Estrogens are essential for female reproduction and overall well-being, and estrogens in the circulation are largely synthesized in ovarian granulosa cells. Using primary cultures of ovarian granulosa cells from gonadotropin-primed immature rats, we have recently discovered that pituitary FSH and ovarian cytokine transforming growth factor beta 1 (TGFb1) induce calcineurin-mediated dephosphorylation-activation of cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC2) to modulate the expression of Star, Cyp11a1, and Hsd3b leading to increased production of progesterone. This study explored the role of calcineurin and CRTC2 in FSH and TGFb1 regulation of Cyp19a1 expression in granulosa cells. Ovarian granulosa cells treated with FSH displayed increased aromatase protein at 24 h post-treatment, which subsided by 48 h, while TGFb1 acting through its type 1 receptor augmented the action of FSH with a greater and longer effects. It is known that the ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptor NR5A subfamily liver receptor homolog 1 (LRH1/NR5A2) and steroidogenic factor 1 (SF1/NR5A1), and that the Nr5a2 promoter also has a potential CREB-binding site. Herein, we demonstrate that FSHCTGFb1 increased LRH1 and SF1 protein levels, and their binding to the Cyp19a1 PII-promoter evidenced, determined by chromatin immunoprecipitation analysis. Moreover, pretreatment with calcineurin auto-inhibitory peptide (CNI) abolished the FSHCTGFb1-upregulated but not FSH-upregulated aromatase activity at 48 h, and the corresponding mRNA changes in Cyp19a1, and Nr5a2 and Nr5a1 at 24 h. In addition, FSH and TGFb1 increased CRTC2 binding to the Cyp19a1 PII-promoter and Nr5a2 promoter at 24 h, with CREB bound constitutively. In summary, the results of this study indicate that calcineurin and CRTC2 have important roles in mediating FSH and TGFb1 collateral Key Words Journal of Molecular EndocrinologyResearch W-A LAI and others Calcineurin and CRTC2 in ovary Cyp19a1 Nr5a expression 53:2 259-270

8-Oxoguanine, through its ability to mispair bases other than cytosine, is assumed to be one of the most potent premutagenic lesions in nuclear DNA damaged by reactive oxygen radicals. In this study, we examine whether the presence of residual 8-oxoguanine can be detected in mammalian cells after exposure to ionizing radiation. MOLT-4 human leukemia cells and CHO-K1 Chinese hamster cells were acutely irradiated in vitro with 0, 0.2, 0.4, 0.6 and 1.0 Gy gamma radiation at room temperature. The amounts of 8-oxoguanine and total DNA in the cell nucleus were detected by fluorescein-isothiocyanate (FITC)-labeled avidin, which binds specifically and directly to 8-oxoguanine, and propidium iodide, respectively. The intensity ratios between these two fluorescent dyes were then taken as indices to measure the content of 8-oxoguanine within individual cells. We found an apparent dose-dependent increase in the amount of 8-oxoguanine accumulated in cells of both lines. Moreover, the content of 8-oxoguanine decreased from 2 to 20 h after irradiation in CHO-K1 cells, which may reflect the time-dependent repair processes at the 8-oxoguanine lesions. This novel approach may provide a sensitive tool for in situ measurement of 8-oxoguanine in cells or even in the human body after exposure to ionizing radiation.

The present study investigated the modulatory role of transforming growth factor beta 1 (TGFbeta1) on the secretion of matrix metalloproteinases (MMPs) and tested whether the altered secretion of MMPs could directly affect the invasive behavior of ovarian cancer cells. To this aim, human ovarian cancer SKOV3 cells were treated once with vehicle or various concentrations of TGFbeta1 for 24 h. Gelatinase activities in conditioned media were analyzed by zymography and densitometry. TGFbeta1 dose-dependently stimulated the secretion of a 68-kDa gelatinase, which was characterized as an MMP because its activity was inhibited by a metalloproteinase inhibitor 1,10-phenanthroline, and by a synthetic MMP inhibitor BB3103. In addition, we used aminophenylmercuric acetate (APMA) to activate latent gelatinases. APMA time-dependently decreased the activity of 68-kDa gelatinase, and increased the activities of 64- and 62-kDa gelatinolytic bands. The 68-kDa gelatinase was further characterized as MMP2 (gelatinase A) by immunoblotting analysis. We then tested TGFbeta1 effect on the invasive potential of SKOV3 cells as assessed by the migration ability through reconstituted basement membrane, and further investigated whether TGFbeta1 may act through modulating the MMP activity to affect ovarian cancer cell invasion. The results show that TGFbeta1 stimulated the invasive behavior of SKOV3 cells, and that MMP inhibitor BB3103 abrogated this effect of TGFbeta1. In conclusion, this study indicates that TGFbeta1 may act partly through stimulating the secretion of MMP in promoting the invasive behavior of human ovarian cancer cells. Furthermore, this work supports the idea that specific MMP inhibitors of the hydroxamate class could be therapeutically useful in controlling cancer cell invasion/metastasis.

Cholesterol is an essential structural component of mammalian cell membrane and a precursor for the synthesis of steroid hormones and bile acids ( 1 ). During ovarian steroid hormone synthesis, cholesterol is fi rst transported into mitochondrial inner membrane facilitated by the steroidogenic acute regulatory protein (StAR), and then converted to the important sex steroid progesterone under sequential actions of the mitochondrial enzyme P450 cholesterol side chain-cleavage enzyme (P450scc) and endoplasmic reticulum enzyme 3 ␤ -hydroxysteroid dehydrogenase (3 ␤ -HSD). Progesterone could be further enzymatically processed into androgens and estrogens ( 2 ). Cellular cholesterol could be derived from the de novo synthesis pathway or from circulating lipoproteins. For steroidogenic cells, lipoproteins are the major source that provides suffi cient cholesterol to meet the demand of steroid hormone synthesis ( 3 ). Aside from the ubiquitous LDL receptor (LDLR)-mediated endocytic uptake of LDL-cholesterol adapted by most cell types, steroidogenic cells additionally utilize HDL-derived cholesterol through scavenger receptor class B member I Abstract Cellular cholesterol is known to be under homeostatic control in nonsteroidogenic cells, and this intrigued us to understand how such control works in ster oidogenic cells that additionally use cholesterol for steroid hormone synthesis. We employed primary culture of rat ovarian granulosa cells to study how steroidogenic cells adapt to acquire suffi cient cholesterol to meet the demand of active steroidogenesis under the stimulation of gonadotropin follicle-stimulating hormone (FSH) and cytokine transforming growth factor (TGF) ␤ 1. We found that TGF ␤ 1 potentiated FSH to upregulate scavenger receptor class B member I (SR-BI) and LDL receptor (LDLR), both functional in uptaking cholesterol as hHDL 3 NSC99-2320-B-010-013-MY3 (to J.-J.H.) and NSC98-2311-B-002-005-MY3 (to F.-C.K.) Abbreviations: 25-OHC, 25-hydroxycholesterol; 3 ␤ -HSD, 3 ␤ -hydroxysteroid dehydrogenase; ACTH, adrenocorticotropic hormone; AMG, aminoglutethimide; CE, cholesteryl ester; ChIP, chromatin immunoprecipitation; FSH, follicle-stimulating hormone; LDLR, LDL receptor; LRH-1, liver receptor homolog-1; P450scc, P450 cholesterol side chain-cleavage enzyme; SCAP, SREBP cleavage-activating protein; SR-BI, scavenger receptor class B member I; SRE, sterol response element; SREBP, sterol regulatory element-binding protein; StAR, steroidogenic acute regulatory protein; TGF ␤ 1, transforming growth factor ␤ 1. This study was supported by National Science Council of Taiwan Grants

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