Machine learning identifies the role of SMAD6 in the prognosis and drug susceptibility in bladder cancer.

BACKGROUND: Bladder cancer (BCa) is among the most prevalent malignant tumors affecting the urinary system. Due to its highly recurrent nature, standard treatments such as surgery often fail to significantly improve patient prognosis. Our research aims to predict prognosis and identify precise therapeutic targets for novel treatment interventions. METHODS: We collected and screened genes related to the TGF-ß signaling pathway and performed unsupervised clustering analysis on TCGA-BLCA samples based on these genes. Our analysis revealed two novel subtypes of bladder cancer with completely different biological characteristics, including immune microenvironment, drug sensitivity, and more. Using machine learning classifiers, we identified SMAD6 as a hub gene contributing to these differences and further investigated the role of SMAD6 in bladder cancer in the single-cell transcriptome data. Additionally, we analyzed the relationship between SMAD6 and immune checkpoint genes. Finally, we performed a series of in vitro assays to verify the function of SMAD6 in bladder cancer cell lines. RESULTS: We have revealed two novel subtypes of bladder cancer, among which C1 exhibits a worse prognosis, lower drug sensitivity, a more complex tumor microenvironment, and a 'colder' immune microenvironment compared to C2. We identified SMAD6 as a key gene responsible for the differences and further explored its impact on the molecular characteristics of bladder cancer. Through in vitro experiments, we found that SMAD6 promoted the prognosis of BCa patients by inhibiting the proliferation and migration of BCa cells. CONCLUSION: Our study reveals two novel subtypes of BCa and identifies SMAD6 as a highly promising therapeutic target.

Homozygous SMAD6 variants in two unrelated patients with craniosynostosis and radioulnar synostosis.

BACKGROUND: SMAD6 encodes an intracellular inhibitor of the bone morphogenetic protein (BMP) signalling pathway. Until now, rare heterozygous loss-of-function variants in SMAD6 were demonstrated to increase the risk of disparate clinical disorders including cardiovascular disease, craniosynostosis and radioulnar synostosis. Only two unrelated patients harbouring biallelic SMAD6 variants presenting a complex cardiovascular phenotype and facial dysmorphism have been described. CASES: Here, we present the first two patients with craniosynostosis harbouring homozygous SMAD6 variants. The male probands, both born to healthy consanguineous parents, were diagnosed with metopic synostosis and bilateral or unilateral radioulnar synostosis. Additionally, one proband had global developmental delay. Echocardiographic evaluation did not reveal cardiac or outflow tract abnormalities. MOLECULAR ANALYSES: The novel missense (c.[584T>G];[584T>G], p.[(Val195Gly)];[(Val195Gly)]) and missense/splice-site variant (c.[817G>A];[817G>A], r.[(817g>a,817delins[a;817+2_817+228])];[(817g>a,817delins[a;817+2_817+228])], p.[(Glu273Lys,Glu273Serfs*72)];[(Glu273Lys,Glu273Serfs*72)]) both locate in the functional MH1 domain of the protein and have not been reported in gnomAD database. Functional analyses of the variants showed reduced inhibition of BMP signalling or abnormal splicing, respectively, consistent with a hypomorphic mechanism of action. CONCLUSION: Our data expand the spectrum of variants and phenotypic spectrum associated with homozygous variants of SMAD6 to include craniosynostosis.

miR-92a-3p-inspired shRNA exhibits pro-chondrogenic and chondrocyte protective effects in osteoarthritis treatment through targeting SMAD6/7.

INTRODUCTION: Osteoarthritis (OA) compromises patients' quality of life and requires further study. Although miR-92a-3p was reported to possess chondroprotective effects, the underlying mechanism requires further clarification. The objectives of this study were to elucidate the mechanism by which miR-92a-3p alleviates OA and to examine the efficacy of shRNA-92a-3p, which was designed based on mature miR-92a-3p. MATERIALS AND METHODS: TargetScan and luciferase reporter assay were used to predict the target of miR-92a-3p. Adipose-derived stem cells (ADSCs) were transfected with miR-92a-3p/miR-NC mimic for the analysis of chondrogenic biomarkers and SMAD proteins. ADSCs and osteoarthritic chondrocytes were transduced with shRNA-92a-3p for the analysis of chondrogenic biomarkers and SMAD proteins. OA was surgically induced in C57BL/6JJcl mice, and ADSCs with/without shRNA-92a-3p transduction were intra-articularly injected for the assessment of cartilage damage. RESULTS: SMAD6 and SMAD7 were predicted as direct targets of miR-92a-3p by TargetScan and luciferase reporter assay. Transfection of the miR-92a-3p mimic resulted in a decrease in SMAD6 and SMAD7 levels and an increase in phospho-SMAD2/3, phospho-SMAD1/5/9, SOX9, collagen type II, and aggrecan levels in ADSCs. Furthermore, shRNA-92a-3p decreased SMAD6 and SMAD7 levels, and increased phospho-SMAD2/3, phospho-SMAD1/5/9, SOX9, collagen type II, and aggrecan levels in ADSCs and osteoarthritic chondrocytes. Additionally, ADSC-shRNA-92a-3p-EVs reduced the rate of decrease of SOX9, collagen type II, and aggrecan in osteoarthritic chondrocytes. In mice with surgically induced OA, shRNA-92a-3p-treated ADSCs alleviated cartilage damage more effectively than nontreated ADSCs. CONCLUSIONS: miR-92a-3p and shRNA-92a-3p exhibit therapeutic effects in treating OA by targeting SMAD6 and SMAD7, thereby enhancing TGF-ß signaling.

Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development.

BMP signaling is crucial to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here, we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo. At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. Mechanistically, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial cell junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a 'Goldilocks' pathway in vascular biology that requires a certain signaling amplitude, regulated by SMAD6, to function properly.

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

SMAD6 inhibits granulosa cell proliferation and follicle growth rate in carrier and noncarrier heifers of the Trio allele.

In brief: Follicle selection is a key event in monovular species. In this manuscript, we demonstrate the role of SMAD6 in promoting decreased granulosa cell proliferation and follicle growth rate in carriers vs noncarriers of the Trio allele and after vs before follicle deviation. Abstract: Cattle are generally considered a monovular species; however, recently, a bovine high fecundity allele, termed the Trio allele, was discovered. Carriers of Trio have an elevated ovulation rate (3-5), while half-sibling noncarriers are monovular. Carriers of the Trio allele have overexpression in granulosa cells of SMAD6, an inhibitor of oocyte-derived regulators of granulosa cell proliferation and differentiation. In experiment 1, follicle size was tracked for each follicle during a follicular wave. Follicle growth rate was greater before vs after follicle deviation in both carriers and noncarriers. Additionally, follicle growth rate was consistently less in carriers vs noncarriers. In experiment 2, we collected granulosa cells from follicles before and after deviation for evaluation of granulosa cell gene expression. Granulosa cell proliferation was less in carriers vs noncarriers and after vs before follicle deviation (decreased expression of cell cycle genes CCNB1 and CCNA2). The decreased granulosa cell proliferation in noncarriers after deviation was associated with increased SMAD6 expression. Similarly, in experiment 3, decreased expression of SMAD6 in granulosa cells of noncarriers cultured in vitro for 60 h was associated with increased expression of cell cycle genes. This suggests that SMAD6 may not just be inhibiting follicle growth rate in carriers of Trio but may also play a role in the decreased follicle growth after deviation in noncarriers. The hypotheses were supported that (1) follicle growth and granulosa cell proliferation decrease after deviation in both carriers and noncarriers and that (2) granulosa cell proliferation is reduced in carriers compared to noncarriers.

miR-20a-5p regulated SMAD6 to inhibit chondrogenesis of hDPSCs.

OBJECTIVES: Chondrogenic differentiation of human dental pulp stem cells (hDPSCs) is highly promising for cartilage repair. The specific mechanism, however, still needs to be explicated. MATERIALS AND METHODS: In this study, we isolated hDPSCs and transfected cells with lentiviruses containing an over-expression, knock-down, or negative control of miR-20a-5p. Three-D pellet cultures of hDPSCs were used for the chondrogenic induction. Following the pellet culture period, chondrogenesis was assessed by histological and immunohistochemical analysis and expression of chondrogenic-related genes. Dual-luciferase report assay was performed to determine potential targeted genes of miR-20a-5p, and the phosphorylation levels of P65 and IκBα were explored. Animal experiments were performed to determine the effect of miR-20a-5p on cartilage regeneration. RESULTS: miR-20a-5p was showed to repress the expression of SMAD6 to inhibit the chondrogenic differentiation of hDPSCs. Accordingly, the knock-down of miR-20a-5p promoted cartilage regeneration in the osteochondral defects of rats. Mechanically, it is indicated that NF-κB signaling is the potential down-stream network of miR-20a-5p/Smad6 crosstalk during chondrogenic differentiation. CONCLUSIONS: miR-20a-5p could target SMAD6 to activate NF-κB signaling pathway, and thus inhibit chondrogenesis of hDPSCs, which provided promising therapeutic target for cartilage defects clinically.

Down-regulation of hsa-circ-0107593 promotes osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

OBJECTIVES: Increasing evidence indicated circRNAs were involved in stem cells osteogenesis differentiation. Herein, we aimed to clarify the role of hsa-circ-0107593 during the osteogenesis process of human adipose-derived stem cells (hADSCs) and the underlying mechanisms. METHODS: The ring structure of hsa-circ-0107593 was confirmed using RNase R treatment and Sanger sequencing. Nucleoplasmic separation and fluorescence in situ hybridization detected hsa-circ-0107593 distribution. Lentivirus and siRNA were used to modulate the expression of hsa-circ-0107593, and the binding relationship between hsa-circ-0107593 and miR-20a-5p was verified by luciferase assay and RNA immunoprecipitation. We detected the osteogenic activity of hADSCs through alkaline phosphatase staining, alizarin red S staining, real-time polymerase chain reaction (RT-PCR), western blot, and cellular immunofluorescence experiment. In vivo, micro-computed tomography was performed to analyze bone formation around skull defect. RESULTS: RT-PCR results exhibited that hsa-circ-0107593 was downregulated while miR-20a-5p was upregulated during hADSCs osteogenesis. In vivo and in vitro experiments results indicated that knocking down hsa-circ-0107593 promoted the osteogenic differentiation of hADSCs, while overexpression of hsa-circ-0107593 showed an inhibitory effect on hADSCs osteogenic differentiation. In vitro experiment results showed hsa-circ-0107593 acted as a hADSCs osteogenic differentiation negative factor for it inhibited the suppressing effect of miR-20a-5p on SMAD6. CONCLUSION: Knocking down hsa-circ-0107593 acts as a positive factor of the osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

Ceramide synthase 3 affects invasion and metastasis of hepatocellular carcinoma via the SMAD6 gene. / ç¥žç»é °èƒºåˆæˆé ¶3通过SMAD6åŸºå› å½±å“è‚ç»†èƒžç™Œçš„ä¾µè¢­å’Œè½¬ç§».

OBJECTIVES: Patients with hepatocellular carcinoma (HCC) have poor prognosis due to lack of early diagnosis and effective treatment. Therefore, there is an urgent need to better understand the molecular mechanisms associated with HCC and to identify effective targets for early diagnosis and treatment. This study is to explore the expression and biological role of ceramide synthase 3 (CerS3) in HCC. METHODS: A total of 159 pairs of HCC tissues and adjacent non-tumor tissues were obtained from the patients underwent radical resection in Shenzhen People's Hospital, and the total RNA and proteins from HCC tissues and adjacent non-tumor tissues were obtained. The expression of CerS3 protein and mRNA in HCC was detected by immunohistochemistry, Western blotting and real-time PCR. In vitro experiments, Hep3B cells were divided into a control vector group and a CerS3 vector group, and the cells were transfected with retroviral vector containing control cDNA or CerS3 cDNA, respectively. HCCLM3 cells were divided into a normal control shRNA group and a CerS3 shRNA group, and the cells were transfected with lentiviral vectors containing normal control shRNA or CerS3 shRNA, respectively. MTT, EdU, Transwell and scratch method were used to detect cell proliferation, migration and invasion. RNA sequencing was performed to determine the downstream signal of CerS3. RESULTS: Compared with the corresponding adjacent tissues,the mRNA and protein levels of CerS3 were elevated in the HCC tissues, with significant difference (both P<0.05). The Univariate and multivariate analysis showed that the overall survival rate was significantly correlated with the presence of venous invasion (95% CI 1.8-9.2, P<0.01), TNM stage (95% CI 2.3-5.2, P<0.05), poor histological grade (95% CI 1.4-6.8, P<0.05), and CerS3 (95% CI 1.5-3.9, P<0.05). Furthermore, the high CerS3 expression levels in tumor tissues were significantly associated with shorter overall survival rates compared with the low CerS3 expression (P<0.05). Compared with the vector control group, the Hep3B cell viability, EdU positive cells, and migration and invasion cell numbers in the CerS3 vector group were significantly increased (all P<0.05). Compared with the shRNA normal control group, the HCCLM3 cell viability, EdU positive cells, and numbers of migrating and invasive cells in the CerS3 shRNA group were significantly lower (all P<0.05). The RNA sequencing confirmed that the small mothers against decapentaplegic family member 6 (SMAD6) gene as an oncogenic gene could promote the HCC metastasis. CONCLUSIONS: Clinically, the overexpression of CerS3 is closely related to poor clinical features and poor prognosis. Functionally, CerS3 participates in the proliferation, invasion and metastasis of liver cancer cells via activating SMAD6 gene.

Genetic variants in bone morphogenetic proteins signaling pathway might be involved in palatal rugae phenotype in humans.

This study investigated, if genetic variants in BMP2, BMP4 and SMAD6 are associated with variations in the palatal rugae pattern in humans. Dental casts and genomic DNA from 75 patients were evaluated. Each patient was classified as follows: total amount of rugae; bilateral symmetry in the amount, length and shape of the palatal rugae; presence of secondary or fragmentary palatal rugae; presence of unifications; predominant shape; and predominant direction of the palatal rugae. The genetic variants in BMP2 (rs1005464 and rs235768), BMP4 (rs17563) and SMAD6 (rs2119261 and rs3934908) were genotyped. Genotype distribution was compared between palatal rugae patterns using the chi-square test (alpha = 0.05). The allele A was associated with the presence of secondary or fragmentary rugae for rs1005464 (OR = 2.5, 95%CI 1.1-6.3; p = 0.014). Secondary or fragmentary rugae were associated with the G allele in rs17563 (OR = 2.1, 95%CI 1.1-3.9; p = 0.017). rs17563 was also associated with rugae unification (p = 0.017 in the additive model). The predominant shape (wavy) was associated with rs2119261 (p = 0.023 in the additive model). The left-right symmetry of the length of primary rugae was associated with rs3934908 in the recessive model (OR = 3.6, 95%CI 1.2-11.7; p = 0.025). In conclusion, genetic variants in the BMP pathway impacted on palatal rugae pattern.

Identification, molecular evolution, and expression analysis of the transcription factor Smad gene family in lamprey.

Transcription factor small mothers against decapentaplegic (Smad) family SMAD proteins are the essential intracellular signal mediators and transcription factors for transforming growth factor ß (TGF-ß) signal transduction pathway, which usually exert pleiotropic actions on cell physiology, including immune response, cell migration and differentiation. In this study, the Smad family was identified in the most primitive vertebrates through the investigation of the transcriptome data of lampreys. The topology of phylogenetic tree showed that the four Smads (Smad1, Smad3, Smad4 and Smad6) in lampreys were subdivided into four different groups. Meanwhile, homology analysis indicated that most Smads were conserved with typical Mad Homology (MH) 1 and MH2 domains. In addition, Lethenteron reissneri Smads (Lr-Smads) adopted general Smads folding structure and had high tertiary structural similarity with human Smads (H-Smads). Genomic synteny analysis revealed that the large-scale duplication blocks were not found in lamprey genome and neighbor genes of lamprey Smads presented dramatic differences compared with jawed vertebrates. Importantly, quantitative real-time PCR analysis demonstrated that Smads were widely expressed in lamprey, and the expression level of Lr-Smads mRNA was up-regulated with different pathogenic stimulations. Moreover, depending on the weighted gene co-expression network analysis (WGCNA), four Lr-Smads were identified as two meaningful modules (green and gray). The functional analysis of these two modules showed that they might have a correlation with ployI:C. And these genes presented strong positive correlation during the immune response from the results of Pearson's correlation analysis. In conclusion, our results would not only enrich the information of Smad family in jawless vertebrates, but also lay the foundation for immunity in further study.

Hsa-miR-186-5p regulates TGFß signaling pathway through expression suppression of SMAD6 and SMAD7 genes in colorectal cancer.

TGFß signaling is a known pathway to be involved in colorectal cancer (CRC) progression and miRNAs play crucial roles by regulating different components of this pathway. Hence, finding the link between miRNAs and the pathway could be beneficial for CRC therapy. Array data indicated that miR-186-5p is a differentially expressed miRNA in colorectal Tumor/Normal tissues and bioinformatics tools predicted SMAD6/7 (inhibitory SMADs) as bona fide targets of this miRNA. Here, we intended to investigate the regulatory effect of the miR-186-5p expression on TGFß signaling in CRC. Firstly, the miR-186-5p overexpression in HCT116 cells resulted in a significant reduction of SMAD6/7 expression, measured through RT-qPCR. Then, the direct interactions of miR-186-5p with SMAD6/7 3'UTRs were supported through dual luciferase assay. Furthermore, miR-186-5p overexpression suppressed proliferation, cell viability, and migration while, it increased apoptosis in CRC cells, assessed by cell cycle, MTT, scratch and Annexin V/PI apoptosis assays. Consistently, miR-186-5p overexpression resulted in reduced CyclinD1 protein using western blot, and also resulted in increased P21 and decreased c-Myc expression. Overall, these results introduced miR-186-5p as a cell cycle suppressor through downregulation of SMAD6/7 expression. Thus, miR-186-5p might be served as a novel tumor suppressive biomarker and therapeutic target in CRC treatment.

A high level of lncFGD5-AS1 inhibits epithelial-to-Mesenchymal transition by regulating the miR-196a-5p/SMAD6/BMP axis in gastric Cancer.

BACKGROUND: Long non-coding RNA (lncRNA) was a vital factor in the progression and initiation of human cancers. This study found a new lncRNA, FGD5-AS1, which can inhibit EMT process, proliferation, and metastasis in vitro and in vivo. METHODS: qRT-PCR was employed to test the expression of lncFGD5-AS1 in 30 gastric cancer patients' cancer tissue and para-cancer tissue. Overexpressed lncFGD5-AS1 cells shown sharply decrease of proliferation, migration, and epithelial-mesenchymal transition (EMT). miR-196a-5p/SMAD6 was confirmed as downstream molecular mechanism of lncFGD5-AS1 by expression correlation analysis and mechanism experiments. In vivo study illustrated overexpression of lncFGD5-AS1 suppression tumor growth. RESULTS: LncFGD5-AS1 served as a ceRNA of miR-196a-5p to release its inhibition on SMAD6, a conventional inhibitor on the BMP pathway. Comparing with normal gastric cancer cells, FGD5-AS1 overexpressed group had fewer migration cells, lower cell viability, and lower EMT transformation rate. Meanwhile, xenografts nude mice injecting with overexpressed-FGD5-AS1 cells also shown smaller tumor weight and volume. CONCLUSION: In conclusion, this research supported the first evidence that FGD5-AS1 suppressed proliferation and metastasis in gastric cancer by regulating miR-196a-5p/SMAD6/BMP axis and suggested a potential therapeutic candidate for gastric cancer.

Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling.

Bone morphogenetic proteins (BMPs) secreted by a variety of cell types are known to play essential roles in cell differentiation and matrix formation in the bone, cartilage, muscle, blood vessel, and neuronal tissue. BMPs activate intracellular effectors via C-terminal phosphorylation of Smad1, Smad5, and Smad9, which relay the signaling by forming a complex with Smad4 and translocate to the nucleus for transcriptional activation. Smad6 inhibits BMP signaling through diverse mechanisms operative at the membrane, cytosolic, and nuclear levels. However, the mechanistic underpinnings of Smad6 functional diversity remain unclear. Here, using a biochemical approach and cell differentiation systems, we report a cytosolic mechanism of action for Smad6 that requires arginine methylation at arginine 81 (R81) and functions through association with Smad1 and interference with the formation of Smad1-Smad4 complexes. By mutating the methylated arginine residue, R81, and by silencing the expression of protein arginine methyltransferase 1, we show that protein arginine methyltransferase 1 catalyzes R81 methylation of Smad6 upon BMP treatment, R81 methylation subsequently facilitates Smad6 interaction with the phosphorylated active Smad1, and R81 methylation facilitates Smad6-mediated interruption of Smad1-Smad4 complex formation and nuclear translocation. Furthermore, Smad6 WT but not the methylation-deficient R81A mutant inhibited BMP-responsive transcription, attenuated BMP-mediated osteogenic differentiation, and antagonized BMP-mediated inhibition of cell invasion. Taken together, our results suggest that R81 methylation plays an essential role in Smad6-mediated inhibition of BMP responses.

SMAD6 transduces endothelial cell flow responses required for blood vessel homeostasis.

Fluid shear stress provided by blood flow instigates a transition from active blood vessel network expansion during development, to vascular homeostasis and quiescence that is important for mature blood vessel function. Here we show that SMAD6 is required for endothelial cell flow-mediated responses leading to maintenance of vascular homeostasis. Concomitant manipulation of the mechanosensor Notch1 pathway and SMAD6 expression levels revealed that SMAD6 functions downstream of ligand-induced Notch signaling and transcription regulation. Mechanistically, full-length SMAD6 protein was needed to rescue Notch loss-induced flow misalignment. Endothelial cells depleted for SMAD6 had defective barrier function accompanied by upregulation of proliferation-associated genes and down regulation of junction-associated genes. The vascular protocadherin PCDH12 was upregulated by SMAD6 and required for proper flow-mediated endothelial cell alignment, placing it downstream of SMAD6. Thus, SMAD6 is a required transducer of flow-mediated signaling inputs downstream of Notch1 and upstream of PCDH12, as vessels transition from an angiogenic phenotype to maintenance of a homeostatic phenotype.

Nuclear IL-33/SMAD signaling axis promotes cancer development in chronic inflammation.

Interleukin (IL)-33 cytokine plays a critical role in allergic diseases and cancer. IL-33 also has a nuclear localization signal. However, the nuclear function of IL-33 and its impact on cancer is unknown. Here, we demonstrate that nuclear IL-33-mediated activation of SMAD signaling pathway in epithelial cells is essential for cancer development in chronic inflammation. Using RNA and ChIP sequencing, we found that nuclear IL-33 repressed the expression of an inhibitory SMAD, Smad6, by interacting with its transcription factor, RUNX2. IL-33 was highly expressed in the skin and pancreatic epithelial cells in chronic inflammation, leading to a markedly repressed Smad6 expression as well as dramatically upregulated p-SMAD2/3 and p-SMAD1/5 in the epithelial cells. Blocking TGF-ß/SMAD signaling attenuated the IL-33-induced cell proliferation in vitro and inhibited IL-33-dependent epidermal hyperplasia and skin cancer development in vivo. IL-33 and SMAD signaling were upregulated in human skin cancer, pancreatitis, and pancreatitis-associated pancreatic cancer. Collectively, our findings reveal that nuclear IL-33/SMAD signaling is a cell-autonomous tumor-promoting axis in chronic inflammation, which can be targeted by small-molecule inhibitors for cancer treatment and prevention.

Bone morphogenetic protein 9 (BMP9) directly induces Notch effector molecule Hes1 through the SMAD signaling pathway in osteoblasts.

Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix-loop-helix domain, is a well-known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9-mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and ChIP assays revealed that two Smad-binding sites in the 5' upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.

Structural basis for inhibitory effects of Smad7 on TGF-ß family signaling.

Smad6 and Smad7 are classified as inhibitory Smads (I-Smads). They are crucial in the fine-tuning of signals by cytokines of the transforming growth factor-ß (TGF-ß) family. They are negative feedback regulators and principally target the activated type I receptors as well as the activated Smad complexes, but with distinct specificities. Smad7 inhibits Smad signaling from all seven type I receptors of the TGF-ß family, whereas Smad6 preferentially inhibits Smad signaling from the bone morphogenetic protein (BMP) type I receptors, BMPR1A and BMPR1B. The target specificities are attributed to the C-terminal MH2 domain. Notably, Smad7 utilizes two alternative molecular surfaces for its inhibitory function against type I receptors. One is a basic groove composed of the first α-helix and the L3 loop, a structure that is shared with Smad6 and receptor-regulated Smads (R-Smads). The other is a three-finger-like structure (consisting of residues 331-361, 379-387, and the L3 loop) that is unique to Smad7. The underlying structural basis remains to be elucidated in detail. Here, we report the crystal structure of the MH2 domain of mouse Smad7 at 1.9 Å resolution. The three-finger-like structure is stabilized by a network of hydrogen bonds between residues 331-361 and 379-387, thus forming a molecular surface unique to Smad7. Furthermore, we discuss how Smad7 antagonizes the activated Smad complexes composed of R-Smad and Smad4, a common partner Smad.

HNF1B inhibits cell proliferation via repression of SMAD6 expression in prostate cancer.

Prostate cancer is the most common malignancy in men in developed countries. In previous study, we identified HNF1B (Hepatocyte Nuclear Factor 1ß) as a downstream effector of Enhancer of zeste homolog 2 (EZH2). HNF1B suppresses EZH2-mediated migration of two prostate cancer cell lines via represses the EMT process by inhibiting SLUG expression. Besides, HNF1B expression inhibits cell proliferation through unknown mechanisms. Here, we demonstrated that HNF1B inhibited the proliferation rate of prostate cancer cells. Overexpression of HNF1B in prostate cancer cells led to the arrest of G1 cell cycle and decreased Cyclin D1 expression. In addition, we re-explored data from ChIP-sequencing (ChIP-seq) and RNA-sequencing (RNA-seq), and demonstrated that HNF1B repressed Cyclin D1 via direct suppression of SMAD6 expression. We also identified CDKN2A as a HNF1B-interacting protein that would contribute to HNF1B-mediated repression of SMAD6 expression. In summary, we provide the novel mechanisms and evidence in support HNF1B as a tumour suppressor gene for prostate cancer.

MiR-186 promotes the apoptosis of glioma U87 cells by down-regulating the expression of Smad6.

OBJECTIVE: MiRNA family gene is an evolutionarily conserved non-coding small RNA that directly participates in a variety of physiological processes and cancer development via regulating gene expression in the biological level of transcription. To research the specific mechanism by which miR-186 regulates apoptosis within gliomas. PATIENTS AND METHODS: RT-qPCR was performed to verify the transcriptional level of miR-186 within glioma tissues and glioma cells. miRanda and Dual-Luciferase assay were performed to predict and confirm that Smad6 gene is an effective target of miR-186 within glioma. The expression of Smad6 protein was tested by Western blot following cell effective transfection. Apoptosis of gliomas was analyzed by inverted fluorescence microscopy and flow cytometry. RESULTS: The mRNA level of miR-186 was suppressed within glioma tissues and glioma U87 cells. MiR-186 is associated with apoptosis in glioma. Overexpression of miR-186 promoted U87 cell apoptosis, whereas suppression of miR-186 had the opposite effect. Besides, miR-186 directly targeted Smad6 and suppress its expression in glioma. The expression of Smad6 affected the regulation of miR-186 on glioma cell apoptosis, restoration of Smad6 rescued apoptosis of glioma U87 cells induced by miR-186 mimics, whereas inhibition of Smad6 promoted apoptosis. CONCLUSIONS: As noted above, miR-186 exerts a tumor-suppressing effect by targeting Smad6. We propose that miR-186 can be used as a novel biomarker for glioma diagnosis in the future, or as a new pharmacy target in the cure of gliomas.