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.

MiR-664-3p suppresses osteoblast differentiation and impairs bone formation via targeting Smad4 and Osterix.

Osteoporosis is a metabolic disorder characterized by low bone mass and deteriorated microarchitecture, with an increased risk of fracture. Some miRNAs have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass. Our miRNA sequencing results showed that miR-664-3p was significantly down-regulated during the osteogenic differentiation of the preosteoblast MC3T3-E1 cells. However, whether miR-664-3p has an impact on bone homeostasis remains unknown. In this study, we identified overexpression of miR-664-3p inhibited the osteoblast activity and matrix mineralization in vitro. Osteoblastic miR-664-3p transgenic mice exhibited reduced bone mass due to suppressed osteoblast function. Target prediction analysis and experimental validation confirmed Smad4 and Osterix (Osx) are the direct targets of miR-664-3p. Furthermore, specific inhibition of miR-664-3p by subperiosteal injection with miR-664-3p antagomir protected against ovariectomy-induced bone loss. In addition, miR-664-3p expression was markedly higher in the serum from patients with osteoporosis compared to that from normal subjects. Taken together, this study revealed that miR-664-3p suppressed osteogenesis and bone formation via targeting Smad4 and Osx. It also highlights the potential of miR-664-3p as a novel diagnostic and therapeutic target for osteoporotic patients.

Structural based screening of potential inhibitors of SMAD4: a step towards personalized medicine for gall bladder and other associated cancers.

Gall bladder cancer (GBC) is an aggressive and most common malignancy of biliary tract lacking effective treatment due to unavailability of suitable biomarkers and therapeutics. SMAD4 is an essential mediator of transforming growth factor-ß pathway involved in various cellular processes like growth, differentiation and apoptosis and also recognized as therapeutic target for GBC and other gastrointestinal tract cancers. In the present study, 3D structure of SMAD4 mutants was optimized through molecular dynamics simulation (MDS) along with wildtype. Furthermore, binding site of protein was predicted through hybrid approach and structural based virtual screening against two drug libraries was performed followed by docking. MDS of top docking score protein-ligand complexes were carried, and binding free energy was rescored. Two potential inhibitors, namely ZINC2098840 and ZINC8789167, were screened that displayed higher binding affinity towards mutant proteins compared with wildtype and both hydrophilic as well as hydrophobic interactions play a crucial role during protein-ligand binding. Current study identified novel and potent inhibitors of SMAD4 mutant that could be used as a drug candidate for the development of personalized medicine for gall bladder and other associated cancers.

Effects of suppressing Smads expression on wound healing in Hyriopsis cumingii.

As a specific pearl mussel in China, Hyriopsis cumingii has enormous economic value. However, the organism damage caused by pearl insertion is immeasurable. TGF-ß/Smad signal transduction pathways are involved in all phases of wound healing. We have previously reported on two cytoplasmic signal transduction factors, Smad3 and Smad5 in mussel H. cumingii (named HcSmads), suggesting their involvements in wound healing. Here, Smad4 was cloned and described. The full length cDNA of HcSmad4 was 2543 bp encoded 515 amino acids. Deduced HcSmad4 protein possessed conserved MH1 and MH2 domains, nuclear location signals (NLS), nuclear exput signals (NES) and Smad activation domain (SAD). Transcripts of Smad3, 4 and 5 were constitutively expressed in all detected tissues, at highest levels in muscles. Furthermore, HcSmad4 mRNA levels were significantly increased at incision site post wounding, and expression of downstream target genes of Smad4, such as HcMMP1, HcMMP19, HcTIMP1 and HcTIMP2 were upregulated to a certain extent. Whatever knocked down HcSmad3/4 or treated by specific inhibitors of Smad 3 (SIS3), expression levels of these genes displayed a significantly downregulated tendency compared with the wound group. In addition, histological evaluation suggested that Smad3 knockdown or SIS3 treatment was accelerated wound healing, and then Smad4 knockdown delayed the process of wound healing in mussels. These data implicate that Smad3/4 play an important role in tissue repair in mollusks.

SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. METHODS: The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4iΔEC). RESULTS: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. CONCLUSIONS: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

The BMP4-Smad signaling pathway regulates hyperandrogenism development in a female mouse model.

Polycystic ovary syndrome is a common endocrine disorder and a major cause of anovulatory sterility in women at reproductive age. Most patients with polycystic ovary syndrome have hyperandrogenism, caused by excess androgen synthesis. Bone morphogenetic protein 4 (BMP4) is an essential regulator of embryonic development and organ formation, and recent studies have also shown that BMP4 may be involved in female steroidogenesis process. However, the effect of BMP4 on hyperandrogenism remains unknown. Here, using a female mouse model of hyperandrogenism, we found that ovarian BMP4 levels were significantly decreased in hyperandrogenism. Elevated androgens inhibited BMP4 expression via activation of androgen receptors. Moreover, BMP4 treatment suppressed androgen synthesis in theca cells and promoted estrogen production in granulosa cells by regulating the expression of steroidogenic enzymes, including CYP11A, HSD3B2, CYP17A1, and CYP19A1 Consistently, knockdown of BMP4 augmented androgen levels and inhibited estrogen levels. Mechanistically, Smad signaling rather than the p38 MAPK pathway regulated androgen and estrogen formation, thereby mediating the effect of BMP4. Of note, BMP4-transgenic mice were protected against hyperandrogenism. Our observations clarify a vital role of BMP4 in controlling sex hormone levels and offer new insights into intervention for managing hyperandrogenism by targeting the BMP4-Smad signaling pathway.

TGF-ß1 Promotes Hepatocellular Carcinoma Invasion and Metastasis via ERK Pathway-Mediated FGFR4 Expression.

BACKGROUND/AIMS: TGF-ß1 is beneficial during early liver disease but is tumor-progressive during late stages especially for hepatocellular carcinoma (HCC). Thus, exploring the underlying mechanisms may provide information about a potentially therapeutic role of TGF-ß1 in HCC. METHODS: Western blot and real-time quantitative PCR were used to quantify FGFR4 expression in HCC cell lines and a normal liver cell line. After constructing the best silencing FGFR4 expression vector, migration and invasiveness of TGF-ß1 in HCC was studied in vitro and in vivo. Western blot was used to study the mechanism of TGF-ß1 induction on FGFR4 expression with various inhibitors. RESULTS: HepG2 cell lines had the most FGFR4 expression, and data show that silencing FGFR4 suppressed cell proliferation, invasion and migration in HCC induced by TGF-ß1 in vitro and in vivo. Moreover, TGF-ß1 induced FGFR4 expression through the ERK pathway. CONCLUSION: Promoting FGFR4 expression via the ERK pathway, TGF-ß1 contributes to HCC invasion and metastasis.

Tif1γ is essential for the terminal differentiation of mammary alveolar epithelial cells and for lactation through SMAD4 inhibition.

Transforming growth factor ß (TGFß) is widely recognised as an important factor that regulates many steps of normal mammary gland (MG) development, including branching morphogenesis, functional differentiation and involution. Tif1γ has previously been reported to temporally and spatially control TGFß signalling during early vertebrate development by exerting negative effects over SMAD4 availability. To evaluate the contribution of Tif1 γ to MG development, we developed a Cre/LoxP system to specifically invalidate the Tif1g gene in mammary epithelial cells in vivo. Tif1g-null mammary gland development appeared to be normal and no defects were observed during the lifespan of virgin mice. However, a lactation defect was observed in mammary glands of Tif1g-null mice. We demonstrate that Tif1 γ is essential for the terminal differentiation of alveolar epithelial cells at the end of pregnancy and to ensure lactation. Tif1 γ appears to play a crucial role in the crosstalk between TGFß and prolactin pathways by negatively regulating both PRL receptor expression and STAT5 phosphorylation, thereby impairing the subsequent transactivation of PRL target genes. Using HC11 cells as a model, we demonstrate that the effects of Tif1g knockdown on lactation depend on both SMAD4 and TGFß. Interestingly, we found that the Tif1γ expression pattern in mammary epithelial cells is almost symmetrically opposite to that described for TGFß. We propose that Tif1γ contributes to the repression of TGFß activity during late pregnancy and prevents lactation by inhibiting SMAD4.

A comprehensive transcriptomic view on the role of SMAD4 gene by RNAi-mediated knockdown in porcine follicular granulosa cells.

As a key mediator of the transforming growth factor-beta (TGF-ß) signaling pathway, which plays a pivotal role in regulating mammalian reproductive performance, Sma- and Mad-related protein 4 (SMAD4) is closely associated with the development of ovarian follicular. However, current knowledge of the genome-wide view on the role of SMAD4 gene in mammalian follicular granulosa cells (GCs) is still largely unknown. In the present study, RNA-Seq was performed to investigate the effects of SMAD4 knockdown by RNA interference (SMAD4-siRNA) in porcine follicular GCs. A total of 1025 differentially expressed genes (DEGs), including 530 upregulated genes and 495 downregulated genes, were identified in SMAD4-siRNA treated GCs compared with that treated with NC-siRNA. Furthermore, functional enrichment analysis indicated that upregulated DEGs in SMAD4-siRNA treated cells were mainly enriched in cell-cycle related processes, interferon signaling pathway, and immune system process, while downregulated DEGs in SMAD4-siRNA treated cells were mainly involved in extracellular matrix organization/disassembly, pathogenesis, and cell adhesion. In particular, cell cycle and TGF-ß signaling pathway were discovered as the canonical pathways changed under SMAD4-silencing. Taken together, our data reveals SMAD4 knockdown alters the expression of numerous genes involved in key biological processes of the development of follicular GCs and provides a novel global clue of the role of SMAD4 gene in porcine follicular GCs, thus improving our understanding of regulatory mechanisms of SMAD4 gene in follicular development.

AMP-activated protein kinase (AMPK) activation inhibits nuclear translocation of Smad4 in mesangial cells and diabetic kidneys.

Diabetic nephropathy is characterized by diffuse mesangial matrix expansion and is largely dependent on the TGF-ß/Smad signaling pathway. Smad4 is required for TGF-ß signaling; however, its regulation has not been well characterized in diabetic kidney disease. Here, we report that high glucose is sufficient to stimulate nuclear translocation of Smad4 in mesangial cells and that stimulation of the major energy sensor AMP-activated protein kinase (AMPK) has a potent effect to block Smad4 nuclear translocation. Activation of AMPK by 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) inhibited high glucose-induced and TGF-ß stimulation of nuclear Smad4. To identify which of the catalytic α-subunits may be involved, small interfering (si) RNA-based inhibition of AMPK α1- or α2-subunit was employed. Inhibition of either subunit reduced overall AMPK activity and contributed to Smad4 nuclear accumulation. In an animal model of early diabetic kidney disease, induction of diabetes was found to markedly stimulate Smad4 protein levels and enhance nuclear accumulation. AMPK activation with AICAR completely prevented the upregulation of Smad4 and reduced mesangial matrix accumulation. We conclude that stimulation of Smad4 in cell culture and in in vivo models of early diabetic kidney disease is dependent on AMPK.

Nerve growth factor exposure promotes tubular epithelial-mesenchymal transition via TGF-ß1 signaling activation.

Clinical studies showed that renal expression and serum levels of nerve growth factor (NGF) are increased in renal diseases characterized by progressive fibrosis, a pathologic process in which TGF-ß1 mediates most of the key events leading to tubular epithelial-mesenchymal transition (EMT). However, the pathogenic role of high NGF levels has not yet been elucidated. In this study, we found that in tubular renal cells, HK-2, NGF transcriptionally up-regulated TGF-ß1 expression and secretion and enhanced cell motility by activating EMT markers via its receptors, TrkA and p75(NTR). Interestingly, we observed that TGF-ß1-SMAD pathway activation and the up-regulation of EMT markers NGF-induced were both prevented when knockdown of TGF-ß1 gene occurred and that the pretreatment with an antibody anti-NGF reversed the nuclear translocation of pSMAD3/SMAD4 complex. Collectively, our results demonstrated that NGF promotes renal fibrosis via TGF-ß1-signaling activation, suggesting that in kidney diseases high NGF serum levels could contribute to worsen renal fibrosis.

Smad2 and Smad3 cooperate and antagonize simultaneously in vertebrate neurogenesis.

The transforming growth factor beta (TGF-ß) pathway plays key roles in development and cancer. TGF-ß signaling converges on the Smad2 and Smad3 effectors, which can either cooperate or antagonize to regulate their transcriptional targets. Here we performed in vivo and in silico experiments to study how such cooperativity and antagonism might function during neurogenesis. In vivo electroporation experiments in the chick embryo neural tube show that Smad2 and Smad3 cooperate to promote neurogenesis, as well as the transcription of Smad3-specific targets. Knockdown of Smad2 enhances neurogenesis and the transcription of Smad3-specific targets. A mathematical model of the TGF-ß pathway fits the experimental results and predicts that the proportions of the three different trimeric complexes formed dictates the transcriptional responses of the R-Smad proteins. As such, Smad2 targets are activated solely by the Smad2-Smad2-Smad4 complex, whereas Smad3 targets are activated both by Smad2-Smad3-Smad4 and Smad3-Smad3-Smad4 trimers. We have modeled the Smad responses onto arbitrary genes and propose that this mechanism might be extended to additional activities of TGF-ß in development and disease.

Evidence supporting a functional requirement of SMAD4 for bovine preimplantation embryonic development: a potential link to embryotrophic actions of follistatin.

Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-ß signalling pathway.

The transforming growth factor-ß (TGF-ß) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-ß signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-ß transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3'-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-ß to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-ß signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4.

MiR-17-92 cluster regulates cell proliferation and collagen synthesis by targeting TGFB pathway in mouse palatal mesenchymal cells.

Elongation and elevation of palatal shelves, mainly caused by proliferation and extra-cellular matrix synthesis of palatal mesenchymal cells (PMCs), are essential for normal palatal development. Transforming growth factor beta (TGFB) pathway could induce proliferation inhibition and collagen synthesis in PMCs. Recent studies found that miRNA-17-92 (miR-17-92) cluster, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, expressed in the 1st bronchial arch of mouse embryos during the period of palatal shelf elongation and elevation, and directly targeted TGFB pathway in cancer cell lines. Whether miR-17-92 cluster expresses and targets TGFB pathway in PMCs has not yet been studied. Using quantitative real-time RT-PCR, we found that miR-17-92 expressed in PMCs and decreased from embryonic day (E) 12 to E14 in palatal shelves. MTT assay and Western blot showed that miR-17-92 inhibited TGFB1 induced proliferation inhibition and collagen synthesis in PMCs by decreasing TGFBR2, SMAD2, and SMAD4 protein level. Further luciferase assay showed that miR-17 and miR-20a directly targeted 3'UTR of TGFBR2, and that miR-18a directly targeted 3'UTR of SMAD2 and SMAD4. We thus conclude that miR-17-92 cluster could inhibit TGFB pathway induced proliferation inhibition and collagen synthesis in PMCs by directly targeting TGFBR2, SMAD2, and SMAD4.

TGF-ß1 induces proteinase-activated receptor 2 (PAR2) expression in endometriotic stromal cells and stimulates PAR2 activation-induced secretion of IL-6.

BACKGROUND: Proteinase-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is activated by several serine proteases. PAR2 activation in endometriotic stromal cells (ESCs) has been implicated in the development of endometriosis but the regulatory mechanism of PAR2 expression in ESC is unknown. Our objective was to study the mechanism by which PAR2 expression may be regulated in endometriotic lesions. METHODS: Primary cultures of ESCs were treated with transforming growth factor-ß (TGF-ß) 1, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and the expression of PAR2 was examined by real-time quantitative PCR. ESCs pretreated with or without TGF-ß1 were treated with PAR2 agonist peptide (PAR2AP) and the secretion of the pro-endometriotic cytokine, IL-6, was measured using a specific enzyme-linked immunosorbent assay. Effects of TGF-ß type 1 inhibitor, SB431542, and PAR2 small interfering RNA (siRNA) on the TGF-ß1 stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion were also evaluated. To study intracellular signaling, effects of inhibitors of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K) and of Smad4 siRNA on the TGF-ß1-induced PAR2 gene expression were studied. RESULTS: Only TGF-ß1, but neither TNF-α nor IL-1ß, increased gene expression of PAR2. Activation of PAR2 with PAR2AP increased the secretion of IL-6 from ESCs. As expected, TGF-ß1 pretreatment dose-dependently enhanced the PAR2AP-induced increase in IL-6 secretion from ESCs. Treatment of ESCs with the TGF-ß type 1 inhibitor, SB431542, inhibited both TGF-ß1-stimulation of PAR2 gene expression and PAR2AP-induced IL-6 secretion. Transfection of ESCs with PAR2 siRNA produced a similar inhibition of IL-6 secretion. The TGF-ß1-induced increase in PAR2 gene expression was repressed by inhibition of p38 MAPK, p42/44 MAPK or PI3K, but not by knockdown of Smad4 expression. CONCLUSIONS: In view of significant roles of PAR2 and IL-6 in endometriosis, the TGF-ß1-induced increase in PAR2 expression may be an elaborate mechanism that augments the progression of the disease.

TGF-beta1 reduces Wilms' tumor suppressor gene expression in podocytes.

BACKGROUND: Wilms' tumor suppressor gene (WT1) is essential for normal podocyte function, and transforming growth factor (TGF)-beta contributes to focal segmental glomerulosclerosis (FSGS). We aimed to address whether TGF-beta affects WT1 expression in podocytes. METHODS: A human podocyte cell line treated with TGF-beta1 and kidneys in Alb/TGF-beta1-transgenic mice were analyzed for WT1 expression. RESULTS: In cultured podocytes, TGF-beta1 reduced WT1 protein expression determined by western blotting beginning at 8 h and decreased WT1 messenger RNA (mRNA) expression measured by quantitative reverse transcription-polymerase chain reaction beginning at 3 h. Knockdown of Smad4 by small hairpin (sh) RNA partially rescued the TGF-beta1-induced reduction of both WT1 protein and mRNA expressions in the cultured podocytes. TGF-beta1 did not alter luciferase activity of the reporter construct for a human WT1 promoter but reduced that for a human WT1 5' enhancer construct, suggesting that TGF-beta1 may regulate WT1 expression by altering the 5' enhancer activity. In the transgenic mice, WT1 protein expression in podocytes was decreased at 1 and 3 weeks of age, while glomeruloclerosis developed after 3 weeks. CONCLUSION: TGF-beta1 reduces WT1 expression in cultured human podocytes and podocytes in mice before overt glomerulosclerosis begins. The effects are at least partially Smad4 dependent. Our findings identify a novel pathway linking TGF-beta1 to podocyte injury and FSGS. The WT1 reduction may be a useful marker for early podocyte injury.

Smad4 loss is associated with fewer S100A8-positive monocytes in colorectal tumors and attenuated response to S100A8 in colorectal and pancreatic cancer cells.

S100A8 and its dimerization partner S100A9 are emerging as important chemokines in cancer. We previously reported that Smad4-negative pancreatic tumors contain fewer stromal S100A8-positive monocytes than their Smad4-positive counterparts. Here, we studied S100A8/A9-expressing cells in colorectal tumors relating their presence to clinicopathological parameters and Smad4 status. Two-dimensional gel electrophoresis (n = 12) revealed variation in the levels of S100A8 protein in colorectal cancer tumors, whereas immunohistochemical analysis (n = 313) showed variation in the numbers of stromal S100A8-positive and S100A9-positive cells. Loss of Smad4 expression was observed in 42/304 (14%) colorectal tumors and was associated with reduced numbers of S100A8-positive (P = 0.03) but not S100A9-positive stromal cells (P = 0.26). High S100A9 cell counts were associated with large tumor sizes (P = 0.0006) and poor differentiation grade (P = 0.036). However, neither S100A8 nor S100A9 cell counts predicted poor survival, except for patients with Smad4-negative tumors (P = 0.02). To address the impact of environmental S100A8/A9 chemokines on tumor cells, we examined the effects of exogenously added S100A8 and S100A9 proteins on cellular migration and proliferation of colorectal and pancreatic cancer cells. S100A8 and S100A9 enhanced migration and proliferation in Smad4-positive and Smad4-negative cancer cells. However, transient depletion of Smad4 resulted in loss of responsiveness to exogenous S100A8, but not S100A9. S100A8 and S100A9 activated Smad4 signaling as evidenced by phosphorylation of Smad2/3; blockade of the receptor for the advanced glycation end products inhibited this response. In conclusion, Smad4 loss alters the tumor's interaction with stromal myeloid cells and the tumor cells' response to the stromal chemokine, S100A8.

Non-virus-mediated transfer of siRNAs against Runx2 and Smad4 inhibit heterotopic ossification in rats.

Heterotopic ossification of muscles, tendons and ligaments are a common problem affecting patient with trauma or received elective surgery. But the existing preventive or therapeutic methods all have disadvantages. Runt-related protein 2 (Runx2) and Smad4 are two regulators that have important roles in the differentiation of osteoblast. In this study, we attempted to examine the effect of Runx2 and Smad4 on the development of heterotopic ossification in vitro. We constructed non-virus-containing small interference RNAs (siRNAs) against Runx2 and Smad4 and tested it with reverse transcriptase-PCR and western blot. We then analyzed the independent effect of Runx2- and Smad4-specific siRNAs and their cooperative effect on the formation of heterotopic ossification induced by trauma in rats. The effects were measured with computed tomography scanning, hematoxylin and eosin staining and immunohistochemistry. We found that the Runx2- and Smad4-specific siRNAs inhibited the expression of Runx2 and Smad4 at the level of messenger RNA and protein. Runx2 and Smad4 independently inhibited the formation of heterotopic ossification. Moreover, their co-expression significantly enhanced the inhibition of heterotopic ossification compared with the independent effect. We suggest that gene therapy to inhibit Runx2 and Smad4 by RNAi could be a powerful approach to prevent or treat heterotopic ossification.

SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV.

Pancreatic ductal adenocarcinoma (PDAC) represents the eighth frequent solid tumor and fourth leading cause of cancer death. Because current treatments against PDAC are still unsatisfactory, new anticancer strategies are required, including oncolytic viruses. Among these, autonomous parvoviruses (PV), like MVMp (minute virus of mice) and H-1PV are being explored as candidates for cancer gene therapy. Human PDAC cell lines were identified to display various susceptibilities to an infection with H-1PV. The correlation between the integrity of the transcription factor SMAD4, mutated in 50% of all PDAC, and H-1PV permissiveness was particularly striking. Indeed, mutation or deletion of SMAD4 dramatically reduced the activity of the P4 promoter and, consequently, the accumulation of the pivotal NS1 protein. By means of DNA affinity immunoblotting, novel binding sites for SMAD4 and c-JUN transcription factors could be identified in the P4 promoter of H-1PV. The overexpression of wild-type SMAD4 in deficient cell lines (AsPC-1, Capan-1) stimulated the activity of the P4 promoter, whereas interference of endogenous SMAD4 function with a dominant-negative mutant decreased the viral promoter activity in wild-type SMAD4-expressing cells (Panc-1, MiaPaCa-2) reducing progeny virus production. In conclusion, the importance of members of the SMAD family for H-1PV early promoter P4 activity should guide us to select SMAD4-positive PDACs, which may be possible targets for an H-1PV-based cancer therapy.