Rac1 regulates heat shock responses by reorganization of vimentin filaments: identification using MALDI-TOF MS.

Rac1 has been implicated in a wide variety of biological processes, including actin remodeling and various signaling cascades. Here we have examined whether Rac1 might be involved in heat shock-induced cell signaling. We found that Rat2 stable cells expressing a dominant negative Rac1 mutant, RacN17 (Rat2-RacN17), were significantly more tolerant to heat shock than control Rat2 cells, and simultaneously inhibited the activation of SAPK/JNK by heat shock compared to control Rat2 cells. However, no discernible effect was observed in typical heat shock responses including total protein synthesis and heat shock protein synthesis. To identify the proteins involved in this difference, we separated the proteins of both Rat2 and Rat2-RacN17 cell lines after heat shock using two-dimensional gel electrophoresis and identified the differentially expressed proteins by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) after in-gel trypsin digestion. Differentially expressed proteins between two cell lines were identified as vimentin. Rat2-RacN17 cells showed significant changes in vimentin as well as marked changes in vimentin reorganization by heat shock. The vimentin changes were identified as N-terminal head domain cleavage. These results suggest that Rac1 plays a pivotal role in the heat shock-induced signaling cascade by modifying intermediate vimentin filaments.     

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.     

IFN-gamma-induced SOCS-1 regulates STAT6-dependent eotaxin production triggered by IL-4 and TNF-alpha

The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of eotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-alpha. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner.     

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.     

Impaired type I IFN-induced Jak/STAT signaling in FA-C cells and abnormal CD4+ Th cell subsets in Fancc-/- mice

The Fanconi anemia (FA) group C protein, FANCC, interacts with STAT1 following stimulation with IFN-gamma and is required for proper docking of STAT1 at the IFN-gamma receptor alpha-chain (IFN-gammaRalpha, IFN-gammaR1). Consequently, loss of a functional FANCC results in decreased activation of STAT1 following IFN-gamma stimulation. Because type I IFN receptors influence the function of type II receptors, and vice versa, we conducted experiments designed to determine whether type I IFN-induced activation of other STAT proteins is compromised in FA-C cells and found that activation of STAT 1, 3, and 5 is diminished in type I IFN-stimulated cells bearing Fancc-inactivating mutations. We also determined that the reduced activation of STATs was accompanied by significant reduction of type I IFN-induced tyrosine kinase 2 and Jak1 phosphorylation. Because tyrosine kinase 2 plays a role in differentiation of Th cells, we quantified cytokine secretion from CD4+ cells and in vitro generated CD4+ Th cell subsets from splenocytes of Fancc null mice to that of heterozygous mice and discovered reduced CD4+ IFN-gamma secretion in the Fancc-/- mouse, indicating impaired Th1 differentiation. We suggest that Fancc mutations result in a subtle immunological defect owing to the failure of FANCC to normally support Jak/STAT signaling.     

IFN-gamma and STAT1 arrest monocyte migration and modulate RAC/CDC42 pathways

Positive regulation of cell migration by chemotactic factors and downstream signaling pathways has been extensively investigated. In contrast, little is known about factors and mechanisms that induce migration arrest, a process important for retention of cells at inflammatory sites and homeostatic regulation of cell trafficking. In this study, we found that IFN-gamma directly inhibited monocyte migration by suppressing remodeling of the actin cytoskeleton and cell polarization in response to the chemokine CCL2. Inhibition was dependent on STAT1 and downstream genes, whereas STAT3 promoted migration. IFN-gamma altered monocyte responses to CCL2 by modulating the activity of Pyk2, JNK, and the GTPases Rac and Cdc42, and inhibiting CCL2-induced activation of the downstream p21-activated kinase that regulates the cytoskeleton and cell polarization. These results identify a new role for IFN-gamma in arresting monocyte chemotaxis by a mechanism that involves modulation of cytoskeleton remodeling. Crosstalk between Jak-STAT and Rac/Cdc42 GTPase-mediated signaling pathways provides a molecular mechanism by which cytokines can regulate cell migration.     

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.     

Connective tissue growth factor induces collagen I expression in human lung fibroblasts through the Rac1/MLK3/JNK/AP-1 pathway

Connective tissue growth factor (CTGF) plays an important role in lung fibrosis. In this study, we investigated the role of Rac1, mixed-lineage kinase 3 (MLK3), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CTGF-induced collagen I expression in human lung fibroblasts. CTGF caused concentration- and time-dependent increases in collagen I expression. CTGF-induced collagen I expression was inhibited by the dominant negative mutant (DN) of Rac1 (RacN17), MLK3DN, MLK3 inhibitor (K252a), JNK1DN, JNK2DN, a JNK inhibitor (SP600125), and an AP-1 inhibitor (curcumin). Treatment of cells with CTGF caused activation of Rac1, MLK3, JNK, and AP-1. The CTGF-induced increase in MLK3 phosphorylation was inhibited by RacN17. Treatment with RacN17 and the MLK3DN inhibited CTGF-induced JNK phosphorylation. CTGF caused increases in c-Jun phosphorylation and the recruitment of c-Jun and c-Fos to the collagen I promoter. Furthermore, stimulation of cells with the CTGF resulted in increases in AP-1-luciferase activity; this effect was inhibited by Rac1N17, MLK3DN, JNK1DN, and JNK2DN. Moreover, CTGF-induced α-smooth muscle actin (α-SMA) expression was inhibited by the procollagen I small interfering RNA (siRNA). These results suggest for the first time that CTGF acting through Rac1 activates the MLK3/JNK signaling pathway, which in turn initiates AP-1 activation and recruitment of c-Jun and c-Fos to the collagen I promoter and ultimately induces collagen I expression in human lung fibroblasts.     

Thrombin induces NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells by a Rac1-dependent PI3K/Akt pathway

We previously showed that thrombin induces interleukin (IL)-8/CXCL8 expression via the protein kinase C (PKC)α/c-Src-dependent IκB kinase α/β (IKKα/β)/NF-κB signaling pathway in human lung epithelial cells. In this study, we further investigated the roles of Rac1, phosphoinositide 3-kinase (PI3K), and Akt in thrombin-induced NF-κB activation and IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by a PI3K inhibitor (LY294002), an Akt inhibitor (1-L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), and the dominant negative mutants of Rac1 (RacN17) and Akt (AktDN). Treatment of cells with thrombin caused activation of Rac and Akt. The thrombin-induced increase in Akt activation was inhibited by RacN17 and LY294002. Stimulation of cells with thrombin resulted in increases in IKKα/β activation and κB-luciferase activity; these effects were inhibited by RacN17, LY294002, an Akt inhibitor, and AktDN. Treatment of cells with thrombin induced Gβγ, p85α, and Rac1 complex formation in a time-dependent manner. These results imply that thrombin activates the Rac1/PI3K/Akt pathway through formation of the Gβγ, Rac1, and p85α complex to induce IKKα/β activation, NF-κB transactivation, and IL-8/CXCL8 expression in human lung epithelial cells.     

Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.