PMID: 1917967;Abstract:
Glucocorticoid treatment of certain lymphoma cell lines and thymocytes activates a self-destructive pathway of programmed cell death referred to as apoptosis. Calcium and calmodulin (CaM) may be important signals in the apoptotic cascade because an early event is a sustained elevation in cytosolic Ca2+ and CaM inhibitors interfere with the death pathway. In the present study, expression of the CaM gene was examined during glucocorticoid-induced apoptosis in WEHI7.2 lymphocytes. Steady state levels of CaM mRNA were increased up to 10-fold following a 4-6-h exposure of WEHI7.2 cells to 10-6 M dexamethasone. This increase was mediated through the glucocorticoid receptor since the response was not observed in WEHI7.418, a variant line which does not express active glucocorticoid receptor. Induction of CaM mRNA was dose-dependent and highly specific for glucocorticoids, as other steroids were unable to elicit the response. A stringent cell specificity was also observed. Pretreatment of WEHI7.2 lymphocytes with cycloheximide did not interfere with dexamethasone-dependent increases in CaM mRNA levels, and studies with actinomycin D demonstrated that the stability of the transcript was not altered by hormone. Finally, a calmodulin inhibitor elicited a protective effect on WEHI7.2 cells following glucocorticoid exposure. These results indicate that CaM mRNA levels were hormonally controlled in WEHI7.2 lymphocytes and support the putative involvement of CaM in glucocorticoid-induced apoptosis.
PMID: 11572317;Abstract:
Androgen receptor (AR) and glucocorticoid receptor (GR) influence distinct physiologic responses in steroid-responsive cells despite their shared ability to selectively bind in vitro to the same canonical DNA sequence (TGTTCT). While the DNA-binding domains (DBDs) of these receptors are highly conserved, the amino N-terminal domain (NTD) and hormone-binding domain (HBD) are evolutionarily divergent. To determine the relative contribution of these functional domains to steroid-specific effects in vivo, we constructed a panel of AR/GR gene fusions by interchanging the NTD, DBD, and HBD regions of each receptor and measured transcriptional regulatory activities in transfected kidney and prostate cell lines. We found that GR was approximately 10-fold more active than AR when tested with the mouse mammary tumor virus promoter, and that this difference in activity was primarily owing to sequence divergence in the NTDs. We also tested transcriptional activation of the androgen-dependent rat probasin promoter, and in this case, AR was at least twofold more active than GR. Analysis of the chimeric receptors revealed that this difference mapped to the DBD region of the two receptors. Transcriptional repression functions of the wild-type and chimeric receptors were measured using an activator protein 1 (AP-1) transrepression assay and identified the GR HBD as a more potent transrepressor of AP-1 transcriptional activation than the AR HBD. Taken together, our analyses reveal that evolutionary sequence divergence between AR and GR functional domains results in unique promoter-specific activities within biologic systems in which both AR and GR are normally expressed.
PMID: 6775816;Abstract:
When a cloned 6 kb Eco RI-Sal I fragment of mouse ribosomal gene nontranscribed spacer DNA (rDNA NTS) was used to screen a BALB/c mouse gene library, 25% of the recombinant phage hybridized with it. In situ hybridization experiments and characterization of 12 clones selected using this probe supported the idea that sequences homologous to this rDNA NTS region are scattered throughout the genome. Subsequently, sequences homologous to mouse rDNA NTS were found flanking mouse μ, α and γ2b immunoglobulin C(H) genes. One region was localized 3' to the μ coding sequence, an area which has been identified as an intervening sequence between the secreted C(μ) heavy chain terminus and the C terminal portion of the membrane-bound C(μ) heavy chain.
PMID: 6095060;PMCID: PMC368912;
It has been proposed that cell-specific responses to steroid action are the result of coordinate expression of steroid gene networks. Using three different cell systems, we have performed transcriptional analyses to determine if the observed hormone-induced alterations in gene expression are consistent with a limited number of potential target genes in any one cell type. Our results indicate that greater than 95% of the transcripts in dexamethasone-treated rat hepatoma (HTC), or mouse lymphoma (WEH17) cells, are similar to the mRNAs in untreated cells based on subtraction hybridization. In addition, we find that although the castration-induced expression of androgen-regulated transcripts in the rat ventral prostate (RVP) is significantly different between normal and castrated rats (19%), the majority of these mRNAs are accounted for by the over abundance of sulfated glycoprotein-2 sequences. Specifically, analysis of an RVP subtracted cDNA library revealed that sulfated glycoprotein-2 mRNA masked the presence of less abundant differentially expressed sequences, confirming that the actual size of the RVP androgen gene network is small. We conclude that steroid-mediated changes in transcription accurately reflect the expression of a few cell-specific target genes, and thus support the model of steroid gene networks. The potential to characterize key elements which determine both the time course and magnitude of cell-specific hormone responses is discussed.
