Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species

Experimental Hematology 34 (2006) 264–273

Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species Cheng-Hui Hsiaoa, Wei Lia, Tzu-Fang Loua, B. Surendra Baligab, and Betty S. Pace, M.D.a a

University of Texas at Dallas, Department of Molecular and Cell Biology, Richardson, Tex., USA; b University of South Alabama, Department of Pediatrics, USA Medical Center, Mobile, Ala., USA (Received 19 July 2005; revised 28 November 2005; accepted 8 December 2005)

Objective. Several compounds, including butyrate and trichostatin A, have been shown to activate g-gene expression via p38 mitogen-activated protein kinase (MAPK) signaling. In eukaryotic cells, reactive oxygen species (ROS) act as signaling molecules to mediate phosphorylation of tyrosine kinases such as p38 MAPK to regulate gene expression. Therefore, we determined the role of the reactive oxygen species hydrogen peroxide (H2O2) in drugmediated fetal hemoglobin (HbF) induction. Methods. H2O2 levels were measured using 20 ,70 -dichlorofluorescein-diacetate in K562 cells after drug treatments. To confirm a role for H2O2 in HbF induction, studies were completed with the mitochondrial respiratory chain inhibitor myxothiazole, which prevents ROS generation. The ability of myxothiazole to block g-globin mRNA accumulation and HbF induction was measured in K562 cells and burst-forming unit-erythroid colonies respectively using quantitative real-time PCR and alkaline denaturation. Results. Butyrate and trichostastin A stimulated p38 MAPK phosphorylation via a H2O2dependent mechanism. Pretreatment with myxothiazole to inhibit ROS formation or SB203580 to impede p38 MAPK signaling attenuated g-gene activation in K562 cells and HbF induction in erythroid progenitors. However, myxothiazole had no effect on the ability of hydroxyurea to induce HbF. Conclusion. The findings presented herein support a ROS-p38 MAPK cell signaling mechanism for HbF induction by butyrate and trichostatin A. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

The most attractive option for treating sickle cell disease (SCD) is to reactivate the fetal stage–specific g-globin genes to render clinical complications less severe. Drugmediated fetal hemoglobin (HbF) induction has been demonstrated for hydroxyurea (HU) [1], 5-deoxyazacytidine [2], and the histone deacetylase (HDAC) inhibitors butyrate (NaB), trichostatin A (TSA), and adicipin, among others [1–5]. Epigenetic modifications of chromatin, such as DNA methylation and histone hyperacetylation, are important mechanisms for gene silencing or activation [6,7]. Agents such as 5-azacytidine and 5-deoxyazacytidine produce hypomethylation of the cytosine residue in the CpG dinucleotide to produce gene activation in eukaryotic

Offprint requests to: Betty S. Pace, M.D., University of Texas at Dallas, PO Box 830688, Richardson, TX 75083; E-mail: [email protected]

cells [2,8–11]. Elevated HbF levels can be achieved in patients with SCD using hypomethylating agents [2,8] or HDAC inhibitors [12,13]. However, there often is a lack of correlation between in vitro g-gene activation and HbF levels achieved in vivo; therefore, efforts continue to identify additional compounds for human therapy. Studies to elucidate cell signaling mechanisms for drug-mediated g-gene induction are ongoing to establish additional therapeutic approaches. Several laboratories, including our laboratory, have demonstrated an important role for mitogen-activated protein kinase (MAPK) cell signaling in g-gene reactivation by HDAC inhibitors [3–5]. It is believed that phosphorylated tyrosine kinase molecules translocate to the nucleus where they stimulate target transcription factors that bind promoter elements to regulate gene expression [14]. The p38 MAPK pathway has been implicated as a major mediator

0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2005.12.009

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of g-globin promoter activation by this mechanism [15,16]; however, target downstream trans-activators have not been identified. The p38 pathway is activated by stress, hypoxia, and reactive oxygen species (ROS), including superoxide, H2O2 and nitric oxide (NO) formed by one electron reduction of molecular oxygen in the mitochondrial electron transport chain [17,18]. Hydroxyurea is thought to acts as a NO donor to activate cyclic guanosine monophosphate (cGMP) cell signaling [19,20]. Since the other HbF inducers analyzed in our study did not have chemical structures analogous to known NO donors, we tested whether the reactive oxygen species H2O2 could activate p38 MAPK-mediated g-globin induction. To test this mechanism, the ability of NaB, TSA, and HU to stimulate H2O2 in erythroid cells was analyzed using a 20 ,70 -dichlorodihydrofluorescein-diacetate (DCFH-DA) probe. We demonstrated that NaB and TSA increased H2O2 levels in K562 cells. Inhibition of ROS formation by myxothiazole or p38 MAPK signaling by SB203580 attenuated g-gene induction by HDAC inhibitors. By contrast, hemin stimulated H2O2 production; however, g-gene activation occurred independent of p38 MAPK signaling. The data herein support an ROS-p38 MAPK cell signaling mechanism for HbF induction by HDAC inhibitors in human erythroid progenitors.

Methods Tissue culture K562 cells were maintained in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum (Atlanta Biological, Atlanta, GA, USA), penicillin (100 U/mL), and streptomycin (0.1 mg/mL) at 37
C and 5% CO2. Dose response studies were completed in K562 cells treated with menadione (MND; 50–200 mM) and myxothiazole (MYX; 0.3–10 mM), NaB (0.5–4 mM), or TSA (0.1–0.5 mM). Cell viability was determined using 0.4% trypan blue exclusion at the time of harvest. For drug inductions K562 cells (1 3 106 per well) were incubated from 4 to 48 hours with NaB (2 mM), TSA (0.5 mM), hemin (50 mM), MND (200 mM), anisomycin (200 ng/mL), or HU (100 mM). Additional studies after pretreatment with the ROS inhibitor MYX (10 mM) for 30 minutes were completed as well. To inhibit p38 MAPK signaling, cells were pretreated with SB203580 (10 mM) for 1 hour, followed by drug inductions. The inhibitors were protected from light during the entire culture period. NaB, TSA, and hemin were purchased from Sigma (St. Louis, MO, USA) and prepared by standard methods. MYX and MND were purchased from Molecular Probes (Eugene, OR, USA) and prepared in dimethylsulfoxide as 20-mM and 10-mM stock solutions respectively. Anisomycin was prepared in ethanol and stored at 220
C. Control studies were performed with equivalent concentrations of dimethylsulfoxide and ethanol. Fluorescence-activated cell sorting (FACS) analysis Hydrogen peroxide levels were measured by two techniques, FACS and fluorometry. The fluorescent dye DCFH-DA was used to measure H2O2 levels at 504 nm excitation and 529 nm emission

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wavelengths. A 20-mM stock solution of DCFH-DA was prepared in dimethylsulfoxide and cell viability tested at 0.3- to 32-mM concentrations. The relative fluorescence generated by the positive control MND (25–200 mM) was also measured using the DCFHDA probe. Cells were harvested by centrifuge and washed twice with phosphate-buffered saline, and then pellets were resuspended in 1 mL of sheath fluid. FACS analysis was completed on a FACSCalibur (Becton-Dickinson, San Jose, CA, USA) flow cytometer equipped with 488-nm argon and 635-nm red diode lasers. Cells were analyzed using CellQuest software (Becton-Dickinson). Background counts were gated based on light scattering properties in the side- and forward-scatter modes. Ten thousand cells were counted per sample. The mean fluorescence value from the experimental samples was normalized by untreated controls. For each experiment, viability was monitored using a LIVE/DEAD kit (Molecular Probes, Eugene, OR, USA). Approximately 1 3 106 cells were treated with 2 mL of 50-mM calcein AM and 4 mL of 2-mM ethidium homodimer-1 per mL. The cells were incubated for 15 minutes in the dark, after which FACS analysis was completed using 488 nm excitation wavelength to measure the green fluorescence emission of calcein at 530/20 bandpass. The red fluorescence emission of ethidium bromide was gated on cells at 610/ 20 bandpass to exclude debris. The percent live cells were calculated as a fraction of the total cells counted. Fluorometry analysis K562 cells were washed twice in phosphate-buffered saline and then pellets were mixed in 200 mL of lysis buffer (Promega, Madison, WI, USA) and mixed by vortexing. Cellular debris was removed by centrifugation and then 100 mL of supernatant was loaded in a 96-well plate and read on a BIO-TEK fluorometer (Winooski, VT, USA) with 485/20 nm excitation and 530/35 nm emission filters. Total protein concentrations were determined by Bradford assay (BioRad, Hercules, CA, USA) and used for sample normalization. Fluorescence microscopy Aliquots of K562 cells were resuspended in sheath fluid at the time of harvest for cytospin preps. Phase-contrast images were photographed with a CCD camera (1/100-sec exposure) on an Olympus BX 51 phase contrast/epifluorescent microscope equipped with Hoffman Modulation optics. Fluorescence images were photographed through 485/20 nm emission and 540/20 nm excitation filters at 1/4-second exposure. The percent positive cells for each experimental treatment was determined by dividing the ROSD cells by the total number of cells counted in the same phase contrast field. At least 500 cells were counted for each data point. The percentage of cells that were ROSD due to MND-induced apoptosis was subtracted from the total. p38 MAPK Western blot analysis K562 cells were treated with MND in the absence or presence of three inhibitors: 10 mM MYX, 10 mM SB203580, or 100 mM PD98059. The latter two inhibitors were incubated with cells for 1 hour to impede p38 MAPK and extracellular signal–regulated kinase signaling respectively. The influence of exogenous H2O2 (25, 50, and 100 mM) on phosphorylated (p)-p38 levels was tested at 12 hours because the HDAC inhibitors and hemin induced H2O2 formation and g-globin expression at this time point. The level of p38 MAPK was measured by Western blot analysis in the absence

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or presence of MYX, SB203580, or PD98059. Cells were harvested in 100 mL of ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA, USA) and protein concentrations determined by Bradford assay. Protein extracts (200 mg) were resolved in 12.5% sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes. Western blotting was performed with p-p38 or total p38 (t-p38) antibodies (Cell Signaling Technology). Immunoglobulin IgG–horseradish peroxidase–conjugated secondary antibody was used for chemiluminiscent detection (ECL kit, Amersham Biosciences, Piscataway, NJ, USA) of protein bands, which were quantitated by densitometry (ChemiDoc System, BioRad).

Quantitative PCR (qPCR) analysis Total RNA was isolated from samples using RNA Stat-60 (TELTEST ‘‘B’’ Inc., Friendswood, TX, USA) according to the manufacturer’s instructions. cDNA was prepared from total RNA (100 ng) using the Improm-II reverse transcriptase system and 0.5 mg of oligo (dT)15 primer (Promega, Madison, WI, USA). The g-globin and glyceraldehyde-3-phosphate dehydrogenase (GAPD) mRNA levels were measured by qPCR (iCycler iQ, Bio-Rad) using Sybergreen iQ Supermix (BioRad) and 100 nM of each primer set. Standard curves were generated using a Topo7-based plasmid carrying either the g-globin (Topo7-gglobin) or GAPD (Topo7-GAPD) cDNA, which produced 219bp and 230-bp g-globin and GAPD PCR fragments respectively. Tenfold dilutions of Topo7-g-globin (2000–2.0 pg) and Topo7GAPD (200–0.2 pg) were used to generate standard curves. The qPCR studies were performed with previously published g-globin and GAPD primers [21] at an annealing temperature of 55
C.

Burst-forming unit-erythroid (BFU-E) analysis After obtaining informed consent in accordance with the University of South Alabama Institutional Review Board guidelines, peripheral blood samples were drawn from three sickle cell patients for each drug tested. Mononuclear cells were isolated by densitygradient centrifugation (Histopaque-1077, Sigma) and treated with NaB (1 mM), TSA (0.3 mM), hemin (50 mM), or HU (100 mM). Where indicated, studies with MYX (10 mM) pretreatment for 30 minutes followed by the addition of inducers were completed. The mononuclear cells were cultured in methylcellulose as previously published [22]. BFU-E colonies were counted on day 14 and lysed with sterile water, and then half of the sample was used to measure total hemoglobin by spectrometry. HbF levels were measured by alkaline denaturation as previously published [22] and calculated as a percent of total hemoglobin normalized by total protein.

Statistical analysis The data are reported as the mean 6 standard error of the mean (SEM) for at least three to four experiments. Statistical analysis of the raw data was performed by the two-tailed t-test. The Student’s t-test was used to measure differences in samples. A probability of less than 0.05 (p ! 0.05) was considered significant.

Results Cell toxicity produced by combination drug treatments Few studies have been performed to determine the role of ROS in erythroid differentiation and hemoglobin synthesis. The vitamin K analog MND is known to induce ROS in cardiac myocytes [23]. Therefore the ability of MND to generate H2O2 through a mitochondrial electron transportdependent mechanism [24] was tested. Viability studies were performed for MND (50–200 mM) based on studies in cardiac myocytes [23]. K562 cells were treated with MND for 4 hours and then cell viability was performed using trypan blue exclusion. Viability remained above 98% for 50 mM MND, but dropped to approximately 68% at the 200-mM concentration (data not shown). We confirmed mitochondrial-generated ROS using MYX, which inhibits complex III in the electron transport chain. Cell viability remained greater than 95% for 0.3 to 32 mM MYX (data not shown). The accurate measurement of H2O2 was accomplished with DCFH-DA, a nonpolar compound that diffuses across cell membranes and is hydrolyzed in the cytoplasm by esterases to the nonfluorescent metabolite DCFH. Subsequently, H2O2 selectively oxidizes DCFH to the highly fluorescent compound 20 ,70 -dichlorofluorescein (DCF) [25], which can be quantitated by fluorometry methods. To determine toxicity with combined drug studies K562 cells were treated for 4 hours with MND (200 mM) combined with DCFH-DA (20 mM) or MYX (10 mM) alone or all three compounds. As shown in Figure 1, MND produced a 32% decrease in cell viability compared to 98% viability for DCFH-DA or MYX alone. When MND was combined with DCFH-DA or MYX, cell viability was 69% and 67% respectively. Combining DCFH-DA and MYX treatment produced low toxicity (94% viability) compared to all three drugs where viability was 65% (Fig. 1, *

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Figure 1. Menadione produced toxicity in K562 cells. Viability was measured at 4 hrs. using trypan blue exclusion in K562 cells treated with 200 mM menadione (MND), 20 ,70 -dichlorodihydrofluorescein-diacetate (DCFH-DA), or 10 mM myxothiazole (MYX) alone or in the various combinations shown. Cell viability was calculated as the percent of live cells divided by the total for a minimum of 500 cells. A p value of less than 0.05 was considered statistically significant.

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striped bar). Additionally, cell viability studies were completed for K562 cells treated with the experimental HbF inducers. All experimental drugs produced less than 5% toxicity at 4 hours except anisomycin, for which 22% toxicity was observed (data not shown). We concluded that MND was the main contributor to cell death. The percentage of DCFD cells produced by MND alone was subtracted from percentages obtained when DCFH-DA was added to measure H2O2 levels in MND dose-response studies.

Menadione induced H2O2 formation in K562 cells Initial studies were completed to determine the accuracy of H2O2 measurements in K562 cells treated with the known ROS generator MND (25–200 mM). Fluorescence intensity was measured by FACS and fluorometry methods for comparison. The quantitative data for mean fluorescence obtained from FACS analysis showed a threefold (p ! 0.05) increase in H2O2 for 200 mM MND (data not shown). This concentration is similar to that required to generate ROS in other cell types [26]. For the same samples analyzed by fluorometry, a steady increase in relative fluorescence was observed with MND starting at the 50-mM concentration with a maximal fivefold increase (p ! 0.05) in relative fluorescence at 200 mM (Fig. 2A). The higher levels of relative fluorescence observed by fluorometry reflect differences in methods used to harvest cells. From these data we concluded that fluorometry gave better relative fluorescence measurements at lower H2O2 levels. Therefore we used fluorometry to measure H2O2 levels for the remainder of our analysis. Subsequent studies to determine the ability of NaB and TSA to stimulate ROS formation were completed (Fig. 2B and C). For both agents, a dose-dependent increase in H2O2 was produced. The optimal concentrations for NaB (2 mM) and TSA (0.5 mM) were determined. To complement our quantitative data, fluorescent photomicrographs were taken to illustrate DCFH activation and to calculate the percent ROSD cells. Endogenous H2O2 produced lowlevel auto-fluorescence in untreated K562 cells similar to that observed in other eukaryotic cells [27]. Figure 2D shows control studies with untreated and MND-treated cells in the absence of DCFH-DA probe. Due to apoptosis produced by MND, approximately 30% of cells were positive for low-level fluorescence. When DCFH was added with MND, the cell viability ranged from 70 to 74% and 80.5% of cells stained positive (Fig. 2D, MND (D)) with an overall 50.5% increase in fluorescence. For NaB, TSA, hemin, anisomycin, or HU, fluorescence-positive cells were not observed without DCFH-DA. After the addition of probe, fluorescence-positive cells were observed for NaB (29%), TSA (19%), anisomycin (32%), and hemin (60%) treatments. Cell fluorescence remained unchanged for HU treatments at all time points; thus HU served as a negative control (Fig. 2D, HU).

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HDAC inhibitors activated p38 MAPK signaling via ROS generation To confirm that the increased fluorescence observed after drug inductions was due to ROS generation, time course studies were completed in K562 cells with MND (200 mM), NaB (2 mM), TSA (0.5 mM), hemin (50 mM), anisomycin (200 ng/ mL), or HU (100 mM) treatment for 4, 6, 12, and 24 hours. Drug treatments were performed in the absence or presence of 10 mM MYX for 30 minutes. At 4 hours MND, NaB, and hemin produced a 3.0-fold, 2.0-fold, and 2.8-fold increase in relative fluorescence which was inhibited by MYX 50%, 40%, and 65% respectively (Fig. 3A). The p38 MAPK activator anisomycin generated H2O2 1.8-fold that was also inhibited by MYX. By contrast, TSA and HU did not stimulate H2O2 to significant levels above steady state at 4 hours. By 6 hours MND generated higher ROS levels whereas NaB and hemin maintained 2.0- to 2.5-fold increased relative fluorescence (Fig. 3B). Pretreatment with MYX inhibited MND-, NaB-, and hemin-mediated H2O2 production by 48%, 50%, and 56% respectively. In addition, TSA stimulated ROS 1.9-fold that was blocked 50% by MYX while the influence of anisomycin had dissipated. By 12 to 24 hours MND generated H2O2 up to 4.9-fold and MYX blocked the response consistently (Fig. 3C and D). The increase in H2O2 produced by NaB and TSA was maintained at 12 and 24 hours and MYX blocked DCF accumulation by 25 to 50%. Hydroxyurea, ethanol, and dimethylsulfoxide did not stimulate H2O2 at any time point (Fig. 3, data not shown). These data confirm the ability of MND, NaB, TSA, and hemin to generate H2O2. It has been previously demonstrated that p-p38 signaling is required for g-globin induction in K562 cells by HDAC inhibitors [4,16]. Furthermore, in several cell culture systems ROS are known to act as upstream activators of p38 MAPK signaling [17,18]. Thus we speculated that HbF inducers might enhance g-globin expression through a ROS-p38 MAPK-dependent mechanism. To explore this possibility we determined the effects of blocking ROS formation on drug-mediated p38 MAPK phosphorylation and g-gene induction in K562 cells and primary erythroid progenitors. Phosphorylated and total p38 levels were measured by Western blot at 12 hours since MYX blocked maximum ROS formation by the HDAC inhibitors at this time point (Fig. 3C). MYX and SB203580 inhibited p-p38 induction by MND; however, pretreatment with the extracellular signal-regulated kinase inhibitor PD98059 had no effect (Fig. 4A). Exogenous H2O2 (100 mM) has been reported to increase p-p38, which supports its role as an upstream activator. We observed a concentration-dependent increase (25–100 mM) in p38 MAPK phosphorylation by H2O2 (Fig. 4B), which was inhibited by SB203580 treatment (data not shown). Subsequent studies with HbF inducers showed increased p-p38 levels after NaB, TSA, and HU, in contrast to hemin, where p38 MAPK was not activated (Fig. 4C). Moreover, MYX pretreatment blocked p38

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Figure 2. Menadione generated ROS at high levels. K562 cells were treated with MND (25 to 200 mM) and 20 mM DCFH-DA for 4 hours, after which mean fluorescence was measured by fluorometry per a standard protocol (see methods section). (A): Quantitative data for studies performed with MND-treated K562 cells at the concentrations shown. Cell lysates (100 mL) were analyzed by fluorometry to obtain DCF relative fluorescence. (B): Quantitative data for studies performed with NaB-treated K562 cells at the concentrations shown. (C): Quantitative data for studies performed with TSA-treated K562 cells at the concentrations shown. (D): K562 cells (UT) were treated with the different agents including MND (200 mM), NaB (2 mM), TSA (0.5 mM), hemin (HEM, 50 mM), anisomycin (ANI; 200 ng/mL), and hydroxyurea (HU; 100 mM). Cells were prepared in sheath fluid for fluorescence microscopy. Shown are the fluorescent and corresponding phase photomicrographs of the same field.

activation by the HDAC inhibitors; however, p38 activation by HU was not altered. From these studies we concluded that the mechanism for p38 MAPK activation by HDAC inhibitors involves ROS generation. ROS generated by drug inducers is required for g-globin activation Subsequent studies were performed to answer the question whether g-globin induction by NaB and TSA involves

signaling through a ROS-p38 MAPK cell signaling mechanism. Additional studies were completed to determine the effects of SB203580 and MYX on downstream g-globin gene induction. It is known that ROS act as signaling molecules in cells via p38 MAPK signaling [17,18]. Experiments were performed in K562 cells and primary erythroid progenitors to determine the ability of MYX to block g-globin induction by HbF inducers. The g-globin to GAPD mRNA ratio was

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*p<0.05 Figure 3. Butyrate and trichostatin A mediate the generation of H2O2. K562 cells were treated with the different drugs shown for 4 hrs. at the same concentrations used in Figure 2D. For each time point, studies were performed in the absence (2) or presence (D) of MYX (10 mM) pretreatment for 30 min. The level of 20 ,70 -dichlorodihydrofluorescein (DCF) relative fluorescence obtained from untreated (UT) K562 cells was normalized to one and the fold increase in treated samples calculated accordingly. (A): Shown is the relative fluorescence obtained for the different drug treatments. The bars with a single star (*) are significantly different from untreated K562 cells (white bars). Differences between drug-treated cells in the absence or presence of MYX is represented by a star (*) and bracket ( ) combined. (B): Results are shown for the same experimental conditions except cells were grown for 6 hrs. in culture. (C): Data for a 12-hr. treatment period. (D): DCF relative fluorescence obtained after 24 hrs. of treatment with the various agents.

increased 1.9-fold by NaB and 1.8-fold by TSA, which was inhibited 50 to 55% by MYX and 63 to 67% by SB203580 (Fig. 5A). HU induced g-globin twofold, which was not attenuated by MYX; however, SB203580 treatment repressed g-globin expression by 75%, demonstrating a role for p38 MAPK signaling in this mechanism. At 24 and 48 hours MYX and SB203580 blocked g-globin activation by NaB and TSA as well (Fig. 5B and C). Hemin induced a significant 2.9-fold increase in g-gene mRNA, which was blocked 90% by MYX at 24 hours but was unaffected by SB203580. HU induced g-globin in a p38 MAPK-dependent manner but pretreatment with MYX had no effect on g-gene induction by this agent. These studies confirm g-globin induction by a ROS-p38 MAPK-dependent mechanism for NaB and TSA and ROS-p38 MAPK independent mechanism for hemin. MYX treatment inhibited fetal hemoglobin induction in human erythroid progenitors K562 cells have proven to be an invaluable paradigm for studying globin gene regulation; however, definitive evidence that ROS plays a role in g-gene induction was obtained from studies in human progenitors. As such, mononuclear cells were isolated from the peripheral blood of 3 sickle cell patients for each experimental condition.

The erythroid progenitors were grown in methylcellulose cultures as previously published [22]. The ability of MYX to block HbF induction by NaB (1 mM), TSA (0.5 mM), hemin (50 mM), and HU (100 mM) was analyzed. For experiments with NaB and TSA the drug concentrations were decreased to 1 mM and 0.3 mM respectively due to excessive toxicity and BFU-E colony death at higher concentrations. HbF levels were measured in day-14 BFU-E colonies grown in the absence or presence of 10 mM MYX (Table 1). In untreated cells the HbF level was decreased 6.6% by MYX compared to a 21% (p ! 0.05) decrease in NaB-treated and 36.5% (p ! 0.05) in hemin-treated cells. The ability of MYX to inhibit HbF induced by hemin is consistent with our findings in K562 cells. By contrast, HbF induced by TSA was not significantly inhibited by MYX due to the lower 0.3-mM TSA concentration required to decrease BFU-E toxicity. However, DCF fluorescence was not increased in K562 cells at this concentration (Fig. 2C). Due to unacceptable toxicity we were unable to analyze the 0.5-mM concentration. HU induction was not significantly affected by MYX pretreatment. We previously demonstrated inhibition of HbF by SB203580 in human erythroid progenitors [4]. Collectively these results confirmed a role for mitochondrial-generated ROS in HbF induction by NaB and hemin in human erythroid progenitors.

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Figure 4. H2O2 mediated p38 MAPK phosphorylation. (A): Western blot analysis was completed for K562 cells treated with 200 mM MND in the absence (2) and presence (D) of 10 mM MYX for 30 min. to inhibit ROS generation or 10 mM SB203580 (SB) and 100 mM PD98059 (PD) for 1 hr. to inhibit p38 MAPK and extracellular signal-related kinase signaling respectively (see methods for details). A representative gel is shown for the different treatment conditions. Note the absence of p-p38 after MYX and SB203580 treatments. (B): A representative gel is shown for the effect of increasing amounts of exogenous H2O2 on p-p38 MAPK levels in K562 cells. (C): The p-p38 levels were measured after drug treatments in the absence (2) and presence (D) of MYX. The same membrane was stripped and probed with (total) t-p38 antibody as an internal control for sample loading errors.

Discussion MAPK cell signaling pathways are activated by mitogenic stimuli, environmental stress, and inflammatory agents. Many extracellular ligands generate and/or require ROS as second messengers to transmit intracellular signals [28,29]. The stress-activated protein kinase p38 is phosphorylated by ROS generated in response to angiotension II [30] and acute pancreatitis [31]. In addition, hypoxia stimulates an increase in mitochondrial-generated ROS in cardiomyocytes [32] and other cell types [33,34]. Whether ROS mediates p38 MAPK phosphorylation during hypoxia is not known [3,35,36]. The vitamin K analog MND produces ROS through redox cycling to generate superoxide, which is later converted to H2O2 [37]. High ROS levels induced by MND produce oxidant stress and depletion of the antioxidant-reduced glutathione [38,39]. This would be especially detrimental to sickle erythrocytes since most have decreased levels of reduced glutathione at steady state [40]. Excessive ROS cause multiple toxic effects [41,42] through p38 MAPK-mediated apoptosis [43]. Detailed studies were completed to determine the optimal concentration of MND required for increasing ROS levels with minimal toxicity. At 200 mM, MND increased ROS while producing 33% toxicity in K562 cells. The contribution of apoptotic cells to fluorescence measurements was monitored by FACS analysis to quantitate live and dead cells and fluorescence produced in the absence of DCFH-DA by microscopy. After many years of intensive research, the mechanism for drug-mediated HbF induction remains elusive. The studies presented herein were performed to determine whether HDAC inhibitors stimulate ROS formation as a mechanism for p38 MAPK signaling and g-globin induc-

tion. Studies were completed with MYX to inhibit the formation of superoxide at complex III in mitochondria [44]. In K562 cells MYX inhibited both p-p38 signaling and ggene induction, demonstrating a role for ROS in the mechanisms for HbF induction via p38 MAPK signaling. Subsequent studies were performed to determine whether the reactive oxygen specie H2O2 was capable of activating p38 MAPK. Superoxide anions are degraded primarily by superoxide dismutase to H2O2, which can be released in the cytosol and act as a second messenger molecule. In our studies, H2O2 produced a dose-dependent increase in p-p38 in K562 cells that was inhibited by MYX, supporting an important link between ROS and p38 signaling. To define the role of ROS-mediated p38 activation in g-globin induction, a system to measure intracellular H2O2 levels was established to monitor the relative fluorescence produced by H2O2-mediated DCFH activation [25,26]. To confirm that fluorescence was due to ROS, MYX was used to attenuate the formation of superoxide in mitochondria [45]. Increased H2O2 levels were generated by MND, NaB, TSA, and hemin; however, HU did not generate ROS above baseline at any time point tested. ROS [46], such as NO, superoxide, and hydroxyl radicals, respond as second messengers to activate multiple cell signaling pathways including MAPKs and cGMP [47,48]. Nitric oxide also alters the ability of p38 MAPK to activate immediate early genes [51]. Browning et al. [52] demonstrated MEK3 and p38 MAPK activation by NO in human neutrophils and 293T fibroblast, suggesting this mechanism of gene regulation is not confined to certain cell types. Previously published data suggest that HU acts as a NO donor [19] to stimulate cGMP signaling and HbF induction in erythroid progenitors [20,49]. Our data

C.-H. Hsiao et al./ Experimental Hematology 34 (2006) 264–273

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Figure 5. Fetal hemoglobin induction is mediated through ROS/p38 MAPK signaling. K562 cells were treated with the various inducers for 12–48 hrs. Total RNA was isolated and g-globin mRNA levels were measured by quantitative real-time PCR (qPCR). (See methods for details). (A): Shown is the ratio of gglobin to GAPD mRNA for 12 hrs. of drug treatment in the absence (2) or presence (D) of 10 mM MYX (M) or 10 mM SB203580 (SB) pretreatment for 30 min. and 1 hr. respectively. Statistically significant differences in g-globin mRNA levels for drug-treated cells (black and striped bars) compared to untreated cells (white bars) are shown by the (*). The (*) and bracket ( ) combined represent differences in values for HbF-induced cells and MYX or SB203580 pretreatment (white bars vs black or striped bars). Double stars (**) represent statistical differences between MYX- and SB203580-treated samples (HU, striped bar). (B): The mRNA data obtained for K562 cells treated for 24 hrs. with the various agents. (C): The mRNA data obtained for K562 cells treated for 48 hrs.

support a second mechanism for HbF induction by HU via p38 MAPK signaling. We previously demonstrated that zileuton, a structural analog of HU, induced HbF via cGMP signaling independent of NO molecules [20]. Similarly, hemin induced HbF through cGMP signaling [50], which is consistent with our Western blot data, where hemin induced g-globin independent of p38 MAPK signaling. The fact that hemin increased ROS formation but did not activate g-

globin expression via p38 signaling suggests that complex interactions occur between drug inducers and cell signaling pathways (Fig. 6). Our studies with MYX are not sufficiently specific for identifying the individual ROS; therefore, hemin might stimulate other ROS such as nitric oxide capable of activating g-globin expression via cGMP signaling. Additional studies are needed to elucidate this mechanism.

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Acknowledgments Butyrate

ROS

p38

Hemin

ROS

cGMP

NO

cGMP p38

Hydroxyurea

This work was supported by grant RO1 HL69234-02 from the National Heart, Lung, and Blood Institute to Dr. Betty Pace. A special thanks to Dr. Solomon Ofori-Acquah and Ms. Glorias Dixon for reviewing the manuscript and making helpful editing suggestions.

-globin

Figure 6. Mechanisms for g-globin gene activation via cell signaling pathways. Experimental data were presented for a ROS/p38 MAPK pathway signaling mechanism to achieve g-globin activation by NaB and TSA. By contrast, hemin increased ROS but did not activate g-globin through p38 MAPK signaling. This agent most likely activates g-globin via cyclic guanosine monophosphate (cGMP) signaling. Hydroxyurea can act as a nitric oxide (NO) donor to activate cGMP and p38 MAPK signaling to produce g-globin activation and fetal hemoglobin induction.

To confirm the ROS/p38/HbF connection, the level of p-p38 was determined in the absence or presence of MYX by Western blot analysis. Interestingly, MYX blocks p38 MAPK and g-globin activation by NaB and TSA, which supports p38 MAPK activation by ROS to achieve HbF induction. The data presented herein demonstrate the ability of HU to induce HbF via the p38 MAPK in addition to cGMP signaling [50]. Nitric oxide produced by HU most likely activates cGMP signaling. Insights into cell signaling mechanisms will provide new opportunities for therapeutic intervention with small molecules to augment g-gene expression. HbF effectively blocks hemoglobin S polymerization and ameliorates the clinical symptoms of sickle cell disease. Mapping the downstream molecules that interact in the g-promoter will provide specificity and increased efficacy to improve treatment outcomes. Studies in our laboratory and others [53,54] continue to identify the transcription factors that directly interact in the g-globin promoter for this purpose.

Table 1. The role of reactive oxygen species in fetal hemoglobin induction by HDAC inhibitors in burst-forming unit-erythroid colonies Fetal Hemoglobin Levels (%)1 Treatment

(2) MYX

(D) MYX

Untreated 20.9 6 2.30 19.5 Sodium Butyrate 209.5 6 18.4 165.3 Trichostatin A 128 6 19.1 134.8 Hemin 41.3 6 1.86 26.7 Hydroxyurea 264.4 6 42.2 269.1

6 6 6 6 6

3.22 6.4 6.6 3.5 62.17

% Change2 p values 6.6 21.0 4.6 36.5 1.8

0.486 0.017 0.633 0.020 0.912

MYX 5 myxothiazole. 1 The fetal hemoglobin levels for the inducers in the absence (2) or presence (D) of myxothiazole (MYX) are calculated as a percent of total hemoglobin. 2 The % Change was calculated as the difference in fetal hemoglobin levels for progenitors in the absence or presence of MYX divided by the level in the absence of (2) MYX.

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