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

Bronislaw L. Slomiany, Amalia Slomiany

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DOI: 10.4236/ajmb.2011.12006                

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 Tyr⁴¹⁶. Further, we show that the countering effect of ghrelin on the LPS-induced 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 Ser1179. 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-nitrosylation and the increase in its phosphorylation at Tyr⁴¹⁶.

Keywords

Ghrelin; P. Gingivalis; Salivary Acinar Cells; cNOS; cSrc Activation; S-Nitrosylation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Kojima, M., Hosoda, H., Date, Y., Nakazato, M. and Kangawa, K. (1999) Ghrelin is a growth-hormonereleasing acylated peptide from stomach. Nature, 402, 656-660. doi:10.1038/45230
[2]	Kojima, M. and Kangawa, K. (2005) Ghrelin: Structure and function. Physiology Reviews, 85, 495-522. doi:10.1152/physrev.00012.2004
[3]	Groschl, M., Topf, H.G., Bohlender, J., Zenk, J., Klussmann, S., Dotsch, J., Rascher, W. and Rauh, M. (2005) Identification of ghrelin in human saliva: production by the Salivary glands and potential role in proliferation of oral keratinocytes. Clinical Chemistry, 51, 997-1006. doi:10.1373/clinchem.2004.040667
[4]	Slomiany, B.L. and Slomiany, A. (2010) Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis- induced salivary gland acinar cell apoptosis. Inflammopharmacology, 18, 119-125. doi:10.1007/s10787-010-0035-7
[5]	Slomiany, B.L. and Slomiany, A. (2010) Suppression by ghrelin of Porphyromonas gingivalis-induced constitutive nitric oxide synthase S-nitrosylation and apoptosis in Salivary gland acinar cells. Journal of Signal Transduction, 2010, Article ID 280464. doi:10.1155/2010/643642
[6]	Slomiany, B.L. and Slomiany, A. (2010) Role of ghrelin in modulation of S-nitrosylation-dependent Akt inactivation induced in salivary gland acinar cells by Porphyromonas gingivalis. Health, 2, 1448-1455. doi:10.4236/health.2010.212215
[7]	Pierce, K.L., Premont, R.T. and Lefkowitz, R.J. (2002) Seven-transmembrane receptors. Nature Reviews Molecular Cell Biology, 3, 639-650. doi:10.1038/nrm908
[8]	Lodeiro, P., Theodoropoulou, M., Pardo, M., Casanueva, F.F. and Camina, J.P. (2009) c-Src regulates Akt signaling in response to ghrelin via b-arrestin signaling-independent and—dependent mechanism. PLoS ONE, 4, Article ID e4686.
[9]	Roskoski, R. (2004) Src protein-tyrosine kinase structure and regulation Biochemical and Biophysical Research Communications, 324, 1155-1164. doi:10.1016/j.bbrc.2004.09.171
[10]	Lutrell, D.K. and Lutrell, L.M. (2004) Not so strange bedfellows: G-protein-coupled receptors and Src family kinases. Oncogene, 23, 7969-7978. doi:10.1038/sj.onc.1208162
[11]	Chiaguri, P. (2008) Src redox regulation: There is more than meets the eye. Molecules and Cells, 26, 329-337.
[12]	Cayer, M.P., Proulx, M., Ma, X.Z., Sakac, D., Giguere, J.F., Drouin, M., Neron, S., Branch, D.R. and Jung, D. (2009) c-Src tyrosine co-associates with and phosphory- lates signal transducer and activator of transcription 5b which mediates the proliferation of normal human B lymphocytes. Clinical & Experimental Immunology, 156, 419-427. doi:10.1111/j.1365-2249.2009.03917.x
[13]	Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E. and Stamler, J.S. (2005) Protein S-nitrosylation: Purview and parametrs. Nature Reviews Molecular Cell Biology, 6, 150-166. doi:10.1038/nrm1569
[14]	Rahman, M.A., Senga, T., Ito, S., Hyodo, T., Hasegawa, H. and Hamaguchi, M. (2010) S-nitrosylation at cysteine 498 of c-Src tyrosine kinase regulates nitric oxidemediated cell invasion. Journal of Biological Chemistry, 285, 3806-3814. doi:10.1074/jbc.M109.059782
[15]	Akhand, A.A., Pu, M., Senga, T., Kato, M., Suzuki, H., Miyata, T., Hamaguchi, M. and Nakashima, I. (1999) Nitric oxide controls Src kinase activity through a sufhydryl group modification-mediated Tyr-527-independent and Tyr-416-linked mechanism. Journal of Biological Chemistry, 274, 25821-25826. doi:10.1074/jbc.274.36.25821
[16]	Minetti, M., Mallozzi, C. and Stassi, A.M. Di (2002) Peroxynitrite activates kinases of the Src family and upregulates tyrosine phosphorylation signaling. Free Radical Biology & Medicine, 33, 744-754. doi:10.1016/S0891-5849(02)00891-2
[17]	Xu, X., Jhun, B.S., Ha, C.H. and Jin, Z.G. (2008) Molecular mechanisms of ghrelin-mediated endothelial nitric-oxide synthase activation. Endocrinology, 149, 4183- 4192. doi:10.1210/en.2008-0255
[18]	Slomiany, B.L. and Slomiany, A. (2003) Activation of peroxisome proliferator-activated receptor g impedes Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis through phosphatidy-linositol 3-kinase/ERK pathway. (2003) Journal of Physiology and Pharmacology, 54, 3-15.
[19]	Slomiany, B.L. and Slomiany, A. (2005) Role of leptin in modulation of Porphyromonas gingivalis lipopolysaccharide-induced up-regulation of endothelin-1 in salivary gland acinar cells. IUBMB Life, 57, 591-595. doi:10.1080/15216540500215598
[20]	Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S. and Tannenbaum, S.R. (1982) Analysis of nitrte, nitrite, and [15N] nitrate in biological fluids. Analytical Biochemistry, 126, 131-138. doi:10.1016/0003-2697(82)90118-X
[21]	Lee, S., Lin, X., Nam, N.H., Parang, K. and Sun, G. (2003) Determination of the substrate-docking site of protein tyrosine kinase c-terminal Src kinase. Proceedings of the National Academy of Sciences of the United States of America, 100, 14707-14712. doi:10.1073/pnas.2534493100
[22]	Tamiya, S. and Delamere, N.A. (2005) Studies of tyrosine phosphorylation and Src family tyrosine kinases in the lens epithelium. Investigative Ophthalmology & Visual Science, 46, 2076-2081. doi:10.1167/iovs.04-1199
[23]	Jaffrey, S.R., Erdjument-Bromage, H., Ferris, D., Tempst, P. and Snyder, S.H. (2001) Protein S-nitrosylation: A physiological signal for neuronal nitric acid. Nature Cell Biology, 3, 193-197. doi:10.1038/35055104
[24]	Forrester, M.T., Foster, M.W. and Stamler, J.S. (2007) Assessment and application of the biotin switch technique for examining protein S-nitrosylation under conditions of pharmacologically induced oxidative stress. Journal of Biological Chemistry, 282, 13977-13983. doi:10.1074/jbc.M609684200
[25]	Ximenz-Fyvie, L.A., Haffajee, A.D. and Socransky, S. (2000) Micorbial composition of supra- and subgingival plaque in subjects with adult periodontitis. Journal of Clinical Periodontology, 27, 722-732. doi:10.1034/j.1600-051x.2000.027010722.x
[26]	Nonnenmacher, C., Mutters, R. and de Jacoby, L.F. (2001) Microbiological characteristics of subgingival microbiota in adult periodontitis, localized juvenile periodontitis and rapidly progressive periodontitis subjects. Clinical Microbiology and Infection, 7, 213-221. doi:10.1046/j.1469-0691.2001.00210.x
[27]	Wang, P.L. and Ohura, K. (2002) Porphyromonas gingi- valis lipopolysaccharide signaling in gingival fibro-blasts—CD14 and Toll-like receptors. Critical Reviews in Oral Biology and Medicine, 13, 132-142. doi:10.1177/154411130201300204
[28]	Waseem, T., Duxbury, M., Ito, H., Ashley, S.W. and Robinson, M.K. (2008) Exogenous ghrelin modulates release of proinflammatory and anti-inflammatory cytokines in LPS-stimulated macrophages through distinct signaling pathways. Surgery, 143, 334-342. doi:10.1016/j.surg.2007.09.039
[29]	Haynes, M.P., Li, L., Sinha, D., Russell, K.S., Hisamoto, K., Baron, R., Collinge, M., Sessa, W.C. and Bender, J.R. (2003) Src kinase mediates phosphatidylinositol3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. Journal of Biological Chemistry, 278, 2118-2123. doi:10.1074/jbc.M210828200
[30]	Korhonen, R., Lahti, A., Kankaanranta, H. and Moilanen, E. (2005) Nitric oxide production and signaling in inflammation. Current Drug Targets: Inflammation & Allergy, 4, 471-479. doi:10.2174/1568010054526359
[31]	Yu, S.M., Wu, J.F., Lin, T.L. and Kuo, S.C. (328) Inhibition of nitric oxide synthase expression by PPM-18, a novel anit-inflammatory agent, in vitro and in vivo. Biochemical Journal, 328, 363-369.
[32]	Slomiany, B.L. and Slomiany, A. (2011) Ghrelin suppression of Helicobacter pylori induced S-nitrosylation-dependent gastric mucosal Akt inactivation exerts modulatory influence on gastric mucin synthesis. Inflammo-pharmacology,19, 89-97. doi:10.1007/s10787-011-0078-4
[33]	Erwin, P.A., Lin, A.J., Golan, E. and Michel, T. (2005) Receptor-regulated dynamic S-nitrosylation of endothelial nitric-oxide synthase in vascular endothelial cells. Journal of Biological Chemistry, 280, 19888-19894. doi:10.1074/jbc.M413058200