Differential regulation of actin stress fiber assembly and proplatelet formation by alpha2beta1 integrin and GPVI in human megakaryocytes.
ABSTRACT The actin cytoskeleton plays a major role in platelet function. In contrast, its precise role in the function of megakaryocytes (MKs) is less understood but may be important for a chemoattractive response and an efficient proplatelet formation. In the marrow microenvironment, mature MKs are in contact with the extracellular matrix, including fibrillar collagen type I. MKs express alpha2beta1 integrin and the immunoglobulin superfamily member glycoprotein VI (GPVI), the main receptors for collagen. Using function-blocking antibodies or specific ligands, we investigated in primary human MKs how alpha2beta1 integrin and GPVI regulate stress fiber formation, the primary actin structures needed for cell contraction. Stress fiber assembly requires synergistic activation of the MAPK/Erk1/2 pathway and the small guanosine triphosphatase Rho via its effector, Rho-associated coiled-coil kinase (ROCK). alpha2beta1 integrin is crucial for stress fiber formation, whereas GPVI triggers rapid and sustained activation of the Erk1/2 pathway. Strikingly, after a longer adhesion time, proplatelet formation was significantly inhibited by the engagement of alpha2beta1 integrin, not by GPVI, likely through the Rho/ROCK pathway. Thus, proplatelet formation in human MKs could be tightly regulated by differential interactions with their collagen receptors. We propose that this interaction with collagen prevents proplatelet formation within the marrow.
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ABSTRACT: We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK α2β1 develop normally, are fertile, and like their systemic α2β1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet α2β1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of α2β1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of α2 integrin in selected cell types.PLoS ONE 01/2013; 8(1):e55094. · 3.73 Impact Factor
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ABSTRACT: The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding b1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx’’. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted a1b1 and a2b1 integrin mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of a1b1 or a2b1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.PLoS ONE 12/2012; 7(12):e52209. · 3.73 Impact Factor
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ABSTRACT: We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]-ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.Cell Biology International 01/2012; 36(1):39-45. · 1.64 Impact Factor