Santanu Deb

Regulation of Aromatase Expression in Estrogen-Responsive Breast and Uterine Disease: From Bench to Treatment

A single gene encodes the key enzyme for estrogen biosynthesis termed aromatase, inhibition of which effectively eliminates estrogen production. Aromatase inhibitors successfully treat breast cancer and endometriosis, whereas their roles in endometrial cancer, uterine fibroids, and aromatase excess syndrome are less clear. Ovary, testis, adipose tissue, skin, hypothalamus, and placenta express aromatase normally, whereas breast and endometrial cancers, endometriosis, and uterine fibroids overexpress aromatase and produce local estrogen that exerts paracrine and intracrine effects. Tissue-specific promoters distributed over a 93-kilobase regulatory region upstream of a common coding region alternatively control aromatase expression. A distinct set of transcription factors regulates each promoter in a signaling pathway- and tissue-specific manner. Three mechanisms are responsible for aromatase overexpression in a pathologic tissue versus its normal counterpart. First, cellular composition is altered to increase aromatase-expressing cell types that use distinct promoters (breast cancer). Second, molecular alterations in stromal cells favor binding of transcriptional enhancers versus inhibitors to a normally quiescent aromatase promoter and initiate transcription (breast/endometrial cancer, endometriosis, and uterine fibroids). Third, heterozygous mutations, which cause the aromatase coding region to lie adjacent to constitutively active cryptic promoters that normally transcribe other genes, result in excessive estrogen formation owing to the overexpression of aromatase in many tissues.

Ontogeny of placental lactogen-I and placental lactogen-II expression in the developing rat placenta

The purpose of this investigation was to identify the cellular origin, and the temporal and regional characteristics of placental lactogen-I (PL-I) and placental lactogen-II (PL-II) expression during placental development in the rat. PL-I and PL-II mRNA expression were assessed by Northern blot analysis and in situ hybridization. PL-I and PL-II protein expression were determined by Western blot and immunocytochemical analyses. PL-I mRNA was first detected by in situ hybridization at Day 6 of gestation in mural trophoblast giant cells and a day later, PL-I protein was first detected by immunocytochemistry. PL-I immunostaining extended to the polar trophoblast giant cells as gestation advanced. Polar trophoblast giant cell staining for PL-I was not as intense as the mural trophoblast giant cell staining. Northern and Western blot analyses confirmed the asymmetric distribution of PL-I expression. PL-I mRNA migrated as a 1-kb species and PL-I protein migrated as 30- and 36-40-kDa forms. PL-I expression abruptly declined at Day 12, and by Day 13, PL-I was not detectable. PL-II protein was first detectable at Day 11 of gestation and was localized to trophoblast giant cells. PL-II mRNA could be detected at Day 10 of gestation. Northern and Western blot analyses indicated that PL-II expression significantly increased as gestation advanced and that PL-II expression was asymmetrically distributed similar to PL-I. PL-II mRNA migrated as a 1-kb species and PL-II protein migrated as a 25-kDa species. Blastocysts recovered on Day 4 of gestation initially showed no detectable expression of PL-I or PL-II; however, after 2 days of culture PL-I protein expression was detectable. Biochemical characteristics of PL-I synthesized and secreted by blastocyst outgrowths were similar to PL-I synthesized and secreted by Day 10 placental explants. In summary, (1) PL-I and PL-II are produced by trophoblast giant cells of the developing placenta, (2) PL-I and PL-II exhibit distinct temporal and regional patterns of expression during placental morphogenesis, and (3) PL-I expression by blastocyst outgrowths can be induced in vitro, whereas a more complex array of signals appears necessary for induction of PL-II expression.

Differential Expression of the Estrogen Receptors α and β in the Rat Corpus Luteum of Pregnancy: Regulation by Prolactin and Placental Lactogens1

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ERα and ERβ) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ERα and ERβ messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ERα and ERβ) are coexpressed in the rat corpus luteum during pregnancy. Whereas ERα mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ERβ mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examinati...

Estrogen Receptors α and β in Rat Decidua Cells: Cell-Specific Expression and Differential Regulation by Steroid Hormones and Prolactin1

Estradiol is known to play an important role in the growth and differentiation of rat uterine stromal cells into decidual cells. In particular, this hormone with progesterone is necessary for blastocyst implantation and subsequent decidualization in the rat. Although binding experiments have demonstrated the presence of estrogenbinding sites, no evidence exists as to whether the rat decidua expresses both isoforms of the estrogen receptor (ER), a and b. In this investigation, we analyzed the expression of decidual ERa and ERb, studied their regulation by PRL and steroid hormones and examined the ability of decidual ERb to transduce the estradiol signal to the progesterone receptor. Immunocytochemistry, RT-PCR, and Northern blot analysis showed that both ER species are coexpressed in the decidua during pseudopregnancy. Interestingly, these genes were preferentially found in a cell population localized in the antimesometrial site of the uterus where blastocyst implantation takes place. Using decidual cells in primary culture obtained from pseudopregnant rats and a decidua-derived cell line (GG-AD), we show a differential regulation of ERa and ERb by PRL and ovarian steroid hormones. Whereas PRL, estradiol, and progesterone all increased ERb messenger RNA (mRNA) expression in a dose-dependent manner, only PRL up-regulated the mRNA levels of ERa. Estradiol had no effect on ERa expression, whereas progesterone markedly decreased its mRNA levels. Interestingly, progesterone, which up-regulates the ability of PRL to signal to a PRL-regulated gene in mammary-gland derived cells, prevented PRL stimulation of decidual ERa and had no synergistic effect on ERb expression. The use of GG-AD cells, which express only ERb, allowed us to demonstrate that this receptor subtype is functional and transduces estradiol signal to the progesterone receptor. In summary, the results of this investigation have revealed that ERb is expressed in addition to ERa in the rat decidua, and that the expression of both ERs are cell specific and differentially regulated by PRL and steroids. One salient finding of this investigation is that progesterone down-regulates ERa, but concomitantly increases the expression of a functional ERb that mediates estradiol up-regulation of the decidual progesterone receptor. (Endocrinology 141: 3842‐3851, 2000)

The Expression of Interleukin-6 (IL-6), IL-6 Receptor, and gp130-Kilodalton Glycoprotein in the Rat Decidua and a Decidual Cell Line: Regulation by 17β-Estradiol and Prolactin1

The cytokine interleukin 6 (IL-6), a major mediator of immune and acute phase responses of the liver, has been implicated in the termination of pregnancy once expressed in the uterus. This study was undertaken to investigate the expression and regulation of genes encoding IL-6 and IL-6 receptor (IL-6R) in rat decidual tissue. Total RNA obtained from rat decidual tissue on different days of pseudopregnancy was analyzed by RT-PCR using specific primers for IL-6, IL-6R, and 130-kDa glycoprotein (gp130). Ribosomal L19 primers served as an internal control. IL-6R and gp130 were found to be expressed in the decidua throughout development, while no messenger RNA (mRNA) for IL-6 was detected. Interestingly, within several hours of culture, decidual explants acquired the ability to express IL-6. The apparent ability of decidual cells to express IL-6 and its lack of expression in vivo led us to examine whether the IL-6 gene is actively inhibited. Primary decidual cells were cultured in the presence of estradiol, pr...

Antipeptide antibodies reveal structural and functional characteristics of rat placental lactogen-II☆☆☆

The purpose of this investigation was to develop specific immunologic probes to rat placental lactogen-II (PL-II) and to use the immunologic probes to further characterize rat PL-II. Five oligopeptides corresponding to different regions of rat PL-II (amino acids 1-13, 56-70, 89-103, 107-118, 150-164) were chemically synthesized by solid phase methods and purified to homogeneity by reverse phase high performance liquid chromatography. The synthetic peptides were coupled to keyhole limpet hemocyanin (KLH) and the peptide-KLH conjugates were used to immunize rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA), electrophoresis and immunoblotting analyses. Each of the antipeptide antisera showed reactivity with the entire rat PL-II protein; however, the extent of the reactivities of each antiserum with rat PL-II was dependent on the conformational state of rat PL-H. Antisera directed to amino acids 56-70 showed the best reactivity toward each of the conformational states of rat PL-II tested. Antibodies generated to the entire rat PL-II protein specifically recognized the 56-70 amino acid sequence but showed limited reactivity with synthetic peptide corresponding to amino acids 1-13, 89-103, 107-118, and 150-164 of rat PL-II. Antisera to amino acids 56-70 of rat PL-II were specific for PLs as demonstrated by their recognition of rat PL-II, mouse PL-II and human PL and by their lack of reactivity with rat pituitary prolactin and growth hormone and with a series of other synthetic peptides to rat PL-II and rat prolactin-like protein-A. The immunorecognition of human PL was restricted to antipeptide antibodies directed to amino acids 56-70 of rat PL-II. The chemically synthesized peptides representing various regions of rat PL-II did not show significant interactions with prolactin receptors, and antisera directed to the peptides failed to interfere with the binding of either rat PL-II or human PL to prolactin receptors. In summary, we have generated a series of immunologic probes for studying the structure of rat PL-II. The sequence comprising amino acids 56-70 of rat PL-II was shown to make up at least part of an epitope for rat PL-II and to be a region of significant structural homology with mouse PL-II and human PL.

Generation and characterization of antipeptide antibodies to rat cytochrome P-450 side-chain cleavage enzyme

In this report, we describe the generation of immunologic probes to rat P-450scc. Two regions of the P-450scc amino acid sequence were identified (internal domain: amino acids 421-441; carboxy terminal domain: amino acids 509-526), chemically synthesized and used as immunogens in rabbits. Antibody production was monitored by enzyme-linked immunoassay (EIA) and Western blot analyses. Antisera were successfully generated to each of the P-450scc regions that recognized the entire 49 kDa rat P-450scc protein. Antiserum directed to the internal domain of P-450scc showed broad species crossreactivity, whereas antiserum directed to the carboxy terminal domain of P-450scc crossreacted with only rat and mouse. Both antisera were useful for Western blot and immunocytochemical analyses of rat P-450scc expression. In addition to recognizing the major 49 kDa P-450scc protein, each antiserum also recognized lower molecular weight species. Antiserum directed to the internal domain of P-450scc specifically recognized a 42 kDa species, whereas antiserum directed to the carboxy terminal domain specifically recognized an 8 kDa species. We hypothesize that the two lower molecular weight immunoreactive species are generated by proteolytic cleavage of rat P-450scc between the internal and carboxy terminal epitopes.

A Novel Role of Sodium Butyrate in the Regulation of Cancer-associated Aromatase Promoters I.3 and II by Disrupting a Transcriptional Complex in Breast Adipose Fibroblasts

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (–211/–199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPβ, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPβ, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.

Characterization of placental prolactin-like protein-A in intracellular and extracellular compartments

Prolactin-like protein-A (PLP-A) was purified from medium conditioned by junctional zone explants dissected from the rat chorioallantoic placenta via concanavalin A affinity chromatography, sodium dodecyl sulfate-gel electrophoresis and two-dimensional gel electrophoresis. Monomeric PLP-A was found to be heterogeneous and consisted of two major molecular sizes, 29 and 33 kDa. The charge and size heterogeneity attributed to monomeric PLP-A could be accounted for by the addition of N-linked carbohydrate moieties to the PLP-A structure. Monomeric PLP-A lacked lactogenic bioactivities and several attempts to sequence 29 and 33 kDa PLP-A monomers proved unsuccessful. Biochemical characterization of PLP-A species present in various intracellular and extracellular compartments indicated that PLP-A is normally secreted as high molecular weight complexes and that PLP-A species were also targeted to the nucleus. PLP-A species isolated from placental cytosol or from serum of pregnant rats predominantly circulated as disulfide-linked high molecular weight complexes. PLP-A antipeptide antisera showed limited but specific reactivity with the high molecular weight PLP-A species. We have specifically identified a 29 kDa protein species in placental cell nuclei with three antibodies directed to three different regions of the predicted amino acid sequence of PLP-A. The 29 kDa nuclear immunoreactive protein had an equivalent electrophoretic mobility to the 29 kDa PLP-A protein of junctional zone cytosol. The 33 kDa PLP-A protein was not identified in the nucleus. The biological significance of the circulating high molecular weight PLP-A species or the nuclear PLP-A species remains to be determined.

Trophoblast Cell Differentiation: Expression of the Placental Prolactin Family

Normal growth and maturation of a specialized extraembryonic structure, the placenta, are essential for the establishment and maintenance of pregnancy. The placenta acts as the interface between maternal and embryonic compartments, developing in parallel with the embryo and facilitating the acquisition of maternal resources required for embryonic and fetal development. This essential responsibility of the placenta is accomplished in a number of important ways, including (i) regulation of the transport of nutrients and wastes, (ii) an immunologic role in preventing fetal rejection, and (iii) an endocrine/communication/signaling role between fetal and maternal compartments. Consequently, understanding the control of placental development is fundamental to an appreciation of the initiation and maintenance of pregnancy and various abnormalities associated with altered growth and development. The cells responsible for these diverse functions of the placenta are trophoblast cells.