29 research outputs found
Investigation of differential TNFĪ±-induced interleukin-6 gene regulation by synthesis progestins medroxyprogesterone acetate (MPA) and norethindrone acetate (NET-A) in human endocervical epithelial cells and the role of the unliganded glucocorticoid receptor
Includes bibliographical references (pages 127-171).The endocervical mucosae of the female reproductive tract (FRT) not only serve as a physical barrier against microbial infection, but they also express a wide variety of immune mediators. The endocervical epithelial cells express a distinct profile of immune-regulators, which is higher than vaginal and ectocervical epithelial cells. Constant cytokine production would ensure rapid responses to infections and maintenance of the sterility of the upper genital tract. However, overproduction of cytokines could inhibit normal reproductive processes and stimulate excess growth and cell proliferation. The synthetic progestins medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NET-A)) are synthetic steroidal hormones designed to elicit progestational effects similar to those of the endogenous hormone progesterone (P4). They are extensively used as contraceptives and in hormone replacement therapy (HRT). Numerous studies, however, have reported that synthetic progestins affect immune function, increase the risk of sexually transmitted infections (STIs) and also change the morphology of the cervicovaginal mucosa. Despite these findings little is known about the molecular mechanisms of action of MPA and NET, in particular their differential effects on gene expression
Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols
The original publication is available at http://pubs.acs.org/Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERĪ² but not via ERĪ±. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERĪ². HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species. Ā© 2007 American Chemical Society.Publishers' versio
Novel role for receptor dimerization in post-translational processing and turnover of the GRĪ±
CITATION: Wilkinson, L., Verhoog, N. & Louw, A. 2018. Novel role for receptor dimerization in post-translational processing and turnover of the GRĪ±. Scientific Reports, 8:14266, doi:10.1038/s41598-018-32440-z.The original publication is available at https://www.nature.comGlucocorticoids (GCs), acting via the glucocorticoid receptor (GRĪ±), remain the mainstay therapeutic
choice for the treatment of inflammation. However, chronic GC use, aside from generating undesirable
side-effects, results in GRĪ± down-regulation, often coupled to a decrease in GC-responsiveness, which
may culminate in acquired GC resistance. The current study presents evidence for a novel role of the
dimerization state of the GRĪ± in mediating GC-mediated GRĪ± turnover. Through comparing the effects
of dimerization promoting GCs on down-regulation of a transfected human wild type GRĪ± (hGRwt)
or a dimerization deficient GRĪ± mutant (hGRdim), we established that a loss of receptor dimerization
restricts GRĪ± turnover, which was supported by the use of the dimerization abrogating Compound A
(CpdA), in cells containing endogenous GRĪ±. Moreover, we showed that the dimerization state of the
GRĪ± influenced the post-translational processing of the receptor, specifically hyper-phosphorylation
at Ser404, which influenced the interaction of GRĪ± with the E3 ligase, FBXW7Ī±, thus hampering
receptor turnover via the proteasome. Lastly, the restorative effects of CpdA on the GRĪ± pool, in the
presence of Dex, were demonstrated in a combinatorial treatment protocol. These results expand our
understanding of factors that contribute to GC-resistance and may be exploited clinically.https://www.nature.com/articles/s41598-018-32440-zPublisher's versio
Disease- and treatment-associated acquired glucocorticoid resistance
The development of resistance to glucocorticoids (GCs) in therapeutic regimens poses a major threat. Generally, GC resistance is congenital or acquired over time as a result of disease progression, prolonged GC treatment or, in some cases, both. Essentially, disruptions in the function and/or pool of the glucocorticoid receptor Ī± (GRĪ±) underlie this resistance. Many studies have detailed how alterations in GRĪ± function lead to diminished GC sensitivity; however, the current review highlights the wealth of data concerning reductions in the GRĪ± pool, mediated by disease-associated and treatment-associated effects, which contribute to a significant decrease in GC sensitivity. Additionally, the current understanding of the molecular mechanisms involved in driving reductions in the GRĪ± pool is discussed. After highlighting the importance of maintaining the level of the GRĪ± pool to combat GC resistance, we present current strategies and argue that future strategies to prevent GC resistance should involve biased ligands with a predisposition for reduced GR dimerization, a strategy originally proposed as the SEMOGRAMāSEDIGRAM concept to reduce the side-effect profile of GCs
Inhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPĪ²
Corticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPĪ², able to tether to the GR, as well as HNF3Ī± involved in GR signaling, are present. C/EBPĪ², but not HNF3Ī±, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg . Furthermore, knockdown of C/EBPĪ² protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPĪ²ās involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPĪ² and GR to the Cbg promoter, while C/EBPĪ² knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPĪ²
The injectable-only contraceptive medroxyprogesterone acetate, unlike norethisterone acetate and progesterone, regulates inflammatory genes in endocervical cells via the glucocorticoid receptor
Clinical studies suggest that the injectable contraceptive medroxyprogesterone acetate (MPA) increases susceptibility to infections such as HIV-1, unlike the injectable contraceptive norethisterone enanthate (NET-EN). We investigated the differential effects, molecular mechanism of action and steroid receptor involvement in gene expression by MPA as compared to NET and progesterone (P4) in the End1/E6E7 cell line model for the endocervical epithelium, a key point of entry for pathogens in the female genital mucosa. MPA, unlike NET-acetate (NET-A) and P4, increases mRNA expression of the anti-inflammatory GILZ and IĪŗBĪ± genes. Similarly, MPA unlike NET-A, decreases mRNA expression of the pro-inflammatory IL-6, IL-8 and RANTES genes, and IL-6 and IL-8 protein levels. The predominant steroid receptor expressed in the End1/E6E7 and primary endocervical epithelial cells is the glucocorticoid receptor (GR), and GR knockdown experiments show that the anti-inflammatory effects of MPA are mediated by the GR. Chromatin-immunoprecipitation results suggest that MPA, unlike NET-A and P4, represses pro-inflammatory cytokine gene expression in cervical epithelial cells via a mechanism involving recruitment of the GR to cytokine gene promoters, like the GR agonist dexamethasone. This is at least in part consistent with direct effects on transcription, without a requirement for new protein synthesis. Dose response analysis shows that MPA has a potency of ā¼24 nM for transactivation of the anti-inflammatory GILZ gene and ā¼4-20 nM for repression of the pro-inflammatory genes, suggesting that these effects are likely to be relevant at injectable contraceptive doses of MPA. These findings suggest that in the context of the genital mucosa, these GR-mediated glucocorticoid-like effects of MPA in cervical epithelial cells are likely to play a critical role in discriminating between the effects on inflammation caused by different progestins and P4 and hence susceptibility to genital infections, given the predominant expression of the GR in primary endocervical epithelial cells
Evaluation of the phytoestrogenic activity of honeybush (Cyclopia)
Thesis (MSc (Biochemistry))--University of Stellenbosch, 2006.The phytoestrogenic activity of Cyclopia, used to prepare honeybush tea, was evaluated and compared with that of the endogenous estrogen, 17-Ī²-estradiol (E2) and the known phytoestrogen, genistein. Phytoestrogens are plant polyphenols much in demand in the nutraceutical market as they mediate an estrogenic effect through binding to estrogen receptor (ER) subtypes, ERĪ± and ERĪ²
Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols
The original publication is available at http://pubs.acs.org/Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERĪ² but not via ERĪ±. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERĪ². HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species. Ā© 2007 American Chemical Society.Publishers' versio
Acquired Glucocorticoid Resistance Due to Homologous Glucocorticoid Receptor Downregulation: A Modern Look at an Age-Old Problem
For over 70 years, the unique anti-inflammatory properties of glucocorticoids (GCs), which mediate their effects via the ligand-activated transcription factor, the glucocorticoid receptor alpha (GRĪ±), have allowed for the use of these steroid hormones in the treatment of various autoimmune and inflammatory-linked diseases. However, aside from the onset of severe side-effects, chronic GC therapy often leads to the ligand-mediated downregulation of the GRĪ± which, in turn, leads to a decrease in GC sensitivity, and effectively, the development of acquired GC resistance. Although the ligand-mediated downregulation of GRĪ± is well documented, the precise factors which influence this process are not well understood and, thus, the development of an acquired GC resistance presents an ever-increasing challenge to the pharmaceutical industry. Recently, however, studies have correlated the dimerization status of the GRĪ± with its ligand-mediated downregulation. Therefore, the current review will be discussing the major role-players in the homologous downregulation of the GRĪ± pool, with a specific focus on previously reported GC-mediated reductions in GRĪ± mRNA and protein levels, the molecular mechanisms through which the GRĪ± functional pool is maintained and the possible impact of receptor conformation on GC-mediated GRĪ± downregulation