Biomedical subjects

G Shyamala

Publications and source records attributed to G Shyamala.

At least 37 records · Page 2Linked to original sources

PubMed1995

Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities.

Although progesterone has been recognized as essential for the establishment and maintenance of pregnancy, this steroid hormone has been recently implicated to have a functional role in a number of other reproductive events. The physiological effects of progesterone are mediated by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most cases the PR is induced by estrogen, implying that many of the in vivo effects attributed to progesterone could also be the result of concomitantly administered estrogen. Therefore, to clearly define those physiological events that are specifically attributable to progesterone in vivo, we have generated a mouse model carrying a null mutation of the PR gene using embryonic stem cell/gene targeting techniques. Male and female embryos homozygous for the PR mutation developed normally to adulthood. However, the adult female PR mutant displayed significant defects in all reproductive tissues. These included an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development, and an inability to exhibit sexual behavior. Collectively, these results provide direct support for progesterone's role as a pleiotropic coordinator of diverse reproductive events that together ensure species survival.

Animals↗

PubMed1995

Estrogen dependent expression of heat shock transcription factor: implications for uterine synthesis of heat shock proteins.

Transcriptional induction of heat shock protein genes is generally mediated by binding of heat shock transcription factor(s) to the heat shock element present in the promoters of heat shock genes. Although the steady-state levels of heat shock factor mRNAs vary among different tissues, at present virtually nothing is known regarding the cellular signals responsible for their synthesis and hence the observed variations. In this report we demonstrate that the heat shock transcription factor (HSTF or HSF) is under positive regulation by estrogen. The effect of estrogen was observed with both types of heat shock factors (HSF-1 and HSF-2) and occurred at both the mRNA and protein level. Immunolocalization studies emphasized the potential biological importance of these observations whereby the increase in uterine HSF-1 and HSF-2 due to estrogen was found to be associated with the endometrium, the primary tissue component which is targeted for estrogen action. This is the first demonstration of a cellular factor which can regulate HSF-1 and HSF-2 gene expression. The implications of these findings to uterine heat shock protein gene expression are discussed.

Animals↗

PubMed1994

Essential role of endogenous estrogen in directly stimulating mammary growth demonstrated by implants containing pure antiestrogens.

The mammogenic actions of estrogen, although undisputed, lack definition due to uncertainties concerning the relative importance of systemic vs. local actions of the hormone. In addition to its well known, indirect effects on mammary tissue through pituitary intermediaries such as PRL and GH, recent evidence points to, but does not prove, direct estrogen action on mammary targets. The ability of exogenous estrogen to directly and locally stimulate mammary growth in vivo was previously shown in endocrine-ablated animals using small plastic pellets containing estradiol. The more important question of whether the direct action of endogenous estrogen is required for normal mammary growth and morphogenesis in the endocrine-intact animal is now investigated using direct-acting, slow-release plastic implants containing pure antiestrogens (antiestrogens with no estrogenic properties) inserted into the growth region of mammary glands. Local growth inhibition only in the immediate vicinity of the implants and not in other glands in the same mouse demonstrated the requirement of mammary tissues for endogenous, locally acting estrogen. Local actions of antiestrogens on ducts mimicked the ovariectomy-induced loss of systemic estrogen with respect to time course and morphology, with complete inhibition of ductal growth in 14 days. A second effect, in which locally acting antiestrogens simplified the pattern of ductal branching, was observed in both immature and mature animals. Two distinct mitogenic pathways, one governing ductal elongation and the other ductal maintenance, were thus affected. The inhibitory effects of antiestrogen treatment were fully reversible and not accompanied by obvious cytotoxicity. We conclude from these studies of localized estrogen receptor blockade that with respect to ductal mammogenesis, the action of estrogen is direct (acting at the level of the gland itself) and not primarily through the stimulation of pituitary mammogens.

Animals↗

PubMed1993

Identification of a novel glucocorticoid response element within the genome of the human immunodeficiency virus type 1.

We have shown that (HIV-1) replication can be regulated by interaction between glucocorticoid hormones and the viral genome; treatment of acutely infected lymphoid and monocytoid cell lines with cortisol and dexamethasone increased HIV-1 production in culture. The magnitude of this response correlated with glucocorticoid receptor (GR) and GR message in responder and non-responder cell lines. Furthermore, treatment of each of two HIV-infected cell lines with glucocorticoids led to enhancement of HIV-1 gene expression. We have identified a novel intragenic glucocorticoid response element (GRE) within the genome of HIV-1 at position +5002 in the vif open reading frame, as well as a second potential GRE, previously identified by other researchers at position -257 in the HIV-1 negative regulatory element (LTR-NRE). Our data indicate that only the motif at position +5002 represents a true GRE that confers glucocorticoid inducibility to a MMTV-luciferase reporter gene construct. The GRE located at -257 lacked significant functional activity in its native configuration in that it could bind GR but did not transactivate reporter gene constructs. However, this sequence was able to impart glucocorticoid inducibility when inverted or dimerized. These results suggest that as in the case of other retroviruses, HIV-1 has evolved to interface with the GR signal transduction pathway to gain replicative advantage in target cells.

Animals↗

PubMed1993

Relationship between 90-kilodalton heat shock protein, estrogen receptor, and progesterone receptor in human mammary tumors.

To determine, if, as previously observed with rodent uterus, the 90 kilodalton heat shock protein (HSP-90) might be under estrogenic regulation in human mammary tumors, we analyzed its relationship with estrogen and progesterone receptors. A positive relationship between estrogen receptor and HSP-90 was observed when these were normalized to HSP-70 levels (to normalize for the intrinsic variations in each tumor, independent of any hormonal dependence).

Breast Neoplasms↗

PubMed1992

Activation of kappa B-specific proteins by estradiol.

The kappa B enhancer serves as a recognition site for the nuclear transcription factor NF-kappa B and other kappa B-specific proteins which are activated in many cell types in response to a variety of extracellular signals. But a steroid-dependent activation of NF-kappa B or any other kappa B-specific protein has not previously been reported, to our knowledge. In this report we demonstrate that estrogen can activate kappa B-specific protein in its target tissue, uterus. We have done this by analyzing the interaction of nuclear extracts with kappa B enhancers, using DNA mobility shift assays. The activation by estradiol was time dependent, reaching a maximum at approximately 2 hr after steroid treatment, and was not inhibited by prior cycloheximide treatment. The protein-DNA complexes formed in response to estradiol did not contain NF-kappa B and, when compared with other kappa B enhancer motifs, had a higher affinity to the kappa B enhancer corresponding to the PRDII element present in duplicate motifs. These protein-DNA complexes also did not appear to contain estrogen receptor, since antibodies to estrogen receptor were without any effect on either their formation or their mobility. The protein-DNA complexes formed in response to estradiol, however, exhibited a high affinity for the estrogen-responsive element, suggesting the participation of an estrogen-receptor-like molecule in the DNA binding. In contrast, the protein-DNA complexes formed constitutively contained NF-kappa B, had equivalent affinities to various kappa B enhancers, and did not have a high affinity for the estrogen-responsive element. On the basis of these findings, we propose that estrogen-dependent activation of the as-yet-unidentified kappa B-specific protein involves the association of this protein with an estrogen-receptor-related molecule and binding of the resulting complex to PRDII. The high affinity and specificity of this binding to PRDII suggests that this may serve as a composite regulatory element in mediating estrogen-dependent gene expression. The potential significance of such a mechanism for steroid hormone action is discussed.

Amino Acid Sequence↗

PubMed1992

Estrogen dependent regulation of estrogen receptor gene expression in normal mammary gland and its relationship to estrogenic sensitivity.

In the present study, we have examined the relationship between estradiol (E2)-dependent regulation of estrogen receptor (ER) gene expression in normal mammary glands and its relationship to progesterone receptor (PgR) gene expression using tissues from E2-sensitive and -insensitive states. Estradiol caused a time-dependent decrease in ER mRNA levels in E2-sensitive mammary glands reaching a maximum at approx 6 h, at which time the levels of PgR mRNA also reached a maximum. In contrast, in E2-insensitive mammary glands, there was no E2-dependent decrease in ER mRNA at all times tested. Experiments using dissociated cells revealed that although the epithelial cells of mammary glands from both E2-sensitive and -insensitive states contained ER mRNA, in the intact E2-sensitive mammary glands, it was the nonepithelial ER that was decreasing in response to E2. Since the epithelial cells of normal mammary glands are the primary target for E2-dependent PgR synthesis, our studies suggest that a positive correlation between E2-dependent PgR gene expression and E2-dependent downregulation of ER may simply be coincidental and may not bear any true biological relationship.

Animals↗

PubMed1991

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor.

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.

Amino Acid Sequence↗

PubMed1991

Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form.

Progesterone receptors (PgR) are known to exist in two molecular forms commonly designated as 'A' and 'B' forms, and the relative ratio of these two forms has been shown to vary among species. Although the rodent systems were some of the earliest experimental systems used to examine the regulation of PgR, as yet very little is known concerning the molecular composition of PgR in this species. Accordingly, to define the relative ratio of 'A' and 'B' forms in murine PgR, we have analyzed tissue extracts from normal, ovariectomized, and estradiol treated animals by photoaffinity labeling and immunoblotting techniques using a variety of anti-PgR antibodies. Under all experimental conditions, two forms of PgR with approximate molecular weights of 115 kDa ('B' form) and 83 kDa ('A' form) were found. In all tissues examined, the 83 kDa 'A' form was predominant, and this was independent of the hormonal status of the animal and different buffers used to prepare tissue extracts. In uterus the ratio of 'A' to 'B' was 3:1, in vagina it was 2:1, and in mammary glands it more closely resembled the uterus. This leads us to conclude that murine PgR exists predominantly as the 83-kDa 'A' form which may represent a general characteristic of rodent PgR. In this species there may also be some tissue specificity with regard to the absolute ratio of the two forms of PgR.

Affinity Labels↗

PubMed1990

Developmental regulation of murine mammary progesterone receptor gene expression.

Previously we have shown that in normal murine mammary glands progesterone receptor (PgR) levels are modulated as a function of development and differentiation such that lactating mammary glands do not contain detectable levels of PgR as measured by steroid binding. The objective of the present study was to determine whether the lack of steroid binding in lactating mammary glands was due to the absence of receptor protein and if so whether it was accompanied by an alternation in the pattern of PgR gene expression. Accordingly, we have performed an immunological analysis of murine mammary PgR isolated from different developmental states and have also examined these tissues for PgR gene expression. In mammary tissues from all developmental states other than lactation, immunoreactive PgR corresponding to both A and B forms of the protein were detected. Analysis for PgR mRNA revealed multiple species in mammary tissues and the relative order of abundance of the various transcripts and their sizes were approximately 6.9 greater than 8.7 greater than 3.5 greater than 2.7 greater than 4.2. The 6.9 and 8.7 kilobase transcripts accounted for between 70-80% of total mRNA. All five species of mRNA were detected in tissues from nulliparous mice which decreased dramatically during pregnancy, became undetectable during lactation, and were once again detectable in tissues from mice undergoing lactational involution. Experiments designed specifically to examine the effect of estradiol on mammary PgR mRNA revealed that in contrast to tissues from other developmental states, lactating mammary glands were unable to respond to estradiol with an increase in PgR mRNA. Based on these findings and the fact that estrogenic insensitivity of lactating mammary glands coexists with the presence of ER we propose that in this tissue there is an alteration in the estrogen dependent transcriptional regulation of PgR gene expression.

Affinity Labels↗

PubMed1989

Developmental regulation of murine mammary-gland 90 kDa heat-shock proteins.

We have examined the regulation of murine mammary-gland 90 kDa heat-shock protein (hsp-90) as a function of normal development and differentiation. We find that both hsp-90 and amounts of its mRNA are modulated during development and differentiation, with the highest concentrations of mRNA and protein being present in tissues from pregnant and lactating animals respectively. Metabolic labelling experiments with [35S]methionine reveal that the rate of synthesis of hsp-90 also varies among tissues from various developmental states and correlates with the relative hsp-90 mRNA content. These data also suggest that the highest concentration of hsp-90 found in lactating mammary tissues may be due to a greater stability of this protein in this developmental state. The possible significance of the developmental modulation of mammary hsp-90 to mammary steroid-receptor properties is discussed.

Animals↗

PubMed1989

Transferrin receptor and ferritin levels during murine mammary gland development.

Various types of proliferating cell are known to express transferrin receptors which are necessary for transferrin-mediated cellular iron uptake. Neither the mechanism nor the physiological role of transferrin receptor induction has been established with certainty; although it may reflect an increased cellular requirement for iron which is essential for ribonucleotide reductase, a key enzyme of DNA synthesis. The aim of this study was to examine murine mammary gland transferrin-receptor levels during gland development. As compared to virgin controls, total mammary gland transferrin receptors expressed on the basis of DNA, increase during pregnancy and lactation by 29- and 45-fold, respectively. However, on the basis of DNA, mammary gland ferritin, measured by radioimmunoassay, decreased by about 75% and 85% during pregnancy and lactation, respectively, indicating that the increased transferrin receptor levels probably do not lead to intracellular iron accumulation. When epithelial cells from mammary glands of pregnant mice were cultured in vitro transferrin receptor expression correlated with cell proliferation. These results suggest that normal mammary growth which occurs mainly in mammary epithelial cells is associated with a significant increase in transferrin receptor. Since transferrin receptor levels remain high during lactation they are not associated solely with tissue growth, but may also function in transporting iron during milk production.

Animals↗

PubMed1989

Estrogenic regulation of murine uterine 90-kilodalton heat shock protein gene expression.

Murine uterine steady-state protein levels of the 90-kilodalton heat shock protein (HSP90) have been demonstrated recently to be increased by estrogen in a target tissue- and steroid-specific manner (C. Ramachandran, M.G. Catelli, W. Schneider, and G. Shyamala, Endocrinology 123:956-961, 1988). We now report that this regulation occurred with both the HSP86 and HSP84 forms of HSP90 as well as with the 94-kilodalton glucose-regulated protein. At the mRNA level, this response was greatest for HSP86 (15-fold). In contrast, estradiol had no significant effect on HSP70.

Animals↗

PubMed1988

Immunocytochemical study of progesterone receptors in hyperplastic and neoplastic endometrial tissues.

Endometrial carcinoma is the most common genital malignancy in North America. However its pathogenesis, in particular its relationship with hyperplasia is not clear. To understand steroid hormonal interactions in the genesis and growth of human endometrial hyperplasia and carcinoma, we have assayed progesterone receptors in hyperplasia and neoplastic human endometrium by immunocytochemistry. The presence of progesterone receptors in target tissues is known to be a marker of both estrogen and progesterone action. The receptors were identified in fresh-frozen sections using a mouse monoclonal antiprogesterone receptor antibody (alpha PR6). The progesterone receptor content was high in the epithelium of hyperplasia without cytological atypia and low in the epithelium of endometrial intraepithelial neoplasia (hyperplasia with cytological atypia). In carcinomas there was a heterogenous distribution of progesterone receptors in the epithelium but low as compared to hyperplastic endometria without cytological atypia. The stroma contained relatively high progesterone receptor levels irrespective of whether the epithelium was hyperplastic or neoplastic.

Adult↗

PubMed1988

Interaction of murine progesterone receptors with specific monoclonal antibodies to the avian progesterone receptor.

Monoclonal antibodies raised against purified chicken progesterone receptor (PgR) have been described and characterized recently. In this study we have screened these antibodies for cross-reactivity with murine PgR. Of the six anti-PgR antibodies tested, one (alpha PR6) precipitates murine PgR in an assay using protein A-sepharose as an absorbent for the antibody. The antibody is specific for PgR and does not react with the estrogen receptor or the glucocorticoid receptor in the same cytosol. In immunoblot experiments, both alpha PR6 and alpha PR11 recognize a 115,000 Da protein, however, alpha PR11 gives a weaker signal than alpha PR6. In photoaffinity labeling experiments, a 115,000 Da and an 83,000 Da protein covalently bind tritiated R5020 in a receptor-specific way. We conclude that the alpha PR6 antibody can be used as a tool to study the structure and function of the murine PgR.

Affinity Labels↗

PubMed1988

Effects of progesterone, promegestone and RU 486 on glucocorticoid receptor levels in primary cultures of mouse mammary epithelial cells.

Mammary epithelial cells isolated from midpregnant mice and cultured on collagen gels contain glucocorticoid receptors whose levels are modulated by a variety of steroids. In the absence of any added steroid to the cell culture medium, the levels of glucocorticoid receptors in the cells decline during culture, which is counteracted by the addition of a variety of glucocorticoid agonists. The effectiveness of the glucocorticoid in preventing the loss of glucocorticoid receptors is in turn counteracted by the addition of the synthetic progestin promegestone and the synthetic antiglucocorticoid RU 486. Of the two, RU 486 is the most potent in antagonizing the effect of cortisol on the GR levels. Promegestone antagonizes the effect of cortisol, too, although higher concentrations are necessary. Progesterone was without a clear effect either as a glucocorticoid agonist or an antagonist. Progesterone, however, was extensively metabolized by mammary epithelial cells in culture. Based on these observations we conclude that in mammary epithelial cells glucocorticoids positively regulate the metabolism of their own receptors and that antiglucocorticoids, such as RU 486 and progestins, can antagonize that effect.

Animals↗

PubMed1988

Estrogenic regulation of uterine 90-kilodalton heat shock protein.

Recently two lines of evidence have implicated that cellular heat shock proteins (hsp) may play a role in steroid hormonal regulation of target tissues. One is the demonstration that cellular 90K hsp (hsp-90) can complex with steroid receptors in vitro and inhibit their ability to interact with DNA, and second, the demonstration that in avian oviduct sex steroids can regulate the synthesis of hsp-108. As yet, there is no report that sex steroids can regulate hsp-90 synthesis, especially in mammalian tissues. In these studies we have examined the estrogenic regulation of murine uterine hsp-90. We report that ovariectomy reduces the uterine concentration of hsp-90, and estradiol causes a time-dependent increase in uterine hsp-90 as early as 4 h after steroid administration, reaching a maximum increase of 4-fold between 18-24 h. The effect is specific to estrogens and not elicited by other steroid hormones. It is also target tissue specific, such that it is seen with uterus and vagina and does not occur in nontarget tissue for estradiol, such as spleen. The possible physiological significance of estrogenic stimulation of uterine hsp-90 has been discussed.

Androgens↗

PubMed1988

Immunocytochemical study of progesterone receptors in the human endometrium during the menstrual cycle.

Specific mouse monoclonal antibody (alpha PR6) against progesterone receptor was used with an avidin biotin complex technique to localize progesterone receptors in frozen sections of 26 normal cyclic human endometria. Progesterone receptor was detected in the nuclei of epithelial and stromal cells in both the functionalis and basalis layers. In the functionalis, the receptor content increased from the early to the late proliferative phase in both cell components. It remained high in the early secretory phase and decreased in the mid- and late secretory phases, comparatively more rapidly in the epithelium than in the stroma. In the latter, the predecidual cell nuclei were receptor-positive. The menstrual phase endometrium lacked receptors. The basalis was rich in progesterone receptors during the proliferative, early and midsecretory phases in both components and receptor-free during the late secretory and menstrual phases of the cycle. Myometrial smooth muscle cell nuclei contained progesterone receptors, whereas they were absent in endometrial and myometrial vessels. Overall, the epithelial progesterone receptor content seemed to correlate with the endometrial tissue levels of estradiol, possibly reflecting its estrogen sensitivity, whereas the stromal progesterone receptor content during the secretory phase at least, in part, may be constitutively synthetized.

Adult↗