Reactivation of a Hematopoietic Endocrine Program of Pregnancy Contributes to Recovery from Thrombocytopenia

Sumit Bhattacharyya, Jiandie Lin and Daniel I. H. Linzer

Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Daniel Linzer, Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, Illinois 60208. E-mail: dlinzer{at}northwestern.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Regulation of blood platelet levels involves an array of cytokines, including the placental hormone PRL-like protein E (PLP-E). The PLP-E receptor is present on megakaryocytes in pregnant mice, nonpregnant female mice, and male mice. Other known megakaryocytic cytokines do not share the PLP-E receptor, and thus the presence of this receptor in nonpregnant animals suggests that PLP-E may be expressed in tissues other than the placenta. Consistent with this prediction, PLP-E is produced in thrombocytopenic mouse bone marrow, primarily in granulocytes, but not in normal mouse bone marrow. Serum from thrombocytopenic mice, purified thrombopoietin or IL-6, or pregnancy can induce bone marrow cell expression of PLP-E. The induction of PLP-E gene expression in response to thrombocytopenia is physiologically significant, as injection of PLP-E into thrombocytopenic mice restores normal platelet levels with no effect on granulocytes, erythrocytes, and total white blood cell counts. We conclude that inducible expression of PLP-E in bone marrow is part of the mechanism of recovery from thrombocytopenia. These results also suggest a more general concept: that the endocrine program of pregnancy, which in mammals has evolved to support the intrauterine growth and development of the fetus, can also be harnessed to respond to pathophysiology.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THROMBOCYTOPENIA (LOW PLATELET numbers) is of significant clinical concern and results in transfusion of millions of units of platelets annually (1). Platelet levels are determined by the relative rates of platelet production and platelet consumption, with production regulated by the rate of megakaryocyte (MK) differentiation in response to combinations of cytokines. Thrombopoietin (TPO) is the principal cytokine involved in MK development, acting through the c-Mpl receptor at each stage of differentiation from stem cells through the release of platelets from mature MKs (1). Consistent with this role in platelet production, TPO availability to act on early MKs and their precursors is a major determinant in recovery from thrombocytopenia (2, 3, 4, 5, 6, 7). Platelets bind TPO; therefore, as platelet levels fall the concentration of free TPO increases, which in turn stimulates platelet production (8).

Despite the attractiveness of this straightforward model for explaining the mechanism for recovery from thrombocytopenia, TPO acts in concert with other cytokines, and these other cytokines likely contribute in important ways to the recovery process. One of these cytokines in the mouse is a hormone of pregnancy, PRL-like protein E (PLP-E). This hormone was initially identified by a bioinformatics search of expressed sequence tag databases for unknown proteins related to PRL and subsequently shown to be synthesized in the trophoblast giant cells of the placenta (9, 10). With the large changes that occur in hematopoiesis during gestation (11, 12), a reasonable hypothesis is that PLP-E and other placental cytokines superimpose activities on the maternal blood regulatory system that are not present in the normal, nonpregnant adult. Thus, the activity of PLP-E and other related hormones may provide new insights into how blood cell homeostasis can be controlled.

PLP-E induces MK differentiation in primary mouse bone marrow cultures (13). Response to PLP-E requires the gp130 coreceptor (13), suggesting that, similar to many other cytokines, PLP-E signals through a JAK/STAT (Janus family of tyrosine kinases/signal transducer and activator of transcription) pathway. In contrast to its effects on differentiation, PLP-E by itself does not enhance MK proliferation nor does it stimulate colony-forming unit (CFU)-MK growth. In combination with other cytokines, however, including IL-3, IL-3 plus IL-6, or IL-3 plus TPO, PLP-E has a synergistic effect on the induction of CFU-MK (13). Thus, PLP-E can act at multiple steps in the platelet production pathway. Importantly, the ability of PLP-E to enhance CFU-MK growth in response to IL-3 plus IL-6 argues that PLP-E does not simply provide an alternative means of activating gp130-dependent signaling, because IL-6 also acts through this coreceptor.

In contrast to the requirement for other factors in stimulating MK colony formation, PLP-E promotes CFU granulocyte/macrophage (CFU-GM) growth in cultures of mouse bone marrow cells in the absence of other cytokines (14). Using preerythroid cell lines, Müller and colleagues (15) found that PLP-E could also stimulate erythropoiesis and induce STAT activation in these cells. Thus, in addition to its effects on committed MKs, PLP-E either targets multipotential precursors, which then give rise to distinct blood cell types, or PLP-E acts separately on several distinct blood cell lineages.

Although the human equivalent of mouse PLP-E has not been identified from searches of the human genome sequence, the PLP-E receptor is present on human MKs (16). Similar to its effects on mouse MK precursors, PLP-E in combination with TPO, but not PLP-E alone, enhances the growth of human CFU-MK. An effect of PLP-E alone on MK differentiation and on CFU-GM growth, however, was not observed starting with CD34+ human bone marrow cells. Strikingly, in this human cell system PLP-E synergizes with TPO, and with a cocktail of TPO, stem cell factor, and flt3 ligand, to stimulate the growth of CD34+ progenitor cells, the multipotential myeloid precursor CFU-granulocyte, erythrocyte, monocyte, MK, and the precursor burst-forming unit-erythroid (16). Thus, the ability of PLP-E to modulate hematopoiesis is conserved between mouse and human.

The detection of PLP-E effects on blood cells from a variety of individuals, none of whom were pregnant, suggested that in the mouse the receptor for this hormone may also be expressed outside of pregnancy. That point of departure for the current study has led us to investigate whether PLP-E might be involved in platelet production in nonpregnant animals under certain conditions. What we find is that PLP-E can indeed be expressed by cells other than trophoblasts, the major cell type of the placenta, and that this expression is linked to, and functionally important for, the response to thrombocytopenia.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of PLP-E to MKs
A fusion protein containing PLP-E linked to alkaline phosphatase (AP-PLP-E) has been used to detect specific PLP-E binding sites on cells in the pregnant mouse spleen and bone marrow (13). These cells were shown to be MKs based on cell morphology and on the presence of MK-specific marker proteins. To determine whether expression of this receptor on the surface of MKs is restricted to pregnant females, or whether it is expressed more broadly, frozen spleen sections from pregnant female, nonpregnant female, and male mice were prepared and tested for hormone binding. As revealed by staining for heat-stable AP activity, PLP-E binds to MKs present in all three spleen samples (Fig. 1Go).



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Figure 1. PLP-E Binding to MKs

Cross-sections of spleens from a pregnant female, nonpregnant female, and male mouse were incubated with AP-PLP-E and stained for AP activity. PLP-E binding to MKs is seen as darkly stained cells. Magnification, x100.

 
Two possible implications of this result are that PLP-E shares a receptor with a cytokine the production of which is not restricted to pregnancy, or that PLP-E is expressed in tissues other than the placenta and under conditions other than pregnancy. To test the first possibility, various cytokines known to act on MKs were added along with AP-PLP-E to spleen sections, and binding of the fusion protein was assayed by staining for AP activity. Binding is effectively competed by PLP-E, but binding is still detected upon addition of other cytokines, including erythropoietin, TPO, IL-6, IL-11, leukemia inhibitory factor, or stem cell factor (Fig. 2Go). In addition, PRL, GH, IL-3, and ciliary neurotrophic factor fail to compete effectively for PLP-E binding sites (data not shown). This result does not eliminate the possibility that another cytokine exists that also binds the PLP-E receptor, but does suggest that the alternative hypothesis of PLP-E expression in sites other than the placenta should be considered.



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Figure 2. A Unique Receptor for PLP-E

Spleen sections were incubated without competitor or with GST-PLP-E (PLP-E), erythropoietin (EPO), TPO, IL-6, IL-11, leukemia inhibitory factor (LIF), or stem cell factor (SCF). GST-PLP-E competitor was used at 5 µg/ml, whereas other cytokines were added at concentrations of 100 µg/ml. AP-PLP-E was then added and sections were stained for AP activity. PLP-E binding is detected as darkly stained cells in all panels except for the prior incubation with GST-PLP-E. Magnification, x100.

 
PLP-E Expression in Response to Thrombocytopenia
RNA analysis had not revealed any PLP-E-positive tissues except the placenta (9). Possibly, expression does occur but only under certain, induced conditions. Given the effects of PLP-E on MKs, such a condition may be thrombocytopenia. To induce thrombocytopenia, mice were injected with neuraminidase (17). Platelet levels drop rapidly after treatment, and full recovery is not achieved for 5 d (Fig. 3AGo). No expression of PLP-E is seen in liver or spleen of the thrombocytopenic mice (data not shown), but PLP-E mRNA (data not shown) and protein (Fig. 3BGo) are detected in bone marrow extracts of neuraminidase-injected, but not control, mice within 3 d after treatment. The size of this glycoprotein is similar to the largest PLP-E glycoform that accumulates in maternal serum, suggesting that the bone marrow produces mature hormone. Immunostaining of bone marrow cells after neuraminidase injection reveals PLP-E expression in a subset of these cells, but no immunopositive cells are detected in bone marrow cells from saline-injected controls (Fig. 4Go). Most of the immunopositive cells are granulocytes, but PLP-E protein is also detected in other bone marrow cell types.



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Figure 3. Reactivation of PLP-E Expression in the Bone Marrow in Thrombocytopenic Mice

A, Mice were injected with neuraminidase, and platelet levels were measured on that day and succeeding days. B, Total protein from bone marrow cells isolated 3, 4, or 5 d after injection of mice with saline (control) or neuraminidase was fractionated by gel electrophoresis. Proteins were transferred to a filter and probed with an antiserum against PLP-E. Maternal serum from d 12 of pregnancy was used as a positive control for PLP-E protein. All lanes are from a single gel.

 


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Figure 4. Detection of PLP-E Protein in Neuraminidase-Treated Mouse Bone Marrow Cells

Mice were injected with neuraminidase (A and B) or with saline only (C), and bone marrow cells were harvested 4 d later. Cells were attached to glass slides and immunostained with anti-PLP-E (A and C) or with no primary antiserum (B) and counterstained with hematoxylin. Positive staining (dark brown) is detected after neuraminidase treatment (A) but not in the control (C). No immunostaining is detected with bone marrow samples from neuraminidase-treated mice in the absence of the primary anti-PLP-E antiserum (B). PLP-E expressing cells were identified by examination of cell morphology under high magnification.

 
To verify that PLP-E gene activation in the bone marrow is in response to thrombocytopenia, rather than to neuraminidase treatment itself, a second model of thrombocytopenia was analyzed. In mice bearing a homozygous mutation in the mafG gene, platelet production is impaired (18). These mutant mice constitutively express the PLP-E gene in the bone marrow, in contrast to heterozygous siblings (Fig. 5Go). Thus, bone marrow PLP-E expression appears to be consistently induced in response to abnormally low platelet levels.



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Figure 5. PLP-E Expression in the Bone Marrow of mafG Mutant Mice

Total RNA was isolated from the speen and bone marrow (BM) of 2-wk-old littermates, bearing either a heterozygous (+/-) or homozygous (-/-) mutation in the mafG gene. The RNA was analyzed by RT-PCR for the PLP-E mRNA or for the ribosomal protein L19 mRNA as an internal control.

 
That the PLP-E gene is reactivated in response to thrombocytopenia suggests that a stimulatory factor accumulates, or an inhibitory factor decreases, in the serum or bone marrow as platelet levels fall. This factor would then act on bone marrow cells to induce a signal transduction cascade leading to PLP-E gene transcription. To test these predictions, bone marrow cells from normal mice were cultured in the presence of serum or bone marrow extracts from control or thrombocytopenic mice. After 2 d, RNA was prepared and analyzed for PLP-E expression. No PLP-E mRNA is detected in the untreated bone marrow cultures, in cultures maintained with serum or bone marrow extracts from control mice, or in cultures exposed to bone marrow extracts from thrombocytopenic mice. In contrast, PLP-E mRNA is seen in cultures supplemented with serum from thrombocytopenic mice (Fig. 6AGo). Candidates for the PLP-E-inducing serum factor include cytokines that act on bone marrow cells, including TPO, IL-3, and IL-6. Addition of purified TPO to bone marrow cultures from normal mice results in a dose-dependent activation of PLP-E expression, and IL-6 is an even stronger inducer of PLP-E mRNA levels (Fig. 6BGo). Cultures treated with IL-3, which stimulates proliferation in these primary cell cultures (data not shown), have no observable accumulation of PLP-E mRNA, demonstrating specificity in the response to TPO and IL-6.



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Figure 6. Induction of PLP-E Expression

A, Primary bone marrow cell cultures from normal mice were treated for 72 h with no additions, with bone marrow extract or serum from control mice, or with bone marrow (BM) extract or serum from thrombocytopenic mice collected 48 h after induction of thrombocytopenia. B, Primary bone marrow cell cultures from normal mice were treated for 72 h with TPO (50, 25, or 10 ng/ml), IL-3 (10 ng/ml), or IL-6 (10 ng/ml). C, Bone marrow (BM) and placental tissue were isolated on d 10 and d 11 of pregnancy. For all three panels, RNA was then purified and analyzed by RT-PCR for PLP-E mRNA (upper panels) and for the ribosomal protein L19 mRNA control (lower panels). The same results were obtained in two independent experiments.

 
During pregnancy, increased maternal blood volume and increased platelet consumption can bring about a transient, physiological thrombocytopenia. It might be the case, therefore, that PLP-E expression in the bone marrow of thrombocytopenic mice and in the placenta of pregnant mice at midgestation is under similar hormonal control. If so, then the PLP-E gene might be expected to be transcribed in the bone marrow of pregnant mice coincident with expression of this gene in placental trophoblast giant cells. Consistent with this expectation, PLP-E mRNA is found in the bone marrow of pregnant mice at midgestation (Fig. 6CGo).

Effect of PLP-E on Platelet Levels
To determine whether activation of PLP-E gene expression in the bone marrow contributes to recovery from thrombocytopenia, mice were injected with neuraminidase and then injected twice daily with biologically active PLP-E purified from bacterial lysates as a glutathione S-transferase (GST) fusion protein (GST-PLP-E) or with GST alone. Platelet counts were measured after 2 d. PLP-E treatment results in a dose-dependent and significant increase in platelet numbers, and at the highest dose of 30 µg/mouse the platelet level is indistinguishable from that in the control mice, which did not receive neuraminidase (Fig. 7AGo). PLP-E, therefore, is not only capable of stimulating MK differentiation in culture (13) but also of stimulating platelet production in vivo. Neuraminidase treatment had no effect on granulocyte, erythrocyte, or total leukocyte numbers (Fig. 7Go, B–D). Although a nearly 2-fold increase in granulocytes is detected after PLP-E injection (Fig. 7BGo), this change did not reach statistical significance. No change in erythrocyte or total leukocyte numbers follows administration of PLP-E (Fig. 7Go, C and D). Thus, the effects of both neuraminidase and PLP-E are specific for the MK compartment in this system.



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Figure 7. Recovery from Thrombocytopenia in Response to PLP-E

Mice were injected with saline or with neuraminidase, and the neuraminidase-treated animals were also injected twice daily with different amounts of GST-PLP-E (from 5–30 µg) or with GST (30 µg). After 48 h, blood was drawn for determination of the number of platelets (A), granulocytes (B), erythrocytes (C), and total leukocytes (D). Data are the mean ± SE, with n = 3 for the 5 µg GST-PLP-E treatment and n = 4 for all other groups. Statistical significance was determined by a one-way ANOVA followed by a post-hoc Tukey’s test. a vs. b and c, P < 0.001; b vs. c, P < 0.001; a vs. d, P > 0.05; b vs. d, P < 0.001.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Low platelet levels, resulting either from a pharmacological or a genetic model of thrombocytopenia, trigger a response in the bone marrow that includes the reactivation of the PLP-E gene, a gene that we had previously found to be expressed only in trophoblast giant cells of the placenta. In total bone marrow extracts, PLP-E expression is detected after the decline in platelet levels in neuraminidase-treated mice and preceding the return to normal platelet concentrations. Thus, PLP-E is produced in both the appropriate tissue and at the appropriate time to participate in the recovery process. Because only a subset of cells in the bone marrow produce PLP-E, detection of PLP-E protein in extracts is probably delayed relative to the onset of expression. The earliest time of PLP-E synthesis in response to declining platelet levels remains to be determined.

Administration of PLP-E to thrombocytopenic mice induces a dose-dependent increase in platelet levels, demonstrating that PLP-E expression during thrombocytopenia is functionally linked to the recovery. The effective concentration in the bone marrow of the maximal dose of GST-PLP-E, 30 µg/mouse delivered twice daily, is not known. Production of PLP-E as a fusion protein in bacteria offers advantages of high yield, rapid purification, and lack of contaminating mammalian cytokines, but it is important to recognize that GST-PLP-E is not identical with mature PLP-E produced by the placenta or bone marrow. In contrast to the mammalian hormone, the fusion protein lacks N-linked carbohydrate, which may have important consequences on half-life in the circulation and accumulation in the bone marrow, and the N-terminal extension may reduce bioactivity. It is our expectation, therefore, that significantly lower doses of the mature PLP-E glycoprotein hormone compared with GST-PLP-E would be needed to restore platelet levels.

The ability of PLP-E to stimulate recovery from thrombocytopenia does not prove that production of this hormone in the bone marrow is required for recovery in neuraminidase-treated mice. To test the importance of PLP-E in the recovery process, it will be necessary to block PLP-E expression or function. In the future, it should be possible to mutate the PLP-E gene, and a molecular characterization of the PLP-E receptor could lead to the development of an injectable PLP-E binding protein or antagonist.

Most of the PLP-E-positive cells in the thrombocytopenic bone marrow are granulocytes, and we have previously shown that PLP-E is sufficient to stimulate CFU-GM growth in mouse bone marrow cultures (14). A logical prediction therefore would be that, in addition to acting on CFU-MK to stimulate platelet production, PLP-E would act as an autocrine or paracrine factor to stimulate granulocyte production. Nevertheless, the nearly 2-fold increase in granulocytes detected after administration of PLP-E to thrombocytopenic mice is not statistically significant. It may be that homeostatic regulation of granulocyte levels blunts a response to PLP-E that would lead to excessive granulocyte production. Neuraminidase treatment does not cause a decrease in circulating granulocyte numbers, so it remains possible that bone marrow expression of PLP-E would occur and would stimulate granulocyte production under conditions in which circulating granulocyte concentrations fall. PLP-E has also been reported to stimulate erythropoiesis (15), but treatment of thrombocytopenic mice with PLP-E does not cause elevated erythrocyte numbers. As with granulocytes, red blood cell numbers are unaffected by neuraminidase treatment, but the possibility remains that PLP-E is capable of stimulating erythropoiesis in response to anemia.

The evolution of genes encoding placental hematopoietic hormones presumably resulted from the advantage these factors confer for reproductive success by modifying maternal physiology to accommodate the demands of the growing fetus. The contribution of PLP-E to recovery from thrombocytopenia suggests that mammals have also evolved the means to take advantage of the endocrine program of pregnancy as an inducible regulatory system in the nonpregnant state. We do not detect PLP-E expression in the bone marrow of nonpregnant, control mice, suggesting that PLP-E is not involved in the normal homeostatic regulation of platelet production, but instead that PLP-E is part of the response to an acute and severe hematopoietic stress. PLP-E expression may represent just one example of the reactivation of select portions of the extraembryonic genetic program under conditions other than pregnancy. Expression of the closely related angiogenic placental hormone proliferin (19) in wound sites (20) and in certain tumor cells as they develop an angiogenic phenotype (21) supports this general model.

Placental hormones in the PRL family display gestational stage-specific and cell type-specific patterns of expression (22). It is possible that the regulatory elements that establish when and where each hormone is produced in the placenta during pregnancy are also used in nonplacental tissues. Indeed, the detection of PLP-E mRNA in the bone marrow of pregnant mice at the same time that this hormone is produced by the placenta suggests a shared transcriptional regulatory mechanism for the PLP-E gene in these two tissues. The finding that serum from thrombocytopenic mice stimulates PLP-E synthesis in primary bone marrow cultures argues that blood cells and their precursors in the bone marrow have a receptor for a circulating factor that increases in concentration during thrombocytopenia and that triggers these cells to transcribe the PLP-E gene. Two attractive candidates were tested as the possible PLP-E-inducing factor: TPO and IL-6. Platelets sequester TPO; therefore, as platelet levels fall, circulating TPO concentrations increase (8). For IL-6, synthesis and protein levels have been found to increase in response to thrombocytopenia in rodents in some (23, 24) but not all (25) experimental systems. Both TPO and IL-6, but not IL-3, activate PLP-E gene expression in bone marrow cell cultures, with IL-6 having the more pronounced effect. Thus, these cytokines are sufficient to induce PLP-E synthesis, although it is not yet known whether these are the principal inducers present in thrombocytopenic mouse serum. TPO and IL-6 trigger STAT activation (26, 27), and consensus STAT sites are present upstream of the PLP-E gene transcription start site (data not shown). It should be possible to examine the role of these putative regulatory sites in PLP-E expression in the bone marrow cell and trophoblast.

In addition to TPO and IL-6 stimulating PLP-E synthesis, TPO and IL-6 each synergize with PLP-E in stimulating mouse CFU-MK growth (13). Thus, recovery from thrombocytopenia after injection of TPO (2, 3, 4, 5, 6, 7), for example, may, in fact, reflect not just a direct effect of one cytokine on the MK lineage, but instead the up-regulation of expression of PLP-E and subsequent synergistic actions of multiple cytokines.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Mice were maintained on 14-h light,10-h dark cycles with food and water freely available. For hormone binding studies, spleens were isolated from CD1 male and pregnant and nonpregnant female mice (Harlan, Madison, WI). To induce thrombocytopenia, neuraminidase (Clostridium perfringens type VI from Sigma, St. Louis, MO) in sterile 150 mM NaCl was injected ip at 0.05 U per female C57/Bl6 mouse (17). For some mice, neuraminidase injection was followed immediately by ip injection of GST-PLP-E or GST with additional injections at 12-h intervals. Blood was collected from the retroorbital plexus, and samples were analyzed for cell and platelet numbers by the Center for Comparative Medicine clinical pathology facility at Northwestern University Medical School. All procedures were approved by the Animal Care and Use Committee.

Preparation of Fusion Proteins
Expression of an AP-PLP-E fusion protein in CHO cells and the use of conditioned medium for binding assays has been described previously (13). In brief, tissue sections were incubated with appropriate competitor for 30 min at room temperature before incubating with AP-PLP-E for 45 min. Slides were rinsed in HBSS and fixed in 20 mM HEPES (pH 7.4), 60% acetone, 3% formaldehyde. Samples were heated at 65 C for 30 min to inactivate endogenous APs, and the slides were developed with a chromogenic reaction for AP activity. GST and GST-PLP-E were expressed in bacteria and purified by glutathione-agarose chromatography as described previously (13).

Bone Marrow Cell Cultures
Bone marrow was collected from femurs of normal, 8-wk-old C57/Bl6 female mice by flushing with Iscove’s modified Dulbecco’s medium (IMDM) from Life Technologies, Inc. (Gaithersburg, MD). The aspirate was passed through a 21-gauge needle to separate cells, which were then seeded into IMDM and incubated at 37 C for 1 h. The medium and nonadherent cells were collected and centrifuged, and the cell pellet was resuspended at a density of 400,000 cells/ml in IMDM containing 1% BSA, 10 µg/ml insulin, 200 µg/ml transferrin, 2 mM glutamine, and 0.1 mM mercaptoethanol. Cells were transferred to six-well plates and incubated for 3 d with various test substances, including TPO (Sigma), IL-3 (Stem Cells Technology, Vancouver, Canada), IL-6 (Sigma), 10% serum from control or thrombocytopenic mice, or bone marrow extracts from control or thrombocytopenic mice. The bone marrow extracts were prepared by passing the cell suspension collected from femurs through a 0.45-µm membrane, and the filtrate was then concentrated by centrifugation through a 500-Da cut-off filter (Amicon, Inc., Beverly, MA); the entire preparation from one femur was used per well.

RNA and Protein Analysis
Total RNA was purified from isolated bone marrow cells or placenta with Tri-Reagent (Sigma). An RT-PCR (using 2 µg of RNA as template) was used to detect PLP-E mRNA with oligonucleotide primers specific to PLP-E. As an internal control, primers specific for the ribosomal protein L19 mRNA were also included. Reactions included an {alpha}-32P-deoxyribonucleotide to generate radiolabeled products for detection by gel electrophoresis and autoradiography. For immunoblot analysis, equal amounts of protein were fractionated by gel electrophoresis and probed with a rabbit polyclonal antiserum against PLP-E followed by goat-antirabbit antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech, Arlington Heights, IL).

Immunohistochemistry
Bone marrow smears were air dried, fixed in 4% paraformaldehyde, and exposed to rabbit-anti-PLP-E followed by biotinylated goat-antirabbit IgG. Vectastain ABC and peroxidase substrate (Vector Laboratories, Inc., Burlingame, CA) were used for detection. The identities of immunopositive cells were determined by the Special Hematology Laboratory in the Department of Hematology and Oncology, Children’s Memorial Hospital (Chicago, IL).


    ACKNOWLEDGMENTS
 
We thank Carolyn Jahn and Doug Engel for mafG mutant mice, Jack Levin for advice on neuraminidase treatment to induce thrombocytopenia, Andrew Campbell for advice on blood cell counting, and Doug Engel for comments on the work and manuscript.


    FOOTNOTES
 
This work was supported by NIH Grant HD-24518 and by the Robert H. Lurie Comprehensive Cancer Center of Northwestern University.

Abbreviations: AP, Alkaline phosphatase; CFU, colony-forming unit; GM, granulocyte/macrophage; GST, glutathione-S-transferase; IMDM, Iscove’s modified Dulbecco’s medium; MK, megakaryocyte; PLP-E, PRL-like protein E; STAT, signal transducer and activator of transcription; TPO, thrombopoietin.

Received for publication January 8, 2002. Accepted for publication February 19, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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