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© The Rockefeller University Press, 0021-9525/1999//1419 $5.00
The Journal of Cell Biology, Volume 147, Number 7, , 1999 1419-1430

Original Article

Primary Megakaryocytes Reveal a Role for Transcription Factor Nf-E2 in Integrin {alpha}iibβ3 Signaling



Masamichi Shiragaa, Alec Ritchiea, Sallouha Aidoudia, Veronique Barona, David Wilcoxb, Gilbert Whitec, Belen Ybarrondod, George Murphye, Andrew Leavitte, and Sanford Shattila

a Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037
b Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
c Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
d PharMingen, Inc., San Diego, California 92139
e Department of Laboratory Medicine, The University of California at San Francisco, San Francisco, California 94143
Department of Vascular Biology, The Scripps Research Institute, 10550 North Torrey Pines Rd., VB-5, La Jolla, CA 92037.(858) 784-7422(858) 784-7148

shattil{at}scripps.edu

Platelet integrin {alpha}IIbβ3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of {alpha}IIbβ3 signaling remain poorly understood. To better understand this process, we established conditions to study {alpha}IIbβ3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of {alpha}IIbβ3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated β3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of {alpha}IIbβ3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2–deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of {alpha}IIbβ3 is controlled by NF-E2–regulated signaling pathways that mature late in megakaryocyte development and converge at the β3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional {alpha}IIbβ3 signaling.

Key Words: {alpha}IIbβ3 • integrin • megakaryocyte • NF-E2 • signaling

© 1999 The Rockefeller University Press

The first two authors contributed equally to this work.

Abbreviations used in this paper: CAT, chloramphenicol acyltransferase; TPO, thrombopoietin.


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