Gadd45: Biological Overview | References
Gene name - Gadd45
Cytological map position - 43A2-43A2
Function - signaling
Symbol - Gadd45
FlyBase ID: FBgn0033153
Genetic map position - 2R:3,136,668..3,137,508 [+]
Classification - Ribosomal protein L7Ae/L30e/S12e/Gadd45 family
Cellular location - nuclear
The mammalian GADD45 (growth arrest and DNA-damage inducible) gene family is composed of three highly homologous small, acidic, nuclear proteins: GADD45α, GADD45β, and GADD45γ. GADD45 proteins are involved in important processes such as regulation of DNA repair, cell cycle control, and apoptosis. Annotation of the Drosophila genome revealed that it contains a single GADD45-like protein (CG11086; D-GADD45). As its mammalian homologs, D-GADD45 is a nuclear protein; however, D-GADD45 expression is not elevated following exposure to genotoxic and nongenotoxic agents in Schneider cells and in adult flies. The D-GADD45 transcript increased following immune response activation, consistent with previous microarray findings. Since upregulation of GADD45 proteins has been characterized as an important cellular response to genotoxic and nongenotoxic agents, the effect of D-GADD45 overexpression on Drosophila development was characterized. Overexpression of D-GADD45 in various tissues led to different phenotypic responses. Specifically, in the somatic follicle cells overexpression caused apoptosis, while overexpression in the germline affected the dorsal-ventral polarity of the eggshell and disrupted the localization of anterior-posterior polarity determinants. This article focused on the role of D-GADD45 overexpression in the germline and it was found that D-GADD45 caused dorsalization of the eggshell. Since mammalian GADD45 proteins are activators of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways, a genetic interaction was tested in Drosophila. It was found that eggshell polarity defects caused by D-GADD45 overexpression are dominantly suppressed by mutations in the JNK pathway, suggesting that the JNK pathway has a novel, D-GADD45-mediated, function in the Drosophila germline (Peretz, 2007).
The GADD45 gene family is composed of three highly homologous (55-58% overall identity at the amino acid level), small, acidic, nuclear proteins: GADD45α, GADD45β (MyD118), and GADD45γ (CR6, cytokine response gene 6). In recent years, evidence has emerged that the proteins encoded by these genes play similar but not identical roles in terminal differentiation and negative growth control, including growth suppression and apoptotic cell death (Azam, 2001; Zhang, 2001; Vairapandi, 2002; Peretz, 2007 and references therein).
One of the well-described responses to genotoxic and nongenotoxic stresses is the rapid upregulation of different GADD45 proteins, which in turn affect cell-cycle regulation, cell survival, and cell death. It has been shown that all the GADD45 proteins mediate cell-cycle regulation through interactions with PCNA (Kelman, 1998; Azam, 2001), the cyclin-dependent kinase inhibitor p21 (Kearsey, 1995), and the Cdk/cyclin B complex (Zhan, 1999; Jin, 2002; Vairapandi, 2002). The potential role of GADD45 proteins in apoptosis emanates from the observation that GADD45 expression is enhanced during apoptosis following induction by a variety of genotoxic agents. Several studies have shown that GADD45 proteins may play a role in apoptosis via activation of the c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK) signaling pathways (Takekawa; 1998; Harkin, 1999). GADD45 proteins physically interact with the MAPKKK, MTK1 (synonym MEKK4), and the ensuing interactions result in the activation of MTK1. Activated MTK1 is thought to further activate its downstream targets JNK and p38 (Takekawa, 1998). It was shown that the N-terminal of MTK1 auto-inactivates its kinase activity and binding of GADD45 proteins to MTK1 relieves this inhibition (Mita, 2002; Miyake, 2007). It has been proposed that in response to genotoxic stress, p53 is activated, which causes transcriptional upregulation of GADD45α, and GADD45α interacts with MTK1 to initiate the JNK/p38-mediated apoptotic pathway (Peretz, 2007 and references therein).
Several model systems have been used to analyze the role of GADD45 proteins during development. GADD45α-null mice exhibit several phenotypes including genomic instability, increased radiation carcinogenesis, and a low frequency of exencephaly (Hollander, 1999). GADD45γ-deficient mice develop normally and are indistinguishable from their littermates, possibly due to functional redundancy among the GADD45 family members (Hoffeyer, 2001). In the fish, Oryzias latipes, ectopic expression of GADD45γ leads to cell cycle arrest without inducing apoptosis. Loss of function of GADD45γ causes a significant increase in apoptosis, suggesting that GADD45γ is an important component of the molecular pathway that coordinates cell cycle vs. apoptosis decisions during vertebrate development (Candal, 2004). The zebrafish GADD45β genes were found to be periodically expressed as paired stripes in the anterior presomitic mesoderm. Both knockdown and overexpression of GADD45β genes caused somite defects with different consequences for marker gene expression, indicating that the regulated expression of GADD45β genes is required for somite segmentation (Kawahara, 2005). The possible functional redundancy among the GADD45 proteins in these model systems makes the analysis of the molecular function of GADD45 difficult. Annotation of the Drosophila genome revealed that it contains only one GADD45-like protein (Peretz, 2007).
Since upregulation of GADD45 proteins may affect cell cycle regulation, cell survival, and cell death, the effect was studied of D-GADD45 overexpression on D. melanogaster oogenesis. Overexpression of D-GADD45 in the somatic follicle cells led to apoptosis of the entire egg chamber. In contrast, overexpression of D-GADD45 in the germline did not cause apoptosis but affected the dorsal-ventral polarity of the eggshell. Moreover, D-GADD45 also affected anterior-posterior polarity determinants. However, anterior oocyte nuclear migration and bcd localization were unaffected. Finally, it was found that mutations in the MAPK-JNK pathway dominantly suppressed the egg asymmetric defects in D-GADD45 overexpression ovaries, suggesting a novel, D-GADD45-mediated function for the JNK pathway in the germline (Peretz, 2007).
In Drosophila D-GADD45 preserves the nuclear localization property, but unlike its mammalian homologs its expression is not elevated following exposure to different stress stimuli. This result is supported by the Drosophila whole genome microarray analysis which did not identify D-GADD45 as a gene whose expression is increased following various genotoxic and nongenotoxic treatments. Although a number of stress treatments were tried, it is possible that D-GADD45 expression would rise only following exposure to as yet untested stressful conditions (Peretz, 2007).
D-GADD45 was identified as a gene whose expression is induced following microbial infection (De Gregorio, 2001). It was also shown that D-GADD45 expression may be regulated by the NF-BkappaB-like transcription factor, Dorsal, which has an optimal binding site 3 kb upstream to D-GADD45 transcription start site. These results are consistent with those found by De Gregorio (2001) and further strengthen a possible function for D-GADD45 in the immune response. Given that Drosophila is devoid of an adaptive immune system and relies only on innate immune reactions for its defense, D-GADD45 may play an important role during infection (Peretz, 2007).
Ubiquitous overexpression of D-GADD45 was lethal, most likely due to apoptosis, as was directly demonstrated in the follicle cells. However, the results suggest that apoptosis induced by overexpression of D-GADD45 is tissue specific since overexpression of D-GADD45 in other somatic tissues, such as the eye and wing, did not lead to apoptosis. Also, overexpression in the germline did not cause cell death; rather, it affected egg chamber asymmetric development. The apparent phenotypic differences in overexpression of D-GADD45 in the germline as opposed to somatic derived tissues probably reflect the complexity of the biological functions of GADD45, which may be subject to tissue- and/or signal-specific regulation that ultimately dictate their output. Similarly, it has been shown that individual members of the GADD45 family play critical roles in negative growth control in some tissues while in others they are associated with uncontrolled cell growth and tumor development. GADD45α was identified as an important mediator of tumor suppression in human ovarian cancer cells (Jiang, 2003). While in pancreatic ductal adenocarcinoma GADD45α was found to be overexpressed at the mRNA and protein level. Downregulation of GADD45α by means of RNAi reduced proliferation and induced apoptosis in pancreatic cancer cells implying that GADD45α contributes to pancreatic cancer cell proliferation and viability (Schneider, 2006; Peretz, 2007).
Overexpression of D-GADD45 in the germline results in dorsalization of the chorion due to defects in grk localization and translation. The posterior markers osk and Kin:β-gal were mislocalized during mid-oogenesis. In contrast, D-GADD45 overexpression does not affect the localization of anterior end markers such as bcd and Nod:β-gal and also the anterior oocyte nuclear migration is unaffected. Similar results have reported in mutants of squid (sqd) which encodes a heterogeneous nuclear ribonucleoprotein (hnRNP). In these mutants grk mRNA is mislocalized along the anterior ring, leading to dorsalization of the eggshell. Furthermore, loss of sqd function causes an aberrant localization of osk and Kin:β-gal, but does not affect bcd localization and oocyte nucleus migration. It was shown that in sqd mutant oocytes short microtubules (MTs) around the entire oocyte cortex are retained, including at the posterior pole, unlike wild-type MTs which emanate mostly from the anterior. It has been suggested that the primary MT defect in sqd mutants is the failure to eliminate cortical sites of MT nucleation beyond stage 7. It is possible that D-GADD45 overexpression also affects MT organization in the oocyte. This possibility is further supported by the finding that GADD45α interacts with elongation factor 1α (EF-1α), a microtubule-severing protein that plays an important role in maintaining microtubule cytoskeletal stability (Tong, 2005). To test whether D-GADD45 affects MT organization, the ovaries were stained with anti-tubulin. Using this tool, no gross morphological changes were detected in the MT network. Given that the patterning defect seen in D-GADD45 overexpression is weaker than that in sqd mutants, it could be that this kind of staining is not sensitive enough to identify the MT network alterations in D-GADD45 overexpression flies (Peretz, 2007).
A genetic interaction was found between D-GADD45 and proteins of the MAPK-JNK pathway. Mutations in the JNKK, hemipterous, dominantly suppres the dorsalized eggshell phenotype. This genetic interaction is supported by the finding that in human cells GADD45 proteins act as initiators of JNK/p38 signaling via their interaction with the MAPKKK, MTK1 (Takekawa, 1998). It was shown that the N-terminal of MTK1 inhibits its C-terminal kinase domain by preventing the kinase domain from interacting with its substrate, MKK6, and binding of GADD45 proteins relieves this auto-inhibition (Mita, 2002; Miyake, 2007; Peretz, 2007).
Up until now the only roles attributed to the JNK pathway during oogenesis were in the follicle cells and included morphogenesis of the dorsal appendages and the micropyle (Suzanne, 2001). It was also reported that the JNK pathway is involved in the morphogenetic process of dorsal closure during embryogenesis. Surprisingly, it was found that eggshell patterning defects caused by D-GADD45 overexpression are dominantly suppressed in a hep deficient background suggesting an additional role for the JNK pathway in the germline. This novel function may have gone unnoticed in the past while studying JNK loss-of-function alleles due to redundancy with some other pathway. In this study, overexpression of the JNK activator, D-GADD45, may have unmasked this new role during oogenesis (Peretz, 2007).
By using a microarray screen to compare gene responses after sterile laser wounding of wild-type and 'macrophageless' serpent mutant Drosophila embryos, this study showed wound-induced programs that were independent of a pathogenic response, and macrophage dependent genes were distinguished. The evolutionarily conserved nature of this response is highlighted by the finding that one such new inflammation-associated gene, growth arrest and DNA damage-inducible gene 45 (GADD45), is upregulated in both Drosophila and murine repair models. Comparison of unwounded wild-type and serpent mutant embryos also shows a portfolio of 'macrophage-specific' genes, which suggest analogous functions with vertebrate inflammatory cells. Besides identifying the various classes of wound- and macrophage-related genes, these data indicate that sterile injury per se, in the absence of pathogens, triggers induction of a 'pathogen response', which might prime the organism for what is likely to be an increased risk of infection (Stramer, 2008).
Drosophila GADD45 is robustly induced by wounding wild-type embryos, and in situ hybridization showed that this gene was strongly inflammation dependent. The data suggest that Drosophila macrophages might secrete signals that are necessary for full GADD45 induction in the wounded epithelium. There is precedent for a paracrine signalling role for haemocytes during an immune response; after septic injury, an unpaired (Upd)-like cytokine is secreted by haemocytes and is necessary for Jak/Stat signalling in the fat body. Although GADD45 is not a known Jak/Stat target, it is responsive to Toll signalling. However, Toll mutants showed a similar epithelial wound induction of GADD45, and expression of an activated form of the Toll ligand, spatzle (spz), in the epithelium of Drosophila embryos failed to induce GADD45 expression, suggesting that GADD45 induction following wounding is Toll independent (Stramer, 2008).
The data suggest that GADD45 is an 'inflammation-associated' wound response gene in insects. To determine whether this response is conserved in mammals, microarray data was analyzed from an analogous experiment in mice comparing the gene profiles of wounds in the presence and absence of an inflammatory response. These data showed that a murine homologue of Drosophila GADD45 was upregulated rapidly after wounding and that this response was much reduced in PU.1 null mice in which inflammatory cells were missing. The expression of GADD45 protein after injury was examined by western blotting, and rapid and transient induction was shown by 1 day after wounding. Furthermore, immunostaining showed that, as in Drosophila, murine GADD45 was induced in the wound epithelium. This finding provided further evidence for an evolutionarily conserved repair response in flies and vertebrates, and highlights how useful Drosophila might be in elucidating new mechanisms regulating various aspects of vertebrate tissue repair (Stramer, 2008).
Search PubMed for articles about Drosophila Gadd45
Azam, N., et al. (2001). Interaction of CR6 (GADD45) with proliferating cell nuclear antigen impedes negative growth control. J. Biol. Chem. 276: 2766-2774. PubMed ID: 11022036
Candal, E., et al. (2004). Medaka as a model system for the characterisation of cell cycle regulators: a functional analysis of Ol-Gadd45 during early embryogenesis. Mech. Dev. 121: 945-958. PubMed ID: 15210198
De Gregorio, E., et al. (2001). Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc. Natl. Acad. Sci. USA 98(22): 12590-12595. PubMed ID: 11606746
Harkin, D. P., et al. (1999). Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell 97(5): 575-86. PubMed ID: 10367887
Hoffeyer, A., et al. (2001). Gadd45gamma is dispensable for normal mouse development and T-cell proliferation. Mol. Cell. Biol. 9: 3137-3143. PubMed ID: 11287618
Hollander, M. C., et al. (1999). Genomic instability in Gadd45a-deficient mice. Nat. Genet. 23: 176-184. PubMed ID: 10508513
Jiang, F., et al. (2003). G2/M arrest by 1,25-dihydroxyvitamin D3 in ovarian cancer cells mediated through the induction of GADD45 via an exonic enhancer. J. Biol. Chem. 278: 48030-48040. PubMed ID: 14506229
Jin, S., et al. (2002). GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 21(57): 8696-704. PubMed ID: 12483522
Kawahara, A., et al. (2005). Zebrafish GADD45β genes are involved in somite segmentation. Proc. Natl. Acad. Sci. USA 102(2): 361-366. PubMed ID: 15623554
Kelman, Z., and Hurwitz, J. (1998). Protein-PCNA interactions: a DNA-scanning mechanism? Trends Biochem. Sci. 23: 236-238. PubMed ID: 9697409
Kearsey, J. M., et al. (1995). Gadd45 is a nuclear cell cycle regulated protein which interacts with p21Cip1. Oncogene 11(9): 1675-83. PubMed ID: 7478594
Mita, H., et al. (2002). Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding. Mol. Cell. Biol. 22: 4544-4555. PubMed ID: 12052864
Miyake, Z., et al. (2007). Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain. Mol. Cell. Biol. 27(7): 2765-2776. PubMed ID: 17242196
Peretz, G., Bakhrat, A. and Abdu, U. (2007). Expression of the Drosophila melanogaster GADD45 homolog (CG11086) affects egg asymmetric development that is mediated by the c-Jun N-terminal kinase pathway. Genetics 177(3): 1691-702. PubMed ID: 18039880
Schneider, G., et al. (2006). GADD45a is highly expressed in pancreatic ductal adenocarcinoma cells and required for tumor cell viability. Int. J. Cancer 118: 2405-2411. PubMed ID: 16353139
Stramer, B., et al. (2008). Gene induction following wounding of wild-type versus macrophage-deficient Drosophila embryos. EMBO Rep. [Epub ahead of print]. PubMed ID: 18344972
Suzanne, M., Perrimon, N. and Noselli, S. (2001). The Drosophila JNK pathway controls the morphogenesis of the egg dorsal appendages and micropyle. Dev. Biol. 237: 282-294. PubMed ID: 11543614
Takekawa, M., and Saito, H. (1998). A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95: 521-530. PubMed ID: 9827804
Tong, T., et al. (2005). Gadd45a expression induces Bim dissociation from the cytoskeleton and translocation to mitochondria. Mol. Cell. Biol. 25(11): 4488-4500. PubMed ID: 15899854
Vairapandi, M., et al. (2002). GADD45β and GADD45β are cdc2/CyclinB1 kinase inhibitors with a role in S and G2/M cell cycle checkpoints induced by genotoxic stress. J. Cell. Physiol. 192: 327-338. PubMed ID: 12124778
Zhan, Q., et al. (1999). Association with Cdc2 and inhibition of Cdc2/Cyclin B1 kinase activity by the p53-regulated protein Gadd45. Oncogene 18: 2892-2900. PubMed ID: 10362260
Zhang, W., Hoffman, B. and Liebermann, D. A. (2001). Ectopic expression of MyD118/Gadd45/CR6 (Gadd45beta/alpha/gamma) sensitizes neoplastic cells to genotoxic stress-induced apoptosis. Int. J. Oncol. 18: 749-757. PubMed ID: 11251170
date revised: 30 April 2008
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