Shear stress-induced megakaryocyte maturation involves transcription factors p53 and AP-1
Date
2016
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation to release pro/preplatelets into circulating blood. Shear forces promote DNA synthesis, polyploidization, and maturation in Mks, and platelet biogenesis. To investigate mechanisms underlying these Mk responses to shear, we carried out protein expression and transcriptomic analysis on immature and mature stem cell-derived Mks exposed to physiological shear. In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, six AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12.
We show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos expression. JNK and p38 inhibition reduced fibrinogen binding of d12 platelet-like particles (PLPs). AP-1 expression correlated with increased Mk DNA synthesis and polyploidization, which might explain the observed impact of shear on Mks.
While the AP-1 and MAPK studies provide context for shear-induced polyploidization, questions still remain regarding the apoptosis-related processes activated by shear flow (i.e. increased Caspase 3 activation and PS externalization). There was also a limited understanding of the mechanisms that drive Mk microparticle (MkMP) biogenesis. Here we show that transcription-independent p53-induced apoptosis is involved in shear-induced Caspase 3 activation. We demonstrate that in Mks exposed to shear flow, p53 binds to Bax, likely resulting in Bax channel formation in the mitochondrial outer membrane. This is supported by shear-induced release of cytochrome c into the cytoplasm, with subsequent activation of Caspase 9.
We also provide a mechanism by which MkMPs are generated in response to shear flow. The data suggest that transcription-independent p53-induced apoptosis is involved in this process and that by expressing a mutant form of p53 that cannot bind to Bax, the biogenesis of multiple Mk particles (microparticles, platelet-like particles, and pro/pre-platelets) are negatively affected. We also demonstrate that shear-induced phosphatidylserine externalization is a p53-dependent process, but not regulated by transcription-independent p53-induced apoptosis.
Here we elucidate the specific transcriptional and molecular changes that occur in Mks following exposure to shear flow. Our data implicate both the MAPK pathway and a downstream transcription factor, AP-1. We show significant upregulation of AP- 1 transcripts and proteins in immature and mature Mks following exposure to shear. We also found that the JNK pathway was necessary for the shear stress response in both immature and mature Mks and was linked to the observed AP-1 upregulation in response to shear-flow exposure. We also report that shear flow stimulates transcription-independent p53-induced apoptosis (TIPA), resulting in Caspase 9 activations and biogenesis of Mk particles. These are among the first reported data on the molecular and transcriptional mechanotransduction mechanisms in Mk cells.