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Originally published In Press as doi:10.1074/jbc.M600950200 on April 24, 2006

J. Biol. Chem., Vol. 281, Issue 26, 17552-17558, June 30, 2006
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Gadd45a and Gadd45b Protect Hematopoietic Cells from UV-induced Apoptosis via Distinct Signaling Pathways, including p38 Activation and JNK Inhibition*Formula

Mamta Gupta, Shiv Kumar Gupta, Barbara Hoffman, and Dan A. Liebermann1

From the Fels Institute of Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received for publication, January 31, 2006 , and in revised form, April 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gadd45a, Gadd45b, and Gadd45g (Gadd45/MyD118/CR6) are genes that are rapidly induced by genotoxic stress and have been implicated in genotoxic stress-induced responses, notably in apoptosis. Recently, using myeloid-enriched bone marrow (BM) cells obtained from wild-type (WT), Gadd45a-deficient, and Gadd45b-deficient mice, we have shown that in hematopoietic cells Gadd45a and Gadd45b play a survival function to protect hematopoietic cells from DNA-damaging agents, including ultra violet (UV)-induced apoptosis. The present study was undertaken to decipher the molecular paths that mediate the survival functions of Gadd45a and Gadd45b against genotoxic stress induced by UV radiation. It is shown that in hematopoietic cells exposed to UV radiation Gaddd45a and Gadd45b cooperate to promote cell survival via two distinct signaling pathways involving activation of the GADD45a-p38-NF-{kappa}B-mediated survival pathway and GADD45b-mediated inhibition of the stress response MKK4-JNK pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The GADD45 family of genes, including Gadd45a, Gadd45b, and Gadd45g, is rapidly induced by a variety of genotoxic stresses as well as by terminal differentiation and apoptotic cytokines in almost all mammalian cells (1–3). Proteins encoded by Gadd45 genes are remarkably similar, sharing 55–57% overall identity at the amino acid level (4–5). Data accumulated so far suggest that Gadd45 family members serve similar, but not identical, functions along different apoptotic and growth inhibitory pathways. For example, Gadd45b, but not Gadd45a, is activated upon TGFbeta-induced apoptosis (6–7). On the other hand, Gadd45a, but not Gadd45b and Gadd45g, was identified as a target for p53 function (8–10).

In recent years several lines of research have implicated GADD45 gene products in cell cycle arrest (4, 11), DNA repair (12–13), innate immunity (14–15), maintenance of genomic stability (16), and apoptosis (5). There is ample evidence that the functions of GADD45 proteins are mediated via interactions with other cellular proteins implicated in cell cycle regulation and the response of cells to extrinsic stress, including p21, Cdc2/cyclinB1, and the p38/JNK2 stress-induced kinase pathways (17).

GADD45 proteins have been shown to play a pro-apoptotic role via activation of the stress response kinases p38 and JNK (5). In collaboration with Dr. Anita Roberts' group, we have shown that TGFbeta-induced apoptosis of primary hepatocytes involves GADD45b-mediated activation of p38 (7). GADD45a activation of p38 and JNK has been implicated in UV-induced apoptosis of keratinocytes (18). NF-{kappa}B-mediated cell survival was shown to be dependent on suppression of the expression of Gadd45a and Gadd45g (19), where inhibition of NF-{kappa}B in cancer cells resulted in Gadd45a- and Gadd45g-dependent induction of apoptosis via JNK activation. Furthermore, induction of Gadd45a by BRCA-1 has been linked to apoptosis of breast cancer cells (20).

Interestingly, in apparent contradiction to the role GADD45 proteins play in apoptosis, recent reports have suggested that GADD45 proteins also function in cell survival. GADD45b has been implicated in promoting survival of mouse embryo fibroblasts in response to tumor necrosis factor {alpha} treatment (21) and of B cells by mediating the protective effects of CD40 co-stimulation against Fas-induced apoptosis (22). Notably, using Gadd45a null and Gadd454b null mice, we have recently reported that in hematopoietic cells deficiency in either Gadd45a or Gadd45b results in increased apoptosis induced by genotoxic stressors, including ultra-violet (UV) radiation, and the anti-cancer drugs VP-16 and daunorubicin (23). These data indicated that, contrary to the pro-apoptotic function of GADD45 in epithelial and endothelial cells, in hematopoietic cells both Gadd45a and Gadd45b play a survival role against genotoxic stress.

The present study was undertaken to decipher the molecular paths that mediate the survival functions of Gadd45a and Gadd45b against genotoxic stress induced by UV radiation. The findings presented are novel, showing that in hematopoietic cells exposed to UV radiation Gadd45a and Gadd45b cooperate to promote cell survival via two distinct signaling pathways involving activation of p38 and inhibition of JNK activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies—rIL-3 was purchased from R&D Systems; other recombinant cytokines (rIL-6 and rSCF) were generous gifts from Amgen Inc. (Thousand Oaks, CA). Annexin V-fluorescein isothiocyanate was obtained from BD Biosciences (San Diego, CA). TRIzol reagent was purchased from Invitrogen. Polyvinylidene difluoride membrane was purchased from Millipore. The JNK-specific inhibitor SP600215, p38-specific inhibitor SB202190, and I{kappa}B{alpha}-specific inhibitor IKK II were purchased from Calbiochem (La Jolla, CA). Antibodies against p38, JNK, ERK, I{kappa}B{alpha}, MKK3/6, MKK4, and BcL-XL were obtained from Cell Signaling Technologies (Beverly, MA). Phospho-specific antibodies for the active forms of p38, JNK, I{kappa}B{alpha}, MKK3/6, and MKK4 were also obtained from Cell Signaling Technologies. Antibody against phospho-MKK7 was purchased from Upstate Cell Signaling. Antibodies against GADD45a, GADD45b, beta-actin, and cIAP were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).


Figure 1
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  		FIGURE 1.
Increased sensitivity of Gadd45a-deficient BM cells to UV-induced apoptosis is associated with a failure to activate the stress response p38 kinase. BM cells from WT, Gadd45–/–, and Gadd45b–/– mice were exposed to UV radiation (25J/m2), without or with pretreatment with 20 µM SB202190. A, apoptosis was assessed after 24 h in culture by measuring Annexin V staining followed by flow cytometry. Data represent the mean ± S.D. of three experiments. B, cells were collected at indicated times, and protein extracts were analyzed for p38 activity by a kinase assay using ATF-2 as substrate. Proteins were resolved on Western blots, and phosphorylation of ATF-2 was determined with phospho-ATF-2 antibody and total p38 with p38 antibody.

 
Mice—Generation, genotyping, and use of WT, Gadd45a–/–, and Gadd45b–/– mice and age-matched littermates were as described before (23). Mice were housed at the animal facility at Temple University and used at the age of 6–8 weeks with an approved Institutional Animal Care and Use Committee Protocol that followed the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Both Gadd45a–/– and Gadd45b–/– mice were maintained in a mixed (BL6/129VJ) genetic background and propagated using heterozygous breeding pairs.

Bone Marrow Culture—For each experiment indicated, Gadd45a–/–, Gadd45b–/–, and their wild-type littermate mice (6–8-weeks old), were sacrificed and both the femurs were removed. Bone marrow (BM) cells were flushed from each femur and cultured in IMDM. For all indicated experiments, BM cells were cultured in IMDM (with 10% heat-inactivated fetal bovine serum and supplemented with growth factors (rIL-3 (10 ng/ml), rSCF (25 ng/ml), rIL-6 (10 ng/ml)) and cultured for 48 h at 37 °C in humidified atmosphere with 10% CO2 prior to treatment with UV radiation. Cytospin preparations of the BM cells, following culturing as indicated, have shown that >90% of the cell population consisted of myeloid cells at the blast and early intermediate stages of differentiation for all the genotypes.

Retroviral Transduction of BM Cells—Transduction of BM cells was done as previously described (23). Briefly, MIGW-Gadd45a and MIGW-Gadd45b retroviral constructs were generated in this laboratory. Infectious virus particles were produced by transient transfection of amphotropic PhoenixTM retroviral packaging cells (Orbigen, San Diego, CA) with the retroviral plasmids. Retroviral supernatants were collected 48 h after transfection. BM cells were cultured in IMDM medium supplemented with high growth factors Flt-3 ligand (100 ng/ml), rSCF(50 ng/ml), rIL-6 (10 ng/ml), and rIL-3 (25 ng/ml) at 37 °C for 48 h. Subsequently, cells were resuspended in virus supernatant in the presence of polybrene (8 µg/ml) and plated on retronectin-coated plates. Cells were harvested after 72 h and washed, and efficiency of infections was determined by fluorescence-activated cell sorting (FACS) analysis. Cultures were sorted for fluorescent cells (GFP positive) by FACS (BD FACSAria); the sorted cells were collected into the BM culture medium and returned to the CO2 incubator immediately after sorting. After a couple hours of recovery, cells were exposed to UV radiation (25 J/m2), and at indicated time points cell extracts were prepared for Western blot analysis using JNK and p38 phospho-specific antibodies. At 24 h the fraction of apoptotic cells was identified using Annexin V conjugated to PE (fluorescence emission spectra read in FL-2).

Western Blot Analysis—BM cells cultured for 48 h as indicated above were exposed to UVC (25J/m2) and cultured for various times, as indicated in the appropriate figures and figure legends. Cells were washed with cold phosphate-buffered saline and lysed with 1x lysis buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride, and protein content was determined using Bio-Rad protein assay dye reagent. 50 µg of total protein was resolved on 12% SDS-PAGE and transferred onto polyvinylidene difluoride membrane; membrane was then blocked using 5% nonfat dried milk in Tris-buffered saline-T. Blocked membranes were then probed with antibodies as indicated and subsequently incubated with 1:2500 dilution of peroxidase-conjugated secondary antibody. Blots were developed using the ECL chemiluminescence system (Amersham Biosciences). For reprobing, membranes were stripped by incubating at room temperature for 15 min in stripping buffer (Pierce Biotechnology) and reprobed with another antibody.

Kinase Assay—P38, JNK, and ERK kinase activity was determined using a p38 MAPK assay kit, a stress-activated protein kinase (SAPK)/JNK MAPK kit, and an ERK MAPK kit, respectively, from Cell Signaling Technologies as recommended by the manufacturer. Briefly, endogenous phospho-p38 was immunoprecipitated from 200 µg of cell lysates with immobilized anti-phospho-p38, without or with p38 inhibitor, and p38 kinase activity was measured using ATF-2 fusion protein as substrate. The reaction was initiated by the addition of ATP, and phosphorylation of ATF-2 was measured with phospho-ATF-2 antibody. Endogenous JNK/SAPK was pulled down by the c-Jun fusion protein beads in the absence or presence of JNK inhibitor, and the kinase reaction was carried out in the presence of cold ATP. c-Jun phosphorylation was measured using a phospho-c-Jun antibody. A monoclonal phospho-antibody to p44/p42 mitogen-activated protein kinase was used to immunoprecipitate active ERK from lysates. The immunoprecipitate was then incubated with an Elk-1 fusion protein in the presence of cold ATP. Phosphorylation of Elk-1 was measured using a phospho-Elk-1 antibody.


Figure 2
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  		FIGURE 2.
Prolonged JNK activation in Gadd45b–/– BM cells is associated with increased sensitivity to UV-induced apoptosis. BM cells from WT, Gadd45–/–, and Gadd45b–/– mice were exposed to UV radiation (25J/m2), without or with pretreatment with the JNK-specific inhibitor SP600215 (25 µM). A, following the indicated times protein extracts were analyzed for JNK activity by kinase assay with c-Jun as substrate as described under "Experimental Procedures." Western blots were used to measure c-Jun phosphorylation with a phospho-c-Jun antibody and total JNK with JNK antibody. B, after 24 h in culture, cells were harvested, and apoptosis was measured by Annexin V staining followed by flow cytometry. Data represent the mean ± S.D. for three independent experiments. C, following 30 and 60 min in culture, protein extracts were analyzed for MKK4 and MKK7 phosphorylation using phospho-MKK4 and phospho-MKK7 antibody to probe Western blots. Total MKK4 and MKK7 levels were measured with antibody against MKK4 and MKK7, respectively.

 
Assay for Apoptosis—To induce apoptosis, BM cells, following culturing for 48 h as indicated above, were seeded at 1.0 x 106 cells/30 mm and exposed to UVC (25 J/m2), after which cells were cultured for 24 h. Cells were then harvested and washed once with phosphate-buffered saline (pH 7.2); apoptosis was measured by staining with Annexin V-fluorescein isothiocyanate and propidium iodide and analyzed by flow cytometry (FACScan, Cell Quest software; BD Biosciences) as detailed previously (23).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Sensitivity of Gadd45a-deficient BM Cells to UV-induced Apoptosis Is Associated with Failure to Activate the Stress Response p38 Kinase—We previously have shown that Gadd45a–/– and Gadd45b–/– BM cells are more sensitive to UV-induced apoptosis than WT cells (Ref. 23 and Fig. 1A). Because GADD45 proteins have been implicated in activation of the stress response p38 kinase (24),3 it was logical to determine the status of p38 activity following exposure of WT, Gadd45a-deficient, and Gadd45b-deficient BM cells to UV radiation.


Figure 3
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  		FIGURE 3.
Both Gadd45a–/– and Gadd45b–/– BM cells display normal p42/p44 ERK activation in response to UV radiation. BM cells from WT, Gadd45a–/–, and Gadd45b–/– mice were treated with 25J/m2 UV radiation and following the indicated times cells were collected. Protein extracts were analyzed for ERK activity by kinase assay using Elk-1 as substrate, as described under "Experimental Procedures." Phosphorylation of Elk-1 was measured using a phospho-Elk-1 antibody, and total ERK was analyzed with ERK antibody.

 
As shown in Fig. 1B, using an in vitro kinase assay with ATF-2 as substrate to measure p38 activity, in WT BM cells and Gadd45b–/– cells robust P38 activation was observed within 30 min and reached a maximum in 60 min. In contrast, in GADD45a–/– BM cells, p38 was not activated in response to UV radiation. These observations indicate that Gadd45a is essential for activation of p38.

To confirm that the Gadd45a-mediated protection against UV radiation is via activation of p38, we tested the effect of the p38-specific inhibitor SB202190 on the WT and different mutant BM cells. As can be seen in Fig. 1B, SB202190 (20 uM) completely inhibited UV-induced p38 activation in both WT and Gadd45b–/– BM cells. Under the same experimental conditions SB202190 significantly increased UV-induced apoptosis in WT BM cells. In contrast, SB202190 did not significantly increase apoptosis in either Gadd45a–/– or Gadd45b–/– BM cells (Fig. 1B). The results using Gadd45a–/– BM cells is expected, because p38 kinase activity is not induced in these cells. However, the lack of an increase in apoptosis for Gadd45b–/– cells despite the loss of p38 activation was not anticipated and is discussed below.

To confirm the link between GADD45a, p38 kinase activation, and apoptosis, wild-type GADD45a was re-introduced into these Gadd45a-deficient BM cells using high efficiency retroviral transduction (supplemental Fig. S1). Re-expression of GADD45a was observed to restore wild-type phenotypes, including p38 activation and apoptosis.

In conclusion, these observations demonstrate that Gadd45a is essential for activation of p38 MAPK, which plays a protective role in hematopoietic cells following UV treatment.

Prolonged JNK Activation in Gadd45b-deficient BM cells Is Associated with Increased Sensitivity to UV-induced Apoptosis—Gadd45b–/– BM cells displayed increased sensitivity to UV radiation despite activation of p38 kinase. This observation suggested that the survival role of Gadd45b in response to UV radiation involves players other than p38. Because GADD45 proteins have been implicated also in modulation of the activity of the stress response Jun kinase (JNK), the status of JNK activity was determined following exposure of WT, Gadd45a–/–, and Gadd45b –/– BM cells to UV radiation.

As shown in Fig. 2A, where JNK activity was measured using an in vitro kinase assay with c-Jun as the substrate, in WT BM cells JNK activity increased within 30 min following UV irradiation and was maintained up to 60 min, after which it declined to basal level by 2 h. Similar transient JNK activation was observed in Gadd45a–/– BM cells. In sharp contrast, in Gadd45b–/– BM cells JNK activity appeared significantly higher and prolonged, being sustained for up to at least 2 h following UV irradiation. These data are consistent with a role for GADD45b in curtailing JNK activity.


Figure 4
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  		FIGURE 4.
Kinetics of GADD45 protein expression in wild-type BM cells following UV radiation. WT BM cell lysates used to measure p38, JNK, and ERK activities were examined at the indicated times for Gadd45a and Gadd45b protein expression by Western blot, probing with polyclonal antibodies against GADD45a and GADD45b.

 
It was next asked whether limiting JNK protects BM cells from UV irradiation, and conversely, whether prolonged JNK activity increases the sensitivity to UV-induced cell death. This was determined by assessing the effect of blocking JNK activation by the JNK-specific inhibitor SP600125 in the various BM cells. BM cells from WT, Gadd45a–/–, and Gadd45b–/– mice were pretreated with SP600125 (25 µM) prior to UV irradiation. UV-induced activation of JNK was successfully inhibited without deleterious effects on survival of WT and Gadd45a–/– BM cells (Fig. 2, A and B). Most importantly, SP600215 significantly reduced the apoptotic response of the Gadd45b–/– cells to UV treatment, thereby demonstrating that prolonged JNK activity contributed to the enhanced cell death in UV-treated Gadd45b–/– BM cells.

To confirm the link between GADD45b, JNK, and apoptosis, wild-type GADD45b was re-introduced into these Gadd45b-deficient BM cells using high efficiency retroviral transduction (supplemental Fig. S2). Re-expression of GADD45b was observed to restore wild-type phenotypes, including JNK activation and apoptosis.

JNK kinases are activated via phosphorylation by the upstream MAPKK, including MKK4 and MKK7 (25–26). To assess which of these upstream kinases is modulated by Gadd45b, the phosphorylation status of MKK4 and MKK7 was examined in WT, Gadd45a–/–, and Gadd45b–/–cells following UV treatment. Using anti-phospho-MKK4-specific and anti-phospho-MKK7-specific antibodies, it was seen that MKK4 activation was prolonged in Gadd45b–/– BM cells compared with WT and Gadd45a–/– cells following exposure to UV radiation (Fig. 2C). These data indicate that following UV irradiation of BM cells Gadd45b functions to curtail the activity of MKK4, which in turn results in blunting JNK activity. Taken together, these data argue that GADD45b-mediated suppression of the level and duration of JNK activity in BM cells exposed to UV radiation protects the cells from UV-induced cell death.


Figure 5
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  		FIGURE 5.
GADD45a-mediated activation of p38 protection against UV radiation is via NF-{kappa}B. A, BM cells from WT and Gadd45a–/– mice were treated with 25J/m2 UV radiation, and at the indicated times cells were harvested and protein extracts were analyzed for phosphorylation and degradation of I{kappa}B{alpha}, using phospho-I{kappa}B{alpha} and I{kappa}B{alpha} antibody, respectively, to probe a Western blot. B, BM cells from WT mice were treated with 25J/m2 UV radiation, with or without the I{kappa}B inhibitor IKK II (20 uM Wedelolactone). At the indicated times cells were harvested and protein extracts were analyzed for I{kappa}B{alpha} by probing a Western blot with I{kappa}B{alpha} antibody. C, BM cells from WT and Gadd45a–/– littermates were exposed to UV radiation (25J/m2), with or without pretreatment with 20 µM I{kappa}B inhibitor. Following 24 h, apoptosis was measured by Annexin V staining followed by flow cytometry. Data represent the mean ± S.D. for three independent experiments. D, BM cells from WT mice were treated with 25J/m2 UV radiation, with or without p38 inhibitor (20 µM SB202190), and at the indicated times were analyzed by Western blot for phosphorylation and degradation of I{kappa}B{alpha} using phospho-I{kappa}B{alpha} and I{kappa}B{alpha} antibody, respectively. E, BM cells from WT and Gadd45a–/– mice were treated with 25J/m2 UV radiation, alone or in combination with P38-specific inhibitor SB202190 (20µM). At the indicated times, cells were analyzed by Western blotting for c-IAP1 and Bcl-xl expression using specific antibodies.

 
Gadd45a–/– and Gadd45b–/– BM Cells Display Normal p42/p44 ERK Activation in Response to UV Radiation—Because stimulation of ERK p42/p44 MAPK is an important survival pathway (25–26), we also investigated whether Gadd45a and/or Gadd45b survival functions involve activation of ERK. As shown in Fig. 3, using an in vitro kinase assay with Elk as substrate, exposure of wild-type BM cells to UV radiation resulted in ERK activation within 30 min. A similar degree of ERK activation was observed in Gadd45a–/– and Gadd45b–/– BM cells. These results show that UV-induced activation of ERK is not modulated by either Gadd45a or Gadd45b.

Expression Kinetics of GADD45a and GADD45b Proteins in Wild-type BM Cells Exposed to UV Radiation Are Consistent with These Proteins Playing a Role in the Regulation of p38 and JNK Activities—Activation of JNK and p38 was observed within 30–60 min following exposure of BM cells to UV radiation. Thus, it was important to establish that the expression kinetics of GADD45a and GADD45b proteins are consistent with these proteins playing a role in p38 and JNK MAPK activation. As shown in Fig. 4, maximal expression of GADD45a and GADD45b was observed within 20 min following exposure of the BM cells to UV radiation. These data are consistent with the suggested role for Gadd45a and Gadd45b as modulators of p38 and JNK MAPK activities, respectively.

Protective Role of Gadd45a-mediated Activation of p38 Involves Activation of NF-{kappa}B—NF-{kappa}B is a ubiquitous transcription factor with an established role in cell survival (27–28). Our data demonstrate that Gadd45a-mediated activation of p38 plays a role in protection of BM cells against UV-induced apoptosis. Thus, we asked whether the protective role of Gadd45a-mediated activation of p38 involves activation of NF-{kappa}B.

NF-{kappa}B is normally present in the cytoplasm in an inactive state and is bound to members of the I{kappa}B inhibitor protein family, chiefly I{kappa}B{alpha} (Refs. 29 and 30 and references therein). Phosphorylation-dependent degradation of I{kappa}B, which allows translocation of NF-{kappa}B to the nucleus and subsequent activation of NF-{kappa}B target genes, is indicative of NF-{kappa}B activation (29–30). Transient phosphorylation and degradation of I{kappa}B was observed in WT BM cells exposed to UV radiation; phosphorylation and degradation of I{kappa}B was evident within 20 min following UV irradiation, with the level of I{kappa}B increasing by 60 min (Fig. 5A). Similar kinetics were observed in Gadd45b–/– cells (data not shown). In contrast, in Gadd45a–/– BM cells exposed to UV radiation I{kappa}B was neither phosphorylated nor degraded (Fig. 5A). These data demonstrate that Gadd45a is required to activate NF-{kappa}B following UV irradiation.


Figure 6
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  		FIGURE 6.
Schematic diagram indicating that GADD45a and GADD45b cooperate in protecting BM cells from UV-induced apoptosis via two distinct signaling mechanisms involving GADD45a-mediated activation of a p38-NF-{kappa}B survival pathway and GADD45b-mediated inhibition of prolonged pro-death JNK signaling.

 
To examine whether NF-{kappa}B activation plays a survival role following UV treatment of BM cells, the effect of an inhibitor specific to IKK, the kinase involved in phosphorylation of I{kappa}B, on UV-induced apoptosis of WT compared with Gadd45a–/– BM cells was assessed. As shown in Fig. 5, B and C, inhibition of IKK kinase activity, as evident by abrogation of IKK phosphorylation-dependent degradation of I{kappa}B{alpha}, increased apoptosis of WT BM cells to the level observed in the Gadd45a–/– cells. It can be concluded that NF-{kappa}B does participate in protecting again UV-induced apoptosis.

In addition to Gadd45a–/– BM cells being defective in NF-{kappa}B, p38 activation was also found to be impaired. These observations raised the possibility that activation of p38 is required for activation of NF-{kappa}B. To determine whether this is the case, we assessed the effect of the p38 inhibitor SB202190 on phosphorylation and degradation of I{kappa}B. Treatment of BM cells with SB202190 resulted in inhibition of I{kappa}B phosphorylation and degradation following exposure to UV radiation (Fig. 5D). Taken together, these results argue that Gadd45a-mediated activation of p38 protects BM cells against UV-induced apoptosis via activation of the survival factor NF-{kappa}B.

The survival function of NF-{kappa}B is mediated, at least in part, via induction of the anti-apoptotic proteins c-IAP-1 and BcL-XL. Thus, the next logical step was to determine whether c-IAP-1 and BcL-XL are induced in WT BM cells and impaired in the Gadd45a null BM cells. Up-regulation of c-IAP-1 and BcL-XL following exposure of WT BM cells to UV radiation was observed; this up-regulation was completely impaired in Gadd45a–/– cells (Fig. 5E). Furthermore, the p38 inhibitor significantly reduced UV-induced up-regulation of c-IAP-1 and BcL-XL in WT cells. These data further substantiate the notion of the existence of a Gadd45a-p38-NF-{kappa}B signaling cascade that plays a role in protecting BM cells against UV-induced apoptosis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously shown that Gadd45a and Gadd45b protect myeloid hematopoietic cells from genotoxic stress-induced apoptosis (23). Here we investigated the molecular mechanisms used by Gadd45a and Gadd45b to exert their anti-apoptotic activity.

The data obtained revealed the novel mechanisms that mediate the pro-survival functions of Gadd45a and Gadd45b in hematopoietic cells following UV irradiation. We have shown that Gadd45a and Gadd45b cooperate in protecting hematopoietic cells from UV-induced apoptosis via two distinct signaling pathways, one involving activation of a novel Gadd45a-p38-NF-{kappa}B survival pathway and the other in which Gadd45b mediates inhibition of JNK activation.

Previous evidence has shown that Gadd45a mediates activation of p38 by TGFbeta to promote hepatocyte cell death (7). Furthermore GADD45a-mediated activation of p38 has been linked to apoptosis induced by UV radiation in keratinocytes. Our data provide the first evidence for cross-talk between GADD45a-mediated activation of p38 and the NF-{kappa}B pathway, which is known to play a major role in cell survival (27–28). Following UV irradiation GADD45a is essential for p38 activation. In addition, GADD45a-mediated activation of p38 results in phosphorylation and degradation of I{kappa}B, which in turn allows nuclear localization of NF-{kappa}B and activation of its target genes (Fig. 6). This GADD45a-p38-NF-{kappa}B cross-talk pathway differs from another recently documented cross-talk between p38 and NF-{kappa}B that had shown that p38 modulates the transcriptional activity of NF-{kappa}B via phosphorylation of RelA (31). Our data regarding a GADD45a-p38-NF-{kappa}B survival pathway that protects hematopoietic cells from UV-induced apoptosis differ sharply from previous work showing that p38 activation is linked to cell death in endothelial and epithelial cells (18, 20). These data are, however, consistent with other studies showing that activation of p38 is linked to survival in hematopoietic cells (32–33).

Our data indicate that GADD45b-mediated inhibition of UV-induced JNK activity cooperates with p38 activation in promoting hematopoietic cell survival. Previously, GADD45b induction by NF-{kappa}B and subsequent inhibition of JNK activity has been implicated in mouse embryo fibroblast survival in response to tumor necrosis factor {alpha} (21). In contrast, our studies using GADD45b–/– mouse embryo fibroblasts have shown that GADD45b deficiency does not prolong JNK activity in response to tumor necrosis factor {alpha} (34). The reason for this discrepancy is not clear. In this work, using BM cells deficient for GADD45b, we did obtain data indicating that UV-induced GADD45b blunts JNK activity and thereby promotes hematopoietic cell survival. Our data show that in myeloid cells Gadd45b targets MKK4 rather than MKK7 (35) as the upstream regulator of JNK activity. Whereas prolonged JNK activation has been linked to cell death, it has been suggested that transient activation of JNK plays a role in cell survival (36). Whether this may be the case in hematopoietic cells remains to be determined.

In conclusion, we have shown that in hematopoietic cells GADD45a and GADD45b cooperate via utilizing two distinct signaling paths, namely p38 activation and JNK inhibition, to protect hematopoietic cells from genotoxic stress-induced cell death (Fig. 6). Disrupting either pathway increases the apoptotic response of hematopoietic cells to UV treatment. Pretreatment of Gadd45b–/– BM cells with the p38 inhibitor did not alter the apoptotic response to UV radiation, although similar treatment of WT BM significantly increased the apoptotic response. These data suggest that although each pathway is necessary, the protective effects of the two pathways are not additive. Using mice deficient for both Gadd45a and Gadd45b, currently being bred, would further clarify this issue. Taken together, these data and previous observations in which GADD45-mediated activation of p38 and JNK has been implicated in cell death indicate that the nature of the stress stimulus encountered, its magnitude, and Gadd45 interaction with proteins that modulate its function, as well as the specific cell type, ultimately determine whether the outcome will be cell cycle arrest, DNA repair and cell survival, or apoptotic cell death. In this context it will be of interest to determine the mechanisms involved in protecting hematopoietic cells from VP-16 and daunorubicin-induced apoptosis on the one hand and on the other the mechanisms that promote TGFbeta-induced apoptosis.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant RO1 HL 70530-01 (to D. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and supplemental text. Back

1 To whom correspondence should be addressed: The Fels Inst. of Cancer Research and Molecular Biology and Dept. of Biochemistry, Temple University School of Medicine, 3307 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-6903; Fax: 215-707-2805; E-mail: lieberma{at}temple.edu.

2 The abbreviations used are: JNK, c-Jun N-terminal kinase; BM, bone marrow; FACS, fluorescence-activated cell sorter; ERK, extracellular signal-regulated kinase; WT, wild type; MAPK, mitogen-activated protein kinase; IMDM, Iscove's modified Dulbecco's medium. Back

3 D. Liebermann, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Arthur Balliet for help in FACS analysis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zhan, Q., Lord, K. A., Alamo, I., Jr., Hollander, M. C., Carrier, F., Ron, D., Kohn, K. W., Hoffman, B., Liebermann, D. A., and Fornace, A. J., Jr. (1994) Mol. Cell. Biol. 14, 2361–2371[Abstract/Free Full Text]
  2. Fornace, A. J., Jackman, J., Hollander, M. C., Hoffman-Liebermann, B., and Liebermann, D. A. (1992) Ann. N. Y. Acad. Sci. 663, 139–154[Abstract]
  3. Papathanasiou, M. A., Kerr, N. C., Robbins, J. H., McBride, O. W., Alamo, I., Jr., Barrett, S. F., Hickson, I. D., and Fornace, A. J., Jr. (1989) Mol. Cell. Biol. 10, 4196–4203
  4. Zhang, W., Bae, I., Krishnaraju, K., Azam, N., Fan, W., Smith, K., Hoffman, B., and Liebermann, D. A. (1999) Oncogene 18, 4899–4907[CrossRef][Medline] [Order article via Infotrieve]
  5. Takekawa, M., and Saito, H. (1998) Cell 95, 521–530[CrossRef][Medline] [Order article via Infotrieve]
  6. Selvakumaran, M., Lin, H. K., Tjin Tham Sjin, R., Reed, J., Liebermann, D., and Hoffman, B. (1994) Mol. Cell. Biol. 14, 2352–2360[Abstract/Free Full Text]
  7. Yoo, J., Ghiassi, M., Jirmanova, L., Balliet, A. G., Hoffman, B., Fornace, A. J., Jr., Liebermann, D. A., Bottinger, E. P., and Roberts, A. B. (2003) J. Biol. Chem. 278, 43001–43007[Abstract/Free Full Text]
  8. Kastan, M. B., Zhan, Q., El-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992) Cell 71, 587–597[CrossRef][Medline] [Order article via Infotrieve]
  9. Selvakumaran, M., Lin, H. K., Miyashita, T., Wang, H. G., Krajewski, S., Reed, J. C., Hoffman, B., and Liebermann, D. (1994) Oncogene 9, 1791–1798[Medline] [Order article via Infotrieve]
  10. Guillouf, C., Grana, X., Selvakumaran, M., Hoffman, B., Giordano, A., and Liebermann, D. A. (1995) Blood 85, 2691–2698[Abstract/Free Full Text]
  11. Beadling, C., Johnson, K. W., and Smith, K. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2719–2723[Abstract/Free Full Text]
  12. Smith, M. L., Ford, J. M., Hollander, M. C., Bortnick, R. A., Amundson, S. A., Seo, Y. R., Deng, C. X., Hanawalt, P. C., and Fornace, A. J., Jr. (2000) Mol. Cell. Biol. 20, 3705–3714[Abstract/Free Full Text]
  13. Vairapandi, M., Balliet, A. G., Hoffman, B., and Liebermann, D. A. (1996) Oncogene 12, 2579–2594[Medline] [Order article via Infotrieve]
  14. Yang, J., Zhu, H., Murphy, T. L., Ouyang, W., and Murphy, K. M. (2001) Nat. Immunol. 2, 157–164[CrossRef][Medline] [Order article via Infotrieve]
  15. Lu, B., Ferrandino, A. F., and Flavell, R. A. (2004) Nat. Immunol. 5, 38–44[CrossRef][Medline] [Order article via Infotrieve]
  16. Hollander, M. C., Sheikh, M. S., Bulavin, D. V., Lundgren, K., Augeri-Henmueller, L., Shehee, R., Molinaro, T. A., Kim, K. E., Tolosa, E., Ashwell, J. D., Rosenberg, M. P., Zhan, Q., Fernandez-Salguero, P. M., Morgan, W. F., Deng, C. X., and Fornace, A. J., Jr. (1999) Nat. Genet. 23, 176–184[CrossRef][Medline] [Order article via Infotrieve]
  17. Liebermann, D. A., and Hoffman, B. (2002) Leukemia 16, 527–541[CrossRef][Medline] [Order article via Infotrieve]
  18. Hildesheim, J., Bulavin, D. V., Anver, M. R., Alvord, W. G., Hollander, M. C., Vardanian, L., and Fornace, A. J., Jr. (2002) Cancer Res. 62, 7305–7315[Abstract/Free Full Text]
  19. Zerbini, L. F., Wang, Y., Czibere, A., Correa, R. G., Cho, J. Y., Ijiri, K., Wei, W., Joseph, M., Gu, X., Grall, F., Goldring, M. B., Zhou, J. R., and Libermann, T. A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 13618–13623[Abstract/Free Full Text]
  20. Harkin, D. P., Bean, J. M., Miklos, D., Song, Y. H., Truong, V. B., Englert, C., Christians, F. C., Ellisen, L. W., Maheswaran, S., Oliner, J. D., and Haber, D. A. (1999) Cell 97, 575–586[CrossRef][Medline] [Order article via Infotrieve]
  21. De Smaele, E., Zazzeroni, F., Papa, S., Nguyen, D. U., Jin, R., Jones, J., Cong, R., and Franzoso, G. (2001) Nature 414, 308–313[CrossRef][Medline] [Order article via Infotrieve]
  22. Zazzeroni, F., Papa, S., Algeciras-Schimnich, A., Alvarez, K., Melis, T., Bubici, C., Majewski, N., Hay, N., De Smaele, E., Peter, M. E., and Franzoso, G. (2003) Blood 102, 3270–3279[Abstract/Free Full Text]
  23. Gupta, M., Gupta, S. K., Balliet, A. G., Hollander, M. C., Fornace, A. J., Hoffman, B., and Liebermann, D. A. (2005) Oncogene 24, 7170–7179[CrossRef][Medline] [Order article via Infotrieve]
  24. Bulavin, D. V., Kovalsky, O., Hollander, M. C., and Fornace, A. J., Jr. (2003) Mol. Cell. Biol. 23, 3859–3871[Abstract/Free Full Text]
  25. Roux, P. P., and Blenis, J. (2004) Microbiol. Mol. Biol. Rev. 68, 320–344[Abstract/Free Full Text]
  26. Zarubin, T., and Han, J. (2005) Cell Res. 5, 11–18
  27. Karin, M., and Lin, A. (2002) Nat. Immunol. 3, 221–227[CrossRef][Medline] [Order article via Infotrieve]
  28. Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., and Franzoso, G. (2004) J. Cell Sci. 117, 5197–5208[Abstract/Free Full Text]
  29. Ghosh, S., and Karin, M. (2002) Cell 109, S81–96
  30. Baldwin, A. S. (2001) J. Clin. Investig. 107, 241–246[CrossRef][Medline] [Order article via Infotrieve]
  31. Jijon, H., Allard, B., and Jobin, C. (2004) Cell. Signal. 16, 1023–1032[Medline] [Order article via Infotrieve]
  32. Park, J. M., Greten, F. R., Li, Z. W., and Karin, M. (2002) Science 297, 2048–2051[Abstract/Free Full Text]
  33. Platanias, L. C. (2003) Blood 101, 4667–4679[Abstract/Free Full Text]
  34. Amanullah, A., Azam, N., Balliet, A., Hollander, C., Hoffman, B., Fornace, A., and Liebermann, D. (2003) Nature 424, 741–742[Medline] [Order article via Infotrieve]
  35. Papa, S., Zazzeroni, F., Bubici, C., Jayawardena, S., Alvarez, K., Matsuda, S., Nguyen, D. U., Pham, C. G., Nelsbach, A. H., Melis, T., De Smaele, E., Tang, W. J., D'Adamio, L., and Franzoso, G. (2004) Nat. Cell. Biol. 6, 146–153[CrossRef][Medline] [Order article via Infotrieve]
  36. Liu, J., and Lin, A. (2005) Cell Res. 15, 36–42[CrossRef][Medline] [Order article via Infotrieve]

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