Orphan nuclear receptor DAX-1 in human endometrium and its disorders

Abstract

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1) is a recently characterized member of the orphan nuclear receptor family. DAX-1 functions as a global negative regulator of steroid hormone production. It inhibits adrenal 4 binding protein (Ad4BP)/steroidogenic factor-1 (SF-1) pathway-dependent P450arom expression in cultured human endometriotic stromal cells and acts as a corepressor for estrogen receptors (ER). In this study we first examined the localization of DAX-1 in 46 normal cycling endometria, 36 cases of endometrial hyperplasia and 103 cases of endometrial carcinoma by using immunohistochemistry to elucidate the possible involvement of DAX-1 and its correlation to the status of Ad4BP/SF-1, a universal transcription factor of steroidogenesis. We then evaluated DAX-1 mRNA expression, using quantitative reverse transcription-polymerase chain reaction for DAX-1 in 33 cases of endometrial carcinoma for further characterization. We subsequently correlated these findings with various clinicopathological parameters of the cases. Ad4BP/SF-1 immunoreactivity was not detected in any human endometria examined. A significant inverse correlation was detected between the status of DAX-1 immunoreactivity and histological grade (P = 0.0003) in endometrial carcinoma. The labeling index (LI) values of DAX-1 in normal endometrium during the secretory phase (P < 0.0001) and hyperplasia (P < 0.0001) were significantly higher than that of carcinoma. No significant correlations were detected between DAX-1 immunoreactivity and amounts of aromatase mRNA. There was a statistically significant positive correlation between DAX-1 and ERalpha (P = 0.006) and ERbeta LI (P < 0.001). These findings suggest that DAX-1 may inhibit the proliferation and progression of endometrial carcinoma through inhibition of estrogenic actions, possibly by interacting with ER present in carcinoma cells, rather than regulating in situ steroidogenesis.

Figure 1

Immunoblot analysis including immunoabsorption test. (a) Only a single band of 60 kDa was detected for DAX‐1 in the HeLa and H295R control cell lines. Preparations were also incubated with DAX‐1 monoclonal antibody preparations preabsorbed with synthetic antigen peptide as primary antibody. (b) No specific band was detected in these sections.

Figure 2

mRNA in situ hybridization and immunohistochemistry for DAX‐1 in adrenal gland tissue. (a,b) An accumulation of DAX‐1 mRNA hybridization signals was detected predominantly in the cytoplasm of the cortex. (c) Negative controls using a sense probe for DAX‐1 mRNA yielded no significant accumulation of mRNA hybridization signals of this peptide. (d) DAX‐1 immunoreactivity was detected in the cortex. Original magnification ×100 (a,c,d), ×400 (b).

Figure 3

Immunohistochemistry for DAX‐1 and Ad4BP/SF‐1 in normal endometria, endometrial hyperplasia and endometrial carcinoma. DAX‐1 immunoreactivity was detected in the nuclei of both epithelial and stromal cells of normal endometria obtained during (a) the proliferative phase and (b) the secretory phase, and (c) endometrial hyperplasia, and (d) endometrial carcinoma. Ad4BP/SF‐1 immunoreactivity was not detected in (e) normal endometria or (f) endometrial carcinoma. (g) Adrenal gland tissue was the positive control for the immunohistochemical studies. Original magnification × 400.

Figure 4

DAX‐1 labeling index (LI) in (a) endometrial carcinoma, (b) normal endometrium during the proliferative phase, (c) normal endometrium during the secretory phase, and (d) hyperplasia. The DAX‐1 LI in normal endometrium during the secretory phase was significantly higher than that in endometrial carcinoma. Also, the DAX‐1 LI in endometrial hyperplasia was significantly higher than that in endometrial carcinoma. The DAX‐1 LI in the secretory phase was significantly higher than that in the proliferative phase. The DAX‐1 LI in endometrial hyperplasia was significantly higher than that in normal endometrium during the proliferative phase. All values are expressed as mean ± SEM. *P < 0.05; **P < 0.0001 (Scheffe's F‐test).