Biomedical subjects

O K Park-Sarge

Publications and source records attributed to O K Park-Sarge.

At least 19 recordsLinked to original sources

PubMed2005

Contrasting mammalian PTH promoters: identification of transcription factors controlling PTH gene expression.

Parathyroid hormone (PTH) is a key component in the maintenance of calcium and phosphate homeostasis. The steady-state expression of the PTH gene can be modeled as a balance between transcriptional activators and repressors. During renal failure, the gradual loss of kidney function is often accompanied by increased circulating concentrations of PTH and decreased synthesis of 1,25-di-hydroxyvitamin D3 (1,25(OH)2D3). The latter finding results in impaired calcium absorption and the removal of a known repressor of PTH gene transcription. Current regimens for treating secondary hyperparathyroidism associated with renal insufficiency are focused on boosting activities that repress PTH gene transcription or secretion of the hormone, and involve the use of vitamin D and its analogues or calcimimetic agents. However, in recent years, concerns have arisen over the use of the steroid hormone and alternative treatments are being sought. Here, we present new information regarding transcription factors controlling PTH gene expression, which include the specificity proteins (Sp) and the nuclear factor Y (NF-Y) complex. A highly conserved DNA response element for the Sp proteins has been identified in mammalian promoters, while an NF-Y binding site is uniquely positioned in the human promoter. Both of these factors are expressed in the parathyroid gland and their DNA elements appear to be functioning as activators of PTH gene expression. Further elucidation of such pathways may offer novel approaches for treating hyperparathyroidism associated with renal failure via suppression of transcriptional activators. That work is currently in progress.

Gene Expression Regulation↗

PubMed2001

Regulation of heat shock transcription factor 1 by stress-induced SUMO-1 modification.

Heat shock transcription factor 1 (HSF1) mediates the induction of heat shock protein gene expression in cells exposed to elevated temperature and other stress conditions. In response to stress HSF1 acquires DNA binding ability and localizes to nuclear stress granules, but the molecular mechanisms that mediate these events are not understood. We report that HSF1 undergoes stress-induced modification at lysine 298 by the small ubiquitin-related protein called SUMO-1. Antibodies against SUMO-1 supershift the HSF1 DNA-binding complex, and modification of HSF1 in a reconstituted SUMO-1 reaction system causes conversion of HSF1 to the DNA-binding form. HSF1 colocalizes with SUMO-1 in nuclear stress granules, which is prevented by mutation of lysine 298. Mutation of lysine 298 also results in a significant decrease in stress-induced transcriptional activity of HSF1 in vivo. This work implicates SUMO-1 modification as an important modulator of HSF1 function in response to stress.

Cell Compartmentation↗

PubMed2001

Human estrogen receptor beta-specific monoclonal antibodies: characterization and use in studies of estrogen receptor beta protein expression in reproductive tissues.

Investigation of the role of the second, more recently described estrogen receptor, denoted ERbeta, will be critical in understanding the molecular mechanisms underlying tissue-specific gene regulation by estrogens. Expression of ERbeta in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization and size of the endogenous ERbeta protein, due, in part, to the limited availability of human ERbeta-specific antibodies. Thus, our aim was to generate specific antibodies to human ERbeta and use them to determine the tissue-specific distribution and size(s) of the ERbeta protein. To this end, we have cloned three different hybridoma cell lines that produce monoclonal antibodies specific for the hormone-binding domain of human ERbeta. The antibodies, made in mice against human ERbeta amino acids 256-505 (hormone binding domain lacking the F domain), are designated CFK-E12 (E12), CMK-A9 (A9) and CWK-F12 (F12) and were determined to be the IgG gamma1 isotype for E12, and IgG gamma2b for A9 and F12. All three monoclonal antibodies could be used to detect in vitro translated, baculovirus expressed, and cell transfected and expressed ERbeta protein by Western blot analyses, and all failed to detect ERalpha. A9 and F12 were able to immunoprecipitate efficiently the native form of ERbeta protein in the presence and absence of estradiol. Epitope mapping studies indicate that the E12 and F12 antibodies recognize overlapping peptide sequences in the N-terminal region of the hormone-binding domain, a region that is highly conserved among species. Immunocytochemical studies with these antibodies reveal nuclear-specific localization of the ERbeta protein in granulosa cells of the rat ovary. Nuclear ERbeta is also specifically localized in epithelial and some stromal cells of mouse and rat epididymis. Western blot analysis with protein extracts from ovarian granulosa cells of human, rat, mouse, and pig showed a ca. 52 kDa and an additional ca. 62-64 kDa band in these species. These results indicate the presence of two predominant molecular size forms of the ERbeta protein in ovarian granulosa cells and demonstrate the utility of these antibodies for detection of ERbeta in the human and in several other mammalian species.

Animals↗

PubMed2001

Sumo-1 modification regulates the DNA binding activity of heat shock transcription factor 2, a promyelocytic leukemia nuclear body associated transcription factor.

Heat shock transcription factor 2 (HSF2) is a transcription factor that regulates heat shock protein gene expression, but the mechanisms regulating the function of this factor are unclear. Here we report that HSF2 is a substrate for modification by the ubiquitin-related protein SUMO-1 and that HSF2 colocalizes in cells with SUMO-1 in nuclear granules. Staining with anti-promyelocytic leukemia antibodies indicates that these HSF2-containing nuclear granules are PML bodies. Our results identify lysine 82 as the major site of SUMO-1 modification in HSF2, which is located in a "wing" within the DNA-binding domain of this protein. Interestingly, SUMO-1 modification of HSF2 results in conversion of this factor to the active DNA binding form. This is the first demonstration that SUMO-1 modification can directly alter the DNA binding ability of a transcription factor and reveals a new mechanism by which SUMO-1 modification can regulate protein function.

Biological Transport↗

PubMed2001

Gonadotropins decrease estrogen receptor-beta messenger ribonucleic acid stability in rat granulosa cells.

We have previously shown that the preovulatory LH surge down-regulates estrogen receptor-beta (ERbeta) messenger RNA (mRNA) levels selectively in the granulosa cells of preovulatory follicles. To gain insight into the underlying mechanisms, we examined whether the LH-induced loss of ERbeta mRNA expression in rat granulosa cells is attributable to the hormone-induced changes at the level of transcription and/or mRNA degradation. When the rate of ERbeta gene transcription was assessed in cultured granulosa cells, by nuclear run-off assays, we observed only a marginal effect of hCG on ERbeta gene transcription. In contrast, when ERbeta mRNA levels were estimated in granulosa cells that were cultured in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor, we observed a significant inhibitory effect of human CG (hCG) on ERbeta mRNA expression at a magnitude similar to that observed in the absence of DRB. Forskolin (FSK) and 2-O-tetradecanol-phorbol-13-acetate (TPA), pharmacological agents that mimic LH actions in granulosa cells, also showed similar effects. Thus, these results suggest that LH decreases ERbeta mRNA expression in the granulosa cells of preovulatory follicles, primarily by destabilizing the preexisting ERbeta mRNA. We next determined the decay rate of the ERbeta mRNA in granulosa cells that were cultured in the presence of DRB and additional hCG, FSK, or TPA for various time periods, by estimating ERbeta mRNA levels, using semiquantitative RT-PCR assays and subsequent linear regression analyses. The half-life of the ERbeta mRNA in the presence of vehicle was 17.87 +/- 1.2 h (n = 4). hCG dramatically decreased the half-life of the ERbeta mRNA (4.85 +/- 0.49 h, n = 4). Similarly, both FSK and TPA decreased the half-life of the ERbeta mRNA to 3.57 +/- 0.31 h and 4.02 +/- 0.13 h, respectively. We extended these findings by examining whether the LH-induced down-regulation of the ERbeta mRNA is cycloheximide-sensitive. When granulosa cells were cultured in the presence of cycloheximide, a protein synthesis inhibitor, the inhibitory effects of hCG, FSK, and TPA on ERbeta mRNA levels were abolished. Similar results were obtained in the presence or absence of DRB, indicating that the hormone-induced destabilization of the ERbeta mRNA is coupled with translation processes. Taken together, our results demonstrate that LH decreases ERbeta mRNA expression, predominantly at the posttranscriptional level, in a cycloheximide-sensitive manner.

Animals↗

PubMed2001

Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

Animals↗

PubMed2000

Expression of an intracisternal A-particle-like element in rat ovary.

We have isolated a rat intracisternal-A particle element (IAP)-like element (IAP-LE) from ovarian granulosa cells that appears to be identical to the rat EST clone AA964260. The compiled cDNA sequences contain several putative in-frame translation initiation codons with the largest capable of encoding a 365 amino acid protein with a reverse transcriptase domain in the N-terminus as well as a bipartite nuclear localization signal sequence in the middle. Northern blotting shows a major approximately 7 Kb transcript and a minor approximately 5 Kb transcript that are abundantly expressed in the ovary. In situ hybridization histochemistry using ovaries from gonadotropin-treated immature rats and regularly cycling adult rats show that this transcript is predominantly localized to granulosa cells of all healthy follicles, including primary follicles, and to newly-formed and healthy corpora lutea. This cell-specific expression pattern of the IAP-LE gene is distinct from those of the several known retroviral elements, suggesting the potentially novel functional importance of the IAP-LE gene. Taken together, our results demonstrate abundant and cell-specific expression of a novel IAP-LE in rat granulosa cells.

Amino Acid Sequence↗

PubMed2000

Progesterone receptor activation mediates LH-induced type-I pituitary adenylate cyclase activating polypeptide receptor (PAC(1)) gene expression in rat granulosa cells.

We have previously reported that the pituitary adenylate cyclase activating polypeptide (PACAP) gene is regulated in ovarian granulosa cells by the autocrine and/or paracrine interaction between progesterone and its nuclear receptor progesterone receptor (PR). To initiate studies on the functional significance of the progesterone-induced PACAP production in luteinizing granulosa cells, we sought to determine the expression and hormonal regulation of PACAP receptors in the rat ovary. The relative mRNA levels of three known PACAP receptor subtypes (PAC(1), VPAC(1), and VPAC(2)) were determined in ovaries of immature rats treated with gonadotropins, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Results show that all PAC(1), VPAC(1), and VPAC(2) transcripts are expressed at a detectable level in immature rat ovaries. Importantly, the ovarian level of PAC(1), but not VPAC(1) or VPAC(2), mRNA notably changes during gonadotropin challenges. Ovarian PAC(1) mRNA expression decreases during the pregnant mare's serum gonadotropin (PMSG)-induced follicular phase but substantially increases during the human chorionic gonadotropin (hCG)-induced periovulatory period. Because the hCG-induced increase in ovarian PAC(1) mRNA expression is attributable to the hormone-induced PAC(1) mRNA expression in granulosa cells of the preovulatory follicles, we next examined whether hCG regulates PAC(1) mRNA expression by directly acting on granulosa cells. When granulosa cells isolated from PMSG (40 h)-primed immature rats were challenged with hCG (or forskolin), PAC(1), but not VPAC(1) or VPAC(2), mRNA expression significantly increased within 6 h. Because the LH-induced PAC(1) mRNA expression (6 h) proceeds PR activation (3 h) in granulosa cells as the LH-induced PACAP mRNA expression (6 h) does, we further determined the cause-effect relationship among LH, PR activation and PAC(1) receptor gene expression, by examining the effect of PR antagonist, ZK98299, on the ability of LH to increase PAC(1) mRNA levels in luteinizing granulosa cells. Results show that ZK98299 inhibited the stimulatory effect of hCG (or forskolin) on PAC(1) mRNA expression, at the level of all known splice variants of PAC(1) mRNA in granulosa cells. In summary, our results demonstrating that PR activation is critical for the LH-induced PAC(1) gene expression in luteinizing granulosa cells suggest that PR activation regulates the finely tuned expression of the PACAP/PACAP receptor genes in luteinizing granulosa cells and thus dictates the timing of the autocrine and/or paracrine function of PACAP in preovulatory follicles.

Animals↗

PubMed1999

Regulation of protein kinase C delta by estrogen in the MCF-7 human breast cancer cell line.

We have previously shown that estrogen up-regulates expression of protein kinase C (PKC) delta in the rat and rabbit corpus luteum as well as in luteinized rat granulosa primary cell cultures. To determine whether a similar regulation of the PKC delta isoform by estrogen occurred in another estrogen responsive system, we investigated the estrogen receptor positive MCF-7 human breast cancer cells. In a characterization of PKC isoforms in MCF-7 cells we determined that PKC delta was the predominant PKC isoform. However in contrast to the effect of estrogen on PKC delta expression in ovarian cells, estrogen treatment of MCF-7 cells resulted in a significant decrease in PKC delta protein and mRNA expression in a time and dose dependent manner. Treatment of MCF-7 cells with 10(-10)-10(-8) M estrogen for 7 days down-regulated specifically PKC delta mRNA and protein while expression of other PKC isoforms was unchanged. The opposite regulation of PKC delta expression in ovarian and breast cancer cells prompted us to evaluate the type of estrogen receptor present in both cell types. Results showed that luteinized rat granulosa cells expressed predominantly estrogen receptor beta while the MCF-7 cells expressed predominantly estrogen receptor alpha and barely detectable levels of estrogen receptor beta. These results suggest that the differential ability of estrogen to regulate PKC beta expression could potentially be a result of differential signaling through the two estrogen receptor subtypes.

Animals↗

PubMed1999

Characterization of estrogen receptor-beta (ERbeta) messenger ribonucleic acid and protein expression in rat granulosa cells.

We have examined estrogen-responsiveness of ovarian granulosa cells by focusing on estrogen receptor (ER) expression. Estrogen responsiveness was determined by examining the effect of 17beta-estradiol (1-10 nM) on luciferase reporter activity in rat granulosa cells transfected with an ERE-luciferase construct. The results demonstrate an estrogen-induced (approximately 3-fold) increase in luciferase reporter activity, indicating that granulosa cells contain functional estrogen response element (ERE)-binding transcriptional activators. Gel mobility shift assays in combination with ER antibodies show that ERbeta is the predominant ERE-binding protein in granulosa cells. Western blotting results show that granulosa cells contain ERbeta-immunoreactive protein(s) migrating at a size substantially larger than the recombinant protein generated from the originally proposed 485 amino acid open-reading frame. This size discrepancy is not due to granulosa cell expression of ERbeta isoforms with insertions within the coding region because RT-PCR assays revealed products with sizes expected for ERbeta, ERbetaB, and delta3 isoforms. This size discrepancy appears to be due to usage of a well-conserved, upstream in-frame translation initiation codon (ATG436) leading to a 530 amino acid open reading frame. ERbeta messenger RNA (mRNA) characterization using 5'-rapid amplification of complementary DNA ends (5'-RACE) show the presence of two different (P1- and P2-) 5'-ends of rat ERbeta mRNA encoding the full-length ERbeta protein. The generation of the P2-specific exon is likely due to initiation of transcription from an alternative promoter. Both P1- and P2-specific exon-containing ERbeta mRNAs are expressed in granulosa cells, and they are rapidly down-regulated by the cAMP-mediated intracellular signaling pathway in cultured granulosa cells. Taken together, our results show that rat granulosa cells produce two different 3',5'-cAMP-regulated ERbeta mRNA species and that these mRNA species are capable of encoding the full-length ERbeta protein.

Animals↗

PubMed1999

Role of progesterone receptor activation in pituitary adenylate cyclase activating polypeptide gene expression in rat ovary.

It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR activation is critical for successful ovulation. To further understand the molecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa cells. We employed a PCR-based subtraction cloning strategy to screen for genes expressed differentially in granulosa cells that were challenged with forskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activating polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we examined whether PR and PACAP messenger RNA (mRNA) expression is temporally correlated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semi-quantitative RT-PCR assays. However, the peak expression for PR and PACAP mRNAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximide on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PR synthesis is prerequisite for PACAP expression, we examined the effect of ZK98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. This compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent manner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal regulation of ovarian PR and PACAP gene expression in immature rats treated with gonadotropins as well as in adult rats during the preovulatory period by using in situ hybridization and semiquantitative RT-PCR assays. Results show that both PR and PACAP mRNAs are induced in granulosa cells of preovulatory follicles by human CG, but that the PR gene is expressed before the PACAP gene. Taken together, these results demonstrate that PRs mediate the LH-induced PACAP gene expression in rat granulosa cells.

Animals↗

PubMed1998

Differential expression of the estrogen receptors alpha and beta in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens.

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 (ER alpha and ER beta) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ER alpha and ER beta messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ER alpha and ER beta) are coexpressed in the rat corpus luteum during pregnancy. Whereas ER alpha mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ER beta mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examination of ER alpha and ER beta mRNA expression at the cellular level, by in situ hybridization, showed ER alpha expressed in both follicles and corpus luteum, with maximal expression at midpregnancy. In parallel with the RT-PCR studies, ER beta mRNA was similarly expressed throughout pregnancy in the corpus luteum, but it was less abundant when compared with small and growing follicles. Western blot analysis revealed two ER immunoreactive proteins in the nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa moiety, highly expressed at midpregnancy but barely detectable in early and late gestation; and a 61-kDa form that remained developmentally unchanged. Hypophysectomy, performed early in pregnancy, induced a sharp decline in ER alpha mRNA expression but a less-marked reduction in ER beta mRNA levels. PRL treatment reverted the inhibition induced by hypophysectomy in both receptor subtypes. When primary luteinized cells were used to test the effect of PRL, rat placental lactogen I, and rat placental lactogen II on the expression of ER alpha and ER beta mRNA, all these lactogenic hormones stimulated both ER mRNA species in a dose-dependent manner. The regulation of ER mRNA expression was further evaluated in a luteal cell line, termed GG-CL, which apparently expresses only the ER beta mRNA species. Culture of the GG-CL cells, in the presence of PRL, resulted in a dose-related up-regulation of ER beta mRNA expression. In addition, PRL treatment enhanced the binding activity of GG-CL cell nuclear proteins to a classical estrogen response element. Furthermore, in these cells, estradiol treatment induced a dose-dependent up-regulation of the mRNA encoding protein kinase C delta isoform, a well-known estrogen target gene in the corpus luteum of the pregnant rat.

Animals↗

PubMed1997

Estrogen receptor-beta mRNA expression in rat ovary: down-regulation by gonadotropins.

We have examined the expression and regulation of the two estrogen receptor (ER alpha and ER beta) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ER alpha and ER beta genes as single (approximately 6.5-kb) and multiple (ranging from approximately 1.0-kb to approximately 10.0-kb) transcripts, respectively. ER alpha mRNA is expressed at a level lower than ER beta mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ER beta mRNA levels were decreased. Examination of ER beta mRNA expression at the cellular level, by in situ hybridization, showed that ER beta mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ER beta mRNA was observed in a subset of corpora lutea, and that the decrease in ER beta mRNA during proestrous evening is attributable, at least in part, to down-regulation of ER beta mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ER alpha mRNA in the ovary. Although whole ovarian content of ER alpha mRNA was clearly detected by RT-PCR, no apparent modulation of ER alpha mRNA levels was observed during the estrous cycle. Examination of ER alpha mRNA expression at the cellular level, by in situ hybridization, showed that ER alpha mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ER beta mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ER beta mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ER beta mRNA. In situ hybridization for ER beta mRNA shows that small, growing, and preovulatory follicles express ER beta mRNA in the granulosa cells. The preovulatory follicles contain ER beta mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ER beta mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ER beta mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ER beta mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ER alpha and ER beta genes, although ER beta is the predominant form of estrogen receptor in the ovary, 2) ER beta mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ER beta mRNA. These results clearly implicate the physiological importance of ER beta in female reproductive functions.

Age Factors↗

PubMed1996

Progesterone receptors: expression and regulation in the mammalian ovary.

The authors have briefly discussed the molecular structure, regulation, and function of progesterone receptors in the mammalian ovary. Particularly important is the contrast in the regulatory mechanisms of PR induction in the ovary (gonadotropins/membrane receptor mediated) and other well-known progesterone target tissues, such as the uterus and mammary gland (estrogen/nuclear receptor mediated). Future research will focus on how the PR gene responds to these hormonal regulatory signals in this cell-specific manner. Equally important in this discussion has been the mounting evidence indicating that PRs are an essential component of the ovulatory process. The observation that PR-/- knockout mice are incapable of undergoing ovulation, even in response to gonadotropin challenge, further supports the previous physiological evidence indicating that PRs in preovulatory follicles are induced before, and are necessary for, ovulation. Further studies are required to determine the identity of PR-regulated target genes during the periovulatory period. Although our knowledge of PR structure, regulation, and function has increased dramatically during the past decade, many exciting questions remain related to the regulation and function(s) of PRs in the ovary and other tissues.

Animals↗

PubMed1996

Gonadotropins induce rapid phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding protein in ovarian granulosa cells.

The pituitary gonadotropins FSH and LH exert their effects on gonadal target cells, at least in part, through the activation of adenylyl cyclase and the production of the second messenger cAMP. To elucidate further the signal transduction pathways regulating gonadotropin-responsive genes in ovarian granulosa cells, we have investigated the expression of the cAMP response element binding protein (CREB), which mediates many of the effects of cAMP by modulating the transcription of target genes in a cAMP-dependent fashion. In situ hybridization, RNA blot analysis and RT-PCR RNA quantification demonstrated that CREB messenger RNA (mRNA) is expressed at low levels throughout the ovary, and that CREB mRNA levels do not change appreciably after gonadotropin stimulation. Similar results were obtained using immunohistochemistry and Western protein blotting to examine CREB protein in ovaries isolated from immature animals treated with gonadotropins or immunocytochemistry and Western protein blotting to examine the CREB protein in cultured granulosa cells after gonadotropin treatment. In contrast, immunocytochemistry and Western protein blotting using an antipeptide antibody specific to CREB phosphorylated at serine 133 (P-CREB), which is the activated from of the CREB protein, revealed a dramatic increase in the phosphorylated form of CREB within 20 min of gonadotropin treatment of granulosa cells that was transient and was decreased by 60 min after gonadotropin treatment. Stimulation of P-CREB was observed using granulosa cells isolated from immature animals and treated with recombinant human FSH in vitro, or using granulosa cells isolated from immature animals primed with PMSG in vivo and treated with human CG (hCG) in vitro. Stimulation of P-CREB was also observed in ovarian granulosa cells isolated from animals treated with PMSG in vivo. These results indicate that both gonadotropins can induce a rapid and transient phosphorylation of the CREB protein in granulosa cells, leading to the activation of a factor likely to play an important role in the transcription of many gonadotropin-regulated ovarian genes.

Animals↗

PubMed1995

Tissue-dependent expression of heat shock factor 2 isoforms with distinct transcriptional activities.

Heat shock factor 2 (HSF2) functions as a transcriptional regulator of heat shock protein gene expression in mammalian cells undergoing processes of differentiation and development. Our previous studies demonstrated high regulated expression and unusual constitutive DNA-binding activity of the HSF2 protein in mouse testes, suggesting that HSF2 functions to regulate heat shock protein gene expression in spermatogenic cells. The purpose of this study was to test whether HSF2 regulation in testes is associated with alterations in the HSF2 polypeptide expressed in testes relative to other mouse tissues. Our results show that mouse cells express not one but two distinct HSF2 proteins and that the levels of these HSF2 isoforms are regulated in a tissue-dependent manner. The testes express predominantly the 71-kDa HSF2-alpha isoform, while the heart and brain express primarily the 69-kDa HSF2-beta isoform. These isoforms are generated by alternative splicing of HSF2 pre-mRNA, which results in the inclusion of an 18-amino-acid coding sequence in the HSF2-alpha mRNA that is skipped in the HSF2-beta mRNA. HSF2 alternative splicing is also developmentally regulated, as our results reveal a switch in expression from the HSF2-beta mRNA isoform to the HSF2-alpha isoform during testis postnatal developmental. Transfection analysis shows that the HSF2-alpha protein, the predominant isoform expressed in testis cells, is a more potent transcriptional activator than the HSF2-beta isoform. These results reveal a new mechanism for the control of HSF2 function in mammalian cells, in which regulated alternative splicing is used to modulate HSF2 transcriptional activity in a tissue-dependent manner.

Alternative Splicing↗

PubMed1995

Cis-regulatory elements conferring cyclic 3',5'-adenosine monophosphate responsiveness of the progesterone receptor gene in transfected rat granulosa cells.

We have previously shown that both pituitary gonadotropins and forskolin induce progesterone receptor (PR) messenger RNA expression at the level of transcription in granulosa cells of the rat ovary. To determine the DNA regulatory elements that are important for cAMP-induced transcription of the PR gene in the ovary, we examined the cAMP-induced activity of promoter sequences in rat granulosa cells transfected with various fusion constructs containing PRB promoter sequences linked to the luciferase reporter gene. When cells were transfected with a luciferase fusion construct containing the 1375-base pair 5'-flanking region of the rat PRB gene, forskolin treatment substantially increased luciferase activity. Analysis of a series of 5'-deletion mutants indicated that a minimal PRB promoter containing 116 base pairs of upstream sequence (-116/3) was sufficient to increase luciferase activity in response to forskolin in transfected rat granulosa cells. This promoter contains a consensus CCAAT site in reverse orientation (5'-ATTGG-3') and a consensus GC box (5'-GGGGCGGGCC-3'), but no known cAMP-responsive element. Site-specific mutation of the GC box notably decreased both basal and cAMP-induced activity of this minimal PRB promoter. In addition, site-specific mutation of the CCAAT binding site within this proximal promoter of the PRB gene substantially decreased cAMP-induced activity, but did not significantly affect the basal activity of this promoter. Either mutation alone failed to abolish cAMP inducibility. In contrast, double mutation of both the GC box and the CCAAT box completely abolished cAMP inducibility, suggesting that the GC box and the CCAAT box act together to mediate cAMP-induced transcription of the PRB gene. Gel shift analysis shows that the minimal PRB promoter sequences form multiple complexes with nuclear proteins of granulosa cells, all of which are specifically competed by oligonucleotides containing the GC box and the CCAAT box. Taken together, our results suggest a functional role for transcription factors binding the GC box and the CCAAT box in mediating cAMP-induced transcription of the rat PRB promoter in rat granulosa cells.

Animals↗

PubMed1995

Does the rat corpus luteum express the progesterone receptor gene?

To determine whether the progesterone receptor (PR) gene is expressed in rat corpora lutea (CL), PR messenger RNA (mRNA) and protein levels were examined in CL of both cycling and pregnant rats using in situ hybridization, reverse transcription-polymerase chain reaction, and Western blotting analyses. During the estrous cycle, levels of luteal PR mRNA were below the sensitivity of in situ hybridization. Although no signal could be detected in luteal cells, granulosa cells of preovulatory follicles of the same ovary expressed PR mRNA at high levels during the evening of proestrus. Likewise, CL of pregnant rats expressed undetectable levels of PR mRNA, which remained unchanged throughout pregnancy, as determined by in situ hybridization and reverse transcription-polymerase chain reaction. In addition, no PR protein could be detected in rat CL by Western analysis, indicating that luteal expression of the PR gene, if any, is negligible. To determine whether the lack of PR mRNA in the rat CL is due to progesterone-induced down-regulation of PR mRNA or to low levels of estrogen, aminoglutethimide was used to block the synthesis of progesterone in the presence and absence of exogenous estrogen, and PR mRNA levels were examined in the CL as well as the placenta. Inhibition of progesterone synthesis did increase PR mRNA levels in the placenta, and additional estrogen treatment further increased PR mRNA levels in this tissue. In contrast, neither aminoglutethimide alone nor aminoglutethimide plus estrogen induced PR mRNA expression in CL of the same rats. The temperature-sensitive luteal cell line derived from the rat CL, which produces very low levels of progesterone, also did not express PR mRNA. These results indicate that the rat corpus luteum expresses undetectable levels of PR mRNA and protein, which is not attributable to progesterone-induced down-regulation of PR mRNA or to a lack of estrogen-induced up-regulation of PR mRNA in this tissue.

Aminoglutethimide↗