ABSTRACT
The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.
Subject(s)
Dexamethasone/metabolism , Drug Resistance/genetics , Gene Deletion , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/metabolism , Adrenalectomy , Animals , Blotting, Western , Cell Death/drug effects , Cell Extracts , Dexamethasone/pharmacology , Enzyme Induction/drug effects , Glucocorticoids/metabolism , Glucocorticoids/pharmacology , Glucose-6-Phosphatase/genetics , Hepatocytes/drug effects , Hepatocytes/enzymology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclease Protection Assays , Phenotype , Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Glucocorticoid/genetics , Thymus Gland/cytology , Thymus Gland/drug effectsABSTRACT
A role for glucocorticoids in thymopoiesis has been suggested by studies using glucocorticoid receptor (GR) anti-sense transgenic mice, glucocorticoid synthesis inhibitors and GR antagonists. Unfortunately, no consensus has been reached on exactly how glucocorticoids influence T-cell development. The most recent approach, using GR knockout (GR(-/-)) mice, indicates that GR signaling is, in fact, dispensable in this entire process.
Subject(s)
Glucocorticoids/physiology , Stress, Physiological/immunology , T-Lymphocyte Subsets/immunology , Aminoglutethimide/pharmacology , Animals , Cell Differentiation/drug effects , Clonal Deletion/drug effects , DNA, Antisense/genetics , Glucocorticoids/antagonists & inhibitors , Glucocorticoids/deficiency , Glucocorticoids/genetics , Hematopoiesis/drug effects , Hormone Antagonists/pharmacology , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Lymphocyte Activation/drug effects , Metyrapone/pharmacology , Mice , Mice, Knockout , Mice, Transgenic , Mifepristone/pharmacology , Models, Biological , Receptors, Glucocorticoid/deficiency , Receptors, Glucocorticoid/drug effects , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/physiology , Stress, Physiological/physiopathology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/drug effects , Thymus Gland/embryology , Thymus Gland/immunologyABSTRACT
Glucocorticoids are believed to play a role in T cell development and selection, although their precise function is controversial. Glucocorticoid receptor (GR)-deficient mice were used to directly investigate this problem. GR-deficient thymocytes were resistant to dexamethasone-mediated apoptosis, confirming the absence of glucocorticoid responsiveness. An absence of GR signaling had no impact on thymocyte development either in vivo or in vitro. T cell differentiation, including positive selection, was normal as assessed by normal development of CD4+CD8+, alphabetaTCR+CD4+, and alphabetaTCR+CD8+ thymocytes. Negative selection, mediated by the superantigen staphylococcal enterotoxin B (SEB), or anti-CD3/CD28, was also normal in the absence of GR signaling. In contrast to earlier reports, these data demonstrate that GR signaling is not essential for intrathymic T cell development or selection.
