|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Department of Cell and Molecular Biology and
the Department of Pathology, Northwestern University, Chicago, IL.
The megakaryoblastic CHRF-288 cell line was used to investigate
signal transduction pathways responsible for proplateletlike formation
(PPF). The role of fibronectin (FN) and protein kinase C (PKC)
activation in PPF were examined. In the presence of serum and phorbol
12-myristate 13-acetate (PMA), a PKC activator, cells exhibited full
megakaryocytic differentiation, manifested by adhesion, shape change,
increased cell size, polyploidy, PPF, and expression of
CD41+, CD61+, and CD62P+.
The same morphologic and phenotypic features were observed in serum-free cultures in the presence of FN/PMA. Only partial
differentiation occurred when other integrin ligands were
substituted for FN. FN alone induced minimal cell adhesion and
spreading, while PMA alone induced only polyploidy without adhesion.
Signal transduction changes involved the activation of the
extracellular signal-regulated protein kinase 1 (ERK1)/ERK2 as well as
c-Jun amino-terminal kinase 1 (JNK1)/stress-activated protein kinase
(SAPK). Phosphoinositide-3 kinase and p38 were not stimulated
under these conditions. Inhibitors were used to identify the causal
relationship between signaling pathways and PPF. PD98059 and
GF109203X, inhibitors of ERK1/ERK2 pathway and PKC,
respectively, blocked PPF, while adhesion, spreading, and polyploidy
were normal. These studies show that activation of ERK1/ERK2
mitogen-activated protein kinase pathway plays a critical role in PPF.
The elucidation of the signal transduction pathway on megakaryocyte
development and PPF is of crucial importance for understanding this
unique biological process.
(Blood. 2002;99:3579-3584) Megakaryocytopoiesis involves proliferation of
megakaryocyte (MK) progenitors and differentiation. Following a few
mitotic cycles, an aborted mitotic process takes place in which
prophase, metaphase, and initial anaphase stages are normal while late
anaphase and telophase are deficient.1,2 Cytokinesis does
not take place, and endoreplication cycles lead to
polyploidy.3 Nuclear maturation proceeds in concert with
cytoplasmic differentiation and expression of megakaryocytic markers.
Cytoplasmic maturation occurs at different ploidy levels and results in
platelet production (thrombopoiesis). There are different hypotheses
pertaining to platelet formation and release from mature MKs. In one
model, the demarcation membrane system outlines nascent platelets
derived from the interior of the cytoplasm.4,5
Tubular-branching demarcation membranes may be reorganized by a process
of fusion-fission into flat sheets of membrane with subsequent shedding
of platelets into the circulation.6 In another model,
platelets are thought to be released from extruded long cytoplasmic
extensions by rupture of the slender links between
them.7-9 Proplateletlike formation (PPF) in vitro follows
cell adhesion and polyploidization.10,11
Protein kinase C (PKC) is a family of serine/threonine protein
kinases in the cytosol involved in pleiotropic processes such as cell
growth, differentiation, and cytokine secretion.12,13 The
role of PKC signaling in MK differentiation has been well established
over the past 2 decades. In several cell lines, PKC activation by the
agonist phorbol 12-myristate 13-acetate (PMA) induced MK
differentiation, including cell cycle arrest, secretion of cytokines,
up-regulation of MK surface antigens, polyploidization, development of
proplatelet processes, and appearance of demarcation membranes.14-16 Integrins, which are heterodimeric
transmembrane protein receptors, mediate cell membrane-extracellular
matrix interaction and have profound effects on cell division,
differentiation, and survival.17 It has become clear that
integrins not only mediate the physical attachment of cells to
extracellular matrix but also generate a variety of signals to the
interior of the cell18-20 and regulate cell growth,
survival, and gene expression.21-24 Integrin-mediated cell
adhesion has been shown to strongly activate mitogen-activated protein
kinase (MAPK), a key downstream effector of the Raf signaling
pathway in Swiss 3T3/REF52 fibroblasts25 and NIH3T3
fibroblasts.26-27 PKC activation and integrin engagement signaling pathways play different roles in physiological processes and
may generate cross-talk resulting in either up-regulation or
down-regulation, according to the particular demands of different cells.
MAPKs are serine/threonine kinases that are highly conserved in
eukaryotic cells from yeast to human. Several signaling cascades classified as MAPK pathways include extracellular signal-related kinase kinase (MEK)-extracellular signal-regulated protein
kinase 1 (ERK1)/ERK2; MEK-c-Jun amino-terminal kinase
(JNK)/stress-activated protein kinase (SAPK); and MEK-p38. These
pathways have been identified as mediating cell proliferation,
survival, differentiation, and apoptosis. While the ERK pathway
responds mainly to mitogens and growth factors and regulates mammalian
cell proliferation and differentiation, the JNK/SAPK MAPK pathway is
associated with stress and apoptosis.28 The p38
MAPK pathway is activated in response to cellular stress and
inflammation and is involved in many fundamental biological
processes.29-31
Phosphoinositide-3 kinase (PI3K) is known to initiate several signaling
pathways,32 and recently, it has also been found to
stimulate the activation of the ERK1/ERK2 MAPK cascade in some signaling systems as well. Cross-talk and signal integration mechanisms among all these pathways have been described.
While primary MKs have been separated by different
methods,33-36 the yield and degree of purity of the cells
are relatively poor, and consistent PPF is lacking. The study of human
megakaryocytopoiesis and thrombopoiesis requires the development of
either long-term culture systems derived from normal MKs or permanent
cell lines derived from transformed MKs. Therefore, for the purpose of
clarifying the mechanism/signal transduction of PPF, we chose a human
megakaryoblastic CHRF-288 cell line as a culture model that has proved
to be valuable for the study of MKs and MK-associated
functions.37 Our results showed that PPF in
CHRF-288 cells needed 2 critical steps: fibronectin (FN) binding to the
cell and activation of intracellular PKC by PMA. The activation of the
MEK/ERK pathway induced by FN binding and the activation of PKC are
necessary for PPF. Our results suggest substantial overlaps and
cross-talk between the integrin engagement pathway, PKC activation, and
the Ras/MEK/MAPK cascade.
Materials
Cell culture
Morphologic assay Differentiated cells were counted by means of micrometer grids in 1-mm2 areas. Under the 10 × objective lens, the left edge of each culture well was used as the first field; the adjacent 5 fields were counted one by one. Adherent and differentiated cells were stained in situ with Wright Giemsa stain for 15 to 30 minutes, or photographed directly with an LSM510 laser microscope (Zeiss, Jenna, Germany).Electron microscopy Cells or plateletlike particles were fixed in 2% glutaraldehyde, washed with cacodylate buffer, postfixed with 1% osmium tetroxide and uranyl acetate, dehydrated in graded alcohol and propylene oxide, and then embedded with Epon. Ultrathin, 60-nm sections were cut, stained with uranyl acetate and lead citrate to enhance contrast, and examined in a JEOL-100CX electron microscope (Tokyo, Japan).Flow cytometry for phenotypic analysis Cells were counted and viability was assessed by the trypan blue exclusion method. For flow cytometry, cells were washed in PBS containing 1% BSA and then stained for 15 minutes on ice in the dark with fluorescent dye-conjugated monoclonal antibodies specific for MK markers (CD41, CD61, and CD62P) or early myeloid cell markers (CD33 and CD90) (Beckman Coulter, Brea, CA). Corresponding negative controls were fluorescein isothiocyanate-antimouse IgG, phycoerythrin-antimouse IgG, and Cy5-antimouse IgG (Beckman Coulter) used at equivalent IgG concentrations. Each sample contained 5 × 105 cells. Flow cytometric analysis was performed by means of a Coulter Cytometry XL dual laser flow cytometer (Coulter, Hialeah, FL).Ploidy analysis Nonadherent cells were collected by centrifugation. Adherent cells were first dislodged with 0.05% trypsin in 0.33 mM EDTA, and the viable cells counted by trypan blue exclusion. Then, 5 × 105 cells per sample were washed in PBS containing 1% BSA. DNA was stained with 7-amino-actinomycin D (7-AAD) (Calbiochem) following a one-step fixation-permeabilization with the ORTHO PermeaFix reagent (J & J, Raritan, NJ). The ploidy classes were then determined following flow cytometric analysis.Protein extraction and Western blot analysis Cells were collected at 5 minutes, 30 minutes, 1 hour, 2 hours, 24 hours, 48 hours, and 72 hours after PMA treatment in different culture conditions. Nonadherent and dislodged adherent cells were centrifuged at 400g for 5 minutes, and the cell pellets lysed in a buffer containing 30 mM Hepes, 100 mM NaCl, 10 mM benzamidine, 1 mM EDTA, 1% Triton-X-100, and 20 mM NaF, adjusted to pH 7.5. The following protein and phosphatase inhibitors were then added: 1 mM phenylmethyl sulfonyl fluoride, 10 µg/mL aprotinin, 5 µg/mL leupeptin, 2 µg/mL pepstatin, 1 mM Na3VO4. Cell lysates were cleared by centrifugation at 16 000 rpm for 5 minutes at 4°C. The protein concentrations were measured by means of protein/DC Assay (Bio-Rad, Hercules, CA) to ensure equal electrophoretic loading. Lysates were denatured by boiling for 5 minutes in Laemmli sample buffer and loaded at a concentration of 20 µg protein per lane on 10% Tris-glycine iGel. Proteins were then transferred to nitrocellulose membranes and blocked for 16 hours in Tris-buffered saline containing 0.05% Tween 20 and 1% BSA. To ensure equal protein loading, duplicate gels were stained by Ponceau S (Sigma). Antibodies against signal proteins or phosphorylated signal proteins were used to determine the activation of the kinases during cell differentiation. Each antibody was diluted in blocking buffer at the concentration recommended by the supplier and incubated with the blot at room temperature for 2 hours or at 4°C overnight, then incubated with alkaline phosphatase conjugate for 1 hour at room temperature, and color-detected by BCIP/NBT substrate liquid system.Statistical analysis Statistical analysis was performed by means of the 2-tailed Student t test for unpaired data in phenotypic, ploidy, and morphologic analysis.
CHRF-288 cell differentiation in serum-containing medium In the absence of PMA stimulation in serum-containing culture medium, CHRF-288 cells were nonadhesive and proliferated actively (Figure 1A). However, after 5 minutes of exposure to PMA (10 ng/mL), they gradually underwent full differentiation, including adhesion, spreading, size increase, polyploidization, and PPF (Figure 1B), reaching maximal differentiation after 3 days. Thereafter, cells detached from the substrate and underwent apoptosis and cell death. The cells with PPF were characterized by electron microscopy. These elongated cytoplasmic processes (Figure 1C) contained cytoskeletal structures, mitochondria, rough endoplasmic reticulum, macrovesicles, and -granule-like
organelles, but no microtubules were observed. Similar morphology was
observed for the plateletlike particles, and no marginal microtubular
rings, typical of platelets, were seen. The particles did not aggregate
with adenosine 5'-diphosphate or thrombin. The timing of the
differentiation stages was dependent on PMA concentration (1 ng/mL, 10 ng/mL, or 100 ng/mL). The higher the PMA concentration, the more rapid
the differentiation pattern. Without PMA addition, cells showed few
adhesion and shape change patterns (Figure 1A). TPO had no effect on
the differentiation of CHRF cells.
PMA treatment altered the cell surface expression of some membrane
proteins (Table 1). CD33+,
CD90+, and CD62P+ cells significantly
decreased, while CD41+ and CD61+ did not
change. Although cultured CHRF cells show a 2N-4N ploidy pattern in the
absence of PMA (Figure 1D), the addition of PMA led to a significant
increase of the DNA content and ploidy distribution, with the
appearance of 4N to 64N polyploid cells (Figure 1E).
FN is the integrin ligand responsible for full differentiation Different integrin ligands were substituted for serum in the culture medium. While FN alone induced minimal adhesion and shape change, PMA alone induced increased size and polyploidy in the absence of adhesion; FN and PMA costimulation induced full cell differentiation/PPF (Figure 2A). In this culture system, the morphologic features of PPF and functional tests of the particles were similar to the data obtained in serum-containing culture. PMA-treated cells cultured in the presence of LN increased in size without adhesion or PPF. PMA-treated cells cultured in the presence of VN-, CI- and CIV-coated wells showed minimal adhesion and PPF (Figure 2B). No adhesion or differentiation occurred with either BSA or poly-D-lysine-coated culture wells. We determined that the optimal culture model for differentiation and PPF involves seeding CHRF cells at a concentration of 2.5 to 5 × 104 cells per milliliter in X-vivo 20 in the presence of PMA (10 ng/mL) in FN-coated dishes for 3 to 4 days. Using this culture model in a well-defined medium resulted in more consistent PPF than the use of serum in the culture medium.
Activation of ERK1/ERK2 is essential for full differentiation and PPF The differentiation of CHRF cells can be viewed as the result of a balance between stimulatory and inhibitory signals. Western blots were used to identify the activation of signaling molecules related to PPF. When cells were treated with FN alone, a faint phosphorylation signal of ERK1/ERK2 was detected after 1 day of culture (Figure 3A, upper panels). With PMA alone, on the other hand, a weak and quick phosphorylation signal of ERK1/ERK2 was detected within 5 minutes and diminished with time (Figure 3A, middle panel). With both FN and PMA present in the culture, a strong phosphorylation of ERK1/ERK2 occurred. The activation of ERK1/ERK2 was rapid, occurring within 5 minutes of exposure to PMA. The maximum expression of phosphorylated ERK1/ERK2 occurred 30 minutes after PMA treatment, when the cells had firmly adhered to the FN substrate. PMA rapidly and persistently induced the phosphorylation of ERK1/ERK2, but the expression of the kinase tapered with time (Figure 3A, lower panel). Use of the inhibitors PD and GFX in the culture system blocked phosporylation of ERK1/ERK2, as did a high dose of SB (50 µM) (Figure 3B).
To determine a causal relationship between signaling and PPF, we
supplemented our culture model system with various signaling inhibitors
to identify the pathway responsible for differentiation of the CHRF-288
cells (Figure 4). The inhibitors were
added to cells 1 hour before PMA addition. PD98059 (10 µM and50 µM)
and GF109203X (5 µM), inhibitors of upstream of ERK1/ERK2-MAPK and PKC,39 respectively, blocked PPF but not adhesion or size
increase. While a low dose of SB203580 (10 µM), an inhibitor of p38
MAPK40 and JNK/SAPK41 pathways, did not affect
cell differentiation, a high dose (50 µM) of SB blocked the
differentiation. Wortmannin (100 nM and 500 nM), an inhibitor of
PI3K/Akt pathway,39 did not affect cell differentiation. A
high dose of GF109203X (25 µM) was cytotoxic for cells as determined
by the trypan blue exclusion test, and the cell body shrank.
Activation of JNK1/SAPK but not p38 and PI3K Costimulation of cells with FN/PMA induced phosphorylation of JNK1(46 kd)/SAPK within 5 minutes of PMA addition, and this activation lasted at least 2 hours (Figure 5A). Low and high doses of SB (10 and 50 µM) blocked phosphorylation of JNK1 (Figure 5B), but only a high dose of SB blocked PPF (Figure 4C). JNK1/SAPK is therefore not involved in PPF. Neither p38 MAPK nor PI3K/Akt (Ser473 and Thr308) were activated following FN/PMA stimulation (data not shown).
The identification of the signaling pathway responsible for megakaryocytic differentiation, PPF, and platelet release is still debated. In this study, we sought to characterize the MAPK-specific targets in CHRF-288 cells stimulated by FN/PMA. Rapid and prolonged activation of ERK1/ERK2 MAPK was found necessary for PPF. We first investigated a series of integrin ligands for their effects on
PPF.17 As a main component of matrix protein, FN binds to
several megakaryocyte integrins, such as PKC activation with PMA is known to induce a series of biological
processes related to differentiation correlating with the activation of
MAPK.43 In our study, activation of PKC by PMA in the
absence of FN resulted in weak and rapid phosphorylation of ERK1/ERK2;
however, cells increased only in size and ploidy, without either
adhesion or PPF. While the polyploidy pattern is similar in the
presence of PMA alone and of PMA/FN, only the latter induced adhesion
and PPF. Thus, while polyploidy or adhesion is essential for PPF,
neither alone is sufficient to induce this morphologic change. Only
FN/PMA costimulation induced a strong, rapid, and sustained
phosphorylation of ERK1/ERK2 that lasted for at least 72 hours and
correlated with full differentiation, including adhesion, spreading,
size increase, polyploidization, and PPF. Since similar results were
obtained with either serum/PMA or FN/PMA, the integrin ligand FN
appears to be the component in serum responsible for the activation
process leading to cell differentiation and PPF. Even though VN can
also bind to Inhibitors of signaling pathways were used in order to clarify whether there was a causal relationship between ERK1/ERK2 activation and PPF. PD98059, an MEK inhibitor upstream of ERK1/ERK2, blocked the phosphorylation of ERK1/ERK2 as well as PPF, though polyploidy and adhesion were not affected. PPF therefore required not only adhesion to an FN matrix and polyploidy, but also sustained activation of ERK1/ERK2. A causal relationship was therefore demonstrated between ERK1/ERK2 activation and PPF. The phenotypic differences between surface markers of differentiated
and undifferentiated cells are related to the maturation process as
reflected by the decrease of CD90 and CD33, early myeloid surface
markers. The significant decrease of CD62P in differentiated cells may
be due to its translocation to the PPF. CD62P is an Since adhesion and polyploidy are necessary, though not sufficient, for PPF, we investigated stimulation of MAPK pathways other than ERK1/ERK2. The JNK/SAPK pathway is usually activated in response to stress/apoptosis. In this study, phosphorylation of JNK1/SAPK, also induced by FN/PMA costimulation, appeared rapidly in concert with ERK1/ERK2 activation but disappeared after 24 hours, just when cells started to develop PPF. SB203580, an inhibitor of JNK1/SAPK, prevented JNK1/SAPK phosphorylation but did not affect differentiation features of CHRF cells or PPF, when used at a concentration of 10 µM. Thus, though JNK1/SAPK activation correlates with CHRF differentiation, it is not involved in PPF. At a high concentration (50 µM), SB203580 blocked PPF, probably via inhibition of ERK1/ERK2 (Figure 3B). The p38 MAPK is activated when apoptosis, following differentiation, is triggered.40 PI3K, on the other hand, is related to survival, growth, and differentiation.32,39 Since p38 MAPK and PI3K have been reported in some studies to be upstream of the pathway leading to ERK1/ERK2 activation,44,45 we investigated whether this was the case with CHRF cells. In this study, neither kinase was activated following treatment with the FN/PMA combination, and the inhibitors to the respective kinases did not affect cell differentiation or ERK1/ERK2 phosphorylation. Recent reports have shown that TPO activated MK differentiation through the ERK MAPK pathway.46 TPO was ineffective in promoting differentiation features in CHRF cells (data not shown), presumably owing to the lack of the c-Mpl receptor. Only stimulation with the nonphysiological agonist PMA in the presence of FN triggers differentiation. The ultrastructure of PPF and released particles as well as the dysfunction of particles with platelet agonists demonstrates that the CHRF-288 cell line does not produce bona fide platelets. This transformed cell line, however, provides valuable information on the mechanism of MK differentiation, ploidy, and PPF and is useful for a better understanding of molecular events associated with MK development and platelet production.
The authors are grateful to Drs Fern Tablin (University of Califonia at Davis) and Michael A. Lieberman (University of Cincinnati, OH) for the CHRF-288 megakaryoblastic cell line; Mr Phil Lefebvre for helpful discussion; and Dr Mary Dunn for assistance.
Submitted October 9, 2001; accepted January 14, 2002.
Supported in part by grant DAMD17-98-1-8327 from the US Army Breast Cancer Program and a grant from the Rehabilitation Institute of Chicago, both awarded to I.C.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Fang Jiang, Northwestern University, 345 E Superior St, RIC Rm 1407, Chicago, IL 60611; e-mail: f-jiang2{at}northwestern.edu.
1.
Vitrat N, Cohen-Solal K, Pique C, et al.
Endomitosis of human megakaryocytes are due to abortive mitosis.
Blood.
1998;91:3711-3723
2.
Roy L, Coullin P, Vitrat N, et al.
Asymmetrical segregation of chromosomes with a normal metaphase/anaphase checkpoint in polyploid megakaryocytes.
Blood.
2001;97:2238-2247
3.
Nagata Y, Muro Y, Todokoro K.
Thrombopoietin-induced polyploidization of bone marrow megakaryocytes is due to a unique regulatory mechanism in late mitosis.
J Cell Biol.
1997;139:449-457 4. Yamada E. The fine structure of the megakaryocyte in the mouse spleen. Acta Anat (Basel). 1957;29:267-290[Medline] [Order article via Infotrieve].
5.
Zucker-Franklin D, Petursson S.
Thrombocytopoiesis-analysis by membrane tracer and freeze-fracture studies on fresh human and cultured mouse megakaryocytes.
J Cell Biol.
1984;99:390-402
6.
Tavassoli M.
Megakaryocyte-platelet axis and the process of platelet formation and release.
Blood.
1980;55:537-545 7. Behnke O. An electron microscope study of the rat megakaryocyte, II: some aspects of platelet release and microtubules. J Ultrastruct Res. 1969;26:111-129[CrossRef][Medline] [Order article via Infotrieve]. 8. Becker RP, DeBruyn PPH. The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation: a scanning electron microscopic investigation. Am J Anat. 1976;145:183-206[CrossRef][Medline] [Order article via Infotrieve].
9.
Radley JM, Scurfield G.
The mechanism of platelet release.
Blood.
1980;56:996-999 10. Leven RM. Megakaryocyte motility and platelet formation. Scanning Microsc. 1987;1:1701-1709[Medline] [Order article via Infotrieve].
11.
Italiano JE Jr, Lecine P, Shivdasani RA, Hartwig JH.
Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes.
J Cell Biol.
1999;147:1299-1312 12. Ohno S, Akita Y, Hata A, et al. Structural and functional diversities of a family of signal transducing protein kinases, protein kinase C family; two distinct classes of PKC, conventional cPKC and novel nPKC. Adv Enzyme Regul. 1991;31:287-303[CrossRef][Medline] [Order article via Infotrieve].
13.
Nishizuka Y.
Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
Science.
1992;258:607-614 14. Long MW, Smolen JE, Szczepanski P, Boxer LA. Role of phorbol diesters in in vitro murine megakaryocyte colony formation. J Clin Invest. 1984;74:1686-1692. 15. Long MW, Heffner CH, Williams JL, Peters C, Prochownik EV. Regulation of megakarocyte phenotype in human erythroleukemia cells. J Clin Invest. 1990;85:1072-1084. 16. Alitalo R. Induced differentiation of K562 leukemia cells: a model for studies of gene expression in early megakaryoblasts. Leuk Res. 1990;14:501-514[CrossRef][Medline] [Order article via Infotrieve]. 17. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11-15[CrossRef][Medline] [Order article via Infotrieve]. 18. Schwartz MA, Schaller MD, Ginsberg MH. Integrins: emerging paradigms of signal transduction. Annu Rev Cell Dev Biol. 1995;11:549-599[CrossRef][Medline] [Order article via Infotrieve].
19.
Renshaw MW, Price LS, Schwartz MA.
Focal adhesion kinase mediates the integrin signaling requirement for growth factor activation of MAP kinase.
J Cell Biol.
1999;147:611-618 20. Aplin AE, Howe AK, Juliano RL. Cell adhesion molecules, signal transduction and cell growth. Curr Opin Cell Biol. 1999;11:737-744[CrossRef][Medline] [Order article via Infotrieve].
21.
Guadagno TM, Ohtsubo M, Roberts JM, Assoian RK.
A link between cyclin A expression and adhesion-dependent cell cycle progression.
Science.
1993;262:1572-1575
22.
Juliano RL, Haskill S.
Signal transduction from the extracellular matrix.
J Cell Biol.
1993;120:577-585
23.
Fornaro M, Zheng DQ, Languino LR.
The novel structural motif Gln795-Gln802 in the integrin
24.
Boudreau N, Sympson CJ, Werb Z, Bissell MJ.
Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix.
Science.
1995;267:891-893
25.
Chen Q, Kinch MS, Lin TH, Burridge K, Juliano RL.
Integrin-mediated cell adhesion activates mitogen-activated protein kinases.
J Biol Chem.
1994;269:26602-26605 26. Schlaepfer DD, Hanks SK, Hunter T, van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994;372:786-791[Medline] [Order article via Infotrieve].
27.
Lin TH, Chen Q, Howe A, Juliano RL.
Cell anchorage permits efficient signal transduction between Ras and its downstream kinases.
J Biol Chem.
1997;272:8849-8852
28.
Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME.
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science.
1995;270:1326-1331 29. Beyaert R, Cuenda A, Berghe WV, et al. The p38/RK mitogen-activated protein kinase pathway regulates interleukin-6 synthesis in response to tumor necrosis factor. EMBO J. 1996;15:1914-1923[Medline] [Order article via Infotrieve].
30.
Takenaka K, Moriguchi T, Nishida E.
Activation of the protein kinase p38 in the spindle assembly checkpoint and mitotic arrest.
Science.
1998;280:599-602
31.
Wang Y, Huang S, Sah VP, et al.
Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38/mitogen-activated protein kinase family.
J Biol Chem.
1998;273:2161-2168 32. Duronio V, Scheid MP, Ettinger S. Downstream signaling events regulated by phosphatidylinositol 3-kinase activity. Cell Signal. 1998;10:233-239[CrossRef][Medline] [Order article via Infotrieve]. 33. Terstappen LW, Safford M, Loken MR. Flow cytometric analysis of human bone marrow, III: neutrophil maturation. Leukemia. 1990;4:657-663[Medline] [Order article via Infotrieve]. 34. Lefebvre P, Winter JN, Rademaker AW, Goolsby C, Cohen I. In vitro production of megakaryocytes from PIXY321 versus GM-CSF-mobilized peripheral blood progenitor cells. Stem Cells. 1997;15:112-118[Medline] [Order article via Infotrieve].
35.
Levine RF, Fedorko ME.
Isolation of intact megakaryocytes from guinea pig femoral marrow: successful harvest made possible with inhibitors of platelet aggregation; enrichment achieved with a two-step separation technique.
J Cell Biol.
1976;69:159-172 36. Leven RM, Rodriguez A. Immunomagnetic bead isolation of megakaryocytes from guinea-pig bone marrow: effect of recombinant interleukin-6 on size, ploidy and cytoplasmic fragmentation. Br J Haematol. 1991;77:267-273[Medline] [Order article via Infotrieve].
37.
Fugman DA, Witte DP, Jones CLA, Aronow BJ, Lieberman MA.
In vitro establishment and characterization of a human megakaryoblastic cell line.
Blood.
1990;75:1252-1261
38.
Williams SF, Lee WJ, Bender JG, et al.
Selection and expansion of peripheral blood CD34+ cells in autologous stem cell transplantation for breast cancer.
Blood.
1996;87:1687-1691
39.
Wang JF, Park IW, Groopman JE.
Stromal cell-derived factor-1 40. Kanasaki H, Fukunaga K, Takahashi K, Miyazaki K, Miyamoto E. Involvement of p38 mitogen-activated protein kinase activation in bromocriptine-induced apoptosis in rat pituitary GH3 cells. Biol Reprod. 2000;62:1468-1494.
41.
Yue TL, Wang CL, Gu JL, et al.
Inhibition of extracellular signal-regulated kinase enhances ischemia/reoxygenation-induced apoptosis in cultured cardiac myocytes and exaggerates reperfusion injury in isolated perfused heart.
Circ Res.
2000;86:692-699
42.
Borner C, Eppenberger U, Wyss R, Fabbro D.
Continuous synthesis of two protein kinase C-related proteins after down-regulation by phorbol esters.
Proc Natl Acad Sci U S A.
1988;85:2110-2114
43.
Hu X, Moscinski LC, Valkov NI, et al.
Prolonged activation of the mitogen-activated protein kinase pathway is required for macrophage-like differentiation of a human myeloid leukemic cell line.
Cell Growth Differ.
2000;11:191-200 44. Jiang K, Zhong B, Gilvary DL, et al. Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells. Nat Immunol. 2000;1:419-425[CrossRef][Medline] [Order article via Infotrieve].
45.
Ludwig S, Hoffmeyer A, Goebeler M, et al.
The stress inducer arsenite activates mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 via a MAPK kinase 6/p38-dependent pathway.
J Biol Chem.
1998;273:1917-1922
46.
Rojnuckarin P, Drachman JG, Kaushansky K.
Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakarocytes: role in endomitosis.
Blood.
1999;94:1273-1282
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  P. G. Fuhrken, P. A. Apostolidis, S. Lindsey, W. M. Miller, and E. T. Papoutsakis Tumor Suppressor Protein p53 Regulates Megakaryocytic Polyploidization and Apoptosis J. Biol. Chem., June 6, 2008; 283(23): 15589 - 15600. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. V. Vijayan, Y. Liu, W. Sun, M. Ito, and P. F. Bray The Pro33 Isoform of Integrin {beta}3 Enhances Outside-in Signaling in Human Platelets by Regulating the Activation of Serine/Threonine Phosphatases J. Biol. Chem., June 10, 2005; 280(23): 21756 - 21762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Sevinsky, A. M. Whalen, and N. G. Ahn Extracellular Signal-Regulated Kinase Induces the Megakaryocyte GPIIb/CD41 Gene through MafB/Kreisler Mol. Cell. Biol., May 15, 2004; 24(10): 4534 - 4545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. V. Vijayan, Y. Liu, J.-F. Dong, and P. F. Bray Enhanced Activation of Mitogen-activated Protein Kinase and Myosin Light Chain Kinase by the Pro33 Polymorphism of Integrin beta 3 J. Biol. Chem., January 31, 2003; 278(6): 3860 - 3867. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, B. Jian, R. Chu, Z. Lu, Q. Li, J. Dunlop, S. Rosenzweig-Lipson, P. McGonigle, R. J. Levy, and B. Liang Serotonin Mechanisms in Heart Valve Disease II: The 5-HT2 Receptor and Its Signaling Pathway in Aortic Valve Interstitial Cells Am. J. Pathol., December 1, 2002; 161(6): 2209 - 2218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Eto, R. Murphy, S. W. Kerrigan, A. Bertoni, H. Stuhlmann, T. Nakano, A. D. Leavitt, and S. J. Shattil Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling PNAS, October 1, 2002; 99(20): 12819 - 12824. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
3,