Retinoids enhance glucocorticoid-induced apoptosis of T cells by facilitating glucocorticoid receptor-mediated transcription

Abstract

Glucocorticoid-induced apoptosis of thymocytes is one of the first recognized forms of programmed cell death. It was shown to require gene activation induced by the glucocorticoid receptor (GR) translocated into the nucleus following ligand binding. In addition, the necessity of the glucocorticoid-induced, but transcription-independent phosphorylation of phosphatidylinositol-specific phospholipase C (PI-PLC) has also been shown. Here we report that retinoic acids, physiological ligands for the nuclear retinoid receptors, enhance glucocorticoid-induced death of mouse thymocytes both in vitro and in vivo. The effect is mediated by retinoic acid receptor (RAR) alpha/retinoid X receptor (RXR) heterodimers, and occurs when both RARα and RXR are ligated by retinoic acids. We show that the ligated RARα/RXR interacts with the ligated GR, resulting in an enhanced transcriptional activity of the GR. The mechanism through which this interaction promotes GR-mediated transcription does not require DNA binding of the retinoid receptors and does not alter the phosphorylation status of Ser232, known to regulate the transcriptional activity of GR. Phosphorylation of PI-PLC was not affected. Besides thymocytes, retinoids also promoted glucocorticoid-induced apoptosis of various T-cell lines, suggesting that they could be used in the therapy of glucocorticoid-sensitive T-cell malignancies.

Figures

Figure 1

Retinoids promote glucocorticoid-induced apoptosis of thymocytes by RARα/RXR. (a) Dexamethasone acetate induces DNA fragmentation in mouse thymocytes in a dose-dependent manner detected at 6 h following addition. (b) The RARα agonists ATRA, 9cRA, Am580 and CD2081, the RXR agonist LG268, a combination of ATRA with LG268 (0.1 nM), and an RARα antagonist CD2503 all promote dexamethasone (0.1 μM)-induced DNA fragmentation of mouse thymocytes. The amount of DNA degradation in the presence of glucocorticoid alone was 45±4%. (c) The RARγ agonists CD437, CD666 and CD2325 alone induce DNA fragmentation, whereas compounds acting on RXR or RARα do not alter the basal DNA fragmentation (8±3%) of thymocytes detected at 6 h following addition of the retinoids. (d) The RXR agonists 9cRA and LG268 can, but the RARα agonists are unable to promote significantly the dexamethasone acetate (0.1 μM)-induced DNA fragmentation of RARα knockout mouse thymocytes. The amount of DNA degradation in the presence of glucocorticoid alone was 45±4%. Data represent mean±S.D. of three determinations. (e) 9cRA, AM580, CD2503 and LG268 all promote glucocorticoid-induced apoptosis of thymocytes in vitro detected by propidium iodide/Annexin V labeling. (f) Injection of both Am580 (50 μg) and LG268 (50 μg) significantly enhances dexamethasone (0.2 mg)-induced CD4+CD8+ thymocyte apoptosis in vivo determined at 24 h following treatment. Data (one representative experiment out of three) show the number of surviving thymocytes and the distribution of various thymocyte cell populations following in vivo treatments. (g) Glucocorticoid-induced PI-PLC activation is not enhanced by retinoids in mouse thymocytes. Tyrosine-phosphorylated proteins were immunoprecipitated (IP) from cell lysates with agarose-conjugated 4G-10 antibodies, and PI-PLC in the immunoprecipitate was assessed by western blot with anti-PI-PLC antibodies. The results are representative of one of three independent experiments

Figure 2

Retinoids enhance glucocorticoid-induced expression of GILZ in mouse thymocytes. (a) Retinoids do not affect the basal levels of glucocorticoid receptors. Isolated thymocytes (107 cells per ml) were exposed to 0.1 μM dexamethasone acetate alone or together with 0.3 μM ATRA, 9cRA, Am580, or CD2503 for 2 h. Levels of the glucocorticoid receptors were determined by immunoblot analysis. β-Actin was used as loading control. (b–d) Retinoids enhance the glucocorticoid-induced expression of GILZ in a dose-dependent manner. Isolated mouse thymocytes were exposed to 0.1 μM dexamethasone acetate and the indicated concentrations of retinoids. mRNA levels of GILZ were determined 2 h later. Data represent mean±S.D. of three determinations. *Significantly different from glucocorticoid-treated control determined by Student's paired t-test (P<0.05)

Figure 3

RARα/RXR heterodimers mediate the transactivating effects of retinoids. (a) Dexamethasone acetate induces the expression of the pCMX-GRE-luc reporter construct in a dose-dependent manner in COS1 cells transfected transiently. Effect of increasing concentrations of ATRA (b), 9cRA (c), LG268 (d), AM580 (e) and CD2503 (f) on the dexamethasone (0.1 μM)-induced expression of the pCMX-GRE-luc reporter in the presence of the indicated full-length retinoid receptors. (g) Western blot analysis of retinoid receptor expression before and after transient transfections of COS-1 cells. In comparison, the endogenous level of retinoid receptors in the IG3T cell line is also shown. (h) Effect of the combination of AM580 and LG268 on the dexamethasone (0.1 μM)-induced expression of the pCMX-GRE-luc reporter in the presence of the full-length RARα/RXRα receptors. (i) Effect of the indicated concentrations of 9cRA, LG268 and AM580 on the dexamethasone (0.1 μM)-induced expression of the pCMX-GRE-luc reporter in the presence of retinoid receptors not capable of DNA binding. Data represent mean±S.D. of three independent experiments. *Significantly different from glucocorticoid-treated control determined by Student's paired t-test (P<0.05)

Figure 4

The ligated retinoid receptors directly interact with the ligated GR. (a) RARα co-immunoprecipitates with GR only when the ligand of both receptors is present. GR was immunoprecipitated from cell lysates with agarose-conjugated anti-GR antibodies, and GR and RARα in the immunoprecipitate was assessed by western blot with anti-GR and anti-RARα antibodies. The dose of glucocorticoid was 0.1 μM, that of retinoids 0.3 μM, except for LG268, which was 100 nM. The results are representative of one of three independent experiments. (b) Mammalian two-hybrid system reveals a direct interaction between GR and RARα. (c) Mammalian ttwo-hybrid mammalian system reveals a direct interaction between GR and RXRα, which is influenced by the presence of the receptor ligands

Figure 5

Glucocorticoid-induced Ser232 phosphorylation of GR is not affected by retinoids. Thymocytes were exposed to 0.1 μM dexamethasone acetate alone or with 0.3 μM Am580, 0.3 μM CD2503 or 50 nM LG268 for 1 h. GR was immunoprecipitated from cell lysates with agarose-conjugated anti-GR antibodies, and GR and its phosphorylated Ser232 in the immunoprecipitate was assessed by western blot by using specific antibodies (Cell Signaling Antibody No. 4161)

Figure 6

Retinoids enhance glucocorticoid-induced apoptosis of various T-cell lines as well. 9cRA enhances dexamethasone acetate (0.1 μM)-induced apoptosis of (a) IP-12-7T hybridoma, (b) IG3 and (c) CCRF-CEM T cells in a dose-dependent manner determined at 6 h following treatment. (d) RARα and RXRα ligands also enhance dexamethasone acetate (0.1 μM)-induced apoptosis in CCRF-CEM T cells. Data represent mean±S.D. of three determinations. *Significantly different from glucocorticoid-treated control determined by Student's paired t-test (P<0.05)