lead-induced upregulation of the heme-regulated eukaryotic initiation factor 2α kinase is...

ta 1732 (2005) 15–22http://www.elsevier.com/locate/bba

Biochimica et Biophysica Ac

Lead-induced upregulation of the heme-regulated eukaryotic initiation factor2α kinase is compromised by hemin in human K562 cells

Angshuman Sarkar a,1, Abhijeet Kulkarni a, Samit Chattopadhyay b, Devraj Mogare b,Kiran K. Sharma a,2, Kamini Singh b, Jayanta K. Pal a,⁎

a Department of Biotechnology, University of Pune, Pune 411 007, Indiab National Centre for Cell Science, Ganeshkhind, Pune 411 007, India

Received 12 April 2005; received in revised form 10 December 2005; accepted 19 December 2005Available online 19 January 2006

Abstract

Expression and kinase activity of the heme-regulated-eIF-2α kinase or -inhibitor (HRI) are induced during cytoplasmic stresses leading toinhibition of protein synthesis. Using a reporter construct with HRI promoter, we have determined the promoter activity during heat-shock andlead toxicity in human K562 cells. These two conditions induced HRI promoter activity by 2- to 3-fold. Contrary to this, hemin, a suppressor ofHRI kinase activity, downregulated HRI promoter activity and stimulated hemoglobin synthesis. Interestingly, when hemin-treated cells weretransfected and exposed to lead, hemin compromised lead-effect substantially by downregulating HRI promoter activity, HRI transcription andHRI kinase activity. These results together suggest that heme signaling in relation to translation regulation is not only restricted to the cytoplasm(modulating HRI kinase activity) alone but it also spans to the nucleus modulating HRI expression. Hemin may thus be useful for alleviation ofstress-induced inhibition of protein synthesis.© 2005 Elsevier B.V. All rights reserved.

Keywords: Heme-regulated inhibitor; Promoter activity; Expression; Kinase activity; Heat shock; Lead toxicity

1. Introduction

Protein synthesis is regulated in response to environmentalstimuli by covalent modifications, primarily phosphorylation,of components of the translational machinery. Several studieshave suggested that phosphorylation of the subunit (α) ofeukaryotic initiation factor 2 (eIF-2α) plays a central role inregulating the overall rate of protein synthesis in eukaryotes[1]. The phosphorylation of eIF-2α is caused by a family ofeIF-2α kinases [2–7]. Among these, the role of the hemeregulated eIF-2α kinase (also called the heme regulatedinhibitor, HRI) in regulating initiation of protein synthesis inreticulocytes has been well established [4,8,9]. Upon activa-tion, HRI, a Ser/Thr protein kinase, undergoes autopho-

⁎ Corresponding author. Tel.: +91 20 25692248; fax: +91 20 25691821.Email addresses: [email protected], [email protected] (J.K. Pal).

1 Present address: Cold Spring Harbor Laboratory, New York 11724, USA.2 Present address: Department of Biochemistry and Biophysics, University of

Rochester, NY 14642, USA.

0167-4781/$ - see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.bbaexp.2005.12.003

sphorylation and subsequently phosphorylates the 38 kDa αsubunit of eIF-2 at Ser-51 residue [8]. Phosphorylated eIF-2[eIF-2α(P)] binds to eIF-2B, also called the guanine nucleotideexchange factor (GEF) or reversing factor (RF), and forms astable complex that sequesters eIF-2B. Due to unavailability ofeIF-2B, which is required for the exchange of GTP for GDPfor recycling of eIF-2, initiation of protein synthesis ceases[4,8,10]. This kinase gets activated due to heme deficiency ora variety of other conditions, such as heat shock, heavy metaltoxicity, low partial pressure of oxygen, degraded polypep-tides, reactive oxygen species, treatment with N-ethylmalei-mide (NEM) and oxidized glutathione (GSSG) [8–12].

HRI has been purified and extensively characterized fromrabbit reticulocyte lysate. HRI is a dimer of 92 kDa polypeptideand it has a sedimentation co-efficient of 6.6S [8,10]. There aretwo heme binding domains in HRI: one at the N terminal regionand the other in the insertion domain [13].

Rabbit HRI cDNA of 2.7 kb was cloned in 1991 [14]. In vitrotranslation of mRNA transcribed from HRI cDNAyielded a 90-kDa polypeptide which had eIF-2α kinase activity and specific

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reactivity against a monoclonal antibody raised against purifiedrabbit HRI [15]. Although HRI was thought to be erythroid-specific [15,16], presence of HRI transcript was shown in anumber of non-erythroid tissues in rat using rat brain HRI cDNAas the probe [17]. Subsequently, HRI cDNAs have been clonedfrom mouse [18] and human [19]. Further, cDNAs of eIF-2αkinases distantly-related or unrelated to HRI have also beencloned from a number of other organisms, namely, Schizosac-charomyces pombe [20], Plasmodium falciparum [21], Dro-sophila [22], Bombyx mori [23]. Attempts have also been madetowards understanding the regulation of HRI gene expression inmouse [24]. To this effect, molecular cloning and characteriza-tion of the promoter of mouse HRI (mHRI promoter) showedthat it lacks a TATA element, but contains an initiator core (Inr)element and three SP1 consensus-binding motifs. The corepromoter element appeared to be 159 nucleotides long.

Among various conditions and reagents that activate HRI andinhibit protein synthesis, heavy metal exposure and heat shockare chosen here for our studies. Earlier, we have shown that HRIwhich is almost undetectable in reticulocyte lysates of normalrabbits increases up to 24-fold during drug- and also lead-induced anemia [12,25]. Interestingly, under these conditions,the level of HRI transcript increased only 2- to 3-fold. Further,we have shown that there is a 2- to 3-fold increase in HRIexpression as well as its kinase activity in K562 and HeLa cellsduring exposure to lead acetate and heat shock. This increase inHRI expression and its activity was concurrent with a significantdecrease in cell proliferation and cell viability of K562 cells [26].These results together suggested that various cytoplasmicstresses cause increased activity as well as overexpression ofHRI (both at the levels of transcription and translation). In thisreport, we present data on the regulation of promoter activity andexpression of HRI during two cytoplasmic stresses, namely,heat-shock and lead toxicity, and during hemin exposure.

2. Materials and methods

2.1. Materials

All the cell culture media and reagents used in the present study, namely,DMEM, HBSS, FBS, Antibiotic-antimycotic solution (100×), were purchasedfrom either Sigma Chemical Co. (USA) or Life Technologies (Gibco BRL,USA). Lipofectamine 2000 was purchased from Invitrogen (USA). First strandcDNA synthesis kit for RT-PCR was purchased from Roche MolecularBiochemicals (Germany). Anti-phospho-eIF2α (Ser51) and -eIF2α polyclonalantibodies were purchased from Cell Signaling Technology (USA). BMChemiluminescence Western Blotting kit (Mouse/Rabbit) was purchased fromRoche Molecular Biochemicals (Germany). All the restriction enzymes andDNA modifying enzymes were purchased from either New England Biolabs(USA) or Roche Molecular Biochemicals (Germany). Custom made primerswere purchased from Gibco BRL (USA). Vectors (pGEMT-Easy, pSP73 andpGL3-enhancer, pRL-CMV) were purchased from Promega (USA). Humanerythroid K562 and human embryonic kidney 293 cell lines were obtained fromthe cell repository at the National Centre for Cell Science, Pune, India.

2.2. In vitro cell culture and exposure of cells to lead acetate andheat-shock

Human K562 and 293 cells were maintained as continuous culture inDMEM containing 10% FBS at 37 °C and 5% CO2 with antibiotic–antimycotic (1×) solution. These cells were used for various experiments.

K562 cells were exposed to 100 μg/ml of lead acetate for 8 h. For heat-shock,cells cultured at 37 °C were transiently exposed to 42.5 °C for 1 h.

2.3. Cloning of human HRI promoter in pGL3-enhancer vector

Human HRI promoter fragment was cloned in a reporter vector (pGL3-enhancer) plasmid in which the SV40 enhancer is located downstream of fireflyluciferase gene. From the genomic DNA samples of human 293 cells and mouse,HRI promoter fragments (620 bp) were amplified by PCR using HRI promoterspecific-primers (5′-CAA CAG ACA CTC ATATGA AG-3′ and 5′-GAA GTAACA CAA GAG CCA AC-3′). These primers were designed from mouse HRIpromoter data base (Accession No. AF 380347). PCR condition was as follows:95 °C for 2 min, 94 °C for 1 min, 52 °C for 2 min and 72 °C for 1 min for 35cycles. The amplified human promoter fragment was cloned in pGL3-Enhancervector. Positive clones named as pGL3-enhancer (HRI pro) were selected out bydouble digestion using two restriction enzymes, namely, KpnI and HindIII. Oneof these clones was maintained and the plasmid was used for further transfectionexperiments.

2.4. RNA extraction, RT-PCR and quantitative real time PCR analysis

Total cellular RNA was extracted from the cells using TRIZOL reagent asper the manufacturer's (Gibco BRL, USA) protocol. The total RNA wasquantified by spectrophotometric method. After the extraction of total RNAfrom the cells, first strand cDNAwas synthesized from RNA samples using thecDNA synthesis kit for RT-PCR (AMV) from Roche Molecular Biochemicals(Germany), as per specifications provided in the kit. PCR amplification of HRIand β-actin cDNA was carried out using specific primers as described earlier[26]. Further, for quantitative analysis of HRI expression, Real Time PCR wasperformed by icycler iQ thermal cycler system (Bio-rad) using double strandedDNA specific fluorophore SYBR Green I. In a 25 μl PCR reaction 1:25 and1:100 times diluted cDNAwas amplified using 1× iQTN SYBR Green Supermix(Bio-rad) containing 0.4 mM dNTP mix, 1.5 mM MgCl2, 50 pmol of forwardand reverse primer mix, SYBR Green I, 0.5 U iTaq polymerase. Annealingtemperature of 42 °C and 58 °C was used for HRI and β-actin, respectively for40 cycles. Confirmation of single product was determined by 8% PAGE as wellas by melt curve analysis. Quantification of HRI mRNA, described as foldincrease over control was done by the comparative Ct method, where target(HRI) was normalized to the endogenous reference (β-actin). The threshold Ctvalue is the cycle number from logarithmic phase of the PCR curve where anincrease in fluorescence was detected above background. TheΔCt is determinedby subtracting the Ct of β-actin from that of HRI. The fold increase overcontrol=2−ΔΔCt where ΔΔCt=ΔCt control−ΔCt treatment.

2.5. Transfection and luciferase assay

The plasmid used for transfection experiment was prepared by Qiagen Midi-prep kit. Transfection was carried out using Lipofectamine-2000 in DMEMwithout FBS. Cells were cotransfected with pGL3-enhancer plasmid (Promega)containing HRI promoter and pRL-CMV vector plasmid (Promega) whichcontains a cytomegalovirus promoter placed upstream of Renilla luciferasegene. After 5 h of transfection, 10% FBS (final conc.) was added to the medium.After 24 h, half of the medium was replaced by the fresh medium. Cells wereharvested 48 h after transfection and were used for protein extraction using lysisbuffer (provided in the kit) by repeated freeze thawing method. Luciferaseassays were performed using dual Luciferase Assay Reporter system (Promega)as per standard protocol. The activity of Renilla luciferase in protein extracts wasused to normalize any variation in transfection efficiency. The promoter activityin pGL3-enhancer vector containing HRI promoter was calculated as Firefly toRenilla luciferase activity ratio. The assay was done using Fluoroskan AscentLuminometer (Labsystem). Experiments were repeated thrice and results werepresented with error bars.

2.6. Exposure of human K562 cells to hemin

Cells were exposed to hemin chloride as described earlier by Dean et al. [27].In brief, cells were cultured in vitro in DMEM containing 10% FBS and either10, 25 or 50 μM hemin. Every third day, the medium was replaced with the fresh

Fig. 1. HRI promoter activity (Luciferase reporter assay). (A) Differential HRIpromoter activity in erythroid (human K562) and non-erythroid (humanembryonic kidney 293) cells. Relative luciferase activity was measured inRLU after 48 h of transfection of 293 (1) and K562 (2) cells. (B) HRI promoteractivity in K562 cells during heat-shock and lead-exposure. Relative luciferaseactivity was measured in RLU after 48 h of transfection followed by, 1 h oftransient heat-shock at 42.5 °C (2) or 8 h exposure to 100 μg/ml of lead acetate(3); 1, control.

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medium of the same composition. Cells were cultured for two to three passagesin the presence of hemin and visual observations were made for monitoring theaccumulation of haemoglobin.

2.7. Determination of hemin uptake by microwave-induced InductivelyCoupled Plasma-Atomic Emission Spectroscopy (ICP-AES)

Hemin uptake in presence of exogenously added hemin was determined byestimating total iron inside cell as compared to that in control cells by ICP-AESaccording to methods described in [28,29]. In brief, equal number of K562 cells(1.6×107) from the control, and various experimental samples were taken into 5ml conical flasks. Four hundred micro liters of concentrated nitric acid wasadded to each flask and kept at room temperature for overnight. After theaddition of 0.2 ml (30%), H2O2 in each flask, digestion was performed in amicrowave oven (Kenstar, 800W). The digested samples were diluted up to 2 mlwith double distilled water, and further dilution (up to 25 ml) was made in 0.5%nitric acid. An ICP-AES Spectrophotometer (ICPS-7500, Shimadzu) was usedfor the determination of total iron quantity within the cells. The spectropho-tometer was calibrated by using iron standards (0.01 to 0.15 ppm) obtained fromFluka. This experiment was repeated three times.

2.8. SDS PAGE and Western blot analysis

Samples containing equal quantity of proteins were denatured in Laemmlisample buffer for 3–5 min at 100 °C and analysed by SDS PAGE [30].Electrophoresis was carried out at a constant current of 25 mA at roomtemperature. Following SDS PAGE, proteins were electrophoretically trans-ferred to a nitrocellulose membrane [31]. Blots were then processed forimmuno-reaction using anti-Phospho-eIF2α (Ser51) and -eIF2α polyclonalantibodies. In brief, blots were saturated with 2% Blocking reagent (provided inthe kit) for 1 h, and incubated overnight with primary antibody in Tris-bufferedsaline containing 0.1% (v/v) Tween-20 (TBST, pH 7.5) and then with anti-mouse/rabbit IgG-HRP-conjugated secondary antibody for 1 h at roomtemperature. Following each antibody incubation, blots were washed thrice (5min each) in TBST. Blots were developed using the chemiluminescencedetection kit (Roche, Germany). The results were analysed using Bio-rad geldocumentation system (USA).

3. Results

3.1. Differential activity of HRI promoter in erythroidand non-erythroid cells

Two types of cells, namely, erythroid (human K562) andnon-erythroid (human embryonic kidney 293 cells) wereharvested at 48 h after the transfection and total protein wasextracted. Equal quantity of protein was used for measuring therelative luciferase activity (RLU). In K562 cells, HRI-promoterinduced luciferase activity was about 8-fold more (Fig. 1A, lane2) than that in 293 cells (Fig. 1A, lane 1) indicating thereby,erythroid cell-specific regulation of HRI promoter activity.These experiments were performed three times with two batchesin each set. This observation on the promoter activity thusreinforces earlier reports on erythroid-specificity of HRIexpression [9,15,16].

3.2. Heat-shock and lead exposure induce HRI promoteractivity in K562 cells

After 48 h of transfection, one batch of K562 cells wasexposed to transient heat-shock for 1 h at 42.5 °C, and the otherbatch was used as the control. Immediately after the heat-shock,

cells were harvested and protein extracts were prepared fromheat-shocked as well as control (without exposing to heat-shock) cells by repeated freeze thawing method. On exposingthe cells to heat-shock, there was a 3-fold increase in HRIpromoter activity compared to that in the control (Fig. 1B, lane2 vs. lane 1).

We have reported earlier [26] that lead acetate (100 μg/ml)decreases cell viability of K562 cells within 24 h of treatmentand induces HRI expression as well as its kinase activity. It wasalso observed that 8 h exposure was optimum for enhanced HRIexpression [26]. Therefore, after 40 h of transfection, one batchof K562 cells was exposed to 100 μg/ml lead acetate for 8 h.From a set of three individual experiments, it was observed thatupon exposure to lead acetate, there was almost a 3-foldincrease in HRI promoter activity (Fig. 1B, lane 3) as comparedto that in control (lane 1), as has also been observed during heat-shock (Fig. 1B, lane 2).

3.3. Hemin downregulates HRI promoter activity in K562 cells

Hemin is a negative regulator of HRI kinase activity.However, nothing is known with respect to its role in regulationof HRI expression. Therefore, it was of interest to determine theregulation of HRI promoter activity in the presence of hemin.K562 cells were exposed to various concentrations of hemin,namely, 10, 25 and 50 μM hemin for two consecutive passages(total period of 8 days). The cells were washed twice in HBSS.Visual observation of the cell pellets (Fig. 2A) from all the threedifferent concentrations of hemin-treated cells showed that thecells produced haemoglobin in a dose-dependent manner. The


hemin-treated cells appeared red to deep red as the concentra-tion of hemin was increased, indicating a dose-dependentsynthesis of hemoglobin. Untreated control cells did not showany change in colour. To determine HRI promoter activity inthese cells, after culturing them in presence of 10, 25 and 50 μMhemin, for 8 days, they were washed once in HBSS and wereused for transfection. During the transfection, no hemin wasadded in the culture medium. After 24 h of transfection, heminwas added in the medium to get 10 μM, 25 μM and 50 μM finalconcentration. After 48 h of transfection, cells were harvestedand total protein was extracted from each set and relativeluciferase activity was measured. It was interesting to observethat hemin downregulated HRI promoter activity in a dose-dependent manner (Fig. 2B). This experiment was performedthree times in a batch of three sets each time.

3.4. Hemin reduces lead-induced upregulation of HRIpromoter activity

Two batches of K562 cells, one grown in presence of 25 μMhemin continuously for 8 days with one passaging, and the othercultured similarly without hemin (control) were used fortransfection. After 24 h of transfection, the batch grown inpresence of 25 μM hemin was supplemented with 25 μMhemin. Forty hours after transfection, cell samples (+hemin andcontrol) were exposed to 100 μg/ml lead acetate for 8 h. After atotal period of 48 h from the time of transfection, cells wereharvested and relative luciferase activity was measured. Inpresence of 25 μM hemin alone, as expected, HRI promoteractivity was downregulated by almost 2-fold (Fig. 3A, lane 2 vs.

Fig. 2. Hemin-induced downregulation of HRI promoter activity and inductionof hemoglobin synthesis in K562 cells. (A) Photograph of K562 cell pelletshowing hemoglobin synthesis when treated with various concentration ofhemin. (B) HRI promoter activity (Luciferase assay). Human K562 cells weretreated with different concentration of hemin for continuous two passages (8days). After 8 days, cells were harvested and washed in HBSS and cell pelletswere observed under microscope and photographed (A). The cell pellets wereresuspended in HBSS, HRI promoter activity was monitored 48 h aftertransfection (B).

lane 1). While, in the cells treated with 100 μg/ml lead acetate,HRI promoter activity was increased to almost 2.5-fold ascompared to that in control (Fig. 3A, lane 3 vs. lane 1).Interestingly, there was a significant (60%) reduction of HRIpromoter activity in K562 cells treated with 100 μg/ml lead-acetate, that are preincubated with 25 μM hemin as compared tothat in lead exposed cells alone (Fig. 3A, lane 4 vs. lane 3).These results, therefore suggest that hemin interferes with thelead acetate-induced upregulation of HRI promoter activity in asignificant manner.

3.5. Effect of hemin on HRI expression in K562 cells duringlead-exposure

Based on the results of promoter activity, we designedexperiments to determine regulation of HRI expression duringlead treatment in presence and absence of hemin. Among twobatches of K562 cells, one batch was treated with 25 μM heminfor 8 days, and the other batch was used as the control. Cells ofboth the batches were exposed to 100 μg/ml of lead acetate for8 h. HRI expression was determined by RT-PCR analysis.Results of RT-PCR analysis indicated a 2-fold increase (asdetermined by densitometric analysis) in HRI expression in leadacetate exposed cells as compared to that in control (Fig. 3B and3C, lanes 1, 2). Interestingly, increase in HRI expression is onlymarginal in the cells which were preincubated with 25 μMhemin and then exposed to 100 μg/ml lead acetate for 8 h (Fig.3B and C, lane 3).

To further support the RT-PCR analysis data and to analyseHRI expression in more quantitative manner, quantitative RealTime PCR analysis was done which showed 4.5 fold upregula-tion of HRI upon lead acetate treatment (using 1:25 dilution ofthe template). Whereas in hemin-preincubated cells whenexposed to lead acetate, upregulation of HRI expression wasfound to be compromised, and it was only 3.4 fold (Fig. 3D).

To make the PCR reaction more sensitive and to avoidpossible errors, which can occur due to template saturation,higher dilution of template DNA (1:100) was used. In suchexperiment, while the lead acetate exposure induced 6 fold HRIexpression compared to the control, hemin-preincubationcompromised it drastically, and HRI expression was only 1.3fold. Thus, our results indicate that hemin plays a protective roleduring heavy metal toxicity (lead exposure) by downregulatingthe expression of HRI.

3.6. Lead-exposure causes enhanced hemin uptake by K562cells

In order to determine if hemin uptake is modulated duringlead-exposure and thereby resulting differential regulation ofHRI expression, the total iron content of K562 cells exposed todifferent hemin concentrations and to lead acetate wasdetermined. K562 cell samples treated with (a) 0, 25, 50 μMhemin for 8 days, (b) treated with 25 μM hemin for 8 daysfollowed by 100 μg/ml lead acetate exposure for 8 h, were usedfor estimation of iron inside the cells as follows. Total iron in 25μM hemin (hydrolysed) in DMEM was also estimated. The

Fig. 3. Hemin reduces lead-induced upregulation of HRI promoter activity (A) and HRI expression (B–D). (A) Relative luciferase activity (RLU) of K562 cells grownunder various conditions was measured after 48 h of transfection. Bar 1, control cells (untreated); 2, cells preincubated in 25 μM hemin (for 8 days); 3, K562 cells(-hemin) exposed to 100 μg/ml lead-acetate for 8 h; 4, K562 cells preincubated in 25 μM hemin (for 8 days) and subsequently exposed to 100 μg/ml lead acetatefor 8 h. (B) RT-PCR was carried out using 1 μg of total RNA. PCR product was analysed on a 1.2% agarose gel. Lane 1, control; lane 2, lead-exposed; lane 3,lead-exposed after preincubation with 25 μM hemin. (C) Quantification profile of HRI amplificate (230 bp) in gel (B). (D) Real Time PCR profile indicating foldincrease in HRI expression over control, using two dilutions (1:25 and 1:100) of the template cDNA. Three samples are control (open bar), lead acetate exposed(striated bar) and lead acetate exposed cells preincubated with 25 μM hemin (dark bar).


samples were subjected to microwave digestion and analysed byInductively Coupled Plasma-Atomic Emission Spectroscopy(ICP-AES) as detailed in the materials and methods section.These experiments were repeated three times and the results arepresented with standard deviations (Fig. 4). The total heminuptake by the experimental cells was calculated by subtractingthe iron content of the control cells (0 μM hemin, Fig. 4, sample

Fig. 4. Increased uptake of hemin by K562 cells during lead-exposure (ICP-AESanalysis). Total hemin uptake of K562 cells grown in presence of hemin wasestimated by comparing the quantity of total iron inside cells compared to that inthe control by ICP-AES analysis. Sample 1, total iron in control cells (withouthemin-treatment); sample 2, total iron in hydrolysed 25 μM hemin; samples 3, 4and 5, total iron in K562 cells treated with 25 μM hemin, 50 μM hemin, and 25μM hemin followed by lead-exposure (100 μg/ml lead acetate), respectively.Asterisk indicates statistically significant increase in iron-uptake betweensamples 3 and 5 (Student's t test P=0.05).

1). Further, the total hemin uptake by the cells treated with 25μMhemin is quantified in relation to the total iron released from25 μM hemin (Fig. 4, sample 2), considering this value as100%. Hemin (25 μM) treated K562 cells had 73.75% heminuptake (Fig. 4, sample 3) and it increased correspondingly in 50μM hemin treated cells (Fig. 4, sample 4). Incidentally, 25 μMhemin treated cells exposed to 100 μg/ml of lead acetate had92% hemin uptake (Fig. 4, sample 5). Thus, hemin uptake wasfound to be enhanced by 18% during lead toxicity (Fig. 4,sample 5 vs. sample 3).

3.7. Regulation of eIF-2α kinase activity in K562 cells duringlead-exposure

We determined modulation of total eIF-2α kinase activityduring lead-exposure by measuring the eIF-2α phosphorylationby Western blot using a specific antibody that recognizesphosphorylated form of eIF-2α. As seen in Fig. 5, there wasabout 3-fold increase in eIF-2α phosphorylation during leadexposure compared to the control (Fig. 5A; lane 2 vs. lane 1).Interestingly, when the cells were cultured in presence of 25 μMhemin, prior to lead-exposure, there was a significant decreasein eIF-2α phosphorylation (Fig. 5A; lane 4 vs. lane 2). The totalamount of eIF-2α was determined by another antibody whichrecognizes total eIF-2α irrespective of any modification (Fig.5B). These experiments were carried out thrice and thequantification results are presented with standard deviations(Fig. 5C). These results thus indicate that the increased totaleIF-2α kinase activity during lead acetate exposure is hemin-

Fig. 5. Hemin suppresses increased phosphorylation of eIF-2α induced duringlead exposure. (A and B) Western blots of soluble extracts of cell samplesreacted with anti-eIF-2α (P) and anti-eIF-2α antibodies, respectively. (C)Quantification profile of (A and B) from three individual experiments. Samples(cell extracts) loaded in various lanes are, control (lanes 1, 5), lead-exposed(lanes 2, 6), 25 μM hemin-treated (lanes 3, 7) and lead-exposed cells pre-treatedwith 25 μM hemin (lanes 4, 8). In (C), the solid bar and open bar represents totaleIF-2α and phosphorylated eIF-2α [eIF-2α (P)], respectively.


sensitive and therefore is most likely contributed by the heme-regulated eIF-2α kinase (HRI).

4. Discussion

Earlier, we have reported that expression of HRI is modulatedpositively resulting negative regulation of global proteinsynthesis under cytoplasmic stresses, namely, heat-shock andlead toxicity [26]. Conversely, negative modulation has beenobserved in presence of hemin chloride. These observationswere novel and hence prompted us to undertake further studieson the mechanism of regulation of HRI expression and kinaseactivity under various conditions. In the present study, wedetermined HRI promoter activity through transfection experi-ments in human K562 cells using luciferase reporter assay. Wehave also determined expression of HRI and total cellular eIF-2αkinase activity during lead-exposure and hemin-treatment.

Earlier reports in the literature on the erythroid specificity ofHRI [9,15,16] are still debated. It was therefore of interest todetermine HRI promoter activity in erythroid as well as non-erythroid cells. In our study, we obtained a 8-fold higher HRIpromoter activity in K562 cells (erythroid) as compared to thatin 293 cells (non-erythroid). Although it is known that HRImRNA is 10-times more abundant in erythroid cells [17], it isnot absolutely erythroid-specific. HRI expression in variousother tissues and cells has been further shown by others [18,32–34]. Similar conclusion thus can be drawn about HRI promoteractivity ([24], and the present study). It may therefore bepostulated that the transcription factors that may regulate HRIpromoter activity are either overexpressed or more active inerythroid cells. Thus, HRI is not erythroid-specific but is more

abundant in erythroid cells. Although there are a few knownerythroid-specific transcription factors such as, GATA-1, NF-E2and Bcl-x(L), that play a major role in cell survival andproliferation and composition of the erythroid compartments[35], HRI promoter sequence seems to lack binding sites forthese transcription factors [36]. Thus, further investigations arerequired to search for the appropriate transcription factor(s) forHRI expression in erythroid cells.

Data obtained from the present study on regulation of HRIpromoter activity during heat-shock and heavy metal toxicity(lead-exposure) indicated a 3- and 2-fold increase in HRIpromoter activity, respectively, under these conditions. We haveearlier reported that HRI quantity (mRNA and protein) as wellas heme-regulated eIF-2α kinase activity increased duringvarious cytoplasmic stresses, namely, heat-shock, and heavymetal toxicity [12,26]. Similarly, other investigators alsodetermined HRI expression and kinase activity during othercytoplasmic stresses [36]. However, no data were available onthe regulation of HRI promoter activity during such stresses.Therefore, this is the first observation showing increased HRIpromoter activity during heat-shock as well as heavy metaltoxicity in K562 cells. Further, we have shown earlier [26] thatthese stress conditions as used in the present experiments didnot enhance the quantity of major hsps (hsp70 and hsp90), thecontribution of hsps in stabilizing luciferase enzyme and thusincreasing its enzyme activity is less likely. The HRI promoteractivity (analysed by luciferase assay) and its correlation withHRI expression during these stress conditions further supportthe above observations. These data taken together strengthenour earlier hypothesis that during heat-shock and heavy metalexposure of cells/organisms, protein synthesis is severelyaffected by a combined activation of HRI gene expressionand its kinase activity.

The effect of heme on HRI promoter activity appearedinteresting. Hemin downregulated HRI promoter activity in adose-dependent manner: 25 μM and 50 μM caused 2-fold and2.5-fold downregulation, respectively. It was already known thatheme is a positive regulator for β-globin gene expression via thetranscription factor Bach1 in erythroid cells [37]. Further, thereare reports that transcription of few genes like, cytochrome P-450 and CYPIIB1/B2 are negatively regulated by heme [38,39].Our results thus establish inclusion of HRI in the list of geneswhose expression is regulated by heme, in this case, negatively.

Our studies further revealed that hemin reduces lead acetateinduced upregulation of HRI promoter activity in a significantmanner. This also appeared to be true for HRI expression, asdetermined by RT-PCR and quantitative Real Time PCR, aswell as for HRI kinase activity, determined by eIF-2αphosphorylation. The latter can be suppressed significantly by25 μM hemin. Although in the present study, the cells werecultured for several days in presence of hemin to arrive at thecondition when cells undergo differentiation and synthesizeglobin [27]. Hemin does downregulate HRI expression even inshort-term exposure conditions (data not shown). This may betrue for promoter activity as well. Thus, it appears thatdownregulation of HRI expression by hemin precedes hemin-induced differentiation of K562 cells.


However, Lu et al. [36] showed that arsenite activation ofHRI cannot be suppressed by addition of hemin. Thesedifferences could be due to dissimilarity in the nature of theireffect on protein synthesis. Lead acetate interferes with theporphyrin synthesis of human erythroblastic progenitors in vitro[40], however, no other heavy metal, or arsenite is known tocause similar effects.

Heme plays a significant role in many homeostatic andadaptive reactions, including controlling levels of severalproteins that are characteristics of oxidative stress response[41]. In correlation with these data, our ICP-AES analysisrevealed that total quantity of iron concentration in the K562cells preincubated in 25 μM of hemin increased to almost 25%after treatment with 100 μg/ml of lead acetate. This perhapsindicates that under the condition of lead toxicity, K562 cellsincrease their hemin uptake to overcome the stress effects andthereby maintain global protein synthesis. Therefore, it is likelythat hemin could be used as a stress protective, chemotherapeuticreagent to relieve the overall stress effects. It is known that hemeis broken down by heme oxygenase toCO and biliverdinwith therelease of iron. Biliverdin is an anti-oxidant that protects cellsfrom oxidative damage. Further CO also has cyto-protectiveeffects, including the inhibition of HRI activation [33]. Thus, thepossibility that the protective effects of hemin observed by uscould be through these alternative routes could not be eliminated.However, further studies are required to confirm the role ofhemin as a chemotherapeutic agent in living organisms.

Thus, we showed for the first time that stresses, such as, heat-shock and lead toxicity regulate HRI promoter activity in asignificant manner. Further, our results also demonstrated thathemin plays a significant role in regulation of HRI promoteractivity under normal as well as stressed conditions. Results onthe effect of hemin on expression pattern of HRI, its regulationat the promoter level, and on HRI kinase activity suggest thathemin may be a stress protector and may positively regulateglobal protein synthesis during cytoplasmic stresses.

Acknowledgements

Wewould like to thank Prof. B. S. Madhava Rao for allowingus to use ICP-AES facility at the Department of Chemistry,University of Pune. Thanks are also due to Dr. Anita Kar, Schoolof Health Science, University of Pune, Dr. Ruchika Kaul-Ghanekar, and Ms. Dhanashri Godbole for various help duringthis investigation. Financial help in the form of research grantsfrom DBT [BT/PRO/HRD/15/07/97] and CSIR [37(1062)/00/EMR-II], New Delhi to JKP is duly acknowledged. AS wassupported by a Senior Research Fellowship from CSIR, NewDelhi. AK was supported by a Junior Research Fellowship fromBhaba Atomic Research Centre (BARC) under the University ofPune-BARC Collaborative Programme, and subsequently fromthe University Grants Commission, New Delhi.

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