FOLLISTATIN STEADY-STATE MESSENGER-RIBONUCLEIC-ACID LEVELS IN CONFLUENT CULTURES OF A RAT RENAL MESANGIAL CELL-LINE ARE REGULATED BY MULTIPLE FACTORS

The regulation of steady state levels of follistatin (FS) messenger RNA (mRNA) was examined in a rat renal mesangial cell line in tissue culture. A specific P-32-radiolabeled antisense probe was used which corresponds to the 3' end of exon 5 together with the 5' end of exon 6 of the rat FS gene, and which distinguishes between the two different forms of FS mRNA. In addition, a specific S-35-radiolabeled probe for the ubiquitous protein cyclophilin was developed and used as an internal standard. Total RNA was harvested from confluent cell cultures to yield four independent samples per treatment/time point, and equal amounts of RNA from every sample in a given experiment were subjected to S1-nuclease analysis for the estimation of specific mRNA levels. Treatment of the cultured cells with epidermal growth factor (10 nM) caused an 8- to 9-fold increase in the FS mRNA level after 4 h, but no consistent change was observed after treatment with basic fibroblast growth factor (0.28 or 0.56 nM), somatostatin (3.7-73 nM), angiotensin II (0.1-2500 nM), or FS itself (0.29 nM) for between 4 and 48 h. Neither activin (0.5 or 1.2 nM) nor inhibin (0.64 nM) changed the FS mRNA level in the mesangial cell line during a 24-h treatment. FS mRNA levels in the cells also were not affected by a 48-h treatment with the steroids dihydrotestosterone (1-1000 nM), estradiol (1 and 100 nM), and the antiprogesterone RU 486 (1000 nM), whereas 100 nM RU 28362 (a synthetic glucocorticoid) caused a 5- to 6-fold increase and 1000 nM progesterone increased the FS mRNA level up to 3.5-fold above control. Retinoic acid, a vitamin A derivative, significantly increased the FS steady state mRNA level at 3 nM, and at 1000 nM stimulated FS mRNA up to 5-fold within 4 h, whereas incubation of the cells with 30-mu-M prostaglandin E2 for 4 h caused a 10-fold increase. The FS mRNA level increased 3- and 4-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, 13-acetate, and 25-mu-M forskolin, respectively, whereas the calcium ionophore A23187 (1-100-mu-M) caused no change within this timespan. None of the tested hormones had an obvious effect on the ratio of the two different forms of FS mRNA (FS 344:FS 317). From this study we conclude that: 1) glomerular mesangial cells are a likely source of FS in the rat kidney; 2) the FS mRNA level is subjected to regulation by not only peptide/protein but also steroid and eicosanoid hormones; and 3) the protein kinase A- and C-mediated pathways are, but the calcium/calmodulin-dependent kinase-mediated pathways are probably not, involved in regulating the steady state levels of FS mRNA.