Production of follistatin in porcine endothelial cells: differential regulation 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 the highest level of FS expression, but ovariectomy does not cause a permanent 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 messenger RNA (mRNA) and protein; therefore, we studied the regulation of steady state levels of FS mRNA in porcine endothelial cells from aorta (AEC) and brain microvessels (BMVEC) in tissue culture. For detection of FS mRNA, a specific 32P-radiolabeled antisense probe and a S1-nuclease 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 increasing concentrations of FCS and stimulated by 30 micrograms/ml endothelial cell growth supplement. FS mRNA levels in AEC and BMVEC increased approximately 20-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, 13-acetate, whereas 0.5 nmol/ml forskolin tested in AEC for between 4-48 h had no significant effect. Furthermore, 0.1 microM ocadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, caused a significant increase in FS mRNA levels. FS mRNA levels 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 micrograms/ml bacterial lipopolysaccharides (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 synthetic glucocorticoid, inhibited FS mRNA steady state levels in a dose-dependent manner. The induction of FS mRNA with 1 microgram/ml LPS was completely blocked by 100 nM RU 28362, and the stimulatory effects of LPS were 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 proteins were secreted into the culture medium upon stimulation with LPS. None of these treatments had an obvious effect on the ratio of the two different 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 cerebrospinal 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 growth supplement, bacterial LPS, and the glucocorticoid RU 28362; 3) phosphatases and the protein kinase C-dependent, but not the protein kinase A-dependent, pathway are involved in regulating the steady state levels of FS mRNA in AEC and BMVEC; and 4) endothelial cells produce and secrete FS protein and are thus a likely source of FS in serum.