Down-regulation of hepatic lipase expression by elevation of cAMP in human hepatoma but not adrenocortical cells

Expression of hepatic lipase (HL) in the liver is reduced during prolonged fasting. This effect is mainly mediated via catecholamines, which signal through elevation of Ca(i)(2+) as well as cAMP. We have studied the effect of cAMP on HL expression in cell culture. Overnight incubation of HepG2 cells with 10-300microM 8-bromo-cyclic AMP resulted in a dose-dependent, up to 50% reduction in secretion of HL, but had no effect on secretion of alpha(1)-antitrypsin or overall protein synthesis. HL mRNA levels were decreased 1.5 fold, as determined by semi-quantitative and real-time RT-PCR. In HepG2 cells transiently transfected with human HL (-685/+13) or rat HL (-446/+9) promoter-reporter constructs, cAMP induced a similar dose-dependent suppression of HL promoter activity. cAMP responsiveness in HepG2 cells was mediated by a conserved 10-bp response element at -45/-36, that represents a potential binding site for CCAAT/enhancer-binding protein beta (C/EBPbeta). cAMP reduced expression of the 45kDa C/EBPbeta protein and binding of C/EBPbeta to the proximal promoter region of the human HL gene by 50%, as determined by immunoblotting and chromatin immunoprecipitation assay, respectively. In human H295R adrenocortical cells, cAMP failed to suppress HL promoter activity, and only slightly reduced C/EBPbeta expression. We conclude that the fall in HL expression during prolonged fasting may be mediated through elevation of cAMP and lowering of C/EBPbeta expression.     

The intraovarian actions of estrogen receptor-alpha are necessary to repress the formation of morphological and functional Leydig-like cells in the female gonad

The predisposition of the testis and ovary to primarily synthesize testosterone (T) and estradiol (E2), respectively, is due to gonadal-specific cell types that differentially express the various hydroxysteroid (17beta) dehydrogenase (HSD17B) isoforms. In testes, Leydig cells rely on LH stimulation to maintain expression of the type 3 (HSD17B3) isoform, which specifically converts androstenedione to T. In ovaries, thecal interstitial (TI) cells also rely on LH to induce androgen synthesis but lack HSD17B3 and therefore secrete androgens of low biological activity. Therefore, thecal cells may possess a mechanism to repress the Leydig cell phenotype and HSD17B3 expression. E2 is known to inhibit experimentally Leydig cell function and proliferation. In the current study, we provide evidence that E2 prevents the development of functional Leydig-like cells in the murine ovary and that this action is mediated by estrogen receptor (ER) alpha. ERalpha-null (alphaERKO) female mice exhibit testis-like levels of Hsd17b3 expression in the ovaries and male-like levels of plasma T. Herein, we demonstrate that: 1) Hsd17b3 expression in alphaERKO ovaries is a primary effect of the loss of intraovarian ERalpha actions; 2) alphaERKO ovarian cells produce substantial levels of T in vitro, and this is blocked by a HSD17B3-specific inhibitor; 3) Hsd17b3 expression in alphaERKO ovaries is LH regulated and localized to the secondary interstitial (SI)/TI cells; and 4) alphaERKO SI/TI cells possess Leydig-like ultrastructural features. These data indicate that intraovarian ERalpha actions are required to repress Hsd17b3 expression in the ovary and may be important to maintaining a female phenotype in SI/TI cells.     

Divergent effects of retinoic acids on the expression of ERalpha and 17beta-hydroxysteroid dehydrogenase type 2 in endometrial carcinoma cells (RL 95-2)

The effects of E2 are dependent on ERs and local E2 concentration in target cells. Modulation of intracellular E2 concentration involves the action of 17beta-hydroxysteroid dehydrogenase (17HSD) type 2, the enzyme converting E2 to estrone. In the present study, the influence of RAs on the growth of endometrial cancer cell line RL 95-2 as well as the expression of ERs and 17HSD type 2 have been investigated. It was found that RAs repress the growth of RL 95-2 cells, which express all subtypes of RXR and RAR, as examined by RT-PCR. Also, quantitative RT-PCR analysis showed that both ERalpha and ERbeta are present in RL 95-2 cells, and Western blot assay further revealed that ERalpha expression was decreased by all trans-RA treatment. In contrast, RAs induced 17HSD type 2 mRNA expression in a dose- and time-dependent fashion. This stimulatory effect was also detected at the level of in vivo oxidative 17HSD activity in cultured cells. On the other hand, the abundance of 17HSD type 2 mRNA was not altered by RAs in cultured normal epithelial cells isolated from human early- and late-secretory endometrium. The data indicate that RAs have an inhibitory effect on the growth of RL 95-2 cells and a cross-talk with the estrogen pathway in estrogen-responsive endometrial cancer cells.     

Human hydroxysteroid (17-beta) dehydrogenase 1 expression enhances estrogen sensitivity of MCF-7 breast cancer cell xenografts

Hydroxysteroid (17-beta) dehydrogenase 1 (HSD17B1) catalyzes the conversion between estrone (E1) and estradiol (E2). The reaction is reversible in vitro, but the data in cultured cells suggest that E2 production is the predominant reaction in physiological conditions. However, the hypothesis has not been verified in vivo. In the present study, estrogen-dependent MCF-7 human breast cancer cells were stably transfected with an expression plasmid for human HSD17B1. The enzyme efficiently converted E1 to E2 and enhanced the estrogen-dependent growth of cultured MCF-7 cells in the presence of hormonally less active E1. The HSD17B1-expressing cells also formed estrogen-dependent tumors in immunodeficient nude mice. After treating the mice with an appropriate dose of the substrate (E1, 0.1 micromol/kg x d), a marked difference in tumor growth was observed between nontransfected and HSD17B1-transfected MCF-7 cells, mean tumor weights at the end of E1 treatment being 23.2 and 130.4 mg, respectively. Furthermore, estrogen-dependent growth of the HSD17B1-expressing xenografts in the presence of E1 was markedly inhibited by administering 5 micromol/kg x d of a specific HSD17B1 inhibitor. After a 4-wk treatment, the tumor size was reduced by 59.8% as compared with the nontreated tumors, whereas the uterine growth of the mice was not affected by the HSD17B1 inhibitor used. This was in line with the induction of apoptosis of the tumors. The results evidently show that estrogenic response for E1 is enhanced by the local action of HSD17B1 in vivo, and thus, the enzyme is a potential target for pharmacological inhibition of estrogen action.     

Cholesteryl ester transfer protein gene expression is not specifically regulated by CCAAT/enhancer-binding protein in HepG2-cells.

Cholesteryl ester transfer protein (CETP) mediates the exchange of neutral lipids among plasma lipoproteins and is expressed predominantly in liver and intestine. In band shift assays employing nuclear extracts of HepG2 cells we identified C/EBPbeta as the predominant C/EBP isoform involved in binding to the C/EBP consensus sequence within the 5' upstream region of the CETP gene. This was demonstrated by supershift experiments using antibodies specific for C/EBPalpha, C/EBPbeta and C/EBPdelta and an oligonucleotide containing a single point mutation (CAAT-->CTAT) in this site. Expression of a CETP promoter-fragment/luciferase construct in transiently transfected HepG2 and CaCo-2 cells and enhancement of promoter activity by co-transfection with human C/EBPalpha in HepG2 cells could be influenced neither by the mutation in the consensus sequence nor by elimination of this site together with a second potential binding site for C/EBP. Furthermore, transfection of HepG2 with human C/EBPalpha did not influence the synthesis of CETP by these cells. Our results indicate that the expression of C/EBP in HepG2 cells is not able (1) to influence specifically the expression of a transfected CETP promoter dependent reporter through binding to C/EBP sites in the promoter region and (2) to significantly enhance expression of the endogenous CETP gene.     

Estrogen receptors and estrogen-metabolizing enzymes in human ovaries during fetal development

Estrogen action plays a crucial role in many processes throughout the human life span, including development. Estrogens are pivotal in the regulation of female reproduction, but little is known about their role during ovarian development. To better understand estrogen action during ovarian development, the expression of estrogen receptors (ERs)-alpha and -beta and key enzymes regulating estradiol production, 17beta-hydroxysteroid dehydrogenases (17HSDs) types 1, 2, and 7, were analyzed in human fetal ovaries. The expression of ERs was related to the development of ovarian follicles. Before the 26th week of fetal life ERalpha was only occasionally detected, but from then onward, its expression was detected in ovarian follicles. Consistent expression of ERbeta was seen from the 20th week until term. Both ERalpha and ERbeta were localized to the granulosa cells and oocytes. Expression of 17HSD1 and 17HSD7 enzymes, catalyzing the conversion of estrone to more active estradiol, was detected as early as at the 17th week of fetal life. The expression of 17HSD1 displayed a pattern similar to that of ERs and increased toward term, whereas that of 17HSD7 decreased and was negative by the 36th week. 17HSD1 was localized to the granulosa cells, whereas 17HSD7 expression was more diffuse and was found in both granulosa and stromal cells. 17HSD2, converting estradiol to less potent estrone, was negative in all samples studied. The simultaneous appearance of estrogen-converting enzymes and ERs at the time of follicle formation indicates that the machinery for estrogen action exists in fetal ovaries and suggests a possible role for estrogens in the developing ovary.     

Expression of mouse 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 in the ovary, uterus, and placenta: localization from implantation to late pregnancy

Rodent 17beta-hydroxysteroid dehydrogenase/17-ketosteroid reductase type 7 (17HSD/KSR7) catalyzes the conversion of estrone (E1) to estradiol (E2) and is abundantly expressed in the ovaries of pregnant animals in particular. In the present work we demonstrate cell-specific expression of 17HSD/KSR7 in the ovaries, uteri, and placentas of pregnant and nonpregnant mice using in situ hybridization. The results show that mouse 17HSD/KSR7 (m17HSD/KSR7) messenger RNA is distinctly and exclusively expressed in a proportion of corpora lutea (CLs). During pregnancy, expression of m17HSD/KSR7 is most abundant around embryonic day 14.5 (E14.5), when the ovaries are filled with CLs expressing 17HSD/KSR7. In the uterus, m17HSD/KSR7 is first detected on E5.5, when expression surrounds the implantation site on the antimesometrial side. As gestation progresses, m17HSD/KSR7 is expressed in the decidua capsularis on E8 and E9.5, disappearing thereafter from the antimesometrial decidua. On E9 onward, m17HSD/KSR7 messenger RNA expression takes place at the junctional zone of the developing placenta. On E12.5 and E14.5, m17HSD/KSR7 is abundantly expressed in the spongiotrophoblasts, where expression gradually declines toward parturition. In conclusion, m17HSD/KSR7 expression in the CL is related to the life span of the CL. Moreover, spatial and temporal expression of m17HSD/KSR7 in the uterus suggests that locally produced E2 plays a role in implantation and/or decidualization. Finally, the results indicate that mouse placenta is capable of converting E1 to E2 in situ, and that the synthesized E2 may be effective in a paracrine, autocrine, and/or intracrine manner and be involved in placentation.     

AML1-MTG8 leukemic protein induces the expression of granulocyte colony-stimulating factor (G-CSF) receptor through the up-regulation of CCAAT/enhancer binding protein epsilon

The t(8;21) translocation is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). In this translocation, the AML1 (CBFA2/PEBP2aB) gene is disrupted and fused to the MTG8 (ETO) gene. The ectopic expression of the resulting AML1-MTG8 fusion gene product in L-G and 32Dcl3 murine myeloid precursor cells stimulates cell proliferation without inducing morphologic terminal differentiation into mature granulocytes in response to granulocyte-colony stimulating factor (G-CSF). This study found that the ectopic expression of AML1-MTG8 elevates the expression of the G-CSF receptor (G-CSFR). Analysis of the promoter region of the G-CSFR gene revealed that up-regulation of G-CSFR expression by AML1-MTG8 does not depend on the AML1-binding sequence, but on the C/EBP (CCAAT/enhancer binding protein) binding site. The results suggest that the overproduction of G-CSFR is at least partly mediated by C/EBPepsilon, whose expression is activated by AML1-MTG8. The ectopic expression of G-CSFR in L-G cells induced cell proliferation in response to G-CSF, but did not inhibit cell differentiation into mature neutrophils. Overexpression of C/EBPepsilon in L-G cells also stimulated G-CSF-dependent cell proliferation. High expression levels of G-CSFR were also found in the leukemic cells of AML patients with t(8;21). Therefore, G-CSF-dependent cell proliferation of myeloid precursor cells may be implicated in leukemogenesis.