Role of epidermal growth factor receptor transactivation in the activation of cytosolic phospholipase A2 in leptin protection of salivary gland acinar cells against ethanol cytotoxicity

Research Center, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, USA.
Journal of physiology and pharmacology: an official journal of the Polish Physiological Society (Impact Factor: 2.39). 07/2009; 60(2):49-55.
Source: PubMed


A pleiotropic hormone, leptin, secreted into saliva by the acinar cells of salivary glands is an important mediator of the processes of oral mucosal defense. Here, we report on the role of epidermal growth factor receptor (EGFR) transactivation in the signaling events that mediate leptin protection of sublingual salivary gland acinar cells against ethanol cytotoxicity. We show that the protective effect of leptin against ethanol cytotoxicity was associated with the increased EGFR protein tyrosine kinase and cytosolic phospholipase A(2) (cPLA(2)) activity, and characterized by a marked increase in matrix metalloproteinase MMP-9 and arachidonic acid (AA) release, and PGE(2) generation. The loss in countering capacity of leptin against ethanol cytotoxicity was attained with JAK inhibitor AG490, Src inhibitor PP2, and EGFR inhibitor AG1478, as well as ERK inhibitor PD98059. Moreover, the agents evoked also the inhibition in leptin-induced up-regulation in cPLA(2) activity, AA release, and PGE(2) generation. The changes caused by leptin in EGFR phosphorylation, MMP-9, and cPLA(2) activation were susceptible to suppression by metalloprotease inhibitor GM6001, but the production of MMP-9 was not affected by EGFR inhibitor AG1478 or PKC inhibitor Ro318220. These findings point to the involvement of MMP-9 in the event of leptin-induced EGFR transactivation that results in the signaling cascade leading to cPLA(2) activation and up-regulation in PGE(2) generation, thus providing new insights into the mechanism of oral mucosal protection against ethanol toxicity.

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    • "To assess the release of AA from the acinar cells of salivary gland into the incubation medium, aliquots of the cell suspension (1 ml) were labeled in DMEM with 20 μCi of [5,6, 8,9, 11,12,14,15-3H]arachidonic acid for 4 h [27], and resuspended in fresh DMEM free of albumin. The cells were then treated with the indicated dose of the agent of interest or vehicle and incubated for 2 h in the presence of 3% ethanol, and following centrifugation the supernatant was analyzed for the released [3H]arachidonic acid by scintillation spectrometry. "
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    ABSTRACT: Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A(2) (cPLA(2)) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA(2) activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA(2) phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA(2) protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA(2) activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.
    Full-text · Article · Jun 2010 · Advances in Pharmacological Sciences
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    • "The measurement of cPLA2 activity in the acinar cells following various experimental conditions was carried out using cPLA2 assay kit (Cayman). Following experimental treatments, the cells were homogenized in 1 ml of 50 mM HEPES buffer, pH 7.4, containing 1 mM EDTA, centrifuged at 10,000× g for 15 min at 4°C and the supernatants filtered through an Amicon YM30 filter concentrators, followed by 15 min incubation with 5 µM of calcium-independent PLA2 inhibitor, bromoenol lactone, and the aliquots (10 µl) of such prepared cell lysates were subjected to cPLA2 assay (27). "
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    ABSTRACT: Recent advances in identifying the salivary constituents of significance to the maintenance of soft oral tissue integrity have brought to focus the importance of a 28-amino acid peptide hormone, ghrelin. Here, we report on the role of ghrelin in countering the disturbances in salivary mucin synthesis caused by ethanol cytotoxicity in rat sublingual gland acinar cells. We show that the countering effect of ghrelin on mucin synthesis was associated with the increase in NO and PGE2 production, and the enhancement in cytosolic phospholipase A2 (cPLA2) activity. The ghrelin-induced up-regulation in mucin synthesis, like that of cPLA2 activity, was subject to suppression by Src inhibitor, PP2, ERK inhibitor, PD98059, as well as Akt inhibitor, SH-5 and ascorbate. Moreover, the loss in countering effect of ghrelin on the ethanol cytotoxicity and mucin synthesis was attained with cNOS inhibitor, L-NAME as well as COX-1 inhibitor, SC-560. Furthermore, while the effect of L-NAME was also reflected in the inhibition of the acinar cell capacity for NO and PGE2 generation, and cPLA2 S-nitrosylation, the COX-1 inhibitor caused the inhibition in PGE2 only. Our findings demonstrate that ghrelin protection of the acinar cells against ethanol cytotoxicity and the impairment in salivary mucin synthesis involves Src kinase activation of the Akt/cNOS pathway that leads to up-regulation in cNOS activity. We also show that cNOS-derived NO induction of the cPLA2 activation through S-nitrosylation, for the increase in PGE2 generation, is an essential element of the protective mechanism of ghrelin action.
    Full-text · Article · Mar 2010
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    ABSTRACT: Many experimental and clinical studies have demonstrated that elevated leptin concentration in patients with obesity/metabolic syndrome contributes to the pathogenesis of cardiovascular disorders including arterial hypertension, atherosclerosis, restenosis after coronary angioplasty and myocardial hypertrophy. Receptor tyrosine kinases belonging to the ErbB family, especially ErbB1 (epidermal growth factor receptor) and ErbB2 are abundantly expressed in the blood vessels and the heart. EGFR is activated not only by its multiple peptide ligands but also by many other factors including angiotensin II, endothelin-1, norepinephrine, thrombin and prorenin; the phenomenon referred to as "transactivation". Augmented EGFR signaling contributes to abnormalities of vascular tone and renal sodium handling as well as vascular remodeling and myocardial hypertrophy through various intracellular mechanisms, in particular extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinase (PI3K). Recent experimental studies indicate that chronically elevated leptin transactivates the EGFR through the mechanisms requiring reactive oxygen species and cytosolic tyrosine kinase, c-Src. In addition, hyperleptinemia increases ErbB2 activity in the arterial wall. Stimulation of EGFR and ErbB2 downstream signaling pathways such as ERK and PI3K in the vascular wall and the kidney may contribute to the increase in vascular tone, enhanced tubular sodium reabsorption as well as vascular and renal lesions in hyperleptinemic obese subjects.
    No preview · Article · May 2013 · Current pharmaceutical design