Functional activity of the porcine Gnrhr2 gene promoter in testis-derived cells is partially conferred by nuclear factor-κB, specificity protein 1 and 3 (SP1/3) and overlapping early growth response 1/SP1/3 binding sites

• The GnRHR2 promoter is active in cell types derived from extra-pituitary tissues.
• Transcriptional regulation of the porcine GnRHR2 gene in the testis was examined.
• A putative repressor at − 1693/− 709 and activator at − 708/− 489 were identified.
• NF-κB, SP1/3 and overlapping EGR1/SP1/3 sites confer activity in the ST cell line.
• This is the first report of functional elements within the GnRHR2 gene promoter.

Unlike the classical gonadotropin-releasing hormone (GnRH1), the second mammalian isoform (GnRH2) is ubiquitously expressed, suggesting a divergent function. Indeed, we demonstrated that GnRH2 governs LH-independent testosterone secretion in porcine testes via interaction with its receptor (GnRHR2) on Leydig cells. Transient transfections with luciferase reporter vectors containing 3009 bp of 5′ flanking sequence for the porcine Gnrhr2 gene (− 3009pGL3) revealed promoter activity in all 15 cell lines examined, including swine testis-derived (ST) cells. Therefore, ST cells were utilized to explore the molecular mechanisms underlying transcriptional regulation of the porcine Gnrhr2 gene in the testis. Reporter plasmids containing progressive 5′ deletions of the Gnrhr2 promoter indicated that the − 708/− 490 region contained elements critical to promoter activity. Electrophoretic mobility shift assays (EMSAs) with radiolabeled oligonucleotides spanning the − 708/− 490 bp region and ST nuclear extracts, identified specific binding complexes for the − 513/− 490, − 591/− 571 and − 606/− 581 bp segments of promoter. Antibody addition to EMSAs indicated that the p65 and p52 subunits of nuclear factor-κB (NF-κB) comprised the specific complex bound to the oligonucleotide probe for the − 513/− 490 bp promoter region, specificity protein (SP) 1 and 3 bound the − 591/− 571 bp probe and early growth response 1 (EGR1), SP1 and SP3 bound the − 606/− 581 radiolabeled oligonucleotide. Transient transfections with vectors containing mutations of the NF-κB (− 499/− 493), SP1/3 (− 582/− 575) or overlapping EGR1/SP1/3 (− 597/− 587) binding sites reduced luciferase activity by 26%, 61% and 56%, respectively (P < 0.05). Thus, NF-κB, SP1/3 and overlapping EGR1/SP1/3 binding sites are critical to expression of the porcine Gnrhr2 gene in ST cells.