GADD45 Gamma Research Articles

The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation

The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial.We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages.These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level.(1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014) DisclosuresNo relevant conflicts of interest to declare.

Gadd45γ regulates cardiomyocyte death and post-myocardial infarction left ventricular remodelling.

Post-infarction remodelling is accompanied and influenced by perturbations in mitogen-activated protein kinase (MAPK) signalling. The growth arrest and DNA-damage-inducible 45 (Gadd45) proteins are small acidic proteins involved in DNA repair and modulation of MAPK activity. Little is known about the role of Gadd45 in the heart. Here, we explored the potential contribution of Gadd45 gamma (γ) isoform to the acute and late phase of heart failure (HF) after myocardial infarction (MI) and determined the mechanisms underlying Gadd45γ actions. The Gadd45γ isoform is up-regulated in murine cardiomyocytes subjected to simulated ischaemia and in the mouse heart during MI. To mimic the situation observed during MI, we enhanced Gadd45γ content in cardiomyocytes with a single injection of an adeno-associated viral (AAV9) vector encoding Gadd45γ under the cTNT promoter. Gadd45γ overexpression induces cardiomyocyte apoptosis, fibrosis, left ventricular dysfunction, and HF. On the other hand, genetic deletion of Gadd45γ in knockout mice confers resistance to ischaemic injury, at least in part by limiting cardiomyocyte apoptosis. Mechanistically, Gadd45γ activates receptor-interacting protein 1 (RIP1) and caspase-8 in a p38 MAPK-dependent manner to promote cardiomyocyte death. This work is the first to demonstrate that Gadd45γ accumulation during MI promotes the development and persistence of HF by inducing cardiomyocyte apoptosis in a p38 MAPK-dependent manner. We clearly identify Gadd45γ as a therapeutic target in the development of HF.

Comparative analysis of developmentally regulated expressions of Gadd45a, Gadd45b, and Gadd45g in the mouse and marmoset cerebral cortex

The cerebral cortex is an indispensable region that is involved in higher cognitive function in the mammalian brain, and is particularly evolved in the primate brain. It has been demonstrated that cortical areas are formed by both innate and activity-dependent mechanisms. However, it remains unknown what molecular changes induce cortical expansion and complexity during primate evolution. Active DNA methylation/demethylation is one of the epigenetic mechanisms that can modify gene expression via the methylation/demethylation of promoter regions. Three growth arrest and DNA damage-inducible small nuclear proteins, Gadd45 alpha, beta, and gamma, have been identified as regulators of methylation status. To understand the involvement of epigenetic factors in primate cortical evolution, we started by analyzing expression of these demethylation genes in the developing common marmoset (Callithrix jacchus) and mouse (Mus musculus) brain. In the marmoset brain, we found that cortical expression levels of Gadd45 alpha and gamma were reduced during development, whereas there was high expression of Gadd45 beta in some areas of the adult brain, including the prefrontal, temporal, posterior parietal and insula cortices, which are particularly expanded in greater primates and humans. Compared to the marmoset brain, there were no clear regional differences and constant or reduced Gadd45 expression was seen between juvenile and adult mouse brain. Double staining with a neuronal marker revealed that most Gadd45-expressing cells were NeuN-positive neurons. Thus, these results suggest the possibility that differential Gadd45 expression affects neurons, contributing cortical evolution and diversity.

0237: Gadd45 γ promotes cardiac dysfunction by inducing necroptosis pathway in a mouse model of chronic myocardial infarction

Postinfarction remodeling is accompanied and influenced by perturbations in MAPK signaling. The Growth arrest and DNA-damage-inducible 45 (Gadd45) proteins are small acidic proteins involved in DNA repair and modulation of MAPK activities. Despite its relationship with p38 MAPK, little is known about the role of Gadd45 in the heart. Our aim is to explore the potential contribution of Gadd45 gamma (γ) on heart failure development after myocardial infarction (MI). MI was induced in wild-type (WT) versus Gadd45 γ (KO) mice by left anterior descending coronary artery ligation. Pre-MI, WT, and KO hearts had comparable chamber dimensions and ventricular function, but as early as 1 week post-MI, KO mice had significantly better cardiac function than WT mice. The left ventricular dilatation, dysfunction and fibrosis remained better in the KO mice throughout the study (4 weeks). We examined the infarct size following a 24-hour MI and found it to be decreased in the KO mice. In addition, necroptosis activity pathway (RIP1; caspase 8) was reduced in the KO mice. To mimic the Gadd45 γ overexpression observed during MI, we injected 3 10^11 of cTNT-Gadd45 γ AAV9 particles i.v. and studied the cardiac function. Four weeks after the cTNT-Gadd45 γ AAV9 injection, we observed a decrease in the cardiac function compared to the GFP-AAV9 injected control mice. A remodeling process is engaging with more fibrosis. Gadd45 γ overexpression activated the necroptosis pathway through p38 MAPK. Gadd45 γ promotes ischemic injury, accentuates post-MI remodeling and left ventricular dysfunction, and increases the progression to heart failure. The lost of gadd45 γ confers resistance to ischemic injury, at least in part, via limiting necroptosis pathway through a p38 MAPK dependant mechanism. This work, demonstrate the importance of Gadd45 γ/p38 MAPK complex in the development of heart failure, particularly in the balance of the cell death process.

MIS5-1 - The Roles of Growth Arrest DNA Damage-Inducible Gene 45Γ (GADD45Γ) Expression in Sorafenib-Induced Apoptosis in Hepatocellular Carcinoma (HCC) Cells

ABSTRACT Background We previously found that GADD45b, which is associated with cellular stress response and apoptosis regulation, contributes to sorafenib resistance in HCC cells. The present study explored the role of GADD45γ, another GADD45 family protein, in HCC treatment. Methods HCC cell lines tested included Huh-7, HepG2, Hep3B, Huh-7R, HepG2R, the latter two sorafenib-resistant cell lines established by continuously exposure of Huh-7 and HepG2 cells to sorafenib. GADD45γ expression was examined by quantitative real-time PCR (qRT-PCR) and western blotting. Control of GADD45γ transcription was examined by luciferase reporter assay using a series of reporter plasmids with deletions or site-directed mutants of the 5′-flanking region of GADD45γ promoter. Apoptosis was analyzed by flow cytometry and western blotting. Expression of GADD45γ in human HCC tumor tissues was measured by qRT-PCR and immunohistochemistry. Results GADD45γ mRNA and protein levels were increased after sorafenib independent of cellular MEK/ERK activity. GADD45γ induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6-7 µM) than in sorafenib-resistant HCC cells (Hep3B, Huh-7R and HepG2R, IC50 12-15 µM). Knockdown of GADD45γ expression partially abrogated the pro-apoptotic effects of sorafenib in sorafenib-sensitive cells but not sorafenib-resistant cells. The region -449/ - 82 in the 5′-flanking region of GADD45γ promoter was found crucial for GADD45 gamma induction by sorafenib. Decreased GADD45γ expression in HCC tumor tissue was associated with worse overall survival in patients who underwent surgery for HCC. Conclusions Induction of GADD45γ expression contributes to sorafenib-induced apoptosis in HCC cells. The role of GADD45γ as a predictive biomarker for sorafenib sensitivity in HCC warrants further exploration. (Supported by grants NSC-100-2325-B-002 -042, NSC 100-2314-B-002 -058-MY3, DOH100-TD-B-111-001, and NHRI-EX101-9911BC.)

Characterization of Hematopoietic Progenitor Kinase 1 (HPK1) in Multiple Myeloma: a Player in Pathogenesis?,

Abstract 3956Hematopoietic progenitor kinase 1 (HPK1) is a differentially regulated MAP4 kinase (MAP4K) in B lymphocytes (Geisberger et al., 2002; Königsberger et al., 2010). In germinal centres (GCs), B-cell differentiation, selection and Ig-class-switch are modulated by TNF-receptor family members, subsequently activating GC kinases like HPK1 by corresponding adaptor molecules (TRADD and TRAF), or by activated RTKs linked to SH2/SH3 adaptors. Initiating signaling events in the molecular pathogenesis of multiple myeloma (MM) are currently under investigation. A first proteome wide analysis implicated a potential contribution of HPK1 to the pathogenesis of MM demonstrating that selective knock down of HPK1 (MAP4K1) caused a lethal phenotype in MM cells (Tiedemann et al. 2010). Here, we studied expression and regulation of HPK1 in MM and effects on MM phenotype by modulation of HPK1 activity in vitro and in vivo.HPK1 mRNA and protein expression was examined in a series of human MM cell lines and murine MPC-11 MM cells compared to human B-lymphocytes, B-CLL cells and other non-hematological tissues. Subsequently, HPK1 was overexpressed in MPC-11 cells in a kinase-active (HPK1high) and kinase-deficient (K46E) variant. In addition, HPK1 was knocked down by RNAi in MPC-11 cells (HPK1low). Subsequently, we performed analyses of the transcriptome and proteome, in particular with attention to the kinome, in order to further characterize HPK1-modulated MM cells. In addition, transformation of MM phenotype was assayed by thymidine uptake and induced apoptosis in vitro as well as by variances of subcutaneous tumor growth in murine model with regard to the different HPK1 activities in the respective MM cells (HPK1wt, HPK1high, HPK1K46E, HPK1low and corresponding controls).HPK1 expression varied in MM cell lines tested (high: MPC-11, RPMI-8226; mid: U-266, NCI-H929, OPM-2, JJN-3, KMS-11, ANBL-6, MM1.S, OCI-MY5, MM1.R; low: L363, KMS-18, UTMC-2). HPK-1 mRNA and protein expression was found to be significantly (2-5fold) lower in purified CD138+/CD38high cells from bone marrow aspirates of MM patients as in CD19+ peripheral blood B-lymphocytes or CD19+/CD20+/CD5+ B-CLL cells. In cells derived from non-hematological tissues HPK1 was found below the detection limit. For further analyses, MPC-11 cells were generated overexpressing HPK1. Of note, kinase-active HPK1high cells showed decreased sensitivity to induced apoptosis by glucocorticoid receptor inhibition compared to HPK1wt and HPK1K46E cells. In addition, we found a consecutive increased phosphorylation of downstream signaling proteins in HPK1high cells (MEKK1, ASK1 or p38, JNK, TAK1, MEK1/2, p70S6K alpha, MAPKAPK2). Pro-apoptotic effects by hyperactivation of JNK and p38 were blocked by HPK1-induced consecutive overexpression of cell-cycle-associated genes (GADD45 gamma, Cip1, MCL-1). This observation may contribute to inhibition of caspase-dependent pathways and protect myeloma cells from dexamethasone-induced apoptosis. These in vitro findings were correlated with an accelerated tumor growth and higher tumor burden in vivo (194% vs. 100% HPK1high vs. HPK1wt and respective K46E control, day 7). In contrast, HPK1 knock down by siRNA resulted in a decline of phosphorylation of cAMP response element binding protein-kinase (CREB) and TGF beta-activated kinase-1 (TAK1), whereas pJNK1/2, pERK1, p70S6K alpha und pMAPKAPK2 were not modulated compared to HPK1wt cells. Also no significant alteration was observed in HPK1low cells regarding proliferation and apoptosis in vitro.In conclusion, HPK1 is constitutively expressed in MM cells and shows regulatory activity concerning intracellular survival signaling in MM cells. HPK1 may contribute to the malignant phenotype so that a HPK1-targeted approach will be promising. To confirm this hypothesis, a HPK1 knock down approach in vivo is recommended. Disclosures:No relevant conflicts of interest to declare.

Apoptosis of glomerular mesangial cells induced by sublytic C5b‐9 complexes in rats with Thy‐1 nephritis is dependent on Gadd45γ upregulation

The complement C5b-9 complexes can result in cell apoptosis, but the mechanism of sublytic C5b-9-mediated glomerular mesangial cell (GMC) apoptosis in Thy-1 nephritis (Thy-1N) remains largely unclear. The Gadd45 gene is involved in the cellular response to DNA damage and can promote cell apoptosis. In this study, both Gadd45 gamma expression patterns and pathologic changes of renal tissue were examined in rat Thy-1N. Both Gadd45 gamma expression and GMC apoptosis were significantly decreased in Thy-1N rats upon the depletion of complement with cobra venom factor. Our in vitro studies showed that Gadd45 gamma over-expression increased sublytic C5b-9-induced GMC apoptosis, while Gadd45 gamma gene knockdown by siRNA greatly reduced GMC apoptosis. Moreover, Gadd45 gamma gene silencing in vivo markedly inhibited the pathologic changes in the renal tissue of Thy-1N rats. These data suggest that Gadd45 gamma gene expression is involved in regulating GMC apoptosis mediated by sublytic C5b-9 in Thy-1N.

Proteomic analysis of cervical cancer cells treated with adenovirus-mediated MDA-7

Ad.mda-7 inhibited growth and decreased survival in a broad array of human tumor cells, without eliciting detrimental effects in normal cells. This study demonstrates that Ad.mda-7 can effectively impede the proliferation and induce apoptosis of human cervical carcinoma cells, but the underlying mechanisms inducing cell death at protein level are unknown. Using proteome analysis, an investigation aimed at a better understanding of the antiproliferative mechanisms by Ad.mda-7 was carried out in CaSki cervical cancer cells. A total of 43 differentially expressed proteins were visualized by 2-DE and silver stain., 29 proteins of which were identified via matrix-assisted laser desorption/ionization-time of flight mass spectrometry(MALDI-TOF-MS) analysis, 15 were upregulated (eg, Tumor suppressor p53、Apoptosis regulator BAX、Adenylate kinase isoenzyme 1(AK1)、Growth arrest and DNA-damage-inducible protein GADD45 gamma (GADD45γ)) and 14 were downregulated (eg, Eukaryotic translation initiation factor 5A(eIF-5A)、Protein DJ-1、Annexin V、Transcription elongation factor B polypeptide 2 (TCEB2)、TRAF family member-associated NF-kappa-B activator (TRAF2)、c-Myc-responsive protein Rcl (RCL)). Among the identified proteins, the protein and mRNA alterations of six proteins were further confirmed by Western blot and semi-quantitative RT-PCR. Together, at both the mRNA and protein levels, p53, BAX, AK1, GADD45γ, and BCCIP were upregulated, while eIF-5A was downregulated following Ad.mda-7 treatment. Our findings may offer new insights into the antiproliferative mechanisms by Ad.mda-7 and its mode of action in cervical carcinoma cells.

The effect of denervation and reinnervation on PCA cell cycle gene expression

Problem: While it is known that denervation of skeletal muscle can induce atrophy, the effect of denervation on muscle cell cycle genes is unclear. Further, while attempted reinnervation of laryngeal muscle has thus far been unsuccessful in restoring function, cell cycle gene expression might be utilized to predict reinnervation success. The objective of this study was to examine the effects of denervation and reinnervation of the posterior cricoarytenoid (PCA) muscle on key genes involved in the cell cycle. Methods: Female Sprague-Dawley rats were assigned to 1 of 3 groups: control (CON, n = 24); denervation (DEN, n = 24); or reinnervation (REIN, n = 24). DEN and REIN animals had their left recurrent laryngeal nerves transected and ligated at the fourth tracheal ring while REIN animals subsequently had their nerves repaired using 10–0 suture. Animals were then euthanized after 7, 14, and 30 days, and mRNA expression examined using RT-PCR. If significant group differences were seen using one-way ANOVA ( P < 0.05), Tukey post-hoc tests were employed. Results: Expression of p21, p53, GADD45 alpha, GADD45 beta, GADD45 gamma, and cyclin D1 genes were compared. At all time points, cyclin D1 and p53 gene expressions were unchanged with DEN or REIN. However, DEN muscles were found to express significantly higher levels of p21, GADD45 alpha, and GADD45 beta than CON at all time points. Conclusion: Interestingly, while REIN p21, GADD45 alpha, and GADD45 beta expressions were found to be elevated at 7 and 14 days, GADD45 alpha and GADD45 beta levels at 30 days were not significantly different from CON, threreby suggesting a recovery of REIN muscle gene expression at later time points. Significance: Paradoxically, while the restoration of some cell cycle genes to normal levels in REIN muscles might be seen as an indication of successful reinnervation of the PCA, no significant functional recovery was associated. Support: None reported.

GADD45gamma, down-regulated in 65% hepatocellular carcinoma (HCC) from 23 chinese patients, inhibits cell growth and induces cell cycle G2/M arrest for hepatoma Hep-G2 cell lines.

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 gamma (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 gamma cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 y caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 gamma in cell growth control was further confirmed in this paper.

Loss of expression of GADD45 gamma, a growth inhibitory gene, in human pituitary adenomas: implications for tumorigenesis.

The underlying molecular pathogenic mechanisms remain unknown in the majority of human pituitary tumors. GADD45 gamma is a member of a growth arrest and DNA damage-inducible gene family that functions in the negative regulation of cell growth. We have found that the mRNA expression of the GADD45 gamma gene is significantly different between normal human pituitary tissue and clinically nonfunctioning pituitary adenomas using cDNA-representational difference analysis. Although GADD45 gamma mRNA was found in normal human pituitary tissue, it was detectable in only 1 of 18 clinically nonfunctioning pituitary tumors by RT-PCR. Furthermore, this gene was not expressed in the majority of GH- or PRL-secreting pituitary tumors (6 of 8 and 7 of 10, respectively). In colony formation assays, transfection of human GADD45 gamma cDNA into the human pituitary tumor-derived cell line, PDFS, results in a dramatic decrease in cell growth by 88%. GADD45 gamma also reduces colony formation in other pituitary tumor-derived cell lines, AtT20 and GH4, by approximately 60% and 50%, respectively, confirming its function in controlling cell proliferation in the pituitary. These data indicate that GADD45 gamma is a powerful growth suppressor controlling pituitary cell proliferation, and GADD45 gamma represents the first identified gene whose expression is lost in the majority of human pituitary tumors.