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AbstractObjectiveMethodsResultsConclusion\nPLAIN LANGUAGE SUMMARYHeart failure (HF) causes structural and functional changes in the heart, with the pyroptosis-mediated inflammatory response as the core link in HF pathogenesis. E3 ubiquitin ligases participate in cardiovascular disease progression. Here, we explored the underlying molecular mechanisms of E3 ubiquitin ligase Smurf1 in governing HF.HF rat/H9C2 cell models were established by doxorubicin intraperitoneal injections/hypoxia-reoxygenation (H/R), and treated with Smurf1 siRNA and oe-TRIB2 lentivirus plasmids or the NF-κB pathway inhibitor PDTC/si-smurf1, si-TRIB2, protease inhibitor MG132, or lysosomal inhibitor NH4Cl. The cardiac function/cardiac tissue pathological changes/fibrosis in HF rats were evaluated by echocardiography/H&E and Masson staining. GSDMD-N expression was determined by immunohistochemistry. Cell viability/lactate dehydrogenase (LDH) activity/IL-1β and IL-18 levels were measured by CCK-8/LDH kit/ELISA. The interaction between TRIB2 and Smurf1/TRIB2 ubiquitination levels was assessed by co-immunoprecipitation assay. The expression levels of Smurf1 and TRIB2 messenger RNA (mRNA) were determined by RT-qPCR. Levels of Smurf1/TRIB2/the NF-κB pathway-related factors/pyroptosis-related factors and TRIB2 mRNA were determined by Western blot/RT-qPCR.Smurf1 was highly expressed in H/R-induced H9C2 cells/HF rats, while its knockdown up-regulated TRIB2 and repressed the NF-κB pathway, reduced cardiomyocyte pyroptosis, and attenuated HF. Mechanistically, Smurf1 promoted TRIB2 degradation through an ubiquitin-dependent manner and activated the NF-κB pathway under H/R conditions. TRIB2 silencing annulled Smurf1 knockdown-regulated NF-κB pathway and cardiomyocyte pyroptosis. TRIB2 overexpression inactivated the NF-κB pathway and reduced cardiomyocyte pyroptosis, thus retarding HF.Smurf1 was highly expressed in HF rats, which promoted TRIB2 ubiquitination degradation and activated the NF-κB pathway, thereby promoting cardiomyocyte pyroptosis in HF rats.Cardiomyocyte pyroptosis underlies the fundamental cause of heart failure (HF). E3 ubiquitin ligases are involved in cardiomyocyte pyroptosis. Here, we explored the underlying molecular mechanisms of E3 ubiquitin ligase Smurf1 in HF. HF rat and H9C2 cell models were established by doxorubicin intraperitoneal injections and hypoxia-reoxygenation (H/R), respectively. HF rats were treated with Smurf1 siRNA, oe-TRIB2, TRIB2 siRNA lentiviral plasmids, and the NF-κB pathway inhibitor PDTC. H9C2 cells were treated with si-smurf1, si-TRIB2, protease inhibitor MG132, and lysosomal inhibitor NH4Cl, respectively. The cardiac function and myocardial tissue damage in HF rats, GSDMD-N expression, cell viability, lactate dehydrogenase (LDH) activity, IL-1β and IL-18 levels, TRIB2-Smurf1 interaction, and TRIB2 ubiquitination were assessed. Smurf1 was up-regulated in H/R-induced H9C2 cells and HF rats, while its knockdown repressed the NF-κB pathway, reduced cardiomyocyte pyroptosis, and attenuated HF. Smurf1 promoted TRIB2 degradation in an ubiquitin-dependent manner and activated the NF-κB pathway under H/R conditions. TRIB2 silencing annulled Smurf1 knockdown-regulated NF-κB pathway and cardiomyocyte pyroptosis. In vivo experiments verified that TRIB2 inhibited the NF-κB pathway and rat myocardial pyroptosis, thus improving HF. Collectively, Smurf1 was up-regulated in HF rats, which promoted TRIB2 ubiquitination degradation and activated the NF-κB pathway, thereby promoting cardiomyocyte pyroptosis in HF rats. [ABSTRACT FROM AUTHOR]

The calcium-activated phosphatase PPP3/calcineurin dephosphorylates TFEB (transcription factor EB) to trigger its nuclear translocation and the activation of macroautophagic/autophagic targets. However, the detailed molecular mechanism regulating TFEB activation remains poorly understood. Here, we highlighted the importance of SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) in the activation of TFEB for lysosomal homeostasis. SMURF1 deficiency prevents the calcium-triggered ubiquitination of the catalytic subunit of PPP3/calcineurin in a manner consistent with defective autophagic degradation of damaged lysosomes. Mechanically, PPP3CB/CNA2 plays a bridging role in the recruitment of SMURF1 by LGALS3 (galectin 3) upon lysosome damage. Importantly, PPP3CB increases the dissociation of the N-terminal tail (NT) and C-terminal carbohydrate-recognition domain (CRD) of LGALS3, which may promote the formation of open conformers in a PPP3CB dephosphorylation activity-dependent manner. In addition, PPP3CB is ubiquitinated at lysine 146 by the recruited SMURF1 in response to intracellular calcium stimulation. The K63-linked ubiquitination of PPP3CB enhances the recruitment of TFEB. Moreover, TFEB directly interacts with both PPP3CB and the regulatory subunit PPP3R1 which facilitate the conformational correction of TFEB for its activation for the transcription of TFEB-targeted genes. Altogether, our results highlighted a critical mechanism for the regulation of PPP3/calcineurin activity via its ubiquitin ligase SMURF1 in response to lysosomal membrane damage, which may account for a potential target for the treatment of stress-related diseases.Abbreviation AID: autoinhibitory domain; ATG: autophagy related; CD: catalytic domain; CRD: carbohydrate-recognition domain; CsA: cyclosporin A; DMSO: dimethyl sulfoxide; ESCRT: endosomal sorting complexes required for transport; GSK3B: glycogen synthase kinase 3 beta; LAMP1: lysosomal associated membrane protein 1; LGALS3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ML-SA1: mucolipin synthetic agonist 1; MTORC1: mechanistic target of rapamycin kinase complex 1; NT: N-terminal tail; PPP3CB: protein phosphatase 3 catalytic subunit beta; PPP3R1: protein phosphatase 3 regulatory subunit B, alpha; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; VCP/p97: valosin containing protein; YWHA/14-3-3: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein. [ABSTRACT FROM AUTHOR]

SMAD-specific E3 ubiquitin proten ligase 1 (SMURF1) is involved in transforming growth factor (TGF)-β1/Smad pathway-mediated tissue fibrosis. However, its role in pulmonary fibrosis and the related molecular mechanisms are still unclear. This study aims to investigate whether SMURF1 inhibits autophagy and promotes pulmonary fibrosis via SMAD family member 7 (SMAD7) and TGF-β1/SMAD signal pathway. MRC-5 cells were treated with TGF-β1 followed by MURF1-interference. The rate of cell migration was assessed using the cell scratch test. Autophagosomes were analyzed using a transmission electron microscope. mRNA levels of SMURF1, SMAD7, TGF-β1, phosphorylated (p)-SMAD1, p-SMAD3, α-smooth muscle actin (α-SMA), matrix metallopeptidase 7 (MMP7), microtubule-associated protein light chain 3 (LC3) and Beclin1 were evaluated by quantitative real-time PCR (qPCR), Western blotting, and immunofluorescence. The interaction between SMURF1 and SMAD7 was investigated in a co-immunoprecipitation (Co-IP) experiment. We found that after TGF-β1 treatment, the mRNA levels of SMURF1, α-SMA, MMP7, and p-Smad1/3 were increased, and the levels of Beclin1 and LC3 were decreased. Apart from these, cell autophagy was decreased, while the migration ability was increased. After SMURF1-interference, SMURF1, α-SMA and MMP7 mRNA levels were significantly decreased, p-SMAD1 was slightly reduced, and p-Smad3 was not changed. As for Beclin1 and LC3, their transcription increased, cell autophagy increased, and migratory ability decreased. The interaction between SMURF1 and Smad7 was confirmed by Co-IP. In conclusion, SMURF1 may inhibit autophagy and promote lung fibrosis by downregulating SMAD7 and activating the TGF-β1/SMAD pathway. These results may serve as a basis for the development of new therapeutic targets in the pulmonary fibrosis clinic. [ABSTRACT FROM AUTHOR]

The ubiquitin‐proteasome system is a crucial mechanism for regulating protein levels in cells, with substrate‐specific E3 ubiquitin ligases serving as an integral component of this system. Among these ligases are SMAD‐specific E3 ubiquitin‐protein ligase 1 (SMURF1) and SMAD‐specific E3 ubiquitin‐protein ligase 2 (SMURF2), which belong to the neural precursor cell‐expressed developmentally downregulated 4 (NEDD4) subfamily of Homologous to E6‐AP COOH terminus (HECT)‐type E3 ligases. As E3 ligases, SMURFs have critical functions in regulating the stability of multiple proteins, thereby maintaining physiological processes such as cell migration, proliferation, and apoptosis. The occurrence of many diseases is attributed to abnormal cell physiology and an imbalance in cell homeostasis. It is noteworthy that SMURFs play pivotal roles in disease progression, with the regulatory functions being complex and either facilitative or inhibitory. In this review, we elucidate the mechanisms by which SMURF1 and SMURF2 can regulate disease progression in non‐cancerous diseases. These significant findings offer potential novel therapeutic targets for various diseases and new avenues for research on SMURF proteins.Diseases associated with SMURF1/2. The SMURF1/2 protein has been implicated in various diseases affecting multiple systems such as cardiovascular diseases, nervous system diseases, renal diseases, skeletal diseases, liver diseases, diabetes, and viral infection. [ABSTRACT FROM AUTHOR]

Aims: Renal oxidative stress (OSS) is the leading cause of diabetic nephropathy (DN). The silent information regulator 1/forkhead boxo3a (Sirt1/Foxo3a) pathway plays an essential role in regulating the antioxidant enzyme system. In this study, we aimed to investigate the mechanism of connexin32 (Cx32) on the antioxidant enzyme system in DN. Results: In this study, Cx32 overexpression significantly reduced reactive oxygen species generation and effectively inhibited the excessive production of extracellular matrix such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in high-glucose (HG)-induced glomerular mesangial cells. In addition, Cx32 overexpression reversed the downregulation of Sirt1, and promoted the nuclear transcription of Foxo3a, subsequently activating the antioxidant enzymes including catalase and manganese superoxide dismutase (MnSOD), however, Cx32 knockdown showed the opposite effects. A further mechanism study showed that Cx32 promoted the autoubiquitination and degradation of Smad ubiquitylation regulatory factor-1 (Smurf1), thereby reducing the ubiquitination of Sirt1 at Lys335 and the degradation of Sirt1. Moreover, the in vivo results showed that adenovirus-mediated Cx32 overexpression activated the Sirt1/Foxo3a pathway, and inhibited OSS in the kidney tissues, eventually improving the renal function and glomerulosclerosis in diabetic mice. Innovation: This study highlighted the antioxidant role of Cx32-Sirt1-Foxo3a axis to alleviate DN, which is a new mechanism of Cx32 alleviating DN. Conclusion: Cx32 alleviated DN via activating the Sirt1/Foxo3a antioxidant pathway. The specific mechanism was that Cx32 upregulated the Sirt1 expression through reducing the ubiquitination of Lys335 of Sirt1 by inhibiting Smurf1. Antioxid. Redox Signal. 39, 241–261. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) is a traumatic central nervous system disorder that leads to irreversible neurological dysfunction. Emerging evidence has shown that differentially expressed circular RNAs (circRNAs) after SCI is closely associated with the pathophysiological process. Herein, the potential function of circRNA spermine oxidase (circSmox) in functional recovery after SCI was investigated. Methods: Differentiated PC12 cells stimulated with lipopolysaccharide (LPS) were employed as an in vitro model for neurotoxicity research. Levels of genes and proteins were detected by quantitative real‐time PCR and Western blot analysis. Cell viability and apoptosis were determined by CCK‐8 assay and flow cytometry. Western blot analysis was used to detect the protein level of apoptosis‐related markers. The levels of interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α. Dual‐luciferase reporter, RIP, and pull‐down assays were used to confirm the target relationship between miR‐340‐5p and circSmox or Smurf1 (SMAD Specific E3 Ubiquitin Protein Ligase 1). Results: LPS elevated the levels of circSmox and Smurf1, but decreased the levels of miR‐340‐5p in PC12 cells in a dose‐dependent manner. Functionally, circSmox silencing alleviated LPS‐induced apoptosis and inflammation in PC12 cells in vitro. Mechanistically, circSmox directly sponged miR‐340‐5p, which targeted Smurf1. Rescue experiments showed that miR‐340‐5p inhibition attenuated the neuroprotective effect of circSmox siRNA in PC12 cells. Moreover, miR‐340‐5p suppressed LPS‐triggered neurotoxicity in PC12 cells, which was reversed by Smurf1 overexpression. Conclusion: CircSmox enhances LPS‐induced apoptosis and inflammation via miR‐340‐5p/Smurf1 axis, providing an exciting view of the potential involvement of circSmox in SCI pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Macro-autophagy/Autophagy is an evolutionarily well-conserved recycling process to maintain the balance through precise spatiotemporal regulation. However, the regulatory mechanisms of biomolecular condensates by the key adaptor protein p62 via liquid-liquid phase separation (LLPS) remain obscure. Results: In this study, we showed that E3 ligase Smurf1 enhanced Nrf2 activation and promoted autophagy by increasing p62 phase separation capability. Specifically, the Smurf1/p62 interaction improved the formation and material exchange of liquid droplets compared with p62 single puncta. Additionally, Smurf1 promoted the competitive binding of p62 with Keap1 to increase Nrf2 nuclear translocation in p62 Ser349 phosphorylation-dependent manner. Mechanistically, overexpressed Smurf1 increased the activation of mTORC1 (mechanistic target of rapamycin complex 1), in turn leading to p62 Ser349 phosphorylation. Nrf2 activation increased the mRNA levels of Smurf1, p62, and NBR1, further promoting the droplet liquidity to enhance oxidative stress response. Importantly, we showed that Smurf1 maintained cellular homeostasis by promoting cargo degradation through the p62/LC3 autophagic pathway. Conclusions: These findings revealed the complex interconnected role among Smurf1, p62/Nrf2/NBR1, and p62/LC3 axis in determining Nrf2 activation and subsequent clearance of condensates through LLPS mechanism. [ABSTRACT FROM AUTHOR]

The tumor suppressor PTEN mainly inhibits the PI3K/Akt pathway in the cytoplasm and maintains DNA stability in the nucleus. The status of PTEN remains therapeutic effectiveness for chemoresistance of the DNA alkylating agent temozolomide (TMZ) in glioblastoma (GB). However, the underlying mechanisms of PTEN's interconnected role in the cytoplasm and nucleus in TMZ resistance are still unclear. In this study, we report that TMZ-induced PTEN nuclear import depends on PTEN ubiquitylation modification by Smurf1. The Smurf1 suppression decreases the TMZ-induced PTEN nuclear translocation and enhances the DNA damage. In addition, Smurf1 degrades cytoplasmic PTEN K289E (the nuclear-import-deficient PTEN mutant) to activate the PI3K/Akt pathway under TMZ treatment. Altogether, Smurf1 interconnectedly promotes PTEN nuclear function (DNA repair) and cytoplasmic function (activation of PI3K/Akt pathway) to resist TMZ. These results provide a proof-of-concept demonstration for a potential strategy to overcome the TMZ resistance in PTEN wild-type GB patients by targeting Smurf1. [ABSTRACT FROM AUTHOR]

Protein ubiquitination is a precisely controlled enzymatic cascade reaction belonging to the post-translational modification of proteins. In this process, E3 ligases catalyze the binding of ubiquitin (Ub) to protein substrates and define specificity. The neuronally expressed developmentally down-regulated 4 (NEDD4) subfamily, belonging to the homology to E6APC terminus (HECT) class of E3 ligases, has recently emerged as an essential determinant of multiple cellular processes in different tissues, including bone and tooth. Here, we place special emphasis on the regulatory role of the NEDD4 subfamily in the molecular and cell biology of osteogenesis. We elucidate in detail the specific roles, downstream substrates, and upstream regulatory mechanisms of the NEDD4 subfamily. Further, we provide an overview of the involvement of E3 ligases and deubiquitinases in the development, repair, and regeneration of another mineralized tissue—tooth. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) is a typical sensor of intracellular energy metabolism. Our previous study revealed the role of activated AMPK in the suppression of osteogenic differentiation and traumatic heterotopic ossification, but the underlying mechanism remains poorly understood. The E3 ubiquitin ligase Smurf1 is a crucial regulator of osteogenic differentiation and bone formation. We report here that Smurf1 is primarily SUMOylated at a C-terminal lysine residue (K324), which enhances its activity, facilitating ALK2 proteolysis and subsequent bone morphogenetic protein (BMP) signaling pathway inhibition. Furthermore, SUMOylation of the SUMO E3 ligase PIAS3 and Smurf1 SUMOylation was suppressed during the osteogenic differentiation and traumatic heterotopic ossification. More importantly, we found that AMPK activation enhances the SUMOylation of Smurf1, which is mediated by PIAS3 and increases the association between PIAS3 and AMPK. Overall, our study revealed that Smurf1 can be SUMOylated by PIAS3, Furthermore, Smurf1 SUMOylation mediates osteogenic differentiation and traumatic heterotopic ossification through suppression of the BMP signaling pathway. This study revealed that promotion of Smurf1 SUMOylation by AMPK activation may be implicated in traumatic heterotopic ossification treatment. • Smurf1 is modified by SUMOylation at lysine residues K27,K72, and K324. • SUMOylation of Smurf1 enhance its function and suppresses BMP signaling pathways and its role in osteogenic differentiation. • AMPK interacts with Smurf1 and PIAS3 to elevate SUMOylated Smurf1 for a negative control in BMP signaling pathway. • Our study focuses on a novel Smurf1 function and verifies that SUMOylation of Smurf1 could be a potentially effective therapeutic strategy for HO in the future. [ABSTRACT FROM AUTHOR]

• Smurf1 represents a key component in anti-tuberculous host defense. • We hypothesized genetic variants in the Smurf1 gene impact susceptibility to TBM. • The variants of Smurf1 were associated with the incidence of TBM. • The Smurf1 variants were not correlated to the severity or prognosis of TBM. • This indicated an important role for Smurf1 in the immunopathogenesis of TBM. Tuberculous meningitis (TBM) is the most severe form of infection caused by Mycobacterium tuberculosis (Mtb). Smurf1 represents a key component in anti- Mtb autophagic targeting in macrophages and in anti-TB host defense in vivo. We hypothesized that genetic variants in the Smurf1 gene region influence susceptibility to TBM. Using a case-control study design (235 TBM cases, 239 pulmonary TB cases and 478 healthy controls), we evaluated whether 8 haplotype-tagging single nucleotide polymorphisms (SNPs) in the Smurf1 gene are associated with the development of TBM. Even with the most conservative correction, the polymorphism rs6956450 was associated with TBM under a dominant model (odds ratio [OR], 1.653; 95% confidence interval [CI], 1.192–2.294; P = 0.021), the CG haplotype consisting of rs3294 and rs6956450 was positively associated with TBM (P = 0.013) and another haplotype GC remained negatively associated with TBM in Tibetan subgroup (P < 0.001). No correlation was found between rs6956450 and TBM clinical characteristics or prognosis. These results firstly link the variants in the Smurf1 gene region with TBM risk, indicating an important role for Smurf1 in the immunopathogenesis of TBM. Future studies will dissect the mechanism, which may help identify targets or genetic markers to guide diagnosis or host-directed therapy in patients with TBM. [ABSTRACT FROM AUTHOR]

Smad ubiquitination regulatory factor 1 (Smurf1) and Smurf2 are HECT-type E3 ubiquitin ligases, and both Smurfs were initially identified to regulate Smad protein stability in the TGF-β/BMP signaling pathway. In recent years, Smurfs have exhibited E3 ligase-dependent and -independent activities in various kinds of cells. Smurfs act as either potent tumor promoters or tumor suppressors in different tumors by regulating biological processes, including metastasis, apoptosis, cell cycle, senescence and genomic stability. The regulation of Smurfs activity and expression has therefore emerged as a hot spot in tumor biology research. Further, the Smurf1- or Smurf2 -deficient mice provide more in vivo clues for the functional study of Smurfs in tumorigenesis and development. In this review, we summarize these milestone findings and, in turn, reveal new avenues for the prevention and treatment of cancer by regulating Smurfs. [ABSTRACT FROM AUTHOR]

Prostate cancer remains one of the most prevalent cancers and the main causes of cancer‐related deaths in males. Various articles introduced that long noncoding RNAs (lncRNAs) are found in vital functions in the development and progression of cancers. Although SNHG3 (small nucleolar RNA host gene 3) has been investigated in many cancers, now researches on the role and mechanism of SNHG3 in prostate cancer are lacked. In this work, SNHG3 exerted high expression in prostate cancer cell lines. Suppression of SNHG3 inhibited cell proliferation, migration, EMT (epithelial‐mesenchymal transition) process and promoted cell apoptosis. Additionally, it was found that SNHG3 could bind with miR‐577. Subsequently, SMURF1 (Smad ubiquitination regulatory factor 1) was identified as a downstream target of miR‐577 and had a negative correlation with miR‐577. SNHG3 was found to positively regulate SMURF1 expression. Furthermore, rescue assays demonstrated that co‐transfection of pcDNA3.1/SMURF1 reversed the effects of SNHG3 knockdown in cell proliferation, migration, EMT process and cell apoptosis. SNHG3 also promoted tumorigenesis in vivo. All the results above explained that SNHG3 accelerated prostate cancer progression by sponging miR‐577 to up‐regulate SMURF1 expression, suggesting that SNHG3 may act as a biomarker for prostate cancer patients. [ABSTRACT FROM AUTHOR]

Studies on mice have shown that the Smad Ubiquitin Regulatory Factor-1 (SMURF1) gene negatively regulates osteoblast function and the response to bone morphogenetic protein in a dose-dependent fashion (Chan et al. in Mol Cell Biol 27(16):5776-5789, https://doi.org/10.1128/MCB.00218-07, 2007; Yamashita et al. in Cell 121(1):101-113, https://doi.org/10.1016/j.cell.2005.01.035, 2005). In addition, a tumorigenic role for SMURF1 has been implicated due to the interference with apoptosis signals (Nie et al. in J Biol Chem 285(30):22818-22830, https://doi.org/10.1074/jbc.M110.126920, 2010; Wang et al. in Nat Commun 5:4901, https://doi.org/10.1038/ncomms5901, 2014). A 10-year-old girl with a history of severe developmental delay, infantile seizures, and B-cell lymphoma, in remission for approximately 3.5 years, was referred to the metabolic bone clinic for fractures and low bone mineral density. Array comparative genomic hybridization revealed a pathogenic microduplication in chromosome 7 at bands 7q21.3q22.1 that encompasses the SMURF1 gene. The clinical features of this child are congruous with the phenotype as ascribed excess Smurf1 mutations in mice. This is the first case description of osteoporosis in a child secondary to a microduplication involving SMURF1 gene. [ABSTRACT FROM AUTHOR]

Highlights • NDR2 knockdown promotes IL-17-induced inflammation by activation of ERK, p38 and NF-κB. • NDR2 interacts with Smurf1 and MEKK2, and promotes Smurf1-mediated k48-linked ubiquitination of MEKK2. • MEKK2 positively regulates IL-17-induced inflammation while Smurf1 negatively regulates IL-17-induced inflammation. Abstract NDR/LATS kinase family are conserved from yeast to man and their roles in inflammation remains largely unknown. In the present study, we show that knockdown of NDR2 significantly increases IL-17-induced IL-6, CXCL2 and CCL20 expression in Hela and HT-29 cells. Knockdown of NDR2 enhances IL-17-induced MAPK and NF-κB activation. NDR2 interacts with E3 ubiquitin protein ligase Smurf1, promotes Smurf1-mediated K48-linked ubiquitination of MEKK2 and inhibits expression of MEKK2. Consistently, knockdown of Smurf1 increases IL-17-induced IL-6, CXCL2 and CCL20 expression. On the other hand, overexpression of MEKK2 increases IL-17-induced IL-6 expression. These results suggest that NDR2 may play important roles in IL-17-associated inflammation by promoting Smurf1-mediated MEKK2 ubiquitination and degradation. [ABSTRACT FROM AUTHOR]

Abstract Aims CREB (cAMP response element-binding protein)-regulated transcription coactivator (CRTC2) has been reported to act as a coactivator of CREB during gluconeogenesis. The role of CRTC2 in osteoblastic differentiation has not yet been elucidated. The aim of this study is to identify the mechanism of CRTC2 in osteoblast differentiation. Main methods The mRNA expression was determined by RT-PCR and qPCR. Protein levels were measured using Western blot assay. Alkaline phosphatase (ALP) staining was performed to evaluate ALP activity. Alizarin red S (ARS) staining was performed to measure extracellular mineralization. Transcriptional activity was detected using a luciferase assay. Key findings In the present study, TNF-α was found to stimulate CRTC2 expression. However, TNF-α did not increase the gene expression of osteoblast differentiation markers and inhibited BMP2-induced osteoblastic differentiation. Overexpression of CRTC2 decreased the expression of osteogenic genes, ALP activity and extracellular matrix mineralization. Knockdown of CRTC2 restored BMP2-induced osteogenic gene expression and ALP activity. CRTC2 increased Smurf1 mRNA expression, Smurf 1 promoter activity, and protein level. Furthermore, Smurf 1 decreased Smad 1/5/9 protein levels. These results suggest that CRTC2 decreased BMP2-induced osteoblastic differentiation via Smurf 1 expression. Significance Our results indicate that CRTC2 regulates the expression of Smurf1 in osteoblast differentiation. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is a cellular organelle with central roles in maintaining proteostasis. The accumulation of misfolded proteins in the ER lumen causes ER stress. Cells evoke an evolutionarily conserved adaptive signaling network “unfolded protein response” to restore ER homeostasis, however, how the signaling network is delicately orchestrated remains largely unrevealed. Meanwhile, the HECT type E3 ligase Smad ubiquitylation regulatory factor 1 (Smurf1) has been reported to play critical roles in several important biological pathways by targeting distinct substrates for ubiquitylation, including WFS1, a critical mediator of ER stress, whereas the regulation of Smurf1 activity and abundance upon ER stress are poorly understood. Here, we identified Interleukin-1 Receptor Associated Kinase 2 (IRAK2) as a novel modulator of Smurf1 in response to ER stress induced cell death. Mechanistically, IRAK2 phosphorylates Smurf1 at threonine residues to promote its self-degradation by ubiquitylation, resulting in altered cascade of ER effectors to induce apoptosis. Reduced IRAK2 expression correlates with increased Smurf1 abundance in human colorectal cancer cells. Taken together, these findings demonstrate a novel mechanism of interplays for the different branches of ER stress signaling network and highlight IRAK2 as a potential tumor suppressor to counterbalance oncogenic Smurf1. [ABSTRACT FROM AUTHOR]

Smad ubiquitylation regulatory factor 1 (Smurf1) has been identified to play a critical role in bone homeostasis, development, cell cycle regulation and tumorigenesis. However, the role of Smurf1 in macrophage proliferation, apoptosis and migration is still unclear. Here, we show that Smurf1 expression was elevated in LPS-induced RAW264.7 macrophage and mouse embryonic fibroblasts (MEFs). And we found that knockdown of Smurf1 suppresses macrophage proliferation but promotes apoptosis and migration. Furthermore, JNK and p38 MAPK signaling were upregulated in Smurf1-depleted cells. And inhibition of JNK and p38 MAPK signaling in Smurf1 knockdown cells rescue the phenotypes of macrophage proliferation, apoptosis and migration. Therefore, our study suggests that Smurf1 is a new positive regulator for macrophage proliferation and apoptosis, but a negative regulator for macrophage migration. [ABSTRACT FROM AUTHOR]

Protein ubiquitination and deubiquitination enzymes are widely involved in innate immune responses. The ubiquitin specific protease 25 (USP25), a deubiquitinating enzyme, has been demonstrated to play an important role in virus infection and immunity. However, how USP25 is degraded and regulated by E3 ubiquitin ligases remains poorly understood. Here, we identified Smad ubiquitin regulatory factor 1(Smurf1) as a first novel E3 ubiquitin ligase of USP25. Smurf1 overexpression decreases USP25 protein turnover, and the E3 ligase enzymatic activity of Smurf1 is required for USP25 degradation. Additionally, Smurf1-mediated degradation of USP25 is via promoting the K48-linkage polyubiquitination of USP25 in an ubiquitin proteasome dependent pathway. Importantly, USP25 overexpression restricts vesicular stomatitis virus (VSV) replication and the restriction of VSV replication by USP25 is enhanced in Smurf1 stable knock down cells. Therefore, our study firstly identified that Smurf1 negatively regulated the antiviral function mediated by USP25. Our findings revealed a previously unrecognized role of Smurf1 acting on USP25 and also their roles in the regulation of VSV replications. [ABSTRACT FROM AUTHOR]

Glioblastoma, one of the common malignant brain tumors, results in the highly death, but its underlying molecular mechanisms remain unclear. Smurf1, a member of Nedd4 family of HECT-type ligases, has been reported to contribute to tumorigenicity through several important biological pathways. Recently, it was also found to participate in modulate cellular processes, including morphogenesis, autophagy, growth, and cell migration. In this research, we reported the clinical guiding significance of the expression of Smurf1 in human glioma tissues and cell lines. Western blotting analysis discovered that the expression of Smurf1 was increased with WHO grade. Immunohistochemistry levels discovered that high expression of Smurf1 is closely consistent with poor prognosis of glioma. In addition, suppression of Smurf1 can reduce cell invasion and increase the E-cadherin expression, which is a marker of invasion. Our study firstly demonstrated that Smurf1 may promote glioma cell invasion and suppression of the Smurf1 may provide a novel treatment strategy for glioma. [ABSTRACT FROM AUTHOR]

Our previous study indicated that Casein kinase 2 interacting protein-1 (CKIP-1) could promote the activation of the nuclear factor E2-related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway, playing a significant role in inhibiting the fibrosis of diabetic nephropathy (DN). However, the underlying mechanism is still unknown. Here, we investigated whether CKIP-1 affects the polyubiquitination of Nrf2 and its cytosolic inhibitor kelch like ECH-associated protein 1 (Keap1) via mediating Smad ubiquitylation regulatory factor-1 (Smurf1) to promote the activation of the Nrf2/ARE signaling and resist high glucose (HG)-induced renal fibrosis in glomerular mesangial cells (GMCs) and diabetic mice kidneys. Results showed that the expression of Smurf1 increased in HG-induced GMCs, with a paramount upregulation at 1 h. Overexpression of wild-type Smurf1 plasmid further promoted the HG-induced the over-production of fibronectin (FN) and intercellular adhesionmolecule-1 (ICAM-1), and depletion of Smurf1 dramatically reduced the expression of FN and ICAM-1. Overexpression of CKIP-1 decreased the K48-linked polyubiquitination and increased the K63-linked polyubiquitination of Nrf2 as well as enhanced the K48-linked polyubiquitination and reduced K63-linked polyubiquitination of Keap1, promoting the activation of the Nrf2/ARE pathway. Overexpression of Smurf1 increased the K48-linked polyubiquitination and decreased the K63-linked polyubiquitination of Nrf2, and down-regulated the K48-linked polyubiquitination and up-regulated the K63-linked polyubiquitination of Keap1, inhibiting the activation of the Nrf2/ARE pathway. CKIP-1 promoted the degradation of Smurf1 by increasing the ubiquitination of Smurf1. Treatment of CKIP-1 adenovirus infection reduced the Smurf1 levels, promoted the activation of the Nrf2/ARE pathway as well as suppressed the production of reactive oxygen species (ROS), and then improved the failure of renal function of diabetic mice. Experiments above suggested that CKIP-1 affects the polyubiquitination of Nrf2 and Keap1 and promotes the Nrf2-ARE pathway through down-regulating Smurf1 to resist HG-induced up-regulation of FN and ICAM-1 in GMCs and diabetic mice kidneys. [ABSTRACT FROM AUTHOR]

Smad ubiquitylation regulatory factor-1 (Smurf1) is one of C2-WW-HECT domain E3 ubiquitin ligases, it can regulate BMP pathway by mediating ubiquitylation degradation of Smad1/Smad5. Many functions about Smurf1 also are still unknown, especially in retina. This research is about to explore the role of Smurf1 in retina degeneration. Tail vein injection of sodium iodate (NaIO 3) in C57BL/6J mice was the animal model of retina degeneration. In NaIO 3 model, Smurf1 had more expression than normal mice. Specific Smurf1 inhibitor, A01, was injected into vitreous cavity. Results showed that inhibiting Smurf1 could alleviate acute retina injury, such as keeping a better retina structure in living imaging and histologic sections, less cell death and inflammation activation. Tert-butyl hydroperoxide (TBH) was used to establish oxidative stress injury in human retinal pigments epithelial cell line (ARPE-19). Oxidative stress injury gradually caused co-upregulation of Smurf1, TGF-β1 and phosphorylated NF-κB (pNF-κB). TGF-β1 could directly induce Smurf1 expression. Inhibiting Smurf1 had an anti-epithelial mesenchymal transition (anti-EMT) function. Similarly, A01 also could inhibit the expression of pNF-κB, NLRP3 and IL-1β. At last, after searching bioinformatics database, Smurf1 had a possible interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (β-TrCP), another E3 ubiquitin ligases. β-TrCP can mediate ubiquitination degradation of p-IκBα. Lentivirus- SMURF1 was used to overexpress Smurf1, and GS143 was used to inhibit β-TrCP. The results showed Smurf1 could directly induce NF-κB, pNF-κB, and NLRP3 expression, and keep a stable β-TrCP expression. However, inhibiting β-TrCP could cause more NF-κB activation and NLRP3 expression. Therefore, β-TrCP may play a negative role in NF-κB pathway activation. In summary, Smurf1 plays a role in exacerbating oxidative stress injury and inflammation in retina and may become a potential therapeutic target in ROS injury of retina. • The first research to explore the functions of Smurf1 in retina disease. • This research found a crosstalk between TGF-beta pathway and NF kappa B pathway. [ABSTRACT FROM AUTHOR]

Aims MicroRNAs (miRNAs) have been implicated in the pathogenesis of pulmonary hypertension (PH), a multifactorial and progressive condition associated with an increased afterload of the right ventricle leading to heart failure and death. The main aim of this study was to correlate the levels of miR-424(322) with the severity and prognosis of PH and with right ventricle hypertrophy progression. Additionally, we intended to evaluate the mechanisms and signalling pathways whereby miR-424(322) secreted by pulmonary arterial endothelial cells (PAECs) impacts cardiomyocytes. Methods and results Using quantitative real-time PCR, we showed that the levels of circulating miR-424(322) are higher in PH patients when compared with healthy subjects. Moreover, we found that miR-424(322) levels correlated with more severe symptoms and haemodynamics. In the subgroup of Eisenmenger syndrome patients, miR-424(322) displayed independent prognostic value. Furthermore, we demonstrated that miR-424(322) targets SMURF1, through which it sustains bone morphogenetic protein receptor 2 signalling. Moreover, we showed that hypoxia induces the secretion of miR-424(322) by PAECs, which after being taken up by cardiomyocytes leads to down-regulation of SMURF1. In the monocrotaline rat model of PH, we found an association between circulating miR-424(322) levels and the stage of right ventricle hypertrophy, as well as an inverse correlation between miR-424(322) and SMURF1 levels in the hypertrophied right ventricle. Conclusions This study shows that miR-424(322) has diagnostic and prognostic value in PH patients, correlating with markers of disease severity. Additionally, miR-424(322) can target proteins with a direct effect on heart function, suggesting that this miRNA can act as a messenger linking pulmonary vascular disease and right ventricle hypertrophy. [ABSTRACT FROM AUTHOR]

The HECT E3 ligase Smurf1 (Smad ubiquitination regulatory factor 1) plays a critical role in several important biological pathways by targeting many proteins for ubiquitination and degradation, such as Smad1/5, MEKK2 and RhoA. However, the function of Smurf1 in metaphase-to-anaphase transition remains unclear. Here, we show that Smurf1 interacts with and targets Securin, an inhibitor of sister-chromatid separation, for poly-ubiquitination and proteasomal degradation. Further results demonstrate that Securin is a physiological substrate of Smurf1 in MEF cells. Knockdown of Smurf1 results in sister-chromatid separation inhibition and delay of anaphase onset. This study provides the first evidence that Smurf1 functions as a novel regulator for the metaphase-to-anaphase transition. [ABSTRACT FROM AUTHOR]

Smad ubiquitination regulatory factor 1 (Smurf1) is a HECT-type E3 ubiquitin ligase that regulates several important signaling pathways, including the bone morphogenetic protein pathway and the transforming growth factor-beta ( TGF-β) signaling pathway. However, the function of Smurf1 in cell cycle progression remains unclear. Here, we demonstrate that silencing of Smurf1 results in S phase arrest, confirming that Smurf1 is required for S phase progression. Furthermore, we demonstrate that Smurf1-mediated S phase progression is largely dependent on the ubiquitination-dependent degradation of Wee1. This study defines a novel role for Smurf1 in controlling S phase progression by promoting Wee1 degradation. [ABSTRACT FROM AUTHOR]

Heat shock protein 90 (HSP90) molecular chaperone is responsible for the stabilization and biological activity of a diverse set of client proteins. We have previously demonstrated that inhibition of HSP90 by 17-Demethoxy-17-allyaminogeldanmycin (17-AAG) not only reverses the glucocorticoid-induced bone loss but also enhances the basal level of bone mass in mice. Here, we investigate the potential mechanism underlying HSP90-associated osteoblast differentiation and bone formation. Knockdown of HSP90β but not HSP90α or inhibition of HSP90 by 17-AAG or NVP-BEP800 negates the protein levels of large tumor suppressor (LATS), the core kinases of Hippo signaling, resulting in the inactivation of LATS and activation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), in the enhancement of osteoblastic differentiation. In contrast, genetic ablation of Lats1 in mesenchymal stem cells is sufficient to abolish the HSP90 inhibition-induced osteoblastic differentiation and bone formation. Mechanistically, HSP90β but not HSP90α chaperones and prevents the SMAD specific E3 ubiquitin protein ligase 1 (SMURF1)-mediated and ubiquitination-dependent LATS protein proteasomal degradation, whereas 17-AAG abolishes these effects of HSP90β. Thus, these results uncover the HSP90β chaperoning SMURF1-mediated LATS protein proteasomal degradation and the subsequent YAP/TAZ activation as a hitherto uncharacterized mechanism controlling osteoblastic differentiation and bone formation. • The regulation of client proteins by HSP90 is essential for critical cellular processes. • Hsp90β dysregulation is associated with a variety of diseases. • HSP90β chaperoning SMURF1-mediated LATS1 proteasomal degradation. • 17AAG may be used to prevent and treat cancer-induced osteoporosis. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-alpha ( TNFα) plays a pivotal role in inflammation-related osteoporosis through the promotion of bone resorption and suppression of bone formation. Numerous drugs have been produced to treat osteoporosis by inhibiting bone resorption, but they offer few benefits to bone formation, which is what is needed by patients with severe bone loss. Melatonin, which can exert both anti-inflammatory and pro-osteogenic effects, shows promise in overcoming TNFα-inhibited osteogenesis and deserves further research. This study demonstrated that melatonin rescued TNFα-inhibited osteogenesis of human mesenchymal stem cells and that the interactions between SMURF1 and SMAD1 mediated the crosstalk between melatonin signaling and TNFα signaling. Additionally, melatonin treatment was found to downregulate TNFα-induced SMURF1 expression and then decrease SMURF1-mediated ubiquitination and degradation of SMAD1 protein, leading to steady bone morphogenetic protein- SMAD1 signaling activity and restoration of TNFα-impaired osteogenesis. Thus, melatonin has prospects for treating osteoporosis caused by inflammatory factors due to its multifaceted functions on regulation of bone formation, bone resorption, and inflammation. Further studies will focus on unveiling the specific mechanisms by which melatonin downregulates SMURF1 expression and confirming the clinical therapeutic value of melatonin in the prevention and therapy of bone loss associated with inflammation. [ABSTRACT FROM AUTHOR]

Osteoporosis is a common health problem worldwide that affects many older people. Casein kinase 2 interaction protein1 (casein kinase 2 interaction protein 1, CKIP-1) is an important molecule discovered in recent years. It plays an important role in many cell behaviors through its interaction with other molecules. CKIP-1 has been attributed a critical role in regulating bone remodeling. It has close relationship with osteoporosis, and is a target for developing newdrugs for the treatment of osteoporosis. Therefore, in depth research on CKIP-1 will contribute to the prevention of osteoporosis and other bone diseases as well as understanding the pathogenesis of these diseases. This article reviews the relationship between CKIP-1 and osteoporosis. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific peptidase 5 (USP5) has been demonstrated to be critical for the production of Tumor Necrosis Factor-alpha (TNF-α), a pivotal mediator for inflammatory responses. Besides, USP5 regulates p53 activation and DNA repair. However, the mechanism underlying the regulation of USP5, especially its responsible E3 ligase is still unclear. Here we found that Smad ubiquitination regulatory factor 1 (Smurf1) down regulated protein expression of USP5, and the E3 enzyme activity of Smurf1 was required for this function. We also revealed that Smurf1 interacted with USP5 and mediated its degradation via the ubiquitin proteasome pathway. Consequently, Smurf1 inhibited the production of TNF-α through down-regulation of USP5. Taken together, our study for the first time clarified that the E3 ligase Smurf1 regulates USP5 protein stability and USP5-mediated TNF-α production through the ubiquitin proteasome pathway. [ABSTRACT FROM AUTHOR]

TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. [ABSTRACT FROM AUTHOR]

It has been demonstrated previously that F-box protein Fbxl15 targets HECT-type E3 Smurf1 and forms a functionally active SCF complex for ubiquitination and proteasomal degradation. Here we show that another F-box protein Fbxo3, belonging to the FBXO type protein family, also interacts with and targets Smurf1 for poly-ubiquitination and proteasomal degradation. Different from Fbxl15, Fbxo3 targets all the Nedd4 family members for their degradation, indicating that Fbxo3 plays an important role in controlling the stability of Nedd4. Taken together, we show that Smurf1 is an endogenous substrate of Fbxo3. Our study gains further insight into the novel role of Fbxo3 in BMP signaling. [ABSTRACT FROM AUTHOR]

Background Bone morphogenetic protein (BMP) signaling is thought to play key roles in regulating the survival and maintenance of cancer stem cells (CSCs), which contribute to disease recurrences and treatment failures in many malignances, including head and neck squamous cell carcinoma (HNSCC). Intracellular BMP signaling is regulated by SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) during cellular development. However, little is known about the role or regulation of BMP signaling in HNSCC CSCs. Methods Two CSC-like populations, CD44high/BMI1high and CD44high/ALDHhigh, were enriched from HNSCC cell lines and evaluated for the expression of SMURF1 by qRT-PCR, flow cytometry, and immunoblotting. The activation status of BMP signaling in these populations was determined by using immunoblotting to detect phosphorylated SMAD1/5/8 (pSMAD1/5/8) levels. Knockdown of SMURF1 transcripts by RNA interference was used to assess the role of SMURF1 in BMP signaling and CSC maintenance. Loss of CSC-like phenotypes following SMURF1 knockdown was determined by changes in CD44high levels, cellular differentiation, and reduction in colony formation. Results Populations of enriched CSC-like cells displayed decreased levels of pSMAD1/5/8 and BMP signaling target gene ID1 while SMURF1, CD44, and BMI1 were highly expressed when compared to non-CSC populations. Stable knockdown of SMURF1 expression in CSC-like cells increased pSMAD1/5/8 protein levels, indicating the reactivation of BMP signaling pathways. Decreased expression of SMURF1 also promoted adipogenic differentiation and reduced colony formation in a three-dimensional culture assay, indicating loss of tumorigenic capacity. The role of SMURF1 and inhibition of BMP signaling in maintaining a CSC-like population was confirmed by the loss of a CD44high expressing subpopulation in SMURF1 knockdown cells. Conclusions Our findings suggest that inhibition of BMP signaling potentiates the long-term survival of HNSCC CSCs, and that this inhibition is mediated by SMURF1. Targeting SMURF1 and restoring BMP signaling may offer a new therapeutic approach to promote differentiation and reduction of CSC populations leading to reduced drug resistance and disease recurrence. [ABSTRACT FROM AUTHOR]

Highlights: [•] Down-regulation of miR-497 in the more aggressive ovarian cancer cells contributes to cell metastasis. [•] Exogenous expression of miR-497 suppresses ovarian cancer cell migration and invasion. [•] MiR-497 suppresses ovarian cancer cell migration and invasion by targeting oncoprotein SMURF1. [ABSTRACT FROM AUTHOR]

Abstract: Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia. [Copyright &y& Elsevier]

Abstract: It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. [Copyright &y& Elsevier]

Abstract: The Smad ubiquitination regulatory factor 1 (Smurf1) is one of the E3 ubiquitin ligases and is related to multiple biological processes. Despite the various roles played by this protein, there is no report on the function of Smurf1 in neurodegeneration. Hirano bodies (HBs) are intracellular structures within neuronal processes and were first described in the hippocampus of individuals with amyotrophic lateral sclerosis and the parkinsonism–dementia complex of Guam. In addition, the number of HBs increases in the brains of patients with Alzheimer's disease (AD) compared with age-matched non-demented control individuals. In this study, we immunohistochemically demonstrated that Smurf1 localized in HBs in the brains of patients with AD by using plural anti-Smurf1 antibodies, and Smurf1 co-localized with HBs marker proteins by using confocal microscopy. Moreover, we demonstrated that Smurf1 localized in HB-like F-actin aggregates in a cell culture system via treatment with the actin-stabilizing toxin jasplakinolide (jpk). Smurf1 represents a novel protein component of HBs, to be included in an expanding list of HB-associated proteins. [Copyright &y& Elsevier]

Epidermal growth factor (EGF) is a well-known growth factor that induces cancer cell migration and invasion. Previous studies have shown that SMAD ubiquitination regulatory factor 1 (SMURF1), an E3 ubiquitin ligase, regulates cell motility by inducing RhoA degradation. Therefore, we examined the role of SMURF1 in EGF-induced cell migration and invasion using MDA-MB-231 cells, a human breast cancer cell line. EGF increased SMURF1 expression at both the mRNA and protein levels. All ErbB family members were expressed in MDA-MB-231 cells and receptor tyrosine kinase inhibitors specific for the EGF receptor (EGFR) or ErbB2 blocked the EGF-mediated induction of SMURF1 expression. Within the signaling pathways examined, ERK1/2 and protein kinase C activity were required for EGF-induced SMURF1 expression. The overexpression of constitutively active MEK1 increased the SMURF1 to levels similar to those induced by EGF. SMURF1 induction by EGF treatment or by the overexpression of MEK1 or SMURF1 resulted in enhanced cell migration and invasion, whereas SMURF1 knockdown suppressed EGF- or MEK1-induced cell migration and invasion. EGF treatment or SMURF1 overexpression decreased the endogenous RhoA protein levels. The overexpression of constitutively active RhoA prevented EGF- or SMURF1-induced cell migration and invasion. These results suggest that EGFinduced SMURF1 plays a role in breast cancer cell migration and invasion through the downregulation of RhoA. [ABSTRACT FROM AUTHOR]

Since being discovered and intensively studied for over a decade, Smad ubiquitylation regulatory factor-1 (Smurf1) has been linked with several important biological pathways, including the bone morphogenetic protein pathway, the non-canonical Wnt pathway, and the mitogen-activated protein kinase pathway. Multiple functions of this ubiquitin ligase have been discovered in cell growth and morphogenesis, cell migration, cell polarity, and autophagy. Smurf1 is related to physiological manifestations in terms of age-dependent deficiency in bone formation and invasion of tumor cells. Smurf1-knockout mice have a significant phenotype in the skeletal system and considerable manifestations during embryonic development and neural outgrowth. In depth studying of Smurf1 will help us to understand the etiopathological mechanisms of related disorders. Here, we will summarize historical and recent studies on Smurf1, and discuss the E3 ligase-dependent and -independent functions of Smurf1. Moreover, intracellular regulations of Smurf1 and related physiological phenotypes will be described in this review. [ABSTRACT FROM AUTHOR]

Abstract: Smad ubiquitin regulatory factors (Smurfs) belong to the HECT- family of E3 ubiquitin ligases and comprise mainly of two members, Smurf1 and Smurf2. Initially, Smurfs have been implicated in determining the competence of cells to respond to TGF-β/BMP signaling pathway. Nevertheless, the intrinsic catalytic activity has extended the repertoire of Smurf substrates beyond the TGF-β/BMP super family expanding its realm further to epigenetic modifications of histones governing the chromatin landscape. Through regulation of a large number of proteins in multiple cellular compartments, Smurfs regulate diverse cellular processes, including cell-cycle progression, cell proliferation, differentiation, DNA damage response, maintenance of genomic stability, and metastasis. As the genomic ablation of Smurfs leads to global changes in histone modifications and predisposition to a wide spectrum of tumors, Smurfs are also considered to have a novel tumor suppressor function. This review focuses on regulation network and biological functions of Smurfs in connection with its role in cancer progression. By providing a portrait of their protein targets, we intend to link the substrate specificity of Smurfs with their contribution to tumorigenesis. Since the regulation and biological functions of Smurfs are quite complex, understanding the oncogenic potential of these E3 ubiquitin ligases may facilitate the development of mechanism-based drugs in cancer treatment. [Copyright &y& Elsevier]

Axon growth is an essential event during brain development and is extremely limited due to extrinsic and intrinsic inhibition in the adult brain. The E3 ubiquitin ligase Cdh1-anaphase promoting complex (APC) has emerged as an important intrinsic suppressor of axon growth. In this study, we identify in rodents the E3 ligase Smurf1 as a novel substrate of Cdh1-APC and that Cdh1 targets Smurf1 for degradation in a destruction box-dependent manner. We find that Smurf1 acts downstream of Cdh1-APC in axon growth and that the turnover of RhoA by Smurf1 is important in this process. In addition, we demonstrate that acute knockdown of Smurf1 in vivo in the developing cerebellar cortex results in impaired axonal growth and migration. Finally, we show that a stabilized form of Smurf1 overrides the inhibition of axon growth by myelin. Taken together, we uncovered a Cdh1-APC/Smurf1/RhoA pathway that mediates axonal growth suppression in the developing mammalian brain. [ABSTRACT FROM AUTHOR]

Rho GTPases undergo ubiquitylation and degradation via the ubiquitin-proteasome pathway. We now report in the November issue of Developmental Cell that the E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of GTP-bound Rac1. Depletion of HACE1 leads to an increase of Rac1 activity. We have proposed that HACE1 limits Rac1 activity in cells, a regulation that is usurped by some pathogenic bacteria for efficient invasion of host cell monolayers. We here review these indings in parallel with the regulation of RhoA by the ubiquitin and proteasome system (UPS) and discuss the impact of these regulations on the capacity of Rho GTPases to signal. [ABSTRACT FROM AUTHOR]

Abstract: Coronary vessel development requires transfer of mesothelial cells to the heart surface to form the epicardium where some cells subsequently undergo epithelial–mesenchymal transformation (EMT) and invade the subepicardial matrix. Tgfbr3 −/− mice die due to failed coronary vessel formation associated with decreased epicardial cell invasion but the mediators downstream of TGFβR3 are not well described. TGFβR3-dependent endocardial EMT stimulated by either TGFβ2 or BMP-2 requires activation of the Par6/Smurf1/RhoA 1pathway where Activin Receptor Like Kinase (ALK5) signals Par6 to act downstream of TGFβ to recruit Smurf1 to target RhoA for degradation to regulate apical-basal polarity and tight junction dissolution. Here we asked if this pathway was operant in epicardial cells and if TGFβR3 was required to access this pathway. Targeting of ALK5 in Tgfbr3 +/+ cells inhibited loss of epithelial character and invasion. Overexpression of wild-type (wt) Par6, but not dominant negative (dn) Par6, induced EMT and invasion while targeting Par6 by siRNA inhibited EMT and invasion. Overexpression of Smurf1 and dnRhoA induced loss of epithelial character and invasion. Targeting of Smurf1 by siRNA or overexpression of constitutively active (ca) RhoA inhibited EMT and invasion. In Tgfbr3 −/− epicardial cells which have a decreased ability to invade collagen gels in response to TGFβ2, overexpression of wtPar6, Smurf1, or dnRhoA had a diminished ability to induce invasion. Overexpression of TGFβR3 in Tgfbr3 −/− cells, followed by siRNA targeting of Par6 or Smurf1, diminished the ability of TGFβR3 to rescue invasion demonstrating that the Par6/Smurf1/RhoA pathway is activated downstream of TGFβR3 in epicardial cells. [Copyright &y& Elsevier]

The tumor suppressor p53 locates at the key point of cell growth or apoptosis balance, and the expression level of p53 is tightly controlled by ubiquitin ligases including MDM2. Upon DNA damage stresses, p53 was accumulated and activated, leading to cell cycle arrest or apoptosis. We previously showed that Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by interacting with and stabilizing MDM2, and consequently enhancing MDM2-mediated ubiquitylation of p53. However, it is unclear how the Smurf1-MDM2 interaction is regulated in response to DNA damage stress. Here, we show that in response to etoposide treatment Smurf1 dissociates from MDM2, resulting in MDM2 destabilization and p53 accumulation. The negative regulation of Smurf1 on apoptosis is released. Notably, this dissociation is a slow process rather than a rapid response, implicating high expression of Smurf1 might confer the resistance against p53 activation. Consistent with this notion, we observed that Smurf1/2 ligases are highly expressed in colon cancer, esophageal squamous cell carcinoma and pancreatic cancer tissues, suggesting the oncogenic tendency of Smurf1/2. [ABSTRACT FROM AUTHOR]

Abstract: Smurf1-mediated RhoA ubiquitination and degradation plays key roles in regulation of cell polarity and protrusive activity. However, how Smurf1 recognizes RhoA is still not clear. Here we report that the C2 domain of Smurf1 is necessary and sufficient for binding RhoA, and therefore is crucial for targeting RhoA for ubiquitination. In contrast, the C2 domain is dispensable for Smurf1-mediated ubiquitination of Smad1. Consistent with its biochemical specificity, the C2 domain is essential for Smurf1-regulated protrusion formation but not BMP signaling. Therefore, our study reveals the mechanism of the C2 domain of Smurf1 in substrate selection. Structured summary of protein interactions: SMURF1 physically interacts with Smad1 by pull down (View interaction) SMURF1 physically interacts with RhoA by pull down (View interaction) SMURF1 physically interacts with Smad1 by anti tag coimmunoprecipitation (View interaction) SMURF1 physically interacts with RhoA by anti tag coimmunoprecipitation (View interaction) [Copyright &y& Elsevier]

Abstract: Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2. [Copyright &y& Elsevier]

Background/Aims: Mechanical strain plays an important role in osteoblasts differentiation and bone formation but the underlying mechanism remains unclear. The aim of this study was to determine whether Bone Morphogenetic Proteins (BMPs)/Smad signaling pathway is involved in mechanical response in osteoblasts. Methods: MC3T3-E1 cells were exposed to mechanical strain via a four-point bending system. mRNA levels and protein levels of BMP-2, BMP-4, Smad1, Smad5, Smurf1, and Smurf2 were assessed using RT-PCR and immunoblotting. Protein levels of BMP-2 and BMP-4 in the culture medium were also determined using Enzyme-linked Immunosorbent Assay (ELISA). Pretreatment with Noggin and transfection with Smad4 siRNA were carried out to block the BMPs/Smad signaling pathway and MG132 was used to inhibit the proteasome pathway. Results: We found that mechanical strain enhanced alkaline phosphatase (ALP) expression and activated BMPs/Smad signaling pathway. Mechanical strain induced expression of ALP was attenuated by Noggin and by Smad4 siRNA. The protein levels of Smad1 and Smad5, but not their mRNA levels, were up-regulated by mechanical strain. This finding could be explained by the down-regulation of Smurf1. The protein degradation of Smad might be inhibited by mechanical strain through down-regulation of Smuf1 expression. The addition of MG132 further enhanced the mechanical strain induced activation of Smad proteins and the increased expression of ALP. Conclusions: Mechanical strain might promote osteoblasts differentiation through BMPs/Smad signaling pathway. The strain causes a drop in Smurf1 levels, leading to accumulation of Smad proteins and, subsequently, to enhanced BMPs/Smad signaling. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The ubiquitin ligase Smurf1 can target a handful of signaling proteins for ubiquitin-mediated proteasomal destruction or functional modification, including TGF-β receptors, Smads, transcription factors, RhoA and MEKK2. Smurf1 was initially implicated in BMP pathway regulation in embryonic development, but its potential role in vertebrate embryogenesis has yet to be clarified. Here we demonstrate that inhibition of Smurf1 in Xenopus laevis embryos with an antisense morpholino oligonucleotide or a dominant-negative protein disrupts early development, with the nervous system being the principal target. Smurf1 is enriched on the dorsal side of gastrula stage embryos, and blocking Smurf1 disturbs neural folding and neural, but not mesoderm differentiation, enhances BMP/Smad1 signaling, and elevates phospho-Smad1 levels in the dorsal ectoderm. We conclude that in Xenopus embryos, the BMP pathway is a major physiological target of Smurf1, and we propose that in normal development Smurf1 cooperates with secreted BMP antagonists to limit BMP signaling in dorsal ectoderm. Our data also reveal a novel role for Smurf1 and Smad1 in neural plate morphogenesis. [Copyright &y& Elsevier]

Abstract: The Rho-family of small GTPases consists of essential regulators of neurite outgrowth, axonal pathfinding, and dendritic arborization. Previous work has demonstrated in non-neuronal cell types that Smurf1, an E3 ubiquitin ligase, regulates cell polarity and protrusive activity via PKCζ-dependent recruitment to cellular protrusion sites, and subsequent ubiquitination and proteasomal degradation of RhoA. In this study, we show that Smurf1 enhances neurite outgrowth in Neuro2a neuroblastoma cells. We demonstrate that RhoA is ubiquitinated, and that Smurf1 and RhoA physically interact in vivo. Interestingly, Smurf1 overexpression in Neuro2a cells dramatically reduces RhoA protein levels during dibutyric cyclic AMP, but not retinoic acid induced neurite outgrowth. This Smurf1-dependent reduction in RhoA protein levels was abrogated using the general proteasome inhibitor MG132, suggesting that RhoA is targeted for ubiquitination and degradation via Smurf1. Together, our data suggest that localized regulation of different subsets of Rho GTPases by specific guidance signals results in an intracellular asymmetry of RhoA activity, which could regulate neurite outgrowth and guidance. [Copyright &y& Elsevier]

In colorectal carcinoma tissues, the transcription factor Fosl1 activates the expression of Smurf1, which can bind to the tumor suppressor FBXL2 and promote the ubiquitination and degradation of FBXL2, thereby activating the β-catenin nuclear translocation and elevating the expression of downstream genes such as c-Myc and CCND1 to promote the growth and metastasis of colorectal carcinoma cells. [Display omitted] • Fosl1 is highly expressed in CC patients and associated with poor prognosis. • Fosl1 promotes the growth and metastasis of CC cells in vitro and in vivo. • Fosl1 activates the expression of Smurf1 and promotes the degradation of FBXL2. • FBXL2 inhibits the activity of CC cells by blocking the Wnt/β-catenin signaling. • This study can be used as a possible target for the clinical treatment of CC. Colorectal carcinoma (CC), one of the most prevalent digestive cancers with high mortality and morbidity globally, still lacks powerful therapies to improve the prognosis. Here, we established that the expression of fos-like antigen-1 (Fosl1) was elevated in CC tissues versus adjacent tissues. Importantly, high Fosl1 expression was related to dismal prognosis among CC patients. Functional assays displayed that Fosl1 increased the viability, epithelial-to-mesenchymal transition (EMT), migration and invasion of CC cells. Additionally, a xenograft assay showed that silencing of Fosl1 in CC cells retarded lung, liver and kidney metastases in vivo. Further investigation demonstrated that Fosl1 was involved in malignant aggressiveness of CC cells by binding to smad ubiquitination regulatory factor 1 (Smurf1). Mechanistically, Smurf1-induced F-Box and leucine rich repeat protein 2 (FBXL2) ubiquitination resulted in its degradation, while FBXL2 disrupted the activation of the Wnt/β-catenin signaling. In summary, Fosl1 plays a pro-metastatic and carcinogenetic role in CC, and we provided forceful evidence that Fosl1 inhibition might act as a prognostic and therapeutic option in CC. [ABSTRACT FROM AUTHOR]

Transforming growth factor β1 (TGFβ1) is a major mediator in the modulation of osteoblast differentiation. However, the underlying molecular mechanism is still not fully understood. Here, we show that TGFβ1 has a dual stage-dependent role in osteoblast differentiation; TGFβ1 induced matrix maturation but inhibited matrix mineralization. We discovered the underlying mechanism of the TGFβ1 inhibitory role in mineralization using human osteoprogenitors. In particular, the matrix mineralization-related genes of osteoblasts such as osteocalcin (OCN), Dickkopf 1 (DKK1), and CCAAT/enhancer-binding protein beta (C/EBPβ) were dramatically suppressed by TGFβ1 treatment. The suppressive effects of TGFβ1 were reversed with anti-TGFβ1 treatment. Mechanically, TGFβ1 decreased protein levels of C/EBPβ without changing mRNA levels and reduced both mRNA and protein levels of DKK1. The degradation of the C/EBPβ protein by TGFβ1 was dependent on the ubiquitin–proteasome pathway. TGFβ1 degraded the C/EBPβ protein by inducing the expression of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (SMURF1) at the transcript level, thereby reducing the C/EBPβ-DKK1 regulatory mechanism. Collectively, our findings suggest that TGFβ1 suppressed the matrix mineralization of osteoblast differentiation by regulating the SMURF1-C/EBPβ-DKK1 axis. [ABSTRACT FROM AUTHOR]