Activin induction of follicle stimulating hormone is mediated by transforming growth factor beta activated kinase-1 (TAK-1) in pituitary gonadotropes

by Safwat, Nedal Wafik.

SAFWAT, NEDAL WAFIK. Activin Induction of Follicle Stimulating Hormone is Mediated by Transforming Growth Factor Beta Activated Kinase-1 (TAK-1) in Pituitary Gonadotropes (Under the direction of William L. Miller.) Follicle stimulating hormone (FSH) is an essential hormone for female folliculogenesis and plays an important role in male spermatogenesis. The hormone is secreted by pituitary gonadotropes in the anterior pituitary lobe, and its overall production is regulated by expression of the FSH? subunit. The regulation of FSH? subunit is achieved by combined actions of neurocrine, endocrine, and pituitary paracrine/autocrine factors. Activin shown to be produced locally within the pituitary is a potent stimulator of the FSH? subunit. This study used 4.7 kb of the ovine FSH? promoter linked to luciferase (oFSH?Luc) plus a well characterized activins responsive construct, p3TPLuc, to investigate the hypothesis that Smad3, TAK1 (TGF? activated kinase1), or both cause activin-mediated induction of FSH. Over-expression of either Smad3 or TAK1 induced oFSH?Luc in gonadotrope-derived L?T2 cells as much as activin itself. Induction of p3TPLuc by activin is known to require Smad3 activation in many cell types and this was true in L?T2 cells where 10-fold induction by activin (2-8 h after activin treatment) was blocked > 90% by two dominant negative (DN) inhibitors of Smad3 [DN-Smad3 (3SA) and DN-Smad3 (D407E)]. By contrast, 6.5-fold induction of oFSH?Luc by activin (10-24 h after activin treatment) was not blocked by either DN-Smad inhibitor, suggesting that activation of Smad3 did not trigger induction of oFSH?Luc. By contrast, inhibition of TAK1 by a DN-TAK1 construct led to a 50% decrease in activinmediated induction of oFSH?Luc, and a specific inhibitor of TAK1 (5Z-7-Oxozeanol) blocked induction by 100% indicating that TAK1 is necessary for activin induction of oFSH?Luc. Finally, inhibiting p38-mitogen activated protein kinase (p38-MAPK; often activated by TAK1) blocked induction of oFSH?Luc by 60%. In conclusion, the data presented here indicate that activation of TAK1 (and probably p38-MAPK), but not Smad3, is necessary for triggering induction of oFSH? by activin. In addition, a method was developed in our laboratory for purifying primary gonadotropes to study the solitary role of factors regulating FSH? gene. We were able to isolate primary gonadotropes from pituitary with purities higher than 95%. The data show that gonadotropes are able to produce activin and/or activin-like molecule(s), however paracrine factors from pituitary non-gonadotropes play a major role in controlling FSH? at the pituitary level. Overall, the study presented provides an understanding to the TAK1 signaling pathway mediating activin induction of FSH?, and shows that primary gonadotropes rely on paracrine factors to produce activin. Activin Induction of Follicle Stimulating Hormone is Mediated by Transforming Growth Factor Beta Activated Kinase-1 (TAK-1) in Pituitary Gonadotropes by Nedal W. Safwat 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 In Molecular and Structural Biochemistry Raleigh, NC 2006