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Molecular Biology and Genetics |
Gene Response Section, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [J. H., D. V. B., M. C. H., L. V., A. J. F.]; Pathology/Histotechnology Laboratory, Science Applications International Corp., Frederick, Maryland 21702-1201 [M. R. A.]; and Data Management Services, National Cancer Institute at Frederick, Frederick, Maryland 21702-1201 [W. G. A.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One family of genes that is downstream, and perhaps upstream, of the MAPK cascade is the Gadd45 (growth-arrest and DNA damage-inducible) family. This family is composed of three genes: Gadd45a (Gadd45/Gadd45
), Gadd45b (Myd118/Gadd45ß), and Gadd45g (CR6/Gadd45
/OIG37; Refs. 26, 27, 28, 29, 30
). Whereas all of the family members are stress inducible, Gadd45b and Gadd45g appear to have prominent roles in cellular differentiation events (26
, 29
, 31)
, although Gadd45a is a p53-effector and stress-inducible gene (28)
. Gadd45a is an ubiquitously expressed Mr 21,000 acidic protein, which, like p53, has been implicated in many biological processes related to maintenance of genomic stability and apoptosis. Gadd45a (along with Gadd45b and Gadd45g), for instance, has been shown to activate p38 and JNK MAPKs by associating with and activating MEKK4/MTK1 MAPK kinase kinase (32)
, although this issue remains controversial, and has yet to be shown with a genetic approach for both Gadd45a and Gadd45b (33, 34, 35)
. In the same vein, JNK-mediated apoptosis in vitro has been reported to occur via Brca1-induced Gadd45a transactivation (36)
. Gadd45a is also able to associate with proteins involved in cell cycle regulation, such as p21 (Cdkna1), a cyclin-dependent kinase inhibitor (37)
, and Cdc2/cyclinB, a key kinase for G2/M progression (38)
; it also associates with proliferating cell nuclear antigen, which is involved in DNA replication and repair (39
, 40)
. Additionally, Gadd45a binds to core histones in damaged DNA (41)
. The development of Gadd45a-null mice has provided important insights for the in vivo roles of this gene (42, 43, 44)
. To a great extent, the phenotype of Gadd45a-null mice (42)
parallels that of Tp53-null mice (45
, 46)
. Whereas Gadd45a-null mice do not develop spontaneous tumors, these mice have an increased frequency of both ionizing radiation-induced and dimethylbenzanthracene-induced tumors (42
, 43)
. Like p53-deficient cells (47
, 48)
, cells derived from Gadd45a-null mice exhibited genomic instability, single oncogene transformation, loss of normal cellular senescence, increased cellular proliferation, incomplete cytokinesis, centrosome amplification, and reduced DNA repair (42
, 44)
. In the case of apoptosis, Gadd45a-null fibroblasts, thymocytes, and lymphocytes showed proficient apoptosis to a variety of stimuli, such as ionizing (42)
and UV radiation (35)
. Thus, there is substantial but not complete overlap in the cellular roles for Gadd45a and p53.
Although Gadd45a is one of numerous downstream targets of p53 (47) , the fact that: (a) Gadd45 family members are able to activate the p38/JNK MAPK pathway (32 , 49) ; and (b) Tp53-null and Gadd45a-null mice share many similarities (42) , led us to hypothesize that Gadd45a may have some role in the regulation of p53. Because p38 (18) , as well as JNK (19) , can contribute to p53 activation after stresses such as UV radiation, and because Gadd45a can be induced by both p53-dependent (50) and -independent (28) mechanisms, Gadd45a could conceivably contribute to maintaining p53 activity through a p38/JNK MAPK-mediated feedback loop that in turn leads to additional increased expression of Gadd45a. In this report, we used both in vivo skin and in vitro primary keratinocyte culture systems to additionally elucidate the functions of endogenous Gadd45a pertaining to apoptosis, proliferation, and differentiation, all of which can contribute to tumorigenesis if perturbed. The importance (and relevance) of using UVB and solar radiation (290–320 nm and 290–400 nm, respectively) as the genotoxic agent instead of UVC radiation (240–290 nm) and skin as the organ system of choice is based on the fact that UVA and UVB are naturally occurring carcinogens, whereas UVC radiation does not appreciably penetrate the atmosphere, and that epidermal cells are the primary target of solar radiation. Herein we demonstrated that UV-irradiation of murine skin and Gadd45a-null epidermal keratinocytes had compromised p38, JNK, and p53 activation rendering the cells resistant to apoptosis and deficient in G1 and G2 checkpoint control. Whereas wt mice adequately responded to UV-induced stresses, the defects observed in Gadd45a-null mice and/or epidermal keratinocytes ultimately lead to a pronounced increase in susceptibility to UV-induced lesions, including hyperplasias, squamous cell papillomas, and carcinomas comparable with observations reported previously with Tp53-null mice (51) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dermal fibroblasts were harvested from the dermis of the same mice after Dispase treatment. Dermis was digested with collagenase (3.5 mg/ml; Worthington Biomedical Corp.) in DMEM and subsequently subject to two centrifugation steps to first pellet all dermal cells (fibroblasts and follicular keratinocytes, 3 min at 1200 rpm), and second, to eliminate the heavier follicular keratinocyte pellet (two times 3 min at 400 rpm). Dermal fibroblasts remaining in supernatant are seeded in regular flasks and cultured with DMEM/10% fetal bovine serum. Cells were fed every other day.
Cell Cycle Analysis.
Primary keratinocytes and dermal fibroblasts were allowed to reach 
70% confluence before they were UV-irradiated with the specified doses. For G1 checkpoint analysis, cells were pulsed for 3 h with 10 µM BrdUrd 15 h after irradiation. Pulsed cells were in turn harvested by trypsinization followed by centrifugation at 350 x g for 5 min. Cell pellets were fixed in 70% ethanol at -20°C for at least 1 h and hydrolyzed with 2 N HCl/0.5% Triton X-100 for 30 min at room temperature. HCl was in turn neutralized with 0.1 M sodium borate (pH 8.5). After washing the cells with 1x PBS, the pellet was resuspended in blocking solution (PBS containing 0.05% Tween 20/1% BSA) for 30 min, followed by 1-h incubation with FITC-conjugated anti-BrdUrd antibody (BD PharMingen). Lastly, cells were washed with 1x PBS and resuspended in FACS solution (PBS with 5 µg propidium iodide/10 µg/ml RNase).
For G2 checkpoint analysis, cells were fixed at indicated time points after UV irradiation in 70% ethanol. After washing the pellet with 1x PBS, cells were blocked with 2% BSA/0.05% Tween 20 in PBS for 20 min at room temperature. Subsequently, cells were incubated for 1 h with blocking solution containing antihistone H3 antibody (1:1000 dilution; Upstate Biotech). Next, cells were incubated with secondary antibody (Cy2-conjugated antirabbit IgG; Amersham Pharmacia Biotech) for 30 min at room temperature, washed with 1x PBS, and resuspended in FACS solution as described above. Mitotic index corresponds to the fraction of cells in mitosis.
Real-Time PCR.
Total RNA was extracted with Trizol from adherent cells as recommended by the manufacturer (Life Technologies, Inc.). cDNA synthesis was performed according to standard protocols (ThermoScript RT PCR System; Life Technologies, Inc.) on 1 µg of DNase-treated (DNA-free; Ambion) total RNA with a combination of random-hexamer and oligo(dT)20 primer. In turn, the single-stranded cDNA was used as template for real-time PCR performed with an ABI PRISM 7700 sequence detection system. Briefly, the total volume/reaction was 25 µl and composed of 50 ng of single-stranded cDNA template, 1x SYBR Green PCR master mix (Applied Biosystems), and 200 nM primer mix (forward and reverse primers combined). Templates were subject to 40 cycles of: denaturation (94°C, 20 s), annealing (55°C, 20 s), and extension (72°C, 30 s). Primer sets used are as follows: Gadd45, 5'-GGTGAGCC TGAAGAAGGAAGCT-3' (forward), 5'-TTCTTGCAGTGCTTTGTAGTTTTTG-3' (reverse); Gadd45ß, 5'-TACATAT-TTGACAGCCCCCTCA-3' (forward), 5'-CAGAAG GTATCACGGGTAGGGT-3' (reverse); Gadd45, 5'-AGCCGACTGCACTGCTCTTT-3' (forward), 5'-ACGATAGCGTCCTTTAGAA-AATGAA (reverse); and glyceraldehyde-3-phosphate dehydrogenase, 5'-GAA GGTGAAGGTCGGAGTC-3' (forward), 5'-GAAGAT-GGTGATGGGATTTC-3' (reverse).
In Vivo Apoptosis Assay.
Either newborn C57BL6/129 pups or 6-week-old depilated adult C57BL6/129 mice were irradiated with 1000 Jm-2 UVB generated from four Westinghouse FS20 SunLamp bulbs (270–385 nm emission spectrum with peak at 313 nm). The energy emitted by the lamps was measured with a model PMA2100 meter (Solar Light Co.) and a model PMA2106 UVR detector calibrated to register the energy from 282 to 326 nm. Trunk skin was harvested 24 h after irradiation and immediately fixed in 10% neutral buffered formalin. Five µm-thick sections of paraffin-embedded tissues were used for the TUNEL assay according to the manufacturer’s recommendations (TACS2 terminal deoxynucleotidyl transferase-Blue Label In Situ Apoptosis Detection kit; Trevigen). Briefly, end-labeling of fragmented DNA was performed with a reaction mix composed of 2 µl terminal deoxynucleotidyl transferase (15 units/µl), 2 µl cobalt cation (50 mM), 2 µl deoxynucleoside triphosphate mix (0.25 mM each biotinylated nucleotide triphosphate), and 100 µl labeling buffer. Samples were incubated for 60 min in a humidified chamber. Samples were in turn incubated with streptavidin-HRP (1:750 dilution) for 10 min. After conjugation, all of the samples were incubated for precisely the same length of time (2.5 min) with HRP substrate. Lastly, sections were counterstained with Red Counterstain C (Trevigen) and visualized with light microscopy. Ten animals/genotype were used (5 irradiated versus 5 unirradiated).
RNase Protection Assay.
Total RNA was extracted from full thickness adult mouse skin with Trizol (Invitrogen) and subject to the RiboQuant multiprobe RNase protection assay as per manufacturer’s recommendations (BD PharMingen). Briefly, the multiprobes were synthesized by incubating 50 ng of RNase protection assay template set for 60 min at 37°C with [-32P]UTP (3000 Ci/mmol, 10 mCi/ml) and other components supplied in the kit such as transcription buffer, cold nucleotides, T7 polymerase, RNasin, and DTT. The synthesis reaction was terminated with DNase by incubating samples at 37°C for an additional 30 min. Radiolabeled probes (6 x 105 cpm/reaction) were hybridized overnight at 56°C to 20 µg of total RNA and subsequently incubated with RNase A+T1 mixture at 30°C for 45 min, and proteinase K for an additional 15 min at 37°C. After phenol-chloroform extraction and ethanol precipitation, the RNase-digested samples were resuspended in loading buffer and resolved on a 6% polyacrylamide, 8 M urea denaturing gel. Quantitation was performed with a phosphorimager.
Immunohistochemistry.
Newborn mice were irradiated as described above. Trunk skin was harvested 1 h after irradiation and immediately fixed in 10% neutral buffered formalin. Five-µm thick sections were deparaffinized and subject to antigen unmasking with 10 mM sodium citrate buffer (pH 6.0). After unmasking, sections were incubated with 1% hydrogen peroxide for 10 min to eliminate endogenous peroxidase activity. After the quenching step, sections were washed several times in water and 1x PBS, and subsequently blocked for 1 h with 5% goat serum in PBS. Primary antibody (1:100 dilution of F-5 anti-p21 monoclonal antibodies; Santa Cruz Biotech.; 1:50 dilution of Phospho-p38 MAPK polyclonal antibody, 1:20 dilution of Phospho-JNK MAPK monoclonal antibody; Cell Signaling Technology, Inc.; and 1:50 dilution of Gadd45a polyclonal antibody; Santa Cruz Biotech.) was then applied in blocking solution, and sections were incubated overnight at 4°C. The following morning, secondary antibody was added (1:200 dilution of biotinylated antirabbit or antimouse IgG). For HRP conjugation and substrate detection, the Vectastain ABC kit was used as described by the manufacturer (Vector Laboratories). Four (p21) and 8 (p38, JNK, and Gadd45a) animals per genotype were used (2 irradiated per time point and 2 unirradiated).
UV Carcinogenesis and Histopathology.
Hairless mice were generated by crossing SKH1-hairless mice with the established C57BL6/129 Gadd45a-null mouse strain (42)
. Littermates from hairless Gadd45a+/-crosses were used. Animals were age and gender-matched and subjected to three weekly doses of 1000 Jm-2 of UVB (Westinghouse FS40 SunLamp fluorescent tube) and 7300 Jm-2 of UVA (blacklight fluorescent tube; emission spectrum of 310–400 nm with peak at 365 nm) radiation for a total of 52 weeks. For this strain, this exposure corresponds to one minimal erythema dose, and the inclusion of UVA radiation gave a spectrum comparable with solar radiation. Comprehensive histopathology evaluation was performed on skins of 10 mice (5 wt and 5 Gadd45a-null). Entire skins were fixed in 10% buffered neutral formalin. All of the gross lesions on dorsal skin of trunk, pinnae, and tail were embedded in paraffin, sectioned at 5 µm, and stained with H&E.
Statistical Analysis.
Tumor multiplicities between wt and Gadd45a-null mice were compared using the nonparametric Wilcoxon rank-sum test because of the lack of homogeneity of variance between groups on the specific criterion of interest. Probabilities reported are two-sided.
Transient Transfection and CAT Assay.
Cells were transduced with LipofectAMINE 2000 reagent according to manufacturer’s recommendations (Invitrogen). Briefly, 10-cm plates were seeded with 300,000 cells and transfected 2 days later with 10 µg/plate of p53RE-CAT plasmid along with 20 µg of LipofectAMINE 2000/plate. Transfected cells were allowed to rest for 2 more days, after which they were irradiated with the specified doses of UVB. Total cell lysates were prepared 2 days after irradiation, and CAT assays were performed according to established protocols. Two independent experiments were performed in duplicate sets.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Both Gadd45a-null and wt newborn mice were irradiated with a dose ranging from 0.8 to 5 minimum erythema doses in different human skin types (54)
. At the established peak response time of 24 h after irradiation (55)
, their skin was harvested and analyzed by TUNEL assay. Whereas wt mice had appreciable numbers of TUNEL-positive keratinocytes after irradiation in both the basal and nucleated suprabasal keratinocytes, mice lacking Gadd45a were resistant to apoptosis and only rarely showed sunburn/TUNEL-positive cells. Similar results were obtained from adult mice (Fig. 2
, top and middle panels). Histological analysis of the UVB-irradiated adult mouse skin revealed 2.5-fold greater incidence of sunburn cells in wt mice as compared with Gadd45a-null mice. Interestingly, adult wt mice also demonstrated increased sensitivity to UV irradiation in comparison to Gadd45a-null mice as manifested by more pronounced erythema and the emergence of subcorneal pustules and epidermal erosion (Fig. 2
, bottom panels) coincident with elevated inflammatory cytokine responses (Fig. 3)
. Therefore, we conclude that Gadd45a, like p53, is intimately involved in orchestrating events leading to apoptosis in the skin, which may contribute to the observed inflammation in the dermis.
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. Whereas anti-phospho-specific p38 antibodies (which detect activated p38) clearly stained granular keratinocytes in wt mice 15 min to 1 h after UV radiation, p38 activation was not appreciably maintained and/or detected in the epidermis of irradiated Gadd45a-null mice beyond 15 to 30 min after UV radiation (Fig. 4A)
. The suprabasal localization of active p38 in murine skin is in accordance to what has been reported previously (57)
.
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. Whereas wt mice readily showed evidence for persistent JNK activation within the 15–60 min time frame, this was not observed in Gadd45a-null mice. Only transient and modest JNK activation was detected in Gadd45a-null skin at 30 min after UV radiation. Interestingly, unlike p38, active JNK was detected predominantly in the basal layer of the epidermis. Together, maintenance of p38 and JNK activation correlated well with the accumulation of Gadd45a protein in the epidermis 1 h after UV irradiation (Fig. 4C)
and the eventual emergence of TUNEL-positive nuclei of keratinocytes in all of the epidermal layers 24 h after UVR (Fig. 1)
. These results, along with the observation that Gadd45a is required for UV-induced sunburn cells, are consistent with Gadd45a as an upstream component of the MAPK signaling pathway involved in the normal maintenance of both p38 and JNK MAPK activity (18
, 58)
.
UVB Radiation-induced p53 Activation Is Dependent on Gadd45a.
Both p38 and JNK have been implicated in normal p53 activation and p53-mediated apoptosis after UV radiation (18
, 19)
. Furthermore, although it has been estimated that p53 has well over 100 downstream target genes (47)
, the fact that Gadd45a-/- and Tp53-/- epidermal keratinocytes behave very similarly under genotoxic stress conditions suggests that either: (a) Gadd45a is the primary p53-effector gene in skin; and/or (b) that Gadd45a is not only a downstream p53-effector gene but is also, under some circumstances, necessary for p53 activation. To determine the latter, an UVB radiation-induced p53 transcriptional activation assay was performed with wt and Gadd45a-/- primary keratinocytes. UV irradiation of primary keratinocytes transfected with a chloramphenicol acetyltransferase reporter plasmid driven by a p53 response element clearly demonstrated that in the absence of Gadd45a, keratinocytes had a marked reduction in p53 activation (Fig. 5A)
. Whereas pronounced p53 activation was observed in Gadd45a+/+ cells irradiated with UVB (6-fold and 14-fold activation above background for UV doses of 150 and 250 Jm-2, respectively), relatively little or no p53 activation was observed in Gadd45a-/- cells (no activation and 2.5-fold activation above background, respectively, for the two doses; Fig. 5B
). To determine whether the same effects occurred in vivo, p53 activity was indirectly determined by performing immunohistochemistry on UV-irradiated newborn mouse skin with anti-p21WAF1/Cip1-specific antibodies (Fig. 5C)
. Whereas p21 protein levels increase substantially in the nuclei of wt basal keratinocytes 4 h after UV radiation, only modest protein accumulation is detected in Gadd45a-null equivalents, indicative of reduced p53 activity in the absence of Gadd45a protein. The fact that UV radiation-induced p53 activation is dependent on Gadd45a, suggests that whereas Gadd45a is a p53-effector gene, it can also contribute to p53 activation. Consequently, this places Gadd45a both upstream and downstream of p53.
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3-fold greater than the G1:S ratio for irradiated Gadd45a-null cells at 250 Jm-2 (Fig. 6A)
. Whereas both wt and Gadd45a-null cells have a dose-dependent G1 checkpoint response to UV radiation, Gadd45a-null cells are consistently less responsive. Considering that this checkpoint is nearly completely abrogated in p53-null, our findings in Fig. 5
and Fig. 6A
indicate that this checkpoint deficit in Gadd45a-null keratinocytes is because of attenuated p53 signaling. Interestingly, contrary to the observed defects in UV stress-induced G1 checkpoint response, Gadd45a-null keratinocytes respond effectively to classic differentiation and antiproliferative stimuli, such as calcium and transforming growth factor ß1-induced G1 arrest, which play a role in differentiation of suprabasal epidermal keratinocytes (data not shown; Refs. 61, 62, 63, 64, 65, 66
).
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. G2 checkpoint activation was measured by a reduction in the mitotic index in the first 4 h, and thus focuses primarily on cells already in G2 when irradiated. Nearly all of the wt cells (
80%) were arrested by 1 h after UV radiation, and this arrest persisted during the following 3 h of the study. In contrast, only a marginal delay is seen for Gadd45a-null cells at 1 h, and the maximum checkpoint at 2 h was <40%. This weak checkpoint was transient, and the mitotic index returned to unirradiated levels by 4 h. A similar response was seen in Tp53-null keratinocytes where only marginal checkpoint activation is seen in the first 2 h with return to unirradiated levels by 4 h. Whereas G1 checkpoint abrogation is more pronounced in Tp53-null cells (Fig. 6A)
, interestingly the G2 checkpoint deficit is comparable with that of Gadd45a-/- cells. We conclude from these studies that Gadd45a, like p53, plays a pivotal role in sequestering keratinocytes in both G1 and G2 after UV radiation-induced stress. What remained to be seen was whether our results with keratinocytes are applicable to other skin cell types.
Abrogation of UVB Radiation-induced G1 and G2 Checkpoints Is Keratinocyte-specific.
To determine whether the involvement of Gadd45a in UV radiation-induced cell cycle arrest is cell-type specific, primary dermal fibroblasts were tested under similar conditions. In contrast to keratinocytes, Gadd45a-/- dermal fibroblasts did not manifest any G1 or G2 checkpoint deficits after UV radiation relative to Gadd45a+/+ dermal fibroblasts (Fig. 6, C and D
, respectively). On the basis of FACS analysis, both cell types responded equally well, in a dose-dependent manner, to various doses of UVB radiation. The observed differences in response to UV radiation for the two cell types could be because of a redundancy within the Gadd45 gene family, and it is conceivable that one or more Gadd45 genes could compensate for the lack of Gadd45a. To test this possibility, a comparison of the relative mRNA expression levels of the three Gadd45 genes in both primary keratinocytes and dermal fibroblasts was performed by quantitative reverse transcription-PCR (Fig. 6, E and F)
. Interestingly, whereas similar basal levels of Gadd45b expression are detected in keratinocytes and dermal fibroblasts (data not shown), the expression profile for Gadd45a (Fig. 6E)
and Gadd45g (Fig. 6F)
are significantly different for the two cell types. Primary dermal fibroblasts have a 2.6-fold lower expression of Gadd45a and a 10-fold greater expression of Gadd45g relative to primary keratinocytes. The relative expression levels noted for Gadd45b and Gadd45g in wt cells are maintained in Gadd45a-null cells (data not shown), and indicate that no compensatory mechanism exists in Gadd45a-/- keratinocytes and dermal fibroblasts.
Gadd45a Protects the Epidermis against Solar Radiation-induced Carcinogenesis.
Because we demonstrate that: (a) Gadd45a contributes to p53 activation via MAPK signaling; (b) the Gadd45a-null sunburn phenotype mimics that of Tp53-null mice; and (c) it is known that p53 protects against UV radiation-induced squamous cell carcinomas (51
, 67
, 68)
, we were interested in determining whether UV-irradiated Gadd45a-null mice would have increased predisposition to skin tumorigenesis, as do Tp53-null mice (51)
. Gadd45a-null and wt mice littermates were irradiated three times per week for 1 year with a source that mimics solar radiation and subsequently were processed for comprehensive histopathological examination. Overall, relative to wt mice, UV-irradiated Gadd45a-null mice demonstrated a dramatic increase in the total number of proliferative epidermal lesions including premalignant atypical epidermal hyperplasia, squamous cell papillomas, and squamous cell carcinomas (Table 1)
. The atypical hyperplasias consisted of discrete foci of thickened stratum spinosum with disorganization of nuclei and piling up of the basal epithelial cells, with frequent rete ridge protrusions into the dermis. The squamous cell tumors were typical of those normally described in skin carcinogenesis studies (Fig. 7
; Ref. 69
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, for instance, is produced by inflammatory cells and not keratinocytes. Much of the Gadd45a-dependent regulatory effects we observed in keratinocytes appear to be cell type-specific. Whereas we observed a dramatic difference in p38 and JNK activation in keratinocytes exposed to UV radiation, recent reports demonstrated no changes in p38 and JNK MAPK activation after UV irradiation of embryo fibroblasts derived from the same mouse models (35)
. Our results with dermal fibroblasts validate this observation, because Gadd45-null dermal fibroblasts behave similarly to the wt counterparts, and effectively block G1/S and G2/M transition after DNA damage. The fact that Gadd45g is expressed at appreciably higher levels in dermal fibroblasts is one possible explanation for why this particular cell type may be able to compensate for the lack of Gadd45a with proficient G1 and G2 checkpoint activation, in contrast to keratinocytes (Fig. 6, A–D)
. Additionally, our laboratory has reported previously that unlike Tp53-/- thymocytes, Gadd45a-/- thymocytes are as sensitive to DNA damage-induced apoptosis as are wt thymocytes (42)
. Once again, these phenotypic differences can be attributed not only to the fact that Gadd45 family proteins have overlapping functions, but also to our observation that the Gadd45 genes are differentially expressed in different cell types (Fig. 6, E and F)
. A provocative and controversial reported model implicates Gadd45 family proteins as upstream activators of p38 and JNK MAPK (32 , 49) . The Gadd45 genes are known to be stress responsive (28 , 70) . Consequently, stress-inducible Gadd45 family proteins could thus interact with and activate MTK1, which is followed by activation of its downstream targets, p38 and JNK. In contrast, it has been reported that both p38 (35) and JNK (34 , 35) activation not only precedes Gadd45a gene expression, but is also independent of de novo protein synthesis after stresses such as UV radiation. In the case of Gadd45b, its expression has been shown recently to have an antiapoptotic role in embryonic fibroblasts by down-regulating JNK, whereas having no effect on p38 or extracellular signal-regulated kinase activity (33) . In contrast, the activity of JNK and p38 during activation by T-cell receptor signaling has been reported to be markedly reduced in TH1 cells from Gadd45g-null mice. Whereas some of these findings may appear to contradict the former model by maintaining p53-effector genes as downstream targets of p38 and JNK, our data support the notion that Gadd45a is both downstream and upstream of p38/JNK MAPK, and is necessary for sustained MAPK signaling. We demonstrate that whereas the initial activation of both p38 and JNK MAPK is not dependent on Gadd45a, maintenance of MAPK activity beyond 15 to 30 min is dependent on newly synthesized Gadd45a protein. In fact, the effects that we observe with G1/G2-block, p53 activation, and apoptosis are all occurring well within the timeframe we observe de novo Gadd45a synthesis.
The manner in which Gadd45a-/- keratinocytes mimic the phenotype of Tp53-/- keratinocytes is not surprising in the context of Gadd45a being both upstream and downstream of p38/JNK. Whereas it is reasonable to assume that the antiproliferative effects of p38 and JNK are achieved via p53-mediated G1 arrest (32 , 71 , 72) , an increasing body of evidence also implicates p38 in G2 checkpoint regulation. It has been reported recently that initiation of an UV radiation-induced G2/M checkpoint is mediated by p38, which is responsible for the phosphorylation and repression of the G2/M transition initiator Cdc25B phosphatase, and that phosphorylation of this phosphatase is required for 14-3-3-mediated inhibition to occur (25) .
On the basis of our results, along with the information above, we propose a model that delineates how Gadd45a-mediated G1, G2 checkpoint, and apoptosis are achieved via a positive feedback loop with p38 and JNK (Fig. 8)
. Stress MAPK signaling is rapidly activated by UV radiation with induction of the Gadd45a gene by p53 signaling. Gadd45a, as well as Gadd34 (MyD116) and Gadd153 (CHOP), is also stress-inducible in p53-deficient cells via signaling involving p38 and other MAPKs (28
, 47
, 73)
. With increases in the level of Gadd45a after UV radiation, "feedback" signaling loops involving Gadd45a and upstream MAPK components, such as MTK1, maintain strong signaling of p38 and JNK. p53 activation will result in changes in transcription of p53-effector genes such as p21 (Cdkn1a), 14-3-3
, Gadd45a, and others involved in cell cycle control (71)
. p21 will induce G1 arrest, whereas multiple factors, such as 14-3-3 and Gadd45a, will contribute to G2 arrest (74)
. Gadd45a protein will directly contribute to G2 arrest by disrupting Cdc2/cyclin B1 kinase activity and indirectly by blocking Cdc25B phosphatase activity via p38 (25
, 38) . p38/JNK activation by Gadd45a will also indirectly contribute to G1 arrest through p53-mediated events. For example, inhibition of p38 activity has been shown previously to markedly attenuate p53 transcriptional activity and p53-mediated apoptosis after UV radiation (18)
. Whether the cell will undergo either cell cycle arrest or apoptosis may be determined by the severity of the insult as well as cellular context. Whereas transient p38/JNK activation will likely result in cell cycle block, prolonged p38/JNK activation will likely set the apoptotic program in motion. Loss of stress MAPK signaling can lead to inadequate protection against tumorigenesis. Indeed, a recent report clearly demonstrates that p38 MAPK is a critical tumor suppressor protecting against E1A and H-Ras-induced transformation of mouse embryonic fibroblasts (75)
. Moreover, a relatively high frequency of mutations in the Gadd45a gene has been identified recently in pancreatic tumors, additionally strengthening its link to cancer (76)
. Therefore, it is understandable how the absence of Gadd45a predisposes irradiated skin to tumors by enabling damaged and deregulated cells to not only survive the genotoxic insult, but also to proliferate after loss of normal checkpoint controls.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the National Cancer Institute, NIH, under contract No. N01-C0-12400 and N01-C0-12401. 
2 Supplementary data for this article is available at Cancer Research Online (http://cancerres.aacrjournals.org).
3 To whom requests for reprints should be addressed, at Gene Response Section, Center for Cancer Research, National Cancer Institute, NIH, Building 37, Room 6144, 37 Convent Drive, MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 402-0744; Fax: (301) 480-1946; E-mail: af6z{at}nih.gov
4 The abbreviations used are: MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; BrdUrd, bromodeoxyuridine; FACS, fluorescence-activated cell sorter; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; HRP, horseradish peroxidase; wt, wild-type; CAT, chloramphenicol acetyltransferase.
Received 7/ 8/02. Accepted 10/17/02.
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 S. Karger, C. Weidinger, K. Krause, S.-Y. Sheu, T. Aigner, O. Gimm, K.-W. Schmid, H. Dralle, and D. Fuhrer FOXO3a: a novel player in thyroid carcinogenesis? Endocr. Relat. Cancer, March 1, 2009; 16(1): 189 - 199. [Abstract] [Full Text] [PDF]
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 Y. Zhang, E. Calvo, C. Martel, V. Luu-The, F. Labrie, and A. Tchernof Response of the adipose tissue transcriptome to dihydrotestosterone in mice Physiol Genomics, November 12, 2008; 35(3): 254 - 261. [Abstract] [Full Text] [PDF]
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 X. Lu, C. Yang, R. Hill, C. Yin, M. C. Hollander, A. J. Fornace Jr, and T. Van Dyke Inactivation of gadd45a Sensitizes Epithelial Cancer Cells to Ionizing Radiation In vivo Resulting in Prolonged Survival Cancer Res., May 15, 2008; 68(10): 3579 - 3583. [Abstract] [Full Text] [PDF]
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 R. S. M. Shih, S. H. K. Wong, N. W. Schoene, and K. Y. Lei Suppression of Gadd45 Alleviates the G2/M Blockage and the Enhanced Phosphorylation of p53 and p38 in Zinc Supplemented Normal Human Bronchial Epithelial Cells Experimental Biology and Medicine, March 1, 2008; 233(3): 317 - 327. [Abstract] [Full Text] [PDF]
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 A. Fujita, J.R. Sato, H.M. Garay-Malpartida, P.A. Morettin, M.C. Sogayar, and C.E. Ferreira Time-varying modeling of gene expression regulatory networks using the wavelet dynamic vector autoregressive method Bioinformatics, July 1, 2007; 23(13): 1623 - 1630. [Abstract] [Full Text] [PDF]
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 L. Jirmanova, D. Jankovic, A. J. Fornace Jr., and J. D. Ashwell Gadd45{alpha} Regulates p38-Dependent Dendritic Cell Cytokine Production and Th1 Differentiation J. Immunol., April 1, 2007; 178(7): 4153 - 4158. [Abstract] [Full Text] [PDF]
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P. Kumar, I. K. Coltas, B. Kumar, D. B. Chepeha, C. R. Bradford, and P. J. Polverini Bcl-2 Protects Endothelial Cells against {gamma}-Radiation via a Raf-MEK-ERK-Survivin Signaling Pathway That Is Independent of Cytochrome c Release Cancer Res., February 1, 2007; 67(3): 1193 - 1202. [Abstract] [Full Text] [PDF]
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D. Zhang, L. Song, J. Li, K. Wu, and C. Huang Coordination of JNK1 and JNK2 Is Critical for GADD45{alpha} Induction and Its Mediated Cell Apoptosis in Arsenite Responses J. Biol. Chem., November 10, 2006; 281(45): 34113 - 34123. [Abstract] [Full Text] [PDF]
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J. S. Tront, B. Hoffman, and D. A. Liebermann Gadd45a Suppresses Ras-Driven Mammary Tumorigenesis by Activation of c-Jun NH2-Terminal Kinase and p38 Stress Signaling Resulting in Apoptosis and Senescence. Cancer Res., September 1, 2006; 66(17): 8448 - 8454. [Abstract] [Full Text] [PDF]
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 H. J Welters, S. Senkel, L. Klein-Hitpass, S. Erdmann, H. Thomas, L. W Harries, E. R Pearson, C. Bingham, A. T Hattersley, G. U Ryffel, et al. Conditional expression of hepatocyte nuclear factor-1{beta}, the maturity-onset diabetes of the young-5 gene product, influences the viability and functional competence of pancreatic {beta}-cells. J. Endocrinol., July 1, 2006; 190(1): 171 - 181. [Abstract] [Full Text] [PDF]
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M. Gupta, S. K. Gupta, B. Hoffman, and D. A. Liebermann Gadd45a and Gadd45b Protect Hematopoietic Cells from UV-induced Apoptosis via Distinct Signaling Pathways, including p38 Activation and JNK Inhibition J. Biol. Chem., June 30, 2006; 281(26): 17552 - 17558. [Abstract] [Full Text] [PDF]
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Y. Tan, L. Shi, S. M. Hussain, J. Xu, W. Tong, J. M. Frazier, and C. Wang Integrating time-course microarray gene expression profiles with cytotoxicity for identification of biomarkers in primary rat hepatocytes exposed to cadmium Bioinformatics, January 1, 2006; 22(1): 77 - 87. [Abstract] [Full Text] [PDF]
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 L. Liu, E. Tran, Y. Zhao, Y. Huang, R. Flavell, and B. Lu Gadd45{beta} and Gadd45{gamma} are critical for regulating autoimmunity J. Exp. Med., November 21, 2005; 202(10): 1341 - 1348. [Abstract] [Full Text] [PDF]
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 T. Maeda, R. A. Espino, E. G. Chomey, L. Luong, A. Bano, D. Meakins, and V. A. Tron Loss of p21WAF1/Cip1 in Gadd45-deficient keratinocytes restores DNA repair capacity Carcinogenesis, October 1, 2005; 26(10): 1804 - 1810. [Abstract] [Full Text] [PDF]
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 M. C. Hollander, R. T. Philburn, A. D. Patterson, S. Velasco-Miguel, E. C. Friedberg, R. I. Linnoila, and A. J. Fornace Jr. Deletion of XPC leads to lung tumors in mice and is associated with early events in human lung carcinogenesis PNAS, September 13, 2005; 102(37): 13200 - 13205. [Abstract] [Full Text] [PDF]
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 T. Tong, J. Ji, S. Jin, X. Li, W. Fan, Y. Song, M. Wang, Z. Liu, M. Wu, and Q. Zhan Gadd45a Expression Induces Bim Dissociation from the Cytoskeleton and Translocation to Mitochondria Mol. Cell. Biol., June 1, 2005; 25(11): 4488 - 4500. [Abstract] [Full Text] [PDF]
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J. Hildesheim, J. M. Salvador, M. C. Hollander, and A. J. Fornace Jr. Casein Kinase 2- and Protein Kinase A-regulated Adenomatous Polyposis Coli and {beta}-Catenin Cellular Localization Is Dependent on p38 MAPK J. Biol. Chem., April 29, 2005; 280(17): 17221 - 17226. [Abstract] [Full Text] [PDF]
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 J. M. Roper, S. C. Gehen, R. J. Staversky, M. C. Hollander, A. J. Fornace Jr., and M. A. O'Reilly Loss of Gadd45a does not modify the pulmonary response to oxidative stress Am J Physiol Lung Cell Mol Physiol, April 1, 2005; 288(4): L663 - L671. [Abstract] [Full Text] [PDF]
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H. Gao, S. Jin, Y. Song, M. Fu, M. Wang, Z. Liu, M. Wu, and Q. Zhan B23 Regulates GADD45a Nuclear Translocation and Contributes to GADD45a-induced Cell Cycle G2-M Arrest J. Biol. Chem., March 25, 2005; 280(12): 10988 - 10996. [Abstract] [Full Text] [PDF]
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 M. A. Bachelor, S. J. Cooper, E. T. Sikorski, and G. T. Bowden Inhibition of p38 Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Decreases UVB-Induced Activator Protein-1 and Cyclooxygenase-2 in a SKH-1 Hairless Mouse Model Mol. Cancer Res., February 1, 2005; 3(2): 90 - 99. [Abstract] [Full Text] [PDF]
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W.-H. Wang, G. Gregori, R. L. Hullinger, and O. M. Andrisani Sustained Activation of p38 Mitogen-Activated Protein Kinase and c-Jun N-Terminal Kinase Pathways by Hepatitis B Virus X Protein Mediates Apoptosis via Induction of Fas/FasL and Tumor Necrosis Factor (TNF) Receptor 1/TNF-{alpha} Expression Mol. Cell. Biol., December 1, 2004; 24(23): 10352 - 10365. [Abstract] [Full Text] [PDF]
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 S. Papa, F. Zazzeroni, C. G. Pham, C. Bubici, and G. Franzoso Linking JNK signaling to NF-{kappa}B: a key to survival J. Cell Sci., October 15, 2004; 117(22): 5197 - 5208. [Abstract] [Full Text] [PDF]
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S. K. Mak and D. Kultz Gadd45 Proteins Induce G2/M Arrest and Modulate Apoptosis in Kidney Cells Exposed to Hyperosmotic Stress J. Biol. Chem., September 10, 2004; 279(37): 39075 - 39084. [Abstract] [Full Text] [PDF]
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 Y. Matsumura, A. M. Moodycliffe, D. X. Nghiem, S. E. Ullrich, and H. N. Ananthaswamy Resistance of CD1d-/- Mice to Ultraviolet-Induced Skin Cancer Is Associated with Increased Apoptosis Am. J. Pathol., September 1, 2004; 165(3): 879 - 887. [Abstract] [Full Text] [PDF]
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Y.-K. Won, C.-N. Ong, X. Shi, and H.-M. Shen Chemopreventive activity of parthenolide against UVB-induced skin cancer and its mechanisms Carcinogenesis, August 1, 2004; 25(8): 1449 - 1458. [Abstract] [Full Text] [PDF]
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X. Wang, R.-H. Wang, W. Li, X. Xu, M. C. Hollander, A. J. Fornace Jr., and C.-X. Deng Genetic Interactions between Brca1 and Gadd45a in Centrosome Duplication, Genetic Stability, and Neural Tube Closure J. Biol. Chem., July 9, 2004; 279(28): 29606 - 29614. [Abstract] [Full Text] [PDF]
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J. Yoo, M. Ghiassi, L. Jirmanova, A. G. Balliet, B. Hoffman, A. J. Fornace Jr., D. A. Liebermann, E. P. Bottinger, and A. B. Roberts Transforming Growth Factor-{beta}-induced Apoptosis Is Mediated by Smad-dependent Expression of GADD45b through p38 Activation J. Biol. Chem., October 31, 2003; 278(44): 43001 - 43007. [Abstract] [Full Text] [PDF]
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D. V. Bulavin, O. Kovalsky, M. C. Hollander, and A. J. Fornace Jr. Loss of Oncogenic H-ras-Induced Cell Cycle Arrest and p38 Mitogen-Activated Protein Kinase Activation by Disruption of Gadd45a Mol. Cell. Biol., June 1, 2003; 23(11): 3859 - 3871. [Abstract] [Full Text] [PDF]
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S. A. Amundson, R. A. Lee, C. A. Koch-Paiz, M. L. Bittner, P. Meltzer, J. M. Trent, and A. J. Fornace Jr Differential Responses of Stress Genes to Low Dose-Rate {gamma} Irradiation Mol. Cancer Res., April 1, 2003; 1(6): 445 - 452. [Abstract] [Full Text] [PDF]
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) and Gadd45a-null (-/-, 
) mice at the indicated time points after UV radiation were used for RNase protection assays with RiboQuant multiprobe sets. Each time point corresponds to the average of two RNase protection assays performed on two separate animals and normalized to the average of duplicate sets of controls for the respective genotype. IL, interleukin; TNF-
), Gadd45a-/- (
), and Tp53-/- (
) are shown. UV-irradiated data sets were normalized to unirradiated controls for each genotype. G2 checkpoint analysis was performed on (B) keratinocytes and (D) dermal fibroblasts irradiated with 150 Jm-2 of UVB. The mitotic indexes of Gadd45a+/+ (
