title: DAX-1A (NR0B1A) expression levels are extremely low compared to DAX-1 (NR0B1) in human steroidogenic tissues.
abstract: The orphan nuclear receptor DAX-1 (dosage-sensitive sex reversal-AHC critical region on the X chromosome gene 1; NR0B1) is known for its role in human development, specifically sex determination and steroidogenesis. Several recent publications have described an alternatively spliced form of DAX-1 called DAX-1A ( NR0B1A). DAX-1A is encoded by exons 1 and 2A of DAX-1, with exon 2A located within the DAX-1 intron 1. DAX-1A expression has been observed in several tissues, including adrenal gland, ovary, and testis. Transfection studies have further shown that DAX-1A has an inhibitory effect on DAX-1, suggesting a role for DAX-1A in the regulation of adrenal and gonadal differentiation/function. However, the relative level of DAX-1 versus DAX-1A transcripts still remains unclear. Herein, we developed and performed quantitative real-time RT-PCR to measure DAX-1 and DAX-1A mRNA expression levels in H295R human adrenal carcinoma cell lines, human adult and fetal adrenal glands, corpus luteum, testis, whole pre- and postmenopausal ovaries, ovarian follicles, placenta, liver, and kidney. These mRNA expression levels were quantified using DAX-1 and DAX-1A standard curves. In addition, Western blotting analysis was performed to examine both DAX-1 and DAX-1A protein levels in H295R cells, adrenal glands, corpus luteum, and liver. Both DAX-1 and DAX-1A mRNA were detected in all samples of H295R cells, human fetal and adult adrenals, testis, ovary, ovarian follicles, and corpus luteum. However, DAX-1 mRNA levels were significantly higher (> 37-fold) than that seen for DAX-1A (p<0.01). DAX-1A mRNA expression levels were undetectable in human liver, placenta, and kidney. Western blotting analysis results demonstrated that DAX-1 protein was predominantly expressed in H295R cells, human adult adrenal, and corpus luteum. These results suggest that in comparison to DAX-1A, DAX-1 is, by far, the predominant mRNA isoform found in human adrenal glands and gonads.
authors.affiliation: Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
authors.forename: C
authors.initials: C
authors.lastname: Vargas Morris
authors.forename: H
authors.initials: H
authors.lastname: Sasano
authors.forename: W E
authors.initials: WE
authors.lastname: Rainey
mesh_terms: D000311:Adrenal Glands / Q000378:metabolism; D003338:Corpus Luteum / Q000378:metabolism; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005333:Fetus / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D010053:Ovary / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013256:Steroids / Q000096:biosynthesis*; D013737:Testis / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; C539965:NR0B1 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D013256:Steroids