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Keywords:

  • TPO;
  • Mpl receptors;
  • megakaryocytes;
  • PMA

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Thrombopoietin (TPO, c-Mpl ligand) is considered to play an important role in the regulation of megakaryocytopoiesis and platelet production by activating the cytokine receptor c-Mpl. We have examined the binding of 125I-TPO to the human megakaryocytic cell line, CMK, and to primary human megakaryocytes. Scatchard analysis of TPO binding to its cognate receptor in megakaryocytic cells suggested the existence of a single class of c-Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (Kd) of Kd = 223 p M, whereas primary human megakaryocytes exhibited 12 140 receptors per cell and a dissociation constant of Kd = 749 p M. The pretreatment of CMK cells and primary bone marrow megakaryocytes with TPO resulted in a decreased binding of TPO to the c-Mpl receptors. This down-regulation was observed within 3 h and was not inhibited by cycloheximide. Phorbol ester, an activator of protein kinase C, also inhibited TPO binding to the c-Mpl receptors by reducing the number of these receptors. The pretreatment of CMK cells with IL-3, IL-6 and DMSO, all of which induced the differentiation of CMK cells, did not affect the binding of TPO to the c-Mpl receptors. These results suggest an additional mechanism, where protein kinase C may help to regulate the binding of TPO to these cells.

Bone marrow megakaryocytes produce platelets, which are critical for normal haemostasis. Multiple cytokines (IL-1, IL-3, IL-6, IL-11, GM-CSF and erythropoietin) stimulate megakaryocytopoiesis both in vitro and in vivo ( Gordon & Hoffman, 1992; Hoffman, 1989). The ligand for the protooncogene c-Mpl ( Souyri et al, 1990 ; Vigon et al, 1992 ; Methia et al, 1993 ; de Sauvage et al, 1994 ; Lok et al, 1994 ; Bartley et al, 1994 ; Kuter, 1997; Kaushansky et al, 1994 ), called thrombopoietin (TPO), appears to be the major regulator of thrombopoiesis ( de Sauvage et al, 1994 ; Kato et al, 1995 ; Lok et al, 1994 ; Bartley et al, 1994 ; Kuter, 1997; Kaushansky et al, 1994 ). In vitro and in vivo experiments with recombinant TPO indicate that it stimulates both megakaryocyte colony formation and megakaryocyte maturation ( Banu et al, 1995 ; Wendling et al, 1994 ), the formation of CFU-megakaryocytes (CFU-MK) both alone and in combination with early acting factors ( Kaushansky et al, 1994 ; Banu et al, 1995 ), and the production of megakaryocytes and functional platelets from enriched murine or human stem cell populations ( Choi et al, 1995 ). Injection of TPO in mice increases platelet counts 4–6-fold and stimulates a 20-fold increase in bone marrow megakaryocytes ( Lok et al, 1994 ; Kaushansky et al, 1994 ).

The physiological regulation of the early and late stages of megakaryocytopoiesis by TPO was recently demonstrated ( de Sauvage et al, 1996 ). TPO-deficient mice have a > 80% decrease in their platelet and megakaryocyte level, but have normal levels of all the other haemopoietic cell types. Bone marrow from these TPO-deficient mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in megakaryocytes that are present. Mice deficient in c-Mpl exhibit an 85% reduction in peripheral platelet counts and in the numbers of marrow and spleen megakaryocytes ( Gurney et al, 1994 ). These data indicate that TPO is a major physiological regulator of both the proliferation and differentiation of haemopoietic progenitor cells into mature megakaryocytes.

The dynamics of TPO binding to its cognate c-Mpl receptor and its regulation are important; however, the kinetic binding of TPO to megakaryocytes is not well defined. In the present study 125I-labelled recombinant human TPO was used in equilibrium binding experiments to study the binding and internalization of TPO as well as its regulation by cold TPO and phorbol ester (PMA), in both the human megakaryocytic cell line, CMK and primary human megakaryocytes.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Reagents

Recombinant human thrombopoietin (TPO) was expressed and purified as described ( de Sauvage et al, 1994 ). Phorbol ester, PMA (Sigma Chemical Co., St Louis, Mo.) was resolved in DMSO as a stock solution and kept until use. The highest concentration of DMSO (0.1%) did not affect the binding of TPO in this study. Cycloheximide (Sigma) was resolved in phosphate-buffered saline (PBS) at a concentration of 1 mg/ml.

Cells

The CMK cell line was established in our laboratory from a patient with acute megakaryoblastic leukaemia and Down's syndrome ( Sato et al, 1989 ; Komatsu et al, 1989 ). The cells were cultured in RPMI-1640 medium (GIBCO, Grand Island, N.Y.) containing 10% fetal calf serum (FCS) at 37°C in humidified air containing 5% CO2. The CMK cells expressed c-Mpl receptors ( de Sauvage et al, 1994 ) and responded to TPO ( Kaushansky et al, 1994 ). In addition, TPO induced the phosphorylation of the tyrosine kinase JAK-2 and the activation of STAT proteins in the CMK cells ( Gurney et al, 1995 ).

Marrow megakaryocytes

Human bone marrow was obtained by aspiration from the iliac crests of normal donors who gave informed consent according to a protocol approved by the Deaconess Hospital Institutional Review Board. The marrow was separated by centrifugation through Ficoll-Hypaque (Pharmacia, Piscataway, N.J.) at 1000 g at room temperature for 20 min. After three washes with PBS, the cells were resuspended in RPMI-1640 with 10% FCS, seeded onto T-75 tissue flasks (Corning, Corning, N.Y.) and incubated at 37°C in humidified air containing 5% CO2. After 18 h, the nonadherent cells were collected. Human marrow megakaryocytes were isolated by a method employing immunomagnetic beads using anti-human glycoprotein IIIa (CD61) monoclonal antibody as described previously ( Avraham et al, 1992 ). All of the isolated cells were morphologically recognizable as megakaryocytes and the purity of the bone marrow megakaryocytes was 90–95%.

Iodination of TPO

rhTPO was iodinated by the Indirect Iodogen Method ( Jakeman et al, 1992 ), purified by size-exclusion chromatography (SEC), and formulated in 10 m M Tris, 0.15 M NaCl, 0.01% Tween 20, pH = 7.4 (TNT buffer) as described before ( Fielder et al, 1996 , 1995). The specific activity of the radiolabelled material ranged from approximately 1.85 to 3.33 MBq/μg protein.

Binding assay

The assay for specific binding of 125I-TPO to megakaryocytes was essentially performed as described ( Broudy et al, 1994 ). Briefly, CMK cells (1–2 × 107/ml) and primary bone marrow megakaryocytes (1–2 × 105/ml) were suspended in a binding buffer (RPMI-1640 supplemented with 50 m M Hepes (pH 7.4), 1% bovine serum albumin (BSA), 0.1% azide and 1 m M EDTA) containing 125I-TPO (20 p M to 2 n M for CMK cells, 50 p M to 4 n M for primary megakaryocytes) with or without a 100-fold excess of unlabelled TPO, and were incubated for 2 h at 15°C in a shaking incubator. The final incubation volume was 100 μl. After incubation, the cells were sedimented through a phthalate oil mixture to separate the cell-associated 125I-TPO from the free 125I-TPO. In most binding studies, 0.2 n M125I-TPO was used with 20 n M unlabelled TPO for determining the non-specific binding. The radioactivity of the cell pellets was counted by a Packard 5330 γ-ray counter at an efficiency of 74%. The supernatant was also counted for the quantitative binding experiments. There were duplicate determinations for each point. Non-specific binding was defined as bound 125I-TPO that remained in the presence of a 100-fold excess of the unlabelled TPO, except for the competitive binding. The specific binding was determined by subtracting the non-specific binding from the total binding. The specific binding ranged from 1000 to 3000 cpm for CMK cells and from 400 to 700 cpm for primary megakaryocytes when 0.2 n M of 125I-TPO was used in the presence of 20 n M of unlabelled TPO in this system. Scatchard analysis was employed to determine the number of TPO-binding sites. The maximal binding was 1200–1800 cpm/106 cells, and non-specific binding was usually ∼ 20–25% of the binding.

To differentiate the surface-bound TPO from the internalized TPO, the widely used low pH/salt method was employed ( Broudy et al, 1990 ). Briefly, the cells were washed twice, resuspended in 50 m M glycine-HCl buffer (pH 3.0) containing 0.15 M NaCl and then incubated for 5 min on ice and immediately layered onto phthalate oil to separate the cell-associated 125I-TPO from the free 125I-TPO. The radioactivity of the cell pellets was counted by a γ-ray counter. Parallel experiments were performed with a 100-fold excess of unlabelled TPO in the binding assay, and the cells were treated with the high salt (pH 3.0) to determine the non-specifically bound TPO. After the non-specific radioactivity was subtracted, the labelled TPO in the wash was considered surface bound, whereas the labelled TPO resistant to the acid wash was considered internalized. It is important to note that at 15°C, binding of TPO to the cells results in some internalization and degradation of 125I-TPO, which might affect the binding calculations. However, it is unlikely that this would alter the binding calculations derived from the binding experiments.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Binding of 125I-TPO to CMK cells and primary bone marrow megakaryocytes

Fig 1A shows the ability of the unlabelled TPO to compete with 125I-TPO for binding to the c-Mpl receptors on CMK cells. The unlabelled TPO competed effectively with 125I-TPO in a dose-dependent manner. Time course analysis indicated that the maximal specific binding of 125I-TPO to CMK cells was reached after 2 h incubation at 15°C (Fig 1B). Scatchard analysis revealed that CMK cells had a single class of binding sites (Fig 2A). The value of the apparent dissociation constant (Kd) was 223 ± 25  p M (mean ± SD) and the calculated number of binding sites was estimated as 1223 ± 145/cell (mean ± SD) from three separate experiments.

image

Figure 1. 06 cells. Points are the mean ± SD of duplicate determinations. These experiments were repeated twice with similar results.

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image

Figure 2.  h at 15°C. The results in each panel represent data from one out of three experiments. For each concentration of TPO, duplicate measurements were performed and the results of both measurements are presented.

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Bone marrow derived primary megakaryocytes were separated from normal donors, as described in Materials and Methods, and were incubated with different concentrations of 125I-TPO (50 p M to 4 n M) with or without a 100-fold excess of unlabelled TPO for 2 h at 15°C (Fig 2B). Scatchard analysis revealed that the primary megakaryocytes had a single class of binding sites with a Kd of 749 ± 135 p M (mean ± SD) and that the number of binding sites was 12 140 ± 1867/cell (mean ± SD) from three separate experiments.

Effect of TPO pretreatment on 125I-TPO binding to CMK cells and primary bone marrow megakaryocytes

In order to investigate the effects of TPO pretreatment on the binding of 125I-TPO to its cognate receptors, TPO had to be removed from the cell surface before conducting the binding experiments. First, we examined the time course analysis of surface-bound 125I-TPO and interior 125I-TPO in CMK cells labelled with 125I-TPO at 15°C by incubating the cells with culture medium without TPO ( 1 Table I). We found that > 80% of the specific surface-bound TPO was removed from the cell surface within 2 h, whereas 50% of the internalized TPO remained in the cells. Within 3 h, 70% of the internalized TPO, > 90% of the specific surface-bound TPO and > 80% of the specific total-bound TPO were removed from the CMK cells. The decreased level of the total-bound TPO in the primary megakaryocytes was almost at the same level as in the CMK cells ( Table I). When CMK cells were pretreated with 10 ng/ml of 125I-TPO at 37°C, the surface-bound TPO and the total-bound TPO were removed from the CMK cells at the same level as the TPO pretreated at 15°C described in Table I (data not shown). Therefore TPO-pretreated cells were further incubated at 37°C for 3 h in culture medium without TPO to permit internalization and degradation of surface-bound TPO before performing the 125I-TPO binding assay as described ( Broudy et al, 1990 ).

Table 1. Table I. Surface bound 125I-TPO and interior 125I-TPO in CMK cells. CMK cells and primary bone marrow megakaryocytes, separated as described in Materials and Methods, were incubated with 0.2 n M125I-TPO, with or without a 100-fold excess of unlabelled TPO for 2 h at 15°C in the presence of a binding buffer. Cells were washed twice and further incubated (without TPO) at 37°C for the indicated times. Specific total-bound TPO, specific surface-bound TPO and specific interior TPO were determined as described in Materials and Methods. Results are expressed as % of the control. The data are the mean ± SD of duplicate determinations. The experiment was repeated twice with similar results. ND: not done. Thumbnail image of

When CMK cells were pretreated with different concentrations of TPO and further cultured for 3 h to remove the surface-bound TPO, the specific binding of 125I-TPO to the CMK cells greatly decreased in a dose-dependent manner ( Table II). A maximal down-regulation of TPO binding was obtained by pretreatment with 100 ng/ml of TPO for 3 h. The range of inhibition of 125I-TPO binding by TPO-pretreatment was 60–80%, with an average range of 70%, in different experiments. A TPO-induced down-regulation was observed as early as 30 min after incubation and reached the maximal level within 3 h ( Table III). Quantitative analysis revealed that when CMK cells were pretreated with 10 ng/ml of TPO for 3 h, the number of binding sites was decreased to 378/cell without an apparent change in binding affinity (Fig 2).

Table 2. Table II. Concentration-dependence of TPO pretreatment on the binding of 125I-TPO. CMK cells and primary bone marrow megakaryocytes were pretreated with different concentrations of TPO for 16 h at 37°C, washed twice and further incubated (without TPO) for 3 h at 37°C. After cells were washed twice, binding assay was performed as described in Materials and Methods. Results are expressed as % of the control. The data are the mean ± SD of duplicate determinations. The experiment was repeated twice with similar results.Thumbnail image of
Table 3. Table III. Time course of TPO pretreatment on the binding of 125I-TPO to CMK cells. CMK cells were pretreated with or without 10 ng/ml TPO at 37°C for different time periods, washed twice and further incubated (without TPO) at 37°C for 3 h. After cells were washed twice, binding assay was performed as described in Materials and Methods. Results are expressed as % of the control. The data are the mean ± SD of duplicate determinations. The experiment was repeated twice with similar results.Thumbnail image of

In addition, the effect of unlabelled TPO on the binding of 125I-TPO to primary bone marrow megakaryocytes was examined. As shown in Table II, primary bone marrow megakaryocytes demonstrated a decrease in the per cent of specific binding of 125I-TPO when pretreated with TPO (10 ng/ml) for 3 h.

Even though the TPO-induced down-regulation was observed as early as 30 min after incubation, the actual duration in which TPO can stimulate the cells was longer. An incubation period of 3 h was needed to remove the specific surface-bound TPO from the cells. Therefore we have examined whether or not the effect of the TPO-induced down-regulation required new protein synthesis. Cycloheximide at 10 μg/ml, which inhibits most protein synthesis in CMK cells, was added 30 min before TPO pretreatment. The results, shown in Table IV, indicated that cycloheximide did not inhibit the TPO-induced down-regulation effect, suggesting that this TPO-induced effect does not require new protein synthesis. Cycloheximide by itself did not affect the specific-bound 125I-TPO in the CMK cells. When the duration of the cultures was prolonged following TPO treatment, the recovery of TPO binding was slow ( Table IV). The recovery was also observed even in the presence of cycloheximide, although it was very slow. Since the growth of CMK cells was completely inhibited by cycloheximide during that period (data not shown), the recovery of 125I-TPO binding from TPO-induced down-regulation must not require new protein synthesis in part.

Table 4. Table IV. A. Effect of cycloheximide on the TPO-induced down-regulation of binding of 125I-TPO to CMK cells. B. Effect of cycloheximide on the recovery from the TPO-induced down-regulation of binding of 125I-TPO to CMK cells. CMK cells were pretreated with TPO (10 ng/ml) for 3 h at 37°C, washed twice and further incubated (without TPO) with or without cycloheximide (10 μg/ml) at 37°C for the indicated times. After cells were washed twice, binding assay was performed as described in Materials and Methods. Results are expressed as % of the control. The data are the mean ± SD of duplicate determinations. The experiment was repeated twice with similar results.Thumbnail image of

Effects of PMA, DMSO, IL-3 and IL-6 on binding of 125I-TPO

CMK cells were induced to differentiate into mature megakaryocytes by PMA, DMSO, IL-3 and IL-6 ( Sato et al, 1989 ; Komatsu et al, 1989 ; Fuse et al, 1991 ). Preliminary experiments revealed that 125I-TPO binding to the CMK cells was not affected by 100-fold concentrations of both IL-3 and IL-6, indicating that these cytokines do not compete with TPO (data not shown). The pretreatment of the CMK cells with DMSO, IL-3 and IL-6 prior to 125I-TPO binding had no effects on the 125I-TPO binding to the CMK cells ( Table V). Even when the duration of pretreatment was prolonged up to 48 h, a period which is sufficient to induce both proliferation and differentiation of CMK cells, the 125I-TPO binding was not affected.

Table 5. Table V. Effect of pretreatment of PMA, DMSO, IL-6 and IL-3 on the binding of 125I-TPO to CMK cells. CMK cells were pretreated with DMSO, PMA, IL-6 and IL-3 at the indicated concentrations at 37°C for various times. After cells were washed twice, binding assay was performed as described in Materials and Methods. Results are expressed as % of the control. The data are the mean ± SD of duplicate determinations. The experiment was repeated twice with similar results.Thumbnail image of

The pretreatment of the CMK cells with 10 ng/ml of PMA decreased the binding of TPO in a time-dependent manner ( Table V). Quantitative analysis revealed that CMK cells pretreated with 10 ng/ml of PMA for 24 h decreased the number of binding sites to 300/cell without an apparent change of the binding affinity (Fig 2). Treatment with 10 ng/ml PMA for 24 h did not affect cell viability as shown by a trypan blue exclusion assay, although cell growth was inhibited (data not shown). Cycloheximide did not inhibit the PMA-induced decrease of TPO binding (data not shown), indicating that this phenomenon did not require new protein synthesis.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Although the endogenous TPO does not appear to be necessary for the differentiation of precursor cells into megakaryocytes, TPO does amplify this lineage and enhance the development of erythroid and myeloid progenitor cells ( Kuter, 1997). Due to the potential clinical use of TPO in a number of disorders of thrombopoiesis such as aplastic anaemia and thrombocytopenia, it is important to establish the nature and kinetics of the binding of TPO to its cognate receptor. However, the molecular mechanisms of the c-Mpl receptor and its binding to TPO on megakaryocytic cells are not well-defined. Elucidating the molecular mechanisms by which TPO affects haemopoiesis will lead to a better understanding of megakaryocyte growth in both normal and pathologic conditions. In the present study we have demonstrated that TPO binds to the megakaryocytic cell line, CMK, and to primary bone marrow megakaryocytes. The number of binding sites and affinity constant of the CMK cells were 1223 ± 145/cell and 223 ± 25 p M respectively. Primary bone marrow megakaryocytes had a much higher number of binding sites and an affinity constant of 12 140 ± 1867/cell and 749 ± 135 p M respectively. Since human platelets appear to have approximately 25–220 c-Mpl receptors/cell ( Fielder et al, 1995 ), and since one megakaryocyte produces at least 1000 platelets, it is estimated that there is almost no change in receptor numbers during terminal differentiation from megakaryocytes to platelets.

Cytokines down-regulate their own receptors ( Nicola, 1987) similar to hormones ( Catt et al, 1979 ). Although the mechanisms of this phenomenon have not been fully clarified, internalization and differentiation were thought to be the main mechanisms. In the present study, TPO-induced down-regulation was not associated with a differentiation of CMK cells, since IL-3, IL-6 and DMSO, which also induce differentiation of CMK cells ( Sato et al, 1989 ; Komatsu et al, 1989 ; Fuse et al, 1991 ), did not decrease TPO binding to the c-Mpl receptors. Receptors bound with a ligand internalize more rapidly than unoccupied receptors ( Nicola, 1987; Carpentier et al, 1987 ), and these internalized receptors are then recycled back to the cell surface ( Carpentier et al, 1987 ; Brown et al, 1983 ). The numbers of surface receptors decrease until receptor recycling or new receptor synthesis occurs. Internalization as a mechanism of the TPO-induced down-regulation is most likely, but recycling could occur very slowly ( Table IV). Another mechanism was also reported for the c-kit ligand which induced down-regulation through the acceleration of the degradation of the receptor following internalization ( Yee et al, 1993 ). Further studies will be needed to clarify the mechanism of TPO-induced down-regulation.

The mechanisms of down-regulation by phorbol esters are thought to involve the proteolytic cleavage of receptors effecting the release of the ligand binding domain ( Yee et al, 1994 ), immediate stimulation of the early steps in endocytosis, and a more chronic reduction of receptor synthesis ( Schonhorn et al, 1995 ). Since protein kinase C could be activated in a number of disease states ( Bradshaw et al, 1993 ), it will be of interest to assess its activity in disorders of abnormal thrombopoiesis.

It is thought that serum TPO levels are controlled by the platelet mass ( Fielder et al, 1996 ; Kuter & Rosenberg, 1995). However, TPO concentrations in serum are low in some aplastic anaemia patients and in acute phases of idiopathic thrombocytopenic purpura despite their low platelet number ( Emmons et al, 1996 ; our unpublished observation), indicating the possibility that additional mechanisms regulate thrombopoiesis. Interestingly, murine and human thrombopoietin mRNA expression was increased in the bone marrow and not in the liver or kidney under thrombocytopenic conditions, indicating another possible mechanism of regulation of thrombopoiesis ( McCarty et al, 1995 ; Sungaran et al, 1997 ). Also, the regulation of TPO levels by platelets via the c-Mpl receptor on circulating platelets was reported ( Fielder et al, 1995 , 1996). In contrast to platelets from the c-Mpl knockout mice, platelets from normal mice were capable of binding, internalizing and degrading TPO, indicating that circulating TPO levels are regulated by platelets. However, recent studies of experimental thrombocytopenia, particularly in humans, indicate that megakaryocyte mass may be the principal regulator of TPO levels ( Emmons et al, 1996 ). TPO levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction. Our findings that TPO as well as PMA regulate the binding of TPO to megakaryocytes are novel and further support the role of TPO as a regulator of megakaryocytopoiesis.

In summary, we have demonstrated that TPO binding to megakaryocytes and CMK cells is specific and saturable. Following binding, TPO appears to be internalized by these cells causing an apparent TPO-induced down-regulation in c-Mpl number, which may be an important mechanism in regulating thrombopoiesis. Treatment of CMK cells with phorbol ester, an inducer of protein kinase C, also resulted in a down-regulation of TPO-binding. The physiological mechanism(s) involved in the regulation of TPO binding via protein kinase C is an area of great interest for future studies and its elucidation should help in the understanding of normal and abnormal thrombopoiesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This work was supported in part by NIH grants HL51456, HL55445 and HL46668, Ministry of Education, Science and Culture, Japan, and Genentech Inc., South San Francisco, California, U.S.A.

We are grateful to Tee Trac for her typing assistance and Janet Delahanty for proofreading the manuscript.

This paper is dedicated to the memory of Dr Dananagoud Hiregowdara.

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  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References
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