Abstract
Activin acts mitogenically on P19 cells as well as being inhibitory of the differentiation of retinoic acid-treated P19 cells and some neuroblastoma cell lines. Here, we show some lines of evidence that follistatin, an activin-binding protein, is also involved in neural differentiation. Counteracting the activity of activin, addition of follistatin suppresses the anchorage-independent growth of P19 cells in soft agar and stimulates neurite outgrowth of a neuroblastoma cell line, IMR-32 cells. While activin does not seem to be expressed significantly, follistatin is demonstrated in the conditioned medium of these cells. Furthermore, the expression of follistatin in P19 cells is subject to dynamic fluctuations in response to retinoic acid treatment. These neural cells may produce follistatin in a cell stage-specific manner in order to interact with exogenously derived activin.
Highlights
Laboratories,Ajirwmto CO.,Inc.,1-1 Suzuki-cho, Kawasaki-ku,Kawasaki 210, Japan of a RA-regulated mediator which interacts with activin
In the case of ligand blotting, the blots were incubated with Iz5I-activinfor 2 h at room temperature, washed with Tris-buffered saline (TBS) containing 0.1% Triton X-100 for 1 h, and autoradiography wasdeveloped for 4 days. 100 ngof purified porcine follistatin was blotted as a control
Follistatin PromotesDifferentiation of IMR-32 Cells-Among various neuraclell lines investigated (IMR-32,GOTO, NB-1, SK-N-SH, and PC12), IMR-32 cells werefound to differentiateinresponse to porcine follistatin
Summary
Ment, activin and RA differentially activate separate homeo box genes [10]. In cultured mammalian cells, activin inhibits RA-induced differentiation of P19 cells and some types of neuroblastoma cells [11, 12]. While activin does not seem to be expressed Follistatin Treatment of IMR-32 Cells-IMR-32 cells (104/ml)were significantly, follistatin is demonstrated in the condi- plated on 35-mm dishes in 2 ml of a-medium containing 8% FBS. After 5 days, phase-contrast photomicrographs were taken, and thecell number was counted by hemocytometer In another experiment, cells were treated with follistatin (0, 0.03,0.3,3, or 30 nM) alone for 14 days. Cells were treated with follistatin (0, 0.03,0.3,3, or 30 nM) alone for 14 days In this case, medium was changed at day 7 when cells were detached with Ca2+-freephosphatebuffered saline and replated. The middle layer containing 100/well of single cells in 50 pl of a-medium, 10% FBS, and 0.3% agar was overlaid.
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