Phorbol ester (TPA) reduces prostaglandin E2-stimulated cAMP production by desensitization of prostaglandin E2 receptors in a clonal osteoblast-like cell line, MOB 3-4

Abstract

A clonal osteoblast-like cell line, MOB 3-4, increased cAMP production in response to prostaglandin E2 (PGE2) (5–500 ng/ml). The purpose of this study was to show the effects of tumor-promoting phorbol ester (e.g., 12-O-tetradecanoylphorbol 13-acetate, TPA) on basal and PGE2-stimulated cAMP production and the affinity of PGE2 receptors in the cells. Pretreatment with TPA (1 nM–10 μM) for 30 minutes increased basal cAMP production, whereas it markedly reduced the PGE2-stimulated cAMP production in the presence of 0.1 mM isobuthylmethyl xanthine. Both the TPA increase and reduction were dose- and time-dependent. However, TPA exerted no effect on forskolinor cholera toxin-stimulated cAMP production. Copretreatment with TPA and H-7, an inhibitor of protein kinase C (PKC), prevented the TPA-induced increase in basal cAMP production, whereas it did not prevent the reduction of the PGE2-stimulated cAMP production. On the other hand, TPA (0.1–10 μM) decreased3H-PGE2 binding in a dose- and time-dependent manner. Scatchard analysis revealed that TPA decreased the apparent affinity of PGE2 receptors without effect on their apparent number. In addition, 1-oleoyl-2-acetylglycerol (12.6 μM), a synthetic diacylglycerol analog, did not mimic the TPA action on3H-PGE2 binding. Thus, TPA at relatively high concentrations appeared to increase basal cAMP production by a PKC-mediated mechanism, and it appeared to directly act on PGE2 receptors to decrease their apparent affinity and thereby reduce the PGE2-stimulated cAMP production in the clonal osteoblast-like MOB 3-4 cell line.