Leukemia & Lymphoma
ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Modulation of GM-CSF Receptor β-subunit and Interleukin-6 Receptor mRNA Expression in a Human Megakaryocytic Leukemia Cell Line Mutsumi Yasunaga, Ryukichi Ryo & Nobuo Yamaguchi To cite this article: Mutsumi Yasunaga, Ryukichi Ryo & Nobuo Yamaguchi (1992) Modulation of GM-CSF Receptor β-subunit and Interleukin-6 Receptor mRNA Expression in a Human Megakaryocytic Leukemia Cell Line, Leukemia & Lymphoma, 8:4-5, 397-403, DOI: 10.3109/10428199209051020 To link to this article: http://dx.doi.org/10.3109/10428199209051020
Published online: 01 Jul 2009.
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Modulation of GM-CSF Receptor P-subunit and Interleukin-6 Receptor mRNA Expression in a Human Megakaryocytic Leukemia Cell Line MUTSUMI YASUNAGA', RYUKICHI RYO's2 and NOBUO YAMAGUCHI' 'Department of Laboratory Medicine, Kobe University School of Medicine, and 'Blood Transfusion Service, Kobe University Hospital, Kobe, Japan (Received 6 May 1992)
Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of megakaryocyte development. We examined the changes in mRNA expression of granulocyte macrophage colony-stimulating factor receptor P-subunit (GM-CSFR P-subunit), which was a common subunit of a high-affinity interleukin-3 receptor (IL-3R) and a high-affinity GM-CSFR, and interleukin-6 receptor (IL-6R) during megakaryocyte development in a human megakaryocytic leukemia cell line (CMK) which could proliferate and/or differentiate in the presence of 12-0-tetradecanoylphorbol 13-acetate (TPA), IL-3, GM-CSF, and IL-6. We found that GM-CSFR P-subunit mRNA was expressed constitutively in C M K cells and was transiently down-regulated by TPA and IL-6, while the expression of IL-6R mRNA was increased by TPA in association with the differentiation of megakaryocytes. Furthermore, the TPA-induced down-regulation of GM-CSFR P-subunit mRNA expression and its recovery were blocked by cycloheximide (CHX), a protein synthesis inhibitor, suggesting that these modulations required de nouo protein synthesis. These findings imply that multi-lineage cytokines such as GM-CSF and IL-3 may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors, while 1L-6 may be limited in its action to supporting the maturation of more differentiated megakaryocyte progenitor cells. The present study provides evidence that regulation of the expression of cytokine receptor mRNA, which results in changes in receptor densities, may be one of the mechanisms for modulating the responsiveness of megakaryocyte progenitor cells to different cytokines during megakaryocyte development. KEY WORDS:
Granulocyte-macrophage colony-stimulating factor receptor Interleukin-6 receptor mRNA Human megakaryocytic cells
INTRODUCTION Megakaryocytopoiesis is thought to be regulated by the interactions of many different cytokines. Recent studies have shown that multi-lineage cytokines such Address for correspondence: Mutsumi Yasunaga, MD, Department of Laboratory Medicine, Kobe University school of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650, Japan.
as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) act as stimulators in supporting the early development of megakaryocyte progenitor cells'-4, whereas interleukin-6 (IL-6) and interleukin-1 1 (IL-11) induce the terminal differentiation of megakaryocyte progenitor cells including nuclear polyploidization and cytoplaSmiC These findings are Consistent with the two-factor regulatory system proposed by
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Burstein et aL9 and Williams et a/.", in which early and late events during megakaryocyte development are independently controlled by a megakaryocyte colony-stimulating factor (MK-CSF) and a megakaryocyte potentiator factor (Meg-pot, so-called thrombopoietin). However, little is known about the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of maturation. As the biological action of each cytokine on target cells is mediated by a cytokine specific receptor, it is reasonable to postulate that the earlier progenitor cells may express the receptors for multi-lineage cytokines, while the progenitor cells committed to megakaryocytic lineage may possess receptors for IL-6 and IL-11. A cytokine-dependent megakaryocytic cell line should therefore be useful in studying the roles of cytokine receptor mRNA expression during megakaryocyte development. CMK cells, which have been'established by Sato et u1.l' and were used in the present study, differentiate into morphologically and immunologically more mature megakaryocytes under the action of 12-0-tetradecanoylphorbol 13-acetate (TPA), a potent stimulator'2.'3. Furthermore, the proliferation and/or differentiation of this cell line is enhanced by IL-3, IL-6, and GM-CSF'4*' '. Recent molecular cloning of cDNAs encoding a human GM-CSF receptor (GM-CSFR)' 6 , 1 ', IL-3 receptor (IL-3R)lX, and IL-6 receptor (IL-6R)19 has also made it possible to investigate the regulation of these receptors at the mRNA level. To clarify the mechanism of the two-factor regulatory system in megakaryocytopoiesis, the changes in GM-CSFR j-subunit and IL-6R mRNA levels were examined during the differentiation of CMK cells. We found that TPA and IL-6, which induced the differentiation of CMK cells, transiently down-regulated GM-CSFR /?-subunit mRNA expression, whereas TPA up-regulated IL-6R mRNA expression during the course of differentiation. Our findings suggest that megakaryocyte development may be controlled not only by the concentration of responsible cytokines, but also by the corresponding receptor densities on megakaryocyte progenitor cells. MATERIALS AND METHODS Biological reagents and cDNAs
Recombinant human IL-6 was kindly donated by Central Research Laboratories, Ajinomoto Co., Inc. (Yokohama, Japan). TPA and cycloheximide (CHX)
were obtained from Wako Pure Chemicals Industries., Ltd. (Osaka, Japan). GM-CSFR /?-subunit cDNA16 was a generous gift from Dr. A. Miyajima (DNAX Research Institute, Palo Alto, CA). IL-6R cDNALY was kindly provided by Dr. T. Hirano (Osaka University, Osaka, Japan). Glycoprotein IIb (GPIIb) cDNA20 and rat a-tubulin cDNAZ1 were kindly provided by Dr. M. Poncz (The Children's Hospital of Philadelphia, Philadelphia, PA), and Dr. S. Farmer (Boston University, Boston, MA), respectively. Cells and culture
CMK, a human megakaryocytic leukemia cell line22 was maintained in an atmosphere of 5 % CO, at 37°C and cultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories., Inc., Logan, Utah). In some experiments, cells were treated with TPA, CHX, or IL-6 at the optimal concentrations of 10 nM, 10 pg/ml, and 300 U/ml, respectively. For stimulation the cells were grown to approximately 3-5 x lo5 cells/ml before TPA, CHX, or IL-6 was added. RNA purification and Northern blot analysis Northern blot analysis was performed according to the procedures described p r e v i o u ~ l y ~Total ~ . RNA was isolated from 5 x lo7 to lo8 cells by a single-step method24. Total RNA concentrations were estimated from the optical density (OD) at 260 nm. The integrity of samples was confirmed after electrophoresis by ethidium bromide staining of the 28s and 18s ribosomal RNA. RNA was fractioned by formaldehyde/agarose gel electrophoresis, followed by Northern blot transfer to Gene Screen Plus (New England Nuclear Research Products, Boston, MA). Fragments of cDNAs were random-primed with x - ~ ~ P dCTP (Amersham Japan Limited, Tokyo, Japan) for use in hybridization. GPIIb cDNA probe was used as a typical marker of megakaryocyte differentiation, and a-tubulin cDNA probe was used to confirm equivalent gel-loading conditions. Membranes were prehybridized and hybridized overnight at 42°C in a buffer containing 50% deionized formamide, 5 x SSPE, 0.5% SDS, 250pg/ml sonicated salmon sperm DNA, and 5 x Denhardt's solution. The membranes were washed at room temperature in 2 x SSPE and 0.5% SDS for 30 min and then at 58°C in 0.4 x SSPE and 0.5% SDS for 30 min.
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Figure 1 Time-dependent effects of TPA on GM-CSFR 8-subunit, IL-6R, and GPIIb mRNAs in C M K cells. Cells were incubated in the presence or absence of TPA (10 nM). Total RNA was extracted at the indicated times of culture. Northern blot analysis was performed with the GM-CSFR 8-subunit, IL-6R, GPIIb, and a-tubulin cDNA probes as described in “MATERIALS AND METHODS”. A, GM-CSFR /?-subunit; B, IL-6R; C, GPIIb. In A, B, and C, each lane contained 20 pg, 40 pg, 40 pg of total RNA, respectively. Equivalent gel-loading conditions were confirmed using a-tubulin cDNA probe (shown at the bottom in each figure).
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Figure 2 Kinetics of the levels of mRNA expression of GM-CSFR 8-subunit, IL-6R, GPIIb, and a-tubulin induced by addition of TPA to CMK cells. Autoradiographs shown in Figure 1 were densitometrically scanned for quantitation of each mRNA expression. Densitometry values obtained from samples in the absence of TPA were designated as 100% and used for comparison of samples with each other. (O),GM-CSFR p-subunit; (O),IL-6R; (a),GPIIb; (m), a-tubulin.
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and exposed to x-ray films at -80°C with intensifying screens. The levels of autoradiographic signals were scored by densitometric scanning using a Fast Scan 300A (Molecular Dynamics., Sunny Vale, CA).
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Time-dependent effects of TPA on GM-CSFR /?-subunit,IL-6R, and GPIIb mRNA levels in CMK cells As shown in Figure 1, significantly high amounts of GM-CSFR P-subunit mRNA and low amounts of IL-6R mRNA were expressed constitutively in CMK cells. GPIIb mRNA, a typical marker of megakaryocyte differentiation, was detected in the cells cultured in the absence of TPA, and its level was increased by TPA addition (10 nM) in a timedependent fashion up to 48 h (Figure 1). To determine whether expression of GM-CSFR and IL-6R mRNAs was altered in CMK cells treated with TPA, the changes in GM-CSFR P-subunit and IL-6R mRNA levels were observed by Northern blot analysis. GM-CSFR J-subunit mRNA level was decreased rapidly within 4 h after the addition of TPA as compared with that in the untreated cells, with a return to the baseline level by 48 h, but the IL-6R mRNA level had increased after the TPA treatment up to 24 h. Under the same conditions, a-tubulin mRNA level was not changed during the TPAinduced differentiation of CMK cells. These mRNA levels in the experiments were scored with a laser densitometer and the results are illustrated in Figure 2.
Effects of CHX treatment on the TPA-induced changes of GM-CSFR /?-subunitmRNA level in CMK cells To examine the effects of CHX, a protein synthesis inhibitor, on the TPA-induced reduction of GMCSFR p-subunit mRNA and its recovery, CHX (lOpg/ml) was added to the cultures either alone or in combination with TPA. As shown in Figure 3, the TPA-induced down-regulation of GM-CSFR /?subunit mRNA expression in CMK cells was blocked by preincubation with CHX 45 min prior to TPA addition. Furthermore, the recovery of GM-CSFR /?-subunit mRNA level observed by 48 h after TPA addition was also blocked by treatment with CHX. CHX alone did not affect the GM-CSFR mRNA expression. Under the same conditions, a-tubulin mRNA level was not affected by addition of TPA and/or CHX to CMK cells.
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Figure 3 Effects of CHX treatment on GM-CSFR /]-subunit mRNA in C M K cells. C M K cells were incubated in the presence or absence of TPA (10 nM). CHX (10 pg/ml) was added to the cultures either alone or in combination with TPA. Total RNA (20 pg/lane) was sequentially hybridized to GM-CSFR b-subunit or r-tubulin cDNA. Equivalent gel-loading conditions were confirmed using a-tubulin cDNA probe (shown at the bottom in A). Autoradiographs shown in A were densitometrically scanned for quantitation of each mRNA expression as shown in B. Densitometry value obtained from sample in medium alone was designated as 100% and used for comparison of samples with each other. Lane 1, cells cultured in medium alone; lane 2, cells cultured in the presence of TPA for 4 h; lane 3,cells cultured in the presence of TPA for 48 h; lane 4, cells preincubated with CHX 45 min prior to TPA addition, and cultured for 4 h in the presence of TPA; lane 5, cells cultured for 4 h in the presence of TPA, treated with CHX at 4 h, and cultured for 48 h; lane 6, cells cultured for 48 h with CHX alone.
Effects of IL-6 on GM-CSFR /?-subunit,ILdR, and GPIIb mRNA levels in CMK cells GM-CSFR 8-subunit mRNA level was decreased within 24 h after stimulation by IL-6 (300 U/ml) as
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Figure 4 Effects of IL-6 on GM-CSFR P-subunit, IL-6R, and GPIIb mRNAs in C M K cells. Cells were incubated in the presence or absence of IL-6 (300 U/mlj. Total RNA was extracted at the indicated times of culture. Northern blot analysis was performed with GM-CSFR 8-subunit, IL-6R, GPIIb, and a-tubuin cDNA probes as described in "MATERIALS AND METHODS". A, GM-CSFR p-subunit; B, IL-6R: C, GPIIb. In A, B, and C, each lane contained 20pg, 2 0 p g , and 40pg of total RNA, respectively. Equivalent gel-loading conditions were confirmed using a-tubulin cDNA probe (shown at the bottom in each figure).
compared with that in the untreated cells, and returned to the baseline level by 48 h. On the other hand, no significant changes were observed in the level of IL-6R mRNA expression by IL-6 (Figure 4). GPIIb mRNA level was increased by stimulation of IL-6 in a time-dependent fashion up to 48 h. Under the same conditions, a-tubulin mRNA level was not changed.
DISCUSSION Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of maturation. The ability of a cytokine to exert its biological effect is believed to by linked to the expression of the corresponding receptors on cytokine-responsive target cells. Thus the binding of a cytokine to target cells through specific receptors is assumed to be an initial and important event in the regulation of cellular responses. The two-factor regulatory system in megakaryocytopoiesisl 6 . 1 7 may function through the modification of the cytokine-sensitive receptor densities expressed on
megakaryocyte progenitor cells during maturation. In general, the characteristics of cytokine-specific receptors on target cells have been analyzed by observing the pattern of binding of 251-labelled ligands to cells. In our preliminary experiments, neither the apparent numbers of receptors on each CMK cell nor Kd values for GM-CSFR, IL-3R, and IL-6R could be estimated by using 251-labelled GM-SCF (13 TBq/mmol), '251-labelled IL-3 (19 TBq/mmol), and '251-labelled IL-6 (37 TBq/mmol). The reason why the binding assay did not work in this case remains unclear. The numbers of these cytokine receptors on CMK cells may have been too low for detection by the ligands used, or the ligands may have been too rapidly degradated and released by the cells. More sophisticated study will be required to resolve it. However, recent cloning of cDNA encoding GM-CSFR16.", IL-3R" and IL-6R19 allowed us to investigate the regulation of these receptors at the mRNA level. Recently, Miyajima and his colleagues have domonstrated that IL-3R and GM-CSFR share a common /%subunit,which is an essential component to reconstitute high-affinity GM-CSFR and highaffinity IL-3R'69'8. In the present study, the cDNA
M. YASUNAGA, R. RYO AND N. YAMAGUCHI
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encoding the common b-subunit was used to investigate the mechanisms of responses of the earlier megakaryocyte progenitor cells to multi-lineage cytokines such as GM-CSF and IL-3. In addition, cDNA encoding IL-6R was used to study the mechanism of the preferential response of the later megakaryocyte progenitor cells to IL-6. GM-CSFR b-subunit mRNA was found to be expressed constitutively in CMK cells. TPA, which is known to promote the development of CMK cells into the more differentiated megakaryocytes’ was found to downregulate GM-CSFR /-subunit mRNA expression and up-regulate IL-6R mRNA expression in CMK cells. Furthermore, the rapidly down-regulated expression of GM-CSFR /-subunit mRNA gradually recovered to the baseline level, and the recovery appeared to correlate with the increase of GPIIb mRNA level, a typical differential marker of megakaryoFytes. This finding suggests that the responsiveness of megakaryocyte progenitor cells to multi-lineage cytokines may be transiently decreased by reduced receptor densities and multi-lineage cytokines may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors. It should be also noted that the TPA-induced downregulation of GM-CSF ,&subunit mRNA expression is a transient phenomenon, followed by recovery, thus indicating that the reduced responsiveness to multilineage cytokines may be recovered through differentiation, and that multi-lineage cytokines may also be involved in the regulation of the later stage of megakaryocyte differentiation. Previous studies showed that phorbol ester such as TPA downregulated the expression of GM-CSFR in human myeloid ~ e l l s ~Cannistra ~ - ~ ~ . rt uI. provided evidence that phorbol ester exerts the down-regulatory action through rapid internalization of the receptors”. However, our present data demonstrates for the first time that TPA-induced down-regulation of GMCSFR may be partly dependent on the reduced expression of GM-CSFR mRNA. TPA is known to exert its biological action through activation of a protein kinase C2*, but it seems unlikely that this activation directly down-regulates the expression of GM-CSFR b-subunit mRNA. To clarify the mechanism of TPA-induced modulation of GM-CSFR b-subunit mRNA, we observed the effect of CHX, a protein synthesis inhibitor, on the TPA-induced down-regulation and its recovery. Preincubation with CHX resulted in abolition of the down-regulation of GM-CSFR b-subunit mRNA expression; and CHX
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’,
also blocked the recovery from the TPA-induced down-regulation. These results suggest that TPAinduced modulation of GM-CSFR /-subunit mRNA expression requires de novo protein synthesis. IL-6R mRNA, on the other hand, was expressed in small amounts in CMK cells in the absence of TPA; and on addition of TPA the expression increased, suggesting that IL-6 appears to be limited in its action to supporting the maturation of the more differentiated megakaryocyte progenitor cells. TPA is not a physiological regulator for megakaryocytopoiesis. Neither GM-CSF nor IL-6 is known to exert its biological action through a protein Therefore, the kinase C dependent effects of IL-6, a physiological stimulator, on the expression of GM-CSFR, IL-6R, and GPIIb mRNAs were examined in order to clarify the in vivo mechanism of megakaryocytopoiesis. Interestingly, IL-6 was found to possess a similar capacity to TPA for the down-regulation of GM-CSFR p-subunit mRNA expression. This data also confirms our hypothesis that the responsiveness of megakaryocyte progenitor cells to several cytokines during differentiation may depend in part on the receptor densities. Further direct analysis of the relationship between ligand-receptor binding and proliferative responses will be required to define the exact relationship between the densities of GM-CSFR and IL-6R, and the sensitivity of cells to multi-lineage cytokines such as GM-CSF and IL-3, and to IL-6, during megakaryocyte maturation. Our present molecular study, however, provides the first evidence that control of the expression of cytokine receptor mRNA, which would result in changes in receptor densities, may be one of the mechanisms for modulating the responsiveness of megakaryocyte progenitor cells to different cytokines during megakaryocyte development. REFERENCES 1. Quesenberry, P. J., Ihle, J. N . and McGrath, E. (1985).The effect of interleukin 3 and GM-CSA-2 on megakaryocyte and myeloid clonal colony formation. Blood, 65, 2 1 4 7 . 2. Mazur, E. M., Cohen, J. L., Wong, G. G. and Clark, S. C. (1987). Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells. Exp. Hemutol., 15, 1128-33. 3. Teramura, M., Katahira, J., Hoshino, S., Motoji, T., Oshimi, K. and Mizoguchi, H. (1989).Effect of recombinant hemopoietic growth factors on human megakaryocyte colony formation in serum-free cultures. Exp. Hemutol., 17, 101 1-6. 4. Bruno, E., Miller, M. E. and Hoffman, R. (1989). Interacting cytokines regulate in vitro human megakaryocytopoiesis. Blood, 73, 611-7.
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ISSN: 1042-8194 (Print) 1029-2403 (Online) Journal homepage: http://www.tandfonline.com/loi/ilal20
Modulation of GM-CSF Receptor β-subunit and Interleukin-6 Receptor mRNA Expression in a Human Megakaryocytic Leukemia Cell Line Mutsumi Yasunaga, Ryukichi Ryo & Nobuo Yamaguchi To cite this article: Mutsumi Yasunaga, Ryukichi Ryo & Nobuo Yamaguchi (1992) Modulation of GM-CSF Receptor β-subunit and Interleukin-6 Receptor mRNA Expression in a Human Megakaryocytic Leukemia Cell Line, Leukemia & Lymphoma, 8:4-5, 397-403, DOI: 10.3109/10428199209051020 To link to this article: http://dx.doi.org/10.3109/10428199209051020
Published online: 01 Jul 2009.
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Date: 05 November 2015, At: 14:12
0 1992 Harwood Academic Publishers GmbH
Leukemia and Lymphoma, Vol. 8, pp. 397-403 Reprints available directly from the publisher Photocopying permitted by license only
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Modulation of GM-CSF Receptor P-subunit and Interleukin-6 Receptor mRNA Expression in a Human Megakaryocytic Leukemia Cell Line MUTSUMI YASUNAGA', RYUKICHI RYO's2 and NOBUO YAMAGUCHI' 'Department of Laboratory Medicine, Kobe University School of Medicine, and 'Blood Transfusion Service, Kobe University Hospital, Kobe, Japan (Received 6 May 1992)
Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of megakaryocyte development. We examined the changes in mRNA expression of granulocyte macrophage colony-stimulating factor receptor P-subunit (GM-CSFR P-subunit), which was a common subunit of a high-affinity interleukin-3 receptor (IL-3R) and a high-affinity GM-CSFR, and interleukin-6 receptor (IL-6R) during megakaryocyte development in a human megakaryocytic leukemia cell line (CMK) which could proliferate and/or differentiate in the presence of 12-0-tetradecanoylphorbol 13-acetate (TPA), IL-3, GM-CSF, and IL-6. We found that GM-CSFR P-subunit mRNA was expressed constitutively in C M K cells and was transiently down-regulated by TPA and IL-6, while the expression of IL-6R mRNA was increased by TPA in association with the differentiation of megakaryocytes. Furthermore, the TPA-induced down-regulation of GM-CSFR P-subunit mRNA expression and its recovery were blocked by cycloheximide (CHX), a protein synthesis inhibitor, suggesting that these modulations required de nouo protein synthesis. These findings imply that multi-lineage cytokines such as GM-CSF and IL-3 may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors, while 1L-6 may be limited in its action to supporting the maturation of more differentiated megakaryocyte progenitor cells. The present study provides evidence that regulation of the expression of cytokine receptor mRNA, which results in changes in receptor densities, may be one of the mechanisms for modulating the responsiveness of megakaryocyte progenitor cells to different cytokines during megakaryocyte development. KEY WORDS:
Granulocyte-macrophage colony-stimulating factor receptor Interleukin-6 receptor mRNA Human megakaryocytic cells
INTRODUCTION Megakaryocytopoiesis is thought to be regulated by the interactions of many different cytokines. Recent studies have shown that multi-lineage cytokines such Address for correspondence: Mutsumi Yasunaga, MD, Department of Laboratory Medicine, Kobe University school of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650, Japan.
as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) act as stimulators in supporting the early development of megakaryocyte progenitor cells'-4, whereas interleukin-6 (IL-6) and interleukin-1 1 (IL-11) induce the terminal differentiation of megakaryocyte progenitor cells including nuclear polyploidization and cytoplaSmiC These findings are Consistent with the two-factor regulatory system proposed by
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Burstein et aL9 and Williams et a/.", in which early and late events during megakaryocyte development are independently controlled by a megakaryocyte colony-stimulating factor (MK-CSF) and a megakaryocyte potentiator factor (Meg-pot, so-called thrombopoietin). However, little is known about the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of maturation. As the biological action of each cytokine on target cells is mediated by a cytokine specific receptor, it is reasonable to postulate that the earlier progenitor cells may express the receptors for multi-lineage cytokines, while the progenitor cells committed to megakaryocytic lineage may possess receptors for IL-6 and IL-11. A cytokine-dependent megakaryocytic cell line should therefore be useful in studying the roles of cytokine receptor mRNA expression during megakaryocyte development. CMK cells, which have been'established by Sato et u1.l' and were used in the present study, differentiate into morphologically and immunologically more mature megakaryocytes under the action of 12-0-tetradecanoylphorbol 13-acetate (TPA), a potent stimulator'2.'3. Furthermore, the proliferation and/or differentiation of this cell line is enhanced by IL-3, IL-6, and GM-CSF'4*' '. Recent molecular cloning of cDNAs encoding a human GM-CSF receptor (GM-CSFR)' 6 , 1 ', IL-3 receptor (IL-3R)lX, and IL-6 receptor (IL-6R)19 has also made it possible to investigate the regulation of these receptors at the mRNA level. To clarify the mechanism of the two-factor regulatory system in megakaryocytopoiesis, the changes in GM-CSFR j-subunit and IL-6R mRNA levels were examined during the differentiation of CMK cells. We found that TPA and IL-6, which induced the differentiation of CMK cells, transiently down-regulated GM-CSFR /?-subunit mRNA expression, whereas TPA up-regulated IL-6R mRNA expression during the course of differentiation. Our findings suggest that megakaryocyte development may be controlled not only by the concentration of responsible cytokines, but also by the corresponding receptor densities on megakaryocyte progenitor cells. MATERIALS AND METHODS Biological reagents and cDNAs
Recombinant human IL-6 was kindly donated by Central Research Laboratories, Ajinomoto Co., Inc. (Yokohama, Japan). TPA and cycloheximide (CHX)
were obtained from Wako Pure Chemicals Industries., Ltd. (Osaka, Japan). GM-CSFR /?-subunit cDNA16 was a generous gift from Dr. A. Miyajima (DNAX Research Institute, Palo Alto, CA). IL-6R cDNALY was kindly provided by Dr. T. Hirano (Osaka University, Osaka, Japan). Glycoprotein IIb (GPIIb) cDNA20 and rat a-tubulin cDNAZ1 were kindly provided by Dr. M. Poncz (The Children's Hospital of Philadelphia, Philadelphia, PA), and Dr. S. Farmer (Boston University, Boston, MA), respectively. Cells and culture
CMK, a human megakaryocytic leukemia cell line22 was maintained in an atmosphere of 5 % CO, at 37°C and cultured in RPMI 1640 medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% (v/v) fetal bovine serum (Hyclone Laboratories., Inc., Logan, Utah). In some experiments, cells were treated with TPA, CHX, or IL-6 at the optimal concentrations of 10 nM, 10 pg/ml, and 300 U/ml, respectively. For stimulation the cells were grown to approximately 3-5 x lo5 cells/ml before TPA, CHX, or IL-6 was added. RNA purification and Northern blot analysis Northern blot analysis was performed according to the procedures described p r e v i o u ~ l y ~Total ~ . RNA was isolated from 5 x lo7 to lo8 cells by a single-step method24. Total RNA concentrations were estimated from the optical density (OD) at 260 nm. The integrity of samples was confirmed after electrophoresis by ethidium bromide staining of the 28s and 18s ribosomal RNA. RNA was fractioned by formaldehyde/agarose gel electrophoresis, followed by Northern blot transfer to Gene Screen Plus (New England Nuclear Research Products, Boston, MA). Fragments of cDNAs were random-primed with x - ~ ~ P dCTP (Amersham Japan Limited, Tokyo, Japan) for use in hybridization. GPIIb cDNA probe was used as a typical marker of megakaryocyte differentiation, and a-tubulin cDNA probe was used to confirm equivalent gel-loading conditions. Membranes were prehybridized and hybridized overnight at 42°C in a buffer containing 50% deionized formamide, 5 x SSPE, 0.5% SDS, 250pg/ml sonicated salmon sperm DNA, and 5 x Denhardt's solution. The membranes were washed at room temperature in 2 x SSPE and 0.5% SDS for 30 min and then at 58°C in 0.4 x SSPE and 0.5% SDS for 30 min.
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Figure 1 Time-dependent effects of TPA on GM-CSFR 8-subunit, IL-6R, and GPIIb mRNAs in C M K cells. Cells were incubated in the presence or absence of TPA (10 nM). Total RNA was extracted at the indicated times of culture. Northern blot analysis was performed with the GM-CSFR 8-subunit, IL-6R, GPIIb, and a-tubulin cDNA probes as described in “MATERIALS AND METHODS”. A, GM-CSFR /?-subunit; B, IL-6R; C, GPIIb. In A, B, and C, each lane contained 20 pg, 40 pg, 40 pg of total RNA, respectively. Equivalent gel-loading conditions were confirmed using a-tubulin cDNA probe (shown at the bottom in each figure).
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Figure 2 Kinetics of the levels of mRNA expression of GM-CSFR 8-subunit, IL-6R, GPIIb, and a-tubulin induced by addition of TPA to CMK cells. Autoradiographs shown in Figure 1 were densitometrically scanned for quantitation of each mRNA expression. Densitometry values obtained from samples in the absence of TPA were designated as 100% and used for comparison of samples with each other. (O),GM-CSFR p-subunit; (O),IL-6R; (a),GPIIb; (m), a-tubulin.
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and exposed to x-ray films at -80°C with intensifying screens. The levels of autoradiographic signals were scored by densitometric scanning using a Fast Scan 300A (Molecular Dynamics., Sunny Vale, CA).
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Time-dependent effects of TPA on GM-CSFR /?-subunit,IL-6R, and GPIIb mRNA levels in CMK cells As shown in Figure 1, significantly high amounts of GM-CSFR P-subunit mRNA and low amounts of IL-6R mRNA were expressed constitutively in CMK cells. GPIIb mRNA, a typical marker of megakaryocyte differentiation, was detected in the cells cultured in the absence of TPA, and its level was increased by TPA addition (10 nM) in a timedependent fashion up to 48 h (Figure 1). To determine whether expression of GM-CSFR and IL-6R mRNAs was altered in CMK cells treated with TPA, the changes in GM-CSFR P-subunit and IL-6R mRNA levels were observed by Northern blot analysis. GM-CSFR J-subunit mRNA level was decreased rapidly within 4 h after the addition of TPA as compared with that in the untreated cells, with a return to the baseline level by 48 h, but the IL-6R mRNA level had increased after the TPA treatment up to 24 h. Under the same conditions, a-tubulin mRNA level was not changed during the TPAinduced differentiation of CMK cells. These mRNA levels in the experiments were scored with a laser densitometer and the results are illustrated in Figure 2.
Effects of CHX treatment on the TPA-induced changes of GM-CSFR /?-subunitmRNA level in CMK cells To examine the effects of CHX, a protein synthesis inhibitor, on the TPA-induced reduction of GMCSFR p-subunit mRNA and its recovery, CHX (lOpg/ml) was added to the cultures either alone or in combination with TPA. As shown in Figure 3, the TPA-induced down-regulation of GM-CSFR /?subunit mRNA expression in CMK cells was blocked by preincubation with CHX 45 min prior to TPA addition. Furthermore, the recovery of GM-CSFR /?-subunit mRNA level observed by 48 h after TPA addition was also blocked by treatment with CHX. CHX alone did not affect the GM-CSFR mRNA expression. Under the same conditions, a-tubulin mRNA level was not affected by addition of TPA and/or CHX to CMK cells.
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Figure 3 Effects of CHX treatment on GM-CSFR /]-subunit mRNA in C M K cells. C M K cells were incubated in the presence or absence of TPA (10 nM). CHX (10 pg/ml) was added to the cultures either alone or in combination with TPA. Total RNA (20 pg/lane) was sequentially hybridized to GM-CSFR b-subunit or r-tubulin cDNA. Equivalent gel-loading conditions were confirmed using a-tubulin cDNA probe (shown at the bottom in A). Autoradiographs shown in A were densitometrically scanned for quantitation of each mRNA expression as shown in B. Densitometry value obtained from sample in medium alone was designated as 100% and used for comparison of samples with each other. Lane 1, cells cultured in medium alone; lane 2, cells cultured in the presence of TPA for 4 h; lane 3,cells cultured in the presence of TPA for 48 h; lane 4, cells preincubated with CHX 45 min prior to TPA addition, and cultured for 4 h in the presence of TPA; lane 5, cells cultured for 4 h in the presence of TPA, treated with CHX at 4 h, and cultured for 48 h; lane 6, cells cultured for 48 h with CHX alone.
Effects of IL-6 on GM-CSFR /?-subunit,ILdR, and GPIIb mRNA levels in CMK cells GM-CSFR 8-subunit mRNA level was decreased within 24 h after stimulation by IL-6 (300 U/ml) as
MODULATION OF CYTOKINE RECEPTOR mRNAs in MEGAKARYOCYTES
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Figure 4 Effects of IL-6 on GM-CSFR P-subunit, IL-6R, and GPIIb mRNAs in C M K cells. Cells were incubated in the presence or absence of IL-6 (300 U/mlj. Total RNA was extracted at the indicated times of culture. Northern blot analysis was performed with GM-CSFR 8-subunit, IL-6R, GPIIb, and a-tubuin cDNA probes as described in "MATERIALS AND METHODS". A, GM-CSFR p-subunit; B, IL-6R: C, GPIIb. In A, B, and C, each lane contained 20pg, 2 0 p g , and 40pg of total RNA, respectively. Equivalent gel-loading conditions were confirmed using a-tubulin cDNA probe (shown at the bottom in each figure).
compared with that in the untreated cells, and returned to the baseline level by 48 h. On the other hand, no significant changes were observed in the level of IL-6R mRNA expression by IL-6 (Figure 4). GPIIb mRNA level was increased by stimulation of IL-6 in a time-dependent fashion up to 48 h. Under the same conditions, a-tubulin mRNA level was not changed.
DISCUSSION Our present study was designed to clarify the mechanism by which the same megakaryocyte progenitor cells respond to various cytokines at different stages of maturation. The ability of a cytokine to exert its biological effect is believed to by linked to the expression of the corresponding receptors on cytokine-responsive target cells. Thus the binding of a cytokine to target cells through specific receptors is assumed to be an initial and important event in the regulation of cellular responses. The two-factor regulatory system in megakaryocytopoiesisl 6 . 1 7 may function through the modification of the cytokine-sensitive receptor densities expressed on
megakaryocyte progenitor cells during maturation. In general, the characteristics of cytokine-specific receptors on target cells have been analyzed by observing the pattern of binding of 251-labelled ligands to cells. In our preliminary experiments, neither the apparent numbers of receptors on each CMK cell nor Kd values for GM-CSFR, IL-3R, and IL-6R could be estimated by using 251-labelled GM-SCF (13 TBq/mmol), '251-labelled IL-3 (19 TBq/mmol), and '251-labelled IL-6 (37 TBq/mmol). The reason why the binding assay did not work in this case remains unclear. The numbers of these cytokine receptors on CMK cells may have been too low for detection by the ligands used, or the ligands may have been too rapidly degradated and released by the cells. More sophisticated study will be required to resolve it. However, recent cloning of cDNA encoding GM-CSFR16.", IL-3R" and IL-6R19 allowed us to investigate the regulation of these receptors at the mRNA level. Recently, Miyajima and his colleagues have domonstrated that IL-3R and GM-CSFR share a common /%subunit,which is an essential component to reconstitute high-affinity GM-CSFR and highaffinity IL-3R'69'8. In the present study, the cDNA
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encoding the common b-subunit was used to investigate the mechanisms of responses of the earlier megakaryocyte progenitor cells to multi-lineage cytokines such as GM-CSF and IL-3. In addition, cDNA encoding IL-6R was used to study the mechanism of the preferential response of the later megakaryocyte progenitor cells to IL-6. GM-CSFR b-subunit mRNA was found to be expressed constitutively in CMK cells. TPA, which is known to promote the development of CMK cells into the more differentiated megakaryocytes’ was found to downregulate GM-CSFR /-subunit mRNA expression and up-regulate IL-6R mRNA expression in CMK cells. Furthermore, the rapidly down-regulated expression of GM-CSFR /-subunit mRNA gradually recovered to the baseline level, and the recovery appeared to correlate with the increase of GPIIb mRNA level, a typical differential marker of megakaryoFytes. This finding suggests that the responsiveness of megakaryocyte progenitor cells to multi-lineage cytokines may be transiently decreased by reduced receptor densities and multi-lineage cytokines may contribute preferentially to the regulation of the earlier development of megakaryocyte progenitor cells with high densities of multi-lineage cytokine receptors. It should be also noted that the TPA-induced downregulation of GM-CSF ,&subunit mRNA expression is a transient phenomenon, followed by recovery, thus indicating that the reduced responsiveness to multilineage cytokines may be recovered through differentiation, and that multi-lineage cytokines may also be involved in the regulation of the later stage of megakaryocyte differentiation. Previous studies showed that phorbol ester such as TPA downregulated the expression of GM-CSFR in human myeloid ~ e l l s ~Cannistra ~ - ~ ~ . rt uI. provided evidence that phorbol ester exerts the down-regulatory action through rapid internalization of the receptors”. However, our present data demonstrates for the first time that TPA-induced down-regulation of GMCSFR may be partly dependent on the reduced expression of GM-CSFR mRNA. TPA is known to exert its biological action through activation of a protein kinase C2*, but it seems unlikely that this activation directly down-regulates the expression of GM-CSFR b-subunit mRNA. To clarify the mechanism of TPA-induced modulation of GM-CSFR b-subunit mRNA, we observed the effect of CHX, a protein synthesis inhibitor, on the TPA-induced down-regulation and its recovery. Preincubation with CHX resulted in abolition of the down-regulation of GM-CSFR b-subunit mRNA expression; and CHX
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also blocked the recovery from the TPA-induced down-regulation. These results suggest that TPAinduced modulation of GM-CSFR /-subunit mRNA expression requires de novo protein synthesis. IL-6R mRNA, on the other hand, was expressed in small amounts in CMK cells in the absence of TPA; and on addition of TPA the expression increased, suggesting that IL-6 appears to be limited in its action to supporting the maturation of the more differentiated megakaryocyte progenitor cells. TPA is not a physiological regulator for megakaryocytopoiesis. Neither GM-CSF nor IL-6 is known to exert its biological action through a protein Therefore, the kinase C dependent effects of IL-6, a physiological stimulator, on the expression of GM-CSFR, IL-6R, and GPIIb mRNAs were examined in order to clarify the in vivo mechanism of megakaryocytopoiesis. Interestingly, IL-6 was found to possess a similar capacity to TPA for the down-regulation of GM-CSFR p-subunit mRNA expression. This data also confirms our hypothesis that the responsiveness of megakaryocyte progenitor cells to several cytokines during differentiation may depend in part on the receptor densities. Further direct analysis of the relationship between ligand-receptor binding and proliferative responses will be required to define the exact relationship between the densities of GM-CSFR and IL-6R, and the sensitivity of cells to multi-lineage cytokines such as GM-CSF and IL-3, and to IL-6, during megakaryocyte maturation. Our present molecular study, however, provides the first evidence that control of the expression of cytokine receptor mRNA, which would result in changes in receptor densities, may be one of the mechanisms for modulating the responsiveness of megakaryocyte progenitor cells to different cytokines during megakaryocyte development. REFERENCES 1. Quesenberry, P. J., Ihle, J. N . and McGrath, E. (1985).The effect of interleukin 3 and GM-CSA-2 on megakaryocyte and myeloid clonal colony formation. Blood, 65, 2 1 4 7 . 2. Mazur, E. M., Cohen, J. L., Wong, G. G. and Clark, S. C. (1987). Modest stimulatory effect of recombinant human GM-CSF on colony growth from peripheral blood human megakaryocyte progenitor cells. Exp. Hemutol., 15, 1128-33. 3. Teramura, M., Katahira, J., Hoshino, S., Motoji, T., Oshimi, K. and Mizoguchi, H. (1989).Effect of recombinant hemopoietic growth factors on human megakaryocyte colony formation in serum-free cultures. Exp. Hemutol., 17, 101 1-6. 4. Bruno, E., Miller, M. E. and Hoffman, R. (1989). Interacting cytokines regulate in vitro human megakaryocytopoiesis. Blood, 73, 611-7.
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