Focal Adhesion Kinase in Megakaryopoiesis: A Study of Megakaryocyte-Specific FAK Deletion.
Authors: Hitchcock Ian S. , Fox Norma E. , Sear Katherine , Tiedt Ralph , Skoda Radek , Kaushansky Kenneth 
Published: October 2019

Abstract
Megakaryopoiesis is tightly regulated by a number of hematopoietic growth factors to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis, supporting the proliferation and survival of hematopoietic stem cells, driving megakaryocyte differentiation and promoting endomitosis and proplatelet formation. However, recent findings suggest that the chemokines fibroblast growth factor-4 and stromal-derived factor-1 (SDF-1) can partially restore thrombopoiesis in the absence of TPO. These chemokines enhance survival and maturation of megakaryocyte progenitors, as well as platelet release, by promoting progenitor cell movement from the osteoblastic to the vascular niche. However, little is known at present of the molecular mechanisms involved in controlling megakaryocyte motility. Focal adhesion kinase (FAK) is essential for the migration of most cells as it mediates the assembly and disassembly of focal adhesions and we have found it to be highly enriched in megakaryocytes compared to other cells in the bone marrow. Additionally, FAK is activated by SDF-1, a key regulator of chemotaxis, and in this study we found it phosphorylated (activated) by TPO specifically at Y-577 and Y-925. Therefore FAK may be required for megakaryocyte progenitor chemotaxis from the osteoblastic to the vascular niche. In order to determine the potential role of FAK in murine thrombopoiesis we used Cre/loxP technology to conditionally delete fak specifically from megakaryocytes. Mice expressing floxed fak alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter; the progeny failed to express detectable levels of FAK in megakaryocytes by western blotting or immunofluorescence, whilst expression of the gene was unaltered in other tissues, including heart, liver, lung and spleen. While the platelet counts of the mutant mice were normal at steady-state, multiple compensatory mechanisms could be operative. In fact, using megakaryocyte colony assays, we observed a 4-fold increase in the number of colony forming unit-megakaryocytes (CFU-MK) in mice in which fak has been specifically deleted. To clarify the function of FAK in mature megakaryocytes, total bone marrow collected from these and control animals was grown in TPO-containing culture medium for 72 hours and mature megakaryocytes were isolated on a BSA-density gradient. No difference in megakaryocyte adhesion to fibrinogen or fibronectin was found between cells isolated from controls and mutant mice. However, chemotaxis assays using transwell-inserts with an SDF-1α gradient showed a statistically significant increase in chemotaxis in fak null megakaryocytes, compared to controls, suggesting that abnormal cell migration could account for the hematopoietic changes noted in the mice. In summary, we have successfully ablated fak specifically from the megakaryocyte lineage in vivo and are currently using this model to determine a role for this protein in megakaryopoiesis. Specific deletion of FAK in these cells enhances CFU-MK formation and promotes chemotaxis of mature megakaryocytes in response to SDF-1α, which has recently been shown to be required for megakaryocyte motility. Further, we have evidence to suggest that FAK activity is, at least in part, regulated by TPO. Therefore, we propose a previously undescribed role for FAK in megakaryopoiesis and megakaryocyte chemotaxis.