Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells.

Bone morphogenetic protein-15 (BMP-15), an oocyte growth factor belonging to the transforming growth factor-beta superfamily, has recently been shown to be necessary for normal female fertility in mammals. We have previously demonstrated that BMP-15 regulates granulosa cell (GC) proliferation and differentiation; namely, BMP-15 promotes GC mitosis, suppresses follicle-stimulating hormone (FSH) receptor expression, and stimulates kit ligand expression. Although the role of BMP-15 in female reproduction has progressively deserved much attention, there is nothing known to date about the signaling pathway and receptors for BMP-15. Using rat primary GCs and a human GC cell line, COV434, we have now found that administration of BMP-15 causes a rapid and transient phosphorylation, thus activation, of the Smad1/5/8 pathway. BMP-15 also stimulated promoter activity of a selective BMP-responsive reporter construct, further demonstrating the stimulation of Smad1/5/8 signaling by BMP-15. In contrast, BMP-15 stimulation of Smad2 phosphorylation was very weak. To identify the receptors for BMP-15, we utilized recombinant extracellular domains of individual transforming growth factor-beta superfamily receptors and found that activin receptor-like kinase-6 extracellular domain most effectively co-immunoprecipitates with BMP-15, whereas BMP receptor type II extracellular domain was most effective in inhibiting BMP-15 bioactivity on FSH-induced progesterone production and GC thymidine incorporation. We also investigated whether activation of the MAPK pathway is necessary for BMP-15 biological activity and found that the addition of U0126, an inhibitor of ERK1/2 phosphorylation, suppresses BMP-15 activity on GC mitotsis but not on FSH-induced progesterone production, suggesting a selective signaling cascade in GC proliferation and differentiation.