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.     

Maternal Smad3 deficiency compromises decidualization in mice

Transforming growth factor (TGF)‐β and activin, members of TGF‐β superfamily, are abundantly expressed in the endometrium and regulate decidualization of endometrial stroma. Smad2 and Smad3 are receptor‐regulated Smads (R‐Smads) that transduce extracellular TGF‐β/activin/Nodal signaling. In situ hybridization results showed that Smad3 was highly expressed in the decidual zone during the peri‐implantation period in mice. By using artificial decidualization, we found that Smad3 null mice showed partially compromised decidualization. We therefore hypothesized that Smad2 might compensate for the function of Smad3 during the process of decidualization. Smad2 was also highly expressed in the decidual zone and phosphorylated Smad2 was much more abundantly increased in the deciduoma of Smad3 null mice than for wild‐type (WT) mice. We further employed an in vitro uterine stromal cell decidualization model, and found that decidual prolactin‐related protein (dPRP) and cyclin D3, which are well‐known markers for decidual cells, were significantly down‐regulated in Smad3 null decidual cells, and were much more significantly reduced when the expression of Smad2 was simultaneously silenced by its siRNA (P < 0.05). However, the expression levels of dPRP and cyclin D3 remained the same when Smad2 was silenced in WT decidual cells. Collectively, these findings provide evidence for an important role of Smad3 in decidualization and suggest that Smad2 and Smad3 may have redundant roles in decidualization. J. Cell. Biochem. 113: 3266–3275, 2012. © 2012 Wiley Periodicals, Inc.     

Liver fibrogenesis due to cholestasis is associated with increased Smad7 expression and Smad3 signaling

BACKGROUND/AIMS: Profibrogenic TGF-beta signaling in hepatic stellate cells is modulated during transdifferentiation. Strategies to abrogate TGF-beta effects provide promising antifibrotic results, however, in vivo data regarding Smad activation during fibrogenesis are scarce. METHODS: Here, liver fibrosis was assessed subsequent to bile duct ligation by determining liver enzymes in serum and collagen deposition in liver tissue. Activated hepatic stellate cells were identified by immunohistochemistry and immunoblots for alpha smooth muscle actin. Cellular localization of Smad3 and Smad7 proteins was demonstrated by immunohistochemistry. RTPCR for Smad4 and Smad7 was conducted with total RNA and Northern blot analysis for Smad7 with mRNA. Whole liver lysates were prepared to detect Smad2/3/4 and phospho- Smad2/3 by Western blotting. RESULTS: Cholestasis induces TGF-beta signaling via Smad3 in vivo, whereas Smad2 phosphorylation was only marginally increased. Smad4 expression levels were unchanged. Smad7 expression was continuously increasing with duration of cholestasis. Hepatocytes of fibrotic lesions exhibited nuclear staining Smad3. In contrast to this, Smad7 expression was localized to activated hepatic stellate cells. CONCLUSIONS: Hepatocytes of damaged liver tissue display increased TGF-beta signaling via Smad3. Further, negative feedback regulation of TGF-beta signaling by increased Smad7 expression in activated hepatic stellate cells occurs, however does not interfere with fibrogenesis.     

5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.     

Regulation of osteoclastogenesis and RANK expression by TGF-beta1

Transforming growth factor-beta (TGF-beta) has been shown to both inhibit and to stimulate bone resorption and osteoclastogenesis. This may be due, in part, to differential effects on bone marrow stromal cells that support osteoclastogenesis vs. direct effects on osteoclastic precursor cells. In the present study, we used the murine monocytic cell line, RAW 264.7, to define direct effects of TGF-beta on pre-osteoclastic cells. In the presence of macrophage-colony stimulating factor (M-CSF) (20 ng/ml) and receptor activator of NF-kappaB ligand (RANK-L) (50 ng/ml), TGF-beta1 (0.01-5 ng/ml) dose-dependently stimulated (by up to 120-fold) osteoclast formation (assessed by the presence of tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells and expression of calcitonin and vitronectin receptors). In addition, TGF-beta1 also increased steady state RANK mRNA levels in a time- (by up to 3.5-fold at 48 h) and dose-dependent manner (by up to 2.2-fold at 10 ng/ml). TGF-beta1 induction of RANK mRNA levels was present both in undifferentiated RAW cells as well as in cells that had been induced to differentiate into osteoclasts by a 7-day treatment with M-CSF and RANK-L. Using a fluorescence-labeled RANK-L probe, we also demonstrated by flow cytometry that TGF-beta1 resulted in a significant increase in the percentage of RANK+ RAW cells (P < 0.05), as well as an increase in the fluorescence intensity per cell (P < 0.05), the latter consistent with an increase in RANK protein expression per cell. These data thus indicate that TGF-beta directly stimulates osteoclastic differentiation, and this is accompanied by increased RANK mRNA and protein expression.     

TGF-beta control of cell proliferation

This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.     

Wnt-mediated regulation of chondrocyte maturation: modulation by TGF-beta

Wnt proteins are expressed during limb morphogenesis, yet their role and mechanism of action remains unclear during long bone growth. Wnt expression, effects and modulation of signaling events by BMP and transforming growth factor-beta (TGF-beta) were evaluated in chick embryonic chondrocytes. Chondrocyte cell cultures underwent spontaneous maturation with increased expression of colX and this was associated with an increase in the expression of multiple Wnts, including Wnt 4, 5a, 8c, and 9a. Both parathyroid hormone related peptide (PTHrP) and TGF-beta inhibited colX, but had disparate effects on Wnt expression. While TGF-beta strongly inhibited all Wnts, PTHrP did not inhibit either Wnt8c or Wnt9a and had lesser effects on the expression of the other Wnts. BMP-2 induced colX expression, and also markedly increased Wnt8c expression. Overexpression of beta-catenin and/or T cell factor (TCF)-4 also induced the type X collagen promoter. Overexpression of Wnt8c induced maturation, as did overexpression of beta-catenin. The Wnt8c/beta-catenin maturational effects were enhanced by BMP-2 and inhibited by TGF-beta. TGF-beta also inhibited activation of the Topflash reporter by beta-catenin, suggesting a direct inhibitory effect since the Topflash reporter contains only beta-catenin binding sequences. In turn beta-catenin inhibited activation of the p3TP-Luc reporter by TGF-beta, although the effect was partial. Thus, Wnt/beta-catenin signaling is a critical regulator of the rate of chondrocyte differentiation. Moreover, this pathway is modulated by members of the TGF-beta family and demonstrates the highly integrated nature of signals controlling endochondral ossification.     

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.     

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.     

Differential expression of transforming growth factor-beta in the interstitial tissue of testis during aging

Transforming growth factor-betas (TGF-betas) have significant effects on testis development. The pattern of TGF-beta expression in aging testis has not been established to date. We examined age-related changes in the expression of TGF-beta and its receptors in the testis using Western blot analysis. TGF-beta1 expression increased continuously in aging rat testis, whereas no age-associated changes were observed for TGF-beta3. Strong expression of TGF-beta2, as well as type I and II receptors was observed in 12-month-old testis, but following this time, expression decreased dramatically. Interestingly, TGF-beta2 and -beta3 displayed strong and similar expression patterns in liver, regardless of age, suggesting that the down-regulation of TGF-beta2 is testis-specific. We observed significant induction of p53 and p21WAF1 in 18-month-old testis that appeared to correspond with aging. Moreover, caloric restriction (CR) prevented age-related decrease in TGF-beta2 expression. Using immunohistochemistry, we showed that all TGF-beta1, -beta2, and -beta3 proteins are expressed primarily in interstitial cells, which are located in the space between adjoining seminiferous tubules. Our data collectively indicate that aging in the testis is regulated by differential expression of TGF-beta proteins, and decreased levels of TGF-beta2 contribute to the aging process.