Single-cell transcriptomics identifies Gadd45b as a regulator of herpesvirus-reactivating neurons.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for dissecting the complexity of normal and diseased tissues, enabling characterization of cell diversity and heterogeneous phenotypic states in unprecedented detail. However, this technology has been underutilized for exploring the interactions between the host cell and viral pathogens in latently infected cells. Herein, we use scRNA-seq and single-molecule sensitivity fluorescent in situ hybridization (smFISH) technologies to investigate host single-cell transcriptome changes upon the reactivation of a human neurotropic virus, herpes simplex virus-1 (HSV-1). We identify the stress sensor growth arrest and DNA damage-inducible 45 beta (Gadd45b) as a critical antiviral host factor that regulates HSV-1 reactivation events in a subpopulation of latently infected primary neurons. We show that distinct subcellular localization of Gadd45b correlates with the viral late gene expression program, as well as the expression of the viral transcription factor, ICP4. We propose that a hallmark of a "successful" or "aborted" HSV-1 reactivation state in primary neurons is determined by a unique subcellular localization signature of the stress sensor Gadd45b.

GADD45A and GADD45B as Novel Biomarkers Associated with Chromatin Regulators in Renal Ischemia-Reperfusion Injury.

Chromatin regulators (CRs) are essential upstream regulatory factors of epigenetic modification. The role of CRs in the pathogenesis of renal ischemia-reperfusion injury (IRI) remains unclear. We analyzed a bioinformatic analysis on the differentially expressed chromatin regulator genes in renal IRI patients using data from public domains. The hub CRs identified were used to develop a risk prediction model for renal IRI, and their expressions were also validated using Western blot, qRT-PCR, and immunohistochemistry in a murine renal IRI model. We also examined the relationships between hub CRs and infiltrating immune cells in renal IRI and used network analysis to explore drugs that target hub CRs and their relevant downstream microRNAs. The results of machine learning methods showed that five genes (DUSP1, GADD45A, GADD45B, GADD45G, HSPA1A) were upregulated in renal IRI, with key roles in the cell cycle, p38 MAPK signaling pathway, p53 signaling pathway, FoxO signaling pathway, and NF-κB signaling pathway. Two genes from the network, GADD45A and GADD45B (growth arrest and DNA damage-inducible protein 45 alpha and beta), were chosen for the renal IRI risk prediction model. They all showed good performance in the testing and validation cohorts. Mice with renal IRI showed significantly upregulated GADD45A and GADD45B expression within kidneys compared to sham-operated mice. GADD45A and GADD45B showed correlations with plasmacytoid dendritic cells (pDCs) in infiltrating immune cell analysis and enrichment in the MAPK pathway based on the weighted gene co-expression network analysis (WGCNA) method. Candidate drugs that target GADD45A and GADD45B include beta-escin, sertraline, primaquine, pimozide, and azacyclonol. The dysregulation of GADD45A and GADD45B is related to renal IRI and the infiltration of pDCs, and drugs that target GADD45A and GADD45B may have therapeutic potential for renal IRI.

Expression of GADD45B in Renal Tissue of Children with IgA Nephropathy and Correlation with Mesangial Hypercellularity.

Background: We correlated the expression of growth arrest and DNA damage-inducible protein beta (GADD45B) in renal tissue with IgA nephropathy (IgAN) with clinical characteristics and mesangial hypercellularity. Materials and methods: Biopsies from IgAN children were divided into M0 and M1 groups based on the Oxford classification, and biopsies with minimal change disease (MCD) were selected as controls. The mesangial cell proliferation area was evaluated on PAS-stained tissues, and the relative level of GADD45B in renal tissue was assessed by immunohistochemical staining (IHC). Results: Compared with the MCD group, levels of GADD45B in the M0 and M1 groups were significantly higher (p < 0.05). Levels of GADD45B positively correlated with mesangial cell proliferation, proteinuria, and total cholesterol, negatively correlated with Alb levels. Conclusions: It is suggested that high expression of GADD45B may play a regulatory role in mesangial hypercellularity.

FTO knockdown alleviates hypoxia-induced PC12 cell injury by stabilizing GADD45B in an IGF2BP2-dependent manner.

RNA N6-methyladenosine (m6A) level is closely associated with neurodevelopment and central nervous system dysfunctions including spinal cord injury (SCI). M6A level can be dynamically regulated by m6A methyltransferases and demethylases. In this text, the roles of m6A demethylase FTO alpha-ketoglutarate dependent dioxygenase (FTO) in SCI development along with its m6A-dependent regulatory mechanisms were investigated in hypoxia-induced PC12 cell injury model. The results showed that FTO was low expressed in spinal cord tissues of rats after contusive SCI and hypoxia-treated PC12 cells. FTO knockdown alleviated hypoxia-induced PC12 cell injury. FTO loss increased GADD45B expression and m6A level in PC12 cells. GADD45B knockdown weakened the protective effects of FTO depletion on hypoxia-treated PC12 cells. FTO regulated GADD45B expression in an IGF2BP2-dependent manner. In conclusion, FTO knockdown mitigated the injury of hypoxia-induced PC12 cells by up-regulating GADD45B in an IGF2BP2-dependent manner.

Gadd45 in Senescence.

Gadd45a, Gadd45b, and Gadd45g have been implicated in cell cycle arrest, DNA repair, apoptosis, innate immunity, genomic stability, and more recently in senescence. Evidence has accumulated that Gadd45a deficiency results in escape of mouse embryo fibroblasts from senescence, whereas Gadd45b deficiency promotes premature senescence and skin aging. Moreover, recently Gadd45b deficiency was found to promote senescence and attenuate liver fibrosis, whereas Gadd45a was observed to exert a protective effect against hepatic fibrosis. These findings indicate that the Gadd45 stress response proteins play important roles in modulating cellular responses to senescence. Thus, exploring how Gadd45 proteins modulate cellular senescence has the potential to provide new and innovative tools to treat cancer as well as liver disease.

Gadd45b is a novel mediator of depression-like behaviors and neuroinflammation after cerebral ischemia.

BACKGROUND: Poststroke depression (PSD) is an important consequence after stroke, with a negative impact on stroke outcome. Recent evidence points to a modulatory role of Growth arrest and DNA-damage-inducible protein 45 beta (Gadd45b) in depression. Herein, we evaluated the antidepressant efficacy and mechanism underlying the potent therapeutic effects of Gadd45b after cerebral ischemia. METHODS: Adult male Sprague-Dawley rats were subjected to cerebral ischemia by permanent middle cerebral artery occlusion (MCAO). The sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST) were performed after completing MCAO to study the antidepressant-like effects. The expression of brain-derived neurotrophic factor (BDNF) and neuroinflammation were determined in the hippocampus. RESULTS: We showed that Gadd45b knockdown induced depression-like behaviors after cerebral ischemia, including increased immobility time in the FST and TST and reduced sucrose preference. Gadd45b knockdown enhanced the expression of pro-inflammatory cytokines IL-6 and TNF-α, accompanying with decreased protein levels of BDNF in the hippocampus. Moreover, the levels of phosphorylated ERK and CREB, which have been implicated in events downstream of BDNF signaling, were also decreased after cerebral ischemia. CONCLUSION: Hence, the results showed that Gadd45b is a promising drug candidate for treating PSD and possibly other nervous system diseases associated with neuroinflammation. Gadd45b may have therapeutic potential for PSD through BDNF-ERK-CREB pathway and neuroinflammation.

Design, Synthesis and Evaluation of Novel Derivatives of Curcuminoids with Cytotoxicity.

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,ß-unsaturated ß-diketone, α,ß-unsaturated ketone and ß'-hydroxy-α,ß-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC50 values of all the synthesized derivatives, most α,ß-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas ß'-hydroxy-α,ß-unsaturated ketones and α,ß-unsaturated ß-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,ß-unsaturated ketone complex for help in drug design.

TFAP2C increases cell proliferation by downregulating GADD45B and PMAIP1 in non-small cell lung cancer cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.

Matrine inhibits the progression of prostate cancer by promoting expression of GADD45B.

BACKGROUND: Matrine is a naturally occurring alkaloid extracted from the Chinese herb Sophora flavescens. It has been demonstrated to exhibit antiproliferative properties, promote apoptosis, and inhibit cell invasion in a number of cancer cell lines by modulating the NF-κB pathway to downregulate the expression of MMP2 and MM9. It has also been shown to improve the efficacy of chemotherapy when it is combined with other chemotherapy drugs. However, the therapeutic potential of matrine for prostate cancer needs to be further studied. METHODS: We analyzed KEGG pathways of differential gene expression between matrine-treated and untreated prostate cancer cell lines and identified GADD45B as one of major target genes of matrine based on its role in apoptosis and prognosis value for prostate cancer patients in TCGA database. We further analyzed the expression of GADD45B protein in a tissue microarray and mRNA in TCGA database, and tested the synergistic impacts of matrine and GADD45B overexpression on proliferation, apoptosis, migration and invasion of prostate cancer cell DU145. RESULTS: Matrine promoted the expression of GADD45B, a tumor suppressive gene that is involved in the regulation of cell cycle, DNA damage repair, cell survival, aging, apoptosis and other cellular processes through p38/JNK, ROS-GADD45B-p38, or other signal pathways. Although GADD45B is elevated in prostate cancer tissues, levels of GADD45B in prostate tumor tissues are reduced at late stage of tumor invasion, and higher levels of GADD45B predict better survivals of prostate cancer patients. CONCLUSIONS: Matrine may be used to treat prostate cancer patients to increase the levels of GADD45B to inhibit tumor invasion and improve patient survivals.

Cell death caused by the synergistic effects of zinc and dopamine is mediated by a stress sensor gene Gadd45b - implication in the pathogenesis of Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease.

Gadd45b prevents autophagy and apoptosis against rat cerebral neuron oxygen-glucose deprivation/reperfusion injury.

Autophagic (type II) cell death has been suggested to play pathogenetic roles in cerebral ischemia. Growth arrest and DNA damage response 45b (Gadd45b) has been shown to protect against rat brain ischemia injury through inhibiting apoptosis. However, the relationship between Gadd45b and autophagy in cerebral ischemia/reperfusion (I/R) injury remains uncertain. The aim of this study is to investigate the effect of Gadd45b on autophagy. We adopt the oxygen-glucose deprivation and reperfusion (OGD/R) model of rat primary cortex neurons, and lentivirus interference used to silence Gadd45b expression. Cell viability and injury assay were performed using CCK-8 and LDH kit. Autophagy activation was monitored by expression of ATG5, LC3, Beclin-1, ATG7 and ATG3. Neuron apoptosis was monitored by expression of Bcl-2, Bax, cleaved caspase3, p53 and TUNEL assay. Neuron neurites were assayed by double immunofluorescent labeling with Tuj1 and LC3B. Here, we demonstrated that the expression of Gadd45b was strongly up-regulated at 24 h after 3 h OGD treatment. ShRNA-Gadd45b increased the expression of autophagy related proteins, aggravated OGD/R-induced neuron cell apoptosis and neurites injury. ShRNA-Gadd45b co-treatment with autophagy inhibitor 3-methyladenine (3-MA) or Wortmannin partly inhibited the ratio of LC3II/LC3I, and slightly ameliorated neuron cell apoptosis under OGD/R. Furthermore, shRNA-Gadd45b inhibited the p-p38 level involved in autophagy, but increased the p-JNK level involved in apoptosis. ShRNA-Gadd45b co-treatment with p38 inhibitor obviously induced autophagy. ShRNA-Gadd45b co-treatment with JNK inhibitor alleviated neuron cell apoptosis. In conclusion, our data suggested that Gadd45b inhibited autophagy and apoptosis under OGD/R. Gadd45b may be a common regulatory protein to control autophagy and apoptosis.

PPARα activation drives demethylation of the CpG islands of the Gadd45b promoter in the mouse liver.

Growth arrest and DNA damage-inducible beta (GADD45b) plays a pivotal role in many intracellular events in both cell survival- and cell death-related signaling. To date, the study of GADD35b has mainly focused on investigation of its function, as well as interacting molecules. However, studies of Gadd45b gene regulation are limited. In this study, we investigated the transcriptional regulation mechanism of Gadd45b. Since Gadd45b mRNA is highly induced by the PPARα agonist Wy-14,643 in the mouse liver, we analyzed the Gadd45b promoter using an in vivo reporter assay. Interestingly, the naked Gadd45b-luciferase construct strongly induced luciferase activity without any stimulant in our in vivo system. Therefore, we investigated the epigenetic changes in the Gadd45b promoter region using mouse liver genomic DNA, the methylation-specific restriction enzyme (HpaII), and disulfide conversion. Our results showed that two possible CpG methylation sites were methylated and demethylated by Wy-14,643 treatment. This study indicates that epigenetic change at the Gadd45b promoter is critical for Gadd45b induction.

Nonylphenol induces liver toxicity and oxidative stress in rat.

BACKGROUND: Nonylphenol (NP) is one of the most widely used synthetic xenoestrogens in detergents, plastic products, paints and the most important environmental degradation factor. In this study, the effects NP was investigated on hepatic oxidative stress-related gene expression in rats. METHOD: Wistar male rats weighing 150-200 g were divided into control and NP receiving groups. NP was given in three doses (5, 25, and 125 µg/kg). All doses were given by gavage and the experiment continued for a consecutive 35 days. AST, ALT and ALP determined by the colorimetric method. The RNA was extracted from the rats liver tissue and RT- PCR was used to investigate the changes in gene expression. For this purpose, primers and specific probes of HO1 and Gadd45b genes as well as B-actin as control were prepared and the expression of each gene was separately assessed with ABI-7300. Hematoxylin and eosin staining was performed for evaluating of cell death. FINDING: The data from our study indicated nonylphenol increased alkaline phosphatase level but not changed aspartate aminotransferase and alanine aminotransferase in serum. That various doses of NP result in a dose-dependent increase in the expression of HO-1 gene. The intensified expression of HO-1 was statistically significant just at the doses of 25 and 125 µg/kg compared to control group (p < 0.05). In addition, it was shown that different doses of nonylphenol raised the expression of Gadd45b gene and this increase was significantly evident at 5 µg/kg (p < 0.05). Histological evaluation also indicated that NP increased hepatocytes cell death. CONCLUSION: We conclude that NP increased serum alkaline phosphatase, lead to liver damage and can increase the expression of HO1 and Gadd45b genes and may modify the toxic effects on liver through induction of oxidative stress.

Role of Growth Arrest and DNA Damage-Inducible, Beta in Alcohol-Drinking Behaviors.

BACKGROUND: The contribution of epigenetic factors, such as histone acetylation and DNA methylation, to the regulation of alcohol-drinking behavior has been increasingly recognized over the last several years. GADD45b is a protein demonstrated to be involved in DNA demethylation at neurotrophic factor gene promoters, including at brain-derived neurotrophic factor (Bdnf) which has been highly implicated in alcohol-drinking behavior. METHODS: DNA methyltransferase-1 (Dnmt1), 3a, and 3b, and Gadd45a, b, and g mRNA were measured in the nucleus accumbens (NAc) and ventral tegmental areas of high ethanol (EtOH) consuming C57BL/6J (C57) and low alcohol consuming DBA/2J (DBA) mice using quantitative reverse transcriptase polymerase chain reaction (PCR). In the NAc, GADD45b protein was measured via immunohistochemistry and Bdnf9a mRNA using in situ PCR. Bdnf9a promoter histone H3 acetylated at lysines 9 and 14 (H3K9,K14ac) was measured using chromatin immunoprecipitation, and 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) using methylated DNA immunoprecipitation. Alcohol-drinking behavior was evaluated in Gadd45b haplodeficient (+/-) and null mice (-/-) utilizing drinking-in-the-dark (DID) and 2-bottle free-choice paradigms. RESULTS: C57 mice had lower levels of Gadd45b and g mRNA and GADD45b protein in the NAc relative to the DBA strain. C57 mice had lower NAc shell Bdnf9a mRNA levels, Bdnf9a promoter H3K9,K14ac, and higher Bdnf9a promoter 5HMC and 5MC. Acute EtOH increased GADD45b protein, Bdnf9a mRNA, and histone acetylation and decreased 5HMC in C57 mice. Gadd45b +/- mice displayed higher drinking behavior relative to wild-type littermates in both DID and 2-bottle free-choice paradigms. CONCLUSIONS: These data indicate the importance of the DNA demethylation pathway and its interactions with histone posttranslational modifications in alcohol-drinking behavior. Further, we suggest that lower DNA demethylation protein GADD45b levels may affect Bdnf expression possibly leading to altered alcohol-drinking behavior.

Gadd45b is an epigenetic regulator of juvenile social behavior and alters local pro-inflammatory cytokine production in the rodent amygdala.

Precise regulation of the epigenome during perinatal development is critical to the formation of species-typical behavior later in life. Recent data suggests that Gadd45b facilitates active DNA demethylation by recruiting proteins involved in base excision repair (BER), which will catalyze substitution of 5-methyl-cytosine (5mC) for an unmodified cytosine. While a role for Gadd45b has been implicated in both hippocampal and amygdalar learning tasks, to the best of our knowledge, no study has been done investigating the involvement of Gadd45b in neurodevelopmental programming of social behavior. To address this, we used a targeted siRNA delivery approach to transiently knock down Gadd45b expression in the neonatal rat amygdala. We chose to examine social behavior in the juvenile period, as social deficits associated with neurodevelopmental disorders tend to emerge in humans at an equivalent age. We find that neonatal Gadd45b knock-down results in altered juvenile social behavior and reduced expression of several genes implicated in psychiatric disorders, including methyl-CpG-binding protein 2 (MeCP2), Reelin, and brain derived neurotrophic factor (BDNF). We furthermore report a novel role for Gadd45b in the programmed expression of α2-adrenoceptor (Adra2a). Consistent with Gadd45b's role in the periphery, we also observed changes in the expression of pro-inflammatory cytokines interleukin-6 (Il-6) and interleukin-1beta (Il-1beta) in the amygdala, which could potentially mediate or exacerbate effects of Gadd45b knockdown on the organization of social behavior. These data suggest a prominent role for Gadd45b in the epigenetic programming of complex juvenile social interactions, and may provide insight into the etiology of juvenile behavioral disorders such as ADHD, autism, and/or schizophrenia.

The down-regulation of GADD45B leads to a conversion of cellular oxidative phosphorylation to glycolysis and promotes the progression of bladder cancer.

Background: The predominant feature of cancer cells during the process of carcinogenesis is the inclination towards glycolytic metabolism rather than mitochondrial oxidative phosphorylation. Nevertheless, there is a scarcity of research investigating the correlation between bladder cancer and mitochondrial energy metabolism. Methods: A qPCR array comprising 90 genes associated with mitochondrial oxidative phosphorylation was employed to discern metabolic disparities between three sets of bladder cancer tissue and adjacent normal tissue. Wound healing and transwell assays were utilized to assess cell migration and invasion capabilities, respectively. Colony formation assays were conducted to ascertain the tumorigenic potential of the cells. The proliferative capacity of the cells was examined through in vitro CCK-8 assays. Additionally, nude mouse models were established to evaluate the impact of bladder tumor cells on in vivo proliferation. The Seahorse XFe96 Analyzer was utilized to quantify mitochondrial oxidative phosphorylation, while the levels of glucose-6-phosphate and pyruvate were assessed to evaluate glycolysis. Results: Examination of qPCR array data demonstrated a noteworthy inhibition of mitochondrial oxidative phosphorylation in bladder cancer tissue, as evidenced by the down-regulation of a majority of genes associated with mitochondrial energy metabolism. Notably, GADD45B may potentially exert a significant influence on bladder cancer development, warranting further investigation. The down-regulation of GADD45B in bladder cancer cells resulted in impaired mitochondrial respiration and elevated levels of glycolysis, thereby enhancing cell migration and invasion. Conversely, up-regulation of GADD45B had the opposite effect. Furthermore, over-expression of GADD45B inhibited tumor proliferation and tumorigenesis in both in vitro and in vivo settings. Conclusion: These findings from our study indicate that the down-regulation of GADD45B promotes the shift of cell mitochondrial oxidative phosphorylation towards glycolysis, thereby facilitating the progression of bladder cancer.

GADD45B in the ventral hippocampal CA1 modulates aversive memory acquisition and spatial cognition.

AIMS: This study was designed to investigate the role of growth arrest and DNA damage-inducible ß (GADD45B) in modulating fear memory acquisition and elucidate its underlying mechanisms. MAIN METHODS: Adeno-associated virus (AAV) that knockdown or overexpression GADD45B were injected into ventral hippocampal CA1 (vCA1) by stereotactic, and verified by fluorescence and Western blot. The contextual fear conditioning paradigm was employed to examine the involvement of GADD45B in modulating aversive memory acquisition. The Y-maze and novel location recognition (NLR) tests were used to examine non-aversive cognition. The synaptic plasticity and electrophysiological properties of neurons were measured by slice patch clamp. KEY FINDINGS: Knockdown of GADD45B in the vCA1 significantly enhanced fear memory acquisition, accompanied by an upregulation of long-term potentiation (LTP) expression and intrinsic excitability of vCA1 pyramidal neurons (PNs). Conversely, overexpression of GADD45B produced the opposite effects. Notably, silencing the activity of vCA1 neurons abolished the impact of GADD45B knockdown on fear memory development. Moreover, mice with vCA1 GADD45B overexpression exhibited impaired spatial cognition, whereas mice with GADD45B knockdown did not display such impairment. SIGNIFICANCE: These results provided compelling evidence for the crucial involvement of GADD45B in the formation of aversive memory and spatial cognition.

DNA hypomethylation-mediated upregulation of GADD45B facilitates airway inflammation and epithelial cell senescence in COPD.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease typically characterized by chronic airway inflammation, with emerging evidence highlighting the driving role of cellular senescence-related lung aging. Accelerated lung aging and inflammation mutually reinforce each other, creating a detrimental cycle that contributes to disease progression. Growth arrest and DNA damage-inducible (GADD45) family has been reported to involve in multiple biological processes, including inflammation and senescence. However, the role of GADD45 family in COPD remains elusive. OBJECTIVES: To investigate the role and mechanism of GADD45 family in COPD pathogenesis. METHODS: Expressions of GADD45 family were evaluated by bioinformatic analysis combined with detections in clinical specimens. The effects of GADD45B on inflammation and senescence were investigated via constructing cell model with siRNA transfection or overexpression lentivirus infection and animal model with Gadd45b knockout. Targeted bisulfite sequencing was performed to probe the influence of DNA methylation in GADD45B expression in COPD. RESULTS: GADD45B expression was significantly increased in COPD patients and strongly associated with lung function, whereas other family members presented no changes. GADD45B upregulation was confirmed in mice exposed by cigarette smoke (CS) and HBE cells treated by CS extract as well. Moreover, experiments involving bidirectional modulation of GADD45B expression in HBE cells further substantiated its positive regulatory role in inflammatory response and cellular senescence. Mechanically, GADD45B-facilitated inflammation was directly mediated by p38 phosphorylation, while GADD45B interacted with FOS to promote cellular senescence in a p38 phosphorylation-independent manner. Furthermore, Gadd45b deficiency remarkably alleviated inflammation and senescence of lungs in CS-exposed mice, as well as improved emphysema and lung function. Eventually, in vivo and vitro experiments demonstrated that GADD45B overexpression was partially mediated by CS-induced DNA hypomethylation. CONCLUSION: Our findings have shed light on the impact of GADD45B in the pathogenesis of COPD, thereby offering a promising target for intervention in clinical settings.

STAU1 exhibits a dual function by promoting amyloidogenesis and tau phosphorylation in cultured cells.

Staufen-1 (STAU1) is a double-stranded RNA-binding protein (RBP) involved in a variety of pathological conditions. In this study, we investigated the potential role of STAU1 in Alzheimer's disease (AD), in which two hallmarks are well-established as cerebral ß-amyloid protein (Aß) deposition and Tau-centered neurofibrillary tangles. We found that STAU1 protein level was significantly increased in cells that stably express full-length APP and the brain of APP/PS1 mice, an animal model of AD. STAU1 knockdown, as opposed to overexpression, significantly decreased the protein levels of ß-amyloid converting enzyme 1 (BACE1) and Aß. We further found that STAU1 extended the half-life of the BACE1 mRNA through binding to the 3' untranslated region (3'UTR). Transcriptome analysis revealed that STAU1 enhanced the expression of growth arrest and DNA damage 45 ß (GADD45B) upstream of P38 MAPK signaling, which contributed to STAU1-induced regulation of Tau phosphorylation at Ser396 and Thr181. Together, STAU1 promoted amyloidogenesis by inhibiting BACE1 mRNA decay, and augmented Tau phosphorylation through activating GADD45B in relation to P38 MAPK. Targeting STAU1 that acts on both amyloidogenesis and tauopathy may serve as an optimistic approach for AD treatment.

Has-miR-300-GADD45B promotes melanoma growth via cell cycle.

Response to oncogenic factors like UV, GADD45 family in skin participates in scavenging ROS, DNA repair and cell cycle control. Because of this, the previous study of the chronic UVB injury model has found that hsa-miR-300 can conduct intercellular transport by exosomes and target regulation of GADD45B. Whether the hsa-miR-300-GADD45B still regulates tumor development by cell cycle pathway is unclear. Through transcriptomic analysis of primary (n=39) and metastatic (n=102) melanoma, it was confirmed that in metastatic samples, some of the 97 down-regulated genes participate in maintaining skin homeostasis while 42 up-regulated genes were enriched in cancer-related functions. Furthermore, CDKN1A, CDKN2A, CXCR4 and RAD51 in the melanoma pathway, were also differentially expressed between normal skin and melanoma. CDKN1A and CDKN2A were also found to be involved in TP53-dependent cell cycle regulation. In conclusion, it was speculated that CDKN1A, CDKN2A, TP53, GADD45B and hsa-miR-300 may have regulatory relationships. It was demonstrated that there is a bidirectional regulation between hsa-miR-300 and TP53. In addition, miR-300 can regulate CDKN1A by GADD45B/TP53 and promote melanoma growth by accelerating the cell cycle transition from G1/S to G2 phase.