Using RNA interference to identify the different roles of SMAD2 and SMAD3 in NIH/3T3 fibroblast cells

Smad proteins are principal intracellular signaling mediators of transforming growth factor beta (TGF-beta) that regulate a wide range of biological processes. However, the identities of Smad partners mediating TGF-beta signaling are not fully understood. We firstly examined the expression of Smad2 and Smad3 induced by TGF-beta 1 in normal NIH/3T3 cells. The expression of Smad2 and Smad3 was assessed by RT-PCR and Western blotting. The results showed that the expression of Smad2 was increased after treatment with TGF-betaI, but Smad3 was more sensitive to TGF-betaI than Smad2. RNA interference (RNAi) provides a new approach for elucidation of gene function. Use of hairpin siRNA expression vectors for RNAi has provided a rapid and versatile method for assessing gene function in mammalian cells. Here, we have constructed Smad2 and Smad3 hairpin siRNA expression plasmids, and then transfected them into mouse NIH/3T3 cells. Endogenous Smad2 and Smad3 proteins decreased significantly at 48 h after transfection. We found the expression of Smad3 in Smad2-depleted cells was increased, however, the expression of Smad2 in Smad3-depleted cells was not changed. Consistently, the expression of Smad4 mRNA was also attenuated in Smad3-depleted cells. From these data, we suggest that Smad3, but not Smad2, may play a key role in TGF-beta signaling.     

Altered signal transduction in Folr1-/- mouse embryo fibroblasts

Mice lacking the gene for Folr1 (folic acid receptor 1) have an NTD (neural tube defect) that is rescued by maternal folate supplementation. Primary cultures of MEFs (mouse embryonic fibroblasts) were established from these embryos and the effect on various signalling pathways examined. TGFβ1 (transforming growth factor β1) inhibited the proliferation of wild-type and Folr1-/- MEFs, and folate restriction, either in growth medium or through folate uptake, led to further inhibition of growth. This effect may be Smad-independent because reporter assays using the Smad-dependent reporter, p3TP-lux, revealed attenuation of TGFβ1/Smad signalling in Folr1-/- MEFs. Signalling through the canonical Wnt pathway, measured by Wnt-3a stimulated expression of the target gene, Axin2, demonstrated increased activity in Folr1-/- MEFs. Only minor changes in the expression of a panel of TGFβ (transforming growth factor β) and Wnt pathway-associated genes were revealed when Folr1-/- MEFs were compared with wild-type cells. These results demonstrate that under conditions of reduced folate (Folr-/-) signalling, pathways crucial for proper development of the neural tube are significantly altered.     

A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling

Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.     

The TGFbeta/Smad 3-signaling pathway is involved in butyrate-mediated vitamin D receptor (VDR)-expression

Previously, we demonstrated the pivotal role of the vitamin D receptor (VDR) in mediating the butyrate-induced differentiation in colon cancer cells. Smad 3, a downstream component of transforming growth factor-beta (TGFbeta) signaling, has been shown to act as a coactivator of VDR and to possibly regulate the vitamin D signaling pathway. In this study, we demonstrate a distinct impact of the TGFbeta/Smad 3-signaling pathway in the butyrate-mediated VDR expression and induction of differentiation. Butyrate treatment resulted in a significant induction of the phosphorylation level of Smad 3, while the combination of butyrate and a specific TGFbeta1-antibody or a TGFbeta-receptor inhibitor considerably diminished the butyrate-induced upregulation of VDR expression. Using a specific inhibitor, we were also able to demonstrate an involvement of the p38 MAPK in the increase of Smad 3 phosphorylation following butyrate treatment, thus opening the view to further elucidate possible mechanisms mediating the upregulation of VDR expression following butyrate treatment in colon cancer cells.     

Cartilage-derived morphogenetic proteins induce osteogenic gene expression in the C2C12 mesenchymal cell line

Cartilage-derived morphogenetic protein-1, -2, and -3 (CDMP-1, -2, and -3) are members of the bone morphogenetic protein (BMP) family and have been shown to exhibit a variety of biological activities. In the present study, effects of these CDMPs on the temporal and spatial expression of genes in the pluripotent mesenchymal cell line C2C12 were examined. Cells cultured in the presence of CDMPs lost the characteristic elongated shape of myoblasts. At the molecular level, CDMP treatment did not change the mRNA expression of MyoD, aggrecan, Six1, and tendin. Scleraxis mRNA level was reduced by CDMP treatment. CDMP-1 and -3, but not CDMP-2, stimulated expression of osteogenic markers, such as alkaline phosphatase (AP), osteocalcin (OC), BSP, and type I collagen, in a dose- and time-dependent manner. With few exceptions, the three CDMPs changed, with different potencies, the expression profile of different members of the BMP family in a similar temporal pattern. Except at the late phase of treatment, CDMP treatment did not change the expression of ActR-IA, BMPR-IA, BMPR-IB, BMPR-II, and ALK-7 mRNAs. Based on the current data, the CDMPs appear to be able to stimulate the C2C12 cells to differentiate into the osteoblast pathway.     

Rho activation is required for transforming growth factor-beta-induced epithelial-mesenchymal transition in lens epithelial cells

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. We have recently shown that TGF-beta-induced EMT in lens epithelial cells depends on PI3 kinase/Akt signal pathway. In this report, we suggest Smad3 is necessary for TGF-beta-induced EMT by showing that the expression of dominant-negative Smad3 blocks the expression of alpha-smooth muscle actin (alpha-SMA) and morphological changes. We also show that TGF-beta induces a biphasic change in Rho activity, and that Y27632, a selective inhibitor of Rho effector ROCK, inhibits TGF-beta-induced EMT in vitro and in vivo. We finally show that Smad3 activation and Rho signal activation is independent each other. All of these findings suggest that Rho/ROCK activation together with Smad3 is necessary for TGF-beta-induced EMT in lens epithelial cells.     

Inhibition of connective tissue growth factor/CCN2 expression in human dermal fibroblasts by interleukin‐1α and β

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein induced by transforming growth factor (TGF)‐β and intimately involved with tissue repair and overexpressed in various fibrotic conditions. We previously showed that keratinocytes in vitro downregulate TGF‐β‐induced expression of CTGF in fibroblasts by an interleukin (IL)‐1 α‐dependent mechanism. Here, we investigated further the mechanisms of this downregulation by both IL‐1α and β. Human dermal fibroblasts and NIH 3T3 cells were treated with IL‐1α or β in presence or absence of TGF‐β1. IL‐1 suppressed basal and TGF‐β‐induced CTGF mRNA and protein expression. IL‐1α and β inhibited TGF‐β‐stimulated CTGF promoter activity, and the activity of a synthetic minimal promoter containing Smad 3‐binding CAGA elements. Furthermore, IL‐1α and β inhibited TGF‐β‐stimulated Smad 3 phosphorylation, possibly linked to an observed increase in Smad 7 mRNA expression. In addition, RNA interference suggested that TGF‐β activated kinase1 (TAK1) is necessary for IL‐1 inhibition of TGF‐β‐stimulated CTGF expression. These results add to the understanding of how the expression of CTGF in human dermal fibroblasts is regulated, which in turn may have implications for the pathogenesis of fibrotic conditions involving the skin. J. Cell. Biochem. 110: 1226–1233, 2010. Published 2010 Wiley‐Liss, Inc.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

BMP2具有诱导C3H10T1/2细胞成脂肪分化的能力

探讨骨形态发生蛋白2（BMP2）诱导鼠胚胎间充质干细胞C3H10T1/2成脂肪分化能力,为临床脂肪代谢疾病的治疗提供理论基础．培养多潜能的间充质干细胞C3H10T1/2,用20 μg/ml BMP2对其诱导一定时间后,RT-PCR检测是否存在BMP信号通路中关键分子BMP受体BMPR I, BMPR Ⅱ及Smad 1/5/8的表达.Western印迹检测Smad 蛋白及MAPK 信号通路中p38磷酸化水平变化,QRT PCR检测成脂肪标志基因aP2以及成脂肪相关转录因子PPARγ,C/EBPα,C/EBPβ表达水平,同时用油红O染色,观测C3H10T1/2细胞成脂肪分化情况．经BMP2诱导后,C3H10T1/2细胞成脂肪分化标志(油红O染色)显著增加,Smad 蛋白及p38磷酸化水平有所上升,同时成脂肪标志基因aP2以及成脂肪相关转录因子PPARγ,C/EBPα,C/EBPβ表达水平各有一定程度提高．BMP2具有诱导C3H10T1/2细胞成脂肪分化能力,其成脂肪分化呈现对BMP2作用的时间依赖性．     

Opposing effects by glucocorticoid and bone morphogenetic protein-2 in fetal rat bone cell cultures

Glucocorticoid in excess produces bone loss in vivo. Consistent with this, it reduces the stimulatory effect of transforming growth factor β (TGF-β) on collagen synthesis in osteoblast-enriched cultures in vitro, where it also suppresses TGF-β binding to its type I receptors. Analogous studies with bone morphogenetic protein-2 (BMP-2) show directly opposite results. These findings prompted us to assess the effect of glucocorticoid on BMP-2 activity in cultured bone cells, and whether either agent had a dominant influence on TGF-β binding or function. BMP-2 activity was retained in part in osteoblast-enriched cultures pre-treated or co-treated with cortisol, and was fully evident when glucocorticoid exposure followed BMP-2 treatment. In addition, BMP-2 suppressed the effects of cortisol on TGF-β activity, on TGF-β binding, and on gene promoter activity directed by a glucocorticoid sensitive transfection construct. While BMP-2 also alters the function of less-differentiated bone cells, it only minimally prevented cortisol activity in these cultures. Our studies indicate that BMP-2 can oppose certain effects by cortisol on differentiated osteoblasts, and may reveal useful ways to diminish glucocorticoid-dependent bone wasting. J. Cell. Biochem. 67:528–540, 1997. © 1997 Wiley-Liss, Inc.     

Role of Smad3, acting independently of transforming growth factor‐β, in the early induction of Wnt‐β‐catenin signaling by parathyroid hormone in mouse osteoblastic cells

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt‐β‐catenin signaling pathway via the transforming growth factor (TGF)‐β signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF‐β‐independent in stimulating the osteoblast phenotype and PTH‐induced Wnt‐β‐catenin signaling. For this, the TGF‐β receptor type 1 [activin receptor‐like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total β‐catenin and reduced phosphorylated β‐catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3‐E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total β‐catenin and reduction of phosphorylated β‐catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF‐β receptor signaling. On the other hand, MC3T3‐E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated β‐catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3‐E1 cell clones in which wild‐type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the β‐catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast β‐catenin levels via Smad3 independently of, and dependently on, TGF‐β in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285–294, 2009. © 2009 Wiley‐Liss, Inc.