Porcine Niemann Pick-C1 protein is expressed in steroidogenic tissues and modulated by cAMP.
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)(2)cAMP 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.
Gévry
Nicolas
N
Centre de Recherche en Reproduction Animale, Faculté de Médecine Vétérinaire, Université de Montréal, St. Hyacinthe, Québec J2S 7C6, Canada.
Lacroix
Dan
D
Song
Jian-Hua
JH
Pescador
Nazario
N
Dobias
Mira
M
Murphy
Bruce D
BD
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000096:biosynthesis*; D002460:Cell Line; D002478:Cells, Cultured; D003338:Corpus Luteum / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology*; D015342:DNA Probes; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D000077296:Niemann-Pick C1 Protein; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015290:Second Messenger Systems / Q000502:physiology; D013256:Steroids / Q000096:biosynthesis*; D013552:Swine; D014018:Tissue Distribution; D014162:Transfection
D000963:Antimetabolites; D002352:Carrier Proteins; D015342:DNA Probes; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D012333:RNA, Messenger; D013256:Steroids; D000242:Cyclic AMP