Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3β-hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skin disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS on STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N- and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (ΔC2 (1–559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect on STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with an STS–FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.
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T Sugawara, E Nomura, and N Hoshi
T Sugawara, E Nomura, and S Fujimoto
Endometrial carcinoma is a common malignancy of the genital tract. In the present study, we examined the expression of human steroidogenic acute regulatory (StAR) protein, P450 side-chain cleavage enzyme (P450 scc), 17alpha-hydroxylase/17,20-desmolase (P45017alpha) and aromatase (P450 arom) in endometrial carcinoma cells to clarify the ability of these cells to produce steroid hormones. The results of RT-PCR analysis showed that StAR, P450 scc and P45017alpha genes were expressed in endometrial carcinoma cells. To examine the protein expression of StAR and P450 scc, we performed Western blotting using extracts from carcinoma cells. StAR protein and P450 scc were detected in both HHUA and HOUA-1 cells. The production of pregnenolone in HHUA cells increased 2.4-fold with transfection of a StAR expression vector and 4.3-fold with transfection of an F2 side-chain cleavage system. The RT-PCR product of 3beta-hydroxysteroid dehydrogenase was present in endometrial carcinoma cells. In endometrial carcinoma cells, the production of progesterone also increased with over-expression of StAR and the F2 system. Results of steroid metabolic assays showed that endometrial carcinoma cells produced not only 17alpha-hydroxyprogesterone but also androstenedione. Endometrial carcinoma cells express enzymes that are associated with the production of steroid hormones. Locally produced steroid hormones may have effects on tumor proliferation and tumorigenesis.