Smad Family Proteins Research Articles

TGF-β signals the formation of a unique NF1/Smad4-dependent transcription repressor-complex in human diploid fibroblasts

We earlier reported the formation of a unique nuclear NF1/Smad complex in serum-restricted fibroblasts that acts as an NF1-dependent repressor of the human adenine nucleotide translocase-2 gene (ANT2) [K. Luciakova, G. Kollarovic, P. Barath, B.D. Nelson, Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex, Biochem. J. 412 (2008) 123–130]. In the present study, we show that TGF-β, like serum-restriction: (a) induces the formation of NF1/Smad repressor complexes, (b) increases binding of the complexes to the repressor elements (Go elements) in the ANT2 promoter, and (c) inhibits ANT2 expression. Repression of ANT2 by TGF-β is eliminated by mutating the NF1 binding sites in the Go repressor elements. All of the above responses to TGF-β are prevented by inhibitors of TGF-β RI and MAPK p38. These inhibitors also prevent NF1/Smad4 repressor complex formation and repression of ANT2 expression in serum-restricted cells, suggesting that similar signaling pathways are initiated by TGF-β and serum-restriction. The present finding that NF1/Smad4 repressor complexes are formed through TGF-β signaling pathways suggests a new, but much broader, role for these complexes in the initiation or maintenance of the growth-inhibited state.

Abstract 2425: Mechanisms of neuroblastoma metastasis to bone

Abstract The molecular pathways leading to neuroblastoma (NBL) development and progression are understudied despite the fact this tumor is responsible for 15% of all childhood cancer deaths. Bone is one of the most common sites of metastasis in NBL, and over 90% of children with NBL bone metastasis will die. Therefore, the goal of this study is to elucidate the cell-signaling pathways responsible for the destruction of bone. Parathyroid hormone related protein (PTHrP) and Transforming Growth Factor Beta both are involved in osteoclastogenesis. PTHrP travels through the circulation to the bones, where it triggers a chain of event that encourages the dissolution of bone minerals by osteoclast. Moreover, PTHrP results in the mobilization of calcium ions, which travel back via the circulatory system allowing osteoclast differentiation to go unchecked, therefore providing further destruction to the bone microenvironment. Transforming Growth Factor Beta (TGF-β) is a protein which controls proliferation, cellular differentiation and other functions in most cells. Since TGF-β and the SMAD family of proteins modulate the activity TGF-β and its association with osteoclasts, we formulated the hypothesis that osteoclastogenesis occurs during NBL bone metastasis through PTHrP and TGF-β. NBL is a heterogeneous tumor consisting of neuronal (n) and stromal (s) cells, with metastatic cells predominantly of the n type. In our study, we focused on four NBL cell lines: IMR-32 (n), SH-SY5Y (n), SK-N-AS (s), SK-N-SH (s). Conditioned media collected from IMR32 NBL cells induces osteoclast precursors to fuse into multinucleated, TRAP positive cells, indicative of osteoclast differentiation. Whole cell lysates were collected and Western immunoblotting performed for PTHrP. We report that the more tumorigenic cells, SH-SY5Y and IMR32, express higher levels of PTHrP than the s lines SK-N-AS and SK-N-SH. These data indicate that PTHrP may be involved in NBL bone metastasis. We are currently analyzing the effects of TGF-β on NBL and the role of these proteins in NBL-induced osteoclastogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2011-2425

Identification and expression of amphioxus AmphiSmad1/5/8 and AmphiSmad4

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily. In this study, we identified two novel members of the Smad family, termed as AmphiSmad1/5/8 and AmphiSmad4, from Chinese amphioxus. Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains. Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1, 5 and 8 subgroup of the R-Smad subfamily, and AmphiSmad4 in the Co-Smad subfamily. The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord, myotome and alimentary canal, and may help to establish the specification of dorsal-ventral axis of amphioxus. Moreover, AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined, suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.

Rap1-GTP Augments Activation of Smad and p38 Mediated Signaling Downstream of TGF-β Receptor In T Lymphocytes

AbstractAbstract 956Rap1A, a member of the Ras superfamily, was discovered as a gene product that reverted K-Ras-induced transformation. Although it was initially thought that Rap1A (thereafter referred to as Rap1) opposes Ras-induced transformation by competing for common downstream effector(s), it has now become apparent that Ras and Rap1 proteins operate in different signaling networks and mediate distinct functions. In lymphocytes Rap1 is activated by BCR and TCR mediated signals and is involved in inside-out activation of integrins. To understand the role of Rap1 in T cell responses we generated transgenic (Tg) mice that express the active Rap1 mutant Rap1E63 in T cells. Rap1E63-Tg and littermate control mice had no statistically significant difference in absolute thymocyte numbers and differentiation profiles of double positive and single positive thymocytes. In contrast to thymocyte numbers, Rap1E63-Tg mice had reduced T cell numbers in peripheral lymphoid organs. In peripheral lymph nodes and spleen, the total T cell numbers were decreased to approximately one third to one forth of those in littermate controls. Strikingly, Rap1E63-Tg mice had a 4-fold increase in the CD4+CD103+ T cell fraction in spleens and lymph nodes compared to their littermate controls. CD103 defines a subset of peripherally generated Treg with potent suppressive function. CD4+CD103+ Treg cells in RapE63-Tg mice expressed CD45RBlow, CD44high, CD54high, CD62Llow and LFA-1high, findings consistent with an effector memory phenotype. TGF-β is the strongest stimulus for induction of CD103 expression. To examine whether Rap1 affected TGF-β-mediated signaling in T cells, we used stable Jurkat T cell lines expressing Rap1E63, Jurkat T cell lines, in which endogenous Rap1 was depleted by shRNA (Rap1-KD), and primary T cells from Rap1E63-Tg mice and Rap1-KO mice. TGF-β signaling involves the interaction of two membrane-bound receptors, TGF-βRI and II. TGF-β binds the exracellular domain of TGF-βRII, which then recruits TGF-βRI resulting in transphosphorylation. TGF-βRI activation propagates downstream signaling via the Smad family proteins Smad2 and Smad3 and also via p38 MAPK, in a Smad-independent manner. Incubation of Rap1E63 Jurkat T cells with TGF-β resulted in enhanced and sustained phosphorylation of Smad2 and Smad3, which was observed with very low concentrations of TGF-β that were incapable of inducing detectable phosphorylation of Smad2 or Smad3 in control cells. In contrast, diminished level and duration of Smad2 and Smad3 phosphorylation was observed in Rap1-KD Jurkat cells. Activation of p38 downstream of TGF-βRI displayed a dramatically augmented and sustained induction, whereas the opposite effect was observed in Rap1-KD cells. Similar patterns of responses to those observed in Rap1E63 Jurkat T cells and in Rap1-KD cells were observed in primary mouse T cells isolated from Rap1E63-Tg mice and Rap1 deficient mice, respectively. Assessment of TGF-β binding on TGF-β surface receptors by using biotinylated TGF-β revealed that Rap1E63 expressing cells displayed significantly enhanced TGF-β binding compared to their relevant controls, whereas reduced TGF-β binding was observed in Rap1 deficient cells. These results indicate that active Rap1 modulates TGF-β receptor binding and enhances TGF-β mediated signaling, gene transcription, and functional outcome. Our data reveal a novel and unexpected mechanism by which Rap1 regulates T cell responses that may have important implications on TGF-β-mediated T cell homeostasis and maintenance of immune quiescence. Disclosures:No relevant conflicts of interest to declare.

SMAD4 Is Required for Development of Maximal Endotoxin Tolerance

Initial exposure of monocytes/macrophages to LPS induces hyporesponsiveness to a second challenge with LPS, a phenomenon termed LPS tolerance. Molecular mechanisms responsible for endotoxin tolerance are not well defined. We and others have shown that IL-1R-associated kinase (IRAK)-M and SHIP-1 proteins, negative regulators of TLR4 signaling, increase in tolerized cells. TGF-beta1, an anti-inflammatory cytokine, is upregulated following LPS stimulation, mediating its effect through SMAD family proteins. Using a monocytic cell line, THP1, we show that LPS activates endogenous SMAD4, inducing its migration into the nucleus and increasing its expression. Secondary challenge with high dose LPS following initial low-dose LPS exposure does not increase IRAK-M or SHIP1 protein expression in small hairpin (sh)SMAD4 THP-1 cells compared with control shLUC THP1 cells. TNF-alpha concentrations in culture supernatants after second LPS challenge are higher in shSMAD4 THP-1 cells than shLUC THP1 cells, indicating failure to induce maximal tolerance in absence of SMAD4 signaling. Identical results are seen in primary murine macrophages and mouse embryonic fibroblasts, demonstrating the biological significance of our findings. TGF-beta1 treatment does not increase IRAK-M or SHIP1 protein expression in shSMAD4 THP-1 cells, whereas it does so in shLUC THP1 cells, indicating that TGF-beta1 regulates IRAK-M and SHIP1 expression through a SMAD4-dependent pathway. Knockdown of endogenous SHIP1 by shSHIP1 RNA decreases native and inducible IRAK-M protein expression and prevents development of endotoxin tolerance in THP1 cells. We conclude that in THP-1 cells and primary murine cells, SMAD4 signaling is required for maximal induction of endotoxin tolerance via modulation of SHIP1 and IRAK-M.

Transcriptional Control of Human Antigen R by Bone Morphogenetic Protein

Human antigen R (HuR) is an RNA-binding protein with protective activities against cellular stress. This study considers the mechanisms by which HuR transcriptional regulation occurs in renal proximal tubule cells. Under basal conditions, HuR mRNA is expressed in two forms: one that contains a approximately 20-base 5'-untranslated region (UTR) sequence and one that contains a approximately 150-base, G+C-rich 5'-UTR that is inhibitory to translation. Recovery from cellular stresses such as thapsigargin and ATP depletion induced increased expression of the shorter, more translatable transcript and decreased expression of the longer form. Analysis of HuR upstream regions revealed sequences necessary for regulation of the shorter mRNA. Within the long, G+C-rich 5'-UTR exist multiple copies of the alternate Smad 1/5/8-binding motif GCCGnCGC. Recovery from ATP depletion induced increases in Smad 1/5/8 levels; further, gel shift and chromatin immunoprecipitation analyses demonstrated the ability of these Smads to bind to the relevant motif in the HuR 5'-UTR. Transfection of exogenous Smad 1 increased HuR mRNA expression. Finally, HuR mRNA expression driven by the Smad-binding sites was responsive to BMP-7, a protein with known protective effects against ischemic injury in kidney. These data suggest that transcriptional induction of a readily translatable HuR mRNA may be driven by a mechanism known to protect the kidney from injury and provides a novel pathway through which administration of BMP-7 may attenuate renal damage.

Crystal structure of the MH2 domain of Drosophila Mad

The decapentaplegic(Dpp), a member of the TGF-beta superfamily, plays a pivotal role in the control of proliferation, global patterning and induction of specific cell fates during Drosophila development. Mother against Dpp(Mad) is the founding member of the conserved Smad protein family which specifically transduces the intracellular TGF-beta signaling cascade. Here we report the 2.80 A structure of the MH2 domain of Mad(Mad-MH2) that was readily superposed to the mammal Smad-MH2 structures. This unphosphorylated Mad-MH2 forms a symmetric homotrimer in crystals, consistent with the result of the size-exclusion chromatography that Mad-MH2 exhibited a propensity for concentration-dependent oligomerization prior to phosphorylation. Structural analysis revealed that the formation of homotrimeric Mad-MH2 is mainly mediated by contacts involving the extreme C-terminal SSVS motif, and is strengthened by phosphorylation of the last two Ser residues which was confirmed by the gel filtration analysis of the pseudophosphorylated Mad-MH2(DVD). Intriguingly, the homotrimer within an asymmetric unit only possesses two ordered C-terminal tails, reminiscent of the arrangement of the R-Smad/Smad4 complexes, indicating that the subunit with a flexible SSXS motif would be readily replaced by Co-Smad to form a functional heterotrimer.

Structural Basis for the Antiproliferative Activity of the Tob-hCaf1 Complex

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

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-β (TGF-β) 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-β 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-β/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.

Growth-dependent repression of human adenine nucleotide translocator-2 (ANT2) transcription: evidence for the participation of Smad and Sp family proteins in the NF1-dependent repressor complex

NF1 (nuclear factor 1) binds to two upstream elements of the human ANT2 (adenine nucleotide translocator-2) promoter and actively represses expression of the gene in growth-arrested diploid skin fibroblasts [Luciakova, Barath, Poliakova, Persson and Nelson (2003) J. Biol. Chem. 278, 30624-30633]. ChIP (chromatin immunoprecipitation) and co-immunoprecipitation analyses of nuclear extracts from growth-arrested and growth-activated diploid cells demonstrate that NF1, when acting as a repressor, is part of a multimeric complex that also includes Smad and Sp-family proteins. This complex appears to be anchored to both the upstream NF1-repressor elements and the proximal promoter, Sp1-dependent activation elements in growth-arrested cells. In growth-activated cells, the repressor complex dissociates and NF1 leaves the promoter. As revealed by co-immunoprecipitation experiments, NF1-Smad4-Sp3 complexes are present in nuclear extracts only from growth-inhibited cells, suggesting that the growth-state-dependent formation of these complexes is not an ANT2 promoter-specific event. Consistent with the role of Smad proteins in the repression complex, TGF-beta (transforming growth factor-beta) can fully repress ANT2 transcription in normally growing fibroblasts. Finally, pull-down experiments of in vitro transcribed/translated NF1 isoforms by GST (glutathione transferase)-Smad and GST-Smad MH fusion proteins indicate direct physical interactions between members of the two families. These findings suggest a possible functional relationship between the NF1 and Smad proteins that has not been previously observed.

MH1 domain of SMAD4 binds N-terminal residues of the homeodomain of Hoxc9

Smad family proteins mediate signaling initiated by bone morphogenetic proteins (BMPs). Upon BMP stimulation, the Smads such as Smad4 can interact directly with Hox proteins and suppress their DNA-binding activity. Although the interaction between the MAD-homology 1 (MH1) domain of Smad4 and Hox was found to regulate the transcription activity of Hox proteins, the molecular mechanism is not well characterized and direct contact residues remain to be elucidated. In the present study, the interaction between the recombinant homeodomain (HD) of Hoxc9 and MH1 domain of Smad4 was investigated with the use of the GST pull-down assay, surface plasmon resonance (SPR) analysis as well as multidimensional nuclear magnetic resonance (NMR) techniques. The Hoxc9-HD was precipitated with the GST-fused Smad4-MH1 but not with GST alone, demonstrating a direct interaction between Hoxc9-HD and Smad4-MH1 in vitro. SPR measurement further confirmed a moderately strong interaction ( K d ≈ 400 nM) between these two domains. Moreover, NMR titration experiments showed that a strong and specific binding occurred between Smad4-MH1 and Hoxc9-HD. NMR triple-resonance experiments and backbone assignments revealed that the N-terminal arm of Hoxc9-HD, spanning the positive-charged DNA-binding segment of Arg190–Arg196, was intimately involved in the interaction with Smad4-MH1. Ala-substitutions of Arg190–Arg196 led to the loss of interaction between Hoxc9-HD and Smad4-MH1 in both GST-pull down assay and SPR analysis; further provided functional evidence for the critical role of this positive-charged region in binding to Smad4-MH1. This suggested that Smad4-MH1 could occupy one of the DNA binding sites of Hoxc9 and consequently inhibits its transcription activity. The above results are in good agreement and yield the first insight into the interaction between the homeodomain of Hox proteins and the conserved MH1 domain of Smad family proteins.

Down-regulation of Caveolin-1, an Inhibitor of Transforming Growth Factor-β Signaling, in Acute Allergen-induced Airway Remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.

The Interferon Regulatory Factor Family in Host Defense: Mechanism of Action

Transcription factors of the interferon regulatory factor (IRF) family commands the entire type I interferon (IFN) system from induction of IFNs to diverse IFN responses, thereby providing a principal basis for host resistance against pathogens. However, the family has various additional roles. Regulating the development of the immune system, IRFs shape the establishment and execution of innate and adaptive immunity. IRFs also regulate growth and differentiation of many cell types, thus playing a role in leukemia and other cancers. In addition, evidence indicates that IRFs confer antiviral mechanisms not directly ascribed to the IFN system. This review deals with the diverse roles of IRFs in host defense and discusses the molecular mechanisms by which they regulate target gene transcription.

Competition between Ski and CREB-binding Protein for Binding to Smad Proteins in Transforming Growth Factor-β Signaling

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

The TGFβ/Smad pathway plays a dual role in restenosis following angioplasty

Purpose TGFβ is a powerful regulator of the cardiovascular system. The Smad protein family transduces the TGFβ signal and has been implicated in the development of intimal hyperplasia(IH)/restenosis. In this study, we explore the role of the receptor-regulated Smad3 and the inhibitory Smad7 in restenosis following vascular injury. Methods & Results Adenoviral vectors that express LacZ, Smad3, or Smad7 were delivered via intra-luminal perfusion immediately following balloon angioplasty of rat carotid arteries. At a dosage of 2.5x109 pfu, over 80% of medial smooth muscle cells (SMCs) became infected with transgene expression persisting for up to 4 weeks. Compared to animals receiving a control vector, AdSmad3 resulted in a thicker neointima, while AdSmad7 decreased IH. At 14 days post-injury, evaluation of SMC proliferation revealed increased PCNA+ SMCs following Smad3 infection but decreased PCNA staining following AdSmad7, suggesting that Smad3 stimulates SMC proliferation and thus the formation of IH. Moreover, AdSmad3 treatment was accompanied by increased SMC matrix protein production. However, despite a larger neointima, the lumen of AdSmad3-treated arteries was preserved and accompanied by expansion of the external elastic lamina, indicating a role for Smad3 in adaptive vascular remodeling. Blocking Smad3 with Smad7 overexpression also limited SMC matrix protein production and attenuated lumen loss in balloon injured arteries, although no effect on vascular remodeling was observed. Conclusions These finding suggest that the TGFβ/Smad pathway plays a dual role in the development of restenosis following angioplasty.

The role of transforming growth factor beta signaling in messenger RNA stability

Transforming growth factor beta (TGF-β) is a biologically multipotent regulatory protein implicated in functions that include the regulation of cellular growth, differentiation, extracellular matrix formation, and wound healing. It also plays a role in the pathologies of Alzheimer's disease, cancer and autoimmune disorders. TGF-β modulates gene expression by affecting transcriptional activation and mRNA turnover rate. Steady-state mRNA levels depend on both the transcriptional activity and mRNA half-life. The stability of mRNA can be modified by the binding of trans-acting factors to cis-elements on the message. These can protect the mRNA from cleavage by RNAses, or they may promote mRNA cleavage. Changes in mRNA stability can lead to changes in the proteome and subsequently in cellular metabolism. The SMAD family of proteins has been implicated in the transduction of the TGF-β signal, where they regulate transcriptional activity. This review attempts to provide new insights into the role played by TGF-β in the regulation of mRNA turnover.

Immobilized rhBMP-2/succinylated type I atelocollagen gene expression of intracellular signaling molecules on ST2 cells

Recombinant human bone morphogenetic protein-2 (rhBMP-2) chemically-bonded to succinylated type I atelocollagen, a biomaterial carrier with a porous structure, was reported to augment cellular activity of ST2 cells. The Smad protein family has been suggested to play an important role in the intracellular signaling pathway of BMP by its binding to receptors on target cells. However, there has been no study analyzing the downstream genes of the rhBMP-2 induced intracellular signal transduction pathway. The purpose of this study was to examine the effect of immobilized rhBMP-2 on gene expression of intracellular signaling molecules on ST2 cells. Our study showed two expression patterns of downstream genes of rhBMP-2 intracellular signal transduction pathway. In the first pattern, BMPR-IA, Smad 1, and Smad 5 genes showed high basic expression before the addition of rhBMP, and the high level of gene expression continued for long period and decreased in the late stage when rhBMP-2 was immobilized. In the second pattern, Smad 6, Smad 7, and Smad 8 genes showed low basic expression before the addition of rhBMP-2 and a continuous increase from the beginning was followed by a decrease in the late stage when rhBMP-2 was immobilized. Our results also showed that intracellular signaling continued for prolonged period when rhBMP-2 was immobilized to succinylated type I atelocollagen. This study indicated that immobilizing rhBMP-2 is an efficient method to increase bone induction.

Homology modeling of the DNA-binding domain of human Smad5: A molecular model for inhibitor design

Members of the Smad protein family function as signal transducers of the transforming growth factor (TGF-beta) superfamily proteins. The human Smad5 protein, a signal transducer downstream of TGF-beta/BMP receptors, is composed of N-terminal DNA binding domain (MH1) and C-terminal protein–protein interaction domain (MH2) connected together by a linker motif. We used homology-modeling techniques to generate a reliable molecular model of the Smad5 MH1 domain based on the crystal structure of Smad3 MH1 domain. Our study presents the structural features of a BMP-regulated, R-Smad subfamily member (consisting of Smad1, Smad5 and Smad8) for the first time. This model provides a structural basis for explaining both functional similarities and differences between Smad3 and Smad5. Also, the structural model of this molecular target would be useful for structure-based inhibitor design because of its high accuracy. The results of our study provide important insights into understanding the structure–function relationship of the members of the Smad protein family and can serve to guide future genetic and biochemical experiments in this area.

Interaction between the bone morphogenetic proteins and Ras/MAP-kinase signalling pathways in lung cancer

Bone morphogenetic proteins (BMPs) are an integral component of the TGFβ superfamily, responsible for regulation of cell proliferation, differentiation, migration and programmed cell death in a variety of cell types. The BMPs transduce their signals directly through the SMAD family of proteins but they also have been reported to interact with the MAPK and Erk pathways. Inactivation of the BMP pathway genes has been implicated as important in several cancers. Recent work has shown that BMP3b is epigenetically inactivated in cancer and suggests that BMP6 can be epigenetically inactivated. We investigated whether BMP6 is epigenetically inactivated in cell lines and whether BMP3b and BMP6 are epigenetically inactivated in non-small-cell lung cancer (NSCLC). We also studied the relationship between BMP methylation and k-ras mutation. Here, we demonstrate that the BMP3b and BMP6 genes are common targets of epigenetic inactivation in NSCLC, and that they are significantly more likely to be concurrently inactivated (P=0.009). Furthermore, this coinactivation of BMP3b and BMP6 is significantly associated with mutation of k-ras codon 12 in lung cancer (P=0.003); those with a k-ras mutation were six times more likely to have concurrent methylation of these BMP loci. Hence, these data suggest that concurrent inactivation of the BMP and activation of the Ras signalling pathways are important in lung carcinogenesis.