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• W2090137967 abstract "The glucocorticoid and mineralocorticoid receptors (GR and MR) share considerable structural and functional homology and bind as homodimers to hormone-response elements. We have shown previously that MR and GR can form heterodimers that inhibit transcription from a glucocorticoid (GC)-responsive gene and that this inhibition was mediated by the N-terminal domain (NTD) of MR. In this report, we examined the effect of NTD-MR on GC-induced apoptosis in the GC-sensitive pre-B lymphoma cell line, 697. In GC-treated 697 cells, we demonstrated that stable expression of NTD-MR blocks apoptosis and inhibits proteolytic processing of pro-caspases-3, -8, and -9 and poly(ADP-ribose) polymerase. Importantly, gel shift and immunoprecipitation analyses revealed a direct association between the GR and amino acids 203–603 of NTD-MR. We observed down-regulation of c-Myc and of the anti-apoptotic proteins Bcl-2 and Bfl-1 as well as high levels of the pro-apoptotic proteins Bax and Bid. Conversely, cells stably expressing NTD-MR exhibited increased expression of Bcl-2 and Bfl-1 and diminished levels of Bid and Bax. These data provide a potential mechanism for the observed inhibition of cytochromec and Smac release from the mitochondria of NTD-MR cells and resultant resistance to GC-induced apoptosis. Thus, NTD-MR may mediate GC effects through heterodimerization with GR and ensuing inhibition of GC-regulated gene transcription. The glucocorticoid and mineralocorticoid receptors (GR and MR) share considerable structural and functional homology and bind as homodimers to hormone-response elements. We have shown previously that MR and GR can form heterodimers that inhibit transcription from a glucocorticoid (GC)-responsive gene and that this inhibition was mediated by the N-terminal domain (NTD) of MR. In this report, we examined the effect of NTD-MR on GC-induced apoptosis in the GC-sensitive pre-B lymphoma cell line, 697. In GC-treated 697 cells, we demonstrated that stable expression of NTD-MR blocks apoptosis and inhibits proteolytic processing of pro-caspases-3, -8, and -9 and poly(ADP-ribose) polymerase. Importantly, gel shift and immunoprecipitation analyses revealed a direct association between the GR and amino acids 203–603 of NTD-MR. We observed down-regulation of c-Myc and of the anti-apoptotic proteins Bcl-2 and Bfl-1 as well as high levels of the pro-apoptotic proteins Bax and Bid. Conversely, cells stably expressing NTD-MR exhibited increased expression of Bcl-2 and Bfl-1 and diminished levels of Bid and Bax. These data provide a potential mechanism for the observed inhibition of cytochromec and Smac release from the mitochondria of NTD-MR cells and resultant resistance to GC-induced apoptosis. Thus, NTD-MR may mediate GC effects through heterodimerization with GR and ensuing inhibition of GC-regulated gene transcription. glucocorticoid receptor cytochrome c glyceraldehyde-3-phosphate dehydrogenase glucocorticoid(s) glucocorticoid-response element inhibitor of apoptosis protein monoclonal antibody mineralocorticoid receptor normal goat serum N-terminal domain N-terminal domain of MR polyclonal antibody poly(ADP-ribose) polymerase phosphate buffered saline triamcinolone acetonide wild type electrophoretic mobility shift analysis terminal dUTP nick-end labeling reverse transcriptase The glucocorticoid and the mineralocorticoid receptors (GR1 and MR) are closely related members of the steroid receptor superfamily, with high sequence homology within the DNA binding and ligand binding domains (1Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1632) Google Scholar). These receptors function as ligand-activated transcription factors that control various aspects of metabolic homeostasis, embryonic development, and physiological stress by binding to specific hormone-response elements in the regulatory regions of target genes (2Perlmann T. Evans R.M. Cell. 1997; 90: 391-397Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In vitro, both the GR and the MR bind to and are activated by the physiological glucocorticoid (GC), cortisol. Although both receptors, when activated, can bind and transactivate glucocorticoid-response elements (GREs) in the promoters of target genes, each has distinct transcriptional activities (3Arriza J.L. Simerly R.B. Swanson L.W. Evans R.M. Neuron. 1988; 1: 887-900Abstract Full Text PDF PubMed Scopus (510) Google Scholar, 4Alnemri E.S. Maksymowych A.B. Robertson N.M. Litwack G. J. Biol. Chem. 1991; 266: 18072-18081Abstract Full Text PDF PubMed Google Scholar). For instance, the GR, but not the MR, self-synergizes at multimerized hormone-response elements (5Liu W. Wang J. Sauter N.K. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12480-12484Crossref PubMed Scopus (203) Google Scholar, 6Rupprecht R. Arriza J.L. Spengler D. Reul J.M. Evans R.M. Holsboer F. Damm K. Mol. Endocrinol. 1993; 7: 597-603Crossref PubMed Scopus (120) Google Scholar), and only the GR can inhibit induction of AP-1-dependent genes by Fos-Jun heterodimers (7Pearce D. Yamamoto K.R. Science. 1993; 259: 1161-1165Crossref PubMed Scopus (397) Google Scholar). In addition, the ability to mediate apoptosis in susceptible cells is exclusive to the GR (8Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4138) Google Scholar).Induction of apoptosis by GCs occurs in numerous cell types including immature lymphocytes and various malignancies of lymphoid origin (9Thompson E.B. Mol. Endocrinol. 1994; 8: 665-673Crossref PubMed Scopus (155) Google Scholar,10Evans-Storms R.B. Cidlowski J.A. J. Steroid Biochem. Mol. Biol. 1995; 53: 1-8Crossref PubMed Scopus (79) Google Scholar). Evidence that the GR is essential for this process has been provided by studies using GR antagonists, such as RU486, that completely block GC-induced cell death and by experiments involving GR knockout mice (11Thompson E.B. Thulasi R. Saeed M.F. Johnson B.H. Ann. N. Y. Acad. Sci. 1995; 761: 261-275Crossref PubMed Scopus (22) Google Scholar, 12Caron-Leslie L.A. Cidlowski J.A. Mol. Endocrinol. 1991; 5: 1169-1179Crossref PubMed Scopus (72) Google Scholar, 13Tronche F. Kellendonk C. Reichardt H.M. Schutz G. Curr. Opin. Genet. & Dev. 1998; 8: 532-538Crossref PubMed Scopus (156) Google Scholar). The effects of mutations in the GR on its ability to cause apoptosis vary among cell lines. In human CEM and Jurkat lymphoid cells, the DNA binding domain and the ligand binding domain of the GR are essential for GC-induced cell death. Specifically, mutations that deleted either of the zinc fingers of the DNA binding domain or substituted amino acids in critical sites within the N-terminal zinc finger completely blocked the lethal response (14Harbour D.V. Chambon P. Thompson E.B. J. Steroid Biochem. 1990; 35: 1-9Crossref PubMed Scopus (29) Google Scholar). Ligand binding domain mutations also inhibited cell death demonstrating a requirement for hormone binding (15Nazareth L.V. Harbour D.V. Thompson E.B. J. Biol. Chem. 1991; 266: 12976-12980Abstract Full Text PDF PubMed Google Scholar). GR mutants lacking the N-terminal transactivation domain did not prevent apoptosis in these cell lines; however, in S49 mouse lymphoma cells, this region was essential for steroid-induced lethality (16Dieken E.S. Meisfeld R.L. Mol. Cell. Biol. 1992; 12: 589-597Crossref PubMed Scopus (106) Google Scholar).Differences in the transcriptional activities of the GR and the MR, including the ability to mediate apoptosis, may be attributed to structural variability within the N-terminal domain (NTD). This region contains a transactivation function, AF-1, which is involved in the transcriptional transactivation of genes, in receptor heterodimerization, and in binding to other transcription factors (17Hollenberg S.M. Evans R.M. Cell. 1988; 55: 899-906Abstract Full Text PDF PubMed Scopus (545) Google Scholar). Moreover, in the absence of a ligand binding domain, this region is constitutively active. The NTDs of GR and MR share only 15% sequence homology and have been shown to have opposite transactivation properties (1Arriza J.L. Weinberger C. Cerelli G. Glaser T.M. Handelin B.L. Housman D.E. Evans R.M. Science. 1987; 237: 268-275Crossref PubMed Scopus (1632) Google Scholar). Specifically, chimeric receptor analyses demonstrated that this region of the MR is inhibitory for GR-mediated regulation of the Na/K-ATPase β1 gene promoter when MR and GR are coexpressed (18Derfoul A. Robertson N.M. Hall D.J. Litwack G. Endocrine. 2000; 13: 287-295Crossref PubMed Scopus (14) Google Scholar). In addition, several laboratories (5Liu W. Wang J. Sauter N.K. Pearce D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12480-12484Crossref PubMed Scopus (203) Google Scholar, 18Derfoul A. Robertson N.M. Hall D.J. Litwack G. Endocrine. 2000; 13: 287-295Crossref PubMed Scopus (14) Google Scholar, 19Trapp T. Rupprecht R. Castren M. Reul J.M. Holsboer F. Neuron. 1994; 13: 1457-1462Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 20Ou X.M. Storring J.M. Kushwaha N. Albert P.R. J. Biol. Chem. 2001; 276: 14299-14307Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) have shown that GR and MR can form heterodimers capable of inhibiting transcription from a GC-responsive gene.Although the functional consequences of MR/GR heterodimerization are not well understood, the possible physiological significance stems from the observed co-localization of the receptors in various tissues and cells including the brain, heart, vascular smooth muscle, and leukocytes (21Trapp T. Holsboer F. Trends Pharmacol. Sci. 1996; 17: 145-149Abstract Full Text PDF PubMed Scopus (130) Google Scholar, 22Zennaro M.C. Farman N. Bonvalet J.P. Lombes M. J. Clin. Endocrinol. Metab. 1997; 82: 1345-1352Crossref PubMed Google Scholar, 23Wickert L. Watzka M. Bolkenius U. Bidlingmaier F. Ludwig M. Eur. J. Endocrinol. 1998; 138: 702-704Crossref PubMed Scopus (21) Google Scholar, 24van Steensel B. van Binnendijk E.P. Hornsby C.D. van der Voort H.T. Krozowski Z.S. de Kloet E.R. van Driel R. J. Cell Sci. 1996; 109: 787-792Crossref PubMed Google Scholar). For example, in the brain, the MR is involved in the excitability of neurons, whereas the GR opposes these effects. Moreover, in the dentate gyrus of the hippocampus, there is evidence that MR activation can protect neurons against acute GR ligand-mediated apoptosis (25Hassan A.H. von Rosenstiel P. Patchev V.K. Holsboer F. Almeida O.F. Exp. Neurol. 1996; 140: 43-52Crossref PubMed Scopus (162) Google Scholar). A recent study has also demonstrated that heterodimerization of MR and GR mediates direct corticosteroid-induced transrepression of the 5-HT1A receptor promoter (20Ou X.M. Storring J.M. Kushwaha N. Albert P.R. J. Biol. Chem. 2001; 276: 14299-14307Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). These studies have prompted us to examine whether the MR can mediate or abrogate apoptotic cell death in the GC-sensitive pre-B lymphoma cell line, 697.RESULTSWe have demonstrated previously (18Derfoul A. Robertson N.M. Hall D.J. Litwack G. Endocrine. 2000; 13: 287-295Crossref PubMed Scopus (14) Google Scholar, 29Derfoul A. Robertson N.M. Lingrel J.B. Hall D.J. Litwack G. J. Biol. Chem. 1998; 273: 20702-20711Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) that the NTD of MR is inhibitory for GR-mediated gene transcriptional regulation. To examine the ability of this region to modulate GR-induced apoptosis, the NTD of MR (NTD-MR) composed of amino acids 1–516, or the NTD of GR (NTD-GR), encompassing amino acids 1–421, was stably expressed in 697 cells. These cells provide a model system for this study because we have shown previously (30Robertson N.M. Zangrilli J. Fernandes-Alnemri T. Friesen P.D. Litwack G. Alnemri E.S. Cancer Res. 1997; 57: 43-47PubMed Google Scholar, 31Alnemri E.S. Fernandes T.F. Haldar S. Croce C.M. Litwack G. Cancer Res. 1992; 52: 491-495PubMed Google Scholar) that they express GR, and that they are exquisitely sensitive to GC-induced cell death. More importantly, immunoblot and RT-PCR analyses of 697 whole cell extracts and of RNA, respectively, have demonstrated that these cells do not express MR (data not shown). As shown in Fig. 1, expression of the NTD-MR/c-Myc or NTD-GR/c-Myc fusion proteins was evaluated by Western blot analysis using a c-Myc mAb (Fig. 1). The predicted 59-kDa recombinant NTD-MR protein was detected in 5 of 6 clones examined (Fig.1 A, lanes 1–6), although no c-Myc-tagged NTD-MR was expressed in the cells transfected with the control vector (lane 7). Expression of the 51-kDa recombinant NTD-GR protein was also detected in stably transfected cells (Fig.1 C, lanes 1–6). Following evidence of expression, the clones with the highest level of NTD-MR (MR-H7 or -A2) or NTD-GR (GR-D6) were used for further experimentation.NTD-MR Inhibits Glucocorticoid-induced Apoptosis in 697 CellsTo examine the effects of NTD-MR or NTD-GR on the kinetics of GC-induced cell death, cells were treated with 1 μm TA for a 48-h time course. Fig. 1 D shows that 697 cells with vector and NTD-GR cells exhibit declining viability within the first 24 h of treatment, with complete cell death occurring by 48 h. Importantly, stable transfection of the NTD-MR in these cells resulted in a strong inhibition of GC-induced cell death (Fig.1 B). To confirm that overexpression of the MR N terminus protects 697 cells from GC-induced apoptosis, DNA isolated from cells at the indicated time points following treatment with 1 μm TA (0, 24, and 48 h) was examined for nuclear fragmentation using the TUNEL assay. As shown in Fig.2, 697 cells with vector, treated with TA for 24 h, exhibited the typical morphologic changes associated with apoptosis such as shrinkage and nuclear blebbing, compared with untreated cells (Fig. 2, A and B). Moreover, the nuclei of these cells exhibited fluorescence indicating the presence of the labeled 3′-OH ends characteristic of apoptotic cells. In contrast, cells stably expressing NTD-MR, which had been treated with TA for the same time period, retained their viability (Fig. 2, C andD).Figure 2Stable expression of NTD-MR inhibits GC-induced apoptosis in 697 cells. 697 cells with vector or NTD-MR (MR-H7) were incubated in the presence or absence of 1 μm TA. At the 24-h time point, aliquots of cell suspensions were processed using the TUNEL assay (green) and counterstained with 4,6-diamidino-2-phenylindole (blue) to detect changes in nuclear morphology.View Large Image Figure ViewerDownload (PPT)NTD-MR Does Not Affect GR Expression or Nuclear TranslocationTo explore the mechanism of inhibition of GC-induced apoptosis by NTD-MR at the receptor level, we first examined GR protein levels by Western blot and GR subcellular localization by immunofluorescence. To compare GR protein levels in 697 and MR-H7 cells, whole cell extracts were prepared from cells harvested at 0, 3, 8, 12, 18, 24, 36, and 48 h of treatment with 1 μmTA. As shown in Fig. 3 A, overexpression of NTD-MR did not alter endogenous levels of GR protein, which remained steady over the 48-h time course and comparable with those levels detected in 697 cells. Immunocytochemical analysis in Fig.3 B revealed that endogenous GR expression in cells stably transfected with NTD-MR (MR-H7) is cytoplasmic. Following treatment of cells with 1 μm TA for 1 h, the GR was detected primarily in the nucleus. These data suggest that the expression of NTD-MR in 697 cells does not prevent translocation of the GR into the nucleus upon hormone binding.Figure 3Subcellular localization and expression levels of endogenous GR in MR-H7 cells treated with GC. A, 697 cells with vector or NTD-MR were treated with 1 μm TA over a 48-h time course, harvested at designated time points, and subjected to SDS-PAGE followed by immunoblotting with a GR pAb. Accuracy of protein loading and transfer was confirmed by stripping and reprobing with a β-actin pAb (Santa Cruz Biotechnology).B, untreated MR-H7 cells (T 0) or cells treated with 1 μm TA for 1 h (T 1) were processed for immunofluorescence using a GR pAb and confocal microscopy.View Large Image Figure ViewerDownload (PPT)NTD-MR Does Not Affect the Ability of GR to Bind DNATo determine whether the expression of NTD-MR interferes with the ability of GR to bind DNA, EMSA was performed using a GR/MR-specific response element as a probe and nuclear extracts from 697 cells with vector or MR-H7 cells treated for an hour with 1 μm TA. In extracts of 697 cells, we detected protein binding to the β1 MRE/GRE corresponding to endogenous GR (Fig.4 A, lane 1). This binding was reduced when a GR antibody was included in the gel shift reaction (Fig. 4 A, lane 2) and was unaffected in the presence of nonspecific serum (Fig. 4 A,lane 3) or c-Myc mAb (Fig. 4 A,lane 4). In extracts of MR-H7 cells, binding of endogenous GR to the β1 MRE/GRE was similar to that seen for 697 cells (Fig. 4 B,lanes 1 and 2). To determine whether the protein-DNA complexes contained the N-terminal MR, we used two different c-Myc mAbs and MR pAbs. In all cases, the protein-DNA complexes were either ablated or shifted to a higher position when these antibodies were used (Fig. 4 B, lanes 3–6) but remained unchanged when a preimmune serum was included in the reaction (Fig.4 B,lane 7). These results indicate that the N terminus of MR is present with the GR in the protein-DNA complex, suggesting the possibility of MR-GR heterodimer formation.Figure 4NTD-MR physically associates with endogenous GR. EMSA was performed using a GR/MR-specific response element as a probe and nuclear extracts from 697 cells with vector or NTD-MR (MR-H7) treated with 1 μm TA for 1 h. A,GR pAb (α GR) or c-Myc mAb (αmyc) was incubated with nuclear extracts 10 min prior to the addition of probe (lanes 2 and 4, respectively). Lane 1contained a buffer control and lane 3 a preimmune nonspecific serum (NSS). The asterisk denotes GR-specific binding, and the arrow indicates the antibody-mediated shifts on DNA-protein complexes. Ab, antibody. B, αMR (lane 4) or αhMRsN (lane 6) pAb was incubated with nuclear extracts 10 min prior to the addition of probe. Similarly, a c-Myc mAb (αmyc) from Invitrogen or from CN Biosciences was incubated with samples in lanes 3 and 5, respectively. Lane 1 contained a buffer control, lane 2 αGR, and lane 7 a preimmune nonspecific serum (NSS). C, schematic representation of NTD-MR derivatives 103, 203, 303, 403, MR-H7, and 603 cloned into the pCMV/myc/nuc vector. D, 697 cells with vector, NTD-MR, or stably expressed NTD-MR derivatives 103, 203, 303, 403, and 603 were treated with 1 μm TA and harvested after 3 h. Cellular lysates were subjected to immunoprecipitation with c-Myc mAb and analyzed by Western blot using a GR pAb (top) or a c-Myc mAb (bottom) as a control for the amount of immunoprecipitated protein. In lane 3, lysate from cells transfected with full-length GR served as a positive control.View Large Image Figure ViewerDownload (PPT)Interaction between GR and Amino Acids 203–603 of NTD-MRWe have shown previously (18Derfoul A. Robertson N.M. Hall D.J. Litwack G. Endocrine. 2000; 13: 287-295Crossref PubMed Scopus (14) Google Scholar, 29Derfoul A. Robertson N.M. Lingrel J.B. Hall D.J. Litwack G. J. Biol. Chem. 1998; 273: 20702-20711Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) that when co-expressed, the MR and the GR are able to form heterodimers. To determine whether the inhibitory effects of NTD-MR on GR function are the result of a direct interaction with the GR, we performed immunoprecipitation analysis. 697 cells stably expressing NTD-MR (MR-H7) or NTD derivatives (1–103, 1–203, 1–303, 1–403, and 1–603) (Fig. 4 C) were treated with 1 μm TA for 3 h. Cell lysates were subjected to immunoprecipitation using a c-Myc mAb. As shown in Fig. 4 D(top), Western blot analysis with a GR pAb demonstrated that endogenous GR physically associates with the NTD of the MR. Specifically, this interaction occurred within the N-terminal region encompassing amino acids 203–603 (lanes 5–9), because no binding was detected with the deletion derivative 1–103 (lane 4). As a control for the amount of immunoprecipitated protein, the membrane was stripped and successively reprobed with a c-Myc mAb (bottom).Effect of NTD-MR on GR Transcriptional ActivityTo test the effect of GR/NTD-MR heterodimers on GR transcriptional activity in 697 and MR-H7 cells, we evaluated the effect of TA on GR-mediated regulation of the endogenous c-myc gene. Suppression of c-Myc mRNA has been reported previously (31Alnemri E.S. Fernandes T.F. Haldar S. Croce C.M. Litwack G. Cancer Res. 1992; 52: 491-495PubMed Google Scholar) when GR-sensitive cells were treated with GC. To examine the expression of c-Myc at the mRNA level, 697 cells with vector or NTD-MR were cultured in the presence of 1 μm TA for 48 h, and RNA was prepared from cells harvested at time 0, 4, 16, 24, and 48 h. As determined by semi-quantitative RT-PCR, treatment of 697 cells with GC caused a 7-fold repression of c-Myc transcription by 24 h as compared with untreated cells (time 0) (Fig.5 A). This repression was blocked by NTD-MR in MR-H7 cells as shown in Fig. 5 A. GAPDH levels were unaffected by TA treatment in both cell lines. These data were confirmed by Northern blot analysis using a c-Myc-specific cDNA probe in Fig. 5 B, which shows that expression of c-Myc mRNA was dramatically reduced in 697 cells treated with TA after 4 h. Within the period of the experiment, the level of the c-Myc mRNA reached its lowest point at 24 h after TA treatment. However, in the MR-H7 cells, c-Myc mRNA levels remained constant throughout the time course.Figure 5Effect of TA on the expression of c-Myc mRNA in 697 and MR-H7 cells. A, gel electrophoresis of semi-quantitative RT-PCR using 1 μg of total cellular RNA from 697 cells with vector or NTD-MR (MR-H7) harvested at indicated time points throughout a 48-h incubation with 1 μm TA. GAPDH was included as an internal control. Fold induction (FI) of c-Myc mRNA expression is indicated.B, total cellular RNA was isolated from 697 cells with vector (lanes 1–5) or NTD-MR (MR-H7) (lanes 6–10) at 0–48 h after TA (1 μm) treatment. Equal amounts of RNA (15 μg/lane) were fractionated on an agarose-formaldehyde gel (top) and transferred by blotting onto Hybond-N nylon membranes as described under “Experimental Procedures.” The c-Myc mRNA was identified by hybridization using a 1-kb 32P-labeled human c-Myc probe (bottom). The locations of the 28 S and 18 S rRNAs are indicated. The periods of TA treatment (h) are indicated at the top of each lane.View Large Image Figure ViewerDownload (PPT)NTD-MR Inhibits Proteolytic Processing of Caspases and PARPA common end point to apoptosis is a network of caspases whose activation is required for the irreversible commitment to cell death. To determine whether NTD-MR expression inhibits caspase activation in 697 cells, enzymatic cleavage of endogenous caspases during GC-induced apoptosis was assessed. 697 cells stably expressing NTD-MR (MR-H7) or vector alone were cultured in the presence or absence of 1 μm TA during a 48-h time course, and whole cell lysates were examined by Western blot analysis. As shown in Fig.6 A, proteolytic processing of the proform of the apical caspases-8 (55 kDa) and -9 (46 kDa) and the downstream effector caspase-3 (32 kDa) was observed in 697 cells treated with TA. In contrast, in MR-H7 cells treated with TA, the inactive proenzyme forms of caspases-8, -9, and -3 remained intact, revealing that GC-induced caspase cleavage was abolished. Further evidence of this inhibition was seen when a downstream target of activated caspase-3, PARP, was examined for enzymatic degradation. Immunoblot analysis in Fig. 6 B shows that following treatment of 697 cells with TA, the “death substrate” PARP (116 kDa) was proteolyzed with generation of its signature 85-kDa cleavage product, although in MR-H7 cells, PARP cleavage was inhibited.Figure 6Stable expression of NTD-MR inhibits caspase activation and PARP cleavage during GC-induced apoptosis. Western blot analysis of caspase-8, caspase-9, caspase-3, and PARP status after treatment with TA. A, 697 cells with vector or NTD-MR (MR-H7) were treated with 1 μm TA for a 48-h time course. Samples containing whole cell extracts from 1 × 106 cells were subjected to SDS-PAGE followed by immunoblotting with a mAb to caspase-8 and pAbs to caspase-9 and active caspase-3. B, for PARP analysis, samples (50 μg) were electrophoresed on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose membrane, and probed with anti-PARP mAb. Molecular mass markers are indicated (kDa).View Large Image Figure ViewerDownload (PPT)Effect of NTD-MR on Bcl-2 Family MembersIt has been shown that GC-induced apoptosis is regulated by members of the Bcl-2 family upstream of caspase activation (32Brunet C.L. Gunby R.H. Benson R.S. Hickman J.A. Watson A.J. Brady G. Cell Death Differ. 1998; 5: 107-115Crossref PubMed Scopus (110) Google Scholar, 33Hakem A. Sasaki T. Kozieradzki I. Penninger J.M. J. Exp. Med. 1999; 189: 957-968Crossref PubMed Scopus (94) Google Scholar, 34Feinman R. Koury J. Thames M. Barlogie B. Epstein J. Siegel D.S. Blood. 1999; 93: 3044-3052Crossref PubMed Google Scholar). To address potential mechanisms involved in N-terminal MR inhibition of GC-induced apoptosis, we evaluated the expression levels of the anti-apoptotic proteins, Bcl-2 and Bfl-1. As determined by semi-quantitative RT-PCR, treatment of 697 cells with 1 μm TA caused an 18-fold repression of Bcl-2 transcription by 16 h; however, in MR-H7 cells, Bcl-2 mRNA levels increased nearly 2-fold over the 48-h time course (Fig. 7 A). We also observed a 6-fold down-regulation of Bfl-1 mRNA in 697 cells within 4 h of treatment, whereas Bfl-1 mRNA expression in MR-H7 cells was up-regulated 8-fold (Fig. 7 A). These results were corroborated by Western blot analyses of cytosolic and mitochondrial extracts. Following GC treatment of 697 cells, mitochondrial levels of Bcl-2 were decreased, although cytosolic levels increased slightly (Fig. 7 B). In contrast, mitochondrial Bcl-2 levels were dramatically increased in MR-H7 cells (Fig. 7 B). Furthermore, mitochondrial and cytosolic Bfl-1 expression levels were non-detectable in 697 cells following GC treatment, whereas in MR-H7 cells, the expression levels of Bfl-1 in the mitochondrial fraction increased slightly at 24 h and in the cytosolic fraction were increased at 48 h (Fig. 7 B).Figure 7Effect of GC on Bcl-2 and Bfl-1 mRNA expression and protein levels in 697 and MR-H7 cells. A, gel electrophoresis of semi-quantitative RT-PCR using 1 μg of total cellular RNA from 697 cells with vector or (NTD-MR) MR-H7 harvested at indicated time points throughout a 48-h incubation with 1 μm TA. RT-PCR was performed as described under “Experimental Procedures” using the following primer pairs: Bcl-2 forward, 5′-ATGGCGCACGCTGGGAGAACGGGGTACGAC-3′, and Bcl-2 reverse, 5′-TCACTTGTGGCTCAGATAGGCACCCAGGGT-3′; Bfl-1 forward, 5′-ATGACAGACTGTGAATTTGGATATATTTAC-3′, and Bfl-1 reverse 5′-TCAACAGTATTGCTTCAGGAGAGATAGCAT-3′. GAPDH was included as an internal control. Fold induction (FI) of Bcl-2 and Bfl-1 mRNA expression is indicated. B, mitochondrial and cytosolic extracts from 697 cells with vector or NTD-MR (MR-H7) treated with 1 μm TA for a 48-h time course were subjected to SDS-PAGE followed by immunoblotting with a mAb to Bcl-2 (BD Biosciences) and a pAb to Bfl-1 (Santa Cruz Biotechnology). To ensure that the same content of mitochondrial and cytosolic protein was loaded in each case, the membrane was stripped and successively reprobed with a VDAC pAb (Santa Cruz Biotechnology) as a marker for the mitochondrial fraction and a β-actin pAb as a marker for the cytosolic fraction.View Large Image Figure ViewerDownload (PPT)A recent study (35Werner A.B. de Vries E. Tait S.W. Bontjer I. Borst J. J. Biol. Chem. 2002; 277: 22781-22788Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) has demonstrated that Bfl-1 can prevent the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins like Bid and blocking its collaboration with Bax or Bak in the plane of the mitochondrial membrane. Thus, we examined the localization and protein levels of the pro-apoptotic proteins Bid and Bax in 697 and MR-H7 cells treated with 1 μm TA. Western blot analysis in Fig. 8 A shows that in 697 cells, both cytosolic and mitochondrial levels of Bid were dramatically higher than in MR-H7 cells. Moreover, in MR-H7 cells, Bid was located primarily within the mitochondria, whereas in 697 cells it was detected in the cytosol as well. Bax protein levels did not seem to be significantly different between the two cell lines; however, Bax was only faintly detectable in the cytosol of MR-H7 cells at 48 h (Fig. 8 A).Figure 8A, localization of Bid, Bax, Cyt c, and Smac in GC-treated 697 and MR-H7 cells. Mitochondrial and cytosolic extracts from 697 cells with vector or NTD-MR (MR-H7) treated with 1 μm TA for a 48-h time course were subjected to SDS-PAGE followed by immunoblotting with a pAb to Bid (BIOSOURCE), a pAb to Bax (Santa Cruz Biotechnology), a mAb to Cyt c (BD Biosciences), and a pAb to Smac/DIABLO (Upstate Biotechnology, Inc.). To ensure that the same content of mitochondrial and cytosolic protein was loaded in ea" @default.
• W2090137967 created "2016-06-24" @default.
• W2090137967 creator A5062814155 @default.
• W2090137967 creator A5072750067 @default.
• W2090137967 creator A5080212717 @default.
• W2090137967 creator A5081465158 @default.
• W2090137967 creator A5090210226 @default.
• W2090137967 date "2002-11-01" @default.
• W2090137967 modified "2023-10-13" @default.
• W2090137967 title "Inhibition of Glucocorticoid-induced Apoptosis in 697 Pre-B Lymphocytes by the Mineralocorticoid Receptor N-terminal Domain" @default.
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