Dissection of inhibitory Smad proteins: both N- and C-terminal domains are necessary for full activities of Xenopus Smad6 and Smad7

Smad6 and Smad7 comprise a subclass of vertebrate Smads that antagonize, rather than transduce, TGF-β family signaling. These Anti-Smads can block BMP signaling, as evidenced by their ability to induce a secondary dorsal axis when misexpressed ventrally in Xenopus embryos. Smad7 inhibits additional TGF-β related pathways, and causes spina bifida when misexpressed dorsally. We have performed structure-function analyses to identify domains of Anti-Smads that are responsible for their shared and unique activities. We find that the C-terminal domain of Smad7 displays strong axis inducing activity but cannot induce spina bifida. The isolated N-terminal domain of Smad7 is inactive but restores the ability of the C-terminus to cause spina bifida when the two are co-expressed. By contrast, the N- and C-terminal domains of Smad6 have weak axis inducing activity when expressed individually, but show full activity when co-expressed. Chimeric analysis demonstrates that the C-terminal domain of Smad7, but not Smad6, can induce spina bifida when fused to the N-terminal domain of either Smad6 or Smad7. Thus, although the C-terminal domain is the primary determinant of the intrinsic activity of Xenopus Anti-Smads, the N-terminal domain is essential for full activity, is interchangeable between Smad6 and 7, and can function in trans.     

Smad7 Inhibits Mesoderm Formation and Promotes Neural Cell Fate inXenopusEmbryos

We report the isolation and characterization of a new inhibitory Smad inXenopus,which we have designated asXenopusSmad7. Smad7 is present at fairly constant levels throughout early development and at blastula stages enriched in the ventral side of the animal hemisphere. The induction of mesoderm by TGF-β-like signals is mediated by receptor ALK-4 and we show that Smad7 blocks signaling of ALK-4 in a graded fashion: lower levels of Smad7 block activation of dorsal mesoderm genes and higher levels block all mesoderm genes expression. Smad7 is able to directly activate neural markers in explants in the absence of mesoderm or endoderm. This neural-inducing activity of Smad7 may be due to inhibition of BMP-4 signaling because Smad7 can also block BMP-4-mediated mesoderm induction. Thus, Smad7 acts as a potent inhibitor of mesoderm formation and also activates the default neural induction pathway.     

Smad7 Protein Interacts with Receptor-regulated Smads (R-Smads) to Inhibit Transforming Growth Factor-β (TGF-β)/Smad Signaling

TGF-β is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes. TGF-β signaling is spatiotemporally fine-tuned. As a key negative regulator of TGF-β signaling, Smad7 exerts its inhibitory effects by blocking receptor activity, inducing receptor degradation or interfering with Smad-DNA binding. However, the functions and the molecular mechanisms underlying the actions of Smad7 in TGF-β signaling are still not fully understood. In this study we report a novel mechanism whereby Smad7 antagonizes TGF-β signaling at the Smad level. Smad7 oligomerized with R-Smad proteins upon TGF-β signaling and directly inhibited R-Smad activity, as assessed by Gal4-luciferase reporter assays. Mechanistically, Smad7 competes with Smad4 to associate with R-Smads and recruits the E3 ubiquitin ligase NEDD4L to activated R-Smads, leading to their polyubiquitination and proteasomal degradation. Similar to the R-Smad-Smad4 oligomerization, the interaction between R-Smads and Smad7 is mediated by their mad homology 2 (MH2) domains. A positive-charged basic region including the L3/β8 loop-strand module and adjacent amino acids in the MH2 domain of Smad7 is essential for the interaction. These results shed new light on the regulation of TGF-β signaling by Smad7.     

Study of interaction between Smad7 and DNA by single-molecule force spectroscopy

Smad7 is an antagonist of TGF-β 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.     

Expressions of inhibitory Smads, Smad6 and Smad7, are differentially regulated by TPA in human lung fibroblast cells

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 (G?6983), but not by another (G?6976). 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 G?6983, but not by G?6976. Additionally, G?6983 but not G?6976 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.     

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process     

Establishment of murine Smad5 double knockout ES cells and the studies on their properties

Smad5 is an intracellular transducer of TGF-β signals. Targeted disruption of murineSmad5 gene resulted in embryonic lethal. To study the function ofSmad5 in organgenesis, we generatedSmad5 double knockout ES cells by homologous recombination. We deleted theneo gene of theSmad5 targeted ES cells using Cre-LoxP system.Smad5 double knockout ES cells were obtained by transfecting the targeted ES cells using the same targeting construct. The results of chimeric study showed thatSmad5 might play an important role during the development of heart and neural tube.Smad5 double knockout ES cells formed teratoma when injected subcutaneously into nude mice. They differentiated into several types of cells, including neural cells, muscle cells, chondrocytes, endothelial cells and glandaceous cells.Smad5 double knockout ES cells are useful for studying the function ofSmad5 mediated TGF-β during the organgenesis and thein vitro differentiation of ES cells.     

Smad7 deficiency decreases iron and haemoglobin through hepcidin up‐regulation by multilayer compensatory mechanisms

To maintain iron homoeostasis, the iron regulatory hormone hepcidin is tightly controlled by BMP‐Smad signalling pathway, but the physiological role of Smad7 in hepcidin regulation remains elusive. We generated and characterized hepatocyte‐specific Smad7 knockout mice (Smad7^Alb/Alb), which showed decreased serum iron, tissue iron, haemoglobin concentration, up‐regulated hepcidin and increased phosphor‐Smad1/5/8 levels in both isolated primary hepatocytes and liver tissues. Increased levels of hepcidin lead to reduced expression of intestinal ferroportin and mild iron deficiency anaemia. Interestingly, we found no difference in hepcidin expression or phosphor‐Smad1/5/8 levels between iron‐challenged Smad7^Alb/Alb and Smad7^flox/flox, suggesting other factors assume the role of iron‐induced hepcidin regulation in Smad7 deletion. We performed RNA‐seq to identify differentially expressed genes in the liver. Significantly up‐regulated genes were then mapped to pathways, revealing TGF‐β signalling as one of the most relevant pathways, including the up‐regulated genes Smad6, Bambi and Fst (Follistatin). We found that Smad6 and Bambi—but not Follistatin—are controlled by the iron‐BMP–Smad pathway. Overexpressing Smad6, Bambi or Follistatin in cells significantly reduced hepcidin expression. Smad7 functions as a key regulator of iron homoeostasis by negatively controlling hepcidin expression, and Smad6 and Smad7 have non‐redundant roles. Smad6, Bambi and Follistatin serve as additional inhibitors of hepcidin in the liver.     

Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.     

Smurf1 regulates the inhibitory activity of Smad7 by targeting Smad7 to the plasma membrane

Smad ubiquitin regulatory factor 1 (Smurf1), a HECT-type E3 ubiquitin ligase, interacts with inhibitory Smad7 and induces cytoplasmic localization of Smad7. Smurf1 then associates with transforming growth factor-beta type I receptor (TbetaR-I) and enhances the turnover of this receptor. However, the mechanisms of the nuclear export and plasma membrane localization of the Smurf1.Smad7 complex have not been elucidated. We show here that Smurf1 targets Smad7 to the plasma membrane through its N-terminal conserved 2 (C2) domain. Both wild-type Smurf1 (Smurf1(WT)) and Smurf1 lacking the C2 domain (Smurf1(deltaC2)) bound to Smad7 and translocated nuclear Smad7 to the cytoplasm. However, unlike Smurf1(WT), Smurf1(deltaC2) did not move to the plasma membrane and failed to recruit Smad7 to the cell surface TbetaR-II.TbetaR-I complex. Moreover, although Smurf1(deltaC2) induced ubiquitination of Smad7, it failed to induce the ubiquitination and degradation of TbetaR-I and did not enhance the inhibitory activity of Smad7. Thus, these results suggest that the plasma membrane localization of Smad7 by Smurf1 requires the C2 domain of Smurf1 and is essential for the inhibitory effect of Smad7 in the transforming growth factor-beta signaling pathway.     

Establishment of murine Smad5 double knockout ES cells and the studies on their properties

Smad5 is an intracellular transducer of TGF-β signals. Targeted disruption of murine Smad5 gene resulted in embryonic lethal. To study the function of Smad5 in organgenesis, we generated Smad5 double knockout ES cells by homologous recombination. We deleted the neo gene of the Smad5 targeted ES cells using Cre-LoxP system. Smad5 double knockout ES cells were obtained by transfecting the targeted ES cells using the same targeting construct. The results of chimeric study showed that Smad5 might play an important role during the development of heart and neural tube. Smad5 double knockout ES cells formed teratoma when injected subcutaneously into nude mice. They differentiated into several types of cells, including neural cells, muscle cells, chondrocytes, endothelial cells and glandaceous cells. Smad5 double knockout ES cells are useful for studying the function of Smad5 mediated TGF- β during the organgenesis and the in vitro differentiation of ES cells.     

Itch E3 Ubiquitin Ligase Positively Regulates TGF-β Signaling to EMT via Smad7 Ubiquitination

TGF-β regulates pleiotropic cellular responses including cell growth, differentiation, migration, apoptosis, extracellular matrix production, and many other biological processes. Although non-Smad signaling pathways are being increasingly reported to play many roles in TGF-β-mediated biological processes, Smads, especially receptor-regulated Smads (R-Smads), still play a central mediatory role in TGF-β signaling for epithelial-mesenchymal transition. Thus, the biological activities of R-Smads are tightly regulated at multiple points. Inhibitory Smad (I-Smad also called Smad7) acts as a critical endogenous negative feedback regulator of Smad-signaling pathways by inhibiting R-Smad phosphorylation and by inducing activated type I TGF-β receptor degradation. Roles played by Smad7 in health and disease are being increasingly reported, but the molecular mechanisms that regulate Smad7 are not well understood. In this study, we show that E3 ubiquitin ligase Itch acts as a positive regulator of TGF-β signaling and of subsequent EMT-related gene expression. Interestingly, the Itch-mediated positive regulation of TGF-β signaling was found to be dependent on Smad7 ubiquitination and its subsequent degradation. Further study revealed Itch acts as an E3 ubiquitin ligase for Smad7 polyubiquitination, and thus, that Itch is an important regulator of Smad7 activity and a positive regulator of TGF-β signaling and of TGF-β-mediated biological processes. Accordingly, the study uncovers a novel regulatory mechanism whereby Smad7 is controlled by Itch.     

Suppression of ADAM8 attenuates angiotensin II-induced cardiac fibrosis and endothelial-mesenchymal transition via inhibiting TGF-β1/Smad2/Smad3 pathways

Endothelial-to-mesenchymal transition (EndMT) is involved in cardiac fibrosis induced by angiotensin II (Ang II). A disintegrin and metalloproteinase 8 (ADAM8), a member of ADAMs family, participates in cell adhesion, proteolysis and various signaling. However, its effects on the development of cardiac fibrosis remain completely unknown. This study aimed to reveal whether ADAM8 aggravates cardiac fibrosis induced by Ang II in vivo and in vitro. The C57BL/6J mice or cardiac endothelial cells were subjected to Ang II infusion to induce fibrosis. The results showed that systolic blood pressure and diastolic blood pressure were significantly increased under Ang II infusion, and ADAM8 was up-regulated. ADAM8 inhibition attenuated Ang II-induced cardiac dysfunction. ADAM8 knockdown suppressed Ang II-induced cardiac fibrosis as evidenced by the down-regulation of CTGF, collagen I, and collagen III. In addition, the endothelial marker (VE-cadherin) was decreased, whilst mesenchymal markers (α-SMA and FSP1) were increased following Ang II infusion. However, ADAM8 repression inhibited Ang II-induced EndMT. Moreover, ADAM8 silencing repressed the activation of TGF-β1/Smad2/Smad3 pathways. Consistent with the results in vivo, we also found the inhibitory effects of ADAM8 inhibition on EndMT in vitro. All data suggest that ADAM8 promotes Ang II-induced cardiac fibrosis and EndMT via activating TGF-β1/Smad2/Smad3 pathways.     

p38 Mitogen-Activated Protein Kinase Affects Transforming Growth Factor-β/Smad Signaling in Human Dental Pulp Cells

Transforming Growth Factor-β (TGF-β) plays an essential role in differentiation of dental pulp cells into odontoblasts during reparative dentine formation. However, the mechanism by which TGF-β stimulates dental repair remains rather obscure. Human dental pulp cells were used as an in vitro model in the present work. We showed that TGF-β signaled through mitogen-activated protein kinases (MAPKs), such as ERK1/2 and p38, along with Smad pathway. Distinct pathways exerted different time response. SB203580, a specific p38 MAPK inhibitor, reduced phosphorylation of Smad3, while it slightly enhanced phosphorylation of Smad2. Increased phosphorylation of ERK1/2 and p38 confirmed that SB203580 did not block activation of TGF-β receptors. In addition, the inhibition of ERK1/2 activity with MEK1/2 inhibitor U0126 increased TGF-β mediated phosphorylation of Smad3. Our results suggest that p38 affects the phosphorylation of Smad2 and Smad3 differentially during TGF-β signaling in human dental pulp cells and ERK1/2 might be involved in the process.     

Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.     

支架蛋白GIT2通过与Smad3相互作用调节其转录活性

G蛋白偶联受体激酶相互作用蛋白2(G protein-coupled receptor kinase interacting proteins 2,GIT2)是一种信号支架蛋白,可募集多种信号通路的关键分子,参与肌动蛋白细胞骨架组装、整合素介导的细胞粘附、G蛋白偶联受体的内化及胞内信号传递等生物学过程. 采用酵母双杂交实验证明,TGF-β1信号通路的转录因子Smad3是GIT2的相互作用蛋白质,内、外源免疫共沉淀实验均证实,GIT2与Smad3存在蛋白质相互作用. 报告基因实验及免疫印迹结果表明,GIT2增加Smad3的转录活性并增强TGF-β1诱导的Smad3的磷酸化.研究还发现,Git2-/-小鼠骨髓间充质干细胞(MSC)的Smad3磷酸化受到抑制,其骨形成相关靶基因的表达水平也低于Git2+/+小鼠. 本研究表明,GIT2通过与Smad3的相互作用调节其转录活性并活化TGF-β1信号通路,可能参与调节骨髓间充质干细胞的分化.