The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200-to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/ C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.The glucocorticoid receptor (GR) is a member of a family of ligand-dependent transcription factors capable of both positive and negative regulation of gene expression (4, 51, 69). In its unactivated form, the GR is part of a large heteromeric complex which includes hsp90 (18,35,45,61,67) and hsp56 (FKBP52) (42,57,61,63,81). Binding of the agonist stimulates receptor activation, dissociation from hsp90 (17, 66), and nuclear translocation, prerequisites for both transactivation and transrepression. For transactivation, specific in vitro binding of the activated GR to the glucocorticoid response element does not appear to be ligand dependent (22,79). However, in the absence of bound ligand, the nuclear form of the GR is a poor activator of target genes (52,65). This suggests that a ligandinduced conformational change, comparable to that inferred to be necessary for activation of the progesterone (2) and estrogen (5) receptors, is required for efficient activation (or derepression) of the transcriptional activating function present in the GR ligand-binding domain (34, 78) and/or for interaction of the receptor with other components of the transcription apparatus. Direct evidence for such a change has been provided by Simons et al., who demonstrated that affinity-labeled GR was less sensitive to proteolytic digestion than the unoccupied GR (74).Several mechanisms for GR-mediated transrepression ha...

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Summary.  Background: Comparisons of platelet RNAs could provide crucial information on platelet function, thrombopoiesis and the etiology of megakaryocyte (MK) or platelet disorders.Objectives: We developed a method for stringent purification of platelets from small blood samples from single donors. Purity of the platelet preparations was verified by an RT‐PCR assay. We tested three methods to identify the differences in RNA between platelet sources.Methods: Differential hybridization to cDNA macro‐arrays and suppressive–subtractive hybridization PCR (SSH‐PCR) were used to compare RNAs from normal platelets to those from a Bernard–Soulier syndrome (BSS) patient. Affymetrix GeneChip U133 plus 2.0 arrays were used to compare male and female platelet RNAs.Results: Macroarrays identified ∼7500 platelet transcripts, but failed to identify differentially expressed transcripts with confidence. SSH‐PCR produced libraries almost exclusively of mitochondrial‐derived transcripts, but included nuclear‐encoded genes that could not be confirmed by immunoblotting of normal and BSS platelet lysates. The Affymetrix platform gave reproducible profiles from our small‐scale purified platelet preparations, whereas a partially purified platelet preparation produced a drastically skewed transcript profile. The microarray analysis identified the heparanase precursor transcript as overexpressed in female platelets, and we observed variable yet consistently higher levels of heparanase protein in female platelets compared with male platelets in four independent donor pairs.Conclusions: This demonstrates for the first time that differential platelet transcript levels can identify changes in expression level of platelet proteins. Combined with our small‐scale platelet preparation method, this establishes a system to compare platelets from the limited clinical sources to help elucidate molecular bases for platelet or megakaryocyte pathologies.

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