Ghrelin-induced cSrc activation through constitutive nitric oxide synthase-dependent S-nitrosylation in modulation of salivary gland acinar cell inflammatory responses to <i>Porphyromonas gingivalis</i>

Abstract

A peptide hormone, ghrelin, recognized for its role in the regulation of nitric oxide production has emerged as an important modulator of oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. As cSrc kinase plays a major role in controlling the activity of nitric oxide synthase (NOS) system, in this study we investigated the influence of P. gingivalis LPS on the processes of Src activation in rat sublingual gland acinar cells. The LPS-induced enhancement in the activity of inducible (i) iNOS and the impairment in constitutive (c) cNOS were reflected in the suppression in cSrc activity and the extent of its phosphorylation at Tyr 416. Further, we show that the countering effect of ghrelin on the LPSinduced changes in cSrc activity and the extent of its phosphorylation was accompanied by a marked reduction in iNOS and the increase in cNOS activation through phosphorylation at Ser 1179 . Moreover, the effect of ghrelin on cSrc activation was associated with the kinase S-nitrosylation that was susceptible to the blockage by cNOS inhibition. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to disturbances in cNOS phosphorylation that exerts the detrimental effect on the processes of cSrc activation through cNOS mediated S-nitrosylation. We also show that the effect of ghrelin on P. gingivalis-induced inflammatory changes are manifested in the enhancement in cSrc activation through S-nitro-sylation and the increase in its phosphorylation at Tyr 416 .

Role of Protein Kinase C<i>δ</i>-Mediated Spleen Tyrosine Kinase (Syk) Phosphorylation on Ser in the Amplification of Oral Mucosal Inflammatory Responses to <i>Porphyromonas gingivalis</i>

“…P. gingivalis used for LPS preparation was cultured from clinical isolates obtained from ATCC No. 33277 [25]. In the experiments evaluating the effect of PKC inhibitors, classical PKC isoforms, Gö6976 and the inhibitor of classical and novel PKC isoforms, GF109203X (Sigma), as well as the inhibitors of JNK, SP600125, ERK, PD98059, and p38, SB202190 (Calbiochem), the cells were first preincubated for 30 min with the indicated dose of the agent or vehicle before the addition of the LPS.…”

Role of Signal-Regulated Changes in Microtubule Stability Dynamics through <i>α</i>-Tubulin Phosphorylation on Ser/Tyr in Modulation of Salivary Gland Matrix Metalloproteinase-9 (MMP-9) Secretion in Response to <i>Porphyromonas gingivalis</i> and Ghrelin

“…P. gingivalis used for LPS preparation was cultured from clinical isolates obtained from ATCC No. 33277 [4]. In the experiments evaluating the effect of ghrelin (rat) and wide spectrum PKC inhibitor, GF109203X (Sigma), Src family protein tyrosine kinase (SFK-PTK) selective inhibitor, PP2, ER-to-Golgi transport inhibitor, Brefeldin A (BFA), microtubule stabilizing agent, tubacin (Tb) and microtubule destabilization agent, nocodazole (Noc), (Calbiochem), the cells were first preincubated for 30 min with the indicated dose of the agent or vehicle before the addition of the LPS.…”

“…The oral mucosal responses to P. gingivalis and its key endotoxin, cell-wall lipopolysaccharide (LPS), are characterized by the disturbances in nitric oxide synthase and cyclooxygenase systems, up-regulation in EGFR and MAPK activation, and induction in the secretion of highly glycosylated endopeptidase, metalloproteinase-9 (MMP-9) [4] [5] [6] [7] [8]. Similarly, to other regulated secretory proteins, the processing of MMP-9 along the endoplasmic reticulum (ER), Golgi, and trans-Golgi network (TGN) remains under a strict control of factors that affect the membrane recruitment and activation of various coat and cargo proteins, including ADPribosylation factors (Arfs) and protein kinase D (PKD), [9] [10] [11] [12].…”

<i>Porphyromonas gingivalis</i>-Stimulated TACE Activation for TGF-<i>α</i> Ectodomain Shedding and EGFR Transactivation in Salivary Gland Cells Requires Rac1-Dependent p38 MAPK Membrane Localization

“…P. gingivalis used for LPS preparation was cultured from clinical isolates obtained from ATCC No. 33277 [6]. In the experiments evaluating the effect of p38 MAPK inhibitor, SB202190, ERK inhibitor, P98059, JNK inhibitor, SP600125, and TACE (TNF-α converting enzyme) inhibitor, TAPI-1 (Calbiochem), Rac1 inhibitor, NSC 23766, and EGFR inhibitor, AG1478 (Sigma), the cells were first preincubated for 30 min with the indicated dose of the agent or vehicle before the addition of the LPS.…”

“…The acinar cells from various experimental treatments were collected by centrifugation and resuspended for 30 min in ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM sodium orthovanadate, 4 mM sodium pyrophosphate, 1 mM PMSF, and 1 mM NaF), containing 1 µg/ml leupeptin and 1 µg/ml pepstatin [6] [25]. Following brief sonication, the lysates were centrifuged at 10,000 g for 10 min, and the supernatants were subjected to protein determination using BCA protein assay kit (Pierce).…”