Cycloheximide inhibits follicle-stimulating hormone β subunit transcription by blocking de novo synthesis of the labile activin type II receptor in gonadotrope cells

• We examined how cycloheximide (CHX) inhibits FSH production in gonadotrope cells.
• We found that activins signal via ACVR2 instead of ACVR2B to control FSH synthesis.
• ACVR2 is rapidly degraded in gonadotropes and CHX blocks its de novo synthesis.
• CHX inhibits FSH synthesis by blocking activin signaling via ACVR2 in gonadotropes.
• ACVR2 degradation seems to be lysosome- and proteasome independent.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), play essential roles in the regulation of vertebrate reproduction. Activins and inhibins have opposing actions on FSH (but not LH) synthesis, either inducing or inhibiting transcription of the FSHβ subunit gene (Fshb). The translational inhibitor cycloheximide (CHX) produces inhibin-like effects in cultured pituitary cells, selectively suppressing FSH production. Using the murine gonadotrope-like cell line, LβT2, as a model, we tested the hypothesis that a component of the activin pathway is highly labile in gonadotrope cells and that its rapid loss following CHX treatment impairs activin-stimulated Fshb transcription. Treatment of cells with CHX for 6 h, but not 1 h, blocked activin A-stimulated Fshb transcription. Pre-treatment of LβT2 cells with CHX for as few as 2–3 h inhibited activin A-stimulated SMAD2/3 phosphorylation without altering total SMAD2/3 protein levels. These data indicated that CHX affects activin signalling upstream of SMAD proteins, most likely at the receptor level. Indeed, CHX rapidly reduced activin A binding to LβT2 cells. We went on to show that activin A signals via the type II receptor ACVR2, rather than ACVR2B, to regulate Fshb transcription and that the receptor has a half life of ~ 2 h in LβT2 cells. The mechanism of ACVR2 turnover remains undefined, but appears to be ligand-, proteasome-, and lysosome-independent. Collectively, these data indicate that CHX produces inhibin-like effects in gonadotropes by preventing de novo synthesis of the highly labile ACVR2, thereby blocking activin signaling to the Fshb promoter.