Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Abstract

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a XcDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.Glucocorticoids induce cytolysis in susceptible lymphocyte populations, including immature thymocytes and certain leukemias and lymphomas. Steroid-induced cell death is thought to occur through the activation of an endogenous suicide process. Glucocorticoid treatment of murine lymphocytes results in severe alterations in cellular metabolism which include an inhibition of glucose transport (50), increased RNA (14) and protein (44) degradation, a rise in intracellular calcium levels (37), and decreased incorporation of thymidine into DNA (9). Glucocorticoid-induced lymphocytolysis is preceded by cell cycle arrest (28) and extreme morphological changes. These include widespread chromatin condensation, which is associated with extensive DNA fragmentation in both human and murine lymphocytes (22, 58). The DNA cleavage appears to result from the activation of a preexisting calcium-dependent endonuclease (17, 59). Glucocorticoid-induced DNA fragmentation and cell death are prevented when RNA and protein syntheses are inhibited (17). In addition, there is genetic evidence in mouse and human cells for the existence of a locus involved in cell lysis (25,26,63 (21,32,53,62). The possibility thus exists that both glucocorticoids and cAMP regulate the expression of genes involved in the lytic process.Glucocorticoids and cAMP are known to regulate the levels of several proteins in lymphocytes. For example, glucocorticoids induce glucocortin in rat thymus (18), glutamine synthetase in th...

Interaction of NPY compounds with the rat glucocorticoid-induced receptor (GIR) reveals similarity to the NPY–Y2 receptor

“…GIR was originally identified as a stress-responsive element from the murine T-cell line WEHI-7TG and normal thymocytes treated with glucocorticoids and forskolin [3,10]. Mouse GIR mRNA was detected at high levels in the brain and thymus, and its distribution in brain was found to be enriched in the dorsal and ventral striatum, the forebrain limbic structures and the hypothalamic nuclei [22].…”

Fkbp51, a Novel T-Cell-Specific Immunophilin Capable of Calcineurin Inhibition

“…The construction of oligo(dT)-primed and randomly primed ZAPII cDNA libraries, using RNA from WEHI-7TG cells treated for 5 h with dexamethasone (1 M) and forskolin (12 M), and the subtractive hybridization procedure used to identify induced genes have been described elsewhere (17). The pBluescript SK(-) plasmid containing cDNA encoding FKBP51 (clone 213) was excised from the ZAPII vector by using an in vivo excision protocol (Stratagene).…”

“…The pBluescript SK(-) plasmid containing cDNA encoding FKBP51 (clone 213) was excised from the ZAPII vector by using an in vivo excision protocol (Stratagene). An approximately 500-nucleotide fragment from the 5Ј end of the 2.2-kb cDNA insert of clone 213 was isolated by restriction enzyme digestion with EcoRI and XhoI, labeled to 10 9 cpm/g with the Multiprime DNA-labeling kit (Amersham), and used as a hybridization probe for library screening as previously described (17). Two libraries, a murine thymus ZAP cDNA library (Stratagene) and a UniZAPII cDNA library prepared from poly(A) ϩ RNA isolated from WEHI-7TG cells treated with dexamethasone (1 M) and forskolin (12 M) for various time intervals (0 min, 15 min, 30 min, 1 h, 2 h, or 5 h), were screened to obtain full-length cDNAs corresponding to clone 213.…”

Transcriptional activation of mouse retrotransposons in vivo: specific expression in steroidogenic cells in response to trophic hormones.

“…This is reflected in the repeated incidental selection of VL30 cDNA clones in substructive screening procedures aimed at selecting inducible genes. Thus, VL30 elements were selected as genes in which expression is tumed on after epidermal growth factor {EGFI stimulation of quiescent mouse embryo cells in culture {Foster et al 1982}; VL30 cDNAs were selected as growth-specific elements expressed in SV40-transformed, nonconfluent cells but not in the confluent, contact-inhibited parental cells {Singh et al 1985); VL30 cDNAs were picked up in a selective procedure aimed at identifying genes in lymphoid cells activated by glucocorticoids (Harrigan et al 1989); VL30 elements were shown to be activated by chemical carcinogens {Hsieh et al 1987), whereas other studies have demonstrated a dramatic (500-fold) induction of VL30 expression under conditions of anoxia {Anderson et al 1989).…”