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

GORE, ARNOLD JESSE. Activin Induction of the Ovine Follicle-Stimulating Hormone βSubunit is Mediated by Smad4 and a Forkhead Box Transcription Factor. (Under the direction of Dr. William L. Miller). Follicle stimulating hormone (FSH) is a α/β glycoprotein produced in all vertebrate pituitary gonadotropes that is required for egg maturation, and enhances the performance and maturation of sperm. Currently, the precise physiological mechanisms that increase FSH are unknown, but are complex since its expression is controlled by at least 6 hormones. The most potent inducer of FSH is activin that directly stimulates transcription of its rate-limiting β-subunit (FSHB) by targeting specific elements within the promoter of the FSHB gene. Activin is widely known to activate the transcription factors Smad2/3. It also activates mitogen-activated protein kinase (MAPK) pathways that regulate gene expression by phosphorylating various transcription factors. Early responses to Smadand MAPKmediated gene regulation typically occur rapidly and transiently (1-4 hrs). This rapid timecourse does not explain progressive activin induction of an ovine FSHB (oFSHB) promoter/reporter construct in LβT2 cells, which peaks at 22 hrs. Therefore, induction of oFSHB by activin likely depends on secondary, or primary and secondary transcription factors. Previously, a DNA sequence was discovered in our laboratory between -171 bp and -159 bp of the oFSHB promoter that is necessary for 68 % of activin induction in LβT2 cells, and is also required for 99.9 % of oFSHB expression in vivo. Thus, the activin-responsive proteins bound to this site are critical and physiologically important mediators of oFSHB synthesis and FSH production. This element consists of a Smad binding element (SBE) juxtaposed downstream to a putative Runx/forkhead box (FOX) protein binding site, consistent with the necessity for secondary factors that are induced over time. This study sought to further characterize this region of the oFSHB promoter and the factors involved in activin induction of oFSHB. Binding studies revealed that Smad4 from LβT2 cells binds a palindromic SBE at a maximal level after 20 hrs of activin, mimicking the progressive kinetics of oFSHB induction. This binding was competed 85 % with a wild-type oFSHB oligonucleotide that encompassed the -162 bp SBE, indicating that Smad4 can bind to the oFSHB promoter. Additionally, overexpression of a dominant negative Smad4 reduced the level of activin induction by 62 %. These data provide strong evidence that Smad4 is involved in activinmediated oFSHB transcription. Other studies ruled out participation of Smad2 and 3 in activin induction of oFSHB, but there is evidence supporting participation of a FOX transcription factor. Inhibition of p38, a kinase involved in oFSHB expression, blocked activin induction of oFSHB only after 8 hrs, dividing the progressive, activin-mediated increase into two phases. This suggested the involvement of an alternate and activin-regulated gene required for the second phase, such as Runx or FOX family members. Focus was directed towards FOX genes since Runx is not usually regulated. One candidate gene, FOXQ1, was identified that was transiently induced up to 4.5-fold by activin at 8 hrs. This correlated directly with the time at which the p38 inhibitor blocked oFSHB induction. Given that Smad4 is not activin-regulated, either transcriptionally or post-translationally, is not an independent transcription factor, and that the physiologically important site from -171 bp to -159 bp consists of FOX/Smad binding sites, FOXQ1 may be necessary for oFSHB synthesis. However, FOXQ1 is only one of 43 FOX family members all of which bind the same DNA sequences. Therefore, other FOX proteins may also be important mediators of oFSHB induction. Further studies are required to prove that FOXQ1, and not another FOX member, is a key driver of FSH production. Activin Induction of the Ovine Follicle-Stimulating Hormone β-Subunit is Mediated by Smad4 and a Forkhead Box Transcription Factor by Arnold Jesse Gore A dissertation submitted to the Graduate Faculty of North Carolina State University In partial fulfillment of the Requirements for the degree of Doctor of Philosophy