Integrin-linked kinase function is required for transforming growth factor beta-mediated epithelial to mesenchymal transition

The role of integrin-linked kinase (ILK) in transforming growth factor beta (TGFbeta)-mediated epithelial to mesenchymal transition was investigated. A stable transfection of dominant-negative ILK results in the prevention of TGFbeta-mediated E-cadherin delocalization. TGFbeta-mediated phosphorylation of Akt at Ser-473 was inhibited by dominant-negative ILK and PI3K inhibitors, LY294002 and wortmannin. Treatment with TGFbeta stimulated induction of Akt and ILK kinase activity in HaCat control cells. This increased ILK activity by TGFbeta was lowered by PI3K inhibitor, LY294002. In addition, PI3K inhibitor, dominant-negative Akt, and dominant-negative ILK could not block TGFbeta-mediated C-terminal phosphorylation of Smad2. Taken together, these data suggest that PI3K-ILK-Akt pathway that is independent of the TGFbeta-induced Smad pathway is required for TGFbeta-mediated epithelial to mesenchymal transition.     

Raptor-rictor axis in TGFbeta-induced protein synthesis

Transforming growth factor-beta (TGFbeta) stimulates pathological renal cell hypertrophy for which increased protein synthesis is critical. The mechanism of TGFbeta-induced protein synthesis is not known, but PI 3 kinase-dependent Akt kinase activity is necessary. We investigated the contribution of downstream effectors of Akt in TGFbeta-stimulated protein synthesis. TGFbeta increased inactivating phosphorylation of Akt substrate tuberin in a PI 3 kinase/Akt dependent manner, resulting in activation of mTOR kinase. mTOR activity increased phosphorylation of S6 kinase and the translation repressor 4EBP-1, which were sensitive to inhibition of both PI 3 kinase and Akt. mTOR inhibitor rapamycin and a dominant negative mutant of mTOR suppressed TGFbeta-induced phosphorylation of S6 kinase and 4EBP-1. PI 3 kinase/Akt and mTOR regulated dissociation of 4EBP-1 from eIF4E to make the latter available for binding to eIF4G. mTOR and 4EBP-1 modulated TGFbeta-induced protein synthesis. mTOR is present in two multi protein complexes, mTORC1 and mTORC2. Raptor and rictor are part of mTORC1 and mTORC2, respectively. shRNA-mediated downregulation of raptor inhibited TGFbeta-stimulated mTOR kinase activity, resulting in inhibition of phosphorylation of S6 kinase and 4EBP-1. Raptor shRNA also prevented protein synthesis in response to TGFbeta. Downregulation of rictor inhibited serine 473 phosphorylation of Akt without any effect on phosphorylation of its substrate, tuberin. Furthermore, rictor shRNA increased phosphorylation of S6 kinase and 4EBP-1 in TGFbeta-independent manner, resulting in increased protein synthesis. Thus mTORC1 function is essential for TGFbeta-induced protein synthesis. Our data also provide novel evidence that rictor negatively regulates TORC1 activity to control basal protein synthesis, thus conferring tight control on cellular hypertrophy.     

PI 3 kinase-dependent Akt kinase and PKCepsilon independently regulate interferon-gamma-induced STAT1alpha serine phosphorylation to induce monocyte chemotactic protein-1 expression

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.     

Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDK1

Akt is a protein serine/threonine kinase that plays an important role in the mitogenic responses of cells to variable stimuli. Akt contains a pleckstrin homology (PH) domain and is activated by phosphorylation at threonine 308 and serine 473. Binding of 3'-OH phosphorylated phosphoinositides to the PH domain results in the translocation of Akt to the plasma membrane where it is activated by upstream kinases such as (phosphoinositide-dependent kinase-1 (PDK1). Over-expression of constitutively active forms of Akt promotes cell proliferation and survival, and also stimulates p70 S6 kinase (p70S6K). In many cells, an increase in levels of intracellular cyclic AMP (cAMP) diminishes cell growth and promotes differentiation, and in certain conditions cAMP is even antagonistic to the effect of growth factors. Here, we show that cAMP has inhibitory effects on the phosphatidylinositol 3-kinase/PDK/Akt signaling pathway. cAMP potently inhibits phosphorylation at threonine 308 and serine 473 of Akt, which is required for the protein kinase activities of Akt. cAMP also negatively regulates PDK1 by inhibiting its translocation to the plasma membrane, despite not affecting its protein kinase activities. Furthermore, when we co-expressed myristoylated Akt and PDK1 mutants which constitutively co-localize in the plasma membrane, Akt activity was no longer sensitive to raised intracellular cAMP concentrations. Finally, cAMP was also found to inhibit the lipid kinase activity of PI3K and to decrease the levels of phosphatidylinositol 3,4,5-triphosphate in vivo, which are required for the membrane localization of PDK1. Collectively, these data strongly support the theory that the cAMP-dependent signaling pathway inhibits Akt activity by blocking the coupling between Akt and its upstream regulators, PDK, in the plasma membrane.     

Phosphorylation of the cyclic AMP response element binding protein mediates transforming growth factor beta-induced downregulation of cyclin A in vascular smooth muscle cells

Transforming growth factor beta (TGFbeta), a multifunctional cytokine associated with vascular injury, is a potent inhibitor of cell proliferation. The current results demonstrate that the TGFbeta-induced growth arrest of vascular smooth muscle cells (VSMCs) is associated with cyclin A downregulation. TGFbeta represses the cyclin A gene through a cyclic AMP (cAMP) response element, which complexes with the cAMP response element binding protein (CREB). The CREB-cyclin A promoter interaction is hindered by TGFbeta, preceded by a TGFbeta receptor-dependent CREB phosphorylation. Induction of CREB phosphorylation with forskolin or 6bnz-cAMP mimics TGFbeta's inhibitory effect on cyclin A expression. Conversely, inhibition of CREB phosphorylation with a CREB mutant in which the phosphorylation site at serine 133 was changed to alanine (CREB-S133A) upregulated cyclin A gene expression. Furthermore, the CREB-S133A mutant abolished TGFbeta-induced CREB phosphorylation, cyclin A downregulation, and growth inhibition. Since we have previously shown that the novel PKC isoform protein kinase C delta (PKCdelta) is activated by TGFbeta in VSMCs, we tested the role of this kinase in CREB phosphorylation and cyclin A downregulation. Inhibition of PKCdelta by a dominant-negative mutant or by targeted gene deletion blocked TGFbeta-induced CREB phosphorylation and cyclin A downregulation. Taken together, our data indicate that phosphorylation of CREB stimulated by TGFbeta is a critical step leading to the inhibition of cyclin A expression and, thus, VSMC proliferation.