Thrombopoietin Promotes b1 Integrin-Mediated Adhesion in Hematopoietic Cells Via the Small GTPase Rap1.

Abstract The interactions between hematopoietic stem cells (HSCs), non-hematopoietic cells and extracellular matrices in the surrounding microenvironment are critical for hematopoietic survival, differentiation and function. Integrins play many essential roles in mediating these interactions, including supporting HSC homing to hematopoietic tissues and maintaining stem cell properties at the osteoblastic and vascular niches in the bone marrow. We and others have previously demonstrated that adhesion of HSCs and mature hematopoietic cells to α4β1 and α5β1 integrin-specific matrices promotes cell survival and enhances differentiation of specific cell lineages. The hematopoietic cytokine thrombopoietin (TPO) is critically important in supporting HSCs both in vitro and in vivo. In vitro, TPO synergizes with other hematopoietic factors to promote proliferation, whilst mice lacking TPO or its receptor, c-Mpl, display greatly reduced numbers of long-term repopulating HSCs. We report on studies designed to elucidate the mechanisms by which TPO mediates hematopoietic progenitor adhesion. Using the cell line UT7-TPO as a model of c-Mpl-expressing hematopoietic progenitors, we found that TPO potently increased cell adhesion to fibronectin (FN) in a dose-dependent manner, with 50ng/ml human (h)TPO resulting in a 6-fold increase in adhesion after 3 hours. These results were confirmed in primary murine megakaryocytes where 50ng/ml hTPO doubled adhesion to FN. Adhesion was prevented by pre-treatment with specific pharmacological inhibitors to Janus kinase (JAK) and phosphoinositide-3 kinase (PI3K), both of which function downstream of TPO. TPO treatment did not increase levels of α4, α5 or β1 integrin at the cell surface and biotinylated-FN binding assays also confirmed that TPO-induced adhesion was not due to changes in integrin affinity. Further investigation revealed that TPO induced dramatic cytoskeletal changes in UT7-TPO cells, with TPO (50ng/ml) treatment resulting in a 3-fold increase in the number of spread cells on FN. These results suggest that TPO-induced adhesion is mediated via rearrangements in cytoskeleton and possibly integrin avidity. Recently the small GTPase Rap1 was shown to stimulate αIIbβ3 integrin-mediated adhesion to fibrinogen in megakaryocytic cell lines and platelets and can modulate the actin cytoskeleton in a number of cell lineages. Therefore, we investigated the role of Rap1 in TPO-induced adhesion. We have found that TPO activates Rap1 in UT7-TPO cells via PI3K and causes a rapid transient recruitment of Rap1 to the plasma membrane. This translocation of Rap1 has previously been attributed to the action of modulators of the actin cytoskeleton, such as profillin and Ena/VASP (enabled/vasodilator-stimulated phosphoprotein), which require Rap1-GTP for their activation. Further, over-expression of Rap1GAP in UT7-TPO cells ablated TPO-mediated adhesion and spreading on FN. These results suggest a mechanism through which Rap1 can influence cytoskeletal changes in these cells and mediate the favorable effects of TPO on hematopoietic cell adhesion. Taken together, our findings suggest that TPO increases cell adhesion to FN not by integrin activation, but via Rap1-mediated cytoskeletal reorganization, providing a further novel role for TPO in the bone marrow microenvironment.