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(The FASEB Journal. 1998;12:531-539.)
© 1998 FASEB

RESEARCH COMMUNICATION

Differential expression of the Kell blood group and CD10 antigens: two related membrane metallopeptidases during differentiation of K562 cells by phorbol ester and hemin

Nathalie Belhacène1,a, Laurence Maulona, Sandrine Guérina, Jean Ehrland Riccia, Bernard Mari,2,a, Yves Colinb, Jean Pierre Cartronb, and Patrick Aubergera,1

a CJF INSERM 96. 05, Activation des Cellules Hématopoietiques, Faculté de Médecine, 06107 Nice Cedex 2, France
b INSERM U76 INTS, 75739 Paris Cedex 15, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The erythroleukemic cell line K562 can undergo further differentiation in erythroid or megakaryocytic lineage depending on the nature of the stimulus. Phorbol ester (PMA) stimulates megakaryocytic development whereas hemin promotes erythroid differentiation of these cells. We have examined the effect of PMA and hemin on the expression of the Kell blood group and CD10 antigens, two related proteins that belong to a family of membrane-bound neutral metalloendopeptidases. We show here that differentiation of K562 cells by PMA in the megakaryocytic lineage results in abolishment of Kell mRNA accumulation and protein expression and, in parallel, the induction of CD10 mRNA accumulation, protein expression, and enzymatic activity. Conversely, differentiation of these cells by hemin in the erythroid lineage is accompanied by an up-regulation of Kell mRNA and protein expression, with no changes in CD10 mRNA and protein expression. Thus, CD10 and Kell can be regarded as specific markers of the differentiation of K562 cells in the megakaryocytic and erythroid lineages, respectively.—Belhacène, N., Maulon, L., Guérin, S., Ricci, J. E., Mari, B., Colin, Y., Cartron, J. P., Auberger, P. Differential expression of the Kell blood group and CD10 antigens: two related membrane metallopeptidases during differentiation of K562 cells by phorbol ester and hemin. FASEB J. 12, 531–539 (1998)


Key Words: protein kinase C • CD10 activity • Kell • cell proliferation • K562 differentiation • PMA • cells • hemin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUMAN ERYTHROID/MEGAKARYOCYTIC cell lines have been used extensively as a model system to study molecular aspects of erythroid and megakaryocytic differentiation (1–5). K562 is an erythroleukemic cell line that can undergo further differentiation in both megakaryocytic and erythroid lineages, depending on the stimulus (2, 4, 5). Phorbol-12 myristate-13 acetate (PMA)4 stimulates megakaryocytic development, resulting in an inhibition of cell proliferation, morphologic changes, and acquisition of platelet/megakaryocyte-specific proteins (4, 6–8). Evidence that the effect of PMA on megakaryocytic differentiation is mediated by protein kinase C (PKC) is supported by a recent study demonstrating that the specific PKC inhibitor GF 109203X suppresses megakaryocytic and stimulates erythroid differentiation in HEL cells (8). On the other hand, treatment of K562 or HEL cells with hemin induces erythroid differentiation, resulting in an enhanced expression of specific erythroid markers such as {gamma}-globin and glycophorin A (8, 9). The molecular mechanisms underlying the hemin effect on K562 cell differentiation have been poorly studied, even though tyrosine kinase activities are thought to be required since tyrosine kinase inhibitors such as herbemycin A and genestein block erythroid differentiation of these cells (10, 11).

CD10 (neprilysin) and the Kell blood group antigens are two members of a family of membrane-bound neutral metalloendopeptidases (12, 13) that also includes endothelin-converting enzymes 1, 2, and 3 and two bacterial enzymes (14, 15). CD10 is identical to the common acute lymphoblastic leukemia antigen (12, 13, 16, 17), a membrane-bound neutral metalloendopeptidase involved in the degradation of numerous bioactive peptides (18–20) and also thought to play an important role in regulating activation (21, 22) and differentiation (23–27) of B and T lymphocytes. It is currently unknown whether Kell shares an endopeptidase activity, but the high degree of conservation found in the amino acid sequences of Kell and CD10 (28), especially in their active site region, suggests that Kell may behave as a functional endopeptidase.

An increase in Kell reactivity upon hemin treatment of K562 cells has previously been established by fluorescence-activated cell sorter analysis (9). In this study, we show that differentiation of K562 cells by PMA in the megakaryocytic lineage is accompanied by a total inhibition of Kell mRNA and protein expression and, conversely, by an induction of CD10 mRNA and protein expression, whereas differentiation of these cells by hemin in the erythroid pathway results in an increase in Kell mRNA and protein accumulation, with no changes in the expression of CD10 mRNA and protein. We conclude that CD10 and Kell can be considered specific markers of megakaryocytic and erythroid differentiation, respectively.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
RPMI 1640 and fetal calf serum (FCS) were purchased from (Gibco BRL, Paisley, U.K.). Phorbol-13 myristate-12 acetate (PMA) and hemin were obtained from (Sigma, St. Quentin Fallavier, France). Biotinylated RAM IgG, PE streptavidin, anti-CD10, anti-CD41, and anti-CD44 monoclonal antibodies were purchased from Immunotech (Marseille, France). The CD10 substrate Suc-Ala-Ala-Phe-pNa, purified aminopeptidase N, and phosphoramidon (a CD10 inhibitor) were obtained from Boehringer (Meylan, France). Peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Dako (Trappes, France). Enhanced chemiluminescent detection system was provided by (Amersham, Les Ulis, France). K50 anti-CD10 monoclonal antibody was a kind gift from Prof. Alain Bernard, INSERM U343, Nice, France.

Cells
The cell line K562 was cultured in RPMI 1640 supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, and 1 mM pyruvate under 5% CO2/95% air in a humidified incubator. To induce differentiation, exponentially growing cells (105 cells/ml) were exposed to different concentrations of either PMA or hemin during several days in culture.

Microscopy
K562 cells were treated for 96 h with 10 ng/ml PMA or left untreated. Morphologic changes characteristic of megakaryocytic differentiation were visualized by optical microscopy using standard optics (Zeiss, Oberkochen, Germany).

Cell surface staining and flow cytometric analysis
If not otherwise indicated, K562 cells were incubated with either 30 µM hemin or 10 ng/ml PMA in 10% FCS RPMI 1640 medium. At the onset of each incubation, cells were washed twice with phosphate-buffered saline (PBS). For simple staining experiments, cells (2x106/ml) were directly labeled with a saturating concentration of FITC-conjugated monoclonal antibodies for 30 min at 4°C in 100 µl of PBS 0.1% bovine serum albumin (BSA). For triple staining experiments, cells (2x106/ml) were labeled first with saturating concentrations of the different antibodies, followed by biotinylated RAM IgG, mouse immunoglobulins, and PE streptavidin. At each staining step, antibodies were added to cells at 4°C for 30 min in 100 µl PBS containing 0.1% BSA. Analysis were performed by flow cytometry on a dual laser Facs Star Plus (Becton Dickinson, Bedford, Mass.). Side and forward scatter of blue laser was used to gate out debris and damaged cells.

Determination of CD10 activity
After treatment with either hemin or PMA, K562 cells were extensively washed in PBS. Cells were resuspended at 107 cells/ml in the same buffer. CD10 activity was determined using the CD10 substrate Suc-Ala-Ala-Phe-pNa. Briefly, 2 x 106 cells were incubated in 200 µl of PBS containing 1 mM of the substrate in the presence 10 µg/ml of purified aminopeptidase N (Boehringer). In some experiments, cells were also pretreated with 10 µM phosphoramidon (Boehringer), a CD10 inhibitor, before determination of CD10 activity. CD10 cleaved the substrate at the Ala-Phe bond. Paranitroaniline was then liberated from Phe-pNA by exogenous aminopeptidase N. At different times, CD10 activity was determined by the measure of absorbance of paranitroaniline at 410 nm. CD10 activity represents phosphoramidon-inhibitable activity (21, 25).

Western blot experiments
Total membrane proteins (200 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) on 8% polyacrylamide gels. The transfer onto Hybond-C membrane (Amersham) was made in 48 mM Tris, 150 mM glycine, using Trans-Blot apparatus (Bio-Rad, Ivry-sur-Seine, France) at 100 V for 1 h at 4°C. The membrane was saturated with 10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 3% BSA, and 0.5% gelatin for 1 h at room temperature and then probed with either an anti-CD10 antibody (K50 1/3000 dilution) or an anti-Kell monoclonal antibody (anti-Kell 5A11) (1/50 dilution) in the same buffer overnight at 4°C. This monoclonal antibody is specifically directed against the amino-terminal cytoplasmic domain of Kell (29). Membranes were washed three times with PBS, 0.5% Tween 20. Bound antibodies were revealed after incubation with peroxidase-conjugated anti-mouse antibodies (1/5000 dilution in saturation buffer) for 1 h at room temperature. Membrane was then washed twice in PBS, 0.5% Tween 20 and finally in 0.1 M Tris, pH 8. Revelation was made with the enhanced chemiluminescent detection system with autoradiography hyperfilms MP (Amersham) (30).

mRNA preparation and Nothern blot analysis
Total RNAs were extracted from K562 cell cultures treated with either hemin or PMA according to the acid guanidinium thiocyanate-phenol-chloroform method using 1 ml RNA extraction solution `RNA-PLUS' (Bioprobe System, Montreuil, France). RNA aliquots (25 µg) were electrophoresed on 1% agarose gel containing 6% formaldehyde under denaturating conditions and tranferred to a nylon membrane (Hybond N+, Amersham). Kell or CD10 cDNA probes were purified and labeled by random priming, using [{alpha}-32P] dCTP (Amersham). Hybridization was performed overnight at 65°C, as previously described (21, 31), and the filters were washed in 2 x SSC/0.1% SDS 10 min at 65°C and then in 0.2 x SSC/0.1% SDS 10 min at 65°C before autoradiography, following the procedure of Church and Gilbert (32).

Cell proliferation assay
Cells (105/well) were incubated in triplicate in the presence or absence of various concentrations of either hemin or PMA for different periods of time at 37°C in 10% FCS RPMI 1640 medium. Cellular proliferation was estimated either by cell counting with a Coulter counter or according to the tetrazolium-bromide precipitation assay (XTT assay; Boehringer).

Benzidine staining
Cell hemoglobinization was analyzed by benzidine staining. Fifty microliters of cells (0.5 to 1x106 cells/ml) was mixed with 10 µl benzidine reagent consisting of 0.6% H2O2, 0.5 mol/l acetic acid, and 0.2% benzidine dihydrochloride. The percentage of benzidine-positive cells (blue cells) was determined by light microscopic examination of 100 cells per sample (33). Each experiment was performed in triplicate, and results were averaged.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PMA induces growth arrest in K562 cells
K562 is an erythroleukemic cell line that can undergo further differentiation in both megakaryocytic and erythroid lineages, depending on the stimulus. Addition of hemin (2 to 50 µM) to cell cultures for various periods of time failed to affect significantly K562 cell proliferation ( Fig. 1 B, D). By contrast, PMA totally inhibited K562 cell proliferation as assessed either by cell counting ( Fig. 1A, C) or the XTT assay (not shown). PMA's effect on cell proliferation was observed after 24 h of incubation and was maintained for more than 8 days ( Fig. 1A). A range of PMA concentrations was also tested on K562 cell proliferation. Half-maximal inhibition was obtained for 0.3 to 1 nM PMA, whereas maximal inhibition of cell growth occurred at 10 nM ( Fig. 1C).



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  		Figure 1. The effect of PMA and hemin on K562 cell growth. Total cell number was determined during 8 days of continuous treatment with 10 ng/ml PMA ({bigtriangleup}) or 30 µM hemin ({circ}) (A, B). Effect of different concentrations of PMA ({bigtriangleup}) and hemin ({circ}) on K562 cell growth (C, D). Results represent three different experiments. Each point is the mean of three determinations.

PMA and hemin induce megakaryocytic and erythroid differentiation of K562 cells, respectively
The effect of PMA and hemin on megakaryocytic and erythroid differentiation of K562 cells was investigated both by morphological changes and by the expression of CD41 (GPIIIa) and benzidine staining, respectively. After 4 days in culture, more than 50% of the cells showed morphological changes characteristic of megakaryocytic differentiation ( Fig. 2). Numerous larger cells were clearly visible in PMA-treated culture, some of them adhering to the sub~stratum. This was also accompanied by an increase in CD41 expression (not shown). To assess erythroid differentiation in K562 cells, we measured benzidine staining after hemin treatment. In an untreated culture, the percentage of benzidine-positive cells was very low ( Fig. 3). There was, however, a small increase (2% at 0 time vs. 4% at 96 h) in the percentage of benzidine cells after 96 h in untreated cultures. In hemin-treated cells, the percentage of benzidine-positive cells increased significantly (15% positive cells at 96 h) ( Fig. 3). After 96 h in the presence of PMA, benzidine-positive cells were virtually undetectable in the cell culture. Finally, erythroid differentiation was already detectable at 5 µM hemin, whereas the maximal effect was observed with 30 µM hemin ( Fig. 3, inset).



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  		Figure 2. PMA induces megakaryocytic differentiation of K562 cells. K562 cells were cultured for 96 h in the presence of 10 ng/ml PMA or left untreated. Light microscopic analysis. x200.



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  		Figure 3. Effect of PMA and hemin on K562 cell differentiation. The fraction of benzidine-positive cells was determined as a function of time. Cells were treated for the time indicated in either the absence ({square}) or presence of 10 ng/ml PMA ({blacksquare}) or 30 µM hemin ({blacktriangleup}). Inset: The fraction of benzidine-positive cells was determined after 4 days of continuous treatment with various concentrations of hemin.

Expression of Kell and CD10 during differentiation of K562 by PMA and hemin
A representative flow cytometric analysis for the expression of Kell, CD10, and CD44 is shown in Fig. 4. CD44 expression (homing adhesion cell molecule) was used here as a positive control of PMA stimulation. CD44 expression drastically increased after 48 h in the presence of PMA, whereas hemin failed to affect expression of this antigen as compared to untreated cells. PMA treatment resulted in a significant increase in CD10 expression and a decrease in Kell expression after 48 h ( Fig. 4). Conversely, the addition of hemin in the cell culture medium was accompanied by an increase in Kell expression at the cell surface, with no change in CD10 expression ( Fig. 4). Altogether, these results indicate that two proteins belonging to the same family of enzymes are differentially regulated during the course of differentiation of K562 cells along the megakaryocytic or the erythroid pathway.



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  		Figure 4. Facs analysis of different markers. Undifferentiated or 72 h differentiated K562 cells were incubated with saturating amount of either anti-CD44, anti-CD10, or anti-Kell monoclonal antibodies (mAb's) or an isotype-matched (IgG1) irrelevant mouse monclonal antibody as negative control for 30 min at 4°C, followed by biotinylated rabbit anti-mouse F(ab')2 fragment of IgG and phycoerythrine-streptavidin. Analyses were performed by flow cytometry on a dual Facs Star Plus.

Expression of Kell and CD10 mRNAs in PMA- and hemin-treated cells
The effect of PMA and hemin on erythroid and megakaryocytic differentiation of K562 cells was further investigated by analysis of the expression of Kell and CD10 mRNA levels. Treatment of K562 cells with PMA strongly decreased the steady-state level of the 3.5 kb Kell transcript after 24 h of incubation ( Fig. 5). After 72 h, Kell mRNA expression was totally undetectable. Conversely, hemin increased the steady-state level of Kell mRNA after 48 h of treatment; thereafter, the level of Kell mRNA remained constant or slightly decreased ( Fig. 5). No difference was observed in total mRNA expression during the kinetic of induction of erythroid or megakaryocytic differentiation (not shown). We also checked for CD10 mRNA expression in K562 cells treated for 72 h in the presence of either PMA or hemin. CD10 mRNA was undetectable in either undifferentiated or 72 h hemin-treated K562 cells ( Fig. 6). By contrast, PMA induced expression of the two characteristic 3.5 and 5.8 kb CD10 transcripts (21), an effect that culminated after 72 h in culture. Finally, Fig. 6also shows that equal amounts of RNA were electrophoresed in each experimental condition.



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  		Figure 5. Expression of Kell mRNA in K562 cells treated with PMA or hemin. Cells were incubated for several days with 10 ng/ml PMA or 30 µM hemin, as indicated, and mRNA levels for Kell were determined by Northern blot hybridization as described in Materials and Methods.



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  		Figure 6. Expression of CD10 mRNA in K562 cells treated with PMA or hemin. Cells were incubated for 72 h with 10 ng/ml PMA or 30 µM hemin, as indicated, and mRNA levels for CD10 were determined by Northern blot hybridization (left panel). Right panel: ethidium bromide coloration of total mRNAs.

Expression of Kell and CD10 proteins during differentiation of K562 cells by PMA and hemin
Expression of Kell protein was assessed by Western blotting on membrane extracts prepared from undifferentiated K562 or cells treated with either PMA or hemin. Kell expression was then determined by using the specific monoclonal antibody 5A11 (29), which is directed against the cytoplasmic domain of Kell. Kell appeared as a 94–100 kDa doublet in undifferentiated K562 cells ( Fig. 7). Hemin significantly increased the amount of Kell protein expressed on the cell surface until 48 h, when the level of Kell declined slightly according to the experiment presented in Fig. 5. Increase in Kell expression was also accompanied by a reduction in the apparent molecular weight of the Kell protein. This can reflect abnormal Kell glycosylation in hemin-treated K562 cells, as is the case for glycophorin A and C (34, 35). PMA induced a drastic inhibition in Kell protein expression after 24 h in culture ( Fig. 7), an observation that correlates with the total down-regulation of Kell mRNA ( Fig. 5). A 38 kDa protein also recognized by the anti-Kell monoclonal antibody was consistently observed by Western blotting ( Fig. 7). Variation of the 38 kDa protein during K562 cell differentiation paralleled that of the 94–100 kDa Kell protein. As the 38 kDa protein is specifically recognized by the anti-Kell monoclonal antibody, it is likely to correspond to either a proteolytically modified or an alternatively spliced form of Kell. Evidence for the second hypothesis came from polymerase chain reaction experiments indicating that in addition to the full-length Kell transcript, a smaller 1.2 kb transcript was always amplified when using mRNAs prepared from undifferentiated K562 cells (not shown).



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  		Figure 7. Kell protein expression during K562 cell differentiation by PMA and hemin. Solubilized membrane proteins (200 µg) from PMA- or hemin-treated K562 cells were subjected to immunoprecipitation with anti-Kell mAb and electrophoresed on 8% SDS-PAGE. Molecular weight markers are also indicated. Arrows indicate the migration of Kell and that of a related 38 kDa protein.

CD10 expression and enzymatic activity during differentiation of K562 cells by PMA and hemin
CD10 protein expression in K562 cell membrane preparations was also assessed by Western blotting using the highly specific monoclonal anti-CD10 antibody (K50) (21). CD10 was barely detectable in untreated K562 cells ( Fig. 8). Hemin failed to increase the threshold level of CD10 expression whatever the duration of exposure ( Fig. 8). Conversely, treatment of cell culture with PMA induced a drastic increase in CD10 expression. The PMA effect was clearly visible at 24 h and culminated at 72 h ( Fig. 8). We next analyzed whether or not CD10 expressed on the surface of K562 cells was a functional molecule. CD10 enzymatic activity was assessed by the release of paranitroaniline from the tripeptide Suc-Ala-Ala-Phe-pNA in the presence of exogenous aminopeptidase N, a feature of neprilysin (21). Figure 9 shows that treatment of K562 cells with PMA increased CD10 enzymatic activity with a time course that parallels the accumulation of CD10 mRNA and protein ( Fig. 6and Fig. 8). Hemin failed to affect CD10 enzymatic activity, which remained undetectable whatever the duration of treatment. PMA's effect on CD10 enzymatic activity was dose dependent, and maximal stimulation occurred with 10 ng/ml PMA (not shown). Such a concentration was also found to inhibit totally K562 cell proliferation ( Fig. 1).



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  		Figure 8. CD10 expression during K562 cell differentiation by PMA and hemin. Solubilized membrane proteins (200 µg) from PMA- or hemin-treated K562 cells were subjected to immunoprecipitation with anti-CD10 mAb and analyzed as described in Fig 6. Arrow indicates the migration of CD10.



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  		Figure 9. Effect of hemin and PMA treatment on CD10 enzymatic activity in K562 cells. Cells were incubated for various times at 37°C in the absence or presence of either 10 ng/ml PMA ({square}) or 30 µM hemin ({circ}). For each incubation, cells were collected and extensively washed in PBS. Endopeptidase 24.11 activity was determined in the presence of 1 mM Suc-Ala-Ala-Phe-pNA and 10 µg/ml purified aminopeptidase N by the measure of absorbance of paranitroaniline at 410 nm. Results are representative of three different experiments. Each point is the mean of four determinations. CD10 activity represents the phosphoramidon inhibitable activity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The human erythroleukemic cell line K562 coexpresses erythroid and megakaryocytic genes, but treatment either with tumor-promoting phorbol esters such as PMA or with hemin shifts these cells toward megakaryocytic or erythroid pathways of differentiation, respectively (1–4). In both cases, differentiation is accompanied by the regulation of various gene products. Differentiation in the megakaryocytic pathway results in silencing of erythroid-specific genes such as {gamma}-globin and activation of megakaryocytic-specific genes (8). Conversely, differentiation in the erythroid pathways leads to up-reg~ulation of {gamma}-globin mRNA (8). The purpose of this study was to 1) investigate whether expression of the Kell blood group antigen, an erythrocyte-specific marker, is regulated during differentiation of the human erythroleukemic cell line K562 by PMA and hemin; and 2) compare the regulation of Kell expression with that of CD10, a related metalloendopeptidase, during the course of K562 cell differentiation.

The different antigens of the Kell blood group system are located on a 93 kDa red cell membrane glycoprotein that shows substantial sequence homologies with a family of metalloendopeptidases that also includes the CD10 antigen and endothelin-converting enzymes 1, 2, and 3 (14, 15, 28). Although Kell is constitutively found on the surface of K562 cells (9) and its level increased after stimulation by hemin as judged by Facs analysis, it was not known whether Kell expression was modulated at the mRNA level during differentiation of these cells by PMA or hemin. We show here that differentiation of K562 cells by PMA in the megakaryocytic pathway is accompanied by a total down-regulation of Kell mRNA accumulation and a concomitant loss of Kell expression on the cell surface. Indeed, the Kell protein became virtually undetectable on the cell surface after 2 days in the presence of PMA.

The disappearance of Kell reactivity at the cell surface correlates well with the appearance of morphological changes characteristic of megakaryocytic differentiation and up-regulation of CD41 molecules ( Fig. 2and not shown). A decrease in the expression of {gamma}-globin, platelet-derived growth factor, and topoisomerase II during PMA differentiation of K562 cells has previously been reported (8, 36, 37). Thus, in addition to {gamma}-globin, Kell represents another example of an erythroid-specific gene whose expression is abolished during differentiation of K562 cells along the megakaryocytic lineage. Conversely, we show that differentiation of K562 cells by hemin in the erythroid pathway results in up-regulation of Kell mRNA accumulation and protein expression on the cell surface. Western blot analysis of membrane preparations from hemin-treated cells indicates selective up-regulation of a Kell protein isoform that migrates faster than the Kell protein immunoprecipitated in undifferentiated K562 cells. Whether or not these different isoforms result from posttranscriptional or posttranslational modifications due to erythroid differentiation remains to be established. However, abnormal protein glycosylation during differentiation of K562 cells by hemin has previously been reported for glycophorin A and C (34, 35).

The results concerning up-regulation of CD10 expression and enzymatic activity upon PMA treatment reported in this study were completely unexpected. K562 cells have previously been shown to lack expression of CD10 (21). This observation was confirmed in the present study because we found that undifferentiated K562 cells failed to express CD10 mRNA and enzymatic activity, even though CD10 protein is barely detectable by Western blot experiments when high amounts of solubilized membrane proteins are used. To the best of our knowledge, such an increase in CD10 protein and transcripts upon phorbol ester treatment is unique. Indeed, in all the cellular models examined to date, phorbol esters have been shown to inhibit CD10 mRNA accumulation. For example, phorbol ester treatment of rabbit synovial fibroblasts and mammary epithelials cells (38), human neutrophils (39), acute lymphoblastic leukemia cells (40, 41), and human thymocytes (26) reduces CD10 transcripts and proteins, whereas other effectors such as glucocorticoids, GM-CSF, and C5a increase CD10 expression (42, 43). CD10 promoter contains two separate regulatory elements that appear to control tissue-specific expression of CD10 (44). Regulatory region 1 contains multiple putative PU.1 binding sites and the ets binding motif. Regulation region 2 contains Sp1 binding sites and a potential consensus retinoblastoma control element (44). These observations are of particular interest, since 1) increased expression of the ets-related transcription factor FLI-1/ERGB has been associated with phorbol ester-induced megakaryocytic differentiation (45) and 2) Sp1 has recently been shown to control thromboxane receptor expression during megakaryocytic differentiation (46). From these observations, it is tempting to speculate that the presence of both ets-related and Sp-1 binding sites in the CD10 promoter may contribute to increased expression of CD10 during differentiation of K562 cells in the megakaryocytic lineage.

CD10 functions to cleave small peptides such as enkephalin, f-MLP, substance P, endothelin, bradikynin, angiotensin, and bombesin (18, 19) and therefore controls the response of numerous important biological peptides in different cell types. CD10 has been involved in physiological processes such as enkephalin-induced analgesia (47), chemotaxis, and migration of neutrophils (48, 49), inhibition of bombesin-induced growth of normal bronchial epithelial cells, and small cell carcinomas of the lung (50, 51). In the hematopoietic system, CD10 regulates the production of interleukin 2 in acute lymphoblastic leukemia cell lines (21, 52) and controls the growth and maturation of B cell progenitors (23, 24), early stages of thymocyte development (26, 27), and the proliferation of human thymic epithelial cells (53). It is not known currently whether CD10 expression represents a consequence or is involved in the megakaryocytic differentiation program. A point of particular interest in our study is the opposite regulation of Kell and CD10 expression during differentiation along the megakaryocytic or erythroid pathways. Although no enzymatic activity has so far been associated with the Kell protein despite a high degree of conservation of amino acids thought to be important for metalloendopeptidase activity, it is possible that loss of Kell protein expression together with a gain in CD10 activity might be critical for megakaryocytic development. Thus, a greater amount of CD10 at the cell surface relative to that of Kell might be important for megakaryocytic differentiation, whereas a greater amount of Kell combined with no expression of CD10 might contribute to shifting the cell along the erythroid lineage. Although speculative, this hypothesis is attractive since the main function of ectopeptidases such as CD10 is the degradation and maturation of bioactive peptides that might be involved in K562 cell differentiation.

In conclusion, our results demonstrate that the CD10 and Kell antigens, two related membrane-bound metallendopeptidases, can be considered as specific markers of the differentiation of K562 cells along the megakaryocytic and erythroid pathways respectively. It remains to be established, however, whether differential regulation of these metallopeptidases is involved in or rather represents a consequence of the differentiation program. Finally, study of the potential role of CD10 in this processs could be addressed by analyzing the effect of specific and long-lasting CD10 inhibitors on the differentiation of K562 cells.


   ACKNOWLEDGMENTS
 
We thank Aurore Grima for artwork. This work was supported by INSERM and by grants from the Federation des Entreprises Françaises dans la Lutte contre le Cancer, the Ligue Nationale contre le Cancer, and the Association Recherche et Transfusion.


   FOOTNOTES
 
1 Current address: INSERM U364, Faculté de Médecine, Ave. de Vallombrose, 06107 Nice Cedex 2, France.

2 Current address: Division of Hematologic Malignancies. Dana Farber Cancer Institute, 44, Binney St., Boston, MA 02115, USA.

1 Correspondence: CJF INSERM 96. 05, Faculté de Medecine, Ave. de Vallombrose 06107 Nice Cedex 2, Paris, France. E-mail: auberger{at}unice.fr

4 Abbreviations: XTT assay, tetrazolium-bromide precipitation assay; PMA, phorbol-12 myristate-13 acetate; PKC, protein kinase C; FCS, fetal calf serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Received for publication October 24, 1997. Accepted for publication December 15, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Martin, P., and Papayannopoulou, T. (1982) HEL cells: a new human erythroleukemia cell line with spontaneous and induced globin expression. Science 216, 1233–1235[Abstract/Free Full Text]
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