Inhibitory SMADs: potential regulators of ovarian function.

Transforming growth factor beta (TGFB) superfamily signaling regulates essential reproductive functions. Dysregulation of TGFB signaling results in cellular and molecular deficiencies in the ovary, leading to reproductive diseases and cancer development. SMAD proteins are canonical TGFB signaling components consisting of receptor-regulated SMADs (SMAD1/2/3/5/9), a common SMAD (SMAD4), and inhibitory SMADs (SMAD6/7). Inhibitory SMADs are negative regulators of TGFB and bone morphogenetic protein signaling, and their reproductive functions are poorly defined. Emerging evidence supports that inhibitory SMADs are potential regulators of ovarian function. Further efforts and new genetic models are needed to unveil the role of inhibitory SMADs in the ovary.

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.

Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.

Modeling and analysis of MH1 domain of Smads and their interaction with promoter DNA sequence motif.

The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta (TGF-beta) superfamily of proteins at the cell surface. The prototypic members of the Smad family, Mad and Sma, were first described in Drosophila and Caenorhabditis elegans, respectively. Related proteins in Xenopus, Humans, Mice and Rats were subsequently identified, and are now known as Smads. Smad protein family members act downstream in the TGF-beta signaling pathway mediating various biological processes, including cell growth, differentiation, matrix production, apoptosis and development. Smads range from about 400-500 amino acids in length and are grouped into the receptor-regulated Smads (R-Smads), the common Smads (Co-Smads) and the inhibitory Smads (I-Smads). There are eight Smads in mammals, Smad1/5/8 (bone morphogenetic protein regulated) and Smad2/3 (TGF-beta/activin regulated) are termed R-Smads, Smad4 is denoted as Co-Smad and Smad6/7 are inhibitory Smads. A typical Smad consists of a conserved N-terminal Mad Homology 1 (MH1) domain and a C-terminal Mad Homology 2 (MH2) domain connected by a proline rich linker. The MH1 domain plays key role in DNA recognition and also facilitates the binding of Smad4 to the phosphorylated C-terminus of R-Smads to form activated complex. The MH2 domain exhibits transcriptional activation properties. In order to understand the structural basis of interaction of various Smads with their target proteins and the promoter DNA, we modeled MH1 domain of the remaining mammalian Smads based on known crystal structures of Smad3-MH1 domain bound to GTCT Smad box DNA sequence (1OZJ). We generated a B-DNA structure using average base-pair parameters of Twist, Tilt, Roll and base Slide angles. We then modeled interaction pose of the MH1 domain of Smad1/5/8 to their corresponding DNA sequence motif GCCG. These models provide the structural basis towards understanding functional similarities and differences among various Smads.

Regulating the stability of TGFbeta receptors and Smads.

Transforming growth factor beta (TGFbeta) controls cellular behavior in embryonic and adult tissues. TGFbeta binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that together regulate gene expression. In this review, mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of TGFbeta are presented. We discuss how the activity and duration of TGFbeta receptor/Smad signaling can be regulated by post-translational modifications that affect the stability of key proteins in the pathway. We highlight links between these mechanisms and human diseases, such as tissue fibrosis and cancer.

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.

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.

Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.

Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.

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.

Bone morphogenetic protein activities are enhanced by 3',5'-cyclic adenosine monophosphate through suppression of Smad6 expression in osteoprogenitor cells.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF)-beta superfamily, and some display potent osteogenic activity both in vivo and in vitro. The BMP signaling cascade involving BMP receptors at the cell membrane and intracellular messengers (Smads) has been elucidated, but the regulatory mechanisms of BMP signaling have not been clarified. We previously found that pentoxifyline (PeTx), a nonspecific inhibitor of phosphodiesterase (PDE), and rolipram, a PDE-4-specific inhibitor, enhance BMP-4-induced osteogenic differentiation of mesenchymal cells, probably through the elevation of intracellular cyclic adenosine monophosphate (cAMP) accumulation and modulation of BMP signaling pathways as enhanced BMP-4 action was reproduced by addition of dibutylyl-cAMP (dbcAMP). However, the precise mechanisms underlying the enhancing effects of those agents on BMP signaling were not completely revealed. As already reported, BMPs utilize a specific intracellular signaling cascade to target genes via R-Smads (Smad1,5,8), Co-Smad (Smad4) and I-Smads (Smad6,7). One possibility for cAMP-mediated effects on BMP signaling might be suppression of I-Smads expression since these proteins form a negative feedback loop in BMP signaling. To examine this possibility, changes in I-Smad (Smad6) expression on addition of dbcAMP or PeTx were examined in a bone-marrow-derived osteogenic cell line (ST2). Alkaline phosphatase activity in ST2 cells was consistently induced by BMP-4 treatment (300 ng/ml), and Smad6 mRNA expression was also induced by BMP-4 treatment. Although concurrent treatment of ST2 cells with BMP-4 and dbcAMP elicited further activation of alkaline phosphatase, addition of dbcAMP reduced BMP-4-induced Smad6 expression in a dose-dependent manner. Furthermore, detection of phosphorylated Smad1/5/8 on Western blotting analysis was prolonged, suggesting prolonged kinase activity of BMP receptors through suppressed expression of Smad6. Elevated intracellular cAMP might thus enhance BMP signaling by suppressing Smad6 induction and prolonging intracellular BMP signaling.