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Porcine Niemann Pick-C1 Protein Is Expressed in Steroidogenic Tissues and Modulated by cAMP

Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Québec, Canada J2S 7C6

Address all correspondence and requests for reprints to: Dr. Bruce D. Murphy, Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 rue Sicotte, St. Hyacinthe, Québec, Canada J2S 7C6. E-mail: murphyb{at}medvet.umontreal.ca

Niemann-Pick C-1 (NPC-1) protein is essential for trafficking of low density lipoprotein-derived cholesterol in mammalian cells. The low density lipoprotein pathway is a major route for supply of cholesterol for steroidogenesis in the adrenals and gonads of many species. We investigated the occurrence and regulation of NPC-1 in porcine tissues, with emphasis on the corpus luteum and on granulosa cells undergoing luteinization in vitro. The porcine open reading frame for NPC-1 predicted a protein of 1278 amino acids (aa). It displayed a domain structure consistent with the human protein, and overall homologies were 89% and 86% with the deduced human and mouse aa sequences, respectively. The mRNA for NPC-1 comprised two transcripts, migrating at 5.0 and 2.2 kb, respectively. Transcripts were detected in a variety of pig tissues and were in highest abundance in steroid-producing organs. NPC-1 mRNA abundance increased with the differentiation of the corpus luteum in vivo and with luteinization of granulosa cells in vitro. Actinomycin D blockade of transcription in luteinized granulosa cells resulted in reduced NPC-1 mRNA and provided a half-life estimate of 20 h. Cycloheximide treatment increased NPC-1 transcript abundance in excess of 5-fold over 24 h. Treatment of luteinized granulosa cells with 1 mM (Bu)₂cAMP increased the abundance of the NPC-1 message by 2- to 4-fold. The 5'-flanking region of the pig sequence displayed consensus sequences for binding transcription factors, including specificity protein-1, cAMP response element-binding protein/activating transcription factor-1, activating protein-1, GATA, modified zinc finger protein-1, transcription factor-11 and a CpG island in the first 400 bp upstream of the ATG transcription initiation site. Transient transfection of 1.86 kb of the 5'-flanking region coupled to the luciferase reporter into three steroidogenic cell lines resulted in constitutive transcription. Treatment with (Bu)₂cAMP for 24 h increased the luciferase signal in all three lines. Thus, three types of evidence indicate that cAMP regulates pig NPC-1 expression. These are the presence of consensus binding sites for cAMP-induced transcription factors (cAMP response element-binding protein/activating transcription factor-1) in the proximal 5'-flanking region of the gene, increases in transcription by the NPC-1 promoter, and increases in NPC-1 mRNA abundance induced by (Bu)₂cAMP. We conclude that NPC-1 is expressed in the steroidogenic tissues of the pig and is regulated by the principal pathway of stimulation of steroidogenesis in the gonads and adrenal, the cAMP-PKA pathway.

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