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S P Jayawickreme

Publications and source records attributed to S P Jayawickreme.

6 recordsLinked to original sources
PubMed1999

Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells.

Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-alpha, IL-1beta, and interferon (IFN)-gamma had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1beta. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-gamma or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-gamma and 10 microM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.

Arachidonate 15-Lipoxygenase↗
PubMed1997

The linoleic acid metabolite, (13S)-hydroperoxyoctadecadienoic acid, augments the epidermal growth factor receptor signaling pathway by attenuation of receptor dephosphorylation. Differential response in Syrian hamster embryo tumor suppressor phenotypes.

In Syrian hamster embryo (SHE) fibroblasts, epidermal growth factor receptor (EGFR) tyrosine kinase activity regulates the metabolism of endogenous linoleic acid to (13S)-hydroperoxyoctadecadienoic acid (13S)-HPODE). (13S)-HPODE stimulates EGF-dependent mitogenesis in a SHE cell phenotype, which expresses tumor suppressor genes (supB+), but was not effective in a variant that does not express these suppressor genes (supB-). In the present study, we have investigated the potential effects of this lipid metabolite on the EGFR signaling pathways in these two SHE cell lines. Treatment of quiescent SHE cells with EGF produced a rapid, transient increase in the tyrosine phosphorylation of EGFR. Dependence on EGF concentration for EGFR tyrosine phosphorylation was similar in both SHE cell lines, but a more prolonged phosphorylation was detected in the supB- variant. Incubation of supB+ cells with (13S)-HPODE and EGF increased EGFR autophosphorylation and tyrosine phosphorylation on several signaling proteins with Src homology-2 domains including GTPase-activating protein. The lipid metabolite did not significantly alter EGF-dependent tyrosine phosphorylation in the supB- variant. Tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was also measured. The addition of (13S)-HPODE increased the extent and duration of MAP kinase tyrosine phosphorylation in supB+ cells but not in the supB- variant. MAP kinase activity in supB+ cells, as measured in immunoprecipitates from cells after the addition of EGF, was increased by the presence of (13S)-HPODE. The addition of (13S)-HPODE did not directly alter EGFR kinase activity or the internalization of the EGFR. However, the addition of (13S)-HPODE to supB+ cells extended the tyrosine phosphorylation of the EGFR in response to EGF. The dephosphorylation of the EGFR was measured directly, and a slower rate was observed in the supB- compared with the supB+ cells. Incubation of the supB+ cells with (13S)-HPODE attenuated the dephosphorylation of the EGFR. Thus, (13S)-HPODE stimulates EGF-dependent mitogenesis and up-regulation of EGF-dependent tyrosine phosphorylation by inhibiting the dephosphorylation of the EGFR. This study shows that a metabolite of an essential dietary fatty acid, linoleic acid, can modulate tyrosine phosphorylation and activity of key signal transduction proteins in a growth factor mitogenic pathway.

Animals↗
PubMed1994

Forskolin stabilizes epsilon subunit-containing acetylcholine receptors.

Fetal muscle-like acetylcholine receptors (AChRs) are composed of alpha, beta, gamma, delta subunits (gamma-AChRs) and have a rapid half life (t1/2), whereas adult muscle-like AChRs are composed of alpha, beta, epsilon, delta subunits (epsilon-AChRs) and have a slow t1/2. Two populations of AChRs, a slowly degrading population and a rapidly degrading population, have been shown to coexist in the postsynaptic membrane after denervation [In: Penn et al. (Eds.), Myasthenia Gravis and Related Disorders, Vol. 681, NY Acad. Sci., 1993, pp. 155-164]. Treatment of rat myotubes or mouse diaphragm muscle in organ culture with forskolin or cAMP analogues causes and increase in the t1/2 of the slowly degrading population of AChRs with no apparent effect on the rapidly degrading population of AChRs19. In this study, we have investigated the effect of forskolin on the cell surface half-lives of mouse gamma-AChRs, epsilon-AChRs and alpha beta delta complexes stably expressed in mouse fibroblasts. Forskolin had no significant effect on the t1/2 of gamma-AChRs or alpha beta delta complexes. The effect of forskolin on surface AChRs (alpha beta gamma delta) expressed in the C2 muscle cell line was similar to its effect on gamma-AChRs expressed in fibroblasts. In contrast, forskolin stabilized the epsilon-AChRs by approximately 2 fold. We show that the epsilon-subunit is phosphorylated in vivo, phosphorylation of epsilon increases with forskolin treatment, and the forskolin effect is reversible. Although the precise role of epsilon-subunit phosphorylation is yet to be determined, our results support the hypothesis that the slowly degrading population of AChRs consists of epsilon-AChRs and the rapidly degrading population of AChRs consists of gamma-AChRs.

Animals↗
PubMed1994

Cyclic AMP-regulated AChR assembly is independent of AChR subunit phosphorylation by PKA.

Forskolin treatment of cells expressing Torpedo acetylcholine receptors leads to enhanced assembly efficiency of subunits, which correlates with increased phosphorylation of the gamma subunit. To determine the role of the two potential protein kinase A sites of the gamma subunit in receptor assembly, cell lines expressing different mutant receptors were established. Mouse fibroblast cell lines stably expressing wild-type Torpedo acetylcholine receptor alpha, beta, delta subunits plus one of three gamma subunit mutations (S353A, S354A, or S353,354A) were established to identify the protein kinase A phosphorylation sites of gamma in vivo, and to determine if increased phosphorylation of the gamma subunit leads to enhanced expression of receptors. We found that both serines (353, 354) in gamma are phosphorylated in vivo by protein kinase A, however, phosphorylation of either or both of these sites does not lead to increased assembly efficiency. We established a cell line expressing alpha, beta, and gamma(S353,354A) subunits only (no delta), and found that the presence of delta (or its phosphorylation) is also not necessary for the observed stimulation by forskolin. alpha beta gamma, alpha gamma, and beta gamma associations were stimulated by forskolin but alpha beta and alpha delta interactions were not. These data imply that the presence of gamma is necessary for forskolin action. We postulate that forskolin may stimulate acetylcholine receptor expression through a cellular protein that is involved in the folding and/or assembly of protein complexes, and that forskolin may regulate the action of such a protein through phosphorylation.

3T3 Cells↗