Epithelial to mesenchymal transition in Madin-Darby canine kidney cells is accompanied by down-regulation of Smad3 expression,leading to resistance to transforming growth factor-beta-induced growth arrest

In normal epithelial cells, transforming growth factor-beta (TGF-beta) typically causes growth arrest in the G(1) phase of the cell cycle and may eventually lead to apoptosis. However, transformed cells lose these inhibitory responses and often instead show an increase in malignant character following TGF-beta treatment. In the canine kidney-derived epithelial cell line, MDCK, synergism between activation of the Raf/MAPK pathway and the resulting autocrine production of TGF-beta triggers transition from an epithelial to a mesenchymal phenotype. During this process, these cells become refractive to TGF-beta-induced cell cycle arrest and apoptosis. TGF-beta signals are primarily transduced to the nucleus through complexes of receptor-regulated Smads, Smad2 and Smad3 with the common mediator Smad, Smad4. Here we show that the transition from an epithelial to mesenchymal phenotype is accompanied by gradual down-regulation of expression of Smad3. Restoration of Smad3 to previous levels of expression restores the cell cycle arrest induced by TGF-beta without reverting the cells to an epithelial phenotype or impacting on the MAPK pathway. Regulation of apoptosis is not affected by Smad3 levels. These data attribute to Smad3 a critical role in the control of cell proliferation by TGF-beta, which is lost following an epithelial to mesenchymal transition.     

Requirement of Ras/MAPK pathway activation by transforming growth factor beta for transforming growth factor beta 1 production in a Smad-dependent pathway

Our previous results have shown that transforming growth factor beta (TGFbeta) rapidly activates Ras, as well as both ERKs and SAPKs. In order to address the biological significance of the activation of these pathways by TGFbeta, here we examined the role of the Ras/MAPK pathways and the Smads in TGFbeta(3) induction of TGFbeta(1) expression in untransformed lung and intestinal epithelial cells. Expression of either a dominant-negative mutant of Ras (RasN17) or a dominant-negative mutant of MKK4 (DN MKK4), or addition of the MEK1 inhibitor PD98059, inhibited the ability of TGFbeta(3) to induce AP-1 complex formation at the TGFbeta(1) promoter, and the subsequent induction of TGFbeta(1) mRNA. The primary components present in this TGFbeta(3)-inducible AP-1 complex at the TGFbeta(1) promoter were JunD and Fra-2, although c-Jun and FosB were also involved. Furthermore, deletion of the AP-1 site in the TGFbeta(1) promoter or addition of PD98059 inhibited the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Collectively, our data demonstrate that TGFbeta(3) induction of TGFbeta(1) is mediated through a signaling cascade consisting of Ras, the MAPKKs MKK4 and MEK1, the MAPKs SAPKs and ERKs, and the specific AP-1 proteins Fra-2 and JunD. Although Smad3 and Smad4 were not detectable in TGFbeta(3)-inducible AP-1 complexes at the TGFbeta(1) promoter, stable expression of dominant-negative Smad3 could significantly inhibit the ability of TGFbeta(3) to stimulate TGFbeta(1) promoter activity. Transient expression of dominant-negative Smad4 also inhibited the ability of TGFbeta(3) to transactivate the TGFbeta(1) promoter. Thus, although the Ras/MAPK pathways are essential for TGFbeta(3) induction of TGFbeta(1), Smads may only contribute to this biological response in an indirect manner.     

Intracellular dynamics of Smad-mediated TGFbeta signaling

The transforming growth factor-beta (TGFbeta) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFbeta-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFbeta signaling system in cells of the developing murine palate.     

Antagonistic effects of Smad2 versus Smad7 are sensitive to their expression level during tooth development

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate cell proliferation, differentiation, and apoptosis, controlling the development and maintenance of most tissues. TGF-beta signal is transmitted through the phosphorylation of Smad proteins by TGF-beta receptor serine/threonine kinase. During early tooth development, TGF-beta inhibits proliferation of enamel organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-beta signaling during tooth development. Attenuation of Smad2 gene expression resulted in significant advancement of embryonic tooth development with increased proliferation of enamel organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic tooth development with increased apoptotic activity within enamel organ epithelium. These findings suggest that different Smads may have differential activities in regulating TGF-beta-mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but not Smad3, altered TGF-beta-mediated tooth development. The results indicate that Smads are critical factors in orchestrating TGF-beta-mediated gene regulation during embryonic tooth development. The effectiveness of TGF-beta signaling is highly sensitive to the level of Smad gene expression.     

Cirrhotic hepatocytes exhibit decreased TGFbeta growth inhibition associated with downregulated Smad protein expression

TGFbeta controls hepatocyte growth through cell cycle arrest and apoptosis, and resistance to TGFbeta is a mechanism of malignant transformation. The aim of this study was to assess differences in TGFbeta-mediated growth inhibition in normal and cirrhotic hepatocytes. Cirrhosis was induced in mice and normal and cirrhotic hepatocytes were isolated by collagenase perfusion and treated with or without TGFbeta (5 ng/ml). DNA synthesis, Smad protein expression, and DNA binding activity were determined. TGFbeta reduced DNA synthesis to a greater degree in normal hepatocytes than in cirrhotic hepatocytes (87% vs. 68%; p<0.05). Smad protein expression was decreased in cirrhotic hepatocytes and Smad 2/3/4 complex formation was suppressed. Furthermore, cirrhotic hepatocytes had decreased DNA binding activity at 120 min following TGFbeta treatment. In conclusion, decreased Smad protein expression may impair TGFbeta-mediated growth inhibition in cirrhotic hepatocytes.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

Expression of Smads during in vitro transdifferentiation of hepatic stellate cells to myofibroblasts

TGFbeta is of crucial importance during transdifferentiation of resting retinoid-storing hepatic stellate cells (HSC) to extracellular matrix producing myofibroblasts (MFB) and consequently, inhibition of TGFbeta signal transduction is an effective means for preventing experimental fibrosis. We have shown that isolated HSC lose TGFbeta-dependent growth control during in vitro activation and that alpha2 (I) collagen production in transdifferentiated MFB is TGFbeta-independent. Furthermore, Smad complexes with SBE binding activity were only detected in early cultures of HSC, although TGFbeta receptor types I and II were significantly expressed in HSC and MFB. In the present report, we compared the expression pattern of TGFbeta downstream mediators, i.e., the Smads, in TGFbeta responsive HSC versus nonresponding MFB. The transdifferentiation process was monitored by morphology and increasing expression of TGFbeta and alpha-smooth muscle actin, and TGFbeta signaling was investigated by (CAGA)(9)-MLP-Luc. The expression level of all Smads remained essentially unchanged both during the activation process and after TGFbeta-treatment. Smad7 was transiently upregulated upon TGFbeta stimulation in quiescent HSC, indicating a negative feed back loop in responsive cells. In contrast, MFB neither displayed TGFbeta-inducible nor constitutively upregulated Smad7 expression. Instead, Smad3 mRNA was increased in MFB. Our data indicate that abrogation of the TGFbeta response in MFB versus HSC is not based on different regulation of Smad expression.     

Inhibition of TGFbeta1-mediated PAI-1 induction by oltipraz through selective interruption of Smad 3 activation

Transforming growth factor-beta1 (TGFbeta1) induces plasminogen activator inhibitor-1 (PAI-1) as a major target protein. PAI-1 is associated with fibrosis, thrombosis, and metabolic disorders. TGFbeta1 induces PAI-1 via phosphorylation and nuclear translocation of Smads. Oltipraz inhibits TGFbeta1 expression and also regenerates cirrhotic liver. Nevertheless, whether oltipraz modulates TGFbeta1-mediated cell signaling is unclear. First, this study examined the effect of oltipraz on PAI-1 expression in cirrhotic rat liver. The cells immunochemically stained with anti-PAI-1 antibody accumulated around and within fibrous nodules in cirrhotic liver, which was notably decreased by oltipraz treatment. Next, whether oltipraz inhibits TGFbeta1-mediated Smads activation or Smad-mediated PAI-1 induction was determined in L929 fibroblasts. Oltipraz inhibited the ability of TGFbeta1 to induce PAI-1, as indicated by repression of TGFbeta1-mediated luciferase induction from the plasmid comprising the human PAI-1 promoter and of TGFbeta1-induced Smad-DNA-binding activity. TGFbeta1 induced nuclear transport of receptor-regulated Smad 2 and Smad 3, of which oltipraz selectively inhibited the transport and phosphorylation of Smad 3, thereby reducing formation of Smad 3/4 complex in the nucleus. In summary, oltipraz inhibits PAI-1 induction via a decrease in the formation of Smad 3/4 complex due to selective interruption of Smad 3 activation, indicating that oltipraz regulates the cellular responses downstream of ligand-activated TGFbeta1 receptor.     

Smad3 in the mammary epithelium has a nonredundant role in the induction of apoptosis, but not in the regulation of proliferation or differentiation by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) regulates proliferation, morphogenesis, and functional differentiation in the mammary gland and plays complex roles in mammary tumorigenesis. Here we show that the signaling mediators Smad1-Smad5 are expressed at all stages of mammary gland development. To begin to investigate which Smads mediate which TGF-beta responses, we have analyzed mammary gland development in Smad3 null mice. Smad3 null virgin females showed delayed mammary gland development. However, this phenotype was secondary to ovarian insufficiency because Smad3 null mammary epithelium developed normally in hormonally supplemented Smad3 null mice or when transplanted into wild-type hosts. Absence of Smad3 had no effect on the ability of TGF-beta to inhibit the growth of mammary epithelial cells in culture, and no compensatory changes in expression or activation of Smad2 were seen in the Smad3 null epithelium. A small but significant decrease in apoptotic cells was seen in involuting glands from Smad3 null transplants. The results suggest that epithelial Smad3 is dispensable for TGF-beta effects on proliferation and differentiation in the mammary gland, but that it contributes in a nonredundant manner to the induction of apoptosis.