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.     

Epidermal growth factor up-regulates transforming growth factor-beta receptor type II in human dermal fibroblasts via p38 mitogen-activated protein kinase pathway

TGF-beta receptors (TbetaRs) are serine/threonine kinase receptors that bind to TGF-beta and propagate intracellular signaling through Smad proteins. TbetaRs are repressed in some human cancers and expressed at high levels in several fibrotic diseases. We demonstrated that epidermal growth factor (EGF) up-regulates type II TGF-beta receptor (TbetaRII) expression in human dermal fibroblasts. EGF-mediated induction of TbetaRII expression was inhibited by the treatment of fibroblasts with a specific p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, whereas MEK inhibitor PD98059 did not block the up-regulation of TbetaRII by EGF. EGF induced the TbetaRII promoter activity, and this induction was significantly blocked by SB203580, but not by PD98059. The overexpression of the dominant negative form of p38alpha or p38beta significantly reduced the induction of TbetaRII promoter activity by EGF. These results indicate that the EGF-mediated induction of TbetaRII expression involves the p38 MAPK signaling pathway. The EGF-mediated induction of TbetaRII expression may participate in a synergistic interplay between EGF and TGF-beta signaling pathway.     

Antagonistic effect of EGF on FAK phosphorylation/dephosphorylation in a cell

Focal adhesion kinase (FAK) plays a key role in the crosstalk of growth factor- and cell adhesion-mediated signaling pathway. In this study, we found that the quantitative change of phosphorylated FAK was bell-shaped time-dependently by EGF stimulation in immortalized human keratinocyte (HaCaT). EGF enhanced FAK phosphorylation and cell spreading in adhering HaCaT cells with low-phosphorylated FAK. On the other hand, spread HaCaT cells having high-phosphorylated FAK changed to round shapes with FAK dephosphorylation 15 min after EGF stimulation. Pharmacological agents, U0126 and PD98059 (mitogen-activated protein kinases (MAPK) kinases (MEK) inhibitors), and AG1478 (an EGF receptor kinase inhibitor) blocked the cell rounding and FAK dephosphorylation. In addition, the EGFR-MAPK signaling pathway had an influence on cell migration by regulating FAK dephosphorylation of keratinocytes in response of EGF, since the MEK inhibitors and AG1478 suppressed EGF-induced cell migration. However, FAK phosphorylation and HaCaT cell spreading were inhibited only by the antagonist of EGF-EGFR binding but not by the MEK inhibitors and AG1478. Taken together, we suggest that EGF is antagonistically involved in both FAK phosphorylation and dephosphorylation with different mechanisms in a cell.     

Perturbation of EGF-activated MEK1 and PKB signal pathways by TGF-beta1 correlates with perturbation of EGF-induced cyclin D1 and DNA synthesis by TGF-beta1 in C3H 10T1/2 cells

In mouse C3H 10T1/2 cells, we previously reported that TGF-beta1 first delays and later potentiates EGF-induced DNA synthesis corresponding to an inhibition of EGF-induced cyclin D1 expression at t = 13 h. We report here that in accord with DNA synthesis kinetics, TGF-beta1 initially suppresses EGF-induced cyclin D1 expression then later releases the inhibition. Furthermore, TGF-beta1 also first decreases and later potentiates the levels of EGF-activated MEK1/MAPK and PKB, indicating the existence of cross talk between TGF-beta 1- and EGF-activated signal transduction pathways. PD98059, the specific inhibitor of MEK1, significantly blocks EGF-induced DNA synthesis, whereas wortmannin, the PI3K inhibitor, exerts a modest inhibitory effect, which suggests that the activation of MEK1-MAPK pathway plays a major role in EGF-induced DNA synthesis and the activation of PI3K-PKB pathway plays a minor role. Upon examination of mechanisms underlying the cross talk, it was discovered that application of TGF-beta1 triggers a rapid association between Raf-1 and catalytic subunits of PKA, which are reported to be able to inactivate Raf-1 upon activation. Therefore, TGF-beta1 may activate PKA to inhibit the EGF-activated MEK1-MAPK pathway. The wortmannin-sensitive phosphorylation at the thr(389) site is necessary for activation of p70s6K, an important kinase involved in mitogen-stimulated protein synthesis. Although we found that EGF-stimulated p70s6K phosphorylates through a MAPK-dependent and a MAPK-independent (wortmannin-sensitive) pathway, TGF-beta1 failed to block EGF-triggered phosphorylation of p70s6K at thr(389) and thr(421)/ser(424) sites, implying that PKB inhibition by TGF-beta1 may result from inhibition of PDK1 activity instead of inhibition of PI3K activity. These data also suggest that TGF-beta1 may selectively perturb certain EGF-activated MAPK pools.     

p21Cip1 and p27Kip1 act in synergy to alter the sensitivity of naive T cells to TGF-beta-mediated G1 arrest through modulation of IL-2 responsiveness

Induction of G(1) arrest by TGF-beta correlates with the regulation of p21(Cip1) and p27(Kip1), members of the Cip/Kip family of cyclin-dependent kinase inhibitors (cki). However, no definitive evidence exists that these proteins play a causal role in TGF-beta(1)-induced growth arrest in lymphocytes. In this report we show the suppression of cell cycle progression by TGF-beta is diminished in T cells from mice deficient for both p21(Cip1) and p27(Kip1) (double-knockout (DKO)) only when activated under conditions of optimal costimulation. Although there is an IL-2-dependent enhanced proliferation of CD8(+) T cells from DKO mice, TGF-beta is able to maximally suppress the proliferation of DKO T cells when activated under conditions of low costimulatory strength. We also show that the induction of p15(Ink4b) in T cells stimulated in the presence of TGF-beta is not essential, as TGF-beta also efficiently suppressed proliferation of T cells from p15(Ink4b-/-) mice. Finally, although these cki are dispensable for the suppression of T cell proliferation by TGF-beta, we now describe a Smad3-dependent down-regulation of cdk4, suggesting a potential mechanism underlying to resistance of Smad3(-/-) T cells to the induction of growth arrest by TGF-beta. In summary, the growth suppressive effects of TGF-beta in naive T cells are a function of the strength of costimulation, and alterations in the expression of cki modify the sensitivity to TGF-beta by lowering thresholds for a maximal mitogenic response.     

Transforming growth factor-beta stimulates parathyroid hormone-related protein and osteolytic metastases via Smad and mitogen-activated protein kinase signaling pathways

Transforming growth factor (TGF)-beta promotes breast cancer metastasis to bone. To determine whether the osteolytic factor parathyroid hormone-related protein (PTHrP) is the primary mediator of the tumor response to TGF-beta, mice were inoculated with MDA-MB-231 breast cancer cells expressing a constitutively active TGF-beta type I receptor. Treatment of the mice with a PTHrP-neutralizing antibody greatly decreased osteolytic bone metastases. There were fewer osteoclasts and significantly decreased tumor area in the antibody-treated mice. TGF-beta can signal through both Smad and mitogen-activated protein (MAP) kinase pathways. Stable transfection of wild-type Smad2, Smad3, or Smad4 increased TGF-beta-stimulated PTHrP secretion, whereas dominant-negative Smad2, Smad3, or Smad4 only partially reduced TGF-beta-stimulated PTHrP secretion. When the cells were treated with a variety of protein kinases inhibitors, only specific inhibitors of the p38 MAP kinase pathway significantly reduced both basal and TGF-beta-stimulated PTHrP production. The combination of Smad dominant-negative blockade and p38 MAP kinase inhibition resulted in complete inhibition of TGF-beta-stimulated PTHrP production. Furthermore, TGF-beta treatment of MDA-MB-231 cells resulted in a rapid phosphorylation of p38 MAP kinase. Thus, the p38 MAP kinase pathway appears to be a major component of Smad-independent signaling by TGF-beta and may provide a new molecular target for anti-osteolytic therapy.     

Modulation of cyclin D1 and its signaling components by the phorbol ester TPA and the tyrosine phosphatase inhibitor vanadate

The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) triggers cell-cycle progression at G1 phase in mouse embryonic fibroblast C3H 10T1/2 cells was examined. TPA treatment resulted in a temporary induction of cyclin D1 peaking at 9 h post stimulation. PD98059 (10 microM), the specific inhibitor of MAPK kinase, completely blocked TPA-stimulated cyclin D1 induction and DNA synthesis, confirming that MAPK activation plays an essential role in TPA-stimulated cell-cycle progression. Although both PKCalpha and PKCepsilon are expressed in C3H 10T1/2 cells, inhibitor studies suggest that PKCepsilon activation is required for the activation of MEK/MAPK signal transduction cascade. p70s6K, an important kinase involved in the regulation of protein synthesis and cell-cycle progression, has been reported to be activated through a PKC-dependent pathway (TPA-activatable) in addition to a PI3K-dependent pathway. Here, we demonstrate for the first time that TPA-stimulated MAPK activation is essential for the phosphorylation of several key residues involved in the activation of p70s6K, namely, thr389, thr421, and ser424. Vanadate, the tyrosine phosphatase inhibitor, triggered a sustained elevation of the level of active MAPK. However, corresponding to a rapid loss of cyclin D1 protein, vanadate treatment resulted in a significant shut out of 3H-thymidine incorporation into DNA regardless of TPA cotreatment. Vanadate treatment also led to the increase of active MEK, increased phosphorylation of p70s6K at thr389, thr421, and ser424 yet without activation of PKB. These data suggest that vanadate can selectively perturb the activation of signaling components which raises the interesting issue as to how vanadate downregulates the cyclin D1 level.