Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: J Biochem Mol Toxicol. 2016 Feb 25;30(6):309–316. doi: 10.1002/jbt.21795

Differential Regulation of Bcl-xL Gene Expression by Corticosterone, Progesterone and Retinoic Acid

Steve J Morrissy 1,1, Haipeng Sun 1,2, Jack Zhang 1, Joshua Strom 1, Qin M Chen 1,*
PMCID: PMC4969002  NIHMSID: NIHMS753146  PMID: 26915917

Abstract

Corticosterone (CT), progesterone (PG), and retinoic acid (RA) are capable of inhibiting Doxorubicin (Dox) from inducing apoptosis in rat cardiomyocytes. Mechanistically, CT, PG and RA induce increases of Bcl-xL protein and mRNA, and activate a 3.2 kb bcl-x gene promoter. CT and RA, but not PG induced the activity of a 0.9 kb bcl-x promoter, containing sequences for AP-1 and NF-kB binding. RA, but not CT or PG, induced NF-kB activation. CT, but not PG or RA induced AP-1 activation, and induction of the 0.9 kb bcl-x reporter by CT was inhibited by dominant negative c-Jun TAM-67 or removal of AP-1 binding site. Therefore, although CT, PG and RA all induce Bcl-xL mRNA and protein, three independent mechanisms are in operation: while CT induces Bcl-xL via AP-1 transcription factor, and RA induces NF-kB activation and bcl-x promoter activity, PG induces Bcl-xL via a mechanism independent of NF-kB or AP-1.

Keywords: Apoptosis, cell survival, nuclear receptor, steroids, retinoids

Introduction

The majority of the myocytes in the myocardium cease cell replication within a few days after the birth of a mammal. In adult myocardium, loss of cardiomyocytes occurs as a result of chemotherapy, cardiac disease and aging, contributing to decline in cardiac contractile function and eventually heart failure [1–9]. Apoptotic-like cell death plays a critical role in cardiomyopathy induced by the antineoplastic drug Doxorubicin (Dox), an anthracycline quinone used for treatment of a variety of leukemia, lymphomas and solid tumors [5, 10, 11]. Although the protocol for administering Dox has been improved to minimize acute cardiac toxicity, a dilated cardiomyopathy may develop two to ten years after. During pathological analysis of the failing hearts, apoptosis has been observed in a significant proportion of cardiomyocytes [5, 10, 11]. Previous works from our laboratory have found that corticosterone (CT) and progesterone (PG) were able to protect cardiomyocytes from Dox induced apoptosis [12, 13]. Among the multiple signaling pathways and genes altered by these steroids, induction of Bcl-xL gene appears to mediate the anti-apoptotic effect [12–20].

Bcl-2 family members are known as a major player in apoptosis. Although members of bcl-2 family share consensus sequence and structural domains, some act to promote apoptosis (pro-apoptotic) whereas others inhibit apoptosis (anti-apoptotic). The relative levels of pro versus anti-apoptotic bcl-2 family members are believed to be a key determinant for cell survival [21, 22]. Bcl-xL, an anti-apoptotic member, is encoded by bcl-x gene, which produces several isoforms of proteins due to alternative mRNA splicing with Bcl-xL transcript as the major product [23–25]. Bcl-x gene promoter contains nuclear receptor binding site upstream of −3 kb region, and consensus elements for binding of Sp1, AP-1, Oct-1, Ets, Rel/NF-kB, STATs and GATA-1 transcription factors [23, 24, 26]. These elements suggest multiple mechanisms of transcriptional regulation of bcl-x gene.

The nuclear receptors accept incoming signals from agonists such as steroids or retinoids to alter gene expression or affect cellular metabolism [27, 28]. The Glucocorticoid Receptor (GR) and Progesterone Receptor (PR) each is encoded by a single gene that produces two isoforms from alternative promoters [29, 30]. These receptors undergo dimerization, can become phosphorylated, and bind to the Hormone Response Element (HRE) in the promoter of downstream target genes. There are two classes of retinoid receptors, retinoic acid receptors (RAR) and retinoid X receptors (RXR), which can form homodimers or heterodimers for binding to the Retinoid Response Elements in the promoter of target genes [31, 32]. We have found that glucocorticoids and progesterone are capable of inhibiting Dox from inducing apoptosis and inducing Bcl-xL expression in a receptor dependent manner [12, 13, 20]. Whether retinoic acid can induce Bcl-xL in cardiomyocytes is not known. We compare the induction of Bcl-xL to determine if these nuclear receptor ligands induce Bcl-xL through distinct mechanisms.

Methods

Cell Culture and Drug Treatment

Cardiomyocytes were prepared from 1 to 2 days old neonatal Sprague-Dawley rats (Harland, Indianapolis, IN) as described [33]. The myocytes were seeded at a density of 0.3 × 106 cells per well in 6-well plates or 7.5 × 104 cells per well in 24-well plates. Cells were cultured in low glucose DMEM with 1 mM pyruvate, 10% fetal bovine serum, 100 units/ml penicillin and 100 units/ml streptomyocin for 3–4 days before being placed in fresh DMEM containing 0.5% FBS and CT, PG or RA. After another 24 hrs, cells were harvested for Western blot analyses or the media were changed to fresh DMEM containing 0.5% FBS and 1 μM Dox. Cells were harvested 16 hours later for caspase activity assays.

Caspase Activity Assay

Detached cells were collected by centrifugation and were combined with attached cells from the same well in 6-well plates. The cells were dissolved in 250 μl of lysis buffer (0.5% Nonidet P-40, 0.5 mM EDTA, 150 mM NaCl, and 50 mM Tris pH 7.5). Cell lysates (50 μl) were incubated for 1 hour at 37°C with 40 μM of N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC, Alexis Biochemicals, San Diego, CA) in 100 μl reaction buffer (10 mM HEPES, pH 7.5, 0.05 M NaCl and 2.5 mM DTT). The released AMC was measured using a 96-well fluorescence plate reader (Cambridge Bioresearch Model 7620) with an excitation wavelength of 365 nm and an emission wavelength of 450 nm.

Western Blot

Cells in 100 mm dishes were lysed by scraping in Laemini buffer [0.5 M Tris, pH 6.8, 2.4% (w/v) SDS, 50% (v/v) glycerol] with freshly added protease inhibitors: 10 μg/ml aprotinin and 1 mM phenylmethanesulfonyl fluoride. Protein concentration was determined using the bicinchoninic acid (BCA) method according to the manufacturer’s instruction (Pierce, Rockford, IL). Proteins were separated by SDS polyacrylamide gel electrophoresis using a mini-Protean II electrophoresis apparatus (Bio-Rad, Richmond, CA) run at 60 volts. The separated proteins were transferred to immobilon-P membranes (Millipore, Bedford, MA) by electrophoresis. The membrane was incubated with primary antibodies, anti-Bcl-xL (Santa Cruz Biotechnology, sc#8392) and then secondary antibodies conjugated with horseradish peroxidase. The bound antibodies were detected using an enhanced chemiluminescent reaction.

RT-PCR

Total RNA extracted using Trizol was used as a template for RT-PCR. Superscript II was used for reverse transcription at 37–50°C for 1 hr. For PCR, the denaturation and primer extension were carried out at 94°C and 72°C. The annealing temperature was calculated based on GC content of the primers, 5′-CCCCAGAAACTGAACCA-3′ (forward) and 5′-AGTTTACCCCATCCCGAAAG-3′ (reverse) as suggested by the oligonucleotide synthesis company (Biosynthesis, Lewisville, TX). The products were detected by real-time PCR (BioRad CFX96) using SYBR Green dye.

Bcl-xL Promoter Luciferase Constructs, Transient Transfection and Luciferase

Cardiomyocytes were seeded at 0.5 ×106 per well in 6-well plates (~960 mm2 culture area per well). At 24 h after plating, cells were incubated 5 h with 0.8 μg DNA of luciferase reporter construct per well using 3 μL of Fugene-6 liposomes (Roche, Mannheim, Germany). The transfection efficiency is generally low (3–10%) in cardiomyocytes and can vary due to the nature of primary culture. To control for transfection efficiency, we cotransfected cardiomyocytes with the pRL-TK plasmid (0.04μg/well), which encodes a Renilla luciferase gene under the control of a Thymidine Kinase promoter. After 5 h incubation under tissue culture condition, cells were replenished with fresh DMEM containing 10% FBS for overnight recovery. After 24 h serum starvation followed by experimental treatments, cells were rinsed with cold PBS and lysed in a passive lysis buffer provided in a luciferase assay kit for measurements of luciferase activity according to the manufacturer’s protocol (Promega, Madison, WI). The luciferase activity was normalized to the Renilla activity and expressed as fold induction over control unless indicated otherwise. All treatments were performed in triplicate.

Plasmid Constructs

The −3.2kb murine bcl-x gene promoter-reporter construct was generously provided by the laboratory of Dr. Gabriel Nunez (University of Michigan, Ann Arbor, MI). The construct encodes a 3.2kb fragment of the Bcl-xL promoter fused to the promoterless pGL2 firefly luciferase promoter [23]. Within 1 kb upstream of transcription start side, the sequence homology between mouse bcl-x and human bcl-x gene is 71%. The cis-elements in this region of promoter is well characterized for human bcl-x gene [24]. The 0.9 kb human bcl-x gene promoter-reporter pGL3 construct was generously provided by the laboratory of Dr. G.T. Bowden (University of Arizona, Tucson, AZ). This construct contains a 905 bp region of the Bcl-xL promoter (gene bank number D30746) fused to the promoterless pGL3 firefly luciferase promoter and was first cloned by Dr. Michael A. Bachelor from genomic DNA of Hela cells when studying the cause of bcl-xL protein increase due to UV-A exposure in keratinocytes [34]. The luciferase reporter construct for NF-κB transcription factor was a generous gift of Dr. Zigang Dong (University of Minnesota, The Hormel Institute, Austin, MN)[35]. The NF-κB reporter construct encodes a 196 bp fragment of the HIV promoter containing 2x NF-κB binding elements driving a luciferase reporter gene. The AP-1 luciferase reporter construct has four consensus AP-1 binding sites from collagenase gene was obtained from Stratagene [36]. The dominant negative c-Jun mutant TAM67 is composed of amino acids 123–331 of c-Jun cloned into the expression vector pcDNA3 [37]. Mutant 0.9 kb bcl-x promoter with eliminated NF-κB or AP-1 binding site was obtained using QuickChange II XL site-directed mutagenesis kit (Stratagene) using the primer of 5′ GGA GTG GGG GCG GGG AGG GAG TGA CTT TCC for NF-κB mutation and 5′CAC TGG TGC TTT CGA TGA AGT ATC TTG GAA for AP-1 mutation. The vectors containing mutated sequence were verifying by DNA sequencing.

Statistics

Stata Statistics/Data Analysis software was used for one-way analysis of variance (ANOVA) with a Bonferroni correction to compare multiple means. A p-value of <0.05 was used as the cutoff for statistical significance. When a mean is statistically different from another, the two means were labeled with two different letters, e.g. a and b. A mean labeled with two letters, e.g. ab, indicates no significant difference between the mean labeled with a or b, although the mean labeled a is significantly different from that labeled with b.

Results

CT, PG and RA Inhibit Dox Induced Caspase-3 Activity in Rat Cardiomyocytes

We have previously shown that CT and PG can decrease Dox induced apoptosis in cardiomyocytes as measured by several markers of apoptosis including caspase-3 activation [12, 13]. Caspase-3 activity serves well as a quantitative measurement for apoptosis [12, 13]. When screening a number of nuclear receptor agonists for cytoprotective effect, we found that all-trans retinoic acid (RA) also inhibited Dox from inducing apoptosis based on morphology examination and caspase assays. Figure 1A shows that CT, PG, and RA at 1 μM concentration all decreased activation of caspase-3 by Dox. The amount of protection was 46 ± 2.0 % for 1 μM CT, 30 ± 1.0 % for 1 μM PG, and 52 ± 0.03 % for 1 μM RA. CT and PG are known to bind to GR and PR, respectively. Mifepristone (MF) is an antagonist of GR and PR, and was able to cancel the cytoprotective effect of CT and PR [12, 13]. To document that the protective effect of RA is not through MF sensitive receptors, we tested the effect of MF on RA induced cytoprotection. As expected, MF was not able to cancel the protective effect of RA against Dox induced caspase activation (Fig 1B), suggesting a GR or PR independent mechanism of cytoprotection.

Fig. 1. CT, PG and RA decrease Dox induced caspase-3 activity.

Fig. 1

Open in a new tab

Primary cultured rat neonatal cardiomyocytes were pretreated 24 hours with 1 μM PG, CT or RA prior to 0.8 μM Dox treatment for 24 hrs (A). MF (1 μM) was added to cells 10 mins prior to addition of 1 μM RA. At 24 hours after incubation with RA, cells were treated with 0.8 μM Dox for 24 hours (B). At the end of 24 hours of Dox treatment, cells were harvested for measurements of caspase activity using DEVD-AMC as a substrate. The data represent means ± standard deviations from triplicates of one representative experiment and were analyzed by ANOVA. A letter indicates significant difference from the means labeled with different letters. The mean labeled “ac” is not significant different from that labeled with “a” or “c”, although the mean labeled with “a” is significant different from that labeled with “c”.

CT, PG and RA Induce Transcriptional Activation of Bcl-xL

We have previously shown that CT and PG can induce the anti-apoptotic gene Bcl-xL [12, 13]. To determine whether RA induces Bcl-xL, we incubated cardiomyocytes with varying doses of RA for 24 hours and then harvested the cells for Western blot analysis. PG and CT were included for comparisons. The level of Bcl-xL protein was increased by all three steroids in a dose dependent manner (Fig 2A). With 1 μM of CT, PG or RA, the induction was 2.14, 2.23, and 4.24 fold respectively. An increase of Bcl-xL mRNA was observed with 1 μM of CT, PG or RA (Fig 2B).

Fig. 2. CT, PG and RA induce the expression of Bcl-xL protein and mRNA.

Fig. 2

Open in a new tab

Primary cultured rat neonatal cardiomyocytes were treated with CT, PG or RA at indicated doses and were harvested at 24 hours after for measurements of Bcl-xL protein using Western blot (30 μg protein/lane, A). Vinculin from RA treated cells was used as a loading control (A). Cells were treated with 1 μM CT, PG or RA for 24 hr before harvesting for measurement of Bcl-xL mRNA using real time RT-PCR with triplicate samples (B). A letter indicates significant difference from the means labeled with different letters.

To determine whether increase of Bcl-xL mRNA is due to transcriptional activation of Bcl-x gene, we tested the effect of CT, PG and RA on a −3.2 kb Bcl-xL promoter using a luciferase reporter construct [23]. MF was included to block Bcl-xL promoter activation by CT or PG. Cardiomyocytes transfected with the reporter plasmid were treated with 1 μM CT, 10 μM PG or 1 μM RA for 24 hours. RA is most effective in activating Bcl-xL promoter-reporter (Fig 3A). The activity of the 3.2 kb bcl-x reporter plasmid was increased by RA (1.63 ± 0.18 fold induction p<0.05) and RA+MF (1.84 ± 0.11 fold induction p<0.05). As expected, MF did not block RA induced bcl-x promoter activation. Although CT and PG induced the activity of 3.2 kb bcl-x promoter and as expected MF blocked bcl-x promoter activation by CT or PG, induction of bcl-xl promoter activity is not statistically significant as compared to control without any drug treatment (Fig 3A).

Fig. 3. Activation of Bcl-x gene promoter by CT, PG or RA.

Fig. 3

Open in a new tab

Primary cultured cardiomyocytes were transfected with 3.2kb (A) or 0.9kb (B) bcl-x promoter-luciferase reporter construct. At 24 hrs after transfection, cells were treated with 1 μM CT, 10 μM PG or 1 μM RA in the absence or presence of 1 μM MF, which was added to cells 10 mins prior to CT, PG or RA. Cells were harvested 24 hours after for measurements of luciferase activities. The data represent means ± standard deviations from triplicates as fold increases when the luciferase activity for control untreated cells was set at 1. A letter indicates significant difference from the means labeled with different letters. The mean labeled “ab” is not significant different from that labeled with “a” or “b”, although the mean labeled with “a” is significant different from that labeled with “b”.

Role of NF-κB and AP-1 in Bcl-x Promoter Activation

Sequence alignment of 1 kb human bcl-x gene promoter with mouse bcl-x gene promoter indicate 71% sequence homology. Several cis-elements have been characterized within this region in human bcl-x gene [24]. There is an NF-κB binding site, GGGGGGACTGCCC, between 522 and 536 bp, and an AP-1 binding site, TGACTTAA, between 508 and 528 bp. To determine whether 0.9 kb promoter of bcl-x is sufficient for activation by CT, PG or RA, primary cardiomyocytes were transfected with 0.9 kb promoter-luciferase reporter construct. The activity of the reporter was increased by CT (1.39 ± 0.05 fold induction p<0.05), RA (1.52 ± 0.10 fold induction p<0.05) and RA+MF (1.48 ± 0.08 fold induction p<0.05), but not PG. MF inhibited the induction of CT, but not RA (Fig 3). Therefore, CT and RA but not PG was able to activate the 0.9 kb bcl-x promoter.

To examine the involvement of NF-κB or AP-1 in Bcl-xL induction, we tested NF-κB activation using NF-κB reporter construct. When NF-κB was transfected into cardiomyocytes, RA was able to activate NF-κB (1.96 ± 0.17 p<0.05), which was not inhibited by MF (Fig 4A). Neither CT nor PG was able to activate NF-κB (Fig 4A). However, when NF-κB binding site was eliminated in the 905 bp bcl-x promoter, RA remained capable of activating the bcl-x promoter (Fig 4B), pointing to additional mechanisms beyond NF-κB in bcl-x promoter activation by RA.

Fig. 4. RA but not CT or PG activates NF-κB cis-elements.

Fig. 4

Open in a new tab

Primary cultured cardiomyocytes were transfected with 2x NF-κB luciferase reporter construct (A) or −0.9 kb bcl-x promoter construct with NF-κB binding site being mutated (B). Cells were treated with 1 μM CT, 10 μM PG, or 1 μM RA in the absence or presence of 1 μM MF for 24 hours before harvesting for measurements of luciferases. Data are expressed as means ± standard deviations of triplicate samples with fold induction when the activity of luciferase for control was set at 1. A letter indicates significant difference from the means labeled with a different letter.

In contrast, when AP-1 involvement was determined by transfection of AP-1 reporter construct, CT but not PG or RA was able to activate AP-1 reporter (Fig 5A). Such activation was sensitive to MF (Fig 5A), supporting a role of GR in AP-1 mediated transcriptional event. TAM67 is a dominant negative mutant of c-Jun, encoding 121–331 amino acids of c-Jun protein. Cotransfection of 0.9 kb bcl-x promoter with TAM67 was able to block CT induced bcl-x promoter activation (Fig 5B). With 0.9 kb bcl-x promoter containing mutations eliminating AP-1 binding, CT was not effective in activating the promoter (Fig 5C). In contrast, RA, which was not able to activate AP-1 reporter, remained capable of activating AP-1 null bcl-x promoter (Fig 5C).

Fig. 5. CT but not PG or RA activates AP-1 cis-elements.

Fig. 5

Open in a new tab

Primary cultured cardiomyocytes were transfected with 4xAP-1 reporter construct (A), wild type 0.9 kb bcl-x promoter-reporter construct (B), or 0.9 kb bcl-x promoter-reporter construct with AP-1 binding site being mutated (C). TAM67 (0.08 μg) was cotransfected with the bcl-x promoter-reporter construct (B). At 24 hours after treatment with 1 μM CT, 10 μM PG, or 1 μM RA in the absence or presence of 1 μM MF, cells were harvested for measurements of luciferases. Data are expressed as means ± standard deviations of triplicates with fold induction when the activity of luciferase for control was set at 1. A letter indicates significant difference from the means labeled with different letters. The mean labeled “ac” is not significant different from that labeled with “a” or “c”, although the mean labeled with “a” is significant different from that labeled with “c”.

Discussion

We found that 3.2 kb or 0.9 kb promoter of bcl-x gene was induced by CT and RA, whereas PG is able to activate 3.2 kb but not 0.9 kb promoter of bcl-x gene. The induction of 0.9 kb bcl-x promoter by CT was blocked by a dominant-negative c-Jun, TAM-67. CT was able to activate AP-1 promoter-reporter and such activation could be inhibited with MF. RA induced Bcl-xL, possibly via NF-κB in conjunction with additional mechanisms. Activation of NF-κB cis-element was detected with RA, but not CT or PG.

In cardiomyocytes, we have previously shown that CT or PG can inhibit apoptosis in a Bcl-xL dependent mechanism [12, 13, 20]. The existence of HREs upstream of 3 kb bcl-x promoter region has been previously described [38]. PG and CT were reported to activate bcl-x promoter through two HREs located in −3040 and −3001 region [26]. The fact that 3.2 kb bcl-x promoter is activated by PG in our study suggests the possibility of HRE involvement.

The discovery of CT inducing bcl-x gene promoter through AP-1 response element is novel. Bcl-x gene promoter contains AP-1 responsive elements [39, 40], and it has been shown that induction of AP-1 can stimulate Bcl-xL transcription [41]. The role of AP-1 in CT induced Bcl-xL has been demonstrated by: 1) CT is capable of activating AP-1 reporter, and 2) expression of TAM67, the dominant negative of c-Jun, was capable of preventing 0.9 kb bcl-x promoter activation by CT. Contradictory to our finding, Jacobs-Helber et. al. [40] discovered that erythroid cells undergoing apoptosis had increased AP-1 activity and decreased Bcl-xL expression. TAM67 expression resulted in an increased level of Bcl-xL [40]. This may reflect differential regulation of Bcl-xL by steroids among different tissues or cell types. In most hematopoitic cells, CT induces apoptosis, while in cardiomyocytes CT inhibits apoptosis [42]. Nevertheless, it appears that whether CT induces or inhibits apoptosis correlates with its ability to inhibit or induce Bcl-xL respectively.

Like CT, whether RA induces or inhibits apoptosis is cell type dependent [43]. In a leukemia T-cell line, retinoic acids induce apoptosis through down regulation of Bcl-xL [44]. In contrast, in naïve T-thymocytes, RA has been shown to increase bcl2a1 expression and decrease apoptosis without affecting the expression of Bcl-xL [45]. Retinoic acids have been shown to mediate cell survival response in neuroblastoma cells, neural crest cells, myeloid leukemia blasts, lung cancer cells, islet-epithelial cells, and olfactory sensory neurons [46–51]. In cardiomyocytes, high glucose induced apoptosis can be inhibited by RA in NF-κB dependent manner [52].

The ability of RA to activate NF-κB suggests the possibility of additional mechanism of cell survival besides Bcl-xL. The transcription factor NF-κB has previously been shown to exhibit antiapoptotic effects through induction of anti-apoptotic genes, including cIAP1, cIAP2 [53–55], xIAP [56], and Bcl-2 homolog Bfl-1/A1 [57, 58]. Nevertheless, NF-κB dependent Bcl-xL expression has been reported in hippocampal neurons or in polymorphonuclear leukocytes [59, 60]. Glasgow, et al., identified NF-κB binding sites in the Bcl-xL promoter that are responsive to p65 containing NF-κB transcription factor [61]. The fact that RA activates NF-κB reporter construct but remains capable of activating bcl-x promoter when NF-κB is mutated suggests multiple mechanisms of transcriptional regulation of bcl-x gene by RA.

Acknowledgments

We thank Dr. Gabriel Nunez (University of Michigan, Ann Arbor, MI) for 3.2kb murine bcl-x promoter-reporter construct, Dr. Michael A. Bachelor for 0.9 kb human bcl-x promoter reporter construct, Dr. Zigang Dong (University of Minnesota) for NF-kB luciferase reporter construct. Works in our laboratory were supported by NIH R01 HL 076530, R01 HL089958, R21ES017473, T32 ES007091, R01 GM 111337, Arizona Biomedical Research Commission (QMC), and Marjorie Hornbeck Estate Research Award (JZ) from University of Arizona Sarver Heart Center.

References

  • 1.Narula J, Haider N, Virmani R, DiSalvo TG, Kolodgie FD, Hajjar RJ, et al. Apoptosis in myocytes in end-stage heart failure. New Engl J Med. 1996;335(16):1182–9. doi: 10.1056/NEJM199610173351603. [DOI] [PubMed] [Google Scholar]
  • 2.Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, Voipio-Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation. 1997;95(2):320–3. doi: 10.1161/01.cir.95.2.320. [DOI] [PubMed] [Google Scholar]
  • 3.Narula J, Pandey P, Arbustini E, Haider N, Narula N, Kolodgie FD, et al. Apoptosis in heart failure: release of cytochrome c from mitochondria and activation of caspase-3 in human cardiomyopathy. Proc Natl Acad Sci USA. 1999;96(14):8144–9. doi: 10.1073/pnas.96.14.8144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Olivetti G, Quaini F, Sala R, Lagrasta C, Corradi D, Bonacina E, et al. Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart. J Mol Cell Cardiol. 2005;28(9):2005–16. doi: 10.1006/jmcc.1996.0193. [DOI] [PubMed] [Google Scholar]
  • 5.Singal PK, Li T, Kumar D, Danelisen I, Iliskovic N. Adriamycin-induced heart failure: mechanism and modulation. Mol Cell Biochem. 2000;207(1–2):77–86. doi: 10.1023/a:1007094214460. [DOI] [PubMed] [Google Scholar]
  • 6.Bromme HJ, Holtz J. Apoptosis in the heart: when and why? Mol Cell Biochem. 1996;163–164:261–75. doi: 10.1007/BF00408667. [DOI] [PubMed] [Google Scholar]
  • 7.James TN. Apoptosis in cardiac disease. Am J Med. 1999;107(6):606–20. doi: 10.1016/s0002-9343(99)00308-3. [DOI] [PubMed] [Google Scholar]
  • 8.Yaoita H, Ogawa K, Maehara K, Maruyama Y. Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. Cardiovasc Res. 2000;45(3):630–41. doi: 10.1016/s0008-6363(99)00349-1. [DOI] [PubMed] [Google Scholar]
  • 9.van Empel VP, Bertrand AT, Hofstra L, Crijns HJ, Doevendans PA, De Windt LJ. Myocyte apoptosis in heart failure. Cardiovasc Res. 2005;67(1):21–9. doi: 10.1016/j.cardiores.2005.04.012. [DOI] [PubMed] [Google Scholar]
  • 10.Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology. 2010;115(2):155–62. doi: 10.1159/000265166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Octavia Y, Tocchetti CG, Gabrielson KL, Janssens S, Crijns HJ, Moens AL. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol. 2012;52(6):1213–25. doi: 10.1016/j.yjmcc.2012.03.006. [DOI] [PubMed] [Google Scholar]
  • 12.Chen Q, Alexander D, Sun H, Xie L, Lin Y, Terrand J, et al. Corticosteroids Inhibit Cell Death Induced by Doxorubicin in Cardiomyocytes: Induction of Anti-apoptosis, Antioxidant and Detoxification Genes. Mol Pharm. 2005;67(6):1861–73. doi: 10.1124/mol.104.003814. [DOI] [PubMed] [Google Scholar]
  • 13.Morrissy S, Xu B, Aguilar D, Chen Q. Inhibition of Apoptosis by Progesterone in Cardiomyocytes. Aging Cell. 2010;95(5):799–809. doi: 10.1111/j.1474-9726.2010.00619.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Sun H, Chen QM. Inhibitors of GSK-3 prevent corticosterone from inducing COX-1 expression in cardiomyocytes. Cardiovasc Tox. 2008;8(2):93–100. doi: 10.1007/s12012-008-9018-y. [DOI] [PubMed] [Google Scholar]
  • 15.Sun H, Sheveleva E, Chen QM. Corticosteroids induce cyclooxygenase 1 expression in cardiomyocytes: role of glucocorticoid receptor and Sp3 transcription factor. Mol Endocrinol. 2008;22(9):2076–84. doi: 10.1210/me.2007-0302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Sun H, Sheveleva E, Xu B, Inoue H, Bowden TG, Chen QM. Corticosteroids induce COX-2 expression in cardiomyocytes: role of glucocorticoid receptor and C/EBP-beta. Am J Physiol Cell Physiol. 2008;295(4):915–22. doi: 10.1152/ajpcell.90646.2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Sun H, Xu B, Inoue H, Chen QM. P38 MAPK mediates COX-2 gene expression by corticosterone in cardiomyocytes. Cell Signalling. 2008;20(11):1952–9. doi: 10.1016/j.cellsig.2008.07.003. [DOI] [PubMed] [Google Scholar]
  • 18.Sun H, Xu B, Sheveleva E, Chen QM. LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism. Tox Applied Pharmacol. 2008;232(1):25–32. doi: 10.1016/j.taap.2008.05.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Morrissy S, Strom J, Purdom-Dickinson S, Chen QM. NAD(P)H:quinone oxidoreductase 1 is induced by progesterone in cardiomyocytes. Cardiovasc Tox. 2012;12(2):108–14. doi: 10.1007/s12012-011-9144-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Xu B, Strom J, Chen QM. Dexamethasone induces transcriptional activation of Bcl-xL gene and inhibits cardiac injury by myocardial ischemia. Eur J Pharmacol. 2011;668(1–2):194–200. doi: 10.1016/j.ejphar.2011.06.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281(5381):1322–6. doi: 10.1126/science.281.5381.1322. [DOI] [PubMed] [Google Scholar]
  • 22.Chao DT, Korsmeyer SJ. BCL-2 family: regulators of cell death. Ann Rev Immunol. 1998;16:395–419. doi: 10.1146/annurev.immunol.16.1.395. [DOI] [PubMed] [Google Scholar]
  • 23.Grillot DA, Gonzalez-Garcia M, Ekhterae D, Duan L, Inohara N, Ohta S, et al. Genomic organization, promoter region analysis, and chromosome localization of the mouse bcl-x gene. J Immunol. 1997;158(10):4750–7. [PubMed] [Google Scholar]
  • 24.Grad JM, Zeng XR, Boise LH. Regulation of Bcl-xL: a little bit of this and a little bit of STAT. Curr Opin Oncol. 2000;12(6):543–9. doi: 10.1097/00001622-200011000-00006. [DOI] [PubMed] [Google Scholar]
  • 25.Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, et al. bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell. 1993;74(4):597–608. doi: 10.1016/0092-8674(93)90508-n. [DOI] [PubMed] [Google Scholar]
  • 26.Viegas LR, Vicent GP, Baranao JL, Beato M, Pecci A. Steroid hormones induce bcl-X gene expression through direct activation of distal promoter P4. J Biol Chem. 2004;279(11):9831–9. doi: 10.1074/jbc.M312402200. [DOI] [PubMed] [Google Scholar]
  • 27.Weatherman RV, Fletterick RJ, Scanlan TS. Nuclear-receptor ligands and ligand-binding domains. Ann Rev Biochem. 1999;68:559–81. doi: 10.1146/annurev.biochem.68.1.559. [DOI] [PubMed] [Google Scholar]
  • 28.Escriva H, Delaunay F, Laudet V. Ligand binding and nuclear receptor evolution. Bioessays. 2000;22(8):717–27. doi: 10.1002/1521-1878(200008)22:8<717::AID-BIES5>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
  • 29.Funder JW. Glucocorticoid and mineralocorticoid receptors: biology and clinical relevance. Ann Rev Med. 1997;48:231–40. doi: 10.1146/annurev.med.48.1.231. [DOI] [PubMed] [Google Scholar]
  • 30.Graham JD, Clarke CL. Physiological action of progesterone in target tissues. Endocrine Rev. 1997;18(4):502–19. doi: 10.1210/edrv.18.4.0308. [DOI] [PubMed] [Google Scholar]
  • 31.Nohara A, Kobayashi J, Mabuchi H. Retinoid X receptor heterodimer variants and cardiovascular risk factors. J Atheroscler Thromb. 2009;16(4):303–18. doi: 10.5551/jat.no786. [DOI] [PubMed] [Google Scholar]
  • 32.Shulman AI, Mangelsdorf DJ. Retinoid x receptor heterodimers in the metabolic syndrome. New Engl J Med. 2005;353(6):604–15. doi: 10.1056/NEJMra043590. [DOI] [PubMed] [Google Scholar]
  • 33.Chen Q, Tu V, Wu Y, Bahl J. Hydrogen Peroxide Dose Dependent Induction of Cell Death or Hypertrophy in Cardiomyocytes. Arch Biochem Biophys. 2000;373(1):242–8. doi: 10.1006/abbi.1999.1558. [DOI] [PubMed] [Google Scholar]
  • 34.Bachelor MA, Bowden GT. Ultraviolet A-induced modulation of Bcl-XL by p38 MAPK in human keratinocytes: post-transcriptional regulation through the 3′-untranslated region. J Biol Chem. 2004;279(41):42658–68. doi: 10.1074/jbc.M406626200. [DOI] [PubMed] [Google Scholar]
  • 35.Huang C, Chen N, Ma WY, Dong Z. Vanadium induces AP-1- and NFkappB-dependent transcription activity. Int J Oncol. 1998;13(4):711–5. doi: 10.3892/ijo.13.4.711. [DOI] [PubMed] [Google Scholar]
  • 36.Tu V, Chen Q. Distinct Roles of ERKs and p38 MAPK in Oxidant Induced Cardiomyocyte Hypertrophy. Cardiovasc Tox. 2003;3(2):119–33. doi: 10.1385/ct:3:2:119. [DOI] [PubMed] [Google Scholar]
  • 37.Tu VC, Sun H, Bowden GT, Chen QM. Involvement of Oxidants and AP-1 in Angiotensin II Activated NFAT3 Transcription Factor. Am J Physiol Cell Physiol. 2007;292(4):C1248–55. doi: 10.1152/ajpcell.00624.2005. [DOI] [PubMed] [Google Scholar]
  • 38.Rocha-Viegas L, Vicent GP, Baranao JL, Beato M, Pecci A. Glucocorticoids repress bcl-X expression in lymphoid cells by recruiting STAT5B to the P4 promoter. J Biol Chem. 2006;281(45):33959–70. doi: 10.1074/jbc.M602408200. [DOI] [PubMed] [Google Scholar]
  • 39.Sevilla L, Zaldumbide A, Pognonec P, Boulukos KE. Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFkappaB, STAT and AP1 transcription factor families. Histol Histopathol. 2001;16(2):595–601. doi: 10.14670/HH-16.595. [DOI] [PubMed] [Google Scholar]
  • 40.Jacobs-Helber SM, Wickrema A, Birrer MJ, Sawyer ST. AP1 regulation of proliferation and initiation of apoptosis in erythropoietin-dependent erythroid cells. Mol Cell Biol. 1998;18(7):3699–707. doi: 10.1128/mcb.18.7.3699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Takeuchi K, Motoda Y, Ito F. Role of transcription factor activator protein 1 (AP1) in epidermal growth factor-mediated protection against apoptosis induced by a DNA-damaging agent. FEBS J. 2006;273(16):3743–55. doi: 10.1111/j.1742-4658.2006.05377.x. [DOI] [PubMed] [Google Scholar]
  • 42.Chen Q, Sun H. Glucocortidoid Pharmacotoxicological Interactions and Modulation of Toxicity. In: Harvey P, Everett D, Springall C, editors. Adrenal Toxicology. Target Organ Toxicology Series. Vol. 26. New York: Informa; 2009. pp. 205–30. [Google Scholar]
  • 43.Noy N. Between death and survival: retinoic acid in regulation of apoptosis. Ann Rev Nutrit. 2010;30:201–17. doi: 10.1146/annurev.nutr.28.061807.155509. [DOI] [PubMed] [Google Scholar]
  • 44.Fujimura S, Suzumiya J, Yamada Y, Kuroki M, Ono J. Downregulation of Bcl-xL and activation of caspases during retinoic acid-induced apoptosis in an adult T-cell leukemia cell line. Hematol J. 2003;4(5):328–35. doi: 10.1038/sj.thj.6200321. [DOI] [PubMed] [Google Scholar]
  • 45.Rasooly R, Schuster GU, Gregg JP, Xiao JH, Chandraratna RA, Stephensen CB. Retinoid x receptor agonists increase bcl2a1 expression and decrease apoptosis of naive T lymphocytes. J Immunol. 2005;175(12):7916–29. doi: 10.4049/jimmunol.175.12.7916. [DOI] [PubMed] [Google Scholar]
  • 46.Ronca F, Yee KS, Yu VC. Retinoic acid confers resistance to p53-dependent apoptosis in SH-SY5Y neuroblastoma cells by modulating nuclear import of p53. J Biol Chem. 1999;274(25):18128–34. doi: 10.1074/jbc.274.25.18128. [DOI] [PubMed] [Google Scholar]
  • 47.Henion PD, Weston JA. Retinoic acid selectively promotes the survival and proliferation of neurogenic precursors in cultured neural crest cell populations. Develop Biol. 1994;161(1):243–50. doi: 10.1006/dbio.1994.1024. [DOI] [PubMed] [Google Scholar]
  • 48.Ketley NJ, Allen PD, Kelsey SM, Newland AC. Modulation of idarubicin-induced apoptosis in human acute myeloid leukemia blasts by all-trans retinoic acid, 1,25(OH)2 vitamin D3, and granulocyte-macrophage colony-stimulating factor. Blood. 1997;90(11):4578–87. [PubMed] [Google Scholar]
  • 49.Garcia-Regalado A, Vargas M, Garcia-Carranca A, Arechaga-Ocampo E, Gonzalez-De la Rosa CH. Activation of Akt pathway by transcription-independent mechanisms of retinoic acid promotes survival and invasion in lung cancer cells. Mol Cancer. 2013;12:44. doi: 10.1186/1476-4598-12-44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Li J, Feng ZC, Yeung FS, Wong MR, Oakie A, Fellows GF, et al. Aldehyde dehydrogenase 1 activity in the developing human pancreas modulates retinoic acid signalling in mediating islet differentiation and survival. Diabetologia. 2014;57(4):754–64. doi: 10.1007/s00125-013-3147-y. [DOI] [PubMed] [Google Scholar]
  • 51.Hagglund M, Berghard A, Strotmann J, Bohm S. Retinoic acid receptor-dependent survival of olfactory sensory neurons in postnatal and adult mice. J Neurosci. 2006;26(12):3281–91. doi: 10.1523/JNEUROSCI.4955-05.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Nizamutdinova IT, Guleria RS, Singh AB, Kendall JA, Jr, Baker KM, Pan J. Retinoic acid protects cardiomyocytes from high glucose-induced apoptosis through inhibition of NF-B signaling pathway. J Cell Physiol. 2013;228(2):380–92. doi: 10.1002/jcp.24142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Shu HB, Takeuchi M, Goeddel DV. The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex. Proc Natl Acad Sci USA. 1996;93(24):13973–8. doi: 10.1073/pnas.93.24.13973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Erl W, Hansson GK, de Martin R, Draude G, Weber KS, Weber C. Nuclear factor-kappa B regulates induction of apoptosis and inhibitor of apoptosis protein-1 expression in vascular smooth muscle cells. Circ Res. 1999;84(6):668–77. doi: 10.1161/01.res.84.6.668. [DOI] [PubMed] [Google Scholar]
  • 55.LaCasse EC, Baird S, Korneluk RG, MacKenzie AE. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene. 1998;17(25):3247–59. doi: 10.1038/sj.onc.1202569. [DOI] [PubMed] [Google Scholar]
  • 56.Stehlik C, de Martin R, Kumabashiri I, Schmid JA, Binder BR, Lipp J. Nuclear factor (NF)-kappaB-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor alpha-induced apoptosis. J Exp Med. 1998;188(1):211–6. doi: 10.1084/jem.188.1.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Grumont RJ, Rourke IJ, Gerondakis S. Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis. Genes Develop. 1999;13(4):400–11. doi: 10.1101/gad.13.4.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Zong WX, Edelstein LC, Chen C, Bash J, Gelinas C. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Develop. 1999;13(4):382–7. doi: 10.1101/gad.13.4.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Tamatani M, Che YH, Matsuzaki H, Ogawa S, Okado H, Miyake S, et al. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFkappaB activation in primary hippocampal neurons. J Biol Chem. 1999;274(13):8531–8. doi: 10.1074/jbc.274.13.8531. [DOI] [PubMed] [Google Scholar]
  • 60.Grutkoski PS, Graeber CT, Ayala A, Simms HH. Paracrine suppression of apoptosis by cytokine-stimulated neutrophils involves divergent regulation of NF-kappaB, Bcl-X(L), and Bak. Shock. 2002;17(1):47–54. doi: 10.1097/00024382-200201000-00009. [DOI] [PubMed] [Google Scholar]
  • 61.Glasgow JN, Wood T, Perez-Polo JR. Identification and characterization of nuclear factor kappaB binding sites in the murine bcl-x promoter. J Neurochem. 2000;75(4):1377–89. doi: 10.1046/j.1471-4159.2000.0751377.x. [DOI] [PubMed] [Google Scholar]

RESOURCES