Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, ...chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24μM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.
Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the HBB gene leading to hemoglobin S production and polymerization under hypoxia conditions leading to vaso-occlusion, ...chronic hemolysis, and progressive organ damage. This disease affects ~100,000 people in the United States and millions worldwide. An effective therapy for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents such as hydroxyurea, the only Food and Drug Administration-approved drug for this purpose. Therefore, the goal of our study was to determine whether salubrinal (SAL), a selective protein phosphatase 1 inhibitor, induces HbF expression through the stress-signaling pathway by activation of p-eIF2α and ATF4 trans-activation in the γ-globin gene promoter. Sickle erythroid progenitors treated with 24μM SAL increased F-cells levels 1.4-fold (p = 0.021) and produced an 80% decrease in reactive oxygen species. Western blot analysis showed SAL enhanced HbF protein by 1.6-fold (p = 0.0441), along with dose-dependent increases of p-eIF2α and ATF4 levels. Subsequent treatment of SCD mice by a single intraperitoneal injection of SAL (5mg/kg) produced peak plasma concentrations at 6 hours. Chronic treatments of SCD mice with SAL mediated a 2.3-fold increase in F-cells (p = 0.0013) and decreased sickle erythrocytes supporting in vivo HbF induction.
Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. ...Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.
Two signaling pathways known to be essential for progression from immature to mature B cells are BAFF receptor (BAFF-R) and the B cell receptor (BCR). Here, we first show that phospholipase C ...(PLC)-γ2 is required for a BAFF-R–mediated survival signal. Then, we have examined the question of whether the reduced number of mature B cells in PLC-γ2−/− mice is caused by a defect in either BCR or BAFF-R signaling. We find that a PLC-γ2 SH2 mutant, which inhibits coupling between BCR and PLC-γ2, fails to restore B cell maturation, despite supporting BAFF-dependent survival. Therefore, our data suggest that the BAFF-R–mediated survival signal, provided by PLC-γ2, is not sufficient to promote B cell maturation, and that, in addition, activation of PLC-γ2 by BCR is required for B cell development.
Scurfy mice develop CD4 T-cell–mediated lymphoproliferative disease leading to death within 4 weeks of age. The scurfy mutation causes loss of function of the foxp3 gene (foxp3sf), which is essential ...for development and maintenance of naturally occurring regulatory CD4 T cells (nTregs). In humans, mutations of the foxp3 gene cause immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX). In most patients with IPEX and also in scurfy mice, T cells show hyperreactivity and levels of Th1- and Th2-associated cytokines are substantially elevated. We report that removal of CD28 expression rescued scurfy mice from early death. Longer-term surviving CD28-deficient scurfy mice still had lymphoproliferative disorder, but their CD4 T cells showed decreased interferon-γ and no sign of interleukin-4 or interleukin-10 hyperproduction. Furthermore, injection of CTLA4-Ig to block CD28-B7 interactions substantially improved the survival of scurfy mice by blocking effector T-cell differentiation. These data support the hypothesis that CD28-B7 interactions play a critical role in the etiology of lethal autoimmune disease in scurfy mice by stimulating the differentiation of antigen-activated naive T cells into effector T cells.
•Higher miR-144 gene expression was observed in peripheral blood reticulocytes of sickle cell disease (SCD) patients with low fetal hemoglobin levels.•NRF2 protein levels are regulated by miR-144 as ...a mechanism of γ-globin gene silencing during erythropoiesis in SCD.
Inherited genetic modifiers and pharmacologic agents that enhance fetal hemoglobin (HbF) expression reverse the clinical severity of sickle cell disease (SCD). Recent efforts to develop novel strategies of HbF induction include discovery of molecular targets that regulate γ-globin gene transcription and translation. The purpose of this study was to perform genome-wide microRNA (miRNA) analysis to identify genes associated with HbF expression in patients with SCD. We isolated RNA from purified reticulocytes for microarray-based miRNA expression profiling. Using samples from patients with contrasting HbF levels, we observed an eightfold upregulation of miR-144-3p (miR-144) and miR-144-5p in the low-HbF group compared with those with high HbF. Additional analysis by reverse transcription quantitative polymerase chain reaction confirmed individual miR-144 expression levels of subjects in the two groups. Subsequent functional studies in normal and sickle erythroid progenitors showed NRF2 gene silencing by miR-144 and concomitant repression of γ-globin transcription; by contrast, treatment with miR-144 antagomir reversed its silencing effects in a dose-dependent manner. Because NRF2 regulates reactive oxygen species levels, additional studies investigated mechanisms of HbF regulation using a hemin-induced oxidative stress model. Treatment of KU812 cells with hemin produced an increase in NRF2 expression and HbF induction that reversed with miR-144 pretreatment. Chromatin immunoprecipitation assay confirmed NRF2 binding to the γ-globin antioxidant response element, which was inhibited by miR-144 mimic treatment. The genome-wide miRNA microarray and primary erythroid progenitor data support a miR-144/NRF2-mediated mechanism of γ-globin gene regulation in SCD.
NRF2 is the master regulator for the cellular oxidative stress response and regulates γ-globin gene expression in human erythroid progenitors and sickle cell disease mice. To explore NRF2 function, ...we established a human β-globin locus yeast artificial chromosome transgenic/NRF2 knockout (β-YAC/NRF2−/−) mouse model. NRF2 loss reduced γ-globin gene expression during erythropoiesis and abolished the ability of dimethyl fumarate, an NRF2 activator, to enhance γ-globin transcription. We observed decreased H3K4Me1 and H3K4Me3 chromatin marks and association of TATA-binding protein and RNA polymerase II at the β-locus control region (LCR) and γ-globin gene promoters in β-YAC/NRF2−/− mice. As a result, long-range chromatin interaction between the LCR DNase I hypersensitive sites and γ-globin gene was decreased, while interaction with the β-globin was not affected. Further, NRF2 loss silenced the expression of DNA methylcytosine dioxygenases TET1, TET2, and TET3 and inhibited γ-globin gene DNA hydroxymethylation. Subsequently, protein-protein interaction between NRF2 and TET3 was demonstrated. These data support the ability of NRF2 to mediate γ-globin gene regulation through epigenetic DNA and histone modifications.
Impact statement
Sickle cell disease is an inherited hemoglobin disorder that affects over 100,000 people in the United States causing high morbidity and early mortality. Although new treatments were recently approved by the FDA, only one drug Hydroxyurea induces fetal hemoglobin expression to inhibit sickle hemoglobin polymerization in red blood cells. Our laboratory previously demonstrated the ability of the NRF2 activator, dimethyl fumarate to induce fetal hemoglobin in the sickle cell mouse model. In this study, we investigated molecular mechanisms of γ-globin gene activation by NRF2. We observed the ability of NRF2 to modulate chromatin structure in the human β-like globin gene locus of β-YAC transgenic mice during development. Furthermore, an NRF2/TET3 interaction regulates γ-globin gene DNA methylation. These findings provide potential new molecular targets for small molecule drug developed for treating sickle cell disease.
Background: Thepulmonary endothelium is a metabolically active monolayer on the luminal surface of the lung vasculature. It regulates a variety of functions including the passage of macromolecules ...and fluid between the blood and lung interstitial tissue. Inflammatory agents such as bacterial endotoxin lipopolysaccharide (LPS) cause lung endothelial cells (EC) dysfunction leading to pulmonary edema predisposing to acute chest syndrome, a leading cause of death in sickle cell disease (SCD); therefore, a drug which repairs the damaged EC barrier function might be of therapeutic potential. Recently we demonstrated that β-nicotinamide adenine dinucleotide (β-NAD) reverses LPS-induced EC barrier dysfunction in a model of acute lung injury (Exp Lung Res 2012, 38, 223-32). In this study, we investigated the protective effect of β-NAD against the damaging effects of LPS using primary ECs from human and sickle cell mouse lungs as well as survival studies using the preclinical sickle cell mouse model.
Methods: Early passaged human lung microvascular EC (HLMVEC) were obtained from Lonza and mouse lung microvascular EC (MLMVEC) were isolated from the Townes knock-in homozygous sickle cell (SS) and heterozygous sickle trait (AS) transgenic mice. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) technique. Western blot was done to measure phosphorylation of proteins involved in the regulation of EC barrier function. Survival studies were performed in SS mice treated with intraperitoneal (IP) LPS (0.05 mg/kg) alone or combined with β-NAD (11 mg/kg); normal saline served as a control. Lungs were harvested at 10-12 hrs from the LPS group and at 24 hrs from saline or LPS/β-NAD groups and stained with Hematoxylin and Eosin for to assess interstitial edema and myeloperoxidase as a marker of neutrophil infiltration.
Results: Barrier function measurements using ECIS of HLMVEC indicate that β-NAD (100 µM) enhanced basal EC barrier function by 50-60%. In addition, β-NAD attenuated LPS-induced EC gap formation and actin stress fibers. In human lung endothelium, myosin light chain phosphatase (MLCP) plays a crucial role in the maintenance of the EC barrier integrity. Furthermore, LPS mediates MLCP inactivation by phosphorylating the MLCP regulatory subunit MYPT1 and cytoskeletal targets ERM (Ezrin, Radixin, and Moesin) and CaD (Caldesmon). Western blot analysis demonstrated that β-NAD reversed LPS-induced phosphorylation of MYPT1, ERM and CaD proteins. To determine a functional role, we performed studies using 10 nM MYPT1 siRNA treatment followed by β-NAD (100 μM) for 5 hrs. We observed a 50% attenuation of β-NAD enhanced barrier function suggesting MYPT1 is involved in the mechanism of action for β-NAD. Subsequent studies with SS-MLMVEC demonstrated β-NAD mediated a 40% improvement in barrier function which was comparable to HLMVEC. Further, LPS treatment for 16 hrs produced a 25% decrease in barrier function in SS-MLMVEC compared to a 7% decrease in AS-MLMVEC suggesting impaired EC barrier function in SS lungs. Finally we performed survival studies to determine the ability of β-NAD to protect against LPS-induced mortality in SS mice. In dose optimization studies of LPS (0.1-1.0 mg/kg/dose), SS mice survived for 10-12 hrs with the 0.05 mg/kg LPS dose. By contrary, all LPS doses tested had no effect on AS mice survival. Based on this result, SS mice were given IP injections of saline (3 mice), LPS (4 mice) or LPS/β-NAD (4 mice) and survival rates were monitored. The saline or LPS/β-NAD treated mice survived for 24 hrs (100%) compared with LPS mice which died by 10-12 hrs (p=0.01) supporting a protective effect of β-NAD. Histological assessment of the lung tissue showed increased interstitial edema by Hematoxylin and Eosin stain and neutrophil infiltration by myeloperoxidase in the LPS-treated lungs; these changes were much less prominent in the lungs from LPS/β-NAD treated SS mice.
Conclusions: Our data support a role of MLCP and its cytoskeletal targets in the protective effects of β-NAD against LPS-induced HLMVEC barrier dysfunction. Studies using SS-MLMVEC suggest that SS mice have impaired barrier function and greater susceptibility to LPS toxicity. However, β-NAD treatment improved survival of SS mice supporting the therapeutic potential of this agent to treat the complication of acute chest syndrome in SCD.
Makala:Georgia Regents University: Employment.
Abstract only
Sickle cell disease (SCD) is an inherited blood disorder caused by a point mutation in the β‐globin gene affecting about 100,000 people in the United States and millions worldwide. ...Individuals living with this disease suffer from hemolytic anemia, progressive organ damage, and acute painful episodes. Fetal hemoglobin (HbF), a major genetic modifier of clinical presentation and progression of SCD, is of great interest due to its ability to inhibit sickle hemoglobin (HbS) polymerization and reducing erythrocyte damage. Variation in HbF levels among sickle cell patients is highly heritable and has been linked to the presence of single nucleotide polymorphisms (SNPs) in several genetic regulatory regions. In this study, DNA isolated from two cohorts of African American SCD patients with high and low HbF levels were analyzed by PCR‐based SNP analysis in the
HBB
,
BCL11A
, and
HBS1L‐MYB
(HMIP) gene regions. The presence of SNP alleles was then correlated with their clinical phenotype data to identify those associated with increased levels of HbF. Three SNPs in the
BCL11A
and
HBB
gene regions were identified in the high HbF patient cohort that may contribute to milder phenotypes in African Americans. To complement the SNP studies, genome wide microRNA (miRNA) data was generated which showed increased miR‐16 levels associated with high HbF. Furthermore, MYB, the negative regulator of γ‐globin expression, is silenced by miR‐16 through binding the 3′‐untranslated region. RT‐qPCR and western blot were used to measure γ‐globin mRNA and HbF protein levels respectively after transfection of miR‐16 mimic into KU812 cells. Preliminary data revealed dose dependent miR‐16 mediated
MYB
suppression and γ‐globin activation. The discovery of regulatory regions that modulate HbF expression and miRNA mediated suppression of negative regulators of γ‐globin, provides expanded understanding of gene regulation, which can be used to define novel treatment options for individuals with SCD.
Support or Funding Information
Sponsored by the Medical College of Georgia Medical Scholars Program
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