Sp1 and SF-1 interact and cooperate in the regulation of human steroidogenic acute regulatory protein gene expression.
Steroidogenic acute regulatory (StAR) protein plays a critical role in the movement of cholesterol from the outer to the inner mitochondrial membrane. Steroidogenic factor 1 (SF-1) controls basal and cAMP-stimulated transcription of the StAR gene. The 1.3-kb StAR promoter has three SF-1 binding sites, and two consensus transcription factor Spl binding sequences near the two most distal SF-1 binding sites. Spl mediates cAMP-dependent transcription of steroidogenic P450 enzyme genes, raising the possibility of Sp1 involvement in cAMP regulation of the StAR gene. However, the mechanism of Spl-mediated, cAMP-stimulated responsiveness is not known. In this study, we elucidated the roles of Sp1 and SF-1 in the regulation of the human StAR gene promoter. We found that there was negligible promoter activity in a pGL2 StAR construct (-235 to +39) in which Spl and SF-1 binding sites were mutated in Y-1 adrenal tumor cells. An Sp1 binding site mutation (pGL2Sp1M) did not support promoter activity, suggesting that Spl cooperates with SF-1 in regulating StAR promoter function. In gel shift assays, the SF-1 binding site formed a complex with an SF-1-GST fusion protein and Spl. Coimmunoprecipitation cross-linking experiments indicated that SF-1 physically interacts with Sp1 in vitro. Finally, a mammalian two-hybrid system was employed to demonstrate that Spl and SF-1 associate in vivo. In conclusion, our data indicate that Spl and SF-1 physically interact and cooperate in the regulation of human StAR promoter activity.
Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Japan. terusuga@med.hokudai.ac.jp
T
T
Sugawara
M
M
Saito
S
S
Fujimoto
D000306:Adrenal Cortex Neoplasms; D000310:Adrenal Gland Neoplasms; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004347:Drug Interactions; D051717:Fushi Tarazu Transcription Factors; D005786:Gene Expression Regulation / Q000187:drug effects*; D018398:Homeodomain Proteins; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000502:physiology*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D000098682:Steroidogenic Acute Regulatory Protein
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; C515057:NR5A1 protein, human; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; C515058:steroidogenic factor 1, mouse; D004247:DNA; D000242:Cyclic AMP