Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2 Sagar Sengupta1 and Bohdan Wasylyk2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch cedex, France The glucocorticoid receptor (GR) and the tumor supressor p53 mediate different stress responses. We have studied the mechanism of their mutual inhibition in normal endothelial cells (HUVEC) in response to hypoxia, a physiological stress, and mitomycin C, which damages DNA. Dexamethasone (Dex) stimulates the degradation of endogenous GR and p53 by the proteasome pathway in HUVEC under hypoxia and mitomycin C treatments, and also in hepatoma cells (HepG2) under normoxia. Dex inhibits the functions of p53 (apoptosis, Bax, and p21WAF1/CIP1 expression) and GR (PEPCK and G-6-Pase expression). Endogenous p53 and GR form a ligand-dependent trimeric complex with Hdm2 in the cytoplasm. Disruption of the p53–HDM2 interaction prevents Dex-induced ubiquitylation of GR and p53. The ubiquitylation of GR requires p53, the interaction of p53 with Hdm2, and E3 ligase activity of Hdm2. These results provide a mechanistic basis for GR and p53 acting as opposing forces in the decision between cell death and survival. [Key Words: Endothelial cells; hypoxia; Bax; p21WAF1/CIP1; PEPCK; G6Pase] Received March 5, 2001; revised version accepted July 26, 2001. The development of most tumors is associated with loss p53levels can also be maintained by ARF, which blocks of function of the tumor suppressor p53. Different physi- nucleo-cytoplasmic shuttling of Mdm2 (Sherr and Weber ological stress, including DNA damage and hypoxia, ac- 2000). Most studies of p53involve DNA damaging drugs tivate p53. p53 is a transcription factor that regulates and radiation. Much less is known about the physiologi- genes involved in growth arrest and apoptosis. p53is cal stress, hypoxia. Under hypoxic conditions, p53is sta- down-regulated by its target gene product Mdm2 (human bilized by mitochondria through a redox-dependent homolog Hdm2). Mdm2 forms an autoregulatory loop mechanism (Chandel et al. 2000) and by HIF-1␣ (An et al. with p53by binding to its N-terminal domain, inhibiting 1998), whereas p53induces degradation of HIF-1␣ (Ravi its transcriptional activity and increasing its degradation et al. 2000). The response to hypoxia in vivo also in- by the ubiquitin proteasome pathway (for reviews, see volves the glucocorticoid receptor (GR) (Bauer et al. Jimenez et al. 1999; Lakin and Jackson 1999; Sionov and 1999). Haupt 1999). Mdm2 is a RING finger-dependent ubiqui- GR is a member of the steroid receptor superfamily tin protein ligase for p53and itself (Argentini et al. 2000; that mediates physiological processes controlled by glu- Fang et al. 2000; Honda and Yasuda 2000). Mdm2 also cocorticoids. In the unbound state GR is located in the inhibits p53by nuclear export through a mechanism in- cytoplasm bound to chaperones. Upon ligand binding the volving either the nuclear export signal (NES) of Mdm2 chaperones dissociate, exposing the NLS that enables (Tao and Levine 1999) or the RING finger of Mdm2 and GR to enter the nucleus and regulate transcription the NES of p53(Boyd et al. 2000; Geyer et al. 2000). The (Cheung and Smith 2000). GR activates transcription by NES of p53is masked in the transcriptionally active het- binding to glucocorticoid response elements (GREs), and erodimer, but is exposed in the monomeric form of p53 represses by binding to negative and composite elements (Stommel et al. 1999). MdmX, a Mdm2 homolog that (Kellendonk et al. 1999). GR mediates stress responses, lacks a NES, stabilizes p53by retention in the nucleus and animals that overexpress GR are more resistant to (Jackson and Berberich 2000; Stad et al. 2000). Nuclear stress and endotoxic shock (Reichardt et al. 2000). Recently, evidence has been growing for cross talk be- tween the p53and GR-mediated responses to stress. p53 1 Present address: Laboratory of Human Carcinogenesis, Building 37, physically interacts with and represses the activities of Room 2C23, 37 Convent Drive, National Cancer Institute, NIH Bethesda, MD 20892, USA. GR (Sengupta et al. 2000b). Modulation of glucocorti- 2Corresponding author. coid-induced apoptosis has been linked to p53gene dos- E-MAIL [email protected]; FAX 33-3-88-65-32-01. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ age in mice (Mori et al. 1999). Glucocorticoids prevent gad.202201. p53-induced apoptosis in immortalized human granulo- GENES & DEVELOPMENT15:2367–2380 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org 2367 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Sengupta and Wasylyk sa cells (Sasson et al. 2001). p53and GR regulate in op- ited around threefold by endogenous GR activated with posite manners the type II hexokinase gene, whose over- Dex, but was not affected by RU486 (Fig. 1D, lanes 7–12). expression is associated with increased glucose catabo- With a constant level of CTS1, exogenous GR inhibited lism in cancer cells (Mathupala et al. 1997). Hypoxia CTS1 activity to a small extent (Fig. 1D, lanes 13–16), leads to p53activation and decreased glucose metabo- and the addition of Dex increased this inhibition (Fig. lism (Riva et al. 1998). Because little is known about the 1D, lanes 17–19). In contrast, GR, in the presence of functional interactions between the signalling pathways RU486, had little effect (Fig. 1D, lanes 20–22). Similar mediated by p53and GR under physiological conditions, results were obtained in Saos-2 cells (data not shown). we have investigated how p53and GR regulate each These results indicate that the region of p53that inter- other under hypoxia. We show that they mutually in- acts with GR in vitro is sufficient for efficient repression hibit each other’s activity by cytoplasmic sequestration by GR in vivo. in a ligand-dependent manner, which leads to increased degradation through recruitment of the E3ubiquitin li- gase Hdm2 and the proteasome pathway. Dex decreases p53 and GR levels and inhibits expression of their target genes in HepG2 cells Results Cytoplasmic p53in neuroblastoma cells is released from its complex with GR and translocates to the nucleus Dexamethasone enhances the interaction of GR following treatment with GR antagonists (Sengupta et with the core domain and NLS of p53 al. 2000b). We investigated whether cytoplasmic p53in other tumor cell types, such as HepG2 (hepatoma) We have reported previously that p53and GR interact in and SA1 (osteosarcoma) (Wasylyk et al. 1999; Lu et al. vivo (Sengupta et al. 2000b). We used in vitro GST pull- 2000), behaved similarly. Under normal growth con- down assays to determine which region of p53interacts ditions, Dex increased the amount of p53and GR in with GR (Fig. 1A, left). The GST fusion proteins were the cytoplasm of both cell lines, although some p53 produced in Escherichia coli, purified on Glutathione always remained nuclear. In contrast, RU486 signifi- Sepharose beads, and equivalent amounts of protein cantly increased the amount of nuclear p53(data not (judged by Coomassie blue staining, Fig. 1B), were used. shown). Interestingly, in HepG2 cells, Dex caused a Full-length p53interacted with GR weakly but specifi- drastic decrease in the protein levels of both p53and GR cally in the absence of ligand (Fig. 1A,C, lanes 3and 6). (Fig. 2A, lanes 1,2). This effect was blocked by the pro- This interaction was enhanced by the agonists Dexa- teasome inhibitor LLnL (Fig. 2A, lanes 3,4), suggesting methasone (Dex) or Cortisol (Cort), and inhibited by the that the decrease is due to enhanced degradation. We antagonist RU486 (Fig. 1C, lanes 2–6). These results investigated whether Dex can inhibit the expression of show that the binding of GR to p53is enhanced by ago- genes downstream from both p53(Bax, Hdm2, and nists in vitro. In the presence of ligand, GR interacted p21WAF1/CIP1) and GR (phospho enol pyruvate carboxy with both the core domain [GST–p53(76–304)] and the kinase, PEPCK; Imai et al. 1990). Dex decreased Bax and NLS [GST–p53(305–320)] of p53, and more strongly with p21WAF1/CIP1 protein levels (Fig. 2B) and promoter activ- a mutant containing both regions [GST–p53(76–320)]. ity (reporter assays; data not shown). Dex did not affect No interaction was detected between GR and either the either the level of Hdm2 protein (Fig. 2B), or the activity N-terminal [GST–p53(1–75)] or C-terminal [GST– of the promoter (reporter assay, data not shown), show- p53(321–362) and GST–p53(363–393)] domains. The mu- ing that Hdm2 expression is less sensitive than Bax or tant containing both interaction domains [GST–p53(76– p21WAF1/CIP1 to changes in p53levels. Dex decreased 320)] interacted more strongly than the full-length pro- PEPCK protein levels (Fig. 2B). These results show that tein, raising the possibility that it lacks sequences that Dex down-regulates both p53and GR protein levels and inhibit the interaction with GR. The interactions be- the expression of their downstream targets in HepG2 tween GR and deletion mutants containing the NLS cells. [GST–p53(76–320) and GST–p53(305–320)] were inhib- ited by a peptide containing the NLS (amino acids 305– 320, Pep, Fig. 1C, lanes 7–11), confirming that the GR– Dex activation of endogenous GR inhibits p53 p53NLS interaction is specific. induction by hypoxia in normal endothelial GR interacts with the central region of p53, suggesting cells (HUVEC) that it should inhibit the Chimeric Tumor Supressor, CTS1, that harbors this domain of p53linked to heter- HepG2 is a tumor cell line with undefined genetic alter- ologous activation and dimerization domains (Conseiller ations.