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Submitted on March 7, 2002
Accepted on January 10, 2003
1 Centre de recherche en reproduction animale (N.Y.G., B.D.M.), Faculté de médecine véterinaire, Université de Montréal, St-Hyacinthe Québec, Canada J2 S7C6; Institute de génétique et biologie moléculaire et cellulaire (E.L., P.S.-C.), Centre Nationale de la Recherche Scientifique, INSERM-ULP, Illkirch, C.U. de Strasbourg, France 67404
* To whom correspondence should be addressed. E-mail: murphyb{at}medvet.umontreal.ca.
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 3'5' cyclic adenosine monophosphate (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 protein kinase A (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 electromobility shift assays (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. Further, 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 (ChIP) 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 of Y-1 cells. 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 co-activator CBP and the phosphorylation and acetylation of histone H-3 to transactivate the NPC-1 promoter.
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