Although much is known about the secretory dynamics and post translational regulation of GnRH, little information is available concerning the mechanisms which control its tissue specific expression. With the use of two GnRH secreting neuronal cell lines derived from transgenic mice, and human placental cell lines immortalized using a temperature sensitive SV40 T antigen mutant, the applicant proposes to delineate tissue specific and species specific mechanisms of transcriptional regulation of GnRH production.
Specific Aim 1 will elucidate the specific cis acting DNA sequences that mediate hypothalamic and placental expression of GnRH. Gene transfer studies with deletion analysis of the rat and human promoter will characterize those DNA regions necessary for tissue specific expression. DNase footprinting and gel retardation assays will further define promoter regions that interact with tissue specific nuclear proteins. Targeted site directed mutagenesis will examine the functional significance of these regions in transient transfection assays. Methylation interference assays of promoter sequences will be used to confirm contacts with proteins.
Specific Aim 2 will identify and characterize the trans acting nuclear protein DNA or protein protein interactions that confer hypothalamic and placental expression of the GnRH promoter. Once the cis acting DNA sequences have been rigorously defined, four strategies will be employed to identify and isolate DNA binding proteins: ligand screening, protein purification, PCR amplification of putative transcription factor family members, and differential display. Molecular cloning by differential display will be used to isolate developmentally regulated transcription factors by comparing expressed mRNAs from hypothalamic cells derived at early (Gn 10 cells) and late (GT1 7 cells) stages of GnRH neuronal migration and placental cells derived from early (SPA 2 9 cells) and late (HPA 1) gestation.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD031191-03
Application #
2403353
Study Section
Reproductive Endocrinology Study Section (REN)
Project Start
1995-07-01
Project End
1998-07-31
Budget Start
1997-07-01
Budget End
1998-07-31
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Salian-Mehta, Smita; Xu, Mei; McKinsey, Timothy A et al. (2015) Novel Interaction of Class IIb Histone Deacetylase 6 (HDAC6) with Class IIa HDAC9 Controls Gonadotropin Releasing Hormone (GnRH) Neuronal Cell Survival and Movement. J Biol Chem 290:14045-56
Salian-Mehta, Smita; Xu, Mei; Pierce, Angela et al. (2014) Loss of Growth arrest specific gene 6 (Gas6) results in altered GnRH neuron migration, delayed vaginal opening and sexual maturation in mice. Mol Cell Endocrinol 393:164-70
Salian-Mehta, S; Xu, M; Knox, A J et al. (2014) Functional consequences of AXL sequence variants in hypogonadotropic hypogonadism. J Clin Endocrinol Metab 99:1452-60
Salian-Mehta, Smita; Xu, Mei; Wierman, Margaret E (2013) AXL and MET crosstalk to promote gonadotropin releasing hormone (GnRH) neuronal cell migration and survival. Mol Cell Endocrinol 374:92-100
Wierman, Margaret E; Xu, Mei; Pierce, A et al. (2012) Extracellular signal-regulated kinase 1 and 2 are not required for GnRH neuron development and normal female reproductive axis function in mice. Neuroendocrinology 95:289-96
Rothman, Micol S; Carlson, Nichole E; Xu, Mei et al. (2011) Reexamination of testosterone, dihydrotestosterone, estradiol and estrone levels across the menstrual cycle and in postmenopausal women measured by liquid chromatography-tandem mass spectrometry. Steroids 76:177-82
Pierce, Angela; Xu, Mei; Bliesner, Brian et al. (2011) Hypothalamic but not pituitary or ovarian defects underlie the reproductive abnormalities in Axl/Tyro3 null mice. Mol Cell Endocrinol 339:151-8
Wierman, Margaret E; Kiseljak-Vassiliades, Katja; Tobet, Stuart (2011) Gonadotropin-releasing hormone (GnRH) neuron migration: initiation, maintenance and cessation as critical steps to ensure normal reproductive function. Front Neuroendocrinol 32:43-52