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.     

Activation of phosphoinositide 3-kinase in response to inflammation and nitric oxide leads to the up-regulation of cyclooxygenase-2 expression and subsequent cell proliferation in mesangial cells

In this study, we showed that nitric oxide (NO) donors induced the mesangial cell proliferation and cyclooxygenase-2 (COX-2) protein expression in murine mesangial cells. An inflammatory condition [lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)] could also induce cell proliferation and significantly enhance inducible nitric oxide synthase (iNOS) and COX-2 expression. Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, inhibited these responses. LPS/IFN-gamma-induced COX-2 expression in mesangial cells could be inhibited by iNOS inhibitor, aminoguanidine. Selective COX-2 inhibitor, NS398, was capable of inhibiting NO donor- or LPS/IFN-gamma-induced mesangial cell proliferation. Both NO donor and LPS/IFN-gamma markedly activated the PI3K activity and the phosphorylation of Akt and nuclear factor (NF)-kappaB DNA binding activity in mesangial cells, which could be inhibited by LY294002 and transfection of dominant-negative vectors of PI3K/p85 and Akt. These results indicate that a PI3K/Akt-dependent pathway involved in the NO-regulated COX-2 expression and cell proliferation in mesangial cells under inflammatory condition.     

Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization

Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.     

Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways.

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-beta (TGF-beta), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-beta-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15(INK4B) genes, indicating that IL-6 interferes with only a subset of TGF-beta activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-beta-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-beta, with the former likely playing a major role in this anti-apoptosis.     

Leishmania inhibits STAT1-mediated IFN-gamma signaling in macrophages: increased tyrosine phosphorylation of dominant negative STAT1beta by Leishmania mexicana

Previous studies have demonstrated that Leishmania donovani attenuates STAT1-mediated signaling in macrophages; however it is not clear whether other species of Leishmania, which cause cutaneous disease, also interfere with macrophage IFN-gamma signaling. Therefore, we determined the effect of Leishmania major and Leishmania mexicana infection on STAT1-mediated IFN-gamma signaling pathway in J774A.1 and RAW264.7 macrophages. We found that both L. major and L. mexicana suppressed IFNgammaRalpha (alpha subunit of interferon gamma receptor) and IFN-gammaRbeta (beta subunit of interferon gamma receptor) expression, reduced levels of total Jak1 and Jak2, and down-regulated IFN-gamma-induced Jak1, Jak2 and STAT1 activation. The effect of L. mexicana infection on Jak1, Jak2 and STAT1 activation was more profound when compared with L. major. Although tyrosine phosphorylation of STAT1alpha was decreased in IFN-gamma stimulated macrophages infected with L. major or L. mexicana, those infected with L. mexicana showed a significant increase in phosphorylation of the dominant negative STAT1beta. These findings indicate that L. major and L. mexicana attenuate STAT1-mediated IFN-gamma signaling in macrophages. Furthermore, they also demonstrate that L. mexicana preferentially enhances tyrosine phosphorylation of dominant negative STAT1beta, which may be one of the several survival mechanisms used by this parasite to evade the host defense mechanisms.