GADD45A: With or without you

The growth arrest and DNA damage inducible (GADD)45 family includes three small and ubiquitously distributed proteins (GADD45A, GADD45B, and GADD45G) that regulate numerous cellular processes associated with stress signaling and injury response. Here, we provide a comprehensive review of the current literature investigating GADD45A, the first discovered member of the family. We first depict how its levels are regulated by a myriad of genotoxic and non‐genotoxic stressors, and through the combined action of intricate transcriptional, posttranscriptional, and even, posttranslational mechanisms. GADD45A is a recognized tumor suppressor and, for this reason, we next summarize its role in cancer, as well as the different mechanisms by which it regulates cell cycle, DNA repair, and apoptosis. Beyond these most well‐known actions, GADD45A may also influence catabolic and anabolic pathways in the liver, adipose tissue and skeletal muscle, among others. Not surprisingly, GADD45A may trigger AMP‐activated protein kinase activity, a master regulator of metabolism, and is known to act as a transcriptional coregulator of numerous nuclear receptors. GADD45A has also been reported to display a cytoprotective role by regulating inflammation, fibrosis and oxidative stress in several organs and tissues, and is regarded an important contributor for the development of heart failure. Overall data point to that GADD45A may play an important role in metabolic, neurodegenerative and cardiovascular diseases, and also autoimmune‐related disorders. Thus, the potential mechanisms by which dysregulation of GADD45A activity may contribute to the progression of these diseases are also reviewed below.


| INTRODUCTION: THE GADD45 FAMILY, A BAND WITH THREE COMPONENTS
The growth arrest and DNA damage inducible (GADD)45 family includes three small and highly acidic proteins named GADD45A (also known as GADD45, GADD45α or DNA damage-inducible transcript [DDIT]1 protein), GADD45B (GADD45β or myeloid differentiation primary response 118, MYD118), and GADD45G (GADD45γ, DDIT2, GADD-related protein 17 kDa, GRP17, or cytokine-responsive protein 6, CR6).These proteins are evolutionarily conserved and highly homologous to each other, sharing a 55-57% similarity at the amino acid level. 1 In humans, the Gadd45 genes are located on three different chromosomes: 1p31.3 (Gadd45a), 19p13.3(Gadd45b), and 9q22.2 (Gadd45g). 2The first member described, Gadd45a, was initially identified as one of several genes whose mRNA levels were rapidly induced in Chinese hamster ovary cells by agents that cause DNA damage, such as UV radiation and X-ray irradiation. 3Later, Gadd45b was found to be a primary response gene transiently expressed in myeloid precursor cells following induction of growth arrest by interleukin (IL)-6 or lipopolysaccharide (LPS). 4nally, Gadd45g was identified as an immediate early response gene in T cells stimulated by IL-2. 5 The GADD45 family members are ubiquitously distributed in adult mammalian tissues, although they have distinctive expression patterns. 1At the subcellular level, they are primarily localized within the cell nucleus, but may also be found in the cytoplasm.These proteins display low abundance in normal quiescent cells, peaking in the G1 phase of the cycle and decreasing during the S phase. 6In fact, their levels are regulated by numerous factors at the transcriptional, posttranscriptional, and posttranslational levels.8][9] Indeed, they are rapidly induced in response to oncogenic stress, as well as by terminal differentiation and apoptotic cytokines, being able to modulate tumor formation.For example, GADD45B is the only member induced by the transforming growth factor (TGF)-β, whereas GADD45A is a selective p53 target. 8To operate as stress sensors, they must physical interact with other cellular proteins.For instance, all three members interact with the DNA replication and repair protein PCNA (proliferating cell nuclear antigen) and the cyclin dependent kinase inhibitor 1 (CDKN1A or p21). 100][11] This interaction accounts for their role in nucleotide excision repair and in G1 and G2 arrest in response to DNA damage. 10GADD45 proteins also bind to nucleosome histones, thereby altering accessibility to DNA, particularly where chromatin is damaged, and enabling their role in DNA repair. 9In fact, they interact with other partners involved in DNA repair, including DNA-(apurinic or apyrimidinic site) endonuclease (APEX1), DNA excision repair protein ERCC5 (also known as xeroderma pigmentosum group G-complementing protein, XPG), and p53 itself. 9togen-activated protein kinases (MAPK) are important regulators of cell proliferation, survival, and differentiation; the extracellular signal-regulated kinase (ERK)1/2 pathway is often associated with cell survival and proliferation, whereas JUN N-terminal kinase (JNK) and p38 MAPK are mostly activated by stimuli that lead to apoptosis or inhibit cell growth.Interestingly, GADD45 proteins associate with the MAPK kinase kinase 4 (MEKK4 or MAP3K4), which in turn activates both p38 and JNK to drive apoptosis and increase cytokine production in response to environmental stressors. 7In contrast, NF-κB-mediated induction of GADD45B blunts JNK activation through its association with the MAPK kinase kinase MAP2K7 (or MKK7). 7Thus, GADD45 proteins may carry out antiapoptotic effects via opposing but complementary mechanisms: the activation of the antiapoptotic p38/NF-κB pathway by GADD45A and the inhibition of the proapoptotic MKK7/JNK pathway by GADD45B. 12Of note, both p38 and JNK are also able to regulate the transcription of Gadd45 genes. 9DD45 proteins perform similar but not identical roles, depending on the cell type and the particular stress response pathway that is specifically activated.They participate in cell cycle arrest and growth, DNA repair, and cell apoptosis, survival, and senescence, hence playing a critical role in the carcinogenic process.However, there are specific dissimilarities among them.Thus, GADD45A and GADD45G are known to induce cell growth arrest and apoptosis, 13 but the role of GADD45B is still controversial and considered cell-and stimulus-specific.Although most data demonstrate a tumor suppressor role, [14][15][16][17] other studies point to an antiapoptotic effect of GADD45B in hematopoietic cells, 14 hepatocytes, 18 or neurons. 191][22][23] Besides this, GADD45B can regulate angiogenesis, cell differentiation and migration, and oxidative stress, and it has recently been demonstrated that o modulates gluconeogenesis and lipid metabolism in the liver, [24][25][26][27] and neuroplasticity and neuroprotection in the nervous system. 28In contrast, GADD45G has been reported to participate in primary sex determination and male fertility, 29 and the development of heart failure. 13Despite their many functional similarities, this review will be focused on the first discovered and most well-characterized member of the family, GADD45A.

| Regulation of GADD45A levels
Gadd45a is expressed in brain, heart, kidney, liver, skeletal muscle, spleen, and lung.A myriad of genotoxic and nongenotoxic stressors may induce its gene expression, including UV and γ radiation, X-rays, hypoxia, peroxynitrite free PALOMER ET AL.
| 1377 radicals, low pH, xenobiotics (arsenic, hexavalent chromium compounds, cisplatin, ethanol, cigarette smoke), oncogenic stress, nutrient depletion, and many soil, air, and water pollutants. 2 Gadd45a expression varies significantly throughout the cell cycle; it is usually low at rest, but it increases significantly during the G1 phase and decreases during the S phase, displaying a pivotal role in both S-phase and G2/M arrest. 30Following exposure to stress, Gadd45a expression is upregulated through the combined action of transcriptional and posttranscriptional derepression, transcription factors activation, and the recruitment of posttranscriptional inducers. 31To further complicate it, GADD45A is a short-lived protein that can also be induced at the posttranslational level, through a process that involves a reduction in its ubiquitination. 32In addition, the activity of GADD45A may be modulated by its nuclear translocation through the carrier protein nucleophosmin 1. 33 The signaling pathways responsible for regulating Gadd45a expression are intricate and involve several transcription factors that are differentially activated depending on the causative stress (Figure 1).Historically, increased Gadd45a gene expression has been mostly recognized as a downstream event of p53 activation, a tumor suppressor protein that plays a crucial role in protecting cells from malignant transformation and contributes to the maintenance of genomic stability. 34Specifically, in response to many agents associated with DNA damage, apoptosis, cell cycle control, and cell injury, p53 stimulates Gadd45a transcription through the binding to a conserved site located within the third intron of its gene. 9,35Not surprisingly, Gadd45a-null mice exhibit a phenotype resembling that of p53-deficient mice, including genomic instability as a consequence of perturbations in G2/M cell cycle progression, and impaired DNA repair. 34For this reason, GADD45A is regarded as the link between the p53-dependent DNA repair and cell cycle checkpoints, which are vital responses to ensure the maintenance of genomic fidelity in the event of DNA damage. 36However, p53 can also contribute to increase Gadd45a levels without binding to its promoter, suggesting the occurrence of other mechanisms involving protein-protein interactions. 37Additionally, several p53-independent pathways are also involved in the transcriptional regulation of Gadd45a.As with other family members, EGR1, forkhead box protein O3a (FOXO3a), NFYA, POU2F1, and the tumor suppressor WT1 are involved in its induction by directly binding to specific motifs within its promoter. 33,35,381, in particular, positively regulates Gadd45a expression through both p53-dependent and p53-independent mechanisms in response to DNA damage or chromosome missegregation. 35FOXO3a, a transcription factor activated in response to oxidative stress, directly induces Gadd45a at the same time it suppresses the expression of Gadd45b. 9FOXO3a is known for its ability to block the transition between G2/M cell cycle phases and to trigger the repair of damaged DNA, and this is probably due to a mechanism involving, at least in part, the FOXO3amediated increase in Gadd45a. 2,39Other growth-arrest-associated regulatory factors that may transcriptionally regulate Gadd45a, either directly or indirectly, include breast cancer type 1 susceptibility protein (BRCA1), CCAAT/ enhancer-binding protein (C/EBP)β, and activating transcription factor 4 (ATF4). 9BRCA1 directly upregulates its transcription in a JUN-and FOS-independent manner, thus ruling out the participation of other immediateresponsive genes. 2,40ATF4, which also has a pivotal role in cellular stress response, enhances Gadd45a transcription following arsenite exposure, leucine starvation, or endoplasmic reticulum (ER) stress, by directly interacting with an ATF/CREB (cAMP response element binding protein)-related binding element within its promoter. 9Another activating transcription factor, ATF2, which is a p38 and JNK target involved in stress-induced apoptosis, may also indirectly stimulate Gadd45a transcription through its binding to POU2F1 and BRCA1. 41DD45A, as well as the other two members of the family, are downstream targets of the NF-κB transcriptional activity involved in regulating cell survival and apoptosis. 14However, the role of this transcription factor in regulating Gadd45a is still controversial.It was first demonstrated that NF-κB inhibition following IKKβ (inhibitor of NF-κB [IκB] kinase β) suppression caused an augment of Gadd45a mRNA expression. 42Since no changes were observed in the activity of several regulators of Gadd45a transcription (AKT/PKB or protein kinase B, FOXO3a, and p53), this study pointed to that the IKKβ/NF-κB signaling pathway regulates GADD45A through a posttranscriptional mechanism involving an increase in its mRNA stability. 42Later studies have demonstrated that NF-κB acts as a pro-survival factor that favors apoptosis escape of cancer cells by indirectly reducing the expression of Gadd45a, in a process that involves an increase in MYC repressor, and the downregulating of nucleolin. 9 I G U R E 1 (See caption on next page).
Nucleolin is a nucleolar protein that increases Gadd45a mRNA stability and acts as a transcriptional coactivator of POU2F1.Supporting the importance of nucleolin, another report demonstrated that the increase in Gadd45a mRNA expression observed in IKKβ-deficient fibroblasts was due to an increase in nucleolin levels. 42On the contrary, NF-κB has been reported to induce cell death in UVB-exposed keratinocytes by activating Gadd45a transcription, in a process mediated by EGR1. 43In fact, it is known that the p65 (RELA) subunit of NF-κB activates the transcription of Gadd45b by directly binding to κB elements in the promoter, but Gadd45a does not contain these response elements in its promoter region. 9Anyway, this differential regulation of GADD45A protein levels by NF-κB might contribute to both pro-and antiapoptotic properties observed for this transcription factor.Another related transcription factor that directly increases Gadd45a expression is AP-1, which in a similar way to NF-κB controls a number of cellular processes including inflammation, differentiation, proliferation, and apoptosis, in response to a variety of stimuli (cytokines, growth factors, stress, and bacterial or viral infection). 33More specifically, p38 and JNK activate the AP-1 subunit JUN to enhance Gadd45a transcription. 9e mRNA levels of Gadd45a are finely tuned by several microRNAs (miRNA; e.g.5][46][47][48][49][50][51][52][53] Downregulation of Gadd45a gene expression caused by these noncoding RNAs has been related to changes in cell proliferation and apoptosis, invasion, migration and epithelial-mesenchymal transition in F I G U R E 1 Regulation of GADD45A levels and its major interacting proteins.GADD45A levels are regulated through the combined action of transcriptional, posttranscriptional and posttranslational mechanisms.At the transcriptional level, a myriad of genotoxic and non-genotoxic stressors may induce Gadd45a gene expression through both direct and indirect mechanisms.Among the first stand out p53, although several p53-independent pathways are also involved in its transcriptional initiation, including AP-1, ATF4, BRCA1, EGR1, FOXO3a, NFYA, POU2F1, and WT1.ATF2 may also indirectly stimulate Gadd45a transcription through its binding to POU2F1 and BRCA1.NF-κB may induce Gadd45a mRNA levels through the activation of EGR1, but may also reduce its expression by increasing the MYC repressor and downregulating the levels of nucleolin, a nucleolar protein that increases mRNA stability and acts as a transcriptional coactivator of POU2F1.To maintain constitutively low mRNA levels in nonstressed cells, Gadd45a is negatively regulated by transcriptional (MYC, ZBRK1) and posttranscriptional repressors (AUF1, TIAR1).AUF1 renders instability to Gadd45a mRNA and favors its degradation, while TIAR1 blocks its translation.MYC and AKT are able to indirectly downregulate Gadd45a expression by inhibiting FOXO3a.Finally, the p50 subunit of NF-κB is able to reduce the ubiquitination and subsequent proteasome-dependent degradation of GADD45A.To carry out its numerous functions, GADD45A must regulate several proteins.Thus, it interacts with partners involved in DNA replication and repair (APEX1, ERCC5, p53, PCNA and nuclear histones), while it associates with CDK1, CDKN1A (p21), EF1, and p53 itself to control cell cycle, proliferation and survival.GADD45A induces MEKK4, which in turn activates both p38 and JNK MAPKs to drive apoptosis and regulate immune functions.Of note, both p38 and JNK are able to regulate the transcription of Gadd45a and the activity of NF-κB.GADD45A has also a role in reducing cell survival through the modulation of the AKT, p38 MAPK and STAT5 signaling pathways.Finally, other less-known activities for GADD45A have also been reported.It negatively regulates p63 oncogene and the GSK3β/β-catenin cascade, which are involved in extracellular matrix remodeling, angiogenesis and the invasion and metastasis of cancer cells.By suppressing STAT3 transcriptional activity, GADD45A also lessens the formation of tumor blood vessels.Created with BioRender.com.AP-1, activator protein-1; ATF4, activating transcription factor 4; AUF1, AU-rich element RNA-binding factor 1; BRCA1, breast cancer type 1 susceptibility protein; CDK1, cyclindependent kinase 1; CDKN1A (p21), cyclin dependent kinase inhibitor 1; EGR1, early growth response protein 1; ERCC5, DNA excision repair protein (or xeroderma pigmentosum group G-complementing protein, XPG); FOXO3a, forkhead box protein O3a; GADD45, growth arrest and DNA damage inducible 45; GSK3β, glycogen synthase kinase 3β; IκB, inhibitor of NF-κB; JNK, JUN (c-Jun) N-terminal kinase; MAPK, mitogen-activated protein kinase; MEKK4, MEK Kinase 4 or MAPK kinase kinase 4; NF-κB, nuclear factor-κB; NFYA, nuclear transcription factor Y subunit α; PCNA, proliferating cell nuclear antigen; PKB, Protein kinase B; POU2F1, POU domain, class 2, transcription factor 1; STAT, signal transducer and activator of transcription; WT1, Wilms tumor protein 1. [Color figure can be viewed at wileyonlinelibrary.com] various types of cancer (hepatocellular carcinoma, high-grade glioma, non-small-cell lung cancer), and even other diseases or conditions (acute myocardial infarction, autoimmunity, diabetic cardiomyopathy, male infertility, preeclampsia).
Once the Gadd45a gene is transcribed, the pre-mRNA is spliced and the resulting mRNA exported out of the nucleus to serve as a template for protein synthesis. 31To maintain constitutively low mRNA levels in non-stressed cells, Gadd45a is negatively influenced by transcriptional and posttranscriptional repressors (Figure 1).This is, for instance, the case of MYC and AKT, which are frequently overexpressed in a variety of cancers and are able to indirectly downregulate Gadd45a expression by inhibiting FOXO3a. 9FOXO proteins are tumor suppressors frequently inactivated in cancer cells through its AKT-mediated phosphorylation and subsequent nuclear exclusion.
Following cell growth stimulation, MYC is rapidly recruited to the chromatin and selectively contributes to repress the FOXO3a-mediated expression of Gadd5a. 54MYC is also able to inhibit both basal and induced transcription of Gadd45a by directly binding to its promoter. 33On the contrary, in follicular thyroid carcinoma, AKT activation and cytoplasmatic accumulation of FOXO3a correlate with increased expression of Gadd45a, suggesting that other transcription factors are regulating Gadd45a in these cells. 55More recently, the zinc finger protein 350 (ZBRK1) has been demonstrated to function as a transcriptional repressor of Gadd45a in resting cells, and genomic methylation of specific cytosine residues of CpG dinucleotides within its promoter also contributes to negatively regulate its transcription. 33Among the posttranscriptional repressors, stand out RNA-binding proteins that may render instability to Gadd45a mRNA and favor its degradation (AUF1; AU-rich element RNA-binding factor 1), or block their translation (TIAR; T-cell-restricted intracellular antigen-1 (TIA-1)-related protein), both preventing the translation of those transcripts that escape the transcriptional repression in non-stressed cells. 33In the presence of stress, AUF1 dissociates from mRNA and this results in the increase of translation.

| GADD45A: The conductor of the cell cycle and apoptotic choral orchestra
In response to certain genotoxic stresses, GADD45A interacts with CDK1 and provokes the dissociation of the CDK1-cyclin B1 complex, that, in turn, results in the inhibition of CDK1 kinase activity and the subsequent cell cycle G2/M arrest and cell growth suppression (Figure 1). 8,56In support of this, the tumor suppressor protein CR6interacting factor 1 (CRIF1) boosts the activity of GADD45 family proteins and, by this means, acts as a negative regulator of the cell cycle through the subsequent inhibition of CDK1. 57In human hepatoma HepG2 cells, GADD45A also induces G2/M arrest through the activation of p38 and JNK signaling pathways. 58GADD45A binds to p21 as well, another p53 target gene that inhibits cyclin dependent kinases implicated in G1/S and G2/M cell cycle arrest, overall suggesting that it is involved in the regulation of cell cycle at different stages. 59A more recent study also depicts a role for the prolonged activation of the GADD45A/p21 pathway in cellular senescence and mitochondrial dysfunction induced by oxidative and other stresses, in a process in which the p38 MAPK is involved. 1On the other hand, ER stress and subsequent induction of the unfolded protein response (UPR) induces cell cycle arrest at the G2/M phase by reducing the amount of cyclin B1. 60 Interestingly, cell death caused by ER stress in mouse embryonic fibroblasts is diminished after knocking down Gadd45a expression due to cell cycle arrest at the G2/M phase, and the UPR branch involved in such effect was demonstrated to be the eukaryotic translation initiation factor 2 (eIF2)α signaling pathway. 60Another branch of the UPR, which is driven by the endonuclease activity of the inositol-requiring enzyme 1 (IRE1), may induce pre-miR-301a degradation and, thus, elevate Gadd45a mRNA expression to switch the ER stressed cells to apoptosis. 61optotic cells display a remarkable increase in GADD45A protein levels, thus pointing to a pivotal role for this protein in the signal transduction of the apoptotic cascade. 33GADD45A may prevent oncogene-induced cell proliferation through direct interaction with p38 MAPK, in a process mediated, at least in part, by p53 activity (Figure 1). 62On the other hand, suppression of JNK activity by NF-κB favors tumorigenesis and metastasis in several cancer types, since it enhances cell survival and allow them to escape from apoptosis 63,64 and, interestingly, GADD45A binds to and activates MEKK4 to inhibit cell growth and induce apoptosis through the activation of the p38 and JNK pathways. 65As a consequence, defective activation of MEKK4 and both JNK and p38 MAPK in Gadd45a knockout mice could account for the reduced apoptosis in keratinocytes that display these mice after UV radiation exposure. 66And the opposite is also true; GADD45A activation, through the stimulation of p38 and JNK pathways, induces apoptosis and regulates nuclear import of CDK1 kinase to promote sustained G2/M arrest. 67,68rther, JNK regulates both the intrinsic and extrinsic apoptotic pathways and, consistent with this, GADD45A is also capable of inducing mitochondrial dysfunction and the JUN-dependent AP-1 transcriptional activity during cell death response. 33Strikingly, both JNK and p38 kinases are able to induce Gadd45a transcription in response to certain genotoxic stressors, thus leading to a positive feedback loop in their apoptotic effect. 33Also, despite p53 is widely recognized as an upstream transcriptional activator of Gadd45a, the latter could contribute to withstand p53 activity through the activation of the JNK and p38 MAPK pathways, sustaining another proapoptotic positive feedback regulation. 33

| GADD45A: A first chorus voice in DNA repair and (de)methylation
GADD45A is regarded as an important regulator in nucleotide excision repair, base excision, and post replication repair, and also contributes to avoid abnormal centrosome duplication and amplification, which are essential steps for an effective mitosis, thus preventing uncontrolled cell transformation and tumorigenesis. 69,70However, GADD45A lacks the catalytic activity necessary to directly carry out such effects, and the mechanisms involved have not been fully elucidated.It is known that its binding to PCNA has an important role in nucleotide excision DNA repair in response to UV radiation, 2,71 in the inhibition of DNA replication, 30 as well as in growth suppression by inhibiting the entry of cells into S phase (Figure 1). 36In particular, GADD45A recruits PCNA to the damaged chromatin sites, and also acts as a downstream mediator of FOXO3a to induce G2/M phase arrest and repair damaged DNA under low level of stress condition. 33On the other hand, GADD45A can form homo-oligomers and hetero-oligomers with the other two members of the family and, although the physiological relevance of this phenomenon is still not completely understood, some studies point to that it could be involved in the modulation of the accessibility to damaged chromatin and subsequent DNA repair process. 72,73In fact, the two distinct regions of GADD45A involved in self-association partially overlap with PCNA binding domains and, thus, might lead to functional consequences. 73Besides PCNA, GADD45A has been reported to interact with several other DNA repairrelated proteins, including histones, single-stranded DNA cytosine deaminase (AID or AICDA), methyl-CpG-binding domain protein 4 (MBD4), G/T mismatch-specific thymine DNA glycosylase (TDG), ERCC5, and the transcription initiation factor TFIID subunit 12 (TAF12) (Figure 1). 70It has been demonstrated that GADD45A binds to damaged chromatin and enables the accessibility of other proteins involved in DNA repair, such as the endonuclease ERCC5, 70 and also participates in base excision repair by hampering the interaction of APEX1 with PCNA, a fact which protects from carcinogenic base damage and contributes to preserve genomic stability. 71A methylation at specific cytosine residues within the regulatory promoter regions of the genes causes chromatin condensation and, as a result, it results in the inhibition of the binding of the transcription machinery.
GADD45A-mediated DNA demethylation allows specific epigenetic activation of genes related to cellular responses to stress, nuclear hormones, or differentiation, but also might account for hypermethylation and subsequent inactivation of tumor suppressor genes in the presence of low GADD45A activity. 70,74In support of a role for GADD45A in active demethylation, its overexpression by using genetic tools or its DNA-damage-induced increase promoted DNA demethylation at specific CpG residues within the regulatory regions of genes encoding for ribosomal proteins (rDNA), whereas Gadd45a downregulation led to hypermethylation and rDNA transcription silencing. 75The latter study further demonstrated that GADD45A-driven demethylation is initiated by promoting association of TAF12 to the rDNA gene, followed by recruitment of the nucleotide excision repair machinery that removes methylated cytosines.Likewise, the tumor suppressor inhibitor of growth protein 1 (ING1) 76 and ten-eleven-translocation (TET) proteins 77 cooperate with GADD45A for targeting active DNA demethylation to specific regions.GADD45A also promotes DNA demethylation by bringing on the interaction between thymine DNA glycosylase and activation-induced deaminase in a multimeric complex, and by promoting the DNA repair machinery, which replaces methylated cytosines by unmethylated nucleotides. 74,78,79Some data also suggest that GADD45A might promote demethylation of damaged double-strand DNA, but also it could hamper remethylation of hemi-methylated sites through the inhibition of the DNA (cytosine-5)-methyltransferase 1 (DNMT1). 80Besides relaxing chromatin through DNA demethylation, GADD45A may also locally modify chromatin accessibility by relaxing topoisomerase and increasing its cleavage activity, an effect that can destabilize histone-DNA interactions. 81,82In any case, and due to unknown reasons, other studies have been unable to quantify any change in DNA demethylation after Gadd45a overexpression or suppression 83 and, thus, whether GADD45A promotes DNA demethylation remains still unsolved.

| GADD45A: A MULTI-INSTRUMENTALIST ARTIST WITH ANTICANCER ACTIVITY
GADD45A is a tumor suppressor that is capable of delaying, or even preventing, tumorigenesis at multiple levels (Figure 2 and Table 1).As stated above, it stands out by its important role in regulating apoptosis and senescence, but it participates in DNA repair and inhibits cell migration as well, by this means avoiding the conversion of damaged cells into malignant cells. 8In agreement with these beneficial effects, GADD45A protein levels are reduced in many human tumors and cancer cell lines, and several tumor suppressors induce its transcription. 9Further, Gadd45a expression levels positively correlate | 1383 with a higher survival rate and a better prognosis in patients with esophageal squamous cell carcinoma, 94 and epigenetic modulation of GADD45a is related to its reduced gene expression in cancer, since its promoter region is hypermethylated in several CpG residues of some breast cancers, gastric cardia adenocarcinoma or prostate cancer. 95,96,114It has even been suggested that serum levels or methylation degree of GADD45A could be used as a biomarkers to predict the response and survival rate after neoadjuvant chemotherapy in gastric cancer patients, 97 or to distinguish patients with benign prostate cancer from malignant. 98Even though genetic mutation and inactivation of this protein is a very infrequent cause of cancer, 14,17,99,100,115 there are some studies that report an association between GADD45A mutations or polymorphisms and the development of cancer.For example, an investigation examining the genotype distribution between patients and age-matched healthy women found out a GADD45A polymorphism (+1506 T > C) that was associated with ovarian cancer susceptibility and prognosis, and for this reason, the authors pointed to that it could be successfully used as a biomarker. 116is same genotype and others (+3204 G > C) have been associated with reduced susceptibility to sporadic and F I G U R E 2 GADD45A: A tumor suppressor that acts at multiple levels.GADD45A is an important regulator of nucleotide excision repair, base excision, and post replication repair, thus preventing tumorigenesis.To carry out such effects, it binds to PCNA and hampers its interaction with APEX1, thus enabling the accessibility of other proteins involved in nucleotide excision DNA repair (such as ERCC5).Besides PCNA, GADD45A may interact with several other DNA repairrelated proteins, including histones, AID, MBD4, TAF12, and TDG.Further, GADD45A can homodimerize and even heterodimerize with the other two members of the family (GADD45B and G) to modulate the accessibility to damaged chromatin and subsequent DNA repair process.On the other hand, GADD45A-mediated DNA demethylation allows specific epigenetic activation of genes related to cellular responses to stress or differentiation.The tumor suppressor ING1 and TET proteins cooperate with GADD45A for targeting active DNA demethylation to specific regions.GADD45A is also able to promote DNA demethylation at specific CpG residues within the regulatory regions of genes encoding for ribosomal proteins (rDNA), in a process which requires TAF12 and subsequent recruitment of the nucleotide excision repair machinery.In a similar way, GADD45A also promotes DNA demethylation by bringing on the interaction between TDG and AID in a multimeric complex.GADD45A can also hamper remethylation through the inhibition of DNMT1.Another antitumoral activity of GADD45A encompasses induction of apoptosis and cellular senescence.In response to certain stresses, GADD45A interacts with and inhibits CDK1, leading to subsequent cell cycle G2/M arrest and cell growth suppression.The binding to MEKK4 and subsequent activation of p38 and JNK signaling pathways seems to be also involved in the induction of the G2/M arrest of the cell cycle by GADD45A.Thus, the stimulation of p38 and JNK pathways regulates nuclear import of CDK1 kinase and activates p53 to promote sustained G2/M arrest and apoptosis.Interestingly, both JNK and p38 kinases are able to induce Gadd45a transcription in response to certain genotoxic stressors, thus leading to a positive feedback loop in their apoptotic effect.GADD45A binds to p21 as well, a CDK inhibitor involved in G1/S and G2/M cell cycle arrest, suggesting that it is involved in the regulation of cell cycle at different stages.In fact, prolonged activation of the GADD45A/p21 pathway induces cellular senescence due to mitochondrial dysfunction and oxidative stress.GADD45A may also induce cell death by activating the eIF2α branch of the UPR, which results in a reduction in cyclin B1 levels, by inhibiting autophagy, or by inducing the JUN-dependent AP-1 transcriptional activity.Finally, GADD45A can initiate apoptosis by physically interacting with the elongation factor EF1.This results in the release of the proapoptotic protein BIM from microtubule protein complexes, which boosts the BAX/cytochrome c-dependent apoptosis.Additional mechanisms may account for the anticancer activities of GADD45A.For instance, it negatively regulates p63 and β-catenin oncogenes, resulting in reduced expression of genes involved in extracellular matrix remodeling such as endopeptidases and MMP.MMP expression inhibition by GADD45A probably involves both p38 MAPK and p53 activation.Pancreatic ductal adenocarcinoma [37, 74]   Radiotherapy and some therapeutic drugs ↑ Induces cancer cell apoptosis and inhibits autophagy.

Adipose tissue
Obesity ↑ Promotes white adipocyte differentiation through the upregulation of the expression of cell cycle related genes and the activation of PPARγ.

Atherosclerosis
Promotes endothelial cell apoptosis and activation, and cardiomyocyte apoptosis.
[ 87-93]   (Continues) non-BRCA1/2 familial breast cancers in Chinese populations. 115Finally, point mutations in exon 4 of GADD45A gene have been related to increased frequency of some human pancreatic carcinomas. 117udies on genetically modified mouse models thoroughly support the anticancer role of GADD45A.Gadd45a deletion in mice increases mutation rate and enhances susceptibility to carcinogenesis in the presence of genotoxic stressors.In particular, Gadd45a knockout mice showed an increased prevalence of ovarian, hepatocellular, and vascular tumors. 34,84,85Other reports demonstrate that suppression of Gadd45a alone hastens lung tumor progression, 86 and render mice more prone to UV-induced skin tumors. 68Further supporting this, GADD45A overexpression in UV-exposed keratinocytes mediates a p38 and JNK MAPK-dependent proapoptotic response that avoids the accumulation of damaged and potentially carcinogenic cells. 33GADD45A has also a tumor suppressor role in chronic myelogenous leukemia, since its downregulation in mice resulted in a more aggressive phenotype and a reduced survival time, through the modulation of the AKT, p38 MAPK and signal transducer and activator of transcription (STAT)5 signaling pathways. 107 breast cancer, the GADD45A levels depend on the hormone receptor status; triple-negative breast cancer tumors display low GADD45A protein levels, whereas luminal A and luminal B subtypes have high levels. 118In this regard, the estrogen receptor (ER)β has a protective role in breast cancer development through the inhibition of cell proliferation, and some findings demonstrate that ERβ causes a G2 cell cycle arrest by inactivating CDK1 through the repression of cyclin B1 and stimulation of Gadd45a expression. 119Other studies demonstrate that GADD45A inhibits mammary tumor onset and growth by inducing the JNK-activated apoptosis and p38-mediated cellular senescence, whereas its suppression has an opposite effect. 85In support of this, the anti-tumorigenic effects of the hypoglycemic peroxisome proliferator-activated receptor (PPAR)γ agonist troglitazone in breast cancer models positively correlated with the increase in Gadd45a mRNA expression and protein levels, the activation of the JNK pathway, and apoptosis. 108On the other hand, breast cancer cases are often associated with mutations in BRCA1 protein, which is implicated in the cellular response to DNA damage that triggers apoptosis through activation of JNK, and also may induce GADD45A through the interaction with other transcription factors to upregulate apoptosis. 109,110In the presence of genotoxic stress, p53 protein induces Gadd45a transcription, but in the absence of DNA damage, BRCA1 represses it. 110Further, GADD45A acts synergistically with BRCA1 to regulate centrosome duplication and increase the genomic stability. 120,121A B L E 1 (Continued) Organ/tissue and disease or condition GADD45A levels GADD45A-associated functions References

Skeletal muscle
Muscle atrophy induced by fasting, aging or disuse ↑ Represses genes involved in anabolic signaling, protein synthesis, glucose uptake, glycolysis, oxygen delivery, mitochondrial biogenesis, citric acid cycle, and oxidative phosphorylation.
[ 51, 113]   Induces the expression of genes involved in autophagy and caspase-mediated proteolysis.
| 1387 An important playmaker in cancer development is NF-κB.NF-κB is constitutively triggered due to the activation of IKK in human pancreatic ductal adenocarcinoma and in several pancreatic cancer cell lines, where it exerts an antiapoptotic role. 111Interestingly, GADD45A levels are reduced by NF-κB in pancreatic cancer cells, 111 and constitutive activation of NF-κB in several types of cancer leads to the repression of GADD45A, probably through MYC induction, an effect that is essential for cancer cell survival and tumor formation. 8,63Accordingly, inhibition of NF-κB boosts GADD45A-dependent induction of apoptosis in cancer cells, hence reducing tumor growth, in a process mediated by JNK activation. 63,111wever, it has also been demonstrated that, in response to some cancer therapeutic drugs (e.g.arsenite), the IKKβ/NF-κB pathway may activate the JNK signaling cascade through the induction of GADD45A. 64This effect would be dependent on the p50 subunit of NF-κB, but independent of p65, and would involve a reduction in the ubiquitination and subsequent proteasome-dependent degradation of GADD45A.In addition, GADD45A may stimulate IκB degradation and NF-κB translocation into the nucleus. 2 Thus, NF-κB might regulate the expression of Gadd45a at the same time it acts as its regulatory target, in a sort of positive feedback loop.Overall data suggest that the IKKβ/NF-κB axis may mediate both proor antiapoptotic effects of the GADD45A/JNK pathway by inducing or inhibiting, respectively, its activity, depending on the triggering stimulus.
Additional mechanisms may account for the proapoptotic effects of GADD45A in cancer cells.For instance, GADD45A may induce apoptosis by physically interacting with the elongation factor (EF)1, a microtubule-severing protein, thus releasing the proapoptotic protein BIM from microtubule protein complexes, which will induce BAX/ cytochrome c-dependent apoptosis. 112Not surprisingly, suppression of endogenous BIM greatly inhibits GADD45A-induced apoptosis.A more recent study also demonstrated that GADD45A may induce cancer cell death and reduce tumor recurrence in several murine models and cancer cell lines through a combined action involving stimulation of apoptosis and inhibition of autophagy, in particular after radiotherapy and chemotherapy treatments, when it is strongly induced. 122Other less-known anticancer activities for GADD45A have also been reported.GADD45A negatively regulates p63 and β-catenin oncogenes. 66β-catenin is a transcription factor that induces the expression of genes involved in extracellular matrix remodeling such as endopeptidases and the matrix metalloproteinases (MMP)3 and MMP9, thus enabling cell proliferation, and the invasion and metastasis of cancer cells.For instance, in medulloblastomas, GADD45A negatively regulates cancer cell invasion and migrationassociated proteins, such as MMP9 and β-catenin, thus reducing epithelial-mesenchymal transition and its metastatic potential. 123Another study demonstrated that GADD45A hinders epidermal tumor cell migration and invasion under UV exposure by inhibiting MMP expression, in a process involving p38 MAPK and p53 activation, and association with the adenomatous polyposis coli (APC) complex. 113On the contrary, and for an unknown reason, GADD45A positively regulates the expression of the MMP9 in non-tumoral keratinocytes. 87In fact, the protein levels of GADD45A and MMP9, an enzyme that also hinders wound healing, are upregulated in skin tissues from patients with diabetic foot ulcers and in diabetic rodents, and in cultured human keratinocytes exposed to advanced glycation end products. 87The same study demonstrated that Gadd45a suppression prevented the increase in Mmp9 transcription by inhibiting demethylation in its promoter, without affecting cell proliferation or apoptosis, while Gadd45a overexpression resulted in the opposite.Finally, angiogenesis is another important factor contributing to tumorigenesis, and GADD45A, by suppressing STAT3 transcriptional activity, reduces the secretion of vascular endothelial growth factor (VEGF) and lessens the formation of tumor blood vessels. 88twithstanding its well-established tumor suppressor properties, an antiapoptotic and pro-survival role has also been described for GADD45A in some malignancies, and evidence exist of a positive correlation between its gene expression and certain tumors development.For instance, it exerts a pro-survival activity in hematopoietic cells exposed to UV radiation and certain anticancer drugs, since its suppression results in defective G2/M arrest and increased apoptosis. 124ditional studies demonstrated that GADD45A boosts the NF-κB survival pathway through the activation of p38 MAPK and subsequent phosphorylation-mediated ubiquitination of its inhibitory protein IκB. 12 Likewise, despite GADD45A suppresses the RAS-driven breast tumorigenesis, it is capable of promoting the MYC-driven breast cancer. 85,89In this latter case, GADD45A activation results in a strong decrease in the angiogenic enzyme MMP10 levels via the glycogen synthase kinase (GSK3)β/β-catenin cascade, which enhances tumor vascularization and growth. 89Further supporting this, Gadd45a downregulation in MYC-driven breast cancer enhances apoptosis or cellular senescence. 85,89GADD45A overexpression also prevents melanoma cell death by inhibiting the mitochondrial apoptotic pathway. 33Finally, Gadd45a gene expression and protein levels are elevated in a vast majority of pancreatic ductal adenocarcinomas, all contributing to worsen their prognosis compared to those patients with low GADD45A levels. 8Studies in vitro have demonstrated that knockdown of Gadd45a impairs proliferation and induces apoptosis of pancreatic cancer cells, suggesting that this protein carries out a pro-survival role in these malignant cells. 1,111On the other hand, cisplatin resistance in some melanomas has been related to increased expression of Gadd45a due to the activation of the ERK1/2 MAPK pathway, while suppression of Gadd45a sensitized melanoma cells to this drug by enhancing chemotherapy-induced DNA damage. 90| GADD45A EFFECTS BEYOND CANCER 4.1 | Central nervous system: Rethinking the role of GADD45A Alzheimer's disease (AD) is characterized by the extracellular deposition of neurotoxic amyloid β-peptide (Aβ) in the brain, which correlates with the activation of AP-1 and NF-κB, and relates to DNA damage due to double-strand breaks, a characteristic of apoptosis.DNA rupture is regarded as an early sign of neuron degeneration that may be frequently detected in the cortex of AD patients.Interestingly, brain tissue samples obtained from AD patients display increased GADD45A protein levels in the hippocampus and the occipital lobe, which colocalizes with neurons upregulating the antiapoptotic protein BCL-2 (B-cell lymphoma protein 2) and positively correlates with DNA damage (Figure 3 and Table 1). 125Previous studies have demonstrated that exposure of human preneuronal NT2 cells to Aβ provoke a strong and rapid increase in the expression of Gadd45a, an indicator of DNA damage and The multiple roles of GADD45A beyond cancer.Brain tissue from AD patients displays increased GADD45A protein levels, which colocalizes with neurons upregulating the antiapoptotic protein BCL-2 and positively correlates with DNA damage.Further, GADD45A has a crucial function in the response to DNA strand breakage induced by Aβ in neurons.On the other hand, ischemia or stimulus that enhance oxidative stress and cell death through the activation of the N-methyl-D-aspartate or kainate receptors are associated with a rise of GADD45A mRNA and protein levels.This increase may be both dependent or independent of p53 transcriptional control, and is aimed to counteract oxidative stress and DNA damage induced by this stimulus, thus improving neuron survival and conferring neuroprotection to the injured brain.In both white and brown adipose tissue, GADD45A induces adipogenesis and lipid accumulation by promoting PPARγ demethylation in the promoter region.In brown adipose tissue, this results in enhanced PPARγ transcription and activity, and the subsequent modulation of its target genes, such as Fabp4, Fas, Pgc-1α, and Ucp1.As a result, increased GADD45A activity reduces mitochondrial biogenesis, cell proliferation, and triglyceride lipolysis, while increases lipid accumulation, overall resulting in reduced glucose uptake and insulin sensitivity.Acetaminophen-induced liver injury (AILI) induces a rapid increase in GADD45A levels in the liver, where it exerts an hepatoprotective effect through the activation of AMPK, a master regulator of metabolism that induces the expression of genes related to energy-producing pathways, such as glucose uptake, glycolysis, glycogenolysis, fatty acid β-oxidation, and lipolysis, while inhibiting anabolic processes, such as the gluconeogenesis and the biosynthesis of fatty acids, cholesterol, and proteins.Liver steatosis induced by a high-fat diet feeding is linked to ER stress, which if not corrected may result in hepatocyte apoptosis and excessive lipids liberation, thus contributing to fat accumulation.This is accompanied by increased expression of Atf4, Chop and Gadd45a in the liver, which is coupled with increased steatosis, fibrosis and inflammation.In this context, the PERK branch of the unfolded protein response activated during ER stress boosts hepatocyte apoptosis via ATF4 by regulating Gadd45a expression, while CHOP itself elicits Gadd45a gene expression to attenuate oxidative stress and ensuing inflammatory signaling, and to induce apoptosis to protect cells from excess ER stress.Finally, during hepatic fibrosis, GADD45A reduces the expression of extracellular matrix proteins and counteracts fibrosis by inhibiting the TGF-β signaling pathway, while it also reduces local inflammation (TNF-α, IL-6 rise) and oxidative stress.Skeletal muscle atrophy may be caused by diverse stresses, such as aging, malnutrition, starvation, muscle disuse, any serious injury.In such process, upregulation of GADD45A levels by several upstream regulators has been recognized, including ATF4, C/EBPβ, FOXO and p53.GADD45A transcriptionally repress genes involved in anabolic signaling, protein synthesis, glucose uptake, glycolysis, oxygen delivery, mitochondrial biogenesis, citric acid cycle, and oxidative phosphorylation, but induces genes involved in autophagy and caspase-mediated proteolysis, overall resulting in muscle atrophy.In addition, GADD45A probably cooperates with other transcriptional regulators (e.g., NF-κB) to drive skeletal muscle atrophy.During atherosclerosis, VSMC growth factors induce a robust increase in Gadd45a during the S phase, and a huge increase after cell division, in a process transcriptionally regulated by p53 and BRCA1.In a similar way, PPARγ activation with the antidiabetic thiazolidinediones induces apoptosis by enhancing POU2F1-mediated transcription of Gadd45a in endothelial cells, monocyte-derived macrophages, and also VSMC, thus reducing neointima formation during the atherosclerotic process.GADD45A might also have an important role during heart disease.Oxidative stress-induced apoptosis in cardiomyocytes is accompanied by a remarkable increased expression of several p53 upstream regulators, and several downstream effectors such as GADD45A.GADD45A is regarded an important contributor toward myocardial apoptosis and ensuing development of heart failure.Finally, GADD45A also acts as an upstream modulator of pathophysiological changes occurring during pre-eclampsia.Placental stresses during pregnancy, including hypoxia, inflammatory cytokine secretion or hypertonic stress, induce GADD45A and its downstream effector p38 MAPK, both contributing to endothelial activation and the pathogenesis of pre-eclampsia through a mechanism involving the regulation of Mmp2 and Mmp9 expression and the inhibition of oxidative stress, which could promote the invasion and migration of trophoblast cells during early pregnancy.Created with BioRender.DNA excision-repair processes. 91,126The authors of these studies concluded that GADD45A has a crucial function in the response to DNA strand breakage induced by Aβ in neurons.
Oxidative stress induced by glutamate plays a key role in cell death during the development of several neuronal-related diseases, and is often associated with an increase of GADD45A mRNA and protein levels (Figure 3). 92In mouse hippocampal neuronal cells, p53 genetic ablation suppressed glutamate-induced increase in GADD45A and the subsequent cell death. 92The latter was also prevented by Gadd45a knockdown.Likewise, excitotoxic injury induced with the N-methyl-D-aspartate receptor agonist quinolinic acid enhanced p53 mRNA and protein expression in brain, together with GADD45A and the proapoptotic BAX, all involved in the response to damage. 93However, GADD45A increase preceded that of p53, suggesting that the latter was not involved in its transcriptional upregulation.GADD45A levels are also induced in neurons of rats with either epileptic seizures or hippocampal oxidative neurotoxicity induced by kainate, thus supporting the hypothesis that GADD45A has a cytoprotective role in the injured brain. 92,101,127Actually, GADD45A is regarded as a specific marker of peripheral nerve injury in adult primary sensory and motor neurons, where it plays a role in protecting neurons against cell death. 102In a similar way, GADD45A protein is induced in the brain following transient ischemia in rodents, probably as a protective response to prevent oxidative stress and genomic DNA damage. 103,127In fact, brain injury that occurs as a consequence of an ischemic event is characterized by the presence of both single-stranded and double-stranded DNA breakage, which is followed by an increase of the GADD45A levels in pyramidal neurons of the affected hemisphere. 104Thus, GADD45A is probably upregulated in response to DNA damage as a mechanism to enhance cell survival after the ischemic insult. 104,128tivity regulated cytoskeletal-associated protein (Arc) is among some of the genes that are transcriptionally demethylated and turned on upon neuronal activation in several neurological and psychiatric disorders.Its expression is associated with synaptic plasticity in learning and memory and in pathological conditions such as depression or AD. 105Interestingly, studies performed in in vitro and in vivo models have reported reduced mRNA expression for all three members of the Gadd45 family in hippocampal neurons, and have pointed to that GADD45 proteins, by demethylating the immediate early activity gene Arc, might become suitable therapeutic targets for pathological conditions such as depression or AD. 105On the other hand, it has been reported that valproic acid, which is utilized in the treatment of bipolar disorder and works by improving the efficiency of neurotransmission with several neurotrophic effects, induces neurite outgrowth through the JNK-paxillin signaling cascade in a process mediated by GADD45A. 106

| Regulatory roles of GADD45A in adipose tissue and the liver
Apart from its most well-known actions as stress sensor, GADD45A may also influence metabolism (Figure 3 and Table 1).Transcriptomic analyzes have found a positive correlation Gadd45a mRNA expression in adipose tissue and fat deposition and obesity. 129GADD45A promotes white adipocyte differentiation and has also a role in regulating energy metabolism of adipose tissue. 130In brown adipose tissue, Gadd45a suppression resulted in enhanced lipolysis, mitochondrial biogenesis, and cell proliferation, but reduced lipogenesis, while its overexpression displayed an opposite effect. 129As a result, Gadd45a knockdown in vivo improved insulin sensitivity, glucose uptake, and energy expenditure. 129The adipogenic effect of GADD45A is due to the upregulation of the expression of cell cycle related genes, but also to the stimulation of PPARγ transcriptional activity over genes related to lipid metabolism (Pparα, fatty acid binding protein 4, fatty acid synthase, and Pparγ itself, among others). 129A very recent study corroborates this adipogenic role for GADD45A, but involving a different mechanism.Thus GADD45A expression positively correlated with subcutaneous fat deposition and obesity in humans and animals, whereas its overexpression in mature adipocytes obtained from porcine and mouse subcutaneous adipose tissue promoted their differentiation and lipid accumulation. 131In consonance with this, GADD45A deletion in mice rendered them resistant to high-fat diet-induced obesity, and boosted the expression of PALOMER ET AL.
| 1391 mitochondria-related genes.Importantly, these effects required the interaction of GADD45A with STAT1 and subsequent inhibition of the latter. 131etaminophen-induced liver injury (AILI), a disorder characterized by hepatic metabolism dysregulation, induces a rapid increase in GADD45A levels in livers of patients and in in vivo and in vitro models, where it exerts an hepatoprotective effect. 132For this reason, it has been suggested that Gadd45a gene expression in the liver might be used as a biomarker to identify those patients with drug-induced liver injury. 133In particular, and within the context of AILI, GADD45A triggers AMP-activated protein kinase (AMPK) activity, a master regulator of metabolism that induces the expression of genes related to energy-producing pathways, such as glucose uptake, glycolysis, glycogenolysis, fatty acid β-oxidation, and lipolysis, while inhibiting anabolic processes, such as the gluconeogenesis and the biosynthesis of fatty acids, cholesterol, and proteins. 132In support of a role for GADD45A, caffeic acid, an organic compound that is capable of attenuating AILI, abrogates the EGR1-mediated increase in GADD45A in the liver. 134l three GADD45 family proteins contain at least one LXXLL signature motif, which is known to be involved in protein-protein interactions. 10Actually, GADD45 proteins are known to interact and activate several nuclear receptors, including constitutive androstane receptor (CAR), ERα, retinoic acid receptor (RAR)α, retinoid X receptor (RXR)α, and the three PPAR subtypes, 10,135 suggesting that they may act as transcriptional coregulators.GADD45 proteins could be important, for instance, in DNA demethylation processes at specific gene promoter locus, since nuclear receptors, once bound, may recruit the demethylation machinery. 70ver steatosis induced by a high-fat diet feeding in mice is linked to ER stress, which if not corrected may result in hepatocyte apoptosis and excessive lipids liberation, thus contributing to fat accumulation.7][138] The increased expression of these genes is aimed to induce apoptosis, to protect cells from excess ER stress. 137A recent study has demonstrated that the PERK (protein kinase R-like ER kinase) branch of the unfolded protein response, which is activated during ER stress, boosts hepatocyte apoptosis via ATF4 by regulating Gadd45a expression, 136 while CHOP itself elicits Gadd45a gene expression to stimulate apoptosis (Figure 3). 1388][139][140] In consonance with this, the GLP-1 receptor agonist semaglutide might be useful in controlling liver steatosis by reducing inflammation, insulin resistance, and ER stress through a mechanism involving GADD45A, and independently of weight loss or any effect on lipogenesis. 136In a rodent model of nonalcoholic steatohepatitis induced by methionine-and choline-deficient diet, Gadd45a suppression boosted hepatic inflammation, fibrosis, and apoptosis, which were accompanied by disrupted fatty acid metabolism and greater oxidative and ER stress. 141The same study demonstrated that ER stress alone was able to induce Gadd45a mRNA expression in hepatocytes, in an attempt to attenuate cellular stress and ensuing inflammatory signaling.
Hepatic fibrosis, which may lead to cirrhosis and hepatocellular carcinoma, is characterized by the accumulation of extracellular matrix proteins, including collagens, proteoglycans, fibronectin, and several glycoproteins.This fibrogenic response is accompanied by increased cell proliferation, hepatic stellate cells activation, hepatocyte apoptosis, and local inflammation (tumor necrosis factor α, TNF-α, and IL-6 rise), overall leading to liver injury and dysfunction.Of note, most of these changes are driven by TGF-β-mediated pathways in a GADD45A-dependent manner (Figure 3).Thus, a significant downregulation of GADD45A, but not GADD45B or GADD45G, together with the activation of the TGF-β signaling pathway was observed in fibrotic liver tissues of mice and isolated hepatic stellate cells with chronic liver injury, while forced overexpression of GADD45A reduced the expression of extracellular matrix proteins and counteracted fibrosis by inhibiting the TGF-β signaling pathway. 142Scavenging of reactive oxygen species and upregulation of antioxidant enzymes contributed to the beneficial effects of GADD45A on the liver.

| A critical mediator of muscle atrophy
Muscle atrophy is a complex and debilitating disorder that may be caused by diverse stresses, such as aging, malnutrition, starvation, muscle disuse, muscle denervation, any serious illness or injury.It is often characterized by a reduction in AKT signaling and an increase in Atf4 expression, GSK-3β signaling, caspase-3 activity, and autophagy.It is worth mentioning that Gadd45a expression, which is widely recognized as a major inducer of muscle atrophy, is induced by fasting, aging or disuse in skeletal muscle, in a process at least partially regulated by ATF4 (Figure 3). 78,143Ensuing increased nuclear GADD45A levels transcriptionally repress genes involved in anabolic signaling, protein synthesis, glucose uptake, glycolysis, oxygen delivery, mitochondrial biogenesis, citric acid cycle, and oxidative phosphorylation, but induces the mRNA levels of genes involved in autophagy and caspase-mediated proteolysis, overall resulting in muscle atrophy derepression (Table 1). 78In fact, GADD45A overexpression in muscle or cultured myotubes also induces atrophy in the absence of any upstream stress. 78terestingly, diabetic patients often develop a specific skeletal muscle pathology characterized by mitochondrial function impairment, oxidative stress and type II fiber atrophy, which are all linked to exercise intolerance. 144In support of a role for GADD45A in such effects, overexpression of this protein in mice promoted atrophy of type II fibers, while Gadd45a expression downregulation by using RNA interference technology provoked the opposite effect. 78In fact, type II fibers are more prone to atrophy also during fasting and denervation. 78F4 is not the only pathway regulating GADD45A in muscle atrophy, but also other potential upstream regulators might be involved, including C/EBPβ, FOXO and p53. 78C/EBPβ may work alone or in cooperation with ATF4 to induce Gadd45a expression and muscle atrophy through the binding to specific sites within the promoter of its gene. 143In addition, GADD45A probably cooperates with other transcriptional regulators (e.g.FOXO itself, NF-κB, and myogenin) to drive skeletal muscle gene expression leading to atrophy. 78In this regard, muscle atrophy due to accelerated catabolism of proteins is rapidly observed after peripheral nerve transection or spinal cord injury, and GADD45A, together with insulin-like growth factor 1 (IGF-1) and myogenin, are strongly regulated during the initial stages of this process. 145In any case, beyond this specific role in myofiber atrophy, GADD45A might also have an antiapoptotic effect on these cells, probably by blocking NF-κB signaling, which is itself activated during atrophy due to denervation. 1454 | GADD45A in cardiovascular diseases: Good or evil?
The pathogenesis of atherosclerosis involves the proliferation of vascular smooth muscle cells (VSMC), a process transcriptionally regulated by the immediately early genes EGR1, FOS/AP-1, and MYC. 146In early stages, apoptosis of VMSC reduces neointimal thickening and hastens atherosclerosis, but in later stages it may confer greater instability to atherosclerotic plaque.It has been hypothesized that, during atherosclerosis, VSMC growth factors would induce an initial and transient downregulation of the basal Gadd45a expression before the beginning of the S phase, which would be followed of a robust increase during the S phase and, lastly, a huge increase after cell division. 146Regulation of the transcriptional activity of p53 and BRCA1 would account for these changes in Gadd45a gene expression (Figure 3).In agreement with this, TNF-α-induced apoptosis of human umbilical vein endothelial cells correlates with increased expression of Gadd45a (Table 1). 147Likewise, PPARγ activation with the antidiabetic thiazolidinediones troglitazone and rosiglitazone induced apoptosis in endothelial cells, monocytederived macrophages, and VSMC, thus reducing neointima formation during the atherosclerotic process. 11,148Using genetic approaches or drug treatments, the authors demonstrated that this caspase-mediated apoptosis occurred through a PPARγ-dependent pathway, and required the activation of p53 and subsequent increase in Gadd45a expression, but was independent of BAX and BCL-2 expression. 11In particular, PPARγ activation by thiazolidinediones led to VSMC apoptosis and growth arrest by inducing POU2F1-mediated transcription of Gadd45a.In contrast, other authors have reported that these thiazolidinedione-induced proapoptotic effects are independent of PPARγ activation, although the reasons for this discrepancy are currently unknown. 148 the other hand, GADD45A acts as an upstream modulator of the pathophysiological changes occurring during pre-eclampsia (Figure 3). 149Placental stresses during pregnancy, including hypoxia, inflammatory cytokine secretion or hypertonic stress, induce GADD45A and its downstream effector p38 MAPK, both contributing to endothelial activation and the pathogenesis of pre-eclampsia. 149,150It has been suggested that silencing Gadd45a could be useful in the prevention of pre-eclampsia through a mechanism involving the regulation of Mmp2 and Mmp9 expression and the inhibition of oxidative stress, which could promote the invasion and migration of trophoblast cells during early pregnancy. 151DD45A might also have an important role during heart disease (Figure 3 and Table 1).Adult mammalian cardiomyocytes are refractory to proliferation and, as a consequence, any change in heart size due to stress, increased/decreased workload or disease must occur through hypertrophy or atrophy of the individual cells.Of note, some of the effector pathways regulating cardiomyocyte size are also involved in the transcriptional regulation of Gadd45a, including those controlled by NFAT, AKT, FOXO and MAPK. 152All these pathways regulate protein turnover and, in general, cell hypertrophy takes place when protein synthesis outweighs degradation, whereas the opposite occurs in atrophy.In consonance with this, a recent work aimed to identify differentially expressed genes between hypertrophic cardiomyopathy patients and healthy controls has demonstrated that GADD45A is a hub protein underlying this disease. 153e PERK-eIF2α-ATF4 branch of the ER also intervenes in regulating cardiomyocyte size, since it induces autophagy to resolve accumulation of misfolded proteins, and, as stated above, ATF4 is also relevant in controlling Gadd45a transcription. 152On the other hand, oxidative stress-induced apoptosis in neonatal rat cardiomyocytes and the heart of mice is accompanied by a remarkable increased expression of several p53 upstream regulators, and several downstream effectors such as GADD45A and PUMA, a p53-regulated modulator of apoptosis. 154,155Thus, GADD45A, together with p53, is regarded an important contributor toward myocardial apoptosis and ensuing development of heart failure.
GADD45A might also have a role in the cardiac disturbances occurring in people with diabetes.This so-called diabetic cardiomyopathy, which is defined as the myocardial dysfunction that develops in diabetic patients and is not directly attributable to other cardiovascular diseases, is characterized by metabolic disturbances that are often accompanied by local inflammation, oxidative stress, mitochondrial dysfunction, ER stress, cardiomyocyte apoptosis, and fibrosis, which affect cardiac output and eventually lead to heart failure.The diabetic heart exhibits an increase in Gadd45a expression, but reduced protein levels, suggesting a strong effect of posttranscriptional mechanisms in regulating GADD45A protein and mRNA levels. 50The same study demonstrated that PPARα, whose activity is often dysregulated in the diabetic heart, interacted with GADD45A, although the functional consequences of these interactions are still unknown.

| Immunomodulatory effects of GADD45A
Activation of naive T lymphocytes into Th1 cells is a complex process crucial in the adaptive immune system, and in which p38 MAPK plays a crucial role, but that is also dependent on dendritic cells through the secretion of diverse cytokines. 156Interestingly, GADD45A regulates the activation of both cell types: it induces p38 MAPK in dendritic cells, which favors Th1 polarization, while in T cells it inhibits the alternative activation pathway of this kinase (Table 1). 156,157As a result, Gadd45a knockout mice display impaired Th1 cell development and production of Th1related cytokines, since deficient p38 MAPK activation in dendritic cells overcomes increased p38 activity in T cells. 156Chronic activation of alternatively activated p38 MAPK in T cells in the absence of GADD45A might be involved, for instance, in autoimmune disease development in mice. 158,159In a similar way, Gadd45a knockout mice are prone to develop an autoimmune disease that resembles human systemic lupus erythematosus (SLE), which suggests that this protein acts as a negative regulator of T cells. 158However, another study performed in T cells from patients with SLE demonstrated that GADD45A might contribute to autoantibody formation and autoimmunity by promoting DNA demethylation of CD11a and CD70 regulatory regions in CD4 + T cells. 160 et al. 20 have examined the association of polymorphisms in the promoter region of the Gadd45a gene in patients with rheumatoid arthritis (RA) or SLE, since these polymorphisms have been related with an increase in the susceptibility to these and other inflammatory diseases.They found out a specific genotype (−589 CC) that is less susceptible to RA in DR4-negative individuals, and that is also related to the disease state in SLE patients.Although Gadd45a gene expression was not modified in peripheral blood mononuclear cells of these RA patients, 20 another study has reported a higher expression of Gadd45a in the adventitia of the ascending aorta of patients with RA compared to non-RA patients (Table 1). 161 patients with psoriasis, Gadd45a is expressed at higher levels in peripheral and in dermal infiltrating T cells, although no differences are found regarding p38 MAPK activity. 157The same authors reported that the GADD45A target Uchl1 (ubiquitin carboxyl terminal esterase L1) was hypermethylated at its promoter in psoriatic skin, and that Uchl1 lower expression fairly correlated with reduced GADD45A levels, overall data pointing to that GADD45A promotes keratinocyte hyper-proliferation and the production of pro-inflammatory mediators in psoriasis patients. 157Likewise, silencing of Gadd45a expression correlates with reduced Uchl1 levels in endothelial cells due to methylation changes in its promoter. 162Of note, UCHL1 is capable of inhibiting NF-κB and inducible nitric oxide synthase activity and, thus, its reduction might contribute to the inflammatory process in this pathology. 157

| CONCLUDING REMARKS
The stress-responsive GADD45 family consists of three proteins (GADD45A, GADD45B and GADD45G) that are ubiquitously distributed in adult mammalian tissues.However, they display distinctive expression patterns and partially overlapping functions, depending on the causative stress.Similar to the other family members, GADD45A is often associated with stress signaling and cellular injury response by means of its ability to control cell cycle arrest and growth, cell migration, and cell apoptosis, survival and senescence.Through these multiple actions, GADD45A has reportedly been recognized as a tumor suppressor capable of delaying, or even preventing, tumor development.
Besides these, the regulation that GADD45A exerts on DNA repair, and even its ability to modulate gene expression through epigenetic mechanisms, might also partly account for its tumor suppressor properties.Anyhow, there are also few studies indicating that GADD45A might operate as a tumor promoter due to some antiapoptotic and pro-survival properties in certain malignancies.Likewise, some authors have reported a positive correlation between the mRNA expression of Gadd45a and the development of specific tumors.Thus, overall data indicate that GADD45A may function as either tumor promoter or suppressor, depending on the oncogenic stimuli and the distinct signaling pathways activated.Further studies are warranted to elucidate the effects and potential benefits of GADD45A modulation during tumorigenesis.
Strikingly, more recent studies point to additional roles for GADD45A, although the involved mechanisms are much less studied.For instance, it regulates catabolic and anabolic pathways in many tissues, thus controlling body's overall metabolism.In adipose tissue, GADD45A regulates adipogenesis, lipid accumulation, and thermogenic metabolism, whereas its overexpression in animal models prevents hepatic inflammation, fibrosis, apoptosis, and oxidative and ER stress, while it improves fatty acid metabolism.In this regard, and due to the impact it has on glucose uptake and insulin sensitivity, some authors have pointed to GADD45A might be therapeutically targeted to counteract some of the effects contributing to obesity and diabetes.The transcriptional regulation of genes involved in metabolism, mitochondrial biogenesis and function, autophagy and proteolysis also account for the pivotal role that GADD45A plays during muscle atrophy.Finally, GADD45A has also been reported to display a cytoprotective effect against oxidative stress and inflammation.In this regard, GADD45A dysregulation in the cardiovascular system has been linked to atherosclerosis, or heart failure development due to diverse causes.