Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells.
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.
Endocrine Research Unit, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, USA.
Natesampillai
N
Sekar
Johannes D
JD
Veldhuis
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D007328:Insulin / Q000494:pharmacology*; D008184:Luteal Cells / Q000187:drug effects / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D011973:Receptors, LDL / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051780:Sterol Regulatory Element Binding Protein 1; D013329:Structure-Activity Relationship; D013552:Swine; D014157:Transcription Factors; D015533:Transcriptional Activation / Q000187:drug effects / Q000502:physiology*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007328:Insulin; D011973:Receptors, LDL; D016329:Sp1 Transcription Factor; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D007986:Luteinizing Hormone