Involvement of protein kinase Cdelta in iron chelator-induced IL-8 production in human intestinal epithelial cells

We have shown that the bacterial iron chelator, deferoxamine (DFO), triggers inflammatory signals, including the production of CXC chemokine IL-8, in human intestinal epithelial cells (IECs) by activating ERK1/2 and p38 kinase pathways. In the present study, we show that PKCdelta, one of the novel protein kinase C (PKC) isoforms, involves in signal transduction pathways leading to DFO-induced IL-8 production. Pretreatment of human intestinal epithelial HT-29 cells with rottlerin showed remarkable inhibition of DFO-induced IL-8 production. In contrast, other PKC inhibitors such as G?6976, G?6983, GF109203X, and staurosporine revealed less or no inhibitory effects on DFO-induced IL-8 production, suggesting a potential role of PKCdelta. Accordingly, DFO caused phosphorylation of PKCdelta in the Thr505 and Ser643 residues in HT-29 cells. Transfection of dominant-negative PKCdelta vector inhibited DFO-induced PKCdelta phosphorylation as well as IL-8 promoter activity. In addition, suppression of endogenous PKCdelta by siRNA significantly reduced DFO-induced IL-8 production. Collectively, these results suggest that PKCdelta plays a pivotal role in signaling pathways leading to iron chelator-induced IL-8 production in human IECs.     

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.     

IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC

Protein kinase C (PKC)-activated signal transduction pathways regulate cell growth and differentiation in many cell types. We have observed that interleukin (IL)-1beta upregulates beta2-adrenergic receptor (beta2-AR) density and beta2-AR mRNA in human airway epithelial cells (e.g., BEAS-2B). We therefore tested the hypothesis that PKC-activated pathways mediate IL-1beta-induced beta-AR upregulation. The role of PKC was assessed from the effects of 1) the PKC activator phorbol 12-myristate 13-acetate (PMA) on beta-AR density, 2) selective PKC inhibitors (calphostin C and Ro-31-8220) on beta-AR density, and 3) IL-1beta treatment on the cellular distribution of PKC isozymes. Recombinant human IL-1beta (0.2 nM for 18 h) increased beta-AR density to 213% of control values (P < 0.001). PMA (1 microM for 18 h) increased beta-AR density to 225% of control values (P < 0.005), whereas Ro-31-8220 and calphostin C inhibited the IL-1beta-induced upregulation of beta-AR in dose-dependent fashion. PKC isozymes detected by Western blotting included alpha, betaII, epsilon, mu, zeta, and lambda/iota. IL-1beta increased PKC-mu immunoreactivity in the membrane fraction and had no effect on the distribution of the other PKC isozymes identified. These data indicate that IL-1beta-induced beta-AR upregulation is mimicked by PKC activators and blocked by PKC inhibitors and appears to involve selective activation of the PKC-mu isozyme. We conclude that signal transduction pathways activated by PKC-mu upregulate beta2-AR expression in human airway epithelial cells.     

Activation and translocation of PKCdelta is necessary for VEGF-induced ERK activation through KDR in HEK293T cells

VEGF-KDR/Flk-1 signal utilizes the phospholipase C-gamma-protein kinase C (PKC)-Raf-MEK-ERK pathway as the major signaling pathway to induce gene expression and cPLA2 phosphorylation. However, the spatio-temporal activation of a specific PKC isoform induced by VEGF-KDR signal has not been clarified. We used HEK293T (human embryonic kidney) cells expressing transiently KDR to examine the activation mechanism of PKC. PKC specific inhibitors and human PKCdelta knock-down using siRNA method showed that PKCdelta played an important role in VEGF-KDR-induced ERK activation. Myristoylated alanine-rich C-kinase substrate (MARCKS) translocates from the plasma membrane to the cytoplasm depending upon phosphorylation by PKC. Translocation of MARCKS-GFP induced by VEGF-KDR stimulus was blocked by rottlerin, a PKCdelta specific inhibitor, or human PKCdelta siRNA. VEGF-KDR stimulation did not induce ERK phosphorylation in human PKCdelta-knockdown HEK293T cells, but co-expression of rat PKCdelta-GFP recovered the ERK phosphorylation. Y311/332F mutant of rat PKCdelta-GFP which cannot be activated by tyrosine-phosphorylation but activated by DAG recovered the ERK phosphorylation, while C1B-deletion mutant of rat PKCdelta-GFP, which can be activated by tyrosine-phosphorylation but not by DAG, failed to recover the ERK phosphorylation in human PKCdelta-knockdown HEK293T cell. These results indicate that PKCdelta is involved in VEGF-KDR-induced ERK activation via C1B domain.     

Protein kinase C delta regulates the phosphorylation of heat shock protein 27 in human hepatocellular carcinoma

We have recently reported that attenuated phosphorylation of heat shock protein (HSP) 27 correlates with tumor progression in patients with hepatocellular carcinoma (HCC). In the present study, we investigated what kind of kinase regulates phosphorylation of HSP27 in human HCC-derived HuH7 cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol, direct activators of protein kinase C (PKC), markedly strengthened the phosphorylation of HSP27. Bisindorylmaleimide I, an inhibitor of PKC, suppressed the TPA-induced levels of HSP27 phosphorylation in addition to its basal levels. Knock down of PKCdelta suppressed HSP27 phosphorylation, as well as p38 mitogen-activated protein kinase (MAPK) phosphorylation. SB203580, an inhibitor of p38 MAPK, suppressed the TPA-induced HSP27 phosphorylation. Our results strongly suggest that activation of PKCdelta regulates the phosphorylation of HSP27 via p38 MAPK in human HCC.     

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.     

IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCdelta

CD86 expression is up-regulated in activated monocytes and macrophages by a mechanism that is not clearly defined. Here, we report that IL-4-dependent CD86 expression requires activation of ERK1/2 and JAK/STAT6 but is negatively regulated by PKCdelta. PMA differentiated U937 monocytic cells when stimulated with IL-4 increased CD11b and CD86 expression by 52- and 98-fold, respectively. PMA+IL-4 treatment also induced a synergistic enhancement of ERK1/2 activation when compared to the effects of PMA and IL-4 alone. Use of the mitogen or extracellular kinase (MEK) inhibitor, PD98059, completely blocked up-regulation of CD11b and CD86 demonstrating the importance of MEK-activated ERK1/2. JAK inhibition with WHI-P154-abrogated IL-4-dependent CD11b and CD86 up-regulation and inhibited STAT6 tyrosine phosphorylation. Importantly, CD11b and CD86 expression were not reliant on IL-4-dependent activation of phosphatidylinositol 3'-kinase (PI 3-kinase). Blockade of PKCdelta activation with rottlerin prevented CD11b expression but lead to a 75- and 213-fold increase in PMA and PMA+IL-4-dependent CD86 expression, respectively. As anticipated, increasing PKCdelta activity with anti-sense reduction of CD45 increased CD11b expression and reduced CD86 expression. Likewise, rottlerin prevented nuclear localization of activated PKCdelta. We conclude from these data that IL-4-dependent CD11b expression relies predominantly on enhanced activation of ERK1/2, while IL-4-dependent CD86 expression utilizes the JAK/STAT6 pathway.     

Fibroblast growth factor activation of the rat PRL promoter is mediated by PKCdelta

Fibroblast growth factors play a critical role in cell growth, development, and differentiation and are also implicated in the formation and progression of tumors in a variety of tissues including pituitary. We have previously shown that fibroblast growth factor activation of the rat PRL promoter in GH4T2 pituitary tumor cells is mediated via MAP kinase in a Ras/Raf-1-independent manner. Herein we show using biochemical, molecular, and pharmacological approaches that PKCdelta is a critical component of the fibroblast growth factor signaling pathway. PKC inhibitors, or down-regulation of PKC, rendered the rat PRL promoter refractory to subsequent stimulation by fibroblast growth factors, implying a role for PKC in fibroblast growth factor signal transduction. FGFs caused specific translocation of PKCdelta from cytosolic to membrane fractions, consistent with enzyme activation. In contrast, other PKCs expressed in GH4T2 cells (alpha, betaI, betaII, and epsilon) did not translocate in response to fibroblast growth factors. The PKCdelta subtype-selective inhibitor, rottlerin, or expression of a dominant negative PKCdelta adenoviral construct also blocked fibroblast growth factor induction of rat PRL promoter activity, confirming a role for the novel PKCdelta isoform. PKC inhibitors selective for the conventional alpha and beta isoforms or dominant negative PKCalpha adenoviral expression constructs had no effect. Induction of the endogenous PRL gene was also blocked by adenoviral dominant negative PKCdelta expression but not by an analogous dominant negative PKCalpha construct. Finally, rottlerin significantly attenuated FGF-induced MAP kinase phosphorylation. Together, these results indicate that MAP kinase-dependent fibroblast growth factor stimulation of the rat PRL promoter in pituitary cells is mediated by PKCdelta.     

IL-32θ negatively regulates IL-1β production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation

It has been well known that IL-32 exerts pro-inflammatory effects on the various inflammatory diseases in clinical studies. Here, we confirmed that IL-32θ, a new isoform of IL-32, decreased the phorbol 12-myristate 13-acetate (PMA)-induced IL-1β expression in THP-1 human myelomonocyte. We previously reported that the IL-32 isoforms control expressions of other cytokines via novel PKCs. Likewise, IL-32θ interacted with PKCδ, and consequently inhibited PKCδ-mediated phosphorylation of PU.1. Moreover, IL-32θ attenuated the localization of PU.1 into the IL-1β promoter region. These findings reveal that IL-32θ reduces PKCδ-mediated phosphorylation of PU.1, resulting in attenuation of IL-1β production.     

IL-3 and IL-4 activate cyclic nucleotide phosphodiesterases 3 (PDE3) and 4 (PDE4) by different mechanisms in FDCP2 myeloid cells

In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-alpha, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-alpha also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-alpha, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-alpha could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).