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.
Key words
DAX-1 - DAX-1A - adrenal - gonadal - real-time RT-PCR - Western blotting analysis
References
-
1
Lalli E, Sassone-Corsi P.
DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation.
Mol Endocrinol.
2003;
17
1445-1453
-
2
Iyer AK, MacCabe ER.
Molecular mechanisms of DAX1 action.
Mol Genet Metab.
2004;
83
60-73
-
3
Hossain A, Li C, Saunders GF.
Generation of two distinct functional isoforms of dosage-sensitive sex reversal-adrenal hypoplasia congenita-critical region on the X chromosome gene 1 (DAX-1) by alternative splicing.
Mol Endocrinol.
2004;
8
1428-1437
-
4
Ho J, Zhang YH, Huang BL, MacCabe ER.
NR0B1A: an alternatively spliced form of NR0B1.
Mol Genet Metab.
2004;
83
330-336
-
5
Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P.
DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis.
Nature.
1997;
390
311-315
-
6
Bae DS, Schaefer ML, Partan BW, Muglia L.
Characterization of the mouse DAX-1 gene reveals evolutionary conservation of a unique amino-terminal motif and widespread expression in mouse tissue.
Endocrinology.
1996;
137
3921-3927
-
7
Hanley NA, Rainey WE, Wilson DI, Ball SG, Parker KL.
Expression profiles of SF-1, DAX1, and CYP17 in the human fetal adrenal gland: potential interactions in gene regulation.
Mol Endocrinol.
2001;
15
57-68
-
8
Aigueperse C, Val P, Pacot C, Darne C, Lalli E, Sassone-Corsi P, Veyssiere G, Jean C, Martinez A.
SF-1 (steroidogenic factor-1). C/EBPß (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.
Mol Endocrinol.
2001;
15
93-111
-
9
Lalli E, Melner MH, Stocco DM, Sassone-Corsi P.
DAX1 blocks steroid production at multiple levels.
Endocrinology.
1998;
139
4237-4243
-
10
Phelan JK, MacCabe ER.
Mutations in NR0B1 (DAX1) and NR5A1 (SF1) responsible for adrenal hypoplasia congenital.
Hum Mutat.
2001;
18
472-487
-
11
Guo W, Burris TP, Zhang YH, Huang BL, Mason J, Copeland KC, Kupfer SR, Pagon RA, McCabe ER.
Genomic sequence of the DAX1 gene: an orphan nuclear receptor responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism.
J Clin Endocrinol Metab.
1996;
81
2481-2486
-
12
Rainey WE, Saner K, Schimmer BP.
Adrenocortical cell lines.
Mol Cell Endocrinol.
2004;
228
23-38
-
13
Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H.
Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression's CT difference” formula.
J Mol Med.
2006;
84
901-910
-
14
Livak KJ, Schmittgen TD.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
Methods.
2001;
25
402-408
-
15
Morrison TB, Weis JJ, Wittwer CT.
Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification.
Biotechniques.
1998;
24
954-962
-
16
Guo W, Burris TP, MacCabe ER.
Expression of DAX-1, the gene responsible for X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism, in the hypothalamic-pituitary-adrenal/gonadal axis.
Biochem Mol Med.
1995;
56
8-13
-
17
Sato Y, Suzuki T, Hidaka K, Sato H, Ito K, Ito S, Sasano H.
Immunolocalization of nuclear transcription factors, DAX-1 and COUP-TF II, in the normal human ovary: correlation with adrenal 4 binding protein/steroidogenic factor-1 immunolocalization during the menstrual cycle.
J Clin Endocrinol Metab.
2003;
88
3415-3520
-
18
Ozisik G, Achermann JC, Meeks JJ, Jameson JL.
SF1 in the development of the adrenal gland and gonads.
Horm Res.
2003;
59
94-98
-
19
Beuschlein F, Keegan CE, Bavers DL, Mutch C, Hutz JE, Shah S, Ulrich-Lai YM, Engeland WC, Jeffs B, Jameson JL, Hammer GD.
SF-1, DAX-1, and acd: molecular determinants of adrenocortical growth and steroidogenesis.
Endocr Res.
2002;
28
597-607
-
20
Shibata H, Kobayashi S, Kurihara I, Saito I, Saruta T.
Nuclear receptors and co-regulators in adrenal tumors.
Horm Res.
2003;
59
85-93
-
21
Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, Mangelsdorf DJ.
Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network.
Cell.
2006;
126
789-799
-
22
Mukai T, Kusaka M, Kawabe K, Goto K, Nawata H, Fujieda K, Morohashi K.
Sexually dimorphic expression of Dax-1 in the adrenal cortex.
Genes Cells.
2002;
7
717-729
-
23
Saito S, Ito K, Suzuki T, Utsunomiya H, Akahira J, Sugihashi Y, Niikura H, Okamura K, Yaegashi N, Sasano H.
Orphan nuclear receptor DAX-1 in human endometrium and its disorders.
Cancer Sci.
2005;
96
645-652
Correspondence
W. E. RaineyPhD
Department of Physiology
Medical College of Georgia
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