Objective: Pituitary homeobox 1 (Ptx1) is critical for normal pituitary development and has more recently been shown to be important for expression of pituitary-specific genes in the adult, including the LHÎČ gene. Separate studies have demonstrated that the orphan nuclear receptors, steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter-transcription factor (COUP-TF), also mediate LHÎČ gene expression. The specific objective of this study was to further characterize transcriptional regulation of the LHÎČ gene by Ptx1, alone and in conjunction with SF-1 and the COUP-TFs. Design: LHÎČ promoter activity was measured in the presence or absence of the transcription factors of interest using a transient transfection-reporter system. Electrophoretic mobility shift assay (EMSA) was used to evaluate Ptx1 binding to LHÎČ gene sequences. Materials and Methods: A) Monkey kidney fibroblast cells (CV-1) were transiently transfected with rat LHÎČ promoter sequences subcloned into the luciferase reporter vector, pXP2. Cells were cotransfected with CMV-driven expression vectors for Ptx1, SF-1 and/or COUP-TF. Results are expressed as fold-change relative to expression in cells receiving equivalent amounts of empty expression vector. Statistical significance was determined by ANOVA followed by Studentâs t test with significance set at p < 0.05. B) EMSA was used to determine the ability of a GST-Ptx1 fusion protein or endogenous Ptx1 (from the mouse gonadotrope cell line αT3â1) to bind to various rat LHÎČ promoter sequences. Results: A) Consistent with previous reports by our group and others, addition of Ptx1 significantly increased expression of region â797/+5 of the rat LHÎČ promoter (6-fold). Introduction of SF-1 also stimulated LHÎČ promoter activity (37-fold), with marked synergy in the presence of both factors (over 200-fold) (p < 0.01). Mutation of a previously identified Ptx1 cis-element at position -101 decreased (2.6-fold), but did not eliminate, the Ptx1 effect in a â207/+5 construct (p < 0.005 versus control). The Ptx1-SF-1 synergy was lost in this mutated construct. 5âdeletion constructs further localized the residual Ptx1 response to nucleotides â82/+5, a region with three potential Ptx-1 cis-elements based on sequence homology to the consensus site. Overexpression of either COUP-TFI or COUP-TFII blunted the Ptx1 response by 75% and 50%, the SF-1 response by 50% and 30%, and the SF-1/Ptx1 response by 85% and 70%, respectively (p < 0.0001 in the presence versus the absence of the COUP-TFs). B) By EMSA, both endogenous Ptx1 and GST-Ptx1 bind to an oligonucleotide spanning the -101 LHÎČ-Ptx1 cis-element as confirmed by supershift of the protein-DNA complex with a Ptx1-specific antibody. The Ptx1 fusion protein also bound to the 3âPtx1 region, although with lower affinity than to the â101 element. Oligonucleotides containing mutations in the putative 3âPtx1 sites demonstrated loss of binding with mutation at position â65, but not at the other two sites. Conclusions: Ptx1 increases transcription of the rat LHÎČ gene via two cis-elements. SF-1 synergizes with Ptx1 at the 5âelement, but apparently not at the 3â element. Overexpression of the COUP-TFs blunts both the SF-1 response and the Ptx1 response, although the mechanism by which this occurs remains to be elucidated. These data further our understanding of both stimulatory and inhibitory control of gonadotropin gene expression. Supported by: R01 HD38089 (LMH, CDH, ZH) and HD19938 (K-HJ, UBK).