Cells[J]. Cardiology 137 (3), 179–187. 10.1159/000464321 (2017). Chen, H. et al. Ghrelin attenuates myocardial fibrosis after acute myocardial infarction via inhibiting endothelial-to mesenchymal transition in rat model[J]. Peptides 111 , 118–126. 10.1016/j.peptides.2018.09.001 (2019). Additional Declarations No competing interests reported. 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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-6878518","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":485827355,"identity":"327ce6d9-67e1-4841-b435-64a8f7ebc876","order_by":0,"name":"Panpan Liu","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Liu","suffix":""},{"id":485827356,"identity":"6e72ff69-92d5-4157-bea8-010ea2e01c1d","order_by":1,"name":"Mingyang Zhang","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Mingyang","middleName":"","lastName":"Zhang","suffix":""},{"id":485827357,"identity":"d5b89ce1-d0c3-47fc-9e7c-6de9c02d7a0a","order_by":2,"name":"Shuhui Wang","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Shuhui","middleName":"","lastName":"Wang","suffix":""},{"id":485827358,"identity":"a8eefaa1-a665-4dbb-8d00-1b1b7bb6cd48","order_by":3,"name":"Zhiheng Liu","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Zhiheng","middleName":"","lastName":"Liu","suffix":""},{"id":485827359,"identity":"2b4cfecd-e1f7-4e7a-b1fd-dc4e6974a46d","order_by":4,"name":"Nana Wang","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Wang","suffix":""},{"id":485827360,"identity":"f8c6b119-2682-460d-a010-ac9966ff6fe0","order_by":5,"name":"Zhiyuan Liu","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Liu","suffix":""},{"id":485827361,"identity":"0cf38e35-1159-4c86-ab3a-ccbd88abd437","order_by":6,"name":"Yiyi Shen","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yiyi","middleName":"","lastName":"Shen","suffix":""},{"id":485827362,"identity":"3c9f50b9-7016-4cd6-9ad2-2a508a6b27e2","order_by":7,"name":"Jiaying Zhang","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jiaying","middleName":"","lastName":"Zhang","suffix":""},{"id":485827363,"identity":"955ef9fb-4087-4e56-bdf5-87c06dea68d2","order_by":8,"name":"Jing Li","email":"","orcid":"","institution":"Children's Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""},{"id":485827364,"identity":"11c79daf-22ac-4c4e-92f3-8e5ff3f29a02","order_by":9,"name":"Ying Liu","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Liu","suffix":""},{"id":485827365,"identity":"4389195a-07dc-4684-85ae-a970734b3aba","order_by":10,"name":"Haitao Lv","email":"","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Haitao","middleName":"","lastName":"Lv","suffix":""},{"id":485827366,"identity":"86a72049-4634-4dd0-a31e-1bb5751134a9","order_by":11,"name":"Guanghui Qian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDACCQaGAwwMNkBWAhCzEa8ljUQtQHCYBC3y0c0bD/zccd7e4HjyA4YPZYcZ+Gc34NdieOdYwcHeM7cTN5x5ZsA449xhBok7BwhomZFjcIC37XaCwY0EA2betsMMBhIJhLUc/Nt2zt7gRvoH5r/EaJGXyDE4zNt2gHHDjRwDZkZitBhIpBUclm1LTpx55k3BwZ5z6TwSNwjZMiN588e3bXb2fMfTNz74UWYtxz+DkC0HGAzgnANAzINfPciWBiQto2AUjIJRMAqwAgAPuUqj425PWAAAAABJRU5ErkJggg==","orcid":"","institution":"Children’s Hospital of Soochow University","correspondingAuthor":true,"prefix":"","firstName":"Guanghui","middleName":"","lastName":"Qian","suffix":""}],"badges":[],"createdAt":"2025-06-12 09:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6878518/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6878518/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86882399,"identity":"e240faa6-0dc6-4d11-9d9c-2ed3954523f6","added_by":"auto","created_at":"2025-07-16 16:41:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118724,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 decrease the turnover and stability of Smad2proteins. (A) Overexpression of Smurf1 dose-dependently resulted in reduced levels of endogenous Smad2 protein. (B) Endogenous Smad2 levels decreased as Myc-Smurf1 concentrations increased In 293T cells. (C) Smad2 immunoblot in HCAECs following siRNA-mediated knockdown of endogenous Smurf1. (D-E) qRT-PCR quantification of Smurf1 and Smad2 mRNA levels in HCAECs expressing Myc-Smurf1. (F) Myc-tagged Smad2 was transiently expressed in 293T cells either alone or in combination with Myc-Smurf1. Following transfection, by using CHX 50 to assess protein degradation for varying time intervals. Protein expression patterns were subsequently evaluated through immunoblot analysis. (J) Densitometric quantification (ImageJ) of Smad2 protein levels in (F). (H) CHX chase assay of endogenous Smad2 in 293T cells transfected with control (si-NC) or Smurf1-targeting siRNA (si-Smurf1). (I) Quantification (ImageJ) of Smad2 levels in (H) All values are expressed as mean ± SEM, n = 3 , \u003csup\u003ens\u003c/sup\u003eP ＞ 0.05, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/55c9d8c54003d145f61b3c63.png"},{"id":86881614,"identity":"f0f1c30c-2648-4e47-abea-5cb1006237fa","added_by":"auto","created_at":"2025-07-16 16:33:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138350,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 interacts with Smad2. (A) 2Myc-Smurf1 and Flag-Smad2 expression vectors were used for transient transfection of 293T cells. Following lysis, protein was immunoprecipitated using anti-Myc magnetic beads and analyzed with Flag antibody. (B) Co-immunoprecipitation was used in 293T cells expressing both Myc-Smurf1 and Flag-Smad2. Cell extracts were precipitated with bead-conjugated Flag antibody and immunoblotting with anti-Myc antibody. (C) HCAEC lysates used anti-Smad2 antibody for immunoprecipitation, followed by immunoblotting with relevant antibodies. (D) Subcellular co-localization was visualized by confocal microscopy in HCAECs, with Smurf1 (green) and Smad2 (red) fluorescence patterns analyzed. Nuclear was stained with DAPI (blue). Scale bars = 100 μm.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/224b715366393c7a8071f28e.png"},{"id":86882554,"identity":"8372dc24-306f-4827-ba57-29c73ef50483","added_by":"auto","created_at":"2025-07-16 16:49:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147841,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 ubiquitylates Smad2. (A-B) Myc-tagged Smurf1 and Flag-tagged Smad2 were used to co-transfect 293T cells, either in the presence or absence of an HA-ubiquitin. Ubiquitination was detected by immunoblotting using HA and ubiquitin antibodies. (C) Expression of wild type smurf1 increased ubiquitination of Smad2 in 293T cells, while the catalytically ineffective C699a mutant had no effect on ubiquitination of Smad2. (D) Depletion of endogenous Smurf1 reduced ubiquitination levels of Smad2 in HCAECs.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/ec10ed5edc38c6f3cbcc5678.png"},{"id":86881612,"identity":"3d441154-73ad-4158-be00-945979f36b8f","added_by":"auto","created_at":"2025-07-16 16:33:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":189426,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 catalyzes K48-linked ubiquitination of Smad2. (A) Endogenous Smad2 ubiquitination in 293T cells overexpressing Smurf1, detected using K48-specific ubiquitin antibodies. (B) 293T cells co-transfected with Flag-Smad2, Myc-Smurf1 and HA-K48 ubiquitin. immunoprecipitates were immunoblotted with HA antibodies. (C) siRNA-mediated Smurf1 knockdown reduces K48-polyubiquitinated Smad2 levels in 293T cells. (D) Transfections included wild-type Smad2 or the following lysine-to-arginine mutants: K19R, K13R, K46R, K63R, K156R, K420R, the double mutant K156R/K383R, and either Flag-tagged Smurf1 or empty vector control. (E) 293T cells were transfected with wild-type Smad2, Smad2\u003csup\u003e K420R\u003c/sup\u003e, Smad2\u003csup\u003e K156R\u003c/sup\u003e, or Smad2 \u003csup\u003eK156R/K383R\u003c/sup\u003e and HA - ubiquitin, in combination with either Flag-Smurf1 or an empty vector control. The isolated proteins were analyzed using an antibody targeting HA to assess ubiquitin modification.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/c9ae4a23f700415e3ea8bc9b.png"},{"id":86881618,"identity":"a90d0f29-7a84-4af5-a77c-92d9a077fa9c","added_by":"auto","created_at":"2025-07-16 16:33:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":543450,"visible":true,"origin":"","legend":"\u003cp\u003eSmurf1 inhibits EndMT by decreasing Smad2 protein. (A-B) The migration ability of HCAECs was measured by wound healing test. Scale bars = 200 μm. (C). The expression levels of Fn1, Snail, and N-cadherin in HCAECs were assessed via western blot following transfection with plasmids encoding WT Smad2, Smad2\u003csup\u003eK420R\u003c/sup\u003e, and either Flag-Smurfl or an empty vector. All values are expressed as mean ± SEM, n = 3, *P \u0026lt; 0.05 and **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/5f2249882df185326c2f72ab.png"},{"id":86882556,"identity":"5fc151cc-f836-42af-88d5-97fca854583b","added_by":"auto","created_at":"2025-07-16 16:49:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2340735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/299baa06-19c7-4fda-898e-d6e790dca74d.pdf"},{"id":86881610,"identity":"e4dce743-307b-4616-a887-d6b39e2dc2b6","added_by":"auto","created_at":"2025-07-16 16:33:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1512382,"visible":true,"origin":"","legend":"","description":"","filename":"suppiy.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6878518/v1/dd7670ac43f7884c19db2329.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ubiquitination of Smad2 by Smurf1 inhibits endothelial-to-mesenchymal transition in human coronary artery endothelial cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndothelial-to-mesenchymal transition (EndMT) is a biological process involved in both normal physiological development and the progression of various diseases, and it is associated with the loss of endothelial barrier integrity\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Initially, EndMT was believed to play a critical role in embryogenesis, particularly during cardiogenesis\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. However, subsequent studies have highlighted its pathological involvement in multiple disease states\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. During EndMT, endothelial cells undergo significant alterations in morphology, function, and gene expression profiles\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. This transition is characterized by the upregulation of mesenchymal markers such as vimentin, α-smooth muscle actin (α-SMA), Fn1, n-cadherin, and Snail, and endothelial markers such as von Willebrand factor (vWF), vascular endothelial cadherin (VE-cadherin), and CD31 are decreased)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThese molecular changes lead to enhanced cellular motility and invasiveness\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Morphologically, endothelial cells transition from a tightly-adherent, cobblestone-like monolayer to an elongated, spindle-shaped mesenchymal phenotype with disrupted intercellular junctions\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. EndMT is mainly triggered by transforming growth factor β (TGF-β) signaling, which operated through both Smad-dependent and Smad-independent pathways\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Among the Smad family proteins, Smad2 constitutes a central mechanistic node in mediating TGF-β-induced EndMT\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Given the pathophysiological relevance of EndMT, therapeutic strategies targeting this process\u0026mdash;or its reversal, mesenchymal-to-endothelial transition (MET)\u0026mdash;may offer promising avenues to restore endothelial function in disease contexts.\u003c/p\u003e\u003cp\u003eUbiquitin-mediated proteolysis is a fundamental post-translational regulatory mechanism that governs a wide array of physiological processes. The ubiquitination cascade involves three sequential enzymatic components: E1 ubiquitin-activating enzymes, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Among the E3 ligases, the Smad ubiquitination regulatory factor (Smurf) family-comprising evolutionarily conserved HECT-domain proteins-plays a critical role in modulating TGF-β and BMP signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Smurf1, a representative HECT-type ubiquitin ligase, has been shown to regulate multiple signaling pathways, including TGF-β1, BMP, MAPK and TLR pathways\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Within the canonical TGF-β/BMP receptor superfamily, ligand binding leads to the phosphorylation and activation of receptor-regulated Smad proteins (R-Smads). Specifically, Smad1, Smad5, and Smad8 are mainly involved in BMP signaling, whereas Smad2 and Smad3 mediate canonical TGF-β-mediated signaling\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Accumulating evidence indicates that Smurf1 preferentially targets Smad1/5, while Smurf2 predominantly regulates Smad2/3\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Although our previous study identified Smad2 as a key regulator of EndMT and found its modulation by the deubiquitinating enzyme USP7\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, it remains unclear whether and how Smad2 is regulated by E3 ligase during EndMT process.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the expression and functional relationship between Smad2 and Smurf1 during EndMT. We found that Smurf1 inhibits EndMT by destabilization Smad2 protein. Mechanistically, Smurf1 interacts with Smad2 and promotes its degradation through enhanced K48-linked polyubiquitination at lysine 420. This post-translation modifications leads to a reduced Smad2 protein levels and subsequent suppression of EndMT in human coronary artery endothelial cells (HCAECs). These findings provide novel insights into the post-translational regulation of Smad2 by Smurf1 and highlight a previously unrecognized role of Smurf1 in modulating TGF-β-induced EndMT.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e1.1. Cell culture\u003c/h2\u003e\u003cp\u003eThis study utilized two distinct cell models: human embryonic kidney 293T cells (HEK293T) and HCAECs (Shanghai Biological Cell Bank, Shanghai, China). Both cell types were maintained in Dulbecco's Modified Eagle Medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS, Gibco #10270-106) and 1% penicillin-streptomycin under standard culture conditions (37\u0026deg;C, 5% CO₂, humidified atmosphere). For experimental interventions, cellular stimulation was achieved using recombinant human TGF-β2 (Proteintech #HZ-1092) at specified concentrations and durations as detailed in subsequent sections.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e1.2. Cell transfection\u003c/h2\u003e\u003cp\u003eFor Smurf1 knockdown in cells, we employed both small interfering RNAs (siRNAs, si-Smurf1; GenePharma) and a CRISPR/sgRNA plasmid (pLenti-Smurf1-sgRNA, L10610; Beyotime, Shanghai, China). To overexpress target genes, we constructed pcDNA3.1-based expression vectors containing full-length coding sequences of Smurf1, Smad2, ubiquitin (Ub), or K48-linked ubiquitin (K48), with pcDNA3.1 vector serving as negative control. HA-tagged ubiquitin and Myc-tagged wild-type Smurf1 (Dr. Lingqiang Zhang, State Key Laboratory of Proteomics); Flag-tagged wild-type Smurf1 (Dr. Chengjiang Gao, Shandong University); as well as HA-tagged K48 ubiquitinatin (Prof. Zheng Hui, Soochow University). Flag-tagged Smad2, and Myc-tagged Smad2 (KeyGEN, Nanjing, China). We used QuikChange Site-directed Mutation Kit (Agilent Technology) to produce a Smurf1 mutant (C699A) with no catalytic activity. Smad2 mutant plasmids (K63R, K19R, K46R, K420R, K13R, K156R, and K156R/K383R) were created by Dr. Wang Yan in our lab. All transfections were performed following manufacturers' protocols, using jetPRIME (101000046, Polyplus-transfection SA) for siRNA delivery and LongTrans (TF07, UcallM, Wuxi, China) for plasmid transfection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e1.3. RNA Isolation and qRT-PCR Quantification\u003c/h2\u003e\u003cp\u003eTRIzol reagent (Invitrogen, USA) was used to extract total cellular RNA. Following isolation, RNA samples underwent DNase I treatment (RNase-free) to remove potential genomic DNA contamination. According to the instructions of commercial reverse transcription kit (Takara, Shiga, Japan), DNA (cDNA) complementary to the isolated RNA was synthesized. Gene expression analysis was then conducted through quantitative real-time PCR (qRT-PCR) employing SYBR Green chemistry (Selleck Chemicals, Houston, TX, USA) on a LightCycler 480 system (Roche Diagnostics, Basel, Switzerland). The housekeeping gene GAPDH was used for normalization of all qRT-PCR data. All quantitative PCR reactions were conducted in technical triplicates to ensure experimental reproducibility and data reliability. The following primer sequences were used for amplification: Smurf1(5-CACAAGCCATTTTCTTTGCT-3.\u0026amp;5-ACTTGGCTGCATATCGAAAG-3), Smad2 (5-TGGGGAAGTTTTTGCCGAGT-3.\u0026amp; 5-ACCGTCTGCCTTCGGTATTC-3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e1.4. Immunoblotting (IB) and immunoprecipitation (IP)\u003c/h2\u003e\u003cp\u003eCellular proteins were isolated using RIPA buffer (Beyotime, Shanghai, China) or NP-40 lysis buffer (Biosharp, China), both add protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). For ubiquitination studies, lysis buffer was additionally supplemented with 10 mM N-ethylmaleimide (NEM) to preserve ubiquitin conjugates. The biocinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China) was used to determine protein concentration. Extracted proteins were immunoprecipitated with bead-conjugated Flag antibody (Selleck Chemicals, USA), anti-Myc magnetic beads (Selleck Chemicals, USA), or protein A/G beads (Merck Millipore) conjugated with Smad2 antibody in a rotating incubator for 2 h at 4\u0026deg;C. Following immunoprecipitation, protein-bound beads were subjected to four consecutive washes with co-IP lysis buffer. Add Laemmli buffer containing 5% β-mercaptoethanol to the protein complex and boil at 100\u0026deg;C for 10 min. The proteins were then separated by SDS-PAGE using either 7.5% or 10% resolving gels (determined by target protein molecular weight) and transferred onto PVDF membranes (Bio-Rad Laboratories, Hercules, CA) using wet transfer conditions. Then, membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature to prevent non-specific binding. Primary antibody incubations were carried out overnight at 4\u0026deg;C with gentle agitation to ensure optimal antigen-antibody interactions. The following primary antibodies were employed for immunoprecipitation and immunoblotting experiments: anti-Smurf1 (Santa Cruz Biotechnology, 4833S; 1:500), anti-Flag (Sigma-Aldrich, F1804; 1:5000), anti-Myc (Abbkine Scientific, A02060; 1:1000), anti-Smad2 (Cell Signaling Technology, 5339; 1:1000), anti-HA (Santa Cruz Biotechnology, sc-7392; 1:2000), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017; 1:1000), anti-GAPDH (Cell Signaling Technology, 188S, 1:5000), β-actin (Proteintech, #78; 1:5000), FNI (Abcam, Cambridge, MA, USA, AB268020, 1:1000), anti-Snail1 (Cell Signaling Technology, 3879S; 1:1000), and anti-N-cadherin (Santa Cruz Biotechnology, sc-393933, 1:500). Membranes were incubated with secondary antibodies for 60 minutes at 25\u0026deg;C with gentle agitation, after primary antibody incubation. Exposure of target proteins using commercially available enhanced chemiluminescence (ECL) substrate systems.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e1.5. Immunofluorescence (IF) assay\u003c/h2\u003e\u003cp\u003ePlace sterile glass coverslips in a 12-well plate and seed the cells at 5\u0026times;10\u0026sup3; cells/well. After relevant transfection of the cells, the medium was discarded, and the cells were gently washed 3 times with PBS, then 1ml of 4% paraformaldehyde was added to fix for 30 min, permeabilized with 0.5% Triton X-100 for 30 min at room temperature, and blocked with 5% BSA solution for 1 h. Subsequently, incubate cells with anti-Smurf1 and Smad2 primary antibodies overnight and then with species-matched fluorescent secondary antibodies (1:500 dilution, 1 h). Counterstain the nuclei with DAPI, and images were using an Olympus microscope (Tokyo, Japan) under standardized exposure conditions to ensure consistency across sample.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e1.6. Cell migration assays\u003c/h2\u003e\u003cp\u003eA scratch wound assay was used to assess the migratory capacity of HCAECs. Inoculate HCAECs using 6-well tissue culture plates and allowed to reach full confluency. After this, change the complete medium to serum-free medium. Using a sterile 200\u0026micro;l pipette tip, a consistent scratch was introduced into each monolayer. Following incubation, Non-adherent cells are removed using a gentle wash with PBS, then replenished with serum-free medium. Wound closure was monitored and photographed at the same predetermined locations at 0, 6, 12, and 24 h post-wounding monitored by phase-contrast microscopy (Olympus, Tokyo, Japan). The relative wound closure rate was calculated by calculating the percentage reduction in the wound area at specified intervals relative to the baseline measurement (0 hour), with ImageJ software used for analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e1.7. Cycloheximide chase assay\u003c/h2\u003e\u003cp\u003eTo assess Smad2 degradation kinetics, transfected cells were analyzed under controlled conditions. Plated cells were transfected upon reaching 60% confluency. Following a 48-hour transfection period, cycloheximide (CHX; 10 \u0026micro;g/ml, Sigma) was administered to block new protein synthesis, with subsequent sample collection at predetermined intervals (0, 8, and 16 hours). Cellular proteins were then extracted and subjected to immunoblotting. To validate experimental reliability, three independent biological replicates were conducted for each condition. The protein degradation rate was calculated by quantifying Smad2 band intensity using ImageJ software (National Institutes of Health, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://downloads.micron.ox.ac.uk/fiji_update/mirrors/fiji-latest/fiji-latest-win64-jdk\u003c/span\u003e\u003cspan address=\"https://downloads.micron.ox.ac.uk/fiji_update/mirrors/fiji-latest/fiji-latest-win64-jdk\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003ezip).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e1.8. Statistical analysis\u003c/h2\u003e\u003cp\u003eTo ensure reproducibility, three independent biological replicates were performed for all experiments, each containing triplicate technical samples. Semi-quantitative analysis was conducted using ImageJ software, followed by statistical processing with GraphPad Prism 8.0 (GraphPad, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://app.graphpad.com/\u003c/span\u003e\u003cspan address=\"https://app.graphpad.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SPSS 26.0 software (IBM SPSS Statistics, USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://souurl.cn/NhIGed\u003c/span\u003e\u003cspan address=\"https://souurl.cn/NhIGed\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Intergroup comparisons were analyzed by one-way ANOVA and multiple comparisons use Tukey's post-hoc test. ns, \u003cem\u003ep\u003c/em\u003e\u0026gt;0.05, *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e1.9. Statement\u003c/h2\u003e\u003cp\u003eAll experimental protocols were approved by institute of pediatric research, Children\u0026rsquo;s Hospital of Soochow University, Suzhou, Jiangsu Province, China. We confirm that all methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Smurf1 represses the expression of Smad2 in protein level\u003c/h2\u003e\u003cp\u003eOur results demonstrate that overexpression of Smurf1 dose-dependently casued reduced levels of endogenous and exogenous Smad2 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), while knockdown of endogenous Smurf1 by siRNA caused Smad2 protein level increased significantly in HCAECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notably, Smurf1 did not alter Smad2 mRNA levels in HCAECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). To further confirm that Smurf1 regulates the stability of Smad2, HCAECs were treated with cycloheximide, a protein synthesis inhibitor and the degradation rate was examined. The results showed that transfection of Smurf1 accelerate the degradation rate of Smad2 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Consistently, depletion of endogenous Smurf1 with siRNA attenuated the degradation rate of Smad2 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These results together demonstrate that Smurf1 mediates time- and dose-dependent proteasomal degradation of Smad2 proteins, and the expression level of Smurf1 is negatively correlated with the Smad2 protein level and degradation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Smurf1 interacts with Smad2\u003c/h2\u003e\u003cp\u003eTo investigate whether Smurf1 interacts with Smad2, coimmunoprecipitation assays were performed. 293T cells were co-transfected with Flag-Smad2 and Myc-Smurf1 constructs. Subsequent co-immunoprecipitation experiments using anti-Myc antibody successfully pulled down the Smurf1 complex, which demonstrated specific binding to Flag-Smad2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Reciprocal coimmunoprecipitation experiments with anti-Flag antibody yielded consistent results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To further validate these findings, we examined potential interactions between endogenous Smad2 and Smurf1 proteins, and we found that endogenous Smad2 physiologically interacted with Smurf1 in HCAECs, indicating an interaction between Smad2 and Smurf1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Furthermore, immunofluorescence staining revealed a co-localization between Smurf1 and Smad2 proteins in HCAECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), demonstrating a colocalization of the two proteins. These findings collectively establish that Smurf1 interacts with Smad2 at the cellular level.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.3. \u003cb\u003eSmurf1 ubiquitylates Smad2\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eGiven Smurf1 is a HECT-type ubiquitin ligase, we next questioned whether Smurf1 affects the ubiquitination level of Smad2. We overexpressed Flag-Smad2 and HA-ub together with or without Myc-Smurf1 in cells. The data showed that Smurf1 overexpression causes both endogenous and exogenous ubiquitination levels of Smad2 increased in 293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026amp;B). This result was further corroborated by the siRNA-mediated knockdown of endogenous Smurf1, which caused a significantly decreased polyubiquitination level of Smad2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Furthermore, to clarify whether the E3 enzyme activity of Smurf1was required for its role in upregulating the ubiquitination level of Smad2, we generated an enzymatically inactive mutant by replacing 699 cysteine residue with alanine (Smurf1-C699A). The results showed that the catalytically inactive mutant Smurf1-C699A failed to increase the ubiquitination level of Smad2, demonstrating that the ubiquitin ligase activity of Smurf1 was required for down-regulating Smad2 protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Together, these results establish Smad2 as a substrate of Smurf1 and demonstrate the essential role of Smurf1's E3 ligase activity in mediating this post-translational modification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Smurf1 catalyzes K48-linked ubiquitination of Smad2\u003c/h2\u003e\u003cp\u003eBased on the results aforementioned, we next questioned what type of ubiquitin signals of Smad2 is regulated by Smurf1. Because K48-linked polyubiquitination is recognized as the degradation signal, targeting substrates for proteasomal destruction through specific recognition by the 26S proteasome complex. To test this hypothesis, we co-transfected Smad2 and Smurf1 plasmids into 293T cells to examine the endogenous K48 polyubiquitin levels of Smad2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Smurf1 overexpression significantly increases the endogenous K48 polyubiquitin levels of Smad2. Additionally, we also generated a mutant HA-K48R plasmid by replacing all the lysine residue with arginine except for the lysine 48 and transfected this mutant plasmid with or without Myc-Smurf1 and Flag-Smad2 into 293T cells. The results showed that Smurf1 overexpression also caused exogenous K48 polyubiquitin levels of Smad2 increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Consistently, Smurf1 knockdown caused a decreased K48-linked polyubiquitination level of Smad2 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo further identify which lysine residue is essential for Smad2 ubiquitination, we generated seven mutants including K13R, K19R, K46R, K56R, K63R, K156R and K383R. To screen the specific lysine residue, we respectively transfected these mutant Smad2 plasmid or wild-type Smad2 plasmid together with Flag-smurf1 plasmid, then we detected the protein level changes of Smad2 proteins in each group. The results showed that Smurf1 overexpression causes a decreased level of Smad2 except for the Smad2 \u003csup\u003eK420R\u003c/sup\u003e, Smad2 \u003csup\u003eK156R\u003c/sup\u003e, and Smad2 \u003csup\u003eK156/383R\u003c/sup\u003e plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), suggesting these lysine residues is required for the ubiquitin modification mediated by Smurf1. Next, cells were transfected with the wild-type Smad2 or the mutant including Smad2\u003csup\u003eK420R\u003c/sup\u003e, - Smad2\u003csup\u003eK156R\u003c/sup\u003e and - Smad2\u003csup\u003eK156R/K183R\u003c/sup\u003e, together with or without Smurf1, the results showed that all these mutants had lower levels of ubiquitination compared with the wild-type Smad2 plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggesting that the three lysine residues, especially Smad2\u003csup\u003eK420R\u003c/sup\u003e are the ubiquitin acceptor residues of Smad2. Taken together, our results demonstrate that Smurf1 mediates K48-linked polyubiquitination of Smad2, and lysine 420 is essential for Smad2 ubiquitination.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Smurf1 antagonizes EndMT by decreasing the expression level of Smad2 protein\u003c/h2\u003e\u003cp\u003eDuring EndMT, cells undergo profound functional and transcriptional changes, characterized by the upregulation of mesenchymal marker proteins and a concurrent downregulation of endothelial markers.These alterations enhance the migratory and invasive capacities of the cells\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Based on this rationale, we next investigated whether Smurf1 and Smad2 regulate EndMT in HCAECs. The plasmids of WT Smad2, Smad2\u003csup\u003eK420R\u003c/sup\u003e and Flag-Smurf1 or empty vector plasmid were respectively used to transfect HCAECs. And the scratch test showed that the over-expression of WT-Smad2 or the co-expression of Smurf1 and Smad2\u003csup\u003ek420R\u003c/sup\u003e could increase the cell migration, while the co-expression of Smurf1 or Smurf1 and WT-Smad2 could reduce the cell migration(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To examine the molecular shifts underlying the morphological changes, we extracted the proteins and conducted immunoblotting assay. The results showed that over-expression of WT-Smad2 or the co-expression of Smurf1 and Smad2k420R caused an increased expression level of FN1 N-cadherin and Snail. Interestingly, the co-expression of Smurf1 or Smurf1 and WT-Smad2 caused the expression level of FN1 N-cadherin and Snail downregulated, suggesting that Smad2-WT promoted the increase of FN1 and snail expression, and slightly up-regulated N-cadherin, however, when Smad2 and Smurf1 were co-transfected, FN1 N-cadherin and Snail were down-regulated to varying degrees, and this down-regulation trend was partially reversed by Smad2K420R mutants(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFirstly, the Smurf family was identified as key regulators of TGF-β/BMP signal transduction. Early studies established their ability to bind and ubiquitinate receptor-regulated Smads and the TGF-β type I receptor (TβRI), targeting these proteins for proteasomal degradation\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Beyond their canonical role in TGF-β/BMP regulation, subsequent research has revealed that Smurfs participate in diverse cellular processes. Notably, multiple lines of evidence indicate that Smurf1/2 regulate fundamental biological functions including cell cycle progression and migratory behavior through TGF-β/BMP-independent mechanisms\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The Smurf family is a key regulator of protein homeostasis, controlling the stability of diverse substrates to modulate essential cellular processes including migration, proliferation, and programmed cell death. Abnormal cell physiology and imbalances in cell homeostasis, often linked to disruptions in these processes, contribute to the development of various diseases\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSmurf1 exhibits complex regulatory functions in TGF-β/BMP signaling cascades, early studies focused predominantly on its E3 ubiquitin ligase activity targeting Smad1/5 for proteasomal degradation, thereby blocking intracellular BMP signals through ubiquitination and proteasomal degradation\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. However, subsequent research has revealed that Smurf1 interactions extend beyond these targets. For instance, Multiple studies have demonstrated that Smurf1 promotes Smad7 ubiquitination and facilitates its cytoplasmic retention. Despite these findings, the regulatory mechanisms of Smurf1 on Smad2 remain unclear and somewhat ambiguous. While Smurf1 significantly affects Smad1 and Smad5 levels, its impact on Smad2 is less pronounced. Specifically, Smurf1 only affects Smad2 protein levels at the highest levels of Smurf1 expression\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Previous studies also have contradictory conclusion. Notably, Smurf1 expression failed to produce any detectable changes in total Smad2/3 abundance or their activated phosphorylated forms (pSmad2/3) in the lens epithelial cell system, further underscoring the selective nature of Smurf1's regulatory role\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Smurf1 significantly decreases endogenous Smad5 protein levels in C2C12 cells, while having no detectable effect on Smad2, Smad3, or Smad7\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In experimental conditions where Smurf1 expression showed minimal impact on Smad2 and Smad3 protein levels, particularly in estrogen receptors α (ERα)-deficient contexts\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Additionally, in kidney disease, hypoxia can lead to the increased protein levels of TGF-β1,Smurf1, and Smad2/3\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. When Smad2 is co-expressed with Smurf1, higher degrees of Smad4 downregulation are observed\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSmurf1 is a key molecule in TGF-β/BMP signaling and other cellular processes, with its dysfunction linked to critical physiological and pathological outcomes. While its effects on Smad1, Smad5, and Smad7 are well-documented, its regulatory impact on Smad2 remains less clea. Future studies should systematically examine Smurf1-mediated Smad2 regulation and its dual roles in homeostasis and disease pathogenesis. Based on the above research objectives, we further explored its mechanism in HCAEC and found that Smurf1 regulates Smad2 levels in a dose-dependent manner, with overexpression of Smurf1 reducing Smad2 levels and knockdown increasing them. Smurf1 shortened the half-life of Smad2, promoting its degradation, while Smurf1 depletion prolonged Smad2 stability. The physical interaction between Smurf1 and Smad2 was consistently demonstrated through complementary approaches: (1) co-immunoprecipitation assays revealed molecular association in protein complexes, and (2) immunofluorescence microscopy confirmed their spatial co-localization in cellular compartments. Furthermore, Smurf1 catalyzed K48-linked ubiquitination of Smad2, targeting it for proteasomal degradation, with lysine 420 identified as the primary ubiquitination site. The E3 ubiquitin ligase function of Smurf1 proved critical for mediating this biological process, as an enzymatically inactive mutant (Smurf1 C699A) failed to ubiquitinate Smad2.\u003c/p\u003e\u003cp\u003eThe TGF-β/Smad signaling pathway represents a pleiotropic regulatory system that plays critical roles in both physiological homeostasis and pathological processes within the cardiovascular system. Such as myocardial infarction (AMI), Kawasaki disease (KD), pulmonary hypertension, atherosclerosis have been found to have pathological processes of EndMT\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. For example, Ubiquitin-specific protease 7 (USP7) promotes EndMT in HCAECs through deubiquitination-mediated stabilization of Smad2 and Smad3 proteins\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Apolipoprotein A-I (ApoA-I) exerts a protective effect on endothelial function by suppressing EndMT in HCAECs, decreasing phosphorylated Smad3 and Smad2\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Consistent with these findings, Ghrelin suppresses TGF-β1-mediated EndMT in HCAECs. This inhibitory effect occurs through the GHSR-1a/AMPK signaling axis, which upregulates Smad7 expression, thereby blocking Smad2/3 phosphorylation\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. This investigation presents the initial experimental confirmation that Smurf1 mediates K48-linked ubiquitination of Smad2 at lysine 420s to restrain EndMT in HCAECs, as evidenced by decreased that of EndMT markers (Fn1, N-cadherin, snail). These findings demonstrate that Smurf1 regulates Smad2 stability through ubiquitination and degradation, providing mechanistic insights into its role in TGF-β signaling and EndMT. This study highlights Smurf1 as a potential target for intervention in diseases involving EndMT, such as fibrosis and cardiovascular disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was funded by the National Natural Science Foundation of China (grant number nos. 82171797, 82371806, 82270529, 82470523).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003ePanpan Liu: Writing\u0026ndash;original draft, Data curation, Methodology. Mingyang Zhang: Visualization, Methodology, Data curation. Shuhui Wang: Investigation, Formal analysis. Zhiheng Liu: Visualization, Data curation, Methodology. Nana Wang: Resources. Zhiyuan Liu: Software. Yiyi Shen: Investigation, Methodology. Jiaying Zhang: Methodology, Data curation. Jing Li, Investigation, Data curation. 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Ghrelin attenuates myocardial fibrosis after acute myocardial infarction via inhibiting endothelial-to mesenchymal transition in rat model[J]. \u003cem\u003ePeptides\u003c/em\u003e \u003cb\u003e111\u003c/b\u003e, 118\u0026ndash;126. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.peptides.2018.09.001\u003c/span\u003e\u003cspan address=\"10.1016/j.peptides.2018.09.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Smurf1, Smad2, EndMT, HCAEC","lastPublishedDoi":"10.21203/rs.3.rs-6878518/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6878518/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSmad2 is a well-established regulator involved in tissue development and in the pathogenesis of endothelial-to-mesenchymal transition (EndMT) mediated by TGF-β signaling. However, the mechanism underlying the regulation of Smad2 in human coronary artery endothelial cells (HCAECs), particularly the identity of the responsible E3 ubiquitin ligase and its role during EndMT remain unclear. In this study, we identified Smad ubiquitination regulatory factor 1 (Smurf1) as a negative regulator of Smad2 protein levels in HCAECs and demonstrated that the E3 ligase activity of Smurf1 is essential for this function. Mechanistically, Smurf1 interacts with Smad2, promoting its ubiquitination, and subsequent proteasomal degradation. Specifically, Smurf1 catalyzes K48-linked polyubiquitination of Smad2 at lysine residues K156, K383 and K420. Functionally, Smad2 was found to promote EndMT in HCAECs, an effect that was partially attenuated either by co-expression of Smurf1 or by mutation of Smad2 at lysine 420 (Smad2 -K420R), which replaces. Taken together, our findings identify, for the first time, specific lysine residues on Smad2 targeted by Smurf1 for K48-linked ubiquitination, and highlight their crucial regulatory role in modulating\u003c/p\u003e\u003cp\u003eEndMT in HCAECs.\u003c/p\u003e","manuscriptTitle":"Ubiquitination of Smad2 by Smurf1 inhibits endothelial-to-mesenchymal transition in human coronary artery endothelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 16:33:28","doi":"10.21203/rs.3.rs-6878518/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-09T18:32:52+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T08:23:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"19657756612394829546946787791700657177","date":"2025-11-07T15:28:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T01:25:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253186387561082652992767537648662805415","date":"2025-09-16T01:54:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309324721792923109152956692809504059003","date":"2025-08-16T15:43:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147497315880553509883285363069624720577","date":"2025-07-20T14:47:49+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T13:05:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-10T11:55:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-10T07:15:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-03T13:04:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-03T13:00:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3dbd5c4a-e380-4cfa-bca0-0398f6ddd5d3","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51575344,"name":"Biological sciences/Cell biology"},{"id":51575345,"name":"Health sciences/Cardiology"},{"id":51575346,"name":"Health sciences/Molecular medicine"},{"id":51575347,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-05-05T15:53:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-16 16:33:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6878518","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6878518","identity":"rs-6878518","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}