Search

Background Nephronophthisis (NPHP) is a kind of ciliopathy. Interstitial fibrosis occurs at the early stage of the disease. TGF-β/Smad is a key signaling pathway in regulating interstitial fibrosis and epithelial-mesenchymal transition (EMT). In this study, we explored the activation of the TGF-β/Smad signaling pathway and EMT in NPHP1-defective MDCK cells to further understand the pathogenesis of NPHP. Methods NPHP1-knockdown (NPHP1KD) MDCK cells were constructed by recombinant lentiviral short hairpin RNA, and NPHP1-knockout (NPHP1KO) MDCK cells were constructed by using the CRISPR/Cas9 technique. The morphology and migration ability were observed under a microscope. Western blotting was used to detect the expression of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), fibroblast-specific protein-1(FSP1), TGF-β1, Smad2, Smad3, p-Smad3, Smad4 and Smad7. The localization of Smad3 was determined by immunofluorescence assay. Results NPHP1KD and NPHP1KO MDCK cells were spindle-shaped and presented EMT-like changes. E-cadherin and β-catenin expression decreased, while α-SMA and FSP1 expression increased; the TGF-β/Smad signaling pathway was activated, Smad2, Smad3, p-Smad3 and Smad4 expression increased, Smad3 translocated to nuclear and Smad7 expression decreased compared with those in wild type MDCK cells. Overexpression of Smad7 reversed these changes to different degrees. Conclusions Our results indicate that NPHP1 defects induce the activation of the TGF-β/Smad signaling pathway and EMT in MDCK cells. These factors may be implicated in the pathogenesis of interstitial fibrosis in NPHP.

There has been an increasing number of researches about microRNAs (miRNAs) in the progression of liver fibrosis from the point of their comprehensive functions in regulating the activation of hepatic stellate cells (HSCs). Among them, it has been reported that miR-212 is up-regulated in activated rat primary HSCs. However, its mechanism has not been determined yet. Here, we confirmed that the level of miR-212-3p was up-regulated in livers of carbon tetrachloride (CCl4)-treated mice compared with the normal control, which is a classical model of chronically damaged fibrotic liver. In vitro, we demonstrated that TGF-β, a master fibrogenic cytokine, could induce the level of miR-212. In turn, overexpression of miR-212 could induce the activation marker of HSC including α-smooth muscle actin (α-SMA) and collagens by activating TGF-β signaling pathway. Furthermore, SMAD7, a dominant suppressor of TGF-β pathway, was identified as a direct target of miR-212-3p. Our results indicate that miR-212-3p facilitates the activation of HSCs and TGF-β pathway by targeting SMAD7, highlighting that it can be served as a novel biomarker or therapeutic target for liver fibrosis.

Metastasis is one of the most decisive factors influencing CRC patient prognosis and current studies suggest that a molecular mechanism known as EMT broadly regulates cancer metastasis. NR2F2 is a key molecule in the development of CRC, but the roles and underlying mechanisms of NR2F2 in TGF-β induced EMT in CRC remain largely unknown. In the current study, we were interested to examine the role of NR2F2 in the TGF-β-induced EMT in CRC. Here, we found NR2F2 was upregulated in CRC cells and promotes TGF-β-induced EMT in CRC. Using comparative miRNA profiling TGF-β pre-treated CRC cells in which NR2F2 had been knocked down with that of control cells, we identified miR-21 as a commonly downregulated miRNA in HT29 cells treated with TGF-β and NR2F2 siRNA, and its downregulation inhibiting migration and invasion of CRC cells. Moreover, we found NR2F2 could transcriptional activated miR-21 expression by binding to miR-21 promoter in HT29 by ChIP and luciferase assay. In the last, our data demonstrated that Smad7 was the direct target of miR-21 in CRC cells. Thus, NR2F2 could promote TGF-β-induced EMT and inhibit Smad7 expression via transactivation of miR-21, and NR2F2 may be a new common therapeutic target for CRC.

Adipocyte accumulation is associated with the development of obesity and obesity-related diseases. Interactions of master transcription factors and signaling cascades are required for adipogenesis. Regulation of excessive adipogenic processes may be an attractive therapeutic for treatment of obesity and obesity-related diseases. In this study, we found that atorvastatin exerts an anti-adipogenic activity in 3T3-L1 pre-adipocytes, and that this activity is elevated in combination with metformin. Expression of the adipogenic master regulators PPARγ and C/EBPα, and their target gene aP2, was suppressed by atorvastatin. Furthermore, atorvastatin treatment resulted in increased activation of the key master regulator of cellular energy homeostasis, AMPK. These biological activities of atorvastatin were elevated in combination with metformin. These anti-adipogenic activities were associated with regulation of the STAT3 and TGF-β signaling cascades, resulting in the regulation of the expression of STAT3 target genes, such as KLF5, p53, and cyclin D1, and TGF-β signaling inhibitory genes, such as SMAD7. Our results suggest that combination therapy with atorvastatin and metformin may have therapeutic potential for the treatment of obesity and obesity-related diseases caused by excessive adipogenesis.

Atherosclerosis is a complicated process comprising inflammation, accumulation of collagen matrix and aberrant DNA methylation. SMAD7 is known to play an important role in fibrosis and inflammation. In recent years, increasing research has concentrated on the connection between DNA methylation and atherosclerosis. The current study was designed to investigate methylation status of some specific gene with a focus on SMAD7 in atherosclerosis and elucidate their relationship. We found that SMAD7 expression was decreased and its promoter region was markedly methylated in atherosclerotic plaques when compared with normal artery walls. Using MALDI-TOF MS, increased DNA methylation levels of SMAD7 promoter at CpG unit 5.8.15.16 were found in peripheral blood of atherosclerosis patients relative to matched normal controls, respectively. Correlation analysis revealed that mean DNA methylation levels of SMAD7 promoter of CpG unit 5.8.15.16 were positively associated with homocysteine levels (r = 0.724, p

Groucho related genes encode transcriptional repressor proteins critical for normal developmental processes. The bone morphogenetic proteins belong to the transforming growth factor-β (TGF-β) superfamily and play important signaling roles in development and disease. However, the regulation of BMP signaling, especially within cells, is largely unknown. In this report, we show that expression of the Groucho related gene Grg4 robustly activates the expression of a BMP reporter gene, as well as enhancing and sustaining the upregulation of the endogenous Id1 gene induced by BMP7. BMP7 administration did not affect the endogenous level of Grg4 nor did it enhance the phosphorylation of receptor activated Smad proteins. Rather, Grg4 expression reduced the levels of the endogenous inhibitory Smad7, thus increasing the transcriptional responses mediated by BMP responsive sequences. The data point to a novel mechanisms for attenuating BMP signaling through altering the ratio of activating versus inhibitory Smad proteins.

Smad7, an inhibitory Smad, acts as a key regulator forming autoinhibitory feedback loop in transforming growth factor-beta (TGF-β) signaling. However, a growing body of evidences suggests that Smad7 is capable of apoptotic function. In the present study, we have demonstrated a proapoptotic function of Smad7 as a negative regulator of survival protein heme oxygenase-1 (HO-1). The HO-1 protein level was elevated in cisplatin-resistant A549 human lung cancer cells and blockade of HO-1 activation sensitized the cells to apoptosis. Interestingly, overexpression of Smad7 decreased HO-1 gene expression and its enzymatic activity. Notably, Smad7 reduced Akt activity and infection with adenovirus expressing a constitutively active form of the Akt reversed the inhibitory effects of Smad7 to HO-1, indicating a negative action mechanism of Smad7 to Akt-HO-1-linked survival pathway. Consistently, Smad7 sensitized A549 cells to cisplatin-induced apoptosis and these effects were dependent on HO-1 and Akt inhibition. Based on these findings, we suggest that targeting Smad7 may be an efficient strategy for overcoming drug-resistance in cancer therapy.

Motile cilia play crucial roles in the maintenance of homeostasis in vivo. Defects in the biosynthesis of cilia cause immotile cilia syndrome, also known as primary ciliary dyskinesia (PCD), which is associated with a variety of complex diseases. In this study, we found that inhibitory Smad proteins, Smad7 and Smad6, significantly promoted the differentiation of mouse embryonic stem (ES) cells into ciliated cells. Moreover, these Smad proteins specifically induced morphologically distinct Musashi1-positive ciliated cells. These results suggest that inhibitory Smad proteins could be important regulators not only for the regulation of ciliated cell differentiation, but also for the subtype specification of ciliated cells during differentiation from mouse ES cells.

This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 low and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96®Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 low, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3.

Transforming growth factors β (TGF-β) pathway has been proven to play important roles in oncogenesis and angiogenesis of gliomas. MiR-132 might be related to TGF-β signaling pathway and high miR-132 expression was reported to be a biomarker of poor prognosis in patients diagnosed with glioma. However, the expression regulation way involved in TGF-β pathway and clinical significance of miR-132 have not been investigated in glioma cells. Here we reported that the mRNA level of miR-132 and TGF-β concentration were both increased in patients with brain glioma. Correlation analysis revealed that TGF-β concentration was positively correlated with mRNA level of miR-132. In addition, the mRNA level of miR-132 was up-regulated by TGF-β in a concentration-dependent and time-dependent manner. Furthermore, we found that miR-132 was involved in modulation of the TGF-β signaling pathway and down-regulation of SMAD7 expression by directly targeting the SMAD7 3'-UTR. MiR-132 was negatively correlated with SMAD7 in patients with brain glioma. Taken together, our results suggest that miR-132 could be stimulated by TGF-β and might enhance the activation of TGF-β signaling through inhibiting SMAD7 expression in glioma cells. These findings contribute to a better understanding of the mechanism of the activation of TGF-β signaling by miR-132.

Chondrogenesis is a critical step in palatogenesis. All-trans retinoic acid (atRA), a vitamin A derivative, is a known teratogenic effector of cleft palate. Here, we evaluated the effects of atRA on the osteo-/chondrogenic differentiation of mouse embryonic palate mesenchymal (MEPM) cells. MEPM cells, in a high-density micromass environment, undergo active chondrogenesis in a manner analogous to that of limb-derived mesenchymal cells, and served as a valid model system to investigate the mechanisms regulating chondrogenesis during palatogenesis. atRA-treated MEPM micromass expressed relatively higher levels of osteoblastic gene markers (alkaline phosphatase and collagen type I) and lower levels of chondrocytic gene markers (collagen type II and aggrecan). As transforming growth factor-beta3 (TGF-beta3) is an essential growth factor for chondrogenesis of embryonic mesenchymal cells both in in vivo and in vitro conditions, we thereby explored the effects of atRA on TGF-beta3 signaling pathway. atRA led to an increase in mRNA expression of TGF-beta3 and an instantaneous decrease in TGF-beta type II receptor (TbetaRII) as determined by real-time RT-PCR. Further study showed that atRA inhibited phosphorylation of Smad2 and Smad3 and increased Smad7 expression. Activation of the Smad pathways by transfection with Smad7deltaC mutant or constitutively active TbetaRII retroviral vector abolished atRA-induced inhibition of chondrogenesis as indicated by Alcian blue staining, indicating that Smad signaling is essential for this response. Taken together, these data for the first time demonstrated a role for RA-induced hypochondrogenesis through regulation of the TGF-beta3 pathway and suggested a role for TbetaRII /Smad in retinoid-induced cleft palate.

Smad6 and Smad7 are inhibitory Smads (I-Smads) with differential inhibitory effects on the regulation of the cellular signalings induced by TGF-beta superfamily. Here, we show that phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) down-regulates Smad6 mRNA expression and up-regulates Smad7 mRNA expression in IMR-90, a human lung fibroblast cell line. These regulations of I-Smads by TPA were suppressed by one PKC inhibitor (Gö6983), but not by another (Gö6976). TPA treatment had little effect on the phosphorylation of novel PKCs (PKCdelta and PKCepsilon), but specifically induced PKCmu phosphorylation, and this effect was inhibited by Gö6983, but not by Gö6976. Additionally, Gö6983 but not Gö6976 inhibited ERK- and JNK-phosphorylation as well as Smad7 promoter activity induced by TPA. MEK inhibitor U0126 inhibited the down-regulation of Smad6 mRNA expression but not the up-regulation of Smad7 mRNA expression. In contrast, JNK inhibitor SP600125 had no such effects. Luciferase reporter analysis revealed that TPA did not induce NF-kappaB activation. In addition, TPA up-regulated Smad7 expression in the presence of NF-kappaB inhibitor TLCK. These findings indicate that TPA down-regulates Smad6 expression presumably via PKCmu-ERK-dependent pathway and up-regulates Smad7 expression via PKCmu-dependent mechanism(s) which need no MAPK and NF-kappaB activation.

Bone morphogenetic proteins (BMPs) are shown to potentiate NGF-induced neuronal differentiation in PC12 phaeochromocytoma cells grown on collagen under low-serum conditions. Whereas, cell bodies remained rounded in control medium or with only BMPs present, addition of BMP4 or BMP6 robustly increased the neuritogenic effect of NGF within 2 days. NGF-increased phosphorylation of p44(Erk1) and p42(Erk2) between 2 and 24h was unaffected by addition of BMP6. PC12 cells transfected with the SBE(4x)-luc reporter showed that BMP4 significantly increased receptor-activated Smad activity. Expression of constitutively active BMP receptor ALK2 activating Smad1 and Smad5 resulted in a strong increase in the SBE(4x)-luc reporter response. Adding the inhibitory Smad7 drastically reduced this signal. In contrast to wild-type (wt) Smad5, a Smad5 variant lacking five Erk phosphorylation sites in the linker region (designated Smad5/5SA) showed a strong background transcriptional activity. A fusion construct (Gal4-Smad5/5SA) was also highly transcriptionally active. Addition of the MEK inhibitor U0126 to PC12 cells expressing Gal4-Smad5/wt did not increase background transcriptional activity. However, upon activation by constitutively active ALK2 both Gal4-Smad5/wt and Gal4-Smad5/5SA strongly stimulated transcription. The data show that serine residues of the linker region of Smad5 reduce spontaneous transcriptional activity and that NGF-activated Erk does not antagonise BMP signalling at this site. Hence, NGF and BMP signals are likely to interact further downstream at the transcriptional level in neuronal differentiation of the PC12 cells.

Smad7 transcription is known to be regulated by TGF-beta to form a negative-feedback loop of TGF-beta-mediated biological responses. In this study, we sought to determine whether other signaling cascades, especially mitogen-activated protein (MAP) kinases, might be involved in the transcriptional regulation of Smad7. Hyperosmolarity (500 mOsm/kg H(2)O) or anisomycin (10 microg/ml) potentiated TGF-beta-induced increases of Smad7 mRNA abundance in normal rat kidney fibroblasts. SB203580 (10 microM) treatment had no effect on basal and TGF-beta-induced Smad7 mRNA abundance, and the overexpression of kinase-negative ATF2 had no effect on Smad7 promoter activity. On the other hand, overexpression of dominant-negative JNK and dominant-negative c-Jun significantly attenuated the TGF-beta-induced increases of Smad7 mRNA abundance and promoter activity, respectively. Mutations of the AP-1 element near the Smad-binding element in the rat Smad7 promoter also completely abolished TGF-beta-induced Smad7 promoter activity. These results suggested that the JNK cascade, not p38 kinase, cooperated with the Smad signaling to induce Smad7 transcription through the AP-1 element. Serum treatment (10%) attenuated the TGF-beta-induced Smad7 mRNA increase, and PD98059 (30 microM) treatment increased the basal and TGF-beta-induced Smad7 promoter activity. Gel shift analysis revealed that serum treatment decreased the amount of nuclear Smad complex that PD98059 treatment was shown to restore. These results indicated that ERK activation negatively regulated Smad7 transcription possibly by inhibiting translocation of Smad complex to nuclei. In conclusion, JNK cascade and ERK cascade are important positive and negative regulators of Smad7 transcription, respectively.

TGFbeta is of crucial importance during transdifferentiation of resting retinoid-storing hepatic stellate cells (HSC) to extracellular matrix producing myofibroblasts (MFB) and consequently, inhibition of TGFbeta signal transduction is an effective means for preventing experimental fibrosis. We have shown that isolated HSC lose TGFbeta-dependent growth control during in vitro activation and that alpha2 (I) collagen production in transdifferentiated MFB is TGFbeta-independent. Furthermore, Smad complexes with SBE binding activity were only detected in early cultures of HSC, although TGFbeta receptor types I and II were significantly expressed in HSC and MFB. In the present report, we compared the expression pattern of TGFbeta downstream mediators, i.e., the Smads, in TGFbeta responsive HSC versus nonresponding MFB. The transdifferentiation process was monitored by morphology and increasing expression of TGFbeta and alpha-smooth muscle actin, and TGFbeta signaling was investigated by (CAGA)(9)-MLP-Luc. The expression level of all Smads remained essentially unchanged both during the activation process and after TGFbeta-treatment. Smad7 was transiently upregulated upon TGFbeta stimulation in quiescent HSC, indicating a negative feed back loop in responsive cells. In contrast, MFB neither displayed TGFbeta-inducible nor constitutively upregulated Smad7 expression. Instead, Smad3 mRNA was increased in MFB. Our data indicate that abrogation of the TGFbeta response in MFB versus HSC is not based on different regulation of Smad expression.

Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.

Smad7 is an antagonist of TGF-beta signaling pathway and the mechanism of its inhibitory effect is of great interest. We recently found that Smad7 could function in the nucleus by binding to the DNA elements containing the minimal Smad binding element CAGA box. In this work, we further applied single-molecule force spectroscopy to study the DNA-binding property of Smad7. Smad7 showed similar binding strength to the oligonucleotides corresponding to the CAGA-containing activin responsive element (ARE) and the PAI-1 promoter, as that of Smad4. However, Smad7 also exhibited a binding activity to the mutant ARE with the CAGA sequence substituted, indicating its DNA-binding specificity is different from other Smads. Moreover, we demonstrated that the MH2 domain of Smad7 had a higher binding affinity to the DNA elements than the full-length Smad7, while the N-terminal domain exhibited an inhibitory effect.

AIMP1 (also known as p43) is a factor associated with a macromolecular aminoacyl-tRNA synthetase (ARS) complex but also plays diverse regulatory roles in various physiological processes. Here, we report that AIMP1 negatively regulates TGF-beta signaling via stabilization of Smurf2. TGF-beta-dependent phosphorylation and nuclear localization of R-Smads, induction of target genes, and growth arrest were increased in AIMP1-deficient or -suppressed cells. In AIMP1-deficient or suppressed cells, the Smurf2 level was decreased. Various binding assays demonstrated the direction interaction of the C-terminal region of AIMP1 directly with the Smad7-binding region of Smurf2. The association of Smurf2 with Smad7 and its ubiquitination were inhibited by AIMP1, thereby protecting its autocatalytic degradation stimulated by Smad7. Thus, this work suggests the novel activity of AIMP1 as a component of negative feedback loop of TGF-beta signaling.

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.

Activated pancreatic stellate cells (PSCs) play major roles in promoting pancreatic fibrosis. We previously reported that angiotensin II (Ang II) enhances activated PSC proliferation through EGF receptor transactivation. In the present study, we elucidated a novel intracellular mechanism by which Ang II stimulates cellular proliferation. TGF-beta1 inhibits activated PSC proliferation via a Smad3 and Smad4-dependent pathway in an autocrine manner. We demonstrated that Ang II inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4. Furthermore, Ang II rapidly induced inhibitory Smad7 mRNA expression. Adenovirus-mediated Smad7 overexpression inhibited TGF-beta1-induced nuclear accumulation of Smad3 and Smad4, and potentiated activated PSC proliferation. PKC inhibitor Go6983 blocked the induction of Smad7 mRNA expression by Ang II. In addition, 12-O-tetradecanoyl-phorbol 13-acetate, a PKC activator, increased Smad7 mRNA expression. These results suggest that Ang II enhances activated PSC proliferation by blocking autocrine TGF-beta1-mediated growth inhibition by inducing Smad7 expression via a PKC-dependent pathway.

Activation of the transforming growth factor-beta (TGF-beta) system has been implicated in the pathological changes of diabetic nephropathy such as renal hypertrophy and accumulation of extracellular matrix. Streptozotocin-induced diabetic mice were used to examine whether the Smad pathway, which transduces the TGF-beta signal, is activated in the diabetic kidney, employing Southwestern histochemistry with labeled Smad-binding element (SBE) oligonucleotides and immunoblotting of nuclear protein extracts for Smad3. Mouse mesangial cells were used to study the role of Smads in mediating the effects of high glucose and TGF-beta on fibronectin expression, using transient transfections of Smad expression vectors and TGF-beta-responsive reporter assays. By Southwestern histochemistry, the binding of nuclear proteins to labeled SBE increased in both glomeruli and tubules at 1, 3, and 6 weeks of diabetes. Likewise, immunoblotting demonstrated that nuclear accumulation of Smad3 was increased in the kidney of diabetic mice. Both increases were prevented by insulin treatment. In mesangial cells, high glucose potentiated the effect of low-dose TGF-beta1 (0.2ng/ml) on the following TGF-beta-responsive constructs: 3TP-Lux (containing AP-1 sites and PAI-1 promoter), SBE4-Luc (containing four tandem repeats of SBE sequence), and the fibronectin promoter. Additionally, Smad3 overexpression increased fibronectin promoter activity, an effect that was enhanced by high ambient glucose or treatment with TGF-beta1 (2ng/ml). The TGF-beta-stimulated activity of the fibronectin promoter was prevented by transfection with either a dominant-negative Smad3 or the inhibitory Smad7. We conclude that hyperglycemia activates the intrarenal TGF-beta/Smad signaling pathway, which then promotes mesangial matrix gene expression in diabetic nephropathy.

Activation of TGF-beta superfamily receptors leads to phosphorylation of Smad proteins which function as transcription factors to regulate gene expression. Previous studies have indicated that Smad5, together with Smad1 and Smad8, participates in signaling downstream of BMP receptors. To characterize the DNA-binding characteristics of Smad5, we used the GST-Smad5 N-terminal fusion protein to select for random oligonucleotide sequences that were able to binds the protein. As a result, we found that Smad5 is able to bind a consensus sequence TGTGC. We further used the Smad7 promoter sequence that contains a Smad-binding element (SBE), GTCTAGAC to determine how mutations in each nucleotide in the SBE affects the binding with Smad5, compared with the binding with Smad1, Smad2, Smad3, Smad4, and Smad8. Interestingly, Smad5, but not Smad1 and Smad8, was able to bind the SBE, at a level similar to the binding by Smad3 and Smad4. However, mutations at the SBE had different effect on the binding with Smad5, compared to that with Smad3 and Smad4. These studies suggest that even though Smad5 falls into the same subfamily with Smad1 and Smad8 in mediating the signaling by BMP receptors, it has an unique DNA-binding property that is similar to Smad3, which specifically transduces signaling for TGF-beta and activin receptors.