A Truncated, Activin-Induced Smad3 Protein Acts as a Transcriptional Repressor of FSHβ Expression in Mouse Pituitary.

Abstract

Activin signals are transduced by cell-surface receptors, which recruit and phosphorylate intracellular Smad2/3. Complexes of Smad2/3 (~52 kDa) and Smad4 translocate to the nucleus to act as transcriptional co-activators of activin-responsive genes. Within the pituitary gonadotrope, Smad2/3 degradation and turnover is believed to be a cellular mechanism for limiting the duration of activin-stimulated FSHβ expression. Immunoblots of pituitary gonadotrope cell (LβT2) lysates revealed a smaller than expected 30-kDa Smad3 protein, which was phosphorylated in response to activin treatment. Full-length (52 kDa) Smad3 was induced after 30 minutes of activin treatment, whereas the smaller Smad3 species accumulated slightly later, between 30 minutes and 1 hour, and then persisted beyond 2 hours. Activin-induced phosphorylation of both large and small phospho-Smad3 proteins was blocked by follistatin treatment or transfection of siRNA directed against Smad3. TGFβ failed to induce the small phospho-Smad3 protein in LβT2 cells. We determined that the small Smad3 protein was neither a proteolytic- nor a proteasome-processed form of Smad3, but a transcription product limited to the gonadotrope. 5' RACE performed with LβT2 RNA and a Smad3 MH2 region-specific primer confirmed the existence of two forms of Smad3 in LβT2 cells. To test the biological function of the small Smad3 protein, we constructed a constitutively active C-terminal fragment of Smad3 protein. This protein antagonized activin-stimulated FSHβ expression in LβT2 cells in a dose-dependent manner. These data explain prior observations in the pituitary cell, where activin-mediated FSHβ-expression is extinguished relatively rapidly compared with other TGFβ-regulated genes. Thus, we speculate that the pituitary gonadotrope may contain an ultra-short feedback loop by which activin activates both an agonist and an intracrine antagonist to limit FSHβ production during the normal reproductive cycle. This work is supported by NIH/NICHD grant R01 HD37096 "Inhibin Actions on Reproductive Target Tissues." (platform)