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Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells
Life Sciences 78 (2006) 1217 – 1224 www.elsevier.com/locate/lifescie
Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells Bahire Kucukkaya 1, Devrim Oz Arslan, Beki Kan * Marmara University School of Medicine, Department of Biophysics, Tibbiye Caddesi No 49, Haydarpasa, 34668, Istanbul, Turkey Received 9 May 2005; accepted 22 June 2005
Abstract Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of Gas, Gai2 and Gaq and the absence of Gai1, Gao and Ga16 in K562 cells. We observed the short form of Gas to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of Gas and Gaq. On the other hand, the level of Gai2 was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells. D 2005 Elsevier Inc. All rights reserved. Keywords: Erythroid differentiation; Hemin; G proteins; ERK1/2 phosphorylation; K562
Introduction Heterotrimeric guanine nucleotide binding proteins (G proteins) which couple many cell surface receptors to effectors on the plasma membrane mediate a series of events which ultimately lead to diverse cellular responses. G proteins are composed of an a-subunit that binds to and hydrolyzes GTP, and a hg-subunit complex. To date, 20 different Ga, 6 Gh and 11 Gg subunits have been described. Ga subunits have been divided into four families (Gs, Gi/o, Gq/11, G12/13) based on the homology at the amino acid level. Their mass is between 39 and 52 kDa (Hepler and Gilman, 1992; Offermans and Simon, 1996). G proteins regulate critical processes such as cell growth, differentiation and development (Malbon, 1997). Studies in human pheochromocytoma PC12 cells
* Corresponding author. Tel./fax: +90 216 3480585. E-mail address: [email protected] (B. Kan). 1 Current address: Maltepe University School of Medicine, Istanbul, Turkey. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.06.041
which were treated with nerve growth factor (Strittmatter et al., 1990) and in the neuroblastoma glioma hybrid NG 10815 cell line, cultured with agents that elevate intracellular levels of cAMP (Mullaney and Milligan, 1989) suggested a role of Gao in development and differentiation of nervous tissue. Changes in the levels of G protein a-subunits such as Gas, Gao and Gai were associated with differentiation of 3T3L1 cells from fibroblasts into adipocytes. As these cells were induced to differentiate to adipocytes, a decline in the level of Gas (Wang et al., 1992), a decline (Gierschik et al., 1986) or an increase (Watkins et al., 1987) in the expression of Gai2 was observed. Constitutive activation of Gas by cholera toxin blocked and oligonucleotide(s) antisense to Gas accelerated the differentiation process (Wang et al., 1992). Interestingly, the modulating effect of Gas on differentiation was independent of adenylyl cyclase activation and cAMP levels (Wang and Malbon, 1996). A subsequent study demonstrated differential expression of Gaq/11 at three different stages of adipogenesis: in confluent preadipocytes, differentiated preadipocytes and mature adipocytes (Denis-Henriot et al.,
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1996). In F9 teratocarcinoma stem cells induced to differentiate with retinoic acid, the inhibitory G protein, Gai repressed (Galvin-Parton et al., 1990), whereas expression of Gas induced differentiation (Gao and Malbon, 1996). G proteins are also known to modulate differentiation of hematopoietic cells. Increase in expression of Gai2 and decrease in expression of Ga16, a G protein specifically expressed in hematopoietic cells, were shown in a promyelocytic cell line, HL-60, in the course of differentiation along the neutrophil pathway (Amatruda et al., 1991). When HL-60 cells were induced to differentiate to mature granulocyte-like cells with retinoic acid a decrease in the level of Gas was observed (Meissner et al., 1996). The content of Gai2 remained unchanged in a human erythroleukemia cell line, HEL, induced to differentiate to megakaryocytes (Ashby et al., 1991); however, the levels of Gai2 and Gai3 increased in a human megakaryoblastic leukemia cell line, MEG-01, induced to differentiate with TPA (Nagata et al., 1995). Terminal differentiation of an erythropoietin-sensitive murine erythroleukemia cell line, RED-1, was associated with a loss of Gai3 and an increase in the cytosolic form of Gai2 (Kesselring et al., 1994). In human myeloid progenitors and mature blood cells Gas, Gai2 and Gaq/11 proteins were expressed at high levels during every stage of granulomonocytic and erythroid differentiation, whereas Ga12 and Ga16 proteins were expressed in a lineage-specific manner in normal myeloid cells (Tenailleau et al., 1997). Taken together, these studies reveal that G protein alpha subunits can regulate cell differentiation in distinct ways, depending on the cell type or tissue under investigation as well as the type of inducer. The mitogen-activated protein kinase ERK1/2 pathway is essential in the control of growth, differentiation and survival in many cellular systems. The nature of the response to ERK activation in different cells depends on the agent used to induce cells to growth or differentiation and the duration of ERK activation (Pouyssegur and Lenormand, 2003). Several hematopoietic growth factors and cytokines such as erythropoietin (EPO) stem cell factor (SCF) and interleukin-3 (IL-3) by acting on receptor tyrosine kinases activate members of the ERK or MAPK family (Platanias, 2003). It is documented that Gq- and Gi-coupled receptors also activate the ERK1/2 pathway (Gutkind, 2000). A role for the ERK/MAPK pathway in erythroid differentiation has been proposed in a number of studies. Shelly et al. (1998) established that inhibition of the basal activity of the ERK/MAPK pathway enhanced the erythroid phenotype of K562 cells. Likewise, Kang et al. (1999) demonstrated that inactivation of ERK by MAPK inhibitors resulted in growth arrest and erythroid differentiation of K562 cells. Treatment of K562 cells with sodium butryrate resulted in a rapid but transient activation of ERK1/2 signaling in one study (Rivero and Adunyah, 1996) but caused inhibition of ERK and activation of p38 MAPK pathway in another (Witt et al., 2000). Recently, the involvement of ERK phosphorylation in both growth and hemin-induced erythroid differentiation of K562 cells has been shown (Woessmann and Mivechi, 2001). The signaling pathways mediating hemin-induced
differentiation of K562 cells have not been well characterized. In this study, we demonstrate alterations in the level of Gas, Gai2 and Gaq proteins and ERK1/2 phosphorylation during erythroid differentiation of K562 cells induced by hemin. Materials and methods Hemin, RPMI 1640 and penicillin/streptomycin solutions, alkaline phosphatase-conjugated goat anti-rabbit IgG and other electrophoresis reagents were purchased from Sigma (St Louis, MO, USA). Fetal calf serum (FCS) was from Harlan Sera-Lab (Leicestershire, UK). G protein antisera RM/1 (recognizing Cterminal sequence RMHLRQYELL of Gas), GC/2 (recognizing C-terminal peptide GCTLSAEERAALERS of Gao), and AS/7Transducin (recognizing C-terminal peptide KENLKDCGLF of Gai12) were purchased from Dupont NEN (Lachine, Quebec, Canada). Gaq antibody (internal 115 – 133) and antibody reacting with the C terminus (360 – 373) of the Ga16 subunit were obtained from Calbiochem (San Diego, CA, USA). Antisera against the Ghg (S217) and Ga16 (AS339) subunits were kindly provided by Dr. Alfred Gilman, University of Texas and Dr. Bernd Nu¨rnberg, Heinrich-Heine University, Dusseldorf, respectively. Phosphorylated ERK1/2 antibody was from Santa Cruz Biotechnology (San Diego, CA, USA). Anti-ERK1/2 antibody was from Promega (Madison, WI, USA). Affinity purified peroxidase-conjugated anti-rabbit IgG was purchased from Jackson ImmunoResearch Europe Ltd. (Cambridgeshire, UK). All other chemicals and enzymes were obtained from commercial sources. Cell preparations K562 and HL-60 cells obtained from ATCC (MD, USA) were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 Ag/ml streptomycin, 25 Ag/ml amphotericin B and 2 mM l-glutamine at 37 -C in humidified air containing 5% CO2. Peripheral blood mononuclear cells (PBMC) were obtained from the fresh heparinized blood of healthy human volunteers. AML cells were prepared from the fresh heparinized blood of patient diagnosed with acute myelocytic leukemia AML. PBM and AML cells were isolated by density gradient centrifugation over Ficoll-Hypaque. Induction of hemoglobin synthesis Cells seeded at 1 105 cells/ml were induced to differentiate with 20 AM hemin (day 0) and were harvested at days 1– 6 after induction. At this time, benzidine staining was carried out to assess the extent of differentiation. Measurement of erythroid differentiation of K562 cells by benzidine staining Erythroid differentiation was scored by staining the cells for hemoglobin synthesis with benzidine reagent. A benzidine dihydrochloride stock solution, 0.2% (w/v), was prepared in
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K562, AML and PBM cells were collected by low speed (400 g) centrifugation and washed two times with phosphatebuffered saline (PBS), pH 7.5. The resulting pellets were resuspended and lysed with 20 strokes in a hand Dounce homogenizer in a buffer containing 20 mM Hepes/KOH, pH 8.0, 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 10 Ag/ml leupeptin and 2 Ag/ml aprotinin. Cell debris was pelleted by centrifugation at 400 g for 10 min and the resulting supernatant was centrifuged at 12 000 g for 60 min. The pellets (crude membrane fractions) were then resuspended and washed two times in the same buffer. The crude membranes were stored at 70 -C. The protein content of the preparation was determined by the method of Lowry et al. (1951). Preparation of K562 cell lysates K562 cells induced to differentiate by hemin for 5 –10 min; 3– 96 h were collected by centrifugation at 250 g for 10 min and washed two times with cold PBS. The pellet was then resuspended and lysed in a buffer containing 25 mM Tris –HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate and 1 mM EDTA at 4 -C for 15 min. The lysate was centrifuged at 14 000 g for 15 min and the resulting supernatant was transferred to a clean tube and stored at 70 -C. Western blot analysis Equal amounts of membrane proteins or lysates prepared from K562, HL-60, AML and PBM cells were resolved by SDS/PAGE on 7.5%, 10% or 12% gels and electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell, 0.45 Am) for 1 h at 80 V as described by Towbin et al. (1979). The membranes were blocked with TBS containing 3% bovine serum albumin (BSA) and 0.05% Tween-20 at room temperature for 90 min. Antibodies against G protein subunits and phosphorylated ERK1/2 were diluted with this blocking solution and reacted with the membranes overnight at 4 -C or 4 h at room temperature. After blots were washed three times with TBS containing 0.05% Tween-20 (TBST), alkaline phosphatase- or peroxidase-conjugated goat anti-rabbit IgG (dilution 1 : 1000 or 1 : 5000, respectively) was added. Detection of the antibody-antigen complex was by BCIP/NBT or ECL chemiluminescent substrate solution (Pierce, Rockford, IL, USA). Densitometric analysis was carried out with Bio-Rad Molecular Analyst software program.
RNA was isolated from hemin-induced and uninduced K562 cells, HL60 cell line, PBMC and PBMC of a patient diagnosed with acute myelocytic leukemia (AML). The cells were lysed with 4 M guanidium thiocyanate and then total RNA was prepared by phenol-chloroform extraction method of Chomczynski and Sacchi (1987) with some minor modifications. mRNA was isolated using the Oligotex mRNA isolation kit from Qiagen (Chatsworth, CA, USA) according to the manufacturer’s instructions. Purity and quantification of RNA were assessed by A260 / A280 ratios. Detection by RT-PCR RNA samples were amplified using specific primers for Ga16, Gas and GAPDH by RT-PCR using Titan-One Tube RT-PCR system (Roche, Mannheim, Germany). Amplification reactions were carried out in a 50 Al volume containing 2 Ag total RNA or 1 Ag mRNA, 0.2 mM of each dNTP, 0.4 AM of each primer, 10 Al 5 RT-PCR buffer provided by the manufacturer, 5 mM DTT, 1.5 mM MgCl2 and 1.0 Al enzyme mix (AMV and Expand High Fidelity PCR-System). The
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0.5 M acetic acid and H2O2 (0.14%) was added immediately before use. 1 ml of cell suspension was washed, resuspended with PBS and mixed with the benzidine solution in a 1 : 0.1 (v/ v) ratio. After 10 min, the cells were counted in a hemocytometer and blue cells were considered positive for hemoglobin (Gopalakrishnan and Anderson, 1979).
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Time (Day) Fig. 1. Growth rate and hemin-induced differentiation of K562 cells in culture. K562 cells seeded at 1 105 cells/ml were grown in RPMI-1640 medium containing 10% Fetal calf serum at 37 -C under humidified air/5% CO2 for five days. Cells grown with (h) or without (>) 20 AM hemin were counted in a hemocytometer after dilution with 0.4% trypan blue (A). Cells were removed daily and washed with phosphate-buffered saline and stained with benzidine solution. The percentage of benzidine-positive cells was scored under microscope (B). Results are mean values T S.D. from three different experiments.
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following primer pairs that resulted in a 305 bp product were used to amplify GAPDH: 5V-GTGAGGAGGGGAGATTCAG-3V and 5V-GCATCCTGGGCTACACTG-3V (Rodenburg et al., 1998). For amplification of Ga16 and Gas, the primers used were Ga16: upstream 5V-CACCACGCTAGCCTGGTCATG-3V and downstream 5V-GCGCCCTTCTTGCTGCCCTCGGG-3V (Mapara et al., 1995); Gas: sense 5V-CCACCTGAATTCTATGAGCATGCC-3V and antisense 5V-GCGTGGGTCCTCTCCGGGCTCGGG-3V (Mullaney et al., 1996), yielding products of 672 and 573 bp, respectively. The cycle parameters were as follows: 30 min at 50 -C and 2 min at 94 -C; 2 min at 94 -C, 30 s at 60 -C, 45 s at 68 -C for 10 cycles; 15 s at 94 -C, 15 s at 65 -C, 45 s at 72 -C for 5 cycles; 15 s at 94 -C, 15 s at 55 -C, 45 s at 72 -C for 30 cycles. PCR products were visualized by 1.5% ethidium bromide staining in agarose gels and then analyzed in a Kodak image analyzer. Negative controls were included in each reaction.
Results K562 cell growth and differentiation induced by hemin K562 cells grown in suspension culture were induced to differentiate with hemin. Fig. 1 shows the proliferation and pattern of differentiation of K562 cells. Cell viability was >95% as determined by the trypan blue dye exclusion assay (Fig. 1A). Hemin induced differentiation of K562 cells by 65% in five days (Fig. 1B). G protein levels during hemin-induced differentiation of K562 cells Immunoblot analysis was used to determine changes in expression patterns of specific G protein subunits during hemin-induced differentiation of K562 cells. Expression of Gaq, Gao, Ga16 and Ghg subunits was studied in crude
A Gαq
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Gαi1-2 Fig. 2. Expression of G proteins in K562 membranes. (A). Membrane fractions of K562 cells were obtained after culturing for 5 days in the presence or absence of hemin. Proteins (50 Ag for Gao blot, 100 Ag for Gaq, Ghg, Ga16 blots) were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with anti-Gaq, anti-Gao, anti-Ghg, or anti-Ga16 antisera and then visualized by NBT/BCIP substrate solution. Lanes: 1, uninduced K562 cells; 2, induced K562 cells; 3, membrane extract from rat brain; 4, PBMC lysates. (B). Blot of K562 cells processed for immunoreaction with anti-Gai1 – 2 antibody. B, membrane extracts from rat brain; U, uninduced; I, induced. (C). Cells were removed at 24 h intervals and the levels Gas and Gai1 – 2 in membrane fractions were determined with specific antisera. Each well contains 5 106 cells.
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Fig. 3. Detection of Gas and Ga16 mRNA in K562 cells by RT-PCR. mRNAs isolated from uninduced (U) and induced (I) K562 cells were amplified with specific primers for Gas and Ga16 and GAPDH as described under Materials and methods. PCR products of 573 (Gas), 672 (Ga16) and 305 (GAPDH) bp were visualized on 1.5% agarose gels stained with ethidium bromide. HL-60 and PBMC were used as positive controls.
membrane fractions obtained from K562 cells treated with hemin for five days and their untreated controls. Gaq and Ghg were expressed in hemin-treated and untreated cells, whereas Gao and Ga16 were not detected in these cells (Fig. 2A). The presence of Gao and Ga16 was shown in extracts of brain and PBM cells, respectively. Expression of the long form of Gas was weak in K562 cells in contrast to brain where it was predominantly expressed. On the other hand, we found the short form of Gas to be highly expressed in these cells (Fig. 2C). Immunoblot analysis using an antibody specific to Gai1 – 2 subunits demonstrated the presence of Gai2 but not of Gai1 (Fig. 2B). To explore whether the levels of Gas and Gai2 were altered during the differentiation of K562 cells, membrane fractions obtained on days 1 – 6 after induction with hemin were analyzed for Gas and Gai2 subunits with Western blot analysis (Fig. 2C). The level of the short isoform of Gas increased on days 5 and 6 post-induction by about 93% and 41%, respectively. After treatment of cells with hemin, we observed a one fold increase in the level of Gai2 on day 3, however this was followed by a decline, to a level 49% lower 0
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than the untreated cells by day 6 (Fig. 2B, C). Upon treatment with hemin, the level of Gaq increased by 22% (Fig. 2A). RT-PCR analysis of Gas and Ga16 mRNA in K562 cells To verify the absence of Ga16 in K562 cells at the level of mRNA, RT-PCR amplification was performed with Ga16specific primers using total RNA and mRNA as templates. Ga16 mRNA expression was not observed in K562 cells; although PCR products of 672 bp were detected in HL-60 and PMB cells, which were used as positive controls (Fig. 3). ERK phosphorylation in hemin-induced in K562 cells To explore the role of ERKs during erythroid differentiation, K562 cells were treated with hemin for various time points and ERK phosphorylation in cell lysates was assessed by monitoring immunoreactivity against antibodies specific to phosphorylated ERK1/2 and ERK1/2. Upon treatment with hemin, ERK phosphorylation increased gradually, reaching 24h
48h
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96h pERK1/2
ERK1/2 Fig. 4. ERK phosphorylation in hemin induced K562 cells. 50 Ag cell lyasates obtained from hemin-induced and uninduced K562 cells at indicated time points were subjected to 12% SDS-gel electrophoresis and then electroblotting. Blots were incubated with specific antibody against phosphorylated ERK1/2 (pERK1/2). To verify the amount of loaded protein, membranes were stripped and reprobed with anti-ERK1/2 antibody. Representative blots from three independent experiments are shown.
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peak values at 24– 48 h and falling back to nearly basal values by 96 h (Fig. 4). Discussion Signal transduction pathways mediated by G proteins determine many of the responses of hematopoeitic cells. The chronic myelogenous erythroleukemia cell line K562 cells can be induced in culture to differentiate along the erythrocytic pathway in response to various agents, including hemin (Lozzio and Lozzio, 1975; Rutherford et al., 1979). The purpose of this study was to investigate changes in expression of heterotrimeric G proteins in response to hemin-induced differentiation of K562 cells and to establish the role of MAPK signaling pathways in erythroid differentiation of K562 cells. Hematopoietic cells are known to express several different G proteins. In order to determine alterations in G proteins accompanying erythroid differentiation of K562 cells, the levels of Gas, Gai1 – 2, Gaq, Gao, Ga16 and Ghg subunits were studied in crude membrane fractions prepared from these cells using Western blot analysis. In line with previous reports on K562 cells (Thomas et al., 1995; Davis et al., 2000), Gao and Ga16 were not detected in K562 cells, although immunoreactivity was demonstrated in brain and PBMC that were used as positive controls, respectively. Ga16 protein, which was first described by Amatruda et al. (1991) is expressed only in hematopoietic cells. The restricted pattern of expression of Ga16 indicated that it might play a major role in hematopoietic cell proliferation and differentiation. Indeed, mRNA studies by Amatruda et al. (1991) demonstrated expression of Ga16 in human T lymphocytes and cells of myelomonocytic origin but not in non-hematopoietic cells and two human B cell lines. Furthermore, the expression of Ga16 mRNA was decreased by 90% upon DMSO-induced differentiation of HL-60 cells into neutrophils (Amatruda et al., 1991). A subsequent study by Mapara et al. (1995) showed that Ga16 expression in progenitor B cells decreased when they maturated into B cells. Specific expression of Ga16 proteins in normal human myeloid progenitors and mature blood cells was explored in a detailed study by Tenailleau et al. (1997) which provided further support to the notion that Ga16 is expressed in a lineagespecific manner. Ga16 protein was detected in all human myeloid cells except red blood cells but the level of expression varied according to the cell lineage and it was down regulated during granulocytic and erythrocytic differentiation (Tenailleau et al., 1997). Ga16 protein was expressed in erythroleukemia cells HEL, TF-1 and K562 cells tested in the same study (Tenailleau et al., 1997), contradicting our findings and those of Amatruda et al. (1991). Next, we used RT-PCR analysis to determine whether Ga16 is expressed at the mRNA level. Ga16 mRNA was absent in K562 cells as opposed to HL-60 and PBM cells for which PCR products of 672 bp were visible on agarose gels. To test the efficiency of RT-PCR analysis, we also amplified total RNA and mRNA for the Gas and GAPDH genes. We were able to demonstrate the amplification of both Gas and GAPDH genes in induced and uninduced K562 cells. Thus, our RT-PCR data lend support to findings demonstrating
the absence of Ga16 mRNA by Northern blot analysis (Amatruda et al., 1991). As expected, we detected Gas, Gai2, Gaq and Ghg subunits in K562 cells by Western blot analysis. Other investigators have previously documented the presence of these G protein subunits in K562 cells (Amatruda et al., 1991; Thomas et al., 1995; Davis et al., 2000). We observed that the level of the short form of Gas (Gas-S) is expressed predominantly. This observation is consistent with those of Tenailleau et al. (1997) in red blood cells. Prior reports indicate that the relative proportions of Gas-L and Gas-S vary in different tissues, raising the possibility that the two isoforms may serve different functions in different cells (Novotny and Svoboda, 1998). To address the possible involvement of Gas and Gai2 in regulation of differentiation of K562 cells by hemin, we immunoblotted for Gas and Gai2 subunits in cells cultured in the presence of hemin for various time points. We found that the Gas-S levels were nearly doubled in five days after hemin treatment. The increase in the long isoform of Gas was not as pronounced. The level of Gai2 increased on the third day following hemin treatment. This was followed with a decline back to and below control levels by the sixth day. The possible role of Gas and Gai2 in differentiation of hematopoietic cells has been previously explored in HL-60 and K562 cells. Gas and Gai2 levels decreased in HL-60 myeloid cells after they were induced to differentiate to mature granulocyte-like cells in the presence of retinoic acid (Meissner et al., 1996). The levels of both isoforms of Gas were either unchanged or decreased after treatment of K562 cells with sodium butyrate for 24 and 48 h, respectively. On the other hand, the levels of Gai2 increased up to 9 fold that of control as K562 cells differentiated to the erythrocytic phenotype in response to treatment with sodium butyrate (Davis et al., 2000). The discrepancy between our findings and those of Davis et al. may well be due to the different inducers used in the two studies. Divergent results on the role of Gas and Gai2 protein expression levels have been previously reported during differentiation of various cell lines. Although Gas and Gai2 displayed opposing effects in different cells, these studies indicate that Gas and Gai2 can play a critical role in the control of cell growth and differentiation. Differentiation of K562 cells by hemin was reported to occur independently of Protein Kinase C (Baliga et al., 1993). On the other hand, in HEL cells inhibition of PKC suppressed megakaryocytic differentiation and promoted ertythroid differentiation, suggesting the presence of a basal level of PKC activity that suppresses erythroid differentiation (Hong et al., 1996). We observed a slight increase for Gaq within five days of treatment with hemin, which argued against a major role of Gaq via its effector Protein Kinase C in differentiation of K562 cells by hemin. It is now well established that activation of MAPK pathway plays a crucial role in both cellular proliferation and differentiation. It has also been argued that the duration and amplitude of the MAPK signal determines the decision between proliferation and differentiation. GPCRs have been shown to activate MAPK pathway via different routes (reviewed in Gutkind, 2000; Kranenburg and Moolenaar, 2001). Gai-coupled receptors preferentially use Ghg-dependent
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pathway via Ras but the corresponding Gai-subunits might also contribute to MAPK activation by lowering cytosolic cAMP levels. Gq-coupled receptors can stimulate MAPK in a PKCdependent and PKC-independent manner. Several reports have recently indicated that the MAPK family is involved in erythroid differentiation induced by butyrate (Rivero and Adunyah, 1996; Witt et al., 2000), PMA (Shelly et al., 1998) or hemin (Woessmann and Mivechi, 2001). Since both Gi- and Gq-coupled receptors can potently stimulate MAPK activation; we examined the phosphorylation of ERK1/2 during hemininduced differentiation of K562 cells. We observed that ERK2 was primarily phosphorylated in K562 cells. Hemin stimulation of K562 cells resulted in a gradual increase of ERK2 phosphorylation until 48 h. However, ERK phosphorylation decreased on 72 and 96 h after treatment with hemin, by which time the cells were differentiated by more than 50%. These findings complement evidence, which demonstrate that MEK inhibitors mimic the effect of inducers of erythroid differentiation (Shelly et al., 1998; Witt et al., 2000). K562 cells can differentiate along the megakaryocytic lineage or erythroid lineage, depending on the inducer. It appears that transient activation of ERK is associated with erythroid differentiation whereas sustained activation is required for megakaryocytic differentiation (Racke et al., 1997). Although hemin is a potent regulator of hematopoietic cell differentiation, unlike other inducers such as DMSO it does not result in growth arrest (Baliga et al., 1993). The gradual increase in ERK2 phosphorylation until 48 h and subsequent decrease after 72 h of hemin treatment can partly be explained with concomitant changes in proliferation of K562 cells: sustained ERK phosphorylation until 48 h of hemin treatment may be correlated with cell proliferation and the subsequent decrease with erythrocytic differentiation. The mechanism of action by which hemin induces erythroid differentiation is still unclear. Results presented here show that changes in G protein a-subunits and MAPK activity are associated with hemin-induced differentiation of K562 cells. Acknowledgements We are grateful to Dr. Alfred Gilman for the anti-Gacommon and Ghg antibodies, Dr. Bernd Nu¨rnberg for the anti-Ga16 antibody and Dr. Azzam A. Maghazachi for the ERK antibodies. This study was supported by the Eczacibasi Scientific Research and Prize Fund, The Scientific and Technical Research Council of Turkey, SBAG-1710 and Marmara University Research Fund. References Amatruda III, T.T., Steele, D.A., Zlepak, V.Z., Simon, M.I., 1991. Ga16 a G protein a subunit specifically expressed in hematopoietic cells. Proceedings of the National Academy of Sciences of the United States of America 88 (13), 5587 – 5591. Ashby, B., Almonor, G.O., Wernick, E., Selak, M.A., 1991. Prostaglandinconcentration-dependent desensitization of adenylate cyclase in human erythroleukemia (HEL) cells is abolished by pertussis toxin and enhanced
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by induction by dimethylsulphoxide. Biochemical Journal 280 (Pt.3), 801 – 804. Baliga, B.S., Mankad, M., Shah, A.K., Mankad, V.N., 1993. Mechanism of differentiation of human erythroleukaemic cell line K562 by hemin. Cell Proliferation 26 (6), 519 – 529. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162 (1), 156 – 159. Davis, M.G., Kawai, Y., Arinze, I.J., 2000. Involvement of Gia2 in sodium butyrate-induced erythroblastic differentiation of K562 cells. Biochemical Journal 346 (Pt. 2), 455 – 461. Denis-Henriot, D., Lacasa, D., Goldsmith, P.K., De Mazancourt, P., Giudicelli, Y., 1996. Site-related differences in G-protein a subunit expression during adipogenesis in vitro: possible key role for Gq/11a in the control of preadipocyte differentiation. Biochemical and Biophysical Research Communications 220 (2), 443 – 448. Galvin-Parton, P.A., Watkins, D.C., Malbon, C.C., 1990. Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells. Journal of Biological Chemistry 265 (29), 17771 – 17779. Gao, P., Malbon, C.C., 1996. Differentiation of F9 teratocarcinoma stem cells to primitive endoderm is regulated by the Gia2/Gsa axis via phospholipase C and not adenylylcyclase. Journal of Biological Chemistry 271 (48), 30692 – 30698. Gierschik, P., Morrow, B., Milligan, G., Rubin, C., Spiegel, A., 1986. Changes in the guanine nucleotide-binding proteins, Gi and Go, during differentiation of 3T3-L1 cells. FEBS Letters 199 (1), 103 – 106. Gopalakrishnan, T.V., Anderson, W.F., 1979. Mouse erythroleukemia cells. In: Jakoby, W.B., Pastan, I.H. (Eds.), Methods in Enzymology, vol. 58. Academic Press Inc., London, pp. 506 – 511. Gutkind, J.S., 2000. Regulation of mitogen-activated protein kinase signaling networks by G protein-coupled receptors. Science’s STKE 40, 1 – 13. Hepler, J.R., Gilman, A.G., 1992. G proteins. Trends in Biochemical Sciences 17 (10), 383 – 387. Hong, Y., Martin, J.F., Vainchenker, W., Erusalimsky, J.D., 1996. Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells. Blood 87 (1), 123 – 131. Kang, C.D., Do, I.R., Kim, K.W., Ahn, B.K., Kim, S.H., Chung, B.S., Jhun, B.H., Yoo, M.A., 1999. Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells. Experimental and Molecular Medicine 31 (2), 76 – 82. Kesselring, F., Spicher, K., Porzig, H., 1994. Changes in G protein pattern and in G protein-dependent signaling during erythropoietin and dimethylsulfoxide-induced differentiation of murine erythroleukemia cells. Blood 84 (12), 4088 – 4098. Kranenburg, O., Moolenaar, W.H., 2001. Ras-MAPK kinase signalling by lysophosphatidic acid and other G protein-coupled receptors agonists. Oncogene 20 (13), 1540 – 1546. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193 (1), 265 – 275. Lozzio, C.B., Lozzio, B.B., 1975. Human chronic myelogenous leukemia cellline with positive Philadelphia chromosome. Blood 45 (3), 321 – 334. Malbon, C.C., 1997. Heterotrimeric G-proteins and development. Biochemical Pharmacology 53 (1), 1 – 4. Mapara, M.Y., Bommert, K., Bargou, R.C., Leng, C., Beck, C., Ludwig, W.D., Gierschik, P., Do¨rken, B., 1995. G protein subunit Ga16 expression is restricted to progenitor B cells during human B-cell differentiation. Blood 85 (7), 1836 – 1842. Meissner, J.D., Brown, G.A., Mueller, W.H., Scheibe, R.J., 1996. Retinoic acidmediated decrease of Gas protein expression: involvement of Gas in the differentiation of HL-60 myeloid cells. Experimental Cell Research 225 (1), 112 – 121. Mullaney, I., Milligan, G., 1989. Elevated levels of the guanine nucleotide binding proteins, Go, are associated with differentiation of neuroblastoma glioma hybrid cells. FEBS Letters 244 (1), 113 – 118. Mullaney, I.I., Carr, C., Milligan, G., 1996. Overexpression of Gsa in NG10815, neuroblastoma glioma cells: effects on receptor regulation of the stimulatory adenylyl cyclase cascade. FEBS Letters 397 (2-3), 325 – 330.
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Nagata, K., Okano, Y., Nozawa, Y., 1995. Identification of heterotrimeric GTPbinding proteins in human megakaryoblastic leukemia cell line, MEG-01, and their alteration during cellular differentiation. Life Sciences 57 (18), 1675 – 1681. Novotny, J., Svoboda, P., 1998. The long ( Gsa-L) and short (Gsa-S) variants of the stimulatory guanine nucleotide-binding protein. Do they behave in an identical way? Journal of Molecular Endocrinology 20 (2), 163 – 173. Offermans, S., Simon, M.I., 1996. Organization of transmembrane signalling by heterotrimeric G proteins. In: Tooze, J., Parker, P.J., Pawson, T. (Eds.), Cancer Surveys. Cold Spring Harbor Laboratory Press, New York, pp. 177 – 1198. Platanias, L.C., 2003. Map kinase signaling pathways and hematologic malignancies. Blood 101 (12), 4667 – 4679. Pouyssegur, J., Lenormand, P., 2003. Fidelity and spatio-temporal control in MAP kinase (ERKs) signalling. European Journal of Biochemistry 270 (16), 3291 – 3299. Racke, F.K., Lewandowska, K., Goueli, S., Goldfarb, A.N., 1997. Sustained activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway is required for megakaryocytic differentiation of K562 cells. Journal of Biological Chemistry 272 (37), 23366 – 23370. Rivero, J.A., Adunyah, S.E., 1996. Sodium butyrate induces tyrosine phosphorylation and activation of MAP kinase (ERK-1) in human K562 cells. Biochemical and Biophysical Research Communications 224 (3), 796 – 801. Rodenburg, R.J.T., van den Hoogen, F.H.J., van de Putte, L.B.A., van Venrooij, W.J., 1998. Peripheral blood monocytes of rheumotoid arthritis patients do not express elevated TNF a, IL-1 h and IL-8 mRNA levels. A comparison of monocyte isolation procedures. Journal of Immunological Methods 221 (1-2), 169 – 175. Rutherford, T.R., Clegg, J.B., Weatherall, D.J., 1979. K562 human leukaemic cells synthesis embryonic haemoglobin in response to haemin. Nature 280 (5718), 164 – 165.
Shelly, C., Petruzzelli, L., Herrera, R., 1998. PMA-induced phenotypic changes in K562 cells: MAPK-dependent and -independent events. Leukemia 12 (12), 1951 – 1961. Strittmatter, S.M., Valenzuela, D., Kennedy, T.E., Neer, E.J., Fishman, M.C., 1990. Goa is major growth cone protein subject to regulation by GAP-43. Nature 344 (6269), 836 – 841. Tenailleau, S., Corre, I., Hermouet, S., 1997. Specific expression of heterotrimeric G proteins G12 and G16 during human myeloid differentiation. Experimental Hematology 25 (9), 927 – 934. Thomas, C.P., Dunn, M.J., Mattera, R., 1995. Ca 2+ signalling in K562 erythroleukemia cells: effect of dimethly solphoxide and role of G proteins in thrombin- and thromboxane A2-activated pathways. Biochemical Journal 312 (Pt. 1), 151 – 158. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacryamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76 (9), 4350 – 4354. Wang, H.-y., Malbon, C.C., 1996. The Gsa/Gia2 axis controls adipogenesis independently of adenylyl cyclase. International Journal of Obesity 20 (Suppl. 3), 26 – 31. Wang, H.-Y., Watkins, D.C., Malbon, C.C., 1992. Antisense oliginucleotides to Gs protein a-subunit sequence accelerate differentiation of fibroblasts to adipocytes. Nature 358 (6384), 334 – 337. Watkins, D.C., Northup, J.K., Malbon, C.C., 1987. Regulation of G proteins in differentiation. Altered ratio of a- to h-subunits in 3T3-L1 cells. Journal of Biological Chemistry 262 (22), 10651 – 10657. Witt, O., Sand, K., Pekrun, A., 2000. Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathway. Blood 95 (7), 2391 – 2396. Woessmann, W., Mivechi, N., 2001. Role of ERK activation in growth and erythroid differentiation of K562 cells. Experimental Cell Research 264 (2), 193 – 200.
Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells Bahire Kucukkaya 1, Devrim Oz Arslan, Beki Kan * Marmara University School of Medicine, Department of Biophysics, Tibbiye Caddesi No 49, Haydarpasa, 34668, Istanbul, Turkey Received 9 May 2005; accepted 22 June 2005
Abstract Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of Gas, Gai2 and Gaq and the absence of Gai1, Gao and Ga16 in K562 cells. We observed the short form of Gas to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of Gas and Gaq. On the other hand, the level of Gai2 was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells. D 2005 Elsevier Inc. All rights reserved. Keywords: Erythroid differentiation; Hemin; G proteins; ERK1/2 phosphorylation; K562
Introduction Heterotrimeric guanine nucleotide binding proteins (G proteins) which couple many cell surface receptors to effectors on the plasma membrane mediate a series of events which ultimately lead to diverse cellular responses. G proteins are composed of an a-subunit that binds to and hydrolyzes GTP, and a hg-subunit complex. To date, 20 different Ga, 6 Gh and 11 Gg subunits have been described. Ga subunits have been divided into four families (Gs, Gi/o, Gq/11, G12/13) based on the homology at the amino acid level. Their mass is between 39 and 52 kDa (Hepler and Gilman, 1992; Offermans and Simon, 1996). G proteins regulate critical processes such as cell growth, differentiation and development (Malbon, 1997). Studies in human pheochromocytoma PC12 cells
* Corresponding author. Tel./fax: +90 216 3480585. E-mail address: [email protected] (B. Kan). 1 Current address: Maltepe University School of Medicine, Istanbul, Turkey. 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.06.041
which were treated with nerve growth factor (Strittmatter et al., 1990) and in the neuroblastoma glioma hybrid NG 10815 cell line, cultured with agents that elevate intracellular levels of cAMP (Mullaney and Milligan, 1989) suggested a role of Gao in development and differentiation of nervous tissue. Changes in the levels of G protein a-subunits such as Gas, Gao and Gai were associated with differentiation of 3T3L1 cells from fibroblasts into adipocytes. As these cells were induced to differentiate to adipocytes, a decline in the level of Gas (Wang et al., 1992), a decline (Gierschik et al., 1986) or an increase (Watkins et al., 1987) in the expression of Gai2 was observed. Constitutive activation of Gas by cholera toxin blocked and oligonucleotide(s) antisense to Gas accelerated the differentiation process (Wang et al., 1992). Interestingly, the modulating effect of Gas on differentiation was independent of adenylyl cyclase activation and cAMP levels (Wang and Malbon, 1996). A subsequent study demonstrated differential expression of Gaq/11 at three different stages of adipogenesis: in confluent preadipocytes, differentiated preadipocytes and mature adipocytes (Denis-Henriot et al.,
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1996). In F9 teratocarcinoma stem cells induced to differentiate with retinoic acid, the inhibitory G protein, Gai repressed (Galvin-Parton et al., 1990), whereas expression of Gas induced differentiation (Gao and Malbon, 1996). G proteins are also known to modulate differentiation of hematopoietic cells. Increase in expression of Gai2 and decrease in expression of Ga16, a G protein specifically expressed in hematopoietic cells, were shown in a promyelocytic cell line, HL-60, in the course of differentiation along the neutrophil pathway (Amatruda et al., 1991). When HL-60 cells were induced to differentiate to mature granulocyte-like cells with retinoic acid a decrease in the level of Gas was observed (Meissner et al., 1996). The content of Gai2 remained unchanged in a human erythroleukemia cell line, HEL, induced to differentiate to megakaryocytes (Ashby et al., 1991); however, the levels of Gai2 and Gai3 increased in a human megakaryoblastic leukemia cell line, MEG-01, induced to differentiate with TPA (Nagata et al., 1995). Terminal differentiation of an erythropoietin-sensitive murine erythroleukemia cell line, RED-1, was associated with a loss of Gai3 and an increase in the cytosolic form of Gai2 (Kesselring et al., 1994). In human myeloid progenitors and mature blood cells Gas, Gai2 and Gaq/11 proteins were expressed at high levels during every stage of granulomonocytic and erythroid differentiation, whereas Ga12 and Ga16 proteins were expressed in a lineage-specific manner in normal myeloid cells (Tenailleau et al., 1997). Taken together, these studies reveal that G protein alpha subunits can regulate cell differentiation in distinct ways, depending on the cell type or tissue under investigation as well as the type of inducer. The mitogen-activated protein kinase ERK1/2 pathway is essential in the control of growth, differentiation and survival in many cellular systems. The nature of the response to ERK activation in different cells depends on the agent used to induce cells to growth or differentiation and the duration of ERK activation (Pouyssegur and Lenormand, 2003). Several hematopoietic growth factors and cytokines such as erythropoietin (EPO) stem cell factor (SCF) and interleukin-3 (IL-3) by acting on receptor tyrosine kinases activate members of the ERK or MAPK family (Platanias, 2003). It is documented that Gq- and Gi-coupled receptors also activate the ERK1/2 pathway (Gutkind, 2000). A role for the ERK/MAPK pathway in erythroid differentiation has been proposed in a number of studies. Shelly et al. (1998) established that inhibition of the basal activity of the ERK/MAPK pathway enhanced the erythroid phenotype of K562 cells. Likewise, Kang et al. (1999) demonstrated that inactivation of ERK by MAPK inhibitors resulted in growth arrest and erythroid differentiation of K562 cells. Treatment of K562 cells with sodium butryrate resulted in a rapid but transient activation of ERK1/2 signaling in one study (Rivero and Adunyah, 1996) but caused inhibition of ERK and activation of p38 MAPK pathway in another (Witt et al., 2000). Recently, the involvement of ERK phosphorylation in both growth and hemin-induced erythroid differentiation of K562 cells has been shown (Woessmann and Mivechi, 2001). The signaling pathways mediating hemin-induced
differentiation of K562 cells have not been well characterized. In this study, we demonstrate alterations in the level of Gas, Gai2 and Gaq proteins and ERK1/2 phosphorylation during erythroid differentiation of K562 cells induced by hemin. Materials and methods Hemin, RPMI 1640 and penicillin/streptomycin solutions, alkaline phosphatase-conjugated goat anti-rabbit IgG and other electrophoresis reagents were purchased from Sigma (St Louis, MO, USA). Fetal calf serum (FCS) was from Harlan Sera-Lab (Leicestershire, UK). G protein antisera RM/1 (recognizing Cterminal sequence RMHLRQYELL of Gas), GC/2 (recognizing C-terminal peptide GCTLSAEERAALERS of Gao), and AS/7Transducin (recognizing C-terminal peptide KENLKDCGLF of Gai12) were purchased from Dupont NEN (Lachine, Quebec, Canada). Gaq antibody (internal 115 – 133) and antibody reacting with the C terminus (360 – 373) of the Ga16 subunit were obtained from Calbiochem (San Diego, CA, USA). Antisera against the Ghg (S217) and Ga16 (AS339) subunits were kindly provided by Dr. Alfred Gilman, University of Texas and Dr. Bernd Nu¨rnberg, Heinrich-Heine University, Dusseldorf, respectively. Phosphorylated ERK1/2 antibody was from Santa Cruz Biotechnology (San Diego, CA, USA). Anti-ERK1/2 antibody was from Promega (Madison, WI, USA). Affinity purified peroxidase-conjugated anti-rabbit IgG was purchased from Jackson ImmunoResearch Europe Ltd. (Cambridgeshire, UK). All other chemicals and enzymes were obtained from commercial sources. Cell preparations K562 and HL-60 cells obtained from ATCC (MD, USA) were cultured in RPMI medium supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 Ag/ml streptomycin, 25 Ag/ml amphotericin B and 2 mM l-glutamine at 37 -C in humidified air containing 5% CO2. Peripheral blood mononuclear cells (PBMC) were obtained from the fresh heparinized blood of healthy human volunteers. AML cells were prepared from the fresh heparinized blood of patient diagnosed with acute myelocytic leukemia AML. PBM and AML cells were isolated by density gradient centrifugation over Ficoll-Hypaque. Induction of hemoglobin synthesis Cells seeded at 1 105 cells/ml were induced to differentiate with 20 AM hemin (day 0) and were harvested at days 1– 6 after induction. At this time, benzidine staining was carried out to assess the extent of differentiation. Measurement of erythroid differentiation of K562 cells by benzidine staining Erythroid differentiation was scored by staining the cells for hemoglobin synthesis with benzidine reagent. A benzidine dihydrochloride stock solution, 0.2% (w/v), was prepared in
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K562, AML and PBM cells were collected by low speed (400 g) centrifugation and washed two times with phosphatebuffered saline (PBS), pH 7.5. The resulting pellets were resuspended and lysed with 20 strokes in a hand Dounce homogenizer in a buffer containing 20 mM Hepes/KOH, pH 8.0, 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF), 10 Ag/ml leupeptin and 2 Ag/ml aprotinin. Cell debris was pelleted by centrifugation at 400 g for 10 min and the resulting supernatant was centrifuged at 12 000 g for 60 min. The pellets (crude membrane fractions) were then resuspended and washed two times in the same buffer. The crude membranes were stored at 70 -C. The protein content of the preparation was determined by the method of Lowry et al. (1951). Preparation of K562 cell lysates K562 cells induced to differentiate by hemin for 5 –10 min; 3– 96 h were collected by centrifugation at 250 g for 10 min and washed two times with cold PBS. The pellet was then resuspended and lysed in a buffer containing 25 mM Tris –HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM PMSF, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate and 1 mM EDTA at 4 -C for 15 min. The lysate was centrifuged at 14 000 g for 15 min and the resulting supernatant was transferred to a clean tube and stored at 70 -C. Western blot analysis Equal amounts of membrane proteins or lysates prepared from K562, HL-60, AML and PBM cells were resolved by SDS/PAGE on 7.5%, 10% or 12% gels and electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell, 0.45 Am) for 1 h at 80 V as described by Towbin et al. (1979). The membranes were blocked with TBS containing 3% bovine serum albumin (BSA) and 0.05% Tween-20 at room temperature for 90 min. Antibodies against G protein subunits and phosphorylated ERK1/2 were diluted with this blocking solution and reacted with the membranes overnight at 4 -C or 4 h at room temperature. After blots were washed three times with TBS containing 0.05% Tween-20 (TBST), alkaline phosphatase- or peroxidase-conjugated goat anti-rabbit IgG (dilution 1 : 1000 or 1 : 5000, respectively) was added. Detection of the antibody-antigen complex was by BCIP/NBT or ECL chemiluminescent substrate solution (Pierce, Rockford, IL, USA). Densitometric analysis was carried out with Bio-Rad Molecular Analyst software program.
RNA was isolated from hemin-induced and uninduced K562 cells, HL60 cell line, PBMC and PBMC of a patient diagnosed with acute myelocytic leukemia (AML). The cells were lysed with 4 M guanidium thiocyanate and then total RNA was prepared by phenol-chloroform extraction method of Chomczynski and Sacchi (1987) with some minor modifications. mRNA was isolated using the Oligotex mRNA isolation kit from Qiagen (Chatsworth, CA, USA) according to the manufacturer’s instructions. Purity and quantification of RNA were assessed by A260 / A280 ratios. Detection by RT-PCR RNA samples were amplified using specific primers for Ga16, Gas and GAPDH by RT-PCR using Titan-One Tube RT-PCR system (Roche, Mannheim, Germany). Amplification reactions were carried out in a 50 Al volume containing 2 Ag total RNA or 1 Ag mRNA, 0.2 mM of each dNTP, 0.4 AM of each primer, 10 Al 5 RT-PCR buffer provided by the manufacturer, 5 mM DTT, 1.5 mM MgCl2 and 1.0 Al enzyme mix (AMV and Expand High Fidelity PCR-System). The
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0.5 M acetic acid and H2O2 (0.14%) was added immediately before use. 1 ml of cell suspension was washed, resuspended with PBS and mixed with the benzidine solution in a 1 : 0.1 (v/ v) ratio. After 10 min, the cells were counted in a hemocytometer and blue cells were considered positive for hemoglobin (Gopalakrishnan and Anderson, 1979).
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Time (Day) Fig. 1. Growth rate and hemin-induced differentiation of K562 cells in culture. K562 cells seeded at 1 105 cells/ml were grown in RPMI-1640 medium containing 10% Fetal calf serum at 37 -C under humidified air/5% CO2 for five days. Cells grown with (h) or without (>) 20 AM hemin were counted in a hemocytometer after dilution with 0.4% trypan blue (A). Cells were removed daily and washed with phosphate-buffered saline and stained with benzidine solution. The percentage of benzidine-positive cells was scored under microscope (B). Results are mean values T S.D. from three different experiments.
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following primer pairs that resulted in a 305 bp product were used to amplify GAPDH: 5V-GTGAGGAGGGGAGATTCAG-3V and 5V-GCATCCTGGGCTACACTG-3V (Rodenburg et al., 1998). For amplification of Ga16 and Gas, the primers used were Ga16: upstream 5V-CACCACGCTAGCCTGGTCATG-3V and downstream 5V-GCGCCCTTCTTGCTGCCCTCGGG-3V (Mapara et al., 1995); Gas: sense 5V-CCACCTGAATTCTATGAGCATGCC-3V and antisense 5V-GCGTGGGTCCTCTCCGGGCTCGGG-3V (Mullaney et al., 1996), yielding products of 672 and 573 bp, respectively. The cycle parameters were as follows: 30 min at 50 -C and 2 min at 94 -C; 2 min at 94 -C, 30 s at 60 -C, 45 s at 68 -C for 10 cycles; 15 s at 94 -C, 15 s at 65 -C, 45 s at 72 -C for 5 cycles; 15 s at 94 -C, 15 s at 55 -C, 45 s at 72 -C for 30 cycles. PCR products were visualized by 1.5% ethidium bromide staining in agarose gels and then analyzed in a Kodak image analyzer. Negative controls were included in each reaction.
Results K562 cell growth and differentiation induced by hemin K562 cells grown in suspension culture were induced to differentiate with hemin. Fig. 1 shows the proliferation and pattern of differentiation of K562 cells. Cell viability was >95% as determined by the trypan blue dye exclusion assay (Fig. 1A). Hemin induced differentiation of K562 cells by 65% in five days (Fig. 1B). G protein levels during hemin-induced differentiation of K562 cells Immunoblot analysis was used to determine changes in expression patterns of specific G protein subunits during hemin-induced differentiation of K562 cells. Expression of Gaq, Gao, Ga16 and Ghg subunits was studied in crude
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Gαi1-2 Fig. 2. Expression of G proteins in K562 membranes. (A). Membrane fractions of K562 cells were obtained after culturing for 5 days in the presence or absence of hemin. Proteins (50 Ag for Gao blot, 100 Ag for Gaq, Ghg, Ga16 blots) were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with anti-Gaq, anti-Gao, anti-Ghg, or anti-Ga16 antisera and then visualized by NBT/BCIP substrate solution. Lanes: 1, uninduced K562 cells; 2, induced K562 cells; 3, membrane extract from rat brain; 4, PBMC lysates. (B). Blot of K562 cells processed for immunoreaction with anti-Gai1 – 2 antibody. B, membrane extracts from rat brain; U, uninduced; I, induced. (C). Cells were removed at 24 h intervals and the levels Gas and Gai1 – 2 in membrane fractions were determined with specific antisera. Each well contains 5 106 cells.
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Fig. 3. Detection of Gas and Ga16 mRNA in K562 cells by RT-PCR. mRNAs isolated from uninduced (U) and induced (I) K562 cells were amplified with specific primers for Gas and Ga16 and GAPDH as described under Materials and methods. PCR products of 573 (Gas), 672 (Ga16) and 305 (GAPDH) bp were visualized on 1.5% agarose gels stained with ethidium bromide. HL-60 and PBMC were used as positive controls.
membrane fractions obtained from K562 cells treated with hemin for five days and their untreated controls. Gaq and Ghg were expressed in hemin-treated and untreated cells, whereas Gao and Ga16 were not detected in these cells (Fig. 2A). The presence of Gao and Ga16 was shown in extracts of brain and PBM cells, respectively. Expression of the long form of Gas was weak in K562 cells in contrast to brain where it was predominantly expressed. On the other hand, we found the short form of Gas to be highly expressed in these cells (Fig. 2C). Immunoblot analysis using an antibody specific to Gai1 – 2 subunits demonstrated the presence of Gai2 but not of Gai1 (Fig. 2B). To explore whether the levels of Gas and Gai2 were altered during the differentiation of K562 cells, membrane fractions obtained on days 1 – 6 after induction with hemin were analyzed for Gas and Gai2 subunits with Western blot analysis (Fig. 2C). The level of the short isoform of Gas increased on days 5 and 6 post-induction by about 93% and 41%, respectively. After treatment of cells with hemin, we observed a one fold increase in the level of Gai2 on day 3, however this was followed by a decline, to a level 49% lower 0
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6h
18h
than the untreated cells by day 6 (Fig. 2B, C). Upon treatment with hemin, the level of Gaq increased by 22% (Fig. 2A). RT-PCR analysis of Gas and Ga16 mRNA in K562 cells To verify the absence of Ga16 in K562 cells at the level of mRNA, RT-PCR amplification was performed with Ga16specific primers using total RNA and mRNA as templates. Ga16 mRNA expression was not observed in K562 cells; although PCR products of 672 bp were detected in HL-60 and PMB cells, which were used as positive controls (Fig. 3). ERK phosphorylation in hemin-induced in K562 cells To explore the role of ERKs during erythroid differentiation, K562 cells were treated with hemin for various time points and ERK phosphorylation in cell lysates was assessed by monitoring immunoreactivity against antibodies specific to phosphorylated ERK1/2 and ERK1/2. Upon treatment with hemin, ERK phosphorylation increased gradually, reaching 24h
48h
72h
96h pERK1/2
ERK1/2 Fig. 4. ERK phosphorylation in hemin induced K562 cells. 50 Ag cell lyasates obtained from hemin-induced and uninduced K562 cells at indicated time points were subjected to 12% SDS-gel electrophoresis and then electroblotting. Blots were incubated with specific antibody against phosphorylated ERK1/2 (pERK1/2). To verify the amount of loaded protein, membranes were stripped and reprobed with anti-ERK1/2 antibody. Representative blots from three independent experiments are shown.
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peak values at 24– 48 h and falling back to nearly basal values by 96 h (Fig. 4). Discussion Signal transduction pathways mediated by G proteins determine many of the responses of hematopoeitic cells. The chronic myelogenous erythroleukemia cell line K562 cells can be induced in culture to differentiate along the erythrocytic pathway in response to various agents, including hemin (Lozzio and Lozzio, 1975; Rutherford et al., 1979). The purpose of this study was to investigate changes in expression of heterotrimeric G proteins in response to hemin-induced differentiation of K562 cells and to establish the role of MAPK signaling pathways in erythroid differentiation of K562 cells. Hematopoietic cells are known to express several different G proteins. In order to determine alterations in G proteins accompanying erythroid differentiation of K562 cells, the levels of Gas, Gai1 – 2, Gaq, Gao, Ga16 and Ghg subunits were studied in crude membrane fractions prepared from these cells using Western blot analysis. In line with previous reports on K562 cells (Thomas et al., 1995; Davis et al., 2000), Gao and Ga16 were not detected in K562 cells, although immunoreactivity was demonstrated in brain and PBMC that were used as positive controls, respectively. Ga16 protein, which was first described by Amatruda et al. (1991) is expressed only in hematopoietic cells. The restricted pattern of expression of Ga16 indicated that it might play a major role in hematopoietic cell proliferation and differentiation. Indeed, mRNA studies by Amatruda et al. (1991) demonstrated expression of Ga16 in human T lymphocytes and cells of myelomonocytic origin but not in non-hematopoietic cells and two human B cell lines. Furthermore, the expression of Ga16 mRNA was decreased by 90% upon DMSO-induced differentiation of HL-60 cells into neutrophils (Amatruda et al., 1991). A subsequent study by Mapara et al. (1995) showed that Ga16 expression in progenitor B cells decreased when they maturated into B cells. Specific expression of Ga16 proteins in normal human myeloid progenitors and mature blood cells was explored in a detailed study by Tenailleau et al. (1997) which provided further support to the notion that Ga16 is expressed in a lineagespecific manner. Ga16 protein was detected in all human myeloid cells except red blood cells but the level of expression varied according to the cell lineage and it was down regulated during granulocytic and erythrocytic differentiation (Tenailleau et al., 1997). Ga16 protein was expressed in erythroleukemia cells HEL, TF-1 and K562 cells tested in the same study (Tenailleau et al., 1997), contradicting our findings and those of Amatruda et al. (1991). Next, we used RT-PCR analysis to determine whether Ga16 is expressed at the mRNA level. Ga16 mRNA was absent in K562 cells as opposed to HL-60 and PBM cells for which PCR products of 672 bp were visible on agarose gels. To test the efficiency of RT-PCR analysis, we also amplified total RNA and mRNA for the Gas and GAPDH genes. We were able to demonstrate the amplification of both Gas and GAPDH genes in induced and uninduced K562 cells. Thus, our RT-PCR data lend support to findings demonstrating
the absence of Ga16 mRNA by Northern blot analysis (Amatruda et al., 1991). As expected, we detected Gas, Gai2, Gaq and Ghg subunits in K562 cells by Western blot analysis. Other investigators have previously documented the presence of these G protein subunits in K562 cells (Amatruda et al., 1991; Thomas et al., 1995; Davis et al., 2000). We observed that the level of the short form of Gas (Gas-S) is expressed predominantly. This observation is consistent with those of Tenailleau et al. (1997) in red blood cells. Prior reports indicate that the relative proportions of Gas-L and Gas-S vary in different tissues, raising the possibility that the two isoforms may serve different functions in different cells (Novotny and Svoboda, 1998). To address the possible involvement of Gas and Gai2 in regulation of differentiation of K562 cells by hemin, we immunoblotted for Gas and Gai2 subunits in cells cultured in the presence of hemin for various time points. We found that the Gas-S levels were nearly doubled in five days after hemin treatment. The increase in the long isoform of Gas was not as pronounced. The level of Gai2 increased on the third day following hemin treatment. This was followed with a decline back to and below control levels by the sixth day. The possible role of Gas and Gai2 in differentiation of hematopoietic cells has been previously explored in HL-60 and K562 cells. Gas and Gai2 levels decreased in HL-60 myeloid cells after they were induced to differentiate to mature granulocyte-like cells in the presence of retinoic acid (Meissner et al., 1996). The levels of both isoforms of Gas were either unchanged or decreased after treatment of K562 cells with sodium butyrate for 24 and 48 h, respectively. On the other hand, the levels of Gai2 increased up to 9 fold that of control as K562 cells differentiated to the erythrocytic phenotype in response to treatment with sodium butyrate (Davis et al., 2000). The discrepancy between our findings and those of Davis et al. may well be due to the different inducers used in the two studies. Divergent results on the role of Gas and Gai2 protein expression levels have been previously reported during differentiation of various cell lines. Although Gas and Gai2 displayed opposing effects in different cells, these studies indicate that Gas and Gai2 can play a critical role in the control of cell growth and differentiation. Differentiation of K562 cells by hemin was reported to occur independently of Protein Kinase C (Baliga et al., 1993). On the other hand, in HEL cells inhibition of PKC suppressed megakaryocytic differentiation and promoted ertythroid differentiation, suggesting the presence of a basal level of PKC activity that suppresses erythroid differentiation (Hong et al., 1996). We observed a slight increase for Gaq within five days of treatment with hemin, which argued against a major role of Gaq via its effector Protein Kinase C in differentiation of K562 cells by hemin. It is now well established that activation of MAPK pathway plays a crucial role in both cellular proliferation and differentiation. It has also been argued that the duration and amplitude of the MAPK signal determines the decision between proliferation and differentiation. GPCRs have been shown to activate MAPK pathway via different routes (reviewed in Gutkind, 2000; Kranenburg and Moolenaar, 2001). Gai-coupled receptors preferentially use Ghg-dependent
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pathway via Ras but the corresponding Gai-subunits might also contribute to MAPK activation by lowering cytosolic cAMP levels. Gq-coupled receptors can stimulate MAPK in a PKCdependent and PKC-independent manner. Several reports have recently indicated that the MAPK family is involved in erythroid differentiation induced by butyrate (Rivero and Adunyah, 1996; Witt et al., 2000), PMA (Shelly et al., 1998) or hemin (Woessmann and Mivechi, 2001). Since both Gi- and Gq-coupled receptors can potently stimulate MAPK activation; we examined the phosphorylation of ERK1/2 during hemininduced differentiation of K562 cells. We observed that ERK2 was primarily phosphorylated in K562 cells. Hemin stimulation of K562 cells resulted in a gradual increase of ERK2 phosphorylation until 48 h. However, ERK phosphorylation decreased on 72 and 96 h after treatment with hemin, by which time the cells were differentiated by more than 50%. These findings complement evidence, which demonstrate that MEK inhibitors mimic the effect of inducers of erythroid differentiation (Shelly et al., 1998; Witt et al., 2000). K562 cells can differentiate along the megakaryocytic lineage or erythroid lineage, depending on the inducer. It appears that transient activation of ERK is associated with erythroid differentiation whereas sustained activation is required for megakaryocytic differentiation (Racke et al., 1997). Although hemin is a potent regulator of hematopoietic cell differentiation, unlike other inducers such as DMSO it does not result in growth arrest (Baliga et al., 1993). The gradual increase in ERK2 phosphorylation until 48 h and subsequent decrease after 72 h of hemin treatment can partly be explained with concomitant changes in proliferation of K562 cells: sustained ERK phosphorylation until 48 h of hemin treatment may be correlated with cell proliferation and the subsequent decrease with erythrocytic differentiation. The mechanism of action by which hemin induces erythroid differentiation is still unclear. Results presented here show that changes in G protein a-subunits and MAPK activity are associated with hemin-induced differentiation of K562 cells. Acknowledgements We are grateful to Dr. Alfred Gilman for the anti-Gacommon and Ghg antibodies, Dr. Bernd Nu¨rnberg for the anti-Ga16 antibody and Dr. Azzam A. Maghazachi for the ERK antibodies. This study was supported by the Eczacibasi Scientific Research and Prize Fund, The Scientific and Technical Research Council of Turkey, SBAG-1710 and Marmara University Research Fund. References Amatruda III, T.T., Steele, D.A., Zlepak, V.Z., Simon, M.I., 1991. Ga16 a G protein a subunit specifically expressed in hematopoietic cells. Proceedings of the National Academy of Sciences of the United States of America 88 (13), 5587 – 5591. Ashby, B., Almonor, G.O., Wernick, E., Selak, M.A., 1991. Prostaglandinconcentration-dependent desensitization of adenylate cyclase in human erythroleukemia (HEL) cells is abolished by pertussis toxin and enhanced
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