Abstract
Luteinizing hormone (LH) and insulin stimulate transcriptional activity of the porcine low-density lipoprotein (LDL) receptor (LDLR) promoter supra-additively in primary cultures of granulosa-luteal cells. The mechanistic basis of this bihormonal interaction is unknown. The pig LDLR gene promoter includes three putative Sp1/Sp3-binding sites and one sterol response element (SRE) site 5′ upstream to the transcriptional start site. To assess the role of SRE-binding protein (SREBP) in LDLR gene regulation, swine granulosa-luteal cells were cotransfected with CMV/SREBP-1a or SREBP-2 and the pLDLR1076/luc promoter. SREBP-1a and SREBP-2 stimulated LDLR gene transcription eight- and fourfold, respectively. LH alone augmented stimulation by SREBP-1 twofold. Conversely, cotransfection of a dominant-negative mutant form of SREBP-1a repressed basal and hormonally stimulated LDLR promoter activity by >80% (P < 0.01). Mutation of the SRE - 167 ATCACCCCATG - 157 to - 167 ATCACCgCATG - 157 bp decreased basal expression by 50% and LH + insulin- and LH + IGF-I-stimulated transcriptional activity by 80% and >90%, respectively (both P > 0.01). Mutations within each of the three flanking putative Sp1/Sp3 sites at -216/-211, -201/-196, and -151/-146 bp in the LDLR gene promoter also reduced basal activity (by >85%) and hormonal responsiveness (>95%, P < 0.05). EMSA confirmed that presumptive SRE-1 and Sp1/Sp3 elements bind respective peptides. Mithramycin, an inhibitor of Sp1/Sp3 protein(s) binding, blocked hormonally induced LDLR promoter expression by 80%. Basal transcription and supra-additive stimulation of porcine LDLR gene transcription by LH and insulin in granulosa-luteal cells require SREBP-1a and Sp1/Sp3-binding elements.
Original language | English (US) |
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Pages (from-to) | E128-E135 |
Journal | American Journal of Physiology - Endocrinology and Metabolism |
Volume | 287 |
Issue number | 1 50-1 |
DOIs | |
State | Published - Jul 2004 |
Keywords
- Gonadotropin
- Insulin
- Insulin-like growth factor I
- Low-density lipoprotein
- Luteinizing hormone
- Ovary
- Promoter
- Sterol response element-binding protein
ASJC Scopus subject areas
- Endocrinology, Diabetes and Metabolism
- Physiology
- Physiology (medical)