The E3 ligase SMURF1 stabilizes p27 via UbcH7 catalyzed K29-linked ubiquitin chains to promote cell migration SMURF1-UbcH7 K29 ubiquitination of p27 and cell migration.

Ubiquitination is a key regulator of protein stability and function. The multifunctional protein p27 is known to be degraded by the proteasome following K48-linked ubiquitination. However, we recently reported that when the ubiquitin-conjugating enzyme UbcH7 (UBE2L3) is overexpressed, p27 is stabilized, and cell cycle is arrested in multiple diverse cell types including eye lens, retina, HEK-293, and HELA cells. However, the ubiquitin ligase associated with this stabilization of p27 remained a mystery. Starting with an in vitro ubiquitination screen, we identified RSP5 as the yeast E3 ligase partner of UbcH7 in the ubiquitination of p27. Screening of the homologous human NEDD4 family of E3 ligases revealed that SMURF1 but not its close homolog SMURF2, stabilizes p27 in cells. We found that SMURF1 ubiquitinates p27 with K29O but not K29R or K63O ubiquitin in vitro, demonstrating a strong preference for K29 chain formation. Consistent with SMURF1/UbcH7 stabilization of p27, we also found that SMURF1, UbcH7, and p27 promote cell migration, whereas knockdown of SMURF1 or UbcH7 reduces cell migration. We further demonstrated the colocalization of SMURF1/p27 and UbcH7/p27 at the leading edge of migrating cells. In sum, these results indicate that SMURF1 and UbcH7 work together to produce K29-linked ubiquitin chains on p27, resulting in the stabilization of p27 and promoting its cell-cycle independent function of regulating cell migration.

Smurf1 polyubiquitinates on K285/K282 of the kinases Mst1/2 to attenuate their tumor-suppressor functions.

Sterile 20-like kinases Mst1 and Mst2 (Mst1/2) and large tumor suppressor 1/2 are core kinases to mediate Hippo signaling in maintaining tissue homeostasis. We have previously demonstrated that Smad ubiquitin (Ub) regulatory factor 1 (Smurf1), a HECT-type E3 ligase, ubiquitinates and in turn destabilizes large tumor suppressor 1/2 to induce the transcriptional output of Hippo signaling. Here, we unexpectedly find that Smurf1 interacts with and polyubiquitinates Mst1/2 by virtue of K27- and K29-linked Ub chains, resulting in the proteasomal degradation of Mst1/2 and attenuation of their tumor-suppressor functions. Among the potential Ub acceptor sites on Mst1/2, K285/K282 are conserved and essential for Smurf1-induced polyubiquitination and degradation of Mst1/2 as well as transcriptional output of Hippo signaling. As a result, K285R/K282R mutation of Mst1/2 not only negates the transcriptional output of Hippo signaling but enhances the tumor-suppressor functions of Mst1/2. Together, we demonstrate that Smurf1-mediated polyubiquitination on K285/K282 of Mst1/2 destabilizes Mst1/2 to attenuate their tumor-suppressor functions. Thus, the present study identifies Smurf1-mediated ubiquitination of Mst1/2 as a hitherto uncharacterized mechanism fine-tuning the Hippo signaling pathway and may provide additional targets for therapeutic intervention of diseases associated with this important pathway.

The role of SMURFs in non-cancerous diseases.

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.

piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

Transmembrane anterior posterior transformation 1 regulates BMP signaling and modulates the protein stability of SMAD1/5.

The bone morphogenetic protein (BMP) signaling pathway plays pivotal roles in various biological processes during embryogenesis and adult homeostasis. Transmembrane anterior posterior transformation 1 (TAPT1) is an evolutionarily conserved protein involved in murine axial skeletal patterning. Genetic defects in TAPT1 result in complex lethal osteochondrodysplasia. However, the specific cellular activity of TAPT1 is not clear. Herein, we report that TAPT1 inhibits BMP signaling and destabilizes the SMAD1/5 protein by facilitating its interaction with SMURF1 E3 ubiquitin ligase, which leads to SMAD1/5 proteasomal degradation. In addition, we found that the activation of BMP signaling facilitates the redistribution of TAPT1 and promotes its association with SMAD1. TAPT1-deficient murine C2C12 myoblasts or C3H/10T1/2 mesenchymal stem cells exhibit elevated SMAD1/5/9 protein levels, which amplifies BMP activation, in turn leading to a boost in the transdifferentiation or differentiation processing of these distinct TAPT1-deficient cell lines changing into mature osteoblasts. Furthermore, the enhancing effect of TAPT1 deficiency on osteogenic differentiation of C3H/10T1/2 cells was observed in an in vivo ectopic bone formation model. Importantly, a subset of TAPT1 mutations identified in humans with lethal skeletal dysplasia exhibited gain-of-function activity on SMAD1 protein levels. Thus, this finding elucidates the role of TAPT1 in the regulation of SMAD1/5 protein stability for controlling BMP signaling.

Smurf1: A possible therapeutic target in dry age-related macular degeneration.

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 (NaIO3) in C57BL/6J mice was the animal model of retina degeneration. In NaIO3 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-ß1 and phosphorylated NF-κB (pNF-κB). TGF-ß1 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-1ß. At last, after searching bioinformatics database, Smurf1 had a possible interaction with beta-transducin repeat containing E3 ubiquitin protein ligase (ß-TrCP), another E3 ubiquitin ligases. ß-TrCP can mediate ubiquitination degradation of p-IκBα. Lentivirus-SMURF1 was used to overexpress Smurf1, and GS143 was used to inhibit ß-TrCP. The results showed Smurf1 could directly induce NF-κB, pNF-κB, and NLRP3 expression, and keep a stable ß-TrCP expression. However, inhibiting ß-TrCP could cause more NF-κB activation and NLRP3 expression. Therefore, ß-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.

Neddylation modification of the U3 snoRNA-binding protein RRP9 by Smurf1 promotes tumorigenesis.

Neddylation is a posttranslational modification that attaches ubiquitin-like protein Nedd8 to protein targets via Nedd8-specific E1-E2-E3 enzymes and modulates many important biological processes. Nedd8 attaches to a lysine residue of a substrate, not for degradation, but for modulation of substrate activity. We previously identified the HECT-type ubiquitin ligase Smurf1, which controls diverse cellular processes, is activated by Nedd8 through covalent neddylation. Smurf1 functions as a thioester bond-type Nedd8 ligase to catalyze its own neddylation. Numerous ubiquitination substrates of Smurf1 have been identified, but the neddylation substrates of Smurf1 remain unknown. Here, we show that Smurf1 interacts with RRP9, a core component of the U3 snoRNP complex, which is involved in pre-rRNA processing. Our in vivo and in vitro neddylation modification assays show that RRP9 is conjugated with Nedd8. RRP9 neddylation is catalyzed by Smurf1 and removed by the NEDP1 deneddylase. We identified Lys221 as a major neddylation site on RRP9. Deficiency of RRP9 neddylation inhibits pre-rRNA processing and leads to downregulation of ribosomal biogenesis. Consequently, functional studies suggest that ectopic expression of RRP9 promotes tumor cell proliferation, colony formation, and cell migration, whereas unneddylated RRP9, K221R mutant has no such effect. Furthermore, in human colorectal cancer, elevated expression of RRP9 and Smurf1 correlates with cancer progression. These results reveal that Smurf1 plays a multifaceted role in pre-rRNA processing by catalyzing RRP9 neddylation and shed new light on the oncogenic role of RRP9.

NEDD4 E3 Ligases: Functions and Mechanisms in Bone and Tooth.

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.

The functions and regulation of Smurfs in cancers.

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-ß/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.

microRNA-125b and its downstream Smurf1/KLF2/ATF2 axis as important promoters on neurological function recovery in rats with spinal cord injury.

The purpose of this study is to investigate the role of microRNA-125b (miR-125b) and its mechanism in spinal cord injury (SCI) by targeting Smurf1. After loss- and gain-function approaches were conducted in SCI rat models and neural stem cells (NSCs) isolated from foetal rats, the Basso-Beattie-Bresnahan (BBB) score was calculated, and related protein expression was determined by Western blot analysis and cell apoptosis by TUNEL staining. NSC viability was detected by CCK-8, migration abilities by Transwell assay and apoptosis by flow cytometry. The relationship between miR-125b, Smurf1 and KLF2 was evaluated by dual-luciferase reporter gene experiments, Co-IP and in vivo ubiquitin modification assays. Inhibition of miR-125b and KLF2 and the up-regulation of Smurf1 and ATF2 were observed in SCI rats. BBB scores were elevated, the expression of Nestin, NeuN, GFAP, NF-200 and Bcl-2 protein was enhanced but that of Bax protein was reduced, and cell apoptosis was inhibited in SCI rats after up-regulating miR-125b or silencing ATF2. Smurf1 was a target gene of miR-125b, which promoted KLF2 degradation through its E3 ubiquitin ligase function, and KLF2 repressed the expression of ATF2 in NSCs. The results in vivo were replicated in vitro. miR-125b overexpression promotes neurological function recovery after SCI.

The N-terminal domain of the non-receptor tyrosine kinase ABL confers protein instability and suppresses tumorigenesis.

Chromosome translocation can lead to chimeric proteins that may become oncogenic drivers. A classic example is the fusion of the BCR activator of RhoGEF and GTPase and the ABL proto-oncogene nonreceptor tyrosine kinase, a result of a chromosome abnormality (Philadelphia chromosome) that causes leukemia. To unravel the mechanism underlying BCR-ABL-mediated tumorigenesis, here we compared the stability of ABL and the BCR-ABL fusion. Using protein degradation, cell proliferation, 5-ethynyl-2-deoxyuridine, and apoptosis assays, along with xenograft tumor analysis, we found that the N-terminal segment of ABL, which is lost in the BCR-ABL fusion, confers degradation capacity that is promoted by SMAD-specific E3 ubiquitin protein ligase 1. We further demonstrate that the N-terminal deletion renders ABL more stable and stimulates cell growth and tumorigenesis. The findings of our study suggest that altered protein stability may contribute to chromosome translocation-induced cancer development.

FOSL1 promotes tumorigenesis in colorectal carcinoma by mediating the FBXL2/Wnt/ß-catenin axis via Smurf1.

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/ß-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.

Overexpression of miR-195-5p reduces osteoporosis through activating BMP-2/SMAD/Akt/RUNX2 pathway via targeting SMURF1.

Osteoporosis (OP) is among the most common frequent chronic metabolic bone diseases in postmenopausal women. Here, the effect and underlying mechanisms of miR-195-5p in OP were investigated both in vivo and in vitro. In this study, the microgravity (MG) environment was simulated in MC3T3-E1 cells, and miR-195-5p overexpression or SMURF1 knockdown model was constructed to test their effects on the proliferation, apoptosis and osteogenic differentiation of MC3T3-E1 cells. Furthermore, an OVX mouse model was constructed in vivo, and adenovirus-loaded miR-195-5p mimics were administered to the mice to overexpress miR-195-5p. HE staining and µCT were adopted to observe pathological changes of femur. The targeted relationship between miR-195-5p and SMURF1 was predicted by bioinformatics analysis and verified by the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) experiment. The results indicated that miR-195-5p was down-regulated in the head of femur of OP mouse model and MC3T3-E1 cells subjected to MG microenvironment. In addition, overexpression of miR-195-5p promoted MC3T3-E1 cell osteogenic differentiation and inhibited apoptosis. Mechanistically, SMURF1 is identified as a target of miR-195-5p, and overexpressing miR-195-5p activates the BMP-2/SMAD/Akt/RUNX2 signal by inhibiting the SMURF1 expression. Moreover, SMURF1 downregulation accelerated the osteogenic differentiation of MC3T3-E1 cells and attenuated MG-mediated apoptosis. In addition, upregulating miR-195-5p reduced osteoporosis in the OVX mouse model, accompanied with SMURF1 downregulation and BMP-2/SMAD/Akt/RUNX2 pathway activation. Collectively, miR-195-5p enhances osteogenic differentiation of osteoclast and relieve OP progression in the mouse model through activation of the BMP-2/SMAD/Akt/RUNX2 axis by targeting SMURF1.

SND1 facilitates the invasion and migration of cervical cancer cells by Smurf1-mediated degradation of FOXA2.

Staphylococcal nuclease domain-containing protein 1 (SND1) is known to be involved in the progression of a variety of human cancers. However, the role of SND1 in cervical cancer remains unclear. Here, we found that the expression of SND1 in cervical cancer tissue was higher than that in normal cervical tissue. Importantly, high SND1 expression was closely associated with tumorigenic phenotype and shorter survival among cervical cancer patients. Functional assays demonstrated that SND1 knockdown inhibited the migration and invasion capabilities of cervical cancer cells in vitro. Additionally, a xenograft assay showed that silencing SND1 in cervical cancer cells suppressed lung metastasis in vivo. Further investigation revealed that knockdown of SND1 inhibited epithelial-to-mesenchymal transition (EMT) of cervical cancer cells by enhancing FOXA2 expression. Moreover, the pro-metastasis effect of SND1 in cervical cancer was at least in part dependent on FOXA2 inhibition. Mechanistically, we found that SND1-induced FOXA2 ubiquitination resulted in degradation, mediated by the E3 ligase enzyme Smurf1. In summary, SND1 plays a crucial role in cervical cancer metastasis, and we provide evidence that SND1 may serve as a prognostic and therapeutic target in cervical cancer.

microRNA-148a-3p in extracellular vesicles derived from bone marrow mesenchymal stem cells suppresses SMURF1 to prevent osteonecrosis of femoral head.

Extracellular vesicle (EV)-associated microRNAs (miRNAs) have been found as the important biomarkers participating in the development of osteonecrosis of the femoral head (ONFH). Consequently, this study sought to examine the underlying mechanism of bone marrow mesenchymal stem cell (BMSC)-derived EVs containing miR-148a-3p in ONFH. The ONFH rat models were established. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were applied to detect miR-148a-3p, Smad ubiquitination regulatory factor 1 (SMURF1), SMAD7 and B-cell CLL/lymphoma 2 (BCL2) expression, followed by determination of relationship between miR-148a-3p and SMURF1. BMSCs were isolated from normal rats and ONFH rats, and EVs were extracted from BMSCs of normal rats. BMSCs from ONFH rats were treated with mimic, inhibitor, small interfering RNA or EVs from miR-148a-3p mimic-treated BMSCs from normal rats (BMSC-EV-miR-148a-3p mimic). Cell Counting Kit-8 and alizarin red staining were utilized to detect cell viability and osteogenic differentiation of BMSCs. ONFH rats were injected with BMSC-EV-miR-148a-3p mimic to explore the function of BMSC-EV-delivered miR-148a-3p in vivo. miR-148a-3p was down-regulated in BMSCs and EVs from ONFH rats following decreased BMSCs viability and osteogenic differentiation. SMURF1 was a target gene of miR-148a-3p, and resulted in ubiquitination and degradation of SMAD7 to decreased BCL2 expression. The proliferation and differentiation of BMSCs were promoted by BMSC-EV-miR-148a-3p mimic or SMURF1 silencing. Additionally, BMSC-EV-miR-148a-3p mimic increased cell proliferation and osteogenic response, diminished SMURF1 expression, and elevated SMAD7 and BCL2 expression in ONFH rats. Collectively, miR-148a-3p overexpressed in BMSC-EVs promoted SMAD7 and BCL2 expression by inhibiting SMURF1, thus alleviating ONFH.

A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of E3 ligases.

Accumulating evidence indicates that a wide range of E3 ubiquitin ligases are involved in the development of many human diseases. Searching for small-molecule modulators of these E3 ubiquitin ligases is emerging as a promising drug discovery strategy. Here, we report the development of a cell-based high-throughput screening method to identify modulators of E3 ubiquitin ligases by integrating the ubiquitin-reference technique (URT), based on a fusion protein of ubiquitin located between a protein of interest and a reference protein moiety, with a Dual-Luciferase system. Using this method, we screened for small-molecule modulators of SMAD ubiquitin regulatory factor 1 (SMURF1), which belongs to the NEDD4 family of E3 ubiquitin ligases and is an attractive therapeutic target because of its roles in tumorigenesis. Using RAS homolog family member B (RHOB) as a SMURF1 substrate in this screen, we identified a potent SMURF1 inhibitor and confirmed that it also blocks SMURF1-dependent degradation of SMAD family member 1 (SMAD1) and RHOA. An in vitro auto-ubiquitination assay indicated that this compound inhibits both SMURF1 and SMURF2 activities, indicating that it may be an antagonist of the catalytic activity of the HECT domain in SMURF1/2. Moreover, cell functional assays revealed that this compound effectively inhibits protrusive activity in HEK293T cells and blocks transforming growth factor ß (TGFß)-induced epithelial-mesenchymal transition (EMT) in MDCK cells, similar to the effects on these processes caused by SMURF1 loss. In summary, the screening approach presented here may have great practical potential for identifying modulators of E3 ubiquitin ligases.

miR-4319 inhibited the development of thyroid cancer by modulating FUS-stabilized SMURF1.

Thyroid cancer, a common type of endocrine system cancer, has witnessed rising incidence and deaths over the past few years. The role of microRNAs is being increasingly discovered in a variety of cancers, including thyroid cancer. miR-4319 has been elucidated in several studies to exert an antitumor function in multiple cancers but has never been explored in thyroid cancer. Our study proposed to explore the function and modulatory mechanism of miR-4319 in thyroid cancer. First, we confirmed the downregulation of miR-4319 in thyroid cancer tissues and cells, and revealed the correlation of miR-4319 expression and clinical features in thyroid cancer. Functional assays illustrated that miR-4319 attenuated proliferation, migration, and epithelial-to-mesenchymal transition (EMT) in thyroid cancer. Mechanistically, we identified through the miRDB database and proved that miR-4319 targeted SMAD specific E3 ubiquitin protein ligase 1 (SMURF1). Furthermore, we discovered that miR-4319 competed with fused in sarcoma (FUS) to bind to SMURF1, inhibiting the stabilization of SMURF1 messenger RNA. Rescue assays suggested that miR-4319 retarded proliferation, migration, and EMT through SMURF1. Collectively, the results of this study showed that miR-4319 inhibited the development of thyroid cancer by modulating FUS-stabilized SMURF1, indicating miR-4319 as a potent biological target for thyroid cancer.

Fragile Bones Secondary to SMURF1 Gene Duplication.

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.

TGFß1 Suppressed Matrix Mineralization of Osteoblasts Differentiation by Regulating SMURF1-C/EBPß-DKK1 Axis.

Transforming growth factor ß1 (TGFß1) is a major mediator in the modulation of osteoblast differentiation. However, the underlying molecular mechanism is still not fully understood. Here, we show that TGFß1 has a dual stage-dependent role in osteoblast differentiation; TGFß1 induced matrix maturation but inhibited matrix mineralization. We discovered the underlying mechanism of the TGFß1 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/EBPß) were dramatically suppressed by TGFß1 treatment. The suppressive effects of TGFß1 were reversed with anti-TGFß1 treatment. Mechanically, TGFß1 decreased protein levels of C/EBPß without changing mRNA levels and reduced both mRNA and protein levels of DKK1. The degradation of the C/EBPß protein by TGFß1 was dependent on the ubiquitin-proteasome pathway. TGFß1 degraded the C/EBPß protein by inducing the expression of the E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (SMURF1) at the transcript level, thereby reducing the C/EBPß-DKK1 regulatory mechanism. Collectively, our findings suggest that TGFß1 suppressed the matrix mineralization of osteoblast differentiation by regulating the SMURF1-C/EBPß-DKK1 axis.

Bortezomib attenuates renal interstitial fibrosis in kidney transplantation via regulating the EMT induced by TNF-α-Smurf1-Akt-mTOR-P70S6K pathway.

Allograft interstitial fibrosis was characterized by massive extracellular matrix deposition caused by activated fibroblasts and myofibroblasts. Epithelial-mesenchymal transition (EMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis. Smad ubiquitination regulatory factor 1 (Smurf1) has been recently reported to be involved in the progression of EMT. Our study was to detect the effect of Bortezomib and Smurf1 in the EMT and allograft interstitial fibrosis. Biomarkers of EMT, as well as Smurf1, were examined in human proximal tubular epithelial cells (HK-2) treated with tumour necrosis factor-alpha (TNF-α) in various doses or at various time points by Western Blotting or qRT-PCR. We knockdown or overexpressed Smurf1 in HK-2 cells. Furthermore, rat renal transplant model was established and intervened by Bortezomib. Allograft tissues from human and rats were also collected and prepared for HE, Masson's trichrome, immunohistochemical staining and western blotting assays. As a result, we found that TNF-α significantly promoted the development of EMT in a time-dependent and dose-dependent manner through Smurf1/Akt/mTOR/P70S6K signalling pathway. More importantly, Bortezomib alleviated the progression of EMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting the production of TNF-α and expression of Smurf1. In conclusion, Smurf1 plays a critical role in the development of EMT induced by TNF-α. Bortezomib can attenuate the Sumrf1-mediated progression of EMT and renal allograft interstitial fibrosis, which could be suggested as a novel choice for the prevention and treatment of renal allograft interstitial fibrosis.