Smad and p38-MAPK signaling mediates apoptotic effects of transforming growth factor-beta1 in human airway epithelial cells

Transforming growth factor-beta1 (TGF-beta1) belongs to a family of multifunctional cytokines that regulate a variety of biological processes, including cell differentiation, proliferation, and apoptosis. The effects of TGF-beta1 are cell context and cell cycle specific and may be signaled through several pathways. We examined the effect of TGF-beta1 on apoptosis of primary human central airway epithelial cells and cell lines. TGF-beta1 protected human airway epithelial cells from apoptosis induced by either activation of the Fas death receptor (CD95) or by corticosteroids. This protective effect was blocked by inhibition of the Smad pathway via overexpression of inhibitory Smad7. The protective effect is associated with an increase in the cyclin-dependent kinase inhibitor p21 and was blocked by the overexpression of key gatekeeper cyclins for the G1/S interface, cyclins D1 and E. Blockade of the Smad pathway by overexpression of the inhibitory Smad7 permitted demonstration of a TGF-beta-mediated proapoptotic pathway. This proapoptotic effect was blocked by inhibition of the p38 MAPK kinase signaling with the inhibitor SB-203580 and was associated with an increase in p38 activity as measured by a kinase assay. Here we demonstrate dual signaling pathways involving TGF-beta1, an antiapoptotic pathway mediated by the Smad pathway involving p21, and an apoptosis-permissive pathway mediated in part by p38 MAPK.     

Activation of the pro-survival phosphatidylinositol 3-kinase/AKT pathway by transforming growth factor-beta1 in mesenchymal cells is mediated by p38 MAPK-dependent induction of an autocrine growth factor

Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine involved in differentiation, growth, and survival of mesenchymal cells while inhibiting growth/survival of most other cell types. The mechanism(s) of pro-survival signaling by TGF-beta1 in mesenchymal cells is unclear. In this report, we demonstrate that TGF-beta1 protects against serum deprivation-induced apoptosis of mesenchymal cells isolated from patients with acute lung injury and of normal human fetal lung fibroblasts (IMR-90). TGF-beta receptor(s)-activated signaling in these cells involves rapid activation of the Smad and p38 MAPK pathways within minutes of TGF-beta1 treatment followed by a more delayed activation of the pro-survival phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway. Pharmacological inhibition of p38 MAPK with SB203580 or expression of a p38 kinase-deficient mutant protein inhibits TGF-beta1-induced PKB/Akt phosphorylation. Conditioned medium from TGF-beta1-treated cells rapidly induces PKB/Akt activation in an SB203580- and suramin-sensitive manner, suggesting p38 MAPK-dependent production of a secreted growth factor that activates this pro-survival pathway by an autocrine/paracrine mechanism. Inhibition of the phosphatidylinositol 3-kinase-PKB/Akt pathway blocks TGF-beta1-induced resistance to apoptosis. These results demonstrate the activation of a novel TGF-beta1-activated pro-survival/anti-apoptotic signaling pathway in mesenchymal cells/fibroblasts that may explain cell-specific actions of TGF-beta1 and provide mechanistic insights into its pro-fibrotic and tumor-promoting effects.     

Differential activation of Smads in HeLa and SiHa cells that differ in their response to transforming growth factor-beta

We assessed the responsiveness of six human cervical cancer cell lines to transforming growth factor (TGF)-beta with p3TP-lux reporter assay and found that HeLa and SiHa cells were highly responsive to TGF-beta. However, when pSBE4-BV/Luc reporter with four Smad binding elements was used, only the SiHa, not the HeLa, cells showed Smad activation. Smad DNA binding activity was relatively more in SiHa than in HeLa cells upon TGF-beta treatment, and the active complex contained Smad 2 and Smad 4. In 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, HeLa cells treated with 5 ng/ml of TGF-beta for 24 h showed proliferation, whereas SiHa cells showed growth inhibition under the same conditions. TGF-beta treatment resulted in G(0)/G(1) arrest with a reduction in S-phase only in SiHa cells. A chemical inhibitor of Smad activation (SB203580) blocked the growth inhibitory effect of TGF-beta in SiHa, whereas the proliferative response in HeLa was unaffected. TGF-beta-induced translocation of phospho-Smad 2 was relatively less in HeLa than in SiHa cells. MAPK activation occurred within 5 min and persisted up to 15 min upon TGF-beta treatment in HeLa but was negligible in SiHa cells. TGF-beta activated JNK in HeLa, but SiHa cells showed a down-regulation of its activity. When an inhibitor of MAPK (U0126) was used, the TGF-beta-mediated proliferative response in HeLa cells was completely abolished. SB203580 did not affect MAPK activation induced by TGF-beta in HeLa cells. We report for the first time an activation, presumably independent of Smad activation, of TGF-beta-dependent MAPK within 5 min of treatment that resulted in cell cycle progression in a cervical adenocarcinoma cell line, HeLa.     

Smad7 abrogates transforming growth factor-beta1-mediated growth inhibition in COLO-357 cells through functional inactivation of the retinoblastoma protein

Smad7 is overexpressed in 50% of human pancreatic cancers. COLO-357 pancreatic cancer cells engineered to overexpress Smad7 are resistant to the actions of transforming growth factor-beta1 (TGF-beta1) with respect to growth inhibition and cisplatin-induced apoptosis but not with respect to modulation of gene expression. To delineate the mechanisms underlying these divergent consequences of Smad7 overexpression, we studied the effects of Smad7 on TGF-beta1-dependent signaling pathways and cell cycle regulating proteins. TGF-beta1 induced the phosphorylation of MAPK, p38 MAPK, and AKT2 irrespective of the levels of Smad7, and inhibitors of these pathways did not alter TGF-beta1 actions on cell growth. By contrast, Smad7 overexpression interfered with TGF-beta1-mediated attenuation of cyclin A and B levels, inhibition of cdc2 dephosphorylation and CDK2 inactivation, up-regulation of p27, and the maintenance of the retinoblastoma protein (RB) in a hypophosphorylated state. Smad7 also suppressed TGF-beta1-mediated inhibition of E2F activity but did not alter TGF-beta1-mediated phosphorylation of Smad2, the nuclear translocation of Smad2/3/4, or DNA binding of the Smad2/3/4 complex. Although Smad7 did not associate with the type I TGF-beta receptor (TbetaRI), SB-431542, an inhibitor of the kinase activity of this receptor, blocked TGF-beta1-mediated effects on Smad-2 phosphorylation. These findings point toward a novel paradigm whereby Smad7 acts to functionally inactivate RB and de-repress E2F without blocking the activation of TbetaRI and the nuclear translocation of Smad2/3, thereby allowing for TGF-beta1 to exert effects in a cancer cell that is resistant to TGF-beta1-mediated growth inhibition.     

Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts

Transforming growth factor-beta (TGF-beta) is a prototypical tumour-suppressor cytokine with cytostatic and pro-apoptotic effects on most target cells; however, mechanisms of its pro-survival/anti-apoptotic signalling in certain cell types and contexts remain unclear. In human lung fibroblasts, TGF-beta1 is known to induce myofibroblast differentiation in association with the delayed activation of focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Here, we demonstrate that FAK and AKT are independently regulated by early activation of SMAD3 and p38 MAPK, respectively. Pharmacologic or genetic approaches that disrupt SMAD3 signalling block TGF-beta1-induced activation of FAK, but not AKT; in contrast, disruption of early p38 MAPK signalling abrogates AKT activation, but does not alter FAK activation. TGF-beta1 is able to activate AKT in cells expressing mutant FAK or in cells treated with an RGD-containing peptide that interferes with integrin signalling, inhibits FAK activation and induces anoikis (apoptosis induced by loss of adhesion signalling). TGF-beta1 protects myofibroblasts from anoikis, in part, by activation of the PI3K-AKT pathway. Thus, TGF-beta1 co-ordinately and independently activates the FAK and AKT protein kinase pathways to confer an anoikis-resistant phenotype to myofibroblasts. Activation of these pro-survival/anti-anoikis pathways in myofibroblasts likely contributes to essential roles of TGF-beta1 in tissue fibrosis and tumour-promotion.     

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.     

TGF-beta inhibits IL-2 production and promotes cell cycle arrest in TCR-activated effector/memory T cells in the presence of sustained TCR signal transduction

TGF-beta signaling is critical for controlling naive T cell homeostasis and differentiation; however, the biological and biochemical changes induced by TGF-beta in effector/memory T cells are poorly defined. We show that although TGF-beta inhibits effector/memory peripheral blood T lymphoblast proliferation and IL-2 production, the intensity and kinetics for TCR-induced global tyrosine phosphorylation are markedly increased compared with that in untreated cells or naive T cells. After TCR ligation, tyrosine phosphorylation of proximal tyrosine kinases and docking proteins like linker for activation of T cells is maintained for >30 min in TGF-beta-primed cells compared with untreated cells where phosphorylation of these targets returned to basal levels by 10 min. Extended phosphorylation of linker for activation of T cells in treated peripheral blood T selectively prolongs ERK 1/2 signaling and phospholipase C-gamma1 activation leading to increased Ca(2+) flux. A kinase/phosphatase imbalance could not account for extended phosphorylation as CD45R, SHP-1, and SHP-2 expression remains unaltered. The contradiction between prolonged signal transduction and inhibition of proliferation is partially explained by the observation that TGF-beta priming results in ERK 1/2-independent p21 induction and decreased cyclin D1 expression leading to accumulation of T cells in G(0)/G(1) phases of the cell cycle and cell cycle arrest. Despite inhibition of T cell function by TGF-beta priming, TCR and cytokine signaling pathways are intact and selectively extended, suggesting that suppression in the effector/memory T cell is mediated by reprogramming signal transduction, rather than its inhibition as in the naive T cell.     

Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-beta

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-beta is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-beta1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-beta1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-beta1 on ASM cells was inhibited by selective inhibitors of TGF-beta receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-kappaB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-beta1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-beta1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-beta1-induced proliferation. Our findings suggest that TGF-beta1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-kappaB is also involved in the TGF-beta1 mitogenic signaling, but Smad pathway does not appear important.