The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are critical modulators of gamete maturation and gonadal steroidogenesis. Recent evidence has demonstrated that the transcription factors, steroidogenic factor-1 (SF-1) and early growth response gene 1 (Egr-1), regulate expression of the LHbeta-subunit gene. The long-term objective of our work is to understand fully the transcription factors and cis-elements which modulate LHbeta gene expression, information which will provide insight into both normal and abnormal reproductive function.
The Specific Aims of this proposal are: 1) to characterize the role of the bicoid-related transcription factor, Ptx1, in mediating tissue-specific LHbeta gene expression, 2) to define the physiologic importance of the LHbeta-Ptx1 cis-element(s) in primary gonadotropes in culture and in vivo, and 3) to identify the molecular mechanisms which mediate cAMP/PKA-induced stimulation of LHbeta gene promoter activity.
In Aim 1, based on a report by Drouin and co-workers, we hypothesize that Ptx1 acts alone and in synergy with SF-1 to increase LHbeta promoter activity, as will be tested by electrophoretic mobility shift assay, transient transfection of immortalized cell lines, and generation of Ptx1 """"""""knockdown"""""""" cell lines.
In Aim 2 A, primary pituitary cell cultures will be infected using a highly efficient, recombinant adenovirus approach in order to define the 5'flanking region which confers gonadotrope-specific expression. The defined gonadotrope-specific region will then be used in reporter constructs as either the wild-type sequence or with a mutation in the Ptx1-site(s) identified in Aim 1. We predict that the presence of a mutation in the Ptx1 site(s) will markedly blunt reporter expression in both cultured primary pituitary cells (Aim 2B) and transgenic mouse lines (Aim 2C), verifying the physiologic importance of Ptx1 to LHbeta gene expression.
In Aim 3, we propose to test the hypothesis that cAMP/PKA-induced increases in LHbeta promoter activity are due to: A) PKA-regulated changes in SF-1 and/or Egr-1 gene expression or post-translational modifications, and/or B) putative AP-2 cis-element(s) in the LHbeta gene promoter sequence. By further characterizing factors which mediate tissue-specific and hormonally-regulated LHbeta gene expression, these studies will contribute to our understanding of normal reproductive physiology, as well as the pathophysiology of disorders including infertility, polycystic ovarian syndrome, hypothalamic hypogonadism, and premature and delayed puberty.
