Regulation of niemann-pick c1 gene expression by the 3'5'-cyclic adenosine monophosphate pathway in steroidogenic cells.
The Niemann Pick-C1 (NPC-1) protein is essential for intracellular transport of cholesterol derived from low-density lipoprotein import in mammalian cells. The role of the protein kinase A (PKA) pathway in regulation of expression of the NPC-1 gene was investigated. NPC-1 promoter activity was induced by treatment with dibutryl cAMP (dbcAMP), alone or in combination with the cAMP response element (CRE) binding protein (CREB) overexpressed in adrenal Y-1 cells. When the catalytic subunit of PKA was overexpressed in Y-1 cells, there were similar increases in NPC-1 promoter activity in the presence of CREB. Responses were attenuated by blockade of the PKA pathway, and in the Kin-8 cell line deficient in PKA. Promoter deletion analysis revealed that this response was present in promoter fragments of 186 bp and larger but not present in the 121-bp fragment. Two promoter regions, one at -430 and one at -120 upstream of the translation initiation site, contained CRE consensus sequences. These bound recombinant CREB in EMSA, confirming their authenticity as CREB response elements. Promoters bearing mutations of both CRE displayed no response to dbcAMP. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), was implicated in NPC-1 transactivation by the presence of SF-1 target sequence that formed a complex with recombinant SF-1 in EMSA. Furthermore, transfection of a plasmid that overexpressed SF-1 into ovarian granulosa cells increased promoter activity in response to dbcAMP, an effect abrogated by mutation of the SF-1 target sequence. Chromatin immunoprecipitation assays demonstrated that the CRE region of the endogenous and transfected NPC-1 promoter associated with both acetylated and phosphorylated histone H-3 and that this association was increased by dbcAMP treatment. Treatment with dbcAMP also increased the association of the CRE region of the promoter with CREB binding protein, which has histone acetyltransferase activity. Together, these results demonstrate a mechanism of regulation of NPC-1 expression by the cAMP-PKA pathway that includes PKA phosphorylation of CREB, recruitment of the coactivator CREB binding protein and the phosphorylation and acetylation of histone H-3 to transactivate the NPC-1 promoter.
Gévry
Nicolas Y
NY
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, 3200 rue Sicotte, St-Hyacinthe, Québec J2S 7C6, Canada.
Lalli
Enzo
E
Sassone-Corsi
Paolo
P
Murphy
Bruce D
BD
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D050882:CREB-Binding Protein; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D051717:Fushi Tarazu Transcription Factors; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000502:physiology*; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000077296:Niemann-Pick C1 Protein; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D018160:Receptors, Cytoplasmic and Nuclear; D020218:Response Elements; D015398:Signal Transduction; D054339:Steroidogenic Factor 1; D013256:Steroids / Q000378:metabolism; D013552:Swine; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D051717:Fushi Tarazu Transcription Factors; D006657:Histones; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; C515057:NR5A1 protein, human; D000077296:Niemann-Pick C1 Protein; D009687:Nuclear Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D013256:Steroids; D015534:Trans-Activators; D014157:Transcription Factors; D003994:Bucladesine; D000242:Cyclic AMP; D050882:CREB-Binding Protein; D017868:Cyclic AMP-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases