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Effect of Recombinant Interleukin-6 and Thrombopoietin on Isolated Guinea Pig Bone Marrow Megakaryocyte Protein Phosphorylation and Proplatelet Formation
Blood Cells, Molecules, and Diseases (1997) 23(13) July 15: 252–268 Article No. MD970142
R.M. Leven, et al.
Effect of Recombinant Interleukin-6 and Thrombopoietin on Isolated Guinea Pig Bone Marrow Megakaryocyte Protein Phosphorylation and Proplatelet Formation Submitted 06/26/97 (communicated by George Brecher, M.D., 07/09/97)
Robert M. Leven1, Barbara Clark1, Fern Tablin2 ABSTRACT: Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 µg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation. Keywords: megkaryocytopoiesis, signal transduction, growth factors, thrombopoietin, platelet formation
INTRODUCTION
karyocyte size, ploidy, cytoplasmic maturation, platelet size and the rate of platelet production (4-12). Various cytokines and growth factors stimulate megakaryocytopoiesis either in vivo, and/or in vitro, which leads to increases in megakaryocyte colony number and size, acetylcholine esterase activity, ploidy, cell size, proplatelet formation and platelet size and count (13-45). The primary physiologic regulator of megakaryocytopoiesis now appears to be thrombopoietin (TPO). TPO was cloned by several groups (40-43) and has been demonstrated to have
The humoral regulation of platelet production has been investigated ever since the observation that the plasma of thrombocytopenic animals contains a thrombocytopoietic activity when injected into test animals (1,2,3). It has long been known from in vivo studies that an understanding of the humoral regulation of megakaryocyto-poiesis must take into account the changes that are observed during the response of animals to thrombocytopenia such as increases in mega1
Department of Anatomy, Rush Medical College, Chicago, IL; Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA. Reprint request to: Robert M. Leven, Ph.D., Department of Anatomy, Rush Medical College, 1653 West Congress Parkway, Chicago, IL 60612. phone (312)942-6779, fax (312)942-5744, email: [email protected] 2
Published by Academic Press Established by Springer-Verlag, Inc. in 1975
1079-9796/97 $25.00 Copyright r 1997 by The Blood Cells Foundation, La Jolla, California, USA All rights of reproduction in any form reserved.
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a broad range of effects leading to the stimulation of megakaryocyte maturation both in vivo and in vitro. These effects include stimulation of increased platelet count, megakaryocyte number, ploidy and size in the marrow, CFU-Meg growth and megakaryocytic expansion of CD34 cells (27,31,39-51). The terminal step of megakaryocyte maturation is the release of the cells’ cytoplasm as proplatelet fragments, a complicated process which may be regulated by both humoral and extracellular matrix components. This process is a result of extensive reorganization of the megakaryocyte cytoskeleton and plasma membrane. Proplatelet formation in vitro has been observed by many investigators (52-60), but there has been no systematic study of the effect of megakaryocytopoiesis stimulating cytokines on this final stage of megakaryocytopoiesis. Therefore as part of this study we have observed the effect of certain cytokines, known to alter megakaryocytopoiesis in vivo and in vitro, on guinea pig bone marrow megakaryocyte proplatelet formation. Although there is an extensive literature on the stimulation of megakaryocytopoiesis by growth factors, there is almost no information on the signaling mechanisms which are triggered in megakaryocytes in response to specific growth factors. It has been demonstrated that TPO stimulation leads to the phosphorylation of Stat3, Stat5, JAK2, Shc, Sos, Vav and C-cbl in Mo7e cells (61,62), Erk1 and Erk2 in UT-7/TPO cells (63), Stat3, Stat5, JAK2 and Shc in platelets (64), mpl, JAK2, SHC, SHPTP-1 and SHPTP-2, vav, MAPkinase, Stat3 and Stat5 in 32D cells (65), JAK2 and TYK2 in Mo7e cells (66), Sch, Vav, Raf-1, Map kinase, MAP kinase and Pim-1 in FD-TPO cells (67). In these studies many alterations in protein phosphorylation have been associated with TPO stimulation, but none have been associated with any event which occurs during the course of normal megakaryocyto-poiesis. Therefore an additional goal of this work was to begin to elucidate the stimulus reponse mechanisms which occur in normal bone marrow megakaryocytes stimulated with IL-6 or TPO.
MATERIALS AND METHODS Materials Male Duncan Hartley guinea pigs (300-350 g) were purchased from Charles River (Wilmington, MA). All cell culture reagents were from GIBCO (Grand Island, NY). Recombinant murine interleukin-3 and interleukin-6 were from R & D Systems (Minneapolis, MN). Rat tail collagen and antibodies to MAP kinase, JAK-1 and JAK-2 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to phosphotyrosine, JAK-3 and Tyk-2 were from Transduction Laboratories (Lexington, KY). Recombinant murine interleukin-1 was the gift of Dr. Janet Plate, Rush-Presbyterian-St. Luke’s Medical Center (Chicago, IL). Recombinant murine c-mpl ligand (TPO) was a gift of Dr. Dan Eaton, Genentech, Inc. (South San Francisco, CA). Genestein, calyculin-A and okadaic acid were purchased from Sigma Chemical Co, St. Louis, MO. 32P-orthophosphate and 3H-thymidine were purchased from Amersham Inc.(Arlington Heights, IL). Megakaryocyte Isolation Guinea pig bone marrow megakaryocytes were isolated by a combination of density gradient centrifugation and positive selection with immunomagnetic beads using a monoclonal antibody to platelet glycoprotein Ib as the primary antibody. This method has been previously described in detail (68). Megakaryocyte Culture for Proplatelet Quantitation Isolated megakaryocytes were cultured as previously described (59) in Dulbecco’s Modified Eagle Medium supplemented with 10% newborn calf serum and penicillin/streptomycin. The cells were suspended in medium at 105 cells/ml and 1.0 ml of cell suspension was cultured in a 35 mm tissue culture dish which contained 1.0 ml of a hydrated gel of rat tail collagen. The collagen gel 253
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orthophosphate was added to the medium at 2.5 mCi/ml and incubation continued for two hours after which either IL-6 or TPO were added to the cultures at 10 ng/ml and incubation continued for an additional 10 minutes unless otherwise indicated.
was formed from a mix of 1 volume of 10X concentrated culture medium, 1 volume of 0.1 N NaOH and 8 volumes of rat tail collagen solubilized in acetic acid. After the collagen solution was placed in the culture dish it was allowed to gel for 30 minutes in a tissue culture incubator with 100% humidity, 5% CO2-95% air atmosphere. The gel was rinsed three times with culture medium before plating the cells. All cytokines were added at the time of plating the cells. The cells were observed at 24 and 48 hours after plating and the number of fragmenting cells was counted after 48 hours in culture. A minimum of 500 cells were counted in each dish to determine the percent of cells undergoing cytoplasmic fragmentation. Each condition was prepared in triplicate in each experiment. The data were pooled from three such experiments and the significance in the difference of the means of each group from its control group was determined by calculating a p value using a Mann-Whitney test. 3H-Thymidine
Immunoprecipitation All immunoprecipitation procedures were performed at 4 °C. Isolated megakaryocytes, either labeled with 32P or unlabeled, were washed in 150 mM NaCl, 50 mM Tris, 1.0 mM ethylene glycolbis-(amino-ethyl ether)N,N,N’N,-tetra-acetic acid, 1.0 mM sodium orthovanadate, 1.0 mM NaF, pH 7.4 (Wash Buffer) three times by centrifugation. The cell pellet after was solubilized in Lysis Buffer (Wash Buffer with 1.0 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml aprotinin, 1.0 µg/ml pepstatin, 1.0 µg/ml leupeptin, 1.0% Triton X-100). The cells were mixed slowly in Lysis Buffer for 15 minutes after which insoluble material was removed by centrifugation at 13,000 x g for 10 minutes. The supernatant cell lysate was used for all immunoprecipitations. Material with nonspecific adherence to protein-A sepharose was removed by incubation with beads for 15 minutes and removal of the beads by centrifugation. Primary antibody was added at an appropriate concentration and the lysate was incubated for 1-12 hours. Either protein-A or protein-G sepharose was used to bind the primary antibody. The beads were removed from the lysate by centrifugation, washed three times in Lysis Buffer and then boiled in sample buffer for polyacrylamide electrophoresis (25 mM Tris, .0025% bromophenol blue, 1.0% sodium dodecyl sulfate, 1.0% mercaptoethanol).
Incorporation
Isolated megakaryocytes were incubated at 105 cells/ml in Dulbecco’s Modified Eagle Medium with 10% fetal calf serum with 10µCi/ml 3H-thymidine. There were no other additions to the control cultures, while the IL-3 cultures had 10 ng/ml IL-3 added at the same time the 3Hthymidine was added. After 30 minutes incubation at 37 °C the cells were washed twice in a 50 fold excess volume of ice cold CATCH buffer. The cell pellet after washing was solubilized in scintillation fluid and the radioactivity was counted in a Packard Tricarb 2200CA liquid scintillation analyzer. The data were averaged from three experiments. In each experiment the samples (IL-3 or Control) were prepared in triplicate. P values were calculated using an unpaired t-test.
Electrophoresis and Western Blotting Proteins in the cell lysate or immuno-precipitates were separated by polyacrylamide gel electrophoresis by the method of Laemmli (69). The gels were either dried for autoradiography or the proteins were electrophoretically transferred to nitrocellulose for immunological detection of proteins. Nonspecific protein binding to the nitrocellu-
Megakaryocyte Culture for Protein Phosphorylation Isolated megakaryocytes were incubated for approximately 16 hours in liquid culture medium, as described above, in polystyrene tubes. 32P254
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lose was blocked with 5% milk powder in PBS for 12-16 hours followed by incubation in primary antibody diluted in TBST (0.15 M NaCL, 10.0 mM Tris, .05% Tween, pH 7.0) for 12-24 hours. A control sample was run in a seperate lane using control antibody instead of the primary antibody. The nitrocellulose was washed three times for 15 minutes in TBST and then incubated with biotin labeled second antibody followed by Extravidinalkaline phosphatase (Sigma Chemical Co, St. Louis), also diluted in TBST. The nitrocellulose was then washed three times for 15 minutes in TBST, pH 9.5 Bands were visualized using the NBT, BCIP color reaction. Autoradiography of dried gels was performed with Kodak Biomax or X-Omat film.
immature megakaryocytic cells by IL-3. Since most cytoplasmic maturation is postmitotic, continued DNA synthesis may slow down the entry into proplatelet formation. To test this we measured the effect of IL-3 on thymidine incorporation into isolated megakaryocytes. Megakaryocytes were incubated with 10 ng/ml IL-3 and thymidine incorporation was measured (Figure 2). The control cultures showed an average incorporation of 817 cpm (S.D 37 cpm) while the IL-3 treated cultures showed an average incorporation of 1136 cpm (S.D 78 cpm). This was a statistcally very significant difference (p 5 .003). As previously reported the phosphodiesterase inhibitor, isobutylmethylxanthine stimulated megakaryocyte proplatelet formation (70). The effect of the phosphatase inhibitors calyculin A and okadaic acid and the tyrosine kinase inhibitor genestein on proplatelet formation were also observed (Table 3). We found that both of the phosphatase inhibitors significantly decreased megakaryocyte proplatelet formation, while genestein was without any effect.
RESULTS Proplatelet Formation Isolated guinea pig bone marrow megakaryocytes cultured for 48 hours on a hydrated type one collagen gel, in the presence of serum undergo cytoplasmic changes that result in the release of cytoplasmic fragments with the appearance of blood platelets (Figure 1). We have previously characterized this proplatelet formation in detail (59). Megakaryocyte fragmentation was observed in cultures incubated with IL-1, IL-3, IL-6 and TPO as described above. As shown in Table 1, neither IL-1 or IL-3 had any significant effect on the percent of megakaryocytes that underwent fragmentation. In contrast, addition of either IL-6 or TPO led to a significant increase in the percent of megakaryocytes that underwent fragmentation. Although the percentage of megakaryocytes that formed proplatelet fragments was increased in response to IL-6 or TPO, the appearance of these proplatelet forming cells was the same as those cells forming proplatelet fragments in control cultures. Unexpectedly, as illustrated in Table 2, the addition of IL-3 together with IL-6 or TPO inhibited the increase in the number of fragmenting megakaryocytes stimulated by these factors in the absence of IL-3. We believed this result may have been due to stimulation of mitotic activity in
Protein Phosphorylation The phosphorylation state of the JAK-1, JAK-2 and MAP kinase were observed in unstimulated megakaryocytes and in megakaryocytes that had been stimulated with IBMX, IL-6 or TPO. Figure 3 demonstrates the phosphorylation of JAK1 after ten minutes incubation with either IL6 (10 ng/ml), TPO (10 ng/ml) or IBMX (1.0 mM). Phosphorylation was markedly increased in IL6 stimulated cells, unchanged in the presence of TPO and markedly decreased in IBMX stimulated cells. JAK2 phosphorylation (Figure 4) and MAP kinase (Figure 5) phosphorylation showed the same pattern as JAK1 phosphorylation under these conditions. IL6, unlike TPO, is a member of the family of growth factors (including IL6, IL11, CNTF and Oncostatin-M) whose receptor interacts with GP130 which functions as part of the signal transduction for the binding of these ligands. Although TPO did not lead to any alteration in JAK1, JAK2 or MAPkinase phosphorylation, we found in subsequent experiments that incubation 255
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A)
B)
Figure 1. Isolated guinea pig bone marrow megakaryocytes cultured on hydrated collagen gel. A) Megakaryocytes after 24 hours in culture. One of the cells (arrow) has formed multiple proplatelet processes. B) Megakaryocytes after 48 hours in culture. Morphogenesis of the cells has progressed to the formation of proplatelet fragments as seen by the cell indicated by the arrow. A) x 800 B) x 600.
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Table 1. Cytokine Effect on Proplatelet Formation. (Percent Megakaryocytes in Proplatelet Formation)
1
Cytokine Concentration (ng/ml) 10
100
IL1
16.1 (2.8)*
16.3 (2.7)
15.2 (1.5)
IL3
13.7 (4.4)
13.5 (4.4)
13.6 (4.7)
IL6
16.8 (3.7)
26.4 (3.2)**
26.7 (2.4)*
TPO
16.6 (2.9)
22.6 (2.8)**
26.8 (2.2)**
CONTROL (No cytokine addition) 15.6 (2.9) Megakaryocytes were cultured as described in the methods to observe proplatelet formation. Cytokines were added to the cultures at the indicated concentrations and the percent of the megakaryocytes that had formed proplatelet processes after 48 hours was counted as described in the methods. *indicates the standard deviation. **indicates value that has a significant difference from the control with no cytokine.
Table 2. Combined Cytokine Effect on Proplatelet Formation. (Percent Megakaryocytes in Proplatelet Formation)
1
IL6 or TPO Concentration (ng/ml) 10
IL3 (10 ng/ml) 1IL6
14.2 (3.0)*
16.9 (3.0)**
18.3 (3.3)**
IL3 (10 ng/ml) 1TPO
13.6 (4.5)
16.1 (4.3)**
16.0 (4.2)**
100
CONTROL (No cytokine addition) 15.4 (2.8) Megakaryocytes were cultured as described in the methods to observe proplatelet formation. Cytokines were added at the indicated concentrations. IL3 was 10 ng/ml on all cultures while IL6 and TPO were added at 1, 10 or 100 ng/ml. *indicates the standard deviation. **indicates value that has a significant difference from cultures with IL6 or TPO alone at the same concentration but with no IL3 from Table 1.
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Table 3. Phosphatase, Tyrosine Kinase and Phosphodiesterase Inhibitor Effects on Proplatelet Formation. (Percent Megakaryocytes in Proplatelet Formation) CALYCULIN A
OKADAIC ACID
GENESTEIN
IBMX
CONTROL (No drug addition)
1.0 nM
10.0 nM
5.6 (2.4)*,**
1.7 (1.3)**
0.1 uM
1.0 uM
4.4 (1.6)**
0.23 (.39)**
10 µg/ml
50 µg/ml
100 µg/ml
16.2 (3.1)
18.3 (4.2)
17.7 (3.7)
0.1 mM
1.0 mM
10.0 mM
18.2 (4.5)
23.0 (3.9)**
32.7 (4.9)**
17.1 (4.1)
Megakaryocytes were cultured as described in the methods to observe proplatelet formation. Reagents were added at the time the culture was initiated at the indicated concentrations and proplatelet formation was counted as described in the methods. *Indicates the standard deviation. **indicates value that has a significant difference from control cultures.
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Figure 2. Purified megakaryocytes were incubated with 10µCi 3H-thymidine/ml for 30 minutes together with 10 ng/ml IL-3 or no addition (control). At the end of the incubation the cells were washed and collected as described in methods, and the radioactivity was measured in a scintillation counter.
Figure 3. JAK1 was immunoprecipitated from lysates of megakaryocytes incubated with 32P-orthophosphate. The megakaryoyctes had been treated for 10 minutes with either IL6 (10 ng/ml), TPO (10 ng/ml) or IBMX (1.0 mM). The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the right. Each drug treated cell population is shown with its accompanying control population of cells. The arrowhead on the right indicates the position of JAK1 on the gels (130 kDA). Lane (a) is the control for the IL6 treated cells which are shown in lane (b). There is a clear increase in the amount of JAK1 phosphorylation in IL6 treated cells compared to the control. Lane (c) is the control for the TPO treated cells which are shown in lane (d). Although longer exposure for autoradiograpy in this experiment shows phosphorylation of JAK1 in the control cells, the addtion of TPO never lead to an increased phosphorylation relative to the control. Lane (e) is the control for the IBMX treated cells which are shown in lane (f). There is a consistent decrease in phosphorylation in IBMX treated cells relative to their control. 259
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Figure 4. JAK2 was immunoprecipitated from lysates of megakaryoyctes incubated with 32P-orthophosphate. The megakaryocytes had been treated for 10 minutes with either IL6 (10 ng/ml), TPO (10 ng/ml) or IBMX (1.0 mM). The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the right. Each drug treated cell population is shown with its accompanying control population of cells. The arrow on the left indicates the postion of JAK2 on the gels (130 kDA). The pattern of phosphorylation is identical to that of JAK1 as seen in Figure 3. Lane (a) is the control for the IL6 treated cells shown in lane (b). There is a clear increase in the phosphorylation of JAK2 in IL6 treated cells compared to the control. Lane (c) is the control for the TPO treated cells shown in lane (d). Again the phosphorylation is unchanged in the TPO treated cells as compared to the control. Lane (e) is the control for the IBMX treated cells shown in lane (f). As with JAK1, there is a consistent decrease in JAK2 phosphorylation.
Figure 5. MAP kinase was immunoprecipitated from lysates of megakaryocytes cultured with either IL-6, TPO or IBMX. The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the left. The arrow on the left indicates the location of 42 kDa on the gel, the location of MAPkinase on these gels. Lane (a) is a control culture and lane (b) is a parallel culture incubated with 10 ng/ml IL-6. As with JAK1 and JAK2 there was a consistent increase in MAP kinase in IL-6 treated cells. Lane (c) is the control for TPO treated cells in lane (d). Although MAPkinase phosphorylation appears decreased in this experiment, this was not always the case and phosphorylation was never increased in TPO treated cells. Lane (e) shows the control for cells treated with 1.0 mM IBMX, lane (f). As with JAK1 and JAK2, phosphorylation was dramatically decreased in the presence of IMBX.
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Figure 6. Control megakaryocytes or megakaryocytes incubated with 10 ng/ml TPO for 10 minutes were analysed using an immunoblot with antibody to phosphotyrosine. The position of the molecular weight standards (200kDa, 92 kDa, 69 kDa and 46 kDa) are shown on the left. Lane (a) are the TPO treated cells and lane (b) are the control cells. There is a distinct phosphotyrosinated band at approximately 130 kDa (arrowhead).
Figure 7. A cell lysate was prepared from 32P-orthophosphate labeled megakaryocytes. Lane (a) shows the same immunoprecipitate prepared from control cells. Lane (b) shows an immunoprecipitate prepared with anti TYK2 antibody from cells treated with 10 ng/ml TPO for 10 minutes. We observed a consistent increase in the phosphorylation of the immunoprecipitated TYK2 from TPO treated megakaryocytes relative to their control cultures.
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Figure 8. Isolated megakaryocytes labeled with 32P-orthophosphate were incubated on a hydrated type I collagen gel. This culture substrate stimulates the formation of megakaryocyte proplatelets. after 30 minutes incubation on the gel cell lysates were collected and immunoprecipitates of MAP kinase, JAK2 and JAK1 were prepared. As a control, a nonadherent megakaryocyte population was prepared by incubating cells in a suspension for the same time period. The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the left. The upper arrowhead marks the position of JAK1 and JAK2 on the gel (130 kDA) and the lower arrowhead marks the position of MAP kinase on the gel (43 kDA). MAP kinase phosphorylation is seen in lane (a) which shows adherent and lane (b) which shows nonadherent megakaryocytes. There was little change in the phosphorylation of MAP kinase. JAK2 phosphorylation is seen in lane (c) which shows adherent and lane (d) which shows nonadherent megakaryocytes. JAK1 phosphorylation is shown in lane (e) which shows the adherent and lane (f) which shows the nonadherent megakaryocytes. For both JAK2 and JAK1 there is a marked decrease in phosphorylation in the adherent megakaryocytes.
of megakaryocytes with 10 ng/ml TPO ligand for ten minutes stimulated tyrosine phosphorylation of a protein of near 130 kDa (Figure 6). Subsequent experiments were performed in which Tyk2 was immunoprecipitated from a lysate of 32P labeled megakaryocytes that had been stimulated with TPO. A clear increase in Tyk2 phosphorylation occurred as a result of TPO stimulation (Figure 7). Although IL-6 and TPO led to increased proplatelet formation and IL-6 stimulation was
associated with an increased phosphorylation of JAK1, JAK2, and MAP kinase, the phosphorylation of these proteins was decreased by isobutylmethylxanthine which also increased proplatelet formation. Therefore we looked at the phosphorylation of JAK1, JAK2 and MAP kinase as a result of incubation on a collagen gel under conditions which lead to proplatelet formation (Figure 8). No cytokines were added to the cultures. Isolated megakaryocytes were labeled for one hour with 32P-orthophosphate and then incubated on a hy-
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drated collagen gel or directly on uncoated culture dishes as a control, for thirty minutes. The cells were then collected from the dishes with lysis buffer at 4 °C. The lysis buffer was cleared of insoluble material by centrifugation and immunoprecipitation was performed as described in the methods. We found that relative to the control cells incubated on plastic, the cells incubated on the collagen gel had a decreased level of phosphorylation of JAK1 and JAK2. While MAP kinase phosphorylation appeared slightly increased in the adherent cells in this figure this was not consistant and generally MAP kinase phosphorylation was unchanged.
esis in vivo (71), or with IL-3, which appears to stimulate megakaryocyte progenitors (72,73) did not increase the proplatelet formation in these cultures. IL-1 is an indirect stimulator of megakaryocytopoiesis (71,74) and IL-3, although capable of supporting CFU-Meg growth has never been demonstrated to directly affect the fragmentation of megakaryocytes into platelets. Due to the synergistic stimulation of CFU-Meg growth by IL-3 with IL-6 or TPO, we expected that the same may occur with the stimulation of proplatelet formation. Surprisingly, IL-3 had an inhibitory effect on the stimulation of proplatelet formation by IL-6 or TPO. Although there is no definitive explantion for this observation our data are consistent with the possibility that IL-3 stimulates continued growth of the small immature megakaryocytes in our cultures, consequently causing a delay of differentiation which would decrease the number of cells in the cultures that are competent to form proplatelets. In order to understand how these growth factors stimulate proplatelet formation, we have begun to look at the signal transduction pathways in megakaryocytes. It was not surprising to find that IL-6 stimulation caused an increase in JAK1 and JAK2 phosphorylation. The JAK kinases are associated with the GP130 signal transduction protein which is part of the IL-6, IL-11, oncostatin M, leukemia inhibitory factor and ciliary neurotrophic factor receptors. IL-6 binding to its receptor can trigger phosphorylation of various signal transduction proteins in different cell types (75-83). It has also been previously demonstrated that IL-6 may lead to activation of MAP kinase as well. Although the JAK/Stat pathway and Ras/Raf/MEKK/MAP kinase pathways had appeared to be distinct signaling pathways in cells, we find that IL-6 may activate both pathways in megakaryocytes. This is consistent with the convergence of these two pathways in IL-6 stimulated megakaryocytes. Not expected was the observation that TPO did not alter the phosphorylation of JAK1, JAK2 or MAPkinase, yet still stimulated an increase in
DISCUSSION Formation of platelets by megakaryocytes is a unique process in mammalian biology which involves reorganization of megakaryocyte membranes, cytoskeletal elements and interaction with the extracellular matrix. In vivo experiments show that, like other blood cells, the production of platelets can be increased either in times of demand in the organism or when artificially stimulated by injection of exogenous cytokines, such as IL-6, IL-11 or TPO. There are multiple effects of these cytokines on megakaryocytes, stimulating the development of megakaryocytic progenitor cells as well as the most mature cells in this lineage. Therefore one challenge in studying the regulation of cytokine stimulation of megakaryocytopoiesis will be to understand which responses of the cells are relevant to the various stages of megakaryocytopoiesis. We have begun this process by focusing on the final stage of megakaryocytopoiesis, the release of cytoplasmic fragments by megakaryocytes; proplatelet formation. In our culture system we found that IL-6 and TPO both increased the number of megakaryocytes that form proplatelets after 48 hours in culture. It is not surprising that addition of IL-1, which may indirectly stimulate megakaryocytopoi-
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hypothesis. In addition phosphatases have been demonstrated to play a role in IL6 signaling and in the regulation of cell morphology. In particular, phosphatase inhibitors have been shown to promote relocation of actin to the cell membrane and to alter microtubule organization, both of which are believed to be involved in megakaryocyte fragmentation. Phosphatase/kinase regulation of platelet cytoskeletal reorganization is associated with changes in the phosphorylation state of several actin and tubulin associated proteins, including actin binding protein (84), coractin (85), VASP (86), and vinculin (87). To better understand the mechanism of platelet formation it will be necessary to identify how protein phosphatase activity may be regulated by IL6 and TPO and how they are related to the alteration of cytoskeletal organization in platelet forming megakaryocytes.
proplatelet formation. As stated in the introduction, TPO has been shown to stimulate increased phosphorylation of several proteins, including JAK1 and JAK2 in leukemic cell lines and human platelets. The difference in our data may be due to the different cell types used in previous studies, none of which used normal primary bone marrow megakaryocytes. Although TPO did not alter the phosphorylation of JAK1 or JAK2 in megakaryocytes, we did find that Tyk2 phosphorylation was altered. Therefore there may be a convergence of the separate signal transduction pathways stimulated by IL-6 and TPO toward later steps regulating the cytoskeletal changes involved in proplatelet formation. While IL-6 and TPO stimulation result in increased phosphorylation of JAK1, JAK2, MAP kinase and Tyk2, it is not clear how or if these changes in phosphorylation are related to the changes in cytoskeletal reorganization associated with proplatelet formation. This is supported by our observation that incubation of cells with the phosphodiesterase inhibitor, IBMX, stimulates proplatelet production as effectively as IL-6 or TPO, yet the phosphorylation of JAK1, JAK2 and MAP kinase were all decreased in megakaryocytes treated with this drug. This implies that rather than phosphorylation, dephosphorylation of these proteins may be associated with proplatelet formation. To investigate this possibility we observed the phosphorylation of JAK1, JAK2 and MAPkinase in megakaryocytes incubated on a hydrated collagen gel which stimulates proplatelet formation. In these cultures JAK1, JAK2 and MAPkinase phosphorylation was also decreased. Therefore IL6 and TPO related phosphorylation of signal transduction proteins, though part of the response of megakaryocytes to these factors, most likely does not directly lead to cytoskeletal changes which are required for platelet formation. Instead, the activation of phosphatases subsequent to tyrosine or serine/ threonine kinases may be a critical step in the signal transduction pathway leading to proplatelet formation. The inhibition of pro-platelet formation by both okadaic acid and calyculin A supports this
ACKNOWLEDGMENTS This work was supported by a grant from the American Cancer Society (R.M.L.).
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Effect of Recombinant Interleukin-6 and Thrombopoietin on Isolated Guinea Pig Bone Marrow Megakaryocyte Protein Phosphorylation and Proplatelet Formation Submitted 06/26/97 (communicated by George Brecher, M.D., 07/09/97)
Robert M. Leven1, Barbara Clark1, Fern Tablin2 ABSTRACT: Guinea pig bone marrow megakaryocytes were isolated and cultured on collagen gels to promote proplatelet formation. In control cultures 15.6% of the cells formed proplatelets. Both IL6 and TPO stimulated dose dependent increases in the percent of proplatelet forming cells up to 26.7% at 100 ng/ml IL6 and 26.8% at 100 ng/ml TPO. IL1 and IL3 had no effect on proplatelet formation. IL3 in combination with IL6 and TPO blocked the increase in proplatelet formation observed with IL6 or TPO alone. IL3 was also found to stimulate thymidine incorporation in megakaryocytes. The role of phosphorylation in proplatelet formation was studied using certain inhibitors. The tyrosine kinase inhibitor genestien had no effect on proplatelet formation at concentrations up to 100 µg/ml. The phosphatase inhibitors calyculin A and okadaic acid both inhibited proplatelet formation. The phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) was a potent stimulator of proplatelet formation. Studies on protein phosphorylation revealed that IL6, but not TPO, stimulated phosphorylation of JAK1, JAK2 and MAP kinase. TPO did stimulate tyrosine phosphorylation of Tyk-2. Although IBMX stimulated proplatelet formation, it inhibited phosphorylation of JAKs and MAP kinase. Adhesion of megakaryocytes to collagen gel also inhibited phosphorylation of JAK1 and JAK2, while MAP kinase phosphorylation was unaffected. These data show that IL6 and TPO stimulate megakaryocyte proplatelet formation. In addition, although these cytokines increase phosphorylation of signal transduction proteins in the JAK/STAT pathway, it appears that a different signal transduction pathway regulated by a combination of phosphatase activity and cAMP levels, leads to proplatelet formation. Keywords: megkaryocytopoiesis, signal transduction, growth factors, thrombopoietin, platelet formation
INTRODUCTION
karyocyte size, ploidy, cytoplasmic maturation, platelet size and the rate of platelet production (4-12). Various cytokines and growth factors stimulate megakaryocytopoiesis either in vivo, and/or in vitro, which leads to increases in megakaryocyte colony number and size, acetylcholine esterase activity, ploidy, cell size, proplatelet formation and platelet size and count (13-45). The primary physiologic regulator of megakaryocytopoiesis now appears to be thrombopoietin (TPO). TPO was cloned by several groups (40-43) and has been demonstrated to have
The humoral regulation of platelet production has been investigated ever since the observation that the plasma of thrombocytopenic animals contains a thrombocytopoietic activity when injected into test animals (1,2,3). It has long been known from in vivo studies that an understanding of the humoral regulation of megakaryocyto-poiesis must take into account the changes that are observed during the response of animals to thrombocytopenia such as increases in mega1
Department of Anatomy, Rush Medical College, Chicago, IL; Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA. Reprint request to: Robert M. Leven, Ph.D., Department of Anatomy, Rush Medical College, 1653 West Congress Parkway, Chicago, IL 60612. phone (312)942-6779, fax (312)942-5744, email: [email protected] 2
Published by Academic Press Established by Springer-Verlag, Inc. in 1975
1079-9796/97 $25.00 Copyright r 1997 by The Blood Cells Foundation, La Jolla, California, USA All rights of reproduction in any form reserved.
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a broad range of effects leading to the stimulation of megakaryocyte maturation both in vivo and in vitro. These effects include stimulation of increased platelet count, megakaryocyte number, ploidy and size in the marrow, CFU-Meg growth and megakaryocytic expansion of CD34 cells (27,31,39-51). The terminal step of megakaryocyte maturation is the release of the cells’ cytoplasm as proplatelet fragments, a complicated process which may be regulated by both humoral and extracellular matrix components. This process is a result of extensive reorganization of the megakaryocyte cytoskeleton and plasma membrane. Proplatelet formation in vitro has been observed by many investigators (52-60), but there has been no systematic study of the effect of megakaryocytopoiesis stimulating cytokines on this final stage of megakaryocytopoiesis. Therefore as part of this study we have observed the effect of certain cytokines, known to alter megakaryocytopoiesis in vivo and in vitro, on guinea pig bone marrow megakaryocyte proplatelet formation. Although there is an extensive literature on the stimulation of megakaryocytopoiesis by growth factors, there is almost no information on the signaling mechanisms which are triggered in megakaryocytes in response to specific growth factors. It has been demonstrated that TPO stimulation leads to the phosphorylation of Stat3, Stat5, JAK2, Shc, Sos, Vav and C-cbl in Mo7e cells (61,62), Erk1 and Erk2 in UT-7/TPO cells (63), Stat3, Stat5, JAK2 and Shc in platelets (64), mpl, JAK2, SHC, SHPTP-1 and SHPTP-2, vav, MAPkinase, Stat3 and Stat5 in 32D cells (65), JAK2 and TYK2 in Mo7e cells (66), Sch, Vav, Raf-1, Map kinase, MAP kinase and Pim-1 in FD-TPO cells (67). In these studies many alterations in protein phosphorylation have been associated with TPO stimulation, but none have been associated with any event which occurs during the course of normal megakaryocyto-poiesis. Therefore an additional goal of this work was to begin to elucidate the stimulus reponse mechanisms which occur in normal bone marrow megakaryocytes stimulated with IL-6 or TPO.
MATERIALS AND METHODS Materials Male Duncan Hartley guinea pigs (300-350 g) were purchased from Charles River (Wilmington, MA). All cell culture reagents were from GIBCO (Grand Island, NY). Recombinant murine interleukin-3 and interleukin-6 were from R & D Systems (Minneapolis, MN). Rat tail collagen and antibodies to MAP kinase, JAK-1 and JAK-2 were from Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to phosphotyrosine, JAK-3 and Tyk-2 were from Transduction Laboratories (Lexington, KY). Recombinant murine interleukin-1 was the gift of Dr. Janet Plate, Rush-Presbyterian-St. Luke’s Medical Center (Chicago, IL). Recombinant murine c-mpl ligand (TPO) was a gift of Dr. Dan Eaton, Genentech, Inc. (South San Francisco, CA). Genestein, calyculin-A and okadaic acid were purchased from Sigma Chemical Co, St. Louis, MO. 32P-orthophosphate and 3H-thymidine were purchased from Amersham Inc.(Arlington Heights, IL). Megakaryocyte Isolation Guinea pig bone marrow megakaryocytes were isolated by a combination of density gradient centrifugation and positive selection with immunomagnetic beads using a monoclonal antibody to platelet glycoprotein Ib as the primary antibody. This method has been previously described in detail (68). Megakaryocyte Culture for Proplatelet Quantitation Isolated megakaryocytes were cultured as previously described (59) in Dulbecco’s Modified Eagle Medium supplemented with 10% newborn calf serum and penicillin/streptomycin. The cells were suspended in medium at 105 cells/ml and 1.0 ml of cell suspension was cultured in a 35 mm tissue culture dish which contained 1.0 ml of a hydrated gel of rat tail collagen. The collagen gel 253
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orthophosphate was added to the medium at 2.5 mCi/ml and incubation continued for two hours after which either IL-6 or TPO were added to the cultures at 10 ng/ml and incubation continued for an additional 10 minutes unless otherwise indicated.
was formed from a mix of 1 volume of 10X concentrated culture medium, 1 volume of 0.1 N NaOH and 8 volumes of rat tail collagen solubilized in acetic acid. After the collagen solution was placed in the culture dish it was allowed to gel for 30 minutes in a tissue culture incubator with 100% humidity, 5% CO2-95% air atmosphere. The gel was rinsed three times with culture medium before plating the cells. All cytokines were added at the time of plating the cells. The cells were observed at 24 and 48 hours after plating and the number of fragmenting cells was counted after 48 hours in culture. A minimum of 500 cells were counted in each dish to determine the percent of cells undergoing cytoplasmic fragmentation. Each condition was prepared in triplicate in each experiment. The data were pooled from three such experiments and the significance in the difference of the means of each group from its control group was determined by calculating a p value using a Mann-Whitney test. 3H-Thymidine
Immunoprecipitation All immunoprecipitation procedures were performed at 4 °C. Isolated megakaryocytes, either labeled with 32P or unlabeled, were washed in 150 mM NaCl, 50 mM Tris, 1.0 mM ethylene glycolbis-(amino-ethyl ether)N,N,N’N,-tetra-acetic acid, 1.0 mM sodium orthovanadate, 1.0 mM NaF, pH 7.4 (Wash Buffer) three times by centrifugation. The cell pellet after was solubilized in Lysis Buffer (Wash Buffer with 1.0 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml aprotinin, 1.0 µg/ml pepstatin, 1.0 µg/ml leupeptin, 1.0% Triton X-100). The cells were mixed slowly in Lysis Buffer for 15 minutes after which insoluble material was removed by centrifugation at 13,000 x g for 10 minutes. The supernatant cell lysate was used for all immunoprecipitations. Material with nonspecific adherence to protein-A sepharose was removed by incubation with beads for 15 minutes and removal of the beads by centrifugation. Primary antibody was added at an appropriate concentration and the lysate was incubated for 1-12 hours. Either protein-A or protein-G sepharose was used to bind the primary antibody. The beads were removed from the lysate by centrifugation, washed three times in Lysis Buffer and then boiled in sample buffer for polyacrylamide electrophoresis (25 mM Tris, .0025% bromophenol blue, 1.0% sodium dodecyl sulfate, 1.0% mercaptoethanol).
Incorporation
Isolated megakaryocytes were incubated at 105 cells/ml in Dulbecco’s Modified Eagle Medium with 10% fetal calf serum with 10µCi/ml 3H-thymidine. There were no other additions to the control cultures, while the IL-3 cultures had 10 ng/ml IL-3 added at the same time the 3Hthymidine was added. After 30 minutes incubation at 37 °C the cells were washed twice in a 50 fold excess volume of ice cold CATCH buffer. The cell pellet after washing was solubilized in scintillation fluid and the radioactivity was counted in a Packard Tricarb 2200CA liquid scintillation analyzer. The data were averaged from three experiments. In each experiment the samples (IL-3 or Control) were prepared in triplicate. P values were calculated using an unpaired t-test.
Electrophoresis and Western Blotting Proteins in the cell lysate or immuno-precipitates were separated by polyacrylamide gel electrophoresis by the method of Laemmli (69). The gels were either dried for autoradiography or the proteins were electrophoretically transferred to nitrocellulose for immunological detection of proteins. Nonspecific protein binding to the nitrocellu-
Megakaryocyte Culture for Protein Phosphorylation Isolated megakaryocytes were incubated for approximately 16 hours in liquid culture medium, as described above, in polystyrene tubes. 32P254
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lose was blocked with 5% milk powder in PBS for 12-16 hours followed by incubation in primary antibody diluted in TBST (0.15 M NaCL, 10.0 mM Tris, .05% Tween, pH 7.0) for 12-24 hours. A control sample was run in a seperate lane using control antibody instead of the primary antibody. The nitrocellulose was washed three times for 15 minutes in TBST and then incubated with biotin labeled second antibody followed by Extravidinalkaline phosphatase (Sigma Chemical Co, St. Louis), also diluted in TBST. The nitrocellulose was then washed three times for 15 minutes in TBST, pH 9.5 Bands were visualized using the NBT, BCIP color reaction. Autoradiography of dried gels was performed with Kodak Biomax or X-Omat film.
immature megakaryocytic cells by IL-3. Since most cytoplasmic maturation is postmitotic, continued DNA synthesis may slow down the entry into proplatelet formation. To test this we measured the effect of IL-3 on thymidine incorporation into isolated megakaryocytes. Megakaryocytes were incubated with 10 ng/ml IL-3 and thymidine incorporation was measured (Figure 2). The control cultures showed an average incorporation of 817 cpm (S.D 37 cpm) while the IL-3 treated cultures showed an average incorporation of 1136 cpm (S.D 78 cpm). This was a statistcally very significant difference (p 5 .003). As previously reported the phosphodiesterase inhibitor, isobutylmethylxanthine stimulated megakaryocyte proplatelet formation (70). The effect of the phosphatase inhibitors calyculin A and okadaic acid and the tyrosine kinase inhibitor genestein on proplatelet formation were also observed (Table 3). We found that both of the phosphatase inhibitors significantly decreased megakaryocyte proplatelet formation, while genestein was without any effect.
RESULTS Proplatelet Formation Isolated guinea pig bone marrow megakaryocytes cultured for 48 hours on a hydrated type one collagen gel, in the presence of serum undergo cytoplasmic changes that result in the release of cytoplasmic fragments with the appearance of blood platelets (Figure 1). We have previously characterized this proplatelet formation in detail (59). Megakaryocyte fragmentation was observed in cultures incubated with IL-1, IL-3, IL-6 and TPO as described above. As shown in Table 1, neither IL-1 or IL-3 had any significant effect on the percent of megakaryocytes that underwent fragmentation. In contrast, addition of either IL-6 or TPO led to a significant increase in the percent of megakaryocytes that underwent fragmentation. Although the percentage of megakaryocytes that formed proplatelet fragments was increased in response to IL-6 or TPO, the appearance of these proplatelet forming cells was the same as those cells forming proplatelet fragments in control cultures. Unexpectedly, as illustrated in Table 2, the addition of IL-3 together with IL-6 or TPO inhibited the increase in the number of fragmenting megakaryocytes stimulated by these factors in the absence of IL-3. We believed this result may have been due to stimulation of mitotic activity in
Protein Phosphorylation The phosphorylation state of the JAK-1, JAK-2 and MAP kinase were observed in unstimulated megakaryocytes and in megakaryocytes that had been stimulated with IBMX, IL-6 or TPO. Figure 3 demonstrates the phosphorylation of JAK1 after ten minutes incubation with either IL6 (10 ng/ml), TPO (10 ng/ml) or IBMX (1.0 mM). Phosphorylation was markedly increased in IL6 stimulated cells, unchanged in the presence of TPO and markedly decreased in IBMX stimulated cells. JAK2 phosphorylation (Figure 4) and MAP kinase (Figure 5) phosphorylation showed the same pattern as JAK1 phosphorylation under these conditions. IL6, unlike TPO, is a member of the family of growth factors (including IL6, IL11, CNTF and Oncostatin-M) whose receptor interacts with GP130 which functions as part of the signal transduction for the binding of these ligands. Although TPO did not lead to any alteration in JAK1, JAK2 or MAPkinase phosphorylation, we found in subsequent experiments that incubation 255
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A)
B)
Figure 1. Isolated guinea pig bone marrow megakaryocytes cultured on hydrated collagen gel. A) Megakaryocytes after 24 hours in culture. One of the cells (arrow) has formed multiple proplatelet processes. B) Megakaryocytes after 48 hours in culture. Morphogenesis of the cells has progressed to the formation of proplatelet fragments as seen by the cell indicated by the arrow. A) x 800 B) x 600.
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Table 1. Cytokine Effect on Proplatelet Formation. (Percent Megakaryocytes in Proplatelet Formation)
1
Cytokine Concentration (ng/ml) 10
100
IL1
16.1 (2.8)*
16.3 (2.7)
15.2 (1.5)
IL3
13.7 (4.4)
13.5 (4.4)
13.6 (4.7)
IL6
16.8 (3.7)
26.4 (3.2)**
26.7 (2.4)*
TPO
16.6 (2.9)
22.6 (2.8)**
26.8 (2.2)**
CONTROL (No cytokine addition) 15.6 (2.9) Megakaryocytes were cultured as described in the methods to observe proplatelet formation. Cytokines were added to the cultures at the indicated concentrations and the percent of the megakaryocytes that had formed proplatelet processes after 48 hours was counted as described in the methods. *indicates the standard deviation. **indicates value that has a significant difference from the control with no cytokine.
Table 2. Combined Cytokine Effect on Proplatelet Formation. (Percent Megakaryocytes in Proplatelet Formation)
1
IL6 or TPO Concentration (ng/ml) 10
IL3 (10 ng/ml) 1IL6
14.2 (3.0)*
16.9 (3.0)**
18.3 (3.3)**
IL3 (10 ng/ml) 1TPO
13.6 (4.5)
16.1 (4.3)**
16.0 (4.2)**
100
CONTROL (No cytokine addition) 15.4 (2.8) Megakaryocytes were cultured as described in the methods to observe proplatelet formation. Cytokines were added at the indicated concentrations. IL3 was 10 ng/ml on all cultures while IL6 and TPO were added at 1, 10 or 100 ng/ml. *indicates the standard deviation. **indicates value that has a significant difference from cultures with IL6 or TPO alone at the same concentration but with no IL3 from Table 1.
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Table 3. Phosphatase, Tyrosine Kinase and Phosphodiesterase Inhibitor Effects on Proplatelet Formation. (Percent Megakaryocytes in Proplatelet Formation) CALYCULIN A
OKADAIC ACID
GENESTEIN
IBMX
CONTROL (No drug addition)
1.0 nM
10.0 nM
5.6 (2.4)*,**
1.7 (1.3)**
0.1 uM
1.0 uM
4.4 (1.6)**
0.23 (.39)**
10 µg/ml
50 µg/ml
100 µg/ml
16.2 (3.1)
18.3 (4.2)
17.7 (3.7)
0.1 mM
1.0 mM
10.0 mM
18.2 (4.5)
23.0 (3.9)**
32.7 (4.9)**
17.1 (4.1)
Megakaryocytes were cultured as described in the methods to observe proplatelet formation. Reagents were added at the time the culture was initiated at the indicated concentrations and proplatelet formation was counted as described in the methods. *Indicates the standard deviation. **indicates value that has a significant difference from control cultures.
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Figure 2. Purified megakaryocytes were incubated with 10µCi 3H-thymidine/ml for 30 minutes together with 10 ng/ml IL-3 or no addition (control). At the end of the incubation the cells were washed and collected as described in methods, and the radioactivity was measured in a scintillation counter.
Figure 3. JAK1 was immunoprecipitated from lysates of megakaryocytes incubated with 32P-orthophosphate. The megakaryoyctes had been treated for 10 minutes with either IL6 (10 ng/ml), TPO (10 ng/ml) or IBMX (1.0 mM). The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the right. Each drug treated cell population is shown with its accompanying control population of cells. The arrowhead on the right indicates the position of JAK1 on the gels (130 kDA). Lane (a) is the control for the IL6 treated cells which are shown in lane (b). There is a clear increase in the amount of JAK1 phosphorylation in IL6 treated cells compared to the control. Lane (c) is the control for the TPO treated cells which are shown in lane (d). Although longer exposure for autoradiograpy in this experiment shows phosphorylation of JAK1 in the control cells, the addtion of TPO never lead to an increased phosphorylation relative to the control. Lane (e) is the control for the IBMX treated cells which are shown in lane (f). There is a consistent decrease in phosphorylation in IBMX treated cells relative to their control. 259
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Figure 4. JAK2 was immunoprecipitated from lysates of megakaryoyctes incubated with 32P-orthophosphate. The megakaryocytes had been treated for 10 minutes with either IL6 (10 ng/ml), TPO (10 ng/ml) or IBMX (1.0 mM). The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the right. Each drug treated cell population is shown with its accompanying control population of cells. The arrow on the left indicates the postion of JAK2 on the gels (130 kDA). The pattern of phosphorylation is identical to that of JAK1 as seen in Figure 3. Lane (a) is the control for the IL6 treated cells shown in lane (b). There is a clear increase in the phosphorylation of JAK2 in IL6 treated cells compared to the control. Lane (c) is the control for the TPO treated cells shown in lane (d). Again the phosphorylation is unchanged in the TPO treated cells as compared to the control. Lane (e) is the control for the IBMX treated cells shown in lane (f). As with JAK1, there is a consistent decrease in JAK2 phosphorylation.
Figure 5. MAP kinase was immunoprecipitated from lysates of megakaryocytes cultured with either IL-6, TPO or IBMX. The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the left. The arrow on the left indicates the location of 42 kDa on the gel, the location of MAPkinase on these gels. Lane (a) is a control culture and lane (b) is a parallel culture incubated with 10 ng/ml IL-6. As with JAK1 and JAK2 there was a consistent increase in MAP kinase in IL-6 treated cells. Lane (c) is the control for TPO treated cells in lane (d). Although MAPkinase phosphorylation appears decreased in this experiment, this was not always the case and phosphorylation was never increased in TPO treated cells. Lane (e) shows the control for cells treated with 1.0 mM IBMX, lane (f). As with JAK1 and JAK2, phosphorylation was dramatically decreased in the presence of IMBX.
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Figure 6. Control megakaryocytes or megakaryocytes incubated with 10 ng/ml TPO for 10 minutes were analysed using an immunoblot with antibody to phosphotyrosine. The position of the molecular weight standards (200kDa, 92 kDa, 69 kDa and 46 kDa) are shown on the left. Lane (a) are the TPO treated cells and lane (b) are the control cells. There is a distinct phosphotyrosinated band at approximately 130 kDa (arrowhead).
Figure 7. A cell lysate was prepared from 32P-orthophosphate labeled megakaryocytes. Lane (a) shows the same immunoprecipitate prepared from control cells. Lane (b) shows an immunoprecipitate prepared with anti TYK2 antibody from cells treated with 10 ng/ml TPO for 10 minutes. We observed a consistent increase in the phosphorylation of the immunoprecipitated TYK2 from TPO treated megakaryocytes relative to their control cultures.
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Figure 8. Isolated megakaryocytes labeled with 32P-orthophosphate were incubated on a hydrated type I collagen gel. This culture substrate stimulates the formation of megakaryocyte proplatelets. after 30 minutes incubation on the gel cell lysates were collected and immunoprecipitates of MAP kinase, JAK2 and JAK1 were prepared. As a control, a nonadherent megakaryocyte population was prepared by incubating cells in a suspension for the same time period. The immunoprecipitates were separated on a 10% polyacrylamide gel. The position of molecular weight standards (200kDa, 92 kDa, 69 kDa, 46 kDa and 30kDa) is indicated on the left. The upper arrowhead marks the position of JAK1 and JAK2 on the gel (130 kDA) and the lower arrowhead marks the position of MAP kinase on the gel (43 kDA). MAP kinase phosphorylation is seen in lane (a) which shows adherent and lane (b) which shows nonadherent megakaryocytes. There was little change in the phosphorylation of MAP kinase. JAK2 phosphorylation is seen in lane (c) which shows adherent and lane (d) which shows nonadherent megakaryocytes. JAK1 phosphorylation is shown in lane (e) which shows the adherent and lane (f) which shows the nonadherent megakaryocytes. For both JAK2 and JAK1 there is a marked decrease in phosphorylation in the adherent megakaryocytes.
of megakaryocytes with 10 ng/ml TPO ligand for ten minutes stimulated tyrosine phosphorylation of a protein of near 130 kDa (Figure 6). Subsequent experiments were performed in which Tyk2 was immunoprecipitated from a lysate of 32P labeled megakaryocytes that had been stimulated with TPO. A clear increase in Tyk2 phosphorylation occurred as a result of TPO stimulation (Figure 7). Although IL-6 and TPO led to increased proplatelet formation and IL-6 stimulation was
associated with an increased phosphorylation of JAK1, JAK2, and MAP kinase, the phosphorylation of these proteins was decreased by isobutylmethylxanthine which also increased proplatelet formation. Therefore we looked at the phosphorylation of JAK1, JAK2 and MAP kinase as a result of incubation on a collagen gel under conditions which lead to proplatelet formation (Figure 8). No cytokines were added to the cultures. Isolated megakaryocytes were labeled for one hour with 32P-orthophosphate and then incubated on a hy-
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drated collagen gel or directly on uncoated culture dishes as a control, for thirty minutes. The cells were then collected from the dishes with lysis buffer at 4 °C. The lysis buffer was cleared of insoluble material by centrifugation and immunoprecipitation was performed as described in the methods. We found that relative to the control cells incubated on plastic, the cells incubated on the collagen gel had a decreased level of phosphorylation of JAK1 and JAK2. While MAP kinase phosphorylation appeared slightly increased in the adherent cells in this figure this was not consistant and generally MAP kinase phosphorylation was unchanged.
esis in vivo (71), or with IL-3, which appears to stimulate megakaryocyte progenitors (72,73) did not increase the proplatelet formation in these cultures. IL-1 is an indirect stimulator of megakaryocytopoiesis (71,74) and IL-3, although capable of supporting CFU-Meg growth has never been demonstrated to directly affect the fragmentation of megakaryocytes into platelets. Due to the synergistic stimulation of CFU-Meg growth by IL-3 with IL-6 or TPO, we expected that the same may occur with the stimulation of proplatelet formation. Surprisingly, IL-3 had an inhibitory effect on the stimulation of proplatelet formation by IL-6 or TPO. Although there is no definitive explantion for this observation our data are consistent with the possibility that IL-3 stimulates continued growth of the small immature megakaryocytes in our cultures, consequently causing a delay of differentiation which would decrease the number of cells in the cultures that are competent to form proplatelets. In order to understand how these growth factors stimulate proplatelet formation, we have begun to look at the signal transduction pathways in megakaryocytes. It was not surprising to find that IL-6 stimulation caused an increase in JAK1 and JAK2 phosphorylation. The JAK kinases are associated with the GP130 signal transduction protein which is part of the IL-6, IL-11, oncostatin M, leukemia inhibitory factor and ciliary neurotrophic factor receptors. IL-6 binding to its receptor can trigger phosphorylation of various signal transduction proteins in different cell types (75-83). It has also been previously demonstrated that IL-6 may lead to activation of MAP kinase as well. Although the JAK/Stat pathway and Ras/Raf/MEKK/MAP kinase pathways had appeared to be distinct signaling pathways in cells, we find that IL-6 may activate both pathways in megakaryocytes. This is consistent with the convergence of these two pathways in IL-6 stimulated megakaryocytes. Not expected was the observation that TPO did not alter the phosphorylation of JAK1, JAK2 or MAPkinase, yet still stimulated an increase in
DISCUSSION Formation of platelets by megakaryocytes is a unique process in mammalian biology which involves reorganization of megakaryocyte membranes, cytoskeletal elements and interaction with the extracellular matrix. In vivo experiments show that, like other blood cells, the production of platelets can be increased either in times of demand in the organism or when artificially stimulated by injection of exogenous cytokines, such as IL-6, IL-11 or TPO. There are multiple effects of these cytokines on megakaryocytes, stimulating the development of megakaryocytic progenitor cells as well as the most mature cells in this lineage. Therefore one challenge in studying the regulation of cytokine stimulation of megakaryocytopoiesis will be to understand which responses of the cells are relevant to the various stages of megakaryocytopoiesis. We have begun this process by focusing on the final stage of megakaryocytopoiesis, the release of cytoplasmic fragments by megakaryocytes; proplatelet formation. In our culture system we found that IL-6 and TPO both increased the number of megakaryocytes that form proplatelets after 48 hours in culture. It is not surprising that addition of IL-1, which may indirectly stimulate megakaryocytopoi-
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hypothesis. In addition phosphatases have been demonstrated to play a role in IL6 signaling and in the regulation of cell morphology. In particular, phosphatase inhibitors have been shown to promote relocation of actin to the cell membrane and to alter microtubule organization, both of which are believed to be involved in megakaryocyte fragmentation. Phosphatase/kinase regulation of platelet cytoskeletal reorganization is associated with changes in the phosphorylation state of several actin and tubulin associated proteins, including actin binding protein (84), coractin (85), VASP (86), and vinculin (87). To better understand the mechanism of platelet formation it will be necessary to identify how protein phosphatase activity may be regulated by IL6 and TPO and how they are related to the alteration of cytoskeletal organization in platelet forming megakaryocytes.
proplatelet formation. As stated in the introduction, TPO has been shown to stimulate increased phosphorylation of several proteins, including JAK1 and JAK2 in leukemic cell lines and human platelets. The difference in our data may be due to the different cell types used in previous studies, none of which used normal primary bone marrow megakaryocytes. Although TPO did not alter the phosphorylation of JAK1 or JAK2 in megakaryocytes, we did find that Tyk2 phosphorylation was altered. Therefore there may be a convergence of the separate signal transduction pathways stimulated by IL-6 and TPO toward later steps regulating the cytoskeletal changes involved in proplatelet formation. While IL-6 and TPO stimulation result in increased phosphorylation of JAK1, JAK2, MAP kinase and Tyk2, it is not clear how or if these changes in phosphorylation are related to the changes in cytoskeletal reorganization associated with proplatelet formation. This is supported by our observation that incubation of cells with the phosphodiesterase inhibitor, IBMX, stimulates proplatelet production as effectively as IL-6 or TPO, yet the phosphorylation of JAK1, JAK2 and MAP kinase were all decreased in megakaryocytes treated with this drug. This implies that rather than phosphorylation, dephosphorylation of these proteins may be associated with proplatelet formation. To investigate this possibility we observed the phosphorylation of JAK1, JAK2 and MAPkinase in megakaryocytes incubated on a hydrated collagen gel which stimulates proplatelet formation. In these cultures JAK1, JAK2 and MAPkinase phosphorylation was also decreased. Therefore IL6 and TPO related phosphorylation of signal transduction proteins, though part of the response of megakaryocytes to these factors, most likely does not directly lead to cytoskeletal changes which are required for platelet formation. Instead, the activation of phosphatases subsequent to tyrosine or serine/ threonine kinases may be a critical step in the signal transduction pathway leading to proplatelet formation. The inhibition of pro-platelet formation by both okadaic acid and calyculin A supports this
ACKNOWLEDGMENTS This work was supported by a grant from the American Cancer Society (R.M.L.).
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