Structure-function relationship of inhibitory Smads: Structural flexibility contributes to functional divergence.

Smads are a small family of eukaryotic transcription regulators that play key roles in the transforming growth factor-beta signaling cascade. Smad6 and Smad7, the inhibitory or I-Smads, inhibit signaling downstream of TGF-beta type I receptors, thereby acting as negative regulators of signaling mediated by TGF-beta superfamily of ligands. Smad6 is known to specifically inhibit BMP type I receptor mediated signaling, while Smad7 is a more general inhibitor, able to block signaling mediated by a set of related TGF-beta type I receptors, including type I receptors for BMP and TGF-beta/Activin. In this study we have sought to understand the structural basis for this functional divergence of I-Smads. We have created homology-based models for the MH1 and MH2 domains of the two I-Smads and have carried out detailed molecular dynamics (MD) simulations of these proteins in explicit solvent to investigate the flexibility of the domains. The molecular models show that the I-Smads have lost many of the secondary structural elements found in the R-Smads, giving rise to longer loops in the tertiary structure of Smad6 and Smad7. Detailed analysis of the structural models and the MD trajectories clearly reveal that compared to Smad6, Smad7 has a more flexible overall folding, marked by the presence of highly flexible amino acid residues in functionally important regions of the protein. Interestingly, three of these residues-Phe411, Lys401, and Cys406, map to L3 loop of Smad7 MH2 domain, which is a critical structural determinant in Smad-type I receptor interactions. The increased structural flexibility of Smad7, arising out of longer, more flexible loops in its MH2 domain, might enable Smad7 to interact with a set of related yet structurally diverse type I receptors. Taken together with experimental evidence published in recent literature that hint at structural factors underlying the generic nature of inhibition by Smad7, our results strongly suggest that structural flexibility could be a prime contributor to the functional differences between Smad6 and Smad7. Additionally, we have been able to use the Smad7 structural model to successfully rationalize the results of in vitro site-specific mutagenesis experiments in published literature. This also provides biological validation for our model. Apart from this, analysis of the MH1 molcular model of Smad6 delineates a basic patch on the surface of the domain that might take part in nonspecific DNA binding by Smad6. This finding is consistent with earlier experimental data and is relevant since the characteristic beta-hairpin DNA binding element of R-Smads is completely absent in the I-Smads. Finally, the molecular models described here can serve to guide future biochemical and genetic studies on I-Smads.

Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells.

Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.

[Effects of blocking two sites of transforming growth factor-ß/Smads signaling on the formation of scar-related proteins in human skin fibroblasts].

OBJECTIVE: To explore the effects of blocking two sites of TGF-ß/Smads signaling on the formation of scar-related proteins in human skin fibroblasts. METHODS: Two lentivirus vectors encoding soluble TGF-ß receptor II (sTßRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTßRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-ß1 for 72 h; sTßRII group, transfected with lenti-sTßRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-ß1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test). RESULTS: (1) HFF-1 cells transfected with lenti-sTßRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTßRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTßRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTßRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05). CONCLUSIONS: In human skin fibroblasts, blockage of two sites of TGF-ß/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-ß1 to a greater extent than that of blocking one single site.

Differences in activation of mouse hepcidin by dietary iron and parenterally administered iron dextran: compartmentalization is critical for iron sensing.

The iron regulatory hormone hepcidin responds to both oral and parenteral iron. Here, we hypothesized that the diverse iron trafficking routes may affect the dynamics and kinetics of the hepcidin activation pathway. To address this, C57BL/6 mice were administered an iron-enriched diet or injected i.p. with iron dextran and analyzed over time. After 1 week of dietary loading with carbonyl iron, mice exhibited significant increases in serum iron and transferrin saturation, as well as in hepatic iron, Smad1/5/8 phosphorylation and bone morphogenetic protein 6 (BMP6), and hepcidin mRNAs. Nevertheless, hepcidin expression reached a plateau afterward, possibly due to upregulation of inhibitory Smad7, Id1, and matriptase-2 mRNAs, while hepatic and splenic iron continued to accumulate over 9 weeks. One day following parenteral administration of iron dextran, mice manifested elevated serum and hepatic iron levels and Smad1/5/8 phosphorylation, but no increases in transferrin saturation or BMP6 mRNA. Surprisingly, hepcidin failed to appropriately respond to acute overload with iron dextran, and a delayed (after 5-7 days) hepcidin upregulation correlated with increased transferrin saturation, partial relocation of iron from macrophages to hepatocytes, and induction of BMP6 mRNA. Our data suggest that the physiological hepcidin response is saturable and are consistent with the idea that hepcidin senses exclusively iron compartmentalized within circulating transferrin and/or hepatocytes.

Ter94 ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial degradation.

The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form (Ci155/Gli(FL)) to a truncated repressor (Ci75/Gli(R)) by proteasomes to mediate Hedgehog (Hh) signaling. The mechanism by which proteasomes distinguish ubiquitinated Ci/Gli to carry out complete versus partial degradation is not known. Here, we show that Ter94 ATPase and its mammalian counterpart, p97, are involved in processing Ci and Gli3 into Ci75 and Gli3(R), respectively. Ter94 regulates the partial degradation of ubiquitinated Ci by Cul1-Slimb-based E3 ligase through its adaptors Ufd1-like and dNpl4. We demonstrate that Cul1-Slimb-based E3 ligase, but not Cul3-Rdx-based E3 ligase, modifies Ci by efficient addition of K11-linked ubiquitin chains. Ter94(Ufd1-like/dNpl4) complex interacts directly with Cul1-Slimb, and, intriguingly, it prefers K11-linked ubiquitinated Ci. Thus, Ter94 ATPase and K11-linked ubiquitination in Ci contribute to the selectivity by proteasomes for partial degradation.

Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts.

Transforming growth factor-beta(1) (TGF-beta(1)) signal and downstream Smads play an important role in tissue fibrosis and matrix remodeling in various etiologies of heart failure. Inhibitory Smad7 (I-Smad7) is an inducible regulatory Smad protein that antagonizes TGF-beta(1) signal mediated via direct abrogation of R-Smad phosphorylation. The effect of ectopic I-Smad7 on net collagen production was investigated using hydroxyproline assay. Adenovirus-mediated I-Smad7 gene (at 100 multiplicity of infection) transfer was associated with significant decrease of collagen synthesis in the presence and absence of TGF-beta(1) in primary rat cardiac myofibroblasts. In I-Smad7-infected cells, we also observed the ablation of TGF-beta(1)-induced R-Smad2 phosphorylation vs. LacZ controls. Overdriven I-Smad7 was associated with significantly increased expression of immunoreactive 65-kDa matrix metalloproteinase-2 (MMP-2) protein in culture medium of myofibroblast compared with LacZ-infected cells. Expression of the 72-kDa MMP-2 variant, e.g., the inactive form, was not altered by exogenous I-Smad7 transfection/overexpression. Furthermore, I-Smad7 overexpression was associated with a significant increase and decrease in expression of p27 and phospho-Rb protein, respectively, as well as reduced [(3)H]thymidine incorporation vs. Ad-LacZ-infected controls. We suggest that negative modulation of R-Smad phosphorylation by ectopic I-Smad7 may contribute to the downregulation of collagen in cardiac myofibroblasts and may suppress the proliferation of these cells. Thus treatments targeting the collagen deposition by overexpression of I-Smad7 may provide a new therapeutic strategy for cardiac fibrosis.

Imbalance of receptor-regulated and inhibitory Smads in lung fibroblasts from bleomycin-exposed rats.

Transforming growth factor (TGF)-beta plays a central role in lung fibrosis, stimulating extracellular matrix deposition. Intracellular signaling of TGF-beta is mediated by Smad proteins. We questioned whether the expression and activation of Smads would be altered in lung fibroblasts from rats exposed to bleomycin, an agent used to provoke an experimental model of lung fibrosis. Fibroblasts were isolated from rat lungs 14 d after intratracheal instillation of bleomycin (BLF) or saline (NLF), and cell cultures established. Whole cell lysates were obtained at baseline, and after stimulation with TGF-beta1 (10 ng/ml). Western blot analysis was performed to measure levels of phosphorylated Smad3 (p-Smad3) and Smad7. Real-time PCR was used to determine changes in Smad7 mRNA after TGF-beta stimulation. We found increased baseline levels of p-Smad3 in BLF versus NLF (P < 0.05). In contrast, baseline levels of Smad7 were comparable. The ratio of stimulatory to inhibitory Smads was increased in BLF compared with NLF (P < 0.05). After stimulation with TGF-beta, levels of p-Smad3 were increased in both groups, with maximal responses at 30 min (P < 0.01). While Smad7 mRNA levels were significantly upregulated (at 1 h) after TGF-beta in both groups, the increase in Smad7 protein was significant in NLF only. We conclude there is sustained activation of Smad signaling in lung fibroblasts isolated from bleomycin-exposed rats, with an imbalance between the levels of p-Smad3 and Smad7. Insufficient levels of the inhibitory Smad7 at baseline, and inadequate response to TGF-beta, may contribute to the fibrotic phenotype characteristic of BLF.

IFN-gamma abrogates profibrogenic TGF-beta signaling in liver by targeting expression of inhibitory and receptor Smads.

BACKGROUND/AIMS: In a randomized open-labeled multicenter trial with patients suffering from chronic HBV infection, we recently identified a benefit of 9-month IFN-gamma treatment resulting in decreased fibrosis scores and a reduced number of alpha-smooth muscle actin-positive hepatic stellate cells (HSCs). Approaches opposing profibrogenic activities of TGF-beta may be amenable in chronic liver disease. According to experimental models, IFN-gamma counteracts several TGF-beta effects. METHODS: The crosstalk of IFN-gamma and TGF-beta signaling relevant for fibrogenesis was investigated in primary cultured rat HSCs and a cell line representing activated HSCs. RESULTS: In vitro studies with HSCs demonstrate that TGF-beta-dependent activation of (CAGA)9-MLP-Luc, a Smad3/4 responsive reporter construct, was significantly decreased by IFN-gamma, indicating a TGF-beta antagonizing function. IFN-gamma induced the activity of the Smad7 promoter and Smad7 protein expression via STAT-1 signaling. In contrast to TGF-beta, IFN-gamma was able to induce Smad7 expression in activated HSCs providing increased protein levels for at least 12h. In addition, expression of Smad2/3 was reduced by IFN-gamma and activation of Smads2/3 was abrogated. CONCLUSIONS: IFN-gamma displays antifibrotic effects in liver cells via STAT-1 phosphorylation, upregulation of Smad7 expression and impaired TGF-beta signaling.