Protection against sepsis-induced lung injury by selective inhibition of protein kinase C-δ (δ-PKC).

Inflammation and proinflammatory mediators are activators of δ-PKC. In vitro, δ-PKC regulates proinflammatory signaling in neutrophils and endothelial and epithelial cells, cells that can contribute to lung tissue damage associated with inflammation. In this study, a specific δ-PKC TAT peptide inhibitor was used to test the hypothesis that inhibition of δ-PKC would attenuate lung injury in an animal model of ARDS. Experimental ARDS was induced in rats via 2CLP, a model of polymicrobial sepsis. Following 2CLP surgery, the δ-PKC TAT inhibitory peptide (2CLP+δ-PKC TAT in PBS) or PBS (2CLP+PBS) was administered intratracheally. Controls consisted of SO, where animals underwent a laparotomy without 2CLP. Twenty-four hours after SO or 2CLP, blood, BALF, and lung tissue were collected. 2CLP induced δ-PKC phosphorylation in the lung within 24 h. Treatment with the δ-PKC TAT inhibitory peptide significantly decreased pulmonary δ-PKC phosphorylation, indicating effective inhibition of δ-PKC activation. Plasma and BALF levels of the chemokines CINC-1 and MIP-2 were elevated in 2CLP + PBS rats as compared with SO rats. Treatment with δ-PKC TAT reduced 2CLP-induced elevations in chemokine levels in BALF and plasma, suggesting that δ-PKC modulated chemokine expression. Most importantly, intratracheal administration of δ-PKC TAT peptide significantly attenuated inflammatory cell infiltration, disruption of lung architecture, and pulmonary edema associated with 2CLP. Thus, δ-PKC is an important regulator of proinflammatory events in the lung. Targeted inhibition of δ-PKC exerted a lung-protective effect 24 h after 2CLP.

Regulation of TNF-induced oxygen radical production in human neutrophils: role of delta-PKC.

In human neutrophils, TNF-elicited O(2)(-) production requires adherence and integrin activation. How this cooperative signaling between TNFRs and integrins regulates O(2)(-) generation has yet to be fully elucidated. Previously, we identified delta-PKC as a critical early regulator of TNF signaling in adherent neutrophils. In this study, we demonstrate that inhibition of delta-PKC with a dominant-negative delta-PKC TAT peptide resulted in a significant delay in the onset time of TNF-elicited O(2)(-) generation but had no effect on Vmax, indicating an involvement of delta-PKC in the initiation of O(2)(-) production. In contrast, fMLP-elicited O(2)(-) production in adherent and nonadherent neutrophils was delta-PKC-independent, suggesting differential regulation of O(2)(-) production. An important step in activation of the NADPH oxidase is phosphorylation of the cytosolic p47phox component. In adherent neutrophils, TNF triggered a time-dependent association of delta-PKC with p47phox, which was associated with p47phox phosphorylation, indicating a role for delta-PKC in regulating O(2)(-) production at the level of p47phox. Activation of ERK and p38 MAPK is also required for TNF-elicited O(2)(-) generation. TNF-mediated ERK but not p38 MAPK recruitment to p47phox was delta-PKC-dependent. delta-PKC activity is controlled through serine/threonine phosphorylation, and phosphorylation of delta-PKC (Ser643) and delta-PKC (Thr505) was increased significantly by TNF in adherent cells via a PI3K-dependent process. Thus, signaling for TNF-elicited O(2)(-) generation is regulated by delta-PKC. Adherence-dependent cooperative signaling activates PI3K signaling, delta-PKC phosphorylation, and delta-PKC recruitment to p47phox. delta-PKC activates p47phox by serine phosphorylation or indirectly through control of ERK recruitment to p47phox.

Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R).

Substance P (SP) is a potent modulator of monocyte/macrophage function. The SP-preferring receptor neurokinin-1 receptor (NK1R) has two forms: a full-length NK1R (NK1R-F) isoform and a truncated NK1R (NK1R-T) isoform, which lacks the terminal cytoplasmic 96-aa residues. The distribution of these receptor isoforms in human monocytes is not known. We previously identified an interaction among SP, NK1R, and HIV viral strains that use the chemokine receptor CCR5 as a coreceptor, suggesting crosstalk between NK1R and CCR5. The purpose of this study was to determine which form(s) of NK1R are expressed in human peripheral blood monocytes and to determine whether SP affects proinflammatory cellular responses mediated through the CCR5 receptor. Human peripheral blood monocytes were found to express NK1R-T but not NK1R-F. SP interactions with NK1R-T did not mobilize calcium (Ca2+), but SP mobilized Ca2+ when the NK1R-F was transfected into monocytes. However, the NK1R-T was functional in monocytes, as SP enhanced the CCR5 ligand CCL5-elicited Ca2+ mobilization, a response inhibited by the NK1R antagonist aprepitant. SP interactions with the NK1R-T also enhanced CCL5-mediated chemotaxis, which was ERK1/2-dependent. NK1R-T selectively activated ERK2 but increased ERK1 and ERK2 activation by CCL5. Activation of NK1R-T elicited serine phosphorylation of CCR5, indicating that crosstalk between CCL5 and SP may occur at the level of the receptor. Thus, NK1R-T is functional in human monocytes and activates select signaling pathways, and the NK1R-T-mediated enhancement of CCL5 responses does not require the NK1R terminal cytoplasmic domain.

Selective roles for alpha-PKC in positive signaling for O-(2) generation and calcium mobilization but not elastase release in differentiated HL60 cells.

Protein kinase C (PKC) isotypes and Ca2+ mobilization have been implicated in phagocytic cell functions such as O(-)(2) generation. Ca/DG-dependent alpha-PKC and beta-PKC have similar substrate specificities and cofactor requirements in vitro. However it is not known if these isotypes play redundant or unique roles in the intact cell. In the present study, a role for alpha-PKC in positive signaling for fMet-Leu-Phe- and PMA-activated O(-)(2) generation was probed using an siRNA strategy in HL60 cells differentiated to a neutrophilic phenotype (dHL60 cells). A selective decrease in alpha-PKC in dHL60 cells attenuated O(-)(2) generation but not degranulation, and reduced ligand-induced phosphorylation of p47phox as previously shown for beta-PKC. However alpha-PKC, unlike beta-PKC, was a positive regulator of fMet-Leu-Phe-triggered Ca2+ uptake via SOCC (Store Operated Calcium Channels). The ability of a selective SOCC inhibitor, MRS1845, to decrease fMet-Leu-Phe induced Ca2+ uptake and O(-)(2) generation confirmed that Ca2+ uptake via SOCC was required for O(-)(2) generation. These results indicate that alpha-PKC and beta-PKC are required for optimal O(-)(2) generation, but play different roles in Ca2+ signaling for phagocytic responses such as O(-)(2) generation.

Regulation of TNF mediated antiapoptotic signaling in human neutrophils: role of delta-PKC and ERK1/2.

TNF is implicated in the suppression of neutrophil apoptosis during sepsis. Multiple signaling pathways are involved in TNF-mediated antiapoptotic signaling; a role for the MAP kinases (MAPK), ERK1/2, and p38 MAPK has been suggested. Antiapoptotic signaling is mediated principally through TNF receptor-1 (TNFR-1), and the PKC isotype-delta (delta-PKC) is a critical regulator of TNFR-1 signaling. delta-PKC associates with TNFR-1 in response to TNF and is required for NFkappaB activation and inhibition of caspase 3. The role of delta-PKC in TNF-mediated activation of MAPK is not known. The purpose of this study was to determine whether the MAPK, ERK1/2, and p38 MAPK are involved in TNF antiapoptotic signaling and whether delta-PKC is a key regulator of MAPK activation by TNF. In human neutrophils, TNF activated both p38 MAPK and ERK1/2 principally via TNFR-1. The MEK1/2 inhibitors PD098059 and U0126, but not the p38 MAPK inhibitor SB203580, decreased TNF antiapoptotic signaling as measured by caspase 3 activity. A specific delta-PKC antagonist, V1.1delta-PKC-Tat peptide, inhibited TNF-mediated ERK1/2 activation, but not p38 MAPK. ERK1/2 inhibition did not alter recruitment of delta-PKC to TNFR-1, indicating delta-PKC is acting upstream of ERK1/2. In HL-60 cells differentiated to a neutrophilic phenotype, delta-PKC depletion by delta-PKC siRNA resulted in inhibition of TNF mediated ERK1/2 activation but not p38 MAPK. Thus, ERK1/2, but not p38 MAPK, is an essential component of TNF-mediated antiapoptotic signaling. In human neutrophils, delta-PKC is a positive regulator of ERK1/2 activation via TNFR-1 but has no role in p38 MAPK activation.

Selective regulation by delta-PKC and PI 3-kinase in the assembly of the antiapoptotic TNFR-1 signaling complex in neutrophils.

TNF is implicated in the attenuation of neutrophil constitutive apoptosis during sepsis. Antiapoptotic signaling is mediated principally through the TNF receptor-1 (TNFR-1). In adherent neutrophils, when beta-integrin signaling is activated, TNF phosphorylates TNFR-1 and activates prosurvival and antiapoptotic signaling. Previously, we identified the delta-PKC isotype and phosphatidylinositol (PI) 3-kinase as critical regulators of TNF signaling in adherent neutrophils. Both kinases associate with TNFR-1 in response to TNF and are required for TNFR-1 serine phosphorylation, NF-kappaB activation, and inhibition of apoptosis. The purpose of this study was to examine the role of delta-PKC and PI 3-kinase in the assembly of TNFR-1 signaling complex that regulates NF-kappaB activation and antiapoptotic signaling. Coimmunoprecipitation studies established that PI 3-kinase, delta-PKC, and TNFR-1 formed a signal complex in response to TNF. delta-PKC recruitment required both delta-PKC and PI 3-kinase activity, whereas PI 3-kinase recruitment was delta-PKC independent, suggesting that PI 3-kinase acts upstream of delta-PKC. An important regulatory step in control of antiapoptotic signaling is the assembly of the TNFR-1-TNFR-1-associated death domain protein (TRADD)-TNFR-associated factor 2 (TRAF2)-receptor interacting protein (RIP) complex that controls NF-kappaB activation. Inhibition of either delta-PKC or PI 3-kinase decreased TNF-mediated recruitment of RIP and TRAF2 to TNFR-1. In contrast, TRADD recruitment was enhanced. Thus delta-PKC and PI 3-kinase are positive regulators of TNF-mediated association of TRAF2 and RIP with TNFR-1. Conversely, these kinases are negative regulators of TRADD association. These results suggest that delta-PKC and PI 3-kinase regulate TNF antiapoptotic signaling at the level of the TNFR-1 through control of assembly of a TNFR-1-TRADD-RIP-TRAF2 complex.

Potentiation of insulin secretion by phorbol esters is mediated by PKC-alpha and nPKC isoforms.

Culturing clonal beta-cells (HIT-T15) overnight in the presence of phorbol ester [phorbol myristate acetate (PMA)] enhanced insulin secretion while causing downregulation of some protein kinase C (PKC) isoforms and most PKC activity. We show here that this enhanced secretion required the retention of PMA in the cell. Hence, it could not be because of long-lived phosphorylation of cellular substrates by the isoforms that were downregulated, namely PKC-alpha, -betaII, and -epsilon, but could be because of the continued activation of the two remaining diacylglycerol-sensitive isoforms delta and mu. The enhanced secretion did not involve changes in glucose metabolism, cell membrane potential, or intracellular Ca2+ handling, suggesting a distal effect. PMA washout caused the loss of the enhanced response, but secretion was then stimulated by acute readdition of PMA or bombesin. The magnitude of this restimulation appeared dependent on the mass of PKC-alpha, which was rapidly resynthesized during PMA washout. Therefore, stimulation of insulin secretion by PMA, and presumably by endogenous diacylglycerol, involves the activation of PKC isoforms delta and/or mu, and also PKC-alpha.

A role for PKC-delta and PI 3-kinase in TNF-alpha-mediated antiapoptotic signaling in the human neutrophil.

The proinflammatory cytokine tumor necrosis factor (TNF)-alpha has been implicated in the attenuation of neutrophil spontaneous apoptosis during sepsis. Antiapoptotic signaling is principally mediated through the p60TNF receptor (p60TNFR). In neutrophils, TNF-alpha is an incomplete secretagogue and requires input from a ligated integrin(s) for neutrophil activation. In adherent neutrophils, TNF-alpha triggers association of both protein kinase C (PKC)-delta and phosphatidylinositol (PI) 3-kinase with the p60TNFR. In this study, a role for PKC-delta and PI 3-kinase in TNF-alpha-mediated antiapoptotic signaling was examined. TNF-alpha inhibited spontaneous apoptosis in fibronectin-adherent neutrophils, and this antiapoptotic signaling was blocked by the PKC-delta inhibitor rottlerin, but not by an inhibitor of Ca(2+)-dependent PKC isotypes, Go-6976. Inhibition of PI 3-kinase by LY-294002 also inhibited TNF-alpha-mediated antiapoptotic signaling. Cycloheximide blocked TNF-alpha-mediated antiapoptotic signaling, suggesting protein synthesis is required. Inhibition of either PKC-delta or PI 3-kinase attenuated TNF-alpha-mediated activation of the antiapoptotic transcription factor NFkappaB. Thus both PKC-delta and PI 3-kinase have essential roles in TNF-alpha-mediated antiapoptotic signaling in adherent neutrophils.