Phorbol ester and epidermal growth factor enhance the expression of two inducible prostaglandin H synthase genes in rat tracheal epithelial cells.
Previous studies from our laboratory suggested that phorbol 12-myristate 13-acetate (TPA) stimulates prostaglandin E2 (PGE2) production by inducing de novo synthesis of prostaglandin H synthase (PHS) in a rat tracheal cell line. We report here an extension of this work to further elucidate the mechanisms by which TPA (and epidermal growth factor) stimulates PGE2 production. We used the rat tracheal cell line EGV6, which has a lower basal level of PGE2 production and responds to TPA and EGF stimulation with a much greater increase in PGE2 synthesis than the previously used cell line, Incubation of EGV6 cultures with TPA or EGF resulted in a time- and dose-dependent increase in PGE2 synthesis up to 40-fold and 6-fold, respectively. Serum also stimulated PGE2 synthesis, while bombesin, retinoic acid, and bacterial lipopolysaccharide did not. PHS protein levels in microsomal preparations from the cells were estimated by Western analysis. Antibodies raised against murine PHS-2 cross reacted with the EGV-6 PHS while several antibody preparations that react with PHS-1 from ram or mouse reacted poorly with the cellular preparation. TPA treatment increased the de novo synthesis of PHS-2 while dexamethasone treatment reduced the response to TPA. Northern blot analysis of mRNA from EGV6 cultures using a ram PHS cDNA revealed a 2.8- and a 4.5- to 4.9-kb (designated 4.9 kb) transcript. Treatment with TPA or EGF increased the expression of both transcripts and this effect was further enhanced by cyclohexamide. To further define the PHS mRNA species of EGV6 cells, two well-characterized murine PHS cDNA probes were used. The constitutive murine PHS cDNA probe hybridized only with the 2.8-kb transcript, and the inducible murine PHS cDNA hybridized only with the 4.9-kb transcript. The rates of induction as well as degradation of the 4.9-kb PHS mRNA were much more rapid than those of the 2.8-kb mRNA species. Dexamethasone partially inhibited the induction of both PHS transcripts by TPA. Southern analysis of genomic EGV6 DNA indicated the presence of two distinct PHS genes in these cells. Taken together these findings indicate that two PHS genes are expressed in rat tracheal epithelial cells. In contrast to the PHS genes expressed in murine (and chicken) fibroblasts in which only the gene coding for the larger mRNA species is transcriptionally regulated, in the rat tracheal cells both genes are positively regulated by TPA and EGF and downregulated by glucocorticoids.
Hamasaki
Y
Y
Laboratory of Molecular Biophysics, National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709.
Kitzler
J
J
Hardman
R
R
Nettesheim
P
P
Eling
T E
TE
D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004848:Epithelium / Q000201:enzymology; D015870:Gene Expression / Q000187:drug effects; D005796:Genes; D066298:In Vitro Techniques; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013997:Time Factors; D014132:Trachea / Q000201:enzymology
D012333:RNA, Messenger; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate