PRODUCTION OF FOLLISTATIN IN PORCINE ENDOTHELIAL-CELLS - DIFFERENTIALREGULATION BY BACTERIAL COMPOUNDS AND THE SYNTHETIC GLUCOCORTICOID RU-28362

Follistatin (FS) is the specific binding protein of activin; it has a broad tissue distribution and is also found in serum. The ovary has th e highest level of FS expression, but ovariectomy does not cause a per manent reduction in the serum FS level. Therefore, the source of FS in serum is still elusive. As a regulatable, nongonadal source of serum FS could influence ovarian and pituitary-derived hormone secretion and thus reproductive function, we searched for a source of extragonadal FS expression that might contribute to the FS protein level in serum. We found that endothelial cells from blood vessels express FS messenge r RNA (mRNA) and protein; therefore, we studied the regulation of stea dy state levels of FS mRNA in porcine endothelial cells from aorta (AE C) and brain microvessels (BMVEC) in tissue culture. For detection of FS mRNA, a specific P-32-radiolabeled antisense probe and a S1-nucleas e protection assay were used. FS steady state levels of AEC decreased with time in culture, i.e. postconfluent AEC had lower FS mRNA levels than confluent cultures, which, in turn, had lower FS mRNA levels than subconfluent cell cultures. FS mRNA levels in AEC were induced by inc reasing concentrations of FCS and stimulated by 30 mu g/ml endothelial cell growth supplement. FS mRNA levels in AFC and BMVEC increased app roximately 20-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, la-acetate, whereas 0.5 nmol/ml forskolin tes ted in AEC for between 4-48 h had no significant effect. Furthermore, 0.1 mu M ocadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, caused a significant increase in FS mRNA levels. FS mRNA leve ls in AEC were not significantly affected by various concentrations of porcine FSH, epidermal growth factor, or retinoic acid for between 4- 48 h. Treatment of the cells with 0.01-10 mu g/ml bacterial lipopolysa ccharides (LPS) caused a dose-dependent increase (up to 10-fold) in FS mRNA steady state level in AEC, whereas 1-1000 nM RU 28362, a synthet ic glucocorticoid, inhibited FS mRNA steady state levels in a dose-dep endent manner. The induction of FS mRNA with 1 mu g/ml LPS was complet ely blocked by 100 nM RU 28362, and the stimulatory effects of LPS wer e only visible after 4 h of treatment, not after 24 or 48 h. The same effects were observed with BMVEC. We, furthermore, analyzed FS protein secretion of AEC by Western blotting and demonstrated that FS protein s were secreted into the culture medium upon stimulation with LPS. Non e of these treatments had an obvious effect on the ratio of the two di fferent forms of FS mRNA (FS 344:FS 317). Besides the expression of FS mRNA in AEC and BMVEC, FS mRNA is also expressed in uncultured plexus choroideus epithel and meninges, and FS protein is found in human cer ebrospinal fluid. From this study it is concluded that 1) endothelial cells from different tissues produce FS mRNA; 2) the FS mRNA levels of AEC and BMVEC are subjected to regulation by FCS, endothelial cell gr owth supplement, bacterial LPS, and the glucocorticoid RU 28362; 3) ph osphatases and the protein kinase C-dependent, but not the protein kin ase A-dependent, pathway are involved in regulating the steady state l evels of FS mRNA in AEC and BMVEC; and 4) endothelial cells produce an d secrete FS protein and are thus a likely source of FS in serum.