Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I 2)

Abstract

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1- 14C]prostaglandin H 2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin L 2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F 1α, the stable hydrolysis product of prostaglandin I 2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I 2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I 2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I 2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E 1 > prostaglandin H 2 > prostaglandin E 2, while prostaglandin D 2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I 2 or prostaglandin H 2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I 2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I 2.