Smad7 Binds Differently to Individual and Tandem WW3 and WW4 Domains of WWP2 Ubiquitin Ligase Isoforms.

WWP2 is an E3 ubiquitin ligase that differentially regulates the contextual tumour suppressor/progressor TGFß signalling pathway by alternate isoform expression. WWP2 isoforms select signal transducer Smad2/3 or inhibitor Smad7 substrates for degradation through different compositions of protein-protein interaction WW domains. The WW4 domain-containing WWP2-C induces Smad7 turnover in vivo and positively regulates the metastatic epithelial-mesenchymal transition programme. This activity and the overexpression of these isoforms in human cancers make them candidates for therapeutic intervention. Here, we use NMR spectroscopy to solve the solution structure of the WWP2 WW4 domain and observe the binding characteristics of Smad7 substrate peptide. We also reveal that WW4 has an enhanced affinity for a Smad7 peptide phosphorylated at serine 206 adjacent to the PPxY motif. Using the same approach, we show that the WW3 domain also binds Smad7 and has significantly enhanced Smad7 binding affinity when expressed in tandem with the WW4 domain. Furthermore, and relevant to these biophysical findings, we present evidence for a novel WWP2 isoform (WWP2C-ΔHECT) comprising WW3-WW4 tandem domains and a truncated HECT domain that can inhibit TGFß signalling pathway activity, providing a further layer of complexity and feedback to the WWP2 regulatory apparatus. Collectively, our data reveal a structural platform for Smad substrate selection by WWP2 isoform WW domains that may be significant in the context of WWP2 isoform switching linked to tumorigenesis.

Structure, molecular dynamics simulation, and docking studies of Dictyostelium discoideum and human STRAPs.

The Serine Threonine kinase Receptor Associated Protein (STRAP) is a WD40 containing protein that provides a platform for protein interactions during cell proliferation and development. Overexpression and misregulation of STRAP contributes to various carcinomas that are now recognized as therapeutic targets especially for colorectal and lung cancers. The present study was undertaken to find an effective drug against this molecule using a simple system like Dictyostelium discoideum; which shares close homology to humans. Using techniques like structural modeling, molecular dynamics (MD) simulation and molecular docking, we found similar structure and dynamic behaviors in both, except for the presence of dissimilar numbers of ß-sheets and loop segments. We identified a novel and potential drug targeted to STRAP. The results obtained allow us to use Dictyostelium as a model system for further in vivo studies. Finally, the results of protein-protein interactions using molecular docking and essential dynamics studies show STRAP to participate in TGF-ß signaling in humans. Further, we show some structural units that govern the interaction of TGFß-RI with STRAP and Smad7 proteins in TGF-ß signaling pathway. In conclusion, we propose that D. discoideum can be used for enhancing our knowledge about STRAP protein.

Total Flavonoids from Leaves of Carya Cathayensis Ameliorate Renal Fibrosis via the miR-21/Smad7 Signaling Pathway.

BACKGROUND/AIMS: Renal tubulointerstitial fibrosis is the most common pathway of progressive kidney injury, leading to end-stage renal disease. At present, no effective prophylactic treatment method is available. This study investigated the anti-fibrotic effects of total flavonoids (TFs) extracted from leaves of Carya Cathayensis in vivo and in vitro, and explored the underlying mechanisms. METHODS: Anti-fibrotic effects of TFs were measured using a mouse model of unilateral ureteral obstruction (UUO) and in transforming growth factor-ß1 (TGF-ß1)-treated mouse tubular epithelial cells (mTECs). mRNA expression and protein levels of Collagen I, Collagen III, and α-smooth muscle actin (α-SMA) were also tested by real-time reverse transcription PCR and western blot analysis. To elucidate the underlying mechanisms, expression of miR-21 was examined in mTECs treated with TFs using miR-21 mimics transfected into mTECs before TGF-ß1 and TFs treatment. Regulation of mothers against decapentaplegic homolog (Smad) signaling by miR-21 was subsequently validated via overexpression and deletion of miR-21 followed by a luciferase assay. RESULTS: TFs treatment attenuated renal fibrosis, and inhibited expression of collagens and α-SMA in the kidneys of mice subjected to UUO. In vitro, the TFs significantly decreased expression of fibrotic markers in TGF-ß1-treated mTECs. Moreover, TFs reduced miR-21 expression in a time- and dose-dependent manner in mTECs, increased expression of Smad7, and decreased phosphorylation of Smad3. Treatment with miR-21 mimics abolished the anti-fibrotic effects of the TFs on the TGF-ß1-treated mTECs. In addition, genetic deletion of miR-21 upregulated expression of Smad7 and suppressed phosphorylation of Smad3, attenuating renal fibrosis in mice. Bioinformatics predictions revealed the potential binding site of miR-21 in the 3'-untranslated region of Smad7, and this was further confirmed by the luciferase assay. CONCLUSION: TFs ameliorate renal fibrosis via a miR-21/Smad7 signaling pathway, indicating a potential therapy for the prevention of renal fibrosis.

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

TGF-ß is a pleiotropic cytokine that regulates a wide range of cellular actions and pathophysiological processes. TGF-ß signaling is spatiotemporally fine-tuned. As a key negative regulator of TGF-ß 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-ß signaling are still not fully understood. In this study we report a novel mechanism whereby Smad7 antagonizes TGF-ß signaling at the Smad level. Smad7 oligomerized with R-Smad proteins upon TGF-ß 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/ß8 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-ß signaling by Smad7.

Coupling of tandem Smad ubiquitination regulatory factor (Smurf) WW domains modulates target specificity.

Smad ubiquitination regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that participates in degradation of TGF-ß receptors and other targets. Smurf2 WW domains recognize PPXY (PY) motifs on ubiquitin ligase target proteins or on adapters, such as Smad7, that bind to E3 target proteins. We previously demonstrated that the isolated WW3 domain of Smurf2, but not the WW2 domain, can directly bind to a Smad7 PY motif. We show here that the WW2 augments this interaction by binding to the WW3 and making auxiliary contacts with the PY motif and a novel E/D-S/T-P motif, which is N-terminal to all Smad PY motifs. The WW2 likely enhances the selectivity of Smurf2 for the Smad proteins. NMR titrations confirm that Smad1 and Smad2 are bound by Smurf2 with the same coupled WW domain arrangement used to bind Smad7. The analogous WW domains in the short isoform of Smurf1 recognize the Smad7 PY peptide using the same coupled mechanism. However, a longer Smurf1 isoform, which has an additional 26 residues in the inter-WW domain linker, is only partially able to use the coupled WW domain binding mechanism. The longer linker results in a decrease in affinity for the Smad7 peptide. Interdomain coupling of WW domains enhances selectivity and enables the tuning of interactions by isoform switching.

SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression.

Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin deficiency causes common iron overload syndromes whereas its overexpression is responsible for microcytic anemias. Hepcidin transcription is activated by the bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways, whereas comparatively little is known about how hepcidin expression is inhibited. By using high-throughput siRNA screening we identified SMAD7 as a potent hepcidin suppressor. SMAD7 is an inhibitory SMAD protein that mediates a negative feedback loop to both transforming growth factor-beta and BMP signaling and that recently was shown to be coregulated with hepcidin via SMAD4 in response to altered iron availability in vivo. We show that SMAD7 is coregulated with hepcidin by BMPs in primary murine hepatocytes and that SMAD7 overexpression completely abolishes hepcidin activation by BMPs and transforming growth factor-beta. We identify a distinct SMAD regulatory motif (GTCAAGAC) within the hepcidin promoter involved in SMAD7-dependent hepcidin suppression, demonstrating that SMAD7 does not simply antagonize the previously reported hemojuvelin/BMP-responsive elements. This work identifies a potent inhibitory factor for hepcidin expression and uncovers a negative feedback pathway for hepcidin regulation, providing insight into a mechanism how hepcidin expression may be limited to avoid iron deficiency.

PRMT5 Enables Robust STAT3 Activation via Arginine Symmetric Dimethylation of SMAD7.

Protein arginine methyltransferase 5 (PRMT5) is the type II arginine methyltransferase that catalyzes the mono- and symmetrical dimethylation of protein substrates at the arginine residues. Emerging evidence reveals that PRMT5 is involved in the regulation of tumor cell proliferation and cancer development. However, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remain largely elusive. Here, it is shown that PRMT5 promotes lung cancer cell proliferation through the Smad7-STAT3 axis. Depletion or inhibition of PRMT5 dramatically dampens STAT3 activation and thus suppresses the proliferation of human lung cancer cells. Furthermore, depletion of Smad7 blocks PRMT5-mediated STAT3 activation. Mechanistically, PRMT5 binds to and methylates Smad7 on Arg-57, enhances Smad7 binding to IL-6 co-receptor gp130, and consequently ensures robust STAT3 activation. The findings position PRMT5 as a critical regulator of STAT3 activation, and suggest it as a potential therapeutic target for the treatment of human lung cancer.

Smad7 antagonizes transforming growth factor beta signaling in the nucleus by interfering with functional Smad-DNA complex formation.

Smad7 plays an essential role in the negative-feedback regulation of transforming growth factor beta (TGF-beta) signaling by inhibiting TGF-beta signaling at the receptor level. It can interfere with binding to type I receptors and thus activation of receptor-regulated Smads or recruit the E3 ubiquitin ligase Smurf to receptors and thus target them for degradation. Here, we report that Smad7 is predominantly localized in the nucleus of Hep3B cells. The targeted expression of Smad7 in the nucleus conferred superior inhibitory activity on TGF-beta signaling, as determined by reporter assay in mammalian cells and by its effect on zebrafish embryogenesis. Furthermore, Smad7 repressed Smad3/4-, Smad2/4-, and Smad1/4-enhanced reporter gene expression, indicating that Smad7 can function independently of type I receptors. An oligonucleotide precipitation assay revealed that Smad7 can specifically bind to the Smad-responsive element via its MH2 domain, and DNA-binding activity was further confirmed in vivo with the promoter of PAI-1, a TGF-beta target gene, by chromatin immunoprecipitation. Finally, we provide evidence that Smad7 disrupts the formation of the TGF-beta-induced functional Smad-DNA complex. Our findings suggest that Smad7 inhibits TGF-beta signaling in the nucleus by a novel mechanism.

Mutational analysis of Smad7 in human cervical cancer.

Our aim was to carry out mutational analysis of Smad7 exon 4 that codes for the Smad7 MH2 domain in human cervical cancer tissues. This study is warranted since genetic abnormalities of components of the transforming growth factor-beta (TGF-beta)/Smad signaling system have been implicated in a variety of human cancers. Further, Smad7 is an important member of the Smad family that functions as a negative feedback regulator of TGF-beta responses. By direct sequencing, we screened 60 histopathologically confirmed human cervical carcinomas for mutations in exon 4 of Smad7 that encodes the MH2 domain of the protein, a region of the protein believed to be critical for receptor interaction. No mutations or aberrations could be identified in any of the 60 analyzed tumor samples. However, we identified a previously reported, heterozygous, silent G to C variant in codon 391 of Smad7 that was found twice in the set of 60 clinical samples. Thirty of the clinical samples analyzed in the study were positive for the presence of high-risk human papillomavirus (HPV) subtypes, while the rest were oncogenic HPV-negative. The two instances of the silent variant was found in oncogenic HPV-negative samples. We report mutational analysis of Smad7 in cervical cancer for the first time. Mutations and variants of Smad7 are unlikely to be of major significance to the pathogenesis of HPV-induced cervical cancer.

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

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.

Expression of Smad protein by normal skin fibroblasts and hypertrophic scar fibroblasts in response to transforming growth factor beta1.

BACKGROUND: Smad proteins are important intracellular mediators of transforming growth factor (TGF)-beta signaling. Little has been known about the specific relationship between TGF-beta and TGF-beta/Smad signaling in hypertrophic scars. OBJECTIVE: The objective was to investigate the expression of Smads and the specific relationship between TGF-beta and TGF-beta/Smad signaling in hypertrophic scars. METHODS: In this study, we initially determined the endogenous protein levels of Smad2 and Smad7 in hypertrophic scar fibroblast (HSFs) and normal skin fibroblast (NSFs). Second, we stimulated HSFs and NSFs with recombinant human TGF-beta1 for 24 hours to determine whether the TGF-beta1 could potentiate its effect by further stimulating the production of Smad by reverse transcription-polymerase chain reaction and Western blot analysis. RESULTS: When compared with NSFs, the endogenous expression of Smad2 in HSFs was up-regulated and TGF-beta1 could further stimulate the production of Smad2. Although the levels of Smad7 were similar between HSFs and NSFs, TGF-beta1 up-regulated the expression of Smad7 for NSFs only, with no discernible effect on HSFs. These changes were paralleled by a significant increase in cytoplasm-to-nuclear translocation of Smad2. CONCLUSION: These data substantiated the model of an autocrine positive loop in hypertrophic scars pathogenesis. The authors have indicated no significant interest with commercial supporters.

Inhibition of Smad7, a negative regulator of TGF-beta signaling, suppresses autoimmune encephalomyelitis.

We studied the role of the Transforming growth factor (TGF)-beta signaling antagonist Smad7 in autoimmune central nervous system (CNS) inflammation by using specific antisense oligonucleotides (Smad7-as). Elevated Smad7 protein expression was found in the spinal cord of SJL/J mice and DA rats with experimental autoimmune encephalomyelitis (EAE) and in effector T cells upon antigen stimulation. Smad7-as specifically decreased Smad7 mRNA and protein in cell lines and in ex-vivo-treated primary mouse lymph node cells (LNC). LNC exposed to Smad7-as during secondary activation showed reduced proliferation and encephalitogenicity. After systemic administration, Smad7-as ameliorated clinical signs of active and adoptively transferred EAE, diminished CNS inflammation, and reduced Smad7 protein levels in the brain. Smad7-as was found to be incorporated by peritoneal macrophages as well as by cells of the liver, kidneys, and peripheral lymph nodes. Importantly, Smad7-as treatment was not toxic and did not increase extracellular matrix formation. Smad7 inhibition thus represents a novel systemic treatment strategy for autoimmune CNS inflammation, targeting TGF-beta signaling without TGF-beta-associated toxicity.

Rational design of cyclic peptides to disrupt TGF-Β/SMAD7 signaling in heterotopic ossification.

The human TGF-ß/SMAD7 signaling has been recognized as an attractive target of heterotopic ossification (HO). Here, we report a successful rational design of cyclic peptides to disrupt the signaling pathway by targeting TGF-ß-receptor complex. The intermolecular interaction between TGF-ß and its cognate receptor is characterized in detail using molecular dynamics simulation, binding energetic analysis, and alanine scanning. With the computational analysis a binding loop of receptor protein is identified that plays an essential role in the peptide-mediated TGF-ß-receptor interaction. Subsequently, the loop is stripped from the protein context to generate a linear peptide segment, which possesses considerable flexibility and intrinsic disorder, and thus would incur a large entropy penalty upon binding to TGF-ß. In order to minimize the unfavorable entropic effect, the linear peptide is cyclized by adding a disulfide bond between the N- and C-terminal cysteine residues of the peptide, resulting in a cyclic peptide. In vitro fluorescence anisotropy assays substantiate that the cyclic peptide can bind tightly to TGF-ß with determined Kd value of 54µM. We also demonstrated that structural optimization can further improve the peptide affinity by site-directed mutagenesis of selected residues based on the computationally modeled complex structure of TGF-ß with the cyclic peptide.

Smad7 Modulates Epidermal Growth Factor Receptor Turnover through Sequestration of c-Cbl.

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration, and tumorigenesis. For the maintenance of homeostasis, EGF signaling should be tightly regulated to prevent the aberrant activation. Smad7 has been known as inhibitory Smad that blocks the signal transduction of transforming growth factor ß. In the process of cell proliferation or transformation, Smad7 has been shown the opposite activities as a promoter or suppressor depending on cell types or microenvironments. We found that the overexpression of Smad7 in human HaCaT keratinocyte cells and mouse skin tissues elevated EGF receptor (EGFR) activity by impairing ligand-induced ubiquitination and degradation of activated receptor, which is induced by the E3 ubiquitin ligase c-Cbl. The C-terminal MH2 region but not MH1 region of Smad7 is critical for interaction with c-Cbl to inhibit the ubiquitination of EGFR. Interestingly, wild-type Smad7, but not Smad6 or mutant Smad7, destabilized the EGF-induced complex formation of c-Cbl and EGFR. These data suggest a novel role for Smad7 as a promoter for prolonging the EGFR signal in keratinocyte and skin tissue by reducing its ligand-induced ubiquitination and degradation.

MiR-21/Smad 7 signaling determines TGF-ß1-induced CAF formation.

How TGF-ß1-mediated signaling pathways are finely tuned to orchestrate the generation of carcinoma-associated fibroblasts (CAFs) is poorly understood. Here, we demonstrate that miR-21 and the signaling of its target Smad 7 determine TGF-ß1-induced CAF formation. In primary cultured fibroblasts, mature miR-21 increases after TGF-ß1 treatment, whereas the Smad 7 protein level decreases. MiR-21 binds to the 3' UTR of Smad7 mRNA and inhibits its translation, rather than causing its degradation. Most importantly, Smad 7 is bound to Smad 2 and 3, which are thought to competitively bind to TGFBR1, and prevents their activation upon TGF-ß1 stimulation. The depletion of miR-21 or the overexpression of Smad 7 blocks TGF-ß1-induced CAF formation, whereas the overexpression of miR-21 or the depletion of Smad 7 promotes CAF formation, even without TGF-ß1 stimulation. Collectively, these findings clearly demonstrate that miR-21 and Smad7 are critical regulators of TGF-ß1 signaling during the induction of CAF formation.

Structural basis for the versatile interactions of Smad7 with regulator WW domains in TGF-ß Pathways.

Transforming growth factor (TGF)-ß and BMP signaling is mediated by Smads 1-5 (R-Smads and Co-Smads) and inhibited by Smad7, a major hub of regulation of TGF-ß and BMP receptors by negative feedback and antagonistic signals. The transcription coactivator YAP and the E3 ubiquitin ligases Smurf1/2 and Nedd4L target R-Smads for activation or degradation, respectively. Pairs of WW domain in these regulators bind PY motifs and adjacent CDK/MAPK and GSK3 phosphorylation sites in R-Smads in a selective and regulated manner. In contrast, here we show that Smad7 binds YAP, Smurf1, Smurf2, and Nedd4L constitutively, the binding involving a PY motif in Smad7 and no phosphorylation. We also provide a structural basis for how regulators that use WW domain pairs for selective interactions with R-Smads, resort to one single versatile WW domain for binding Smad7 to centralize regulation in the TGF-ß and BMP pathways.

Negative Smad expression and regulation in the developing chick limb.

The inhibitory or negative Smads, Smad6 and Smad7, block TGFbeta superfamily signals of both the BMP and TGFbeta classes by antagonizing the intracellular signal transduction machinery. We report the cloning of one Smad6 and two Smad7 (Smad7a and Smad7b) chick homologs and their expression and regulation in the developing limb. Smad6 and Smad7a are expressed in dynamic patterns reflecting the domains of BMP gene expression in the limb. Activation and inhibition of the BMP signaling pathway in limb mesenchyme indicates that negative Smad gene expression is regulated, at least in part, by BMP family signals.

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain.

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.

An expanded WW domain recognition motif revealed by the interaction between Smad7 and the E3 ubiquitin ligase Smurf2.

Smurf2 is an E3 ubiquitin ligase that drives degradation of the transforming growth factor-beta receptors and other targets. Recognition of the receptors by Smurf2 is accomplished through an intermediary protein, Smad7. Here we have demonstrated that the WW3 domain of Smurf2 can directly bind to the Smad7 polyproline-tyrosine (PY) motif. Of particular interest, the highly conserved WW domain binding site Trp, which interacts with target PY motifs, is a Phe in the Smurf2 WW3 domain. To examine this interaction, the solution structure of the complex between the Smad7 PY motif region (ELESPPPPYSRYPMD) and the Smurf2 WW3 domain was determined. The structure reveals that, in addition to binding the PY motif, the WW3 domain binds six residues C-terminal to the PY motif (PY-tail). Although the Phe in the WW3 domain binding site decreases affinity relative to the canonical Trp, this is balanced by additional interactions between the PY-tail and the beta1-strand and beta1-beta2 loop of the WW3 domain. The interaction between the Smurf2 WW3 domain and the Smad7 PY motif is the first example of PY motif recognition by a WW domain with a Phe substituted for the binding site Trp. This unusual interaction allows the Smurf2 WW3 domain to recognize a subset of PY motif-containing proteins utilizing an expanded surface to provide specificity.