Role of protein kinase C isoforms in the regulation of interleukin-13-induced 15-lipoxygenase gene expression in human monocytes

We reported previously that interleukin-13 (IL-13) induces tyrosine phosphorylation/activation of Jak2 and Tyk2 kinases and Stats 1, 3, 5, and 6 in primary human monocytes. We recently revealed that p38 MAPK-mediated serine phosphorylation of both Stat1 and Stat3 is required for the induction of 15-lipoxygenase (15-LO) expression by IL-13. In this study, we present data indicating that another serine/threonine kinase, PKCdelta, is also required for IL-13-induced 15-LO expression. PKCdelta, a member of the novel protein kinase C (PKC) subclass, was rapidly phosphorylated and activated upon exposure to IL-13. Treatment of cells with rottlerin, a PKCdelta inhibitor, blocked IL-13-induced 15-LO mRNA and protein expression, whereas Go6976, an inhibitor of the conventional PKC subclass, had no inhibitory effects. Down-regulation of cellular PKCdelta protein levels by PKCdelta-specific antisense oligodeoxyribonucleotides also inhibited 15-LO expression markedly. IL-13-induced 15-LO expression resulted in significant inhibition of synthesis of the potent chemotactic factor leukotriene B4, and that process was reversed by rottlerin, presumably through the blockage of PKCdelta-dependent 15-LO expression. Furthermore, our data demonstrate that IL-13-mediated activation of PKCdelta and p38 MAPK are independent pathways, because inhibition of one kinase activity had no effect on the other, suggesting that the two pathways act in parallel to regulate the downstream targets necessary for 15-LO expression. Inhibition of PKCdelta activation by rottlerin also markedly attenuated IL-13-induced Stat3 DNA binding activity. Our findings indicate that PKCdelta plays an important role in regulating IL-13-induced 15-LO expression in human monocytes and subsequently modulates the inflammatory responses mediated by 15-LO products.     

Hck is a key regulator of gene expression in alternatively activated human monocytes

IL-13 is a Th2 cytokine that promotes alternative activation (M2 polarization) in primary human monocytes. Our studies have characterized the functional IL-13 receptor complex and the downstream signaling events in response to IL-13 stimulation in alternatively activated monocytes/macrophages. In this report, we present evidence that IL-13 induces the activation of a Src family tyrosine kinase, which is required for IL-13 induction of M2 gene expression, including 15-lipoxygenase (15-LO). Our data show that Src kinase activity regulates IL-13-induced p38 MAPK tyrosine phosphorylation via the upstream kinases MKK3 or MKK6. Our findings also reveal that the IL-13 receptor-associated tyrosine kinase Jak2 is required for the activation of both Src kinase as well as p38 MAPK. Further, we found that Src tyrosine kinase-mediated activation of p38 MAPK is required for Stat1 and Stat3 serine 727 phosphorylation in alternatively activated monocytes/macrophages. Additional studies identify Hck as the specific Src family member, stimulated by IL-13 and involved in regulating both p38 MAPK activation and p38 MAPK-mediated 15-LO expression. Finally we show that the Hck regulates the expression of other alternative state (M2)-specific genes (Mannose receptor, MAO-A, and CD36) and therefore conclude that Hck acts as a key regulator controlling gene expression in alternatively activated monocytes/macrophages.     

Monocyte 15-lipoxygenase expression is regulated by a novel cytosolic signaling complex with protein kinase C delta and tyrosine-phosphorylated Stat3

Our previous studies demonstrated that the IL-13-induced 15-lipoxygenase expression in primary human monocytes is regulated by the activation of both Stat1 and Stat3 and by protein kinase C (PKC)delta. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKCdelta with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKCdelta and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKCdelta association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKCdelta and subsequent PKCdelta-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKCdelta with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis.     

Cutting edge: TCR stimulation by antibody and bacterial superantigen induces Stat3 activation in human T cells.

Recent data show that TCR/CD3 stimulation induces activation of Stat5 in murine T cells. Here, we show that CD3 ligation by mAb and Staphylococcal enterotoxin (SE) induce a rapid, gradually accumulating, long-lasting tyrosine, and serine phosphorylation of Stat3 (but not Stat5) in allogen-specific human CD4+ T cell lines. In contrast, IL-2 induces a rapid and transient tyrosine and serine phosphorylation of Stat3. Compared with IL-2, CD3 ligation induces a delayed Stat3 binding to oligonucleotide probes from the ICAM-1 and IL-2R alpha promoter. CD3-mediated activation of Stat3 is almost completely inhibited by a Src kinase inhibitor (PP1), whereas IL-2-induced Stat3 activation is unaffected. In conclusion, we show that CD3 ligation by mAb and SE triggers a rapid, PP1-sensitive tyrosine and serine phosphorylation of Stat3 in human CD4+ T cells. Moreover, we provide evidence that TCR/CD3 and IL-2 induce Stat3 activation via distinct signaling pathways.     

MCP-1-stimulated chemotaxis of monocytic and endothelial cells is dependent on activation of different signaling cascades

Monocyte chemoattractant protein-1 (MCP-1) is important in attracting monocytes to sites of inflammation. Besides induction of monocyte recruitment, MCP-1 can also affect chemotactic response of endothelial cells. The molecular mechanisms involved in MCP-1-induced cell migration are poorly understood. In the current investigation, we demonstrate activation of p42/44(ERK1/2) and p38 mitogen-activated protein kinases (MAPKs), phosphatydilinositol-3-kinase (PI3K) and Src-kinases in both monocytes and endothelial cells stimulated with MCP-1 in vitro. The response was rapid and time-dependent, detectable within 3 min of MCP-1 stimulation. MCP-1-induced phosphorylation of p42/44(ERK1/2) MAPKs was partially blocked by inhibitor of PI3K LY294002, while phosphorylation of p38 MAPK was diminished to a greater extent in presence of Src-kinase inhibitor PP2. There was a substantial inhibition of monocyte migration upon treatment with inhibitors of p38 MAPK, at the same time inhibition of p42/44(ERK1/2) MAPK activation had no effect. On the contrary, the MCP-1-stimulated chemotaxis of endothelial cells was completely abolished by inhibitors of PI3K and p42/44(ERK1/2), but not by p38 MAPK inhibitors. These results suggest that parallel signal transduction pathways are activated by MCP-1, and that depending on the cell type these pathways differentially contribute to cell chemotactic activity.     

Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.     

Selective activation of p38 mitogen-activated protein kinase cascade in human neutrophils stimulated by IL-1beta.

We investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by IL-1beta. IL-1beta induced phosphorylation and activation of p38 MAPK and phosphorylation of MAPK kinase-3/6 (MKK3/6). Maximal activation of p38 MAPK was obtained by stimulation of cells with 300 U/ml IL-1beta for 10 min. Extracellular signal-regulated kinase (ERK) was faintly phosphorylated and c-Jun N-terminal kinase (JNK) was not phosphorylated by IL-1beta. IL-1beta primed neutrophils for enhanced release of superoxide (O(2)(-)) stimulated by FMLP in parallel with increased phosphorylation of p38 MAPK. IL-1beta also induced O(2)(-) release and up-regulation of CD11b and CD15, and both responses were inhibited by SB203580 (p38 MAPK inhibitor), suggesting that p38 MAPK activation mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15. Combined stimulation of neutrophils with IL-1beta and G-CSF, a selective activator of the ERK cascade, resulted in the additive effects when the priming effect and phosphorylation of p38 MAPK and ERK were assessed. IL-1beta induced phosphorylation of ERK and JNK as well as p38 MAPK in human endothelial cells. These findings suggest that 1) in human neutrophils the MKK3/6-p38 MAPK cascade is selectively activated by IL-1beta and activation of this cascade mediates IL-1beta-induced O(2)(-) release and up-regulation of CD11b and CD15, and 2) the IL-1R-p38 MAPK pathway and the G-CSF receptor-ERK pathway work independently for activation of neutrophils.     

Endothelin-1 induces serine phosphorylation of the adaptor protein p66Shc and its association with 14-3-3 protein in glomerular mesangial cells

Endothelin-1 (ET-1) is a vasoconstrictor peptide known to be a potent mitogen for glomerular mesangial cells (GMC). In the current study, it is demonstrated that ET-1 treatment of GMC results in serine phosphorylation of the 66-kDa isoform of the adapter protein Shc (p66(Shc)). ET-1-induced serine phosphorylation of p66(Shc) requires activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling module and is efficiently inhibited by both a MAPK/ERK kinase (MEK)-selective inhibitor and adenovirus-mediated transfer of a dominant interfering MEK1 mutant. Furthermore, adenovirus-mediated transfer of a constitutively active MEK1 mutant was found to markedly increase p66(Shc) serine phosphorylation. Adenoviruses encoding constitutively active mutants of MAPK kinases 3 and 6 (upstream kinases of p38(MAPK)) and 7 (upstream kinase of c-Jun NH(2)-terminal kinase) failed to induce serine phosphorylation of this adaptor protein. Serine phosphorylation of p66(Shc) resulted in its association with the serine binding motif-containing protein 14-3-3. ET-1-induced phosphorylation of a serine encompassed in the 14-3-3 binding motif of p66(Shc) was confirmed in experiments employing anti-phospho-14-3-3 binding motif antibodies. These studies are the first to demonstrate that G protein-coupled receptors stimulate serine phosphorylation of p66(Shc) and the first to report the formation of a signaling complex between p66(Shc) and 14-3-3.     

The role of p38 mitogen-activated protein kinase in regulating interleukin-10 gene expression in Burkitt's lymphoma cell lines

In malignant B lymphoma cells interleukin-10 (IL-10) expression is frequently upregulated. This effect is thought to support to the malignant transformation of these cells and to be a potential target for pharmacotherapy. To define better the mechanism for upregulation of the IL-10 gene, we tested the association between IL-10 and p38 mitogen-activated protein kinase (MAPK) in several Epstein-Barr virus (EBV) infected and non-infected Burkitt's lymphoma (BL) cell lines. The all BL cell lines expressed IL-10 and IL-10 receptor mRNAs, and produced IL-10. p38 MAPK was constitutively phosphorylated in the cytoplasm of the BL cell lines. We further analyzed molecular effects of p38 MAPK on IL-10 expression in Akata cells. Exogenous IL-10 lead rapidly to phosphorylation of Jak1 and Tyk2 as transducers of signals of IL-10, and promoted growth of Akata cells in a dose-dependent manner. The phosphorylation of cytoplasmic p38 MAPK in Akata cells was reduced by the serine/threonine kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). A specific inhibitor of p38 MAPK, SB203580, blocked simultaneously STAT3 DNA-binding activity, and IL-10 mRNA expression, IL-10 production, and then the cell growth was inhibited. These results indicate that the p38 MAPK pathway is functionally linked to IL-10 gene expression and supports the view that the constitutive activation of cytoplasmic p38 MAPK in BL cells is a step in the upregulation of IL-10 gene expression and lymphomagenesis.     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.