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In rabbit proximal tubular cells, ANG II type 2-receptor (AT2)-induced arachidonic acid release is PLA2 coupled and dependent of G protein βγ (Gβγ) subunits. Moreover, ANG II activates ERK1/2 and transactivates EGFR via a c-Src-dependent mechanism. Arachidonic acid has been shown to mimic this effect, at least in part, by an undetermined mechanism. In this study, we determined the effects of ANG II on fibronectin expression in cultured rabbit proximal tubule cells and elucidated the signaling pathways associated with such expression. We found that ANG II and transfection of Gβγ subunits directly increased fibronectin protein expression, and this increase was inhibited by overexpression of β-adrenergic receptor kinase (βARK)-ct or DN-Src. Moreover, ANG II-induced fibronectin protein expression was significantly abrogated by the AT2 receptor antagonist PD123319. In addition, inhibition of cystolic PLA2 diminished ANG II-induced fibronectin expression. Endogenous arachidonic acid mimicked ANG II-induced fibronectin expression. We also found that overexpression of Gβγ subunits induced c-Src, ERK1/2, and EGFR tyrosine phosphorylation, which can be inhibited by overexpression of βARK-ct or DN-Src. Gβγ also induced c-Src SH2 domain association with the EGFR. Supporting these findings, in rabbit proximal tubular epithelium, immunoblot analysis indicated that βγ expression was significant. Interestingly, arachidonic acid- and eicosatetraenoic acid-induced responses were preserved in the presence of βARK-ct. This is the first report demonstrating the regulation of EGFR, ERK1/2, c-Src, and fibronectin by Gβγ subunits in renal epithelial cells. Moreover, this work demonstrates a role for Gβγ heterotrimeric proteins in ANG II, but not arachidonic acid, signaling in renal epithelial cells., (Copyright © 2014 the American Physiological Society.)

Cyclic stretch-induced cPLA2 mediates ERK 1/2 signaling in rabbit proximal tubule cells. Background. Recent evidence from this laboratory have demonstrated a critical role of phospholipase A2 (PLA2) and arachidonic acid in angiotensin II type 2 (AT2) receptor-mediated kinase activation in renal epithelium independent of phosphoinositide- specific phospholipase C (PLC) and without the necessity of eicosanoid biosynthesis. In the present study, we investigated whether cyclic stress phosphorylates and activates the mitogen-activated protein kinase (MAPK) pathway and whether PLA2 activation mediates mechanotransduction in renal epithelial cells. The rational for studying kidney epithelial cells relates to their similarity to podocytes, which undergo mechanical stretch related to changes in intraglomerular pressure. Methods. To produce strain or stretch, primary cultures of rabbit proximal tubular cell cells are grown in tissue culture wells having a collagen-coated Silastic deformable membrane bottoms and applying vacuum to the well to generate alternating cycles of stretch and relaxation (30 cycles/min). Results. We found that cyclic stretching of rabbit proximal tubular cells caused a time- and intensity-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) in proximal tubular cells as detected by its phosphorylation. In addition, mechanical stretch induced PLA2 activation and a subsequent rapid release of arachidonic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphonate ketone (AACOCF3) attenuated both arachidonic acid release and ERK 1/2 activation by cyclic stretch, supporting the importance of PLA2 as a mediator of mechanotransduction in renal proximal tubular cells. A requirement for extracellular Ca2+ and stretch-activated Ca2+ channels was also documented. Complete inhibition of ERK 1/2 by PD98059, a MAPK kinase (MEK) inhibitor, did not suppress stretch- induced PLA2 activation and arachidonic acid release, suggesting the later events were upstream of ERK 1/2. Cyclic stretch also caused rapid phosphorylation of the EGF receptor kinase and c-Src. Furthermore, arachidonic acid itself induced time- and dose-dependent phosphorylation of c-Src. In addition, the c-Src inhibitor PP2 and selective EGF receptor kinase inhibitor AG1478 attenuated both ERK 1/2 and EGF receptor phosphorylation by cyclic stretch. Conclusion. PLA2 dependence for ERK 1/2 activation in response to cyclic stretch in proximal tubular epithelial cells was established in this report. In addition, these findings indicate cyclic stretch increased the tyrosine phosphorylation of the EGF receptor and c-Src and that c-Src acts upstream of the EGF receptor to mediate its phosphorylation, whereby both are critical for stretch- induced ERK 1/2 activation in rabbit proximal tubular cells. These observations documents for the first time a mechanism of mechanical stretch-induced kinase activation mediated by stretch activated Ca2+ channels and PLA2-dependent release of arachidonic acid. [ABSTRACT FROM AUTHOR]

Arachidonic acid directly activates members of the mitogen-activated protein kinase superfamily in rabbit proximal tubule cells. Background. To explore the roles of eicosanoids in arachidonic acid-induced mitogen-activated protein kinase (MAPK) signal transduction, we have shown that exposure of proximal tubular cells to arachidonic acid induces phosphorylation of c-Jun NH2 -terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two members of the MAPK superfamily. We observed that ketoconazole, an inhibitor of the cytochrome P450 pathway, blocked ERK but not JNK activation. Methods. Direct regulation of arachidonic acid on mitogen-activated protein kinase (MAPK) signaling pathways was evaluated more directly by utilizing specific enzyme inhibitors of the cytochrome P450 metabolic pathway and by comparing the relative efficacy of arachidonic acid versus its cytochrome P450 metabolites (exogenous and endogenous), eicosatetraynoic acid (ETYA), and other fatty acids on the phosphorylation of members of the MAPK superfamily (ERKs, JNK, and p38MAPK ), by utilizing early passage rabbit proximal tubular epithelial cells. Results. Arachidonic acid activated p38MAPK , a third member of the MAPK superfamily, in a time- and concentration-dependent manner. Studies designed to evaluate the ability of arachidonic acid and its cytochrome P450 metabolites (endogenously and exogenously) to stimulate ERKs, JNK, and p38MAPK found four conclusions. First, the metabolites of arachidonic acid generated endogenously by cytochrome P450 2C1 significantly augmented basal ERK activity, whereas the metabolites generated by the 2C2 isozyme significantly augmented basal p38MAPK activity. However, their effects were less profound than arachidonic acid itself. In contrast, there were no significant effects with transfection of either isozyme on basal JNK activity. Second, a variety of exogenous cytochrome P450 products were less potent than arachidonic acid on a molar basis in stimulating the activity of all three MAPKs. Third, ketoconazole and 17-octadecynoic acid, inhibitors of the cytochrome P450 pathway, as well as PPOH and DDMS, inhibitors of the epoxygenase and ω-hydroxylase pathways, respectively, failed to significantly reduce the effects of arachidonic acid to activate ERK and p38MAPK (JNK was not evaluated). Finally, arachidonic acid, its inactive analog ETYA, and other fatty acids with differing chain lengths and degrees of saturation stimulated the activity of all three MAPKs. Conclusions. These observations substantiate a role for arachidonic acid and other fatty acids in signaling linked to the MAPK superfamily in rabbit proximal tubular epithelium without the necessity of conversion to cytochrome P450 metabolites. [ABSTRACT FROM AUTHOR]

Proposes a phospholipase A2 (PLA-2)-dependent mechanism of activation of mitogen-activated protein kinase (MAPK) in proximal tubule epithelium, mediated by arachidonic acid (AA). Experimental procedures used to conduct the study; What is angiotensin II (Ang II); Functions of Ang II; Reference to previous studies describing a protein kinase C dependent mechanism of Ang II; Results and discussion on the study.

Presents information on a study which examined whether arachidonic acid mediates angiotensin II's (Ang II) effect on p21ras. Arachidonic acid and Ang II activate p21ras in renal proximal tubular cells; Information on the Ang II stimulation of p21ras; Results of the study.

Recent studies by our laboratory demonstrate that arachidonic acid activates ERK through c-Src dependent transactivation of the epidermal growth factor receptor (EGFR) in rabbit proximal tubular epithelial cells. We demonstrate here that stimulation with arachidonic acid results in a time- and dose-dependent activation of Akt which was significantly inhibited by PI3- kinase and Akt inhibitors, whereas arachidonic acid- induced activation of c-Src, EGFR, ERK, and p38 kinase was not modified. The p38 kinase inhibitor SB20358 significantly attenuated arachidonic acid- induced ERK and Akt activation, whereas the MEK inhibitor PD98059 had no inhibitory effect on Akt and p38 kinase activation in response to arachidonic acid in these cells. In addition, pertussis toxin (Gi inhibitor) and cholera toxin (Gs inhibitor) were without effect. Akt activation was also inhibited by the EGFR kinase inhibitor AG1478 and the Src kinase family inhibitor PP2, suggesting that the EGFR and c-Src may too be critical in linking arachidonic acid's effect on Akt signaling. Collectively, these findings demonstrate that arachidonic acid is a potent and direct activator of Akt in rabbit proximal tubular epithelium and demonstrate a critical upstream role of c-Src, EGFR and p38 kinase in arachidonic acid-induced Akt activation independent of the classical heterotrimeric G protein coupled receptors pathways. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38SAPK, ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38SAPK and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38SAPK is upstream of ERK1/2 and Akt1, since a p38SAPK inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38SAPK isoform α inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38SAPK provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress. [Copyright &y& Elsevier]