Gadd45b Expression Research Articles

Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice

The growth arrest and DNA damage-inducible 45 (Gadd45) proteins act in many cellular processes. In the liver, Gadd45b (encoding Gadd45β) is the gene most strongly induced early during both compensatory regeneration and drug-induced hyperplasia. The latter response is associated with the dramatic and rapid hepatocyte growth that follows administration of the xenobiotic TCPOBOP (1,4-bis[2-(3,5)-dichoropyridyloxy] benzene), a ligand of the nuclear receptor constitutive androstane receptor (CAR). Here, we have shown that Gadd45b-/- mice have intact proliferative responses following administration of a single dose of TCPOBOP, but marked growth delays. Moreover, early transcriptional stimulation of CAR target genes was weaker in Gadd45b-/- mice than in wild-type animals, and more genes were downregulated. Gadd45β was then found to have a direct role in transcription by physically binding to CAR, and TCPOBOP treatment caused both proteins to localize to a regulatory element for the CAR target gene cytochrome P450 2b10 (Cyp2b10). Further analysis defined separate Gadd45β domains that mediated binding to CAR and transcriptional activation. Although baseline hepatic expression of Gadd45b was broadly comparable to that of other coactivators, its 140-fold stimulation by TCPOBOP was striking and unique. The induction of Gadd45β is therefore a response that facilitates increased transcription, allowing rapid expansion of liver mass for protection against xenobiotic insults.

Gene expression profiling reveals differential effects of sodium selenite, selenomethionine, and yeast-derived selenium in the mouse

The essential trace mineral selenium is an important determinant of oxidative stress susceptibility, with several studies showing an inverse relationship between selenium intake and cancer. Because different chemical forms of selenium have been reported to have varying bioactivity, there is a need for nutrigenomic studies that can comprehensively assess whether there are divergent effects at the molecular level. We examined the gene expression profiles associated with selenomethionine (SM), sodium selenite (SS), and yeast-derived selenium (YS) in the intestine, gastrocnemius, cerebral cortex, and liver of mice. Weanling mice were fed either a selenium-deficient (SD) diet (<0.01 mg/kg diet) or a diet supplemented with one of three selenium sources (1 mg/kg diet, as either SM, SS or YS) for 100 days. All forms of selenium were equally effective in activating standard measures of selenium status, including tissue selenium levels, expression of genes encoding selenoproteins (Gpx1 and Txnrd2), and increasing GPX1 enzyme activity. However, gene expression profiling revealed that SS and YS were similar (and distinct from SM) in both the expression pattern of individual genes and gene functional categories. Furthermore, only YS significantly reduced the expression of Gadd45b in all four tissues and also reduced GADD45B protein levels in liver. Taken together, these results show that gene expression profiling is a powerful technique capable of elucidating differences in the bioactivity of different forms of selenium.Electronic supplementary materialThe online version of this article (doi:10.1007/s12263-011-0243-9) contains supplementary material, which is available to authorized users.

Gadd45a and Gadd45g regulate neural development and exit from pluripotency in Xenopus

Gadd45 genes encode a small family of multifunctional stress response proteins, mediating cell proliferation, apoptosis, DNA repair and DNA demethylation. Their role during embryonic development is incompletely understood. Here we identified Xenopus Gadd45b, compared Gadd45a, Gadd45b and Gadd45g expression during Xenopus embryogenesis, and characterized their gain and loss of function phenotypes. Gadd45a and Gadd45g act redundantly and double Morpholino knock down leads to pleiotropic phenotypes, including shortened axes, head defects and misgastrulation. In contrast, Gadd45b, which is expressed at very low levels, shows little effect upon knock down or overexpression. Gadd45ag double Morphants show reduced neural cell proliferation and downregulation of pan-neural and neural crest markers. In contrast, Gadd45ag Morphants display increased expression of multipotency marker genes including Xenopus oct4 homologs as well as gastrula markers, while mesodermal markers are downregulated. The results indicate that Gadd45ag are required for early embryonic cells to exit pluripotency and enter differentiation.

Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development

Gadd45 proteins have been implicated in the cellular response to physiological or environmental stress and the accompanying cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Although their molecular function is well studied, the expression and role of Gadd45 genes during embryonic development in mice is largely unknown. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. In situ hybridizations on sectioned and whole mouse embryos show most prominent Gadd45a expression in the tip of the closing neural tube, the cranial and dorsal root ganglia and the somites. Mouse Gadd45b is expressed strongly in the chorion, but only weakly in the embryo proper, including somites and limb buds. Murine Gadd45g expression strongly resembles Xenopus and medaka fish expression in primary neuron precursors and post-mitotic neurons, indicating a conserved role for Gadd45g in vertebrate neurogenesis. Additionally, Gadd45 genes show conserved expression during somitogenesis. In summary, Gadd45 genes are expressed in evolutionary conserved, but also divergent domains, which predominantly encompass areas of cell differentiation, consistent with their established function in growth arrest and DNA demethylation.

The role of deltex1 in regulating T cell tolerance (63.20)

Abstract Deltex1 (DTX1) is an E3 ubiquitin ligase and is suggested to be a mediator of Notch signaling. Our previous study found that DTX1 supressses T cell activation and promotes MEKK1 degradation. In this study, we found that T cell activation was partially suppressed by DTX1 with either inactive mutation of RING finger domain or deletion of WWE domain. In addition, we identified another suppressive mechanism of DTX1 by upregulation of Gadd45β, an inhibitor of JNK activation. DTX1 may promote the expression of Gadd45β in the absence of RF domain or WWE domain. Therefore, DTX1 inhibit T cell activation by either E3-dependent or E3-independent mechanisms. To further investigate the physiological function of DTX1 in lymphocyte development and activation, Dtx1-deficient mice were generated. T cell development in thymus and spleen was normal. B cell development in bone marrow was not affected but margine zone B cell population in spleen was reduced in Dtx1-deficient mice. T cell proliferation, cytokine production and MAPK activation were enhanced in Dtx1-deficient T cells. In addition, enhanced autoantibody production, elevated immune complex deposition in kidney and increased leukocyte infiltration in lung and liver were observed in aged Dtx1-deficient mice. These results suggest that DTX1 plays a role in T cell tolerance induction.

Senescence of chondrocytes in aging articular cartilage: GADD45β mediates p21 expression in association with C/EBPβ in senescence-accelerated mice

Growth arrest and DNA damage-inducible protein 45β (GADD45β) is expressed in normal and early osteoarthritic articular cartilage. We recently reported that GADD45β enhances CCAAT/enhancer binding protein β (C/EBPβ) activation in vitro. This study was undertaken in order to determine whether GADD45β is expressed with C/EBPβ in aging articular cartilage. We also investigated whether the synergistic expression of GADD45β and C/EBPβ may be involved in the mechanism of chondrocyte senescence. Senescence-accelerated mice (SAMP1) were used as a model of aging. GADD45β, C/EBPβ, and p21 were analyzed by immunohistochemistry. A luciferase reporter assay using ATDC5 cells was performed in order to examine p21 as a target gene of the GADD45β/C/EBPβ cascade. GADD45β exhibited increased expression in the aging articular cartilage of SAMP1 mice compared to that in control mice. The co-localization of GADD45β and C/EBPβ was confirmed by double immunostaining. The synergistic mechanisms of GADD45β and C/EBPβ on the gene regulation of p21, a molecule related to cellular senescence, were verified by a p21-luciferase reporter assay. Co-expression of C/EBPβ and p21 was confirmed. These observations suggest that the synergism between GADD45β and C/EBPβ may play an important role in cellular senescence in the aging articular cartilage.

Protective effect of quercetin, EGCG, catechin and betaine against oxidative stress induced by ethanol in vitro

There is a need for a nontoxic antioxidant agent to be identified which will prevent alcoholic liver disease (ALD) in alcoholic patients. We tested 4 candidate agents: quercetin, EGCG, catechin and betaine, all of which occur naturally in food. HepG2 cells overexpressing CYP2E1 were subjected to arachidonic acid, iron and 100 mM ethanol with or without the antioxidant agent. All the agents prevented oxidative stress and MDA/4HNE formation induced by ethanol, except for EGCG. Catechin prevented CYP2E1 induction by ethanol. All the agents tended to down-regulate the ethanol-induced increased expression of glutathionine peroxidase 4 (GPX4). All the agents, except catechin, tended to reduce the expression of SOD2 induced by ethanol. Heat shock protein 70 was up-regulated by ethanol alone and betaine tended to prevent this. All 4 agents down-regulated the expression of Gadd45b in the presence of ethanol, which could explain the mechanism of DNA demethylation associated with the up-regulation of the gene expression observed in experimental ALD. In conclusion, the in vitro model of oxidative stress induced by ethanol provided evidence that all 4 agents tested prevented some aspect of liver cell injury caused by ethanol.

Pregnane X Receptor PXR Activates the GADD45β Gene, Eliciting the p38 MAPK Signal and Cell Migration

Pregnane X receptor (PXR) was originally characterized as a transcription factor that induces hepatic drug metabolism by activating cytochrome P450 genes. Here we have now demonstrated a novel function of PXR, that of eliciting p38 mitogen-activated protein kinase (MAPK) phosphorylation for cell migration. Upon xenobiotic activation of ectopic human PXR, human hepatocellular carcinoma HepG2 cells were found to exhibit increased phosphorylation of p38 MAPK and to subsequently change morphology and migrate. p38 MAPK was responsible for the regulation of these morphological changes and cell migration because the p38 MAPK inhibitor SB239063 repressed both. Prior to this phosphorylation, PXR directly activated the early response GADD45β gene by binding to a distal direct repeat 4 site of the GADD45β promoter. Ectopic expression of GADD45β increased p38 MAPK phosphorylation, whereas siRNA knockdown of GADD45β decreased the PXR-induced p38 MAPK phosphorylation, confirming that GADD45β can regulate PXR-induced p38 MAPK phosphorylation in HepG2 cells. These results indicate that PXR activates the GADD45β gene, increasing p38 MAPK phosphorylation, and leading HepG2 cells to change morphology and migrate. The GADD45β gene is a direct target for PXR, eliciting cell signals to regulate various cellular functions.

Induction of DNA Damage-Inducible Gene GADD45β Contributes to Sorafenib-Induced Apoptosis in Hepatocellular Carcinoma Cells

Markers that could accurately predict responses to the general kinase inhibitor sorafenib are needed to better leverage its clinical applications. In this study, we examined a hypothesized role in the drug response for the growth arrest DNA damage-inducible gene 45β (GADD45β), which is commonly underexpressed in hepatocellular carcinoma (HCC) where sorafenib may offer an important new therapeutic option. The anticancer activity of sorafenib-induced GADD45β expression was tested in a panel of HCC cell lines and xenograft models. We found that GADD45β mRNA and protein expression were induced relatively more prominently in HCC cells that were biologically sensitive to sorafenib treatment. GADD45β induction was not found after treatment with either the mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 or the Raf inhibitor ZM336372, suggesting that GADD45β induction by sorafenib was independent of Raf/MEK/ERK signaling activity. However, c-Jun NH2-terminal kinase (JNK) kinase activation occurred preferentially in sorafenib-sensitive cells. Small interfering RNA-mediated knockdown of GADD45βor JNK kinase limited the proapoptotic effects of sorafenib in sorafenib-sensitive cells. We defined the -339/-267 region in the GADD45β promoter containing activator protein-1 and SP1-binding sites as a crucial region for GADD45β induction by sorafenib. Together, our findings suggest that GADD45β induction contributes to sorafenib-induced apoptosis in HCC cells, prompting further studies to validate its potential value in predicting sorafenib efficacy.

Gadd45β expression in chondrosarcoma: A pilot study for diagnostic and biological implications in histological grading

BackgroundAlthough the diagnosis of chondrosarcoma, especially the distinction between enchondroma and low-grade chondrosarcoma or low-grade chondrosarcoma and high-grade chondrosarcoma, is pathologically difficult, differential diagnosis is very important because the treatment strategies for these diseases are completely different. The grading system is crucial in predicting biologic behavior and prognosis, however, exact pathological grading is difficult using only routine examinations because the criteria of the grading system are not necessarily definitive. Growth arrest and DNA damage-inducible protein 45β (GADD45β) is an essential molecule for chondrocytes during terminal differentiation. In the present study, we investigated the immunohistochemical expression of GADD45β in enchondroma, and chondrosarcoma of histological grades I, II, and III, to clarify the diagnostic significance of GADD45β in pathological grading of chondrosarcoma.MethodsTwenty samples (enchondroma = 6, chondrosarcoma grade I = 7, grade II = 6, grade III = 1) were used for immunohistochemical analysis to investigate the expression of GADD45β. Quantitative analysis was performed to compare the number of GADD45β positive cells and pathological grading.ResultsOver 70% of the cells in enchondromas expressed GADD45β. On the other hand, the expression of GADD45β decreased significantly according to the histological grade of chondrosarcoma (grade I: 45%; grade II: 13.8%; and grade III: 3.8%).ConclusionsThe association of GADD45β expression and pathological grading of chondrosarcoma in the present study suggests that the immunohistochemical study of GADD45β may be a specific diagnostic parameter for chondrosarcoma cell differentiation.

Gadd45b Mediates Fas-induced Apoptosis by Enhancing the Interaction between p38 and Retinoblastoma Tumor Suppressor

Gadd45b has been known as a positive mediator of apoptosis induced by certain cytokines and oncogenes. Here, we identified Gadd45b as an effector of Fas-induced apoptosis and found that p38-mediated Rb hyperphosphorylation is one of the mechanisms of Fas-induced apoptosis in murine hepatocyte AML12 cells. Gadd45b has been shown to activate p38 through its physical interaction with MTK1 and induce apoptosis. However, in this study, we have showed that the function of Gadd45b during Fas-induced apoptosis in AML12 cells is different from that reported in previous studies. Depletion of Gadd45b expression did not inhibit the phosphorylation of p38, but it suppressed p38-mediated Rb phosphorylation and apoptosis in response to Fas stimulation by reducing the interaction between p38 and Rb. Ectopic expression of Gadd45b was sufficient to enhance this interaction. These findings suggest that Gadd45b mediates p38-induced Rb phosphorylation by enhancing the interaction between p38 and Rb during Fas-induced apoptosis in murine hepatocytes.

Downregulation of Gadd45β Expression by Hepatitis C Virus Leads to Defective Cell Cycle Arrest

Members of the Gadd45 family play central roles in the cellular response to genotoxic stress and have been implicated in several human cancers, including hepatocellular carcinomas. Chronic infection by hepatitis C virus (HCV) is a major risk factor for the onset and development of primary hepatocellular tumors, although the underlying mechanisms are unclear. Here, we show a novel link between diminished Gadd45beta expression and HCV infection. Inhibited Gadd45beta expression was observed in both nontumoral and tumoral tissues from infected individuals, and in cell lines harboring a HCV replicon and the infectious HCV strain JFH1. Decreased Gadd45beta expression was confirmed in vivo in a transgenic murine model expressing the entire HCV open reading frame. Mechanistically, hypermethylation of the Gadd45beta promoter in the presence of HCV is responsible for this defect. Diminished Gadd45beta expression leads to aberrant cell cycle arrest and diminished DNA excision repair. Together, these results provide a novel insight into the mechanisms involved in HCV-associated hepatocellular carcinomas, showing that reduced Gadd45beta expression may play a contributory role to this process, and providing evidence that HCV may interfere with epigenetic gene expression by altering promoter methylation.

Anti-inflammatory action of insulin via induction of Gadd45-&amp;beta; transcription by the mTOR signaling pathway

Insulin regulates a large number of genes in a tissue-specific manner. We have identified genes modulated by insulin in the liver and in liver-derived cells that had not yet been characterized as insulin regulated, and these previous studies indicate that numerous genes are induced by insulin via the MEK-ERK pathway. We now describe new studies indicating that Gadd45-β can be induced by acute insulin treatment. Although other regulators of Gadd45-β expression may utilize the MEK-ERK pathway, the data indicate that insulin utilizes signaling pathways separate from either MEK-ERK, PI3-K or p38 signaling pathways in the regulation of Gadd45-β transcription. Our findings show that activation of a downstream effector of multiple signaling pathways, mTOR, was required for insulin-induction of Gadd45-β gene transcription. Increased expression of Gadd45-β can inhibit c-Jun N-terminal kinase (JNK) activity. Since TNFα is increased during inflammation, and acts, at least in part, via the JNK signaling pathway, insulin induction of Gadd45-β suggests a mechanism for the anti-inflammatory actions of insulin.

Induction and mechanism of growth arrest DNA damage-inducible gene 45 βin human hepatoma HepG2 cell by hydroxyurea

Objective To identify the proximal promoter of down-expression of growth arrest DNA damage-inducible gene 46 β(GADD45β)and to evaluate the influence of hydroxyurea on HCC cell lines.Methods The proximal promoter fragments(-618/-314)were synthesized in vitro and cloned into pGL3 basic luciferase expression plasmid,then transfected into HepG2 by electroporation.The promoter regions were identified in reference of TRANSFAC database.Following hydroxyurea administration,quantitative real-time PCR was employed to validate the expression changes of GADD45β.The effect of hydroxyurea on promoter activity was further focused on.The influence of cell growth and the expression of Caspase-8,Caspase-9 and Caspase-3 were further determined.Results Using the luciferase assay,several transcription factor binding sites were identified in the -528)and one E2F-1(-470/-436).After hydroxyurea administration,the increasing expression of GADD45β was marked in a dose-dependent manner.The luciferase assay results also demonstrated hydroxyurea.The colony formation and DNA syntheses were inhibited apparently and hydroxyurea could trigger the apoptosis rapidly following treatment.Conclusion Hhydroxyurea could induce the expression of GADD45β,which is specifically down-regulated in HCC.The possible mechanism may be the regulation of transcription factors in GADD45β proximal promoter. Key words: Carcinoma hepatocellular; Hydroxyurea; GADD45β; Promoter

874 EPIGENETIC MODULATION OF TUMOUR SUPPRESSOR GADD45 BETA EXPRESSION BY HCV LEADS TO DEFECTIVE CELL CYCLE ARREST AND MAY PLAY A ROLE IN LIVER CARCINOGENESIS

Background and Aims: Chronic hepatitis C virus (HCV) infection is a major risk factor of hepatocellular carcinoma (HCC). Sustained oxidative stress and elevated DNA damage are considered major contributors to HCC. Gadd45b is a major mediator of the cellular response to DNA damage. Our goal was to study the effect of chronic HCV infection on Gadd45b expression. Methods: Gadd45b expression were analyzed by western blotting or quantitative RT-PCR in: (a) paired non-tumoral and tumoral human liver samples from uninfected patients, and patients chronically infected with hepatitis B virus (HBV) or HCV; (b) Huh7 cell cultures harboring the HCV genotype 1b replicon and the JFH1 infectious strain; (c) liver tissues from 18 month old wild-type (wt) and transgenic mice expressing the full-length HCV open reading frame (FL-N/35). Primary hepatocytes were isolated from 7 month old mice. Promoter hypermethylation was assessed by the effect of 5-azacytidine (a DNA methyltransferase inhibitor) on Gadd45b mRNA expression. Cell cycle arrest and mitotic indices were assessed by the presence of phospho-histone H3 after UV-C irradiation. Results: Gadd45b expression was abrogated in tumoral tissue from HBV-positive or -negative patients, whereas it was found at normal levels in non-tumoral tissues. In contrast, in HCV-infected individuals, Gadd45b was absent from both tumoral and nontumoral tissues. Gadd45b expression was also reduced in Huh7 cells exhibiting HCVRNA replication (although these cells originate from a liver tumor). A 2-fold decrease in levels of Gadd45b mRNA was observed in livers of FL-N/35 mice compared to controls, whereas amounts of related Gadd family members remained unaffected. Activation of the Gadd45b promoter was inhibited in the presence of HCV, with cells isolated from FL-N/35 mice demonstrating a 2-fold reduction in promoter activity compared to wt controls. Reduced murine Gadd45b expression was associated with decreased cell cycle arrest in response to DNA damage, and both Gadd45b expression and cell cycle arrest were restored to wt levels by treatment with 5-azacytidine. Conclusions: These data demonstrate that HCV protein expression is associated with inhibition of Gadd45b expression through hypermethylation of its promoter, adversely altering the DNA damage response and potentially promoting liver carcinogenesis.

Gadd45 is a novel mediator of cardiomyocyte apoptosis induced by ischaemia/hypoxia

Because apoptotic death plays a critical role in cardiomyocyte loss during ischaemic heart injury, a detailed understanding of the mechanisms involved is likely to have a substantial impact on the optimization and development of treatment strategies. The goal of this study was to assess gene profiling during ischaemia/hypoxia and to evaluate the functions of ischaemia/hypoxia-responsive genes in in vivo and in vitro ischaemia/hypoxia-induced cardiomyocyte apoptosis models. DNA microarray analysis and real-time polymerase chain reaction were performed on hearts obtained from an in vivo rat transient ischaemia model and on neonatal rat cardiomyocytes from an in vitro hypoxia model. Three genes, namely Ddit4, Gadd45beta and Atf3, were found to be up-regulated in vivo and in vitro. Using loss-of-function and gain-of-function techniques, the functions of these ischaemia/hypoxia-responsive genes were evaluated. Ischaemia/hypoxia-induced cardiomyocyte apoptosis was remarkably attenuated by the small interfering RNA-mediated down-regulation of Gadd45beta in vivo and in vitro, whereas ectopic Gadd45beta expression significantly aggravated hypoxia-induced apoptosis in vitro. These results suggest that Gadd45beta is a key player in ischaemia/hypoxia-induced apoptotic cardiomyocyte death, and that strategies based on its inhibition might be of benefit in the treatment of acute ischaemic heart disease.

Abstract A241: Searching for extracellular signal-regulated kinase (ERK)-independent molecular targets of sorafenib in hepatocellular carcinoma (HCC)

Abstract We have recently identified Bim, a pro-apoptotic factor regulated by ERK signaling pathway, is an important mediator of sorafenib-induced apoptosis in HCC cells.. In this study we further explored other possible mediators of sorafenib activity in HCC cells that is independent of mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK)/ERK signaling. Huh7 cells were treated with sorafenib or the MEK inhibitor CI-1040, alone or in combination, for 24 hours. The gene expression profiles were analyzed by Affymetrix oligonucleotide microarray analysis using HG_U133A chip, and the candidate genes were selected if (1) they had &amp;gt; 1.2 fold or &amp;lt; 0.8 fold change than control after treatment with sorafenib 10 M and (2) they had been reported to be involved in important carcinogenesis-related cellular signaling pathways, such as apoptosis regulation, cell proliferation/survival, cell cycle control, and angiogenesis. The gene expression was considered MEK/ERK-dependent if the direction of changes was similar after treatment with sorafenib 10 M or CI-1040 1 M; otherwise it was considered MEK/ERK-independent. Thirty candidate genes of interest were identified and the expression patterns were validated by real-time PCR (see table). The 14 genes whose expressions were considered ERK-independent involved apoptosis regulation (GADD45b, BAX, JUN), cell proliferation (PCNA, RPS6KA3, SMAD7, BMP2, DUSP1, DUSP8, IL1RAP, RHEB), cell cycle control (CCNE1, CDC25A), and angiogenesis (VEGF). Validation of the biological significance of these markers is ongoing. The preliminary data revealed that induction of GADD45b and JUN gene expression after sorafenib treatment was correlated with intrinsic or acquired resistance of HCC cells to sorafenib. Our data indicated that MEK/ERK-independent signaling pathways may also contribute to sorafenib-induced apoptosis and growth regulation in HCC cells. The signaling molecules identified in this study can serve as novel therapeutic targets for HCC. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, NSC98-3112-B-002-037) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A241.

C-Terminal Mutation of RUNX1 Deteriorates DNA Damage-Repair Response and Promotes the Development of Acute Myeloid Leukemia.

Abstract 183RUNX1 transcription factor regulates hematopoietic ontogeny and is a frequent target of gene rearrangements in hematological malignancies. In addition to gene rearrangements, loss-of-function mutations of RUNX1 have been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Mutations of RUNX1 have been detected in about 10–20% patients classified as MDS/AML (high-risk MDS and AML following MDS). Although loss-of-function mutations of RUNX1 cause leukemia together with additional cooperating events in mouse models, the mechanisms, by which impaired RUNX1 functions led to the subsequent genetic alterations, remain unclear. Because DNA damage-repair response has an important role for prevention of many types of tumors, including hematological malignancies, we analyzed the role for RUNX1 in DNA repair system. First, we stably expressed a dominant-negative mutant of RUNX1, RUNX1dC, in a murine myeloid cell line 32Dcl3. RUNX1dC lacks the C-terminal 225 amino acids, which was originally found in a patient with MDS and suppresses functions of wild-type (WT) RUNX1 by inhibiting its DNA binding activity. To analyze the roles for RUNX1 in the DNA repair system, we took advantage of in vitro DNA repair assays with DNA cross-linking agents in 32D-neo and 32D-RUNX1dC cells. Since the cells that recovered from DNA damage make colonies after cisplatin exposure, we can evaluate DNA repair ability of test cells with this assay. As a result, clonogenic ability of 32D-RUNX1dC was significantly decreased by the 2-hour exposure of cisplatin (10nM treatment: 93.4% reduction) compared to that of 32D-neo cell (10nM treatment: 58.6% reduction) (p=0.0006). In addition, 32D-RUNX1dC showed significantly lower clonogenic ability than 32D-neo after exposure to UV-B and gamma-ray, respectively. To evaluate DNA-damage accumulation in 32D-neo and 32D-RUNX1dC cells, we performed immunofluorescent microscopic analysis using monoclonal antibodies for (6–4) photoproducts (6–4 PPs) and cyclobutane pyrimidine dimers (CPDs), which are major products of DNA damage induced by UV-B. These types of DNA lesions are repaired by nucleotide excision repair (NER) system. After six hours from UV-B exposure, both 6–4 PPs and CPDs accumulated in 32D-RUNX1dC cells more abundantly than in 32D-neo cells. These results suggest that RUNX1dC attenuates NER in 32D cells, ▪ thereby leading to the sustained accumulations of DNA lesions after exposure to UV-B and cisplatin. To identify the molecule(s) involved in DNA-damage signaling, we profiled expression of 84 genes involved in DNA damage signaling by real-time RT-PCR array. The expression profiling revealed that RUNX1dC repressed Gadd45a, a regulator of NER system in 32D cells. Because genetic alteration of RUNX1 is supposed to occur at a HSC level in MDS and AML, we next evaluated whether RUNX1dC modifies Gadd45 expression in murine Lineage−Sca1+c-Kit+ (LSK) cells. As a result, RUNX1dC-transduced LSK cells showed significantly lower expression of Gadd45a and Gadd45b compared to Mock-transduced LSK cells. Luciferase reporter and chromatin immunoprecipitation assays showed that RUNX1 directly regulates Gadd45a expression via two RUNX1-binding sites neighboring to the p53-binding site in the intron 3 of the human Gadd45a gene. To confirm the roles for endogenous RUNX1 in NER system, we next performed RUNX1-knockdown experiments by short hairpin RNA (shRNA) -mediated gene silencing. RUNX1-shRNA-transduced 32D cells showed significantly lower expression of Gadd45a and Gadd45b than non-silensing-shRNA-transduced 32D cells. As expected, RUNX1-shRNA-transduced 32D cells showed significantly lower clonogenic ability after UV-B exposure than non-silensing-shRNA-transduced 32D cells (p=0.0008). These results suggest that endogenous RUNX1 regulates Gadd45 expression, thereby controlling NER system. Finally, we screened mRNA expression of Gadd45a in the samples from 23 MDS/AML patients, and found that its expression was significantly decreased in MDS/AML patients harboring RUNX1-C-terminal mutation compared to those with WT RUNX1 (p=0.0233). In summary, we here demonstrated that RUNX1 participates in the DNA damage-repair response through transcriptional regulation of Gadd45a. Our study suggests that the impaired RUNX1 function deteriorates NER system and may cause additional mutation(s), which are required for multi-step leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Gadd45β deficiency in rheumatoid arthritis: Enhanced synovitis through JNK signaling

AbstractObjectiveJNK‐mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45β, which is an NF‐κB–regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK‐7. This study was carried out to evaluate whether low Gadd45β expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45β deficiency increases arthritis severity in passive K/BxN murine arthritis.MethodsActivation of the NF‐κB and JNK pathways and Gadd45β expression were analyzed in human synovium and fibroblast‐like synoviocytes (FLS) using quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay, and luciferase reporter constructs. Gadd45β−/− and wild‐type mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis, and clinical signs of arthritis, osteoclast formation, and bone erosion were assessed.ResultsExpression levels of the Gadd45β gene and protein were unexpectedly low in human RA synovium despite abundant NF‐κB activity. Forced Gadd45β expression in human FLS attenuated tumor necrosis factor–induced signaling through the JNK pathway, reduced the activation of activator protein 1, and decreased the expression of MMP genes. Furthermore, Gadd45β deficiency exacerbated K/BxN serum–induced arthritis in mice, dramatically increased signaling through the JNK pathway, elevated MMP3 and MMP13 gene expression in the mouse joints, and increased the synovial inflammation and number of osteoclasts.ConclusionDeficient Gadd45β expression in RA can contribute to activation of JNK, exacerbate clinical arthritis, and augment joint destruction. This process can be mitigated by enhancing Gadd45β expression or by inhibiting the activity of JNK or its upstream regulator, MKK‐7.

Distinct mechanisms are utilized to induce stress sensor gadd45b by different stress stimuli

The GADD45 family of proteins consists of three small proteins, GADD45A, GADD45B, and GADD45G, implicated in modulating the cellular response to genotoxic/physiological stressors. Despite similarities in sequence, structure and function, each gadd45 gene is induced differentially by different stress stimuli. Studies on stress-mediated induction of the gadd45 genes have predominantly focused on gadd45a, with knowledge of gadd45b and gadd45g regulation lacking. To generate a more complete understanding of the regulation of gadd45 genes, a comprehensive analysis of stress-mediated induction of human gadd45b has been carried out using human RKO colorectal carcinoma cells as a model system. Novel data indicate that gadd45b induction in RKO cells is regulated by distinct mechanisms in a stress-specific manner. Methylmethane sulfonate (MMS), a DNA alkylating agent, induces gadd45b transcription through a cohort of both constitutive and inducible bound factors, including NFY, Sp1 and Egr1. In contrast, in a hyperosmotic environment generated with sorbitol, gadd45b mRNA is induced exclusively by mRNA stabilization. These findings indicate that the stress-mediated induction of gadd45b is largely distinct from gadd45a. Furthermore, data obtained provide a novel paradigm for stress-response gene induction, indicating that gadd45b induction by distinct stressors, in the same cell type and under the same experimental settings, is differentially regulated at the level of mRNA transcription or mRNA stability. Importantly, this study also provides the groundwork to further examine the regulation of gadd45b expression in in vivo settings using animal models and tissues obtained from normal individuals and cancer patients prior to and after chemotherapeutic intervention.