Differential expression of human gonadotropin-releasing hormone receptor gene in pituitary and ovarian cells

In terms of regulation of gene expression, gonadotropin-releasing hormone r eceptor (GnRHR) found in extrapituitary tissues has been suggested to be di fferent from that in the pituitary. In the present study, we examined the m olecular basis of this difference using the pituitary alpha T3-1 and ovaria n carcinoma OVCAR-3 cells. As a first step, the different expression levels of GnRHR mRNA in the pituitary and ovarian cells were investigated using s emi-quantitative RT-PCR. Quantitative analysis showed that the expression l evel of hGnRHR is a nine-fold higher in primary pituitary tissues than the primary culture of ovarian carcinomas (PCO), In pituitary alpha T3-1 cells, the expression level of hGnRHR was ten-fold higher than ovarian carcinoma OVCAR-3 cells. The possibility of the differential use of various cell-spec ific promoters in different cells was addressed by transiently transfecting cells with 3'-deletion clones of human GnRHR promoter. Sequential deletion of the 5'-flanking region of the gene revealed the use of two putative pro moters, designated PR1 (-771 to -557) and PR2 (-1351 to -1022). and a negat ive control region (-1022 to -771), in the pituitary and ovarian cells. The same promoters appeared to be utilized for driving the basal promoter acti vities in both alpha T3-1 and OVCAR-3 cells, suggesting that there is no ce ll-specific promoter usage for the human GnRHR gene. Alternatively, the inv olvement of different regulatory protein factors was investigated using ele ctrophoretic gel mobility: shift assays. When end-labeled PR1 was used as a probe, two unique shifted complexes were identified in OVCAR-3 cells when compared to alpha T3-1 cells. One unique protein-DNA complex was observed i n alpha T3-1 cells compared to OVCAR-3 cells when incubated with end-labele d PR2 as a probe. These DNA-protein complexes appeared to be specific, as t hey competed with excess amount of unlabelled competitor PR1 and PR2, respe ctively. In summary, it is unlikely that the use of a cell-specific promote r contributes to the different characteristics of ovarian GnRHR. Our study demonstrates that one mechanism by which cell-specific expression of human GnRHR is achieved is through the binding of distinct and/or combinations of cell-specific regulatory factors to various promoter elements in the 5'-fl anking region of the gene. (C) 2000 Elsevier Science Ireland Ltd. All right s reserved.