Activin induction of the ovine follicle-stimulating hormone beta-subunit is mediated by Smad4 and a forkhead box transcription factor

Abstract

Follicle stimulating hormone (FSH) is a a/b glycoprotein produced in pituitary gonadotropes of all vertebrates and is required for egg maturation, and optimal sperm performance. Activin potently induces FSH by inducing transcription of its rate-limiting b-subunit (FSHB). Four nucleotides of the oFSHB promoter (–168 bp to –165 bp) are necessary for 99.9 % of oFSHB expression in vivo and 70 % of its progressive induction by activin in LbT2 gonadotropes over a 24 hr period. These 4 nucleotides form part of a putative forkhead box (FOX) binding site juxtaposed upstream to a single-copy (4 bp) Smad binding element (SBE), both of which are associated with activin action. Smad4 from LbT2 cells did not bind the wild type oFSHB SBE in electrophoretic mobility shift assays, but did bind a palindromic SBE derived from the oFSHB SBE. Binding increased 2.6-fold over 20 h without or with activin (25 % additional increase with activin) and was competed 85 % with the native oFSHB sequence indicating Smad4 has high affinity for the native promoter. Additionally, a dominant negative inhibitor of Smad4 reduced activin induction of oFSHB in LBT2 cells by 62 %, indicating that Smad4 is important for activin induction. A second Smad, Smad3, bound transiently to the palindromic SBE (6-fold increase by activin at 2 h). Dominant negative inhibition of Smad3 (3SA) and depletion of Smad2 by siRNA did not alter activin induction of oFSHB suggesting Smads 2 and 3 may not be involved. A p38 inhibitor blocked induction of oFSHB after 8 h, dividing the 24 hr induction by activin into two phases. This suggested that an activin-regulated early gene product is required for the second phase of oFSHB induction. It was found that one forkhead gene, FOXQ1, was increased 4.5-fold 8 h after activin treatment which correlated nicely with the second phase of oFSHB induction uncovered by the p38 inhibitor. FOXQ1 is only one of 43 FOX family members, however, so further studies are required to prove that FOXQ1 is a key driver of FSH production and that it partners with Smad4 to induce oFSHB transcription.