scholarly journals Thalidomide induces γ-globin gene expression through increased reactive oxygen species–mediated p38 MAPK signaling and histone H4 acetylation in adult erythropoiesis

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2864-2871 ◽  
Author(s):  
Wulin Aerbajinai ◽  
Jianqiong Zhu ◽  
Zhigang Gao ◽  
Kyung Chin ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Globin Gene ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Histone H4 ◽  
Histone H4 Acetylation ◽  
Dose Dependent ◽  
Globin Gene Expression

Abstract Although thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide's specific effects on γ-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on γ-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34+ cells. We found that thalidomide induced γ-globin mRNA expression in a dose-dependent manner, but had no effect on β-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogen-activated protein kinase (MAPK) signaling pathway in a time- and dose-dependent manner and increased histone H4 acetylation. Pretreatment of cells with the antioxidant enzyme catalase and the intracellular hydroxyl scavenger dimethylthiourea (DMTU) abrogated the thalidomide-induced p38 MAPK activation and histone H4 acetylation. Moreover, pretreatment with catalase and DMTU diminished thalidomide-induced γ-globin gene expression. These data indicate that thalidomide induces increased expression of the γ-globin gene via ROS-dependent activation of the p38 MAPK signaling pathway and histone H4 acetylation.

Thalidomide Induces gamma-Globin Gene Expression in Adult Primary Erythroid Cells through Increased Intracellular Reactive Oxygen Species Mediated Activation of p38 MAPK Signal Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1192-1192
Author(s):  
Wulin Aerbajinai ◽  
Jianqiong Zhu ◽  
Peter Gao ◽  
Kyung Chin ◽  
Griffin P. Rodgers
Keyword(s):  
Gene Expression ◽  
P38 Mapk ◽  
Globin Gene ◽  
Mapk Signaling ◽  
Ros Generation ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Gamma Globin ◽  
Dose Dependent ◽  
Globin Gene Expression

Abstract Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide’s therapeutic effect are still being defined. We hypothesize that thalidomide induce the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin gene expression in a dose-dependent manner. The averaged gamma/gamma+beta percentage ratio was 12.89% ± 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% ± 0.07% in Epo alone (P<0.01). Interestingly, we found that intracellular ROS level was significantly increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner. Thalidomide also increased the histone H3 phospho-acetylation and histone H4 acetylation. In contrast, treatment with the anti-oxidant N-acetylcycteine (NAC) inhibited the thalidomide induced p38 activity and histone H3, H4 acetylation. These data indicate that thalidomide induced the gamma-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.

X-Ray and UV Irradiation and Heat Shock Induce γ-Globin Gene Expression in Erythroid Cells Via P38 MAPK Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 459-459
Author(s):  
Rachel West ◽  
Rodwell Mabaera ◽  
Sarah Conine ◽  
Elizabeth Macari ◽  
William J. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
P38 Mapk ◽  
Globin Gene ◽  
K562 Cells ◽  
Mapk Signaling ◽  
Erythroid Cells ◽  
Globin Mrna ◽  
X Ray ◽  
Globin Gene Expression

Abstract Abstract 459 We have recently presented a cell stress signaling model to explain the ability of a wide variety of mechanistically distinct drugs to induce fetal hemoglobin (HbF) production (Mabaera et al, Exp Hematol 2008). This model proposes that HbF inducing agents, including DNMT and HDAC inhibitors, short-chain fatty acid derivatives and cytotoxic agents induce HbF production by activating intracellular cell stress signaling pathways and that the p38 MAPK (mitogen-activated protein kinase) pathway plays a central role in the induction process. The major alternative to this model is that each class of drugs works through a distinct mechanism, such as global DNA hypomethylation or histone hyperacetylation. If our model is correct, then non-pharmacologic inducers of p38 MAPK signaling, such as X-ray and UV irradiation, heat shock, and osmotic shock should also increase γ-globin gene expression. If these physical stresses do not up-regulate γ-globin gene expression, then our model is likely to be incorrect. To mimic our previous experiments in which we treated differentiating human primary erythroid cells with inducing agents over several days, we exposed K562 cells to physical stresses daily for 5 days at doses that did not cause cell death. To test whether X-irradiation induces the transcription of γ-globin mRNA, cells were irradiated at doses ranging from 0 to 1.0 cGy/day, in the absence and presence of SB203580 (a p38 MAPK inhibitor). Experiments were first performed on K562 cells, and then during in vitro erythroid differentiation of primary human CD34+ cells. These treatments stimulated a strong, dose-dependent increase of γ-globin mRNA in both K562 cells (up to 6-fold over untreated cells) and in primary erythroid cells (up to 5-fold) as measured by quantitative RT-PCR. SB203580 abolished this effect in both cell types. The inhibitor also caused a decrease in γ-globin mRNA expression in untreated control cells, suggesting that p38 MAPK signaling plays a role in basal γ-globin gene expression. The same X-ray doses also induced phosphorylation of the down-stream p38 MAPK target, HSP-27 and up-regulated expression of stress-induced transcription factor genes including GADD34, CHOP (GADD153), and ATF3. Expression of GADD34 and CHOP genes and HSP-27 phosphorylation were inhibited by SB203580. We next tested the ability of UV light (254 nm) administered daily for 5 days to induce γ-globin mRNA in K562 cells. A dose-dependent increase in γ-globin mRNA was observed following exposure to doses as low as 10 J/m2. Cells treated with 35 J/m2 had 2.7-fold higher levels of steady state γ-globin mRNA compared to untreated control cells. This induction was also inhibited by SB203580. Preliminary experiments with heat shock have yielded similar results. Following a single 180-minute exposure to 42°C, K562 cells showed a 2.2-fold increase in γ-globin mRNA compared to untreated cells. Shorter exposures to higher temperatures (e.g., 50°C for 15 minutes) caused an approximately 3-fold increase in γ-globin steady-state mRNA after 24 hours. These 2- to 3-fold increases in expression are similar to those we have previously observed in primary human erythroid cells with 5-azacytidine and butyrate. In none of our experiments did the K562 cells become benzidine positive, indicating that increased γ-globin expression was not the result of activating an erythroid differentiation program. Together with other published studies, these data support the hypothesis that p38 MAPK pathway signaling, whether caused by drugs or physical stress, is a key component of γ-globin gene induction. This in turn suggests that the components of the p38 MAPK pathway could serve as novel targets for the development of new HbF inducing agents. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human fetal globin gene expression is regulated by LYAR

2014 ◽  
Vol 42 (15) ◽  
pp. 9740-9752 ◽  
Author(s):  
Junyi Ju ◽  
Ying Wang ◽  
Ronghua Liu ◽  
Yichong Zhang ◽  
Zhen Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
K562 Cells ◽  
Binding Motif ◽  
Dependent Manner ◽  
Histone H4 ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Globin Gene Expression ◽  
Selection Of

AbstractHuman globin gene expression during development is modulated by transcription factors in a stage-dependent manner. However, the mechanisms controlling the process are still largely unknown. In this study, we found that a nuclear protein, LYAR (human homologue of mouse Ly-1 antibody reactive clone) directly interacted with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. We found that PRMT5 binding on the proximal γ-promoter was LYAR-dependent. The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments. Results of EMSA and ChIP assays confirmed that LYAR bound to a DNA region corresponding to the 5′-untranslated region of the γ-globin gene. We also found that LYAR repressed human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Thus, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γ-globin gene.

The defense response of Caenorhabditis elegans to Cutibacterium acnes SK137 via the TIR-1-p38 MAPK signaling pathway

2021 ◽  
Author(s):  
Ayano Tsuru ◽  
Yumi Hamazaki ◽  
Shuta Tomida ◽  
Mohammad Shaokat Ali ◽  
Eriko Kage-Nakadai
Keyword(s):  
Caenorhabditis Elegans ◽  
Signaling Pathway ◽  
P38 Mapk ◽  
Defense Response ◽  
Mapk Pathway ◽  
Defense Responses ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Healthy Skin ◽  
Cutibacterium Acnes

Abstract Cutibacterium acnes plays roles in both acne disease and healthy skin ecosystem. We observed that mutations in the tir-1/SARM1 and p38 MAPK cascade genes significantly shortened Caenorhabditis elegans lifespan upon Cutibacterium acnes SK137 infection. Antimicrobial molecules were induced by SK137 in a TIR-1-dependent manner. These results suggest that defense responses against SK137 involve the TIR-1-p38 MAPK pathway in Caenorhabditis elegans.

AV-65, a Novel Inhibitor of the Wnt/β-Catenin Signaling Pathway, Inhibits the Proliferation of Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2866-2866
Author(s):  
Hisayuki Yao ◽  
Eishi Ashihara ◽  
Rina Nagao ◽  
Shinya Kimura ◽  
Hideyo Hirai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Signaling Pathway ◽  
Small Molecule ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Cancer Cell Line ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Abstract 2866 Poster Board II-842 Although new molecular targeting agents against multiple myeloma (MM) have been developed, MM still remains an incurable disease. It is important to continue to investigate new therapeutic agents based on the biology of MM cells. β-catenin is the downstream effector of Wnt signaling and it regulates genes implicated in malignant progression. We have demonstrated that blockade of Wnt/β-catenin signaling pathway inhibits the progression of MM by using RNA interference methods with an in vivo mouse model (Ashihara E, et al. Clin Cancer Res 15:2731, 2009.). In this study, we investigated the effects of AV-65, a novel inhibitor of the Wnt/β-catenin signaling pathway, on MM cells. The system to identify a series of small molecule compounds using a biomarker driven approach has been established. A gene expression biomarker signature reporting on the inhibition of Wnt/β-catenin signaling was generated upon treatment of a colon cancer cell line with β-catenin siRNA. This gene expression signatiure was used to screen a small molecule compound library to identify compounds which mimic knockdown of β-catenin and thus potentially inhibit the Wnt/β-catenin signaling pathway. One compound series, LC-363, was discovered from this screen and validated as novel Wnt/β-catenin signaling inhibitors (Strovel JW, et al. ASH meeting, 2007.). We investigated the inhibitory effects of AV-65, one of LC-363 compounds, on MM cell proliferation. AV-65 inhibited the proliferation of MM cells in a time- and a dose-dependent manner and the values of IC50 at 72 hrs were ranging from 11.7 to 82.1 nM. AV-65 also showed an inhibitory effect on the proliferation of RPMI8226/LR-5 melphalan-resistant MM cells (provided from Dr. William S. Dalton). In flow cytometric analysis, apoptotic cells were increased by AV-65 treatment in a time- and a dose-dependent manner. Western blotting analysis showed that β-catenin was ubiquitinated and that the expression of nuclear β-catenin diminished (Figure 1). Moreover, AV-65 suppressed T-cell factor transcriptional activities, resulting in the decrease of c-myc expression. Taken together, AV-65 promotes the degradation of β-catenin, resulting in the induction of apoptosis of MM cells. We next investigated the in vivo effects of AV-65 using an orthotopic MM-bearing mouse model. AV-65 inhibits the growth of MM cells and significantly prolongs the survival rates (Figure 2). In conclusion, AV-65 inhibited the proliferation of MM cells via inhibition of the Wnt/β-catenin signaling pathway. AV-65 is a promising therapeutic agent for treatment of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Glucocorticoid Receptor Recruitment of Histone Deacetylase 2 Inhibits Interleukin-1β-Induced Histone H4 Acetylation on Lysines 8 and 12

2000 ◽  
Vol 20 (18) ◽  
pp. 6891-6903 ◽  
Author(s):  
Kazuhiro Ito ◽  
Peter J. Barnes ◽  
Ian M. Adcock
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Interleukin 1Β ◽  
Dependent Manner ◽  
Histone H4 ◽  
Altered Expression ◽  
Gm Csf ◽  
Low Concentrations ◽  
Histone H4 Acetylation

ABSTRACT We have investigated the ability of dexamethasone to regulate interleukin-1β (IL-1β)-induced gene expression, histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity. Low concentrations of dexamethasone (10−10 M) repress IL-1β-stimulated granulocyte-macrophage colony-stimulating factor (GM-CSF) expression and fail to stimulate secretory leukocyte proteinase inhibitor expression. Dexamethasone (10−7 M) and IL-1β (1 ng/ml) both stimulated HAT activity but showed a different pattern of histone H4 acetylation. Dexamethasone targeted lysines K5 and K16, whereas IL-1β targeted K8 and K12. Low concentrations of dexamethasone (10−10 M), which do not transactivate, repressed IL-1β-stimulated K8 and K12 acetylation. Using chromatin immunoprecipitation assays, we show that dexamethasone inhibits IL-1β-enhanced acetylated K8-associated GM-CSF promoter enrichment in a concentration-dependent manner. Neither IL-1β nor dexamethasone elicited any GM-CSF promoter association at acetylated K5 residues. Furthermore, we show that GR acts both as a direct inhibitor of CREB binding protein (CBP)-associated HAT activity and also by recruiting HDAC2 to the p65-CBP HAT complex. This action does not involve de novo synthesis of HDAC protein or altered expression of CBP or p300/CBP-associated factor. This mechanism for glucocorticoid repression is novel and establishes that inhibition of histone acetylation is an additional level of control of inflammatory gene expression. This further suggests that pharmacological manipulation of of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases.

scholarly journals The Role of Adenosine Monophosphate Activated Protein Kinase Alpha 1 (AMPK) in Gamma-Globin Regulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 413-413
Author(s):  
Alicia Chang ◽  
Yankai Zhang ◽  
Nelda Itzep ◽  
Vivien A Sheehan
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Insulin Signaling ◽  
Globin Gene ◽  
Insulin Signaling Pathway ◽  
Gamma Globin ◽  
Compound C ◽  
Globin Gene Expression

Abstract Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF. Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD. Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively. Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p<0.01) and a three-fold reduction in gamma-globin expression (p<0.001). HSPCs treated with metformin or piceatannol exhibited a 2-3 fold rise in AMPK, FOXO3 and gamma globin gene expression (p<0.001). HSPCs treated with piceatannol and metformin showed an increase in pAMPK at Thr172, the activated form of AMPK. In the presence of a specific AMPK inhibitor, Compound C, metformin and piceatannol, no induction of gamma globin was observed (Figure 1), and pAMPK was reduced to untreated levels. Conclusions: Knockdown of AMPK in HSPCs reduces gamma globin expression and %HbF, supporting the role of AMPK in gamma globin regulation. Drugs known to activate AMPK, metformin and piceatannol, increase gamma globin in SCD patient derived HSPCs. Pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction, and reduces the gamma globin induction of metformin and piceatannol to untreated levels. We therefore conclude that AMPK is a positive regulator of HbF, and that pharmacologic induction of HbF with metformin and piceatannol requires AMPK activity. Further work is needed to establish if FOXO3 and AMPK alone are instrumental in HbF regulation, or if other proteins in the insulin signaling pathway may play a role in HbF regulation. Disclosures No relevant conflicts of interest to declare.

EKLF Is Recruited to the γ-Globin Gene Promoter as a Co-Activator and Is Required for γ-Globin Gene Induction by Short-Chain Fatty Acids.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1771-1771
Author(s):  
Susan P. Perrine ◽  
Rishikesh Mankidy ◽  
Michael S. Boosalis ◽  
James J. Bieker ◽  
Douglas V. Faller
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Induction ◽  
Therapeutic Interventions ◽  
Short Chain ◽  
Dependent Manner ◽  
Erythroid Progenitors ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract The erythroid Kruppel-like factor, EKLF, is an essential transcription factor for mammalian β-type globin gene switching, and specifically activates transcription of the adult β-globin gene through binding of its zinc finger domain to the β-globin promoter. We report now that EKLF is also required for activation of the γ-globin gene by short-chain fatty acid (SCFA) derivatives. We found that specific knockdown of EKLF levels by siRNA prevents SCFA induced-expression of an integrated γ-globin promoter in a stably-expressed mLCRβprRluc AγprFluc cassette, and prevents induction of the endogenous γ-globin gene in primary human erythroid progenitors. In chromatin immunoprecipitation (ChIP) assays, EKLF was found to be actively recruited to the endogenous γ-globin gene promoter with exposure of human erythroid progenitors, and hematopoietic cell lines, to SCFA derivatives. The human SWI/WNF complex is a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure in an ATP-dependent manner. We found that the SWI/SNF complex chromatin-modifying core ATPase BRG1 is also required for γ-globin gene induction by SCFA derivatives. Furthermore, BRG1 is actively recruited to the endogenous γ-globin promoter of human erythroid progenitors with exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings all demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes. Recently. we also identified a γ-globin-specific repressor complex, consisting of NCoR and HDAC3, which is displaced from the proximal γ-globin promoter by exposure to SCFA derivatives prior to activation of transcription (Blood, 108:3179–86, 2006). Collectively, these studies identify critical activating and repressing cofactors regulating γ-globin gene expression, and provide new targets for therapeutic interventions.

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