Delayed platelet recovery following cancer therapy is a significant cause of morbidity and mortality. A better understanding of the mechanisms controlling thrombopoiesis may lead to the development of new strategies to treat patients with thrombocytopenia. Here we propose to investigate the role of type-2 Lactosaminyl structures (type2-LacNAc) synthesized by addition of galactose (Gal) to terminal N-acetylglucosamine (GlcNAc) by the galactosyltransferase ?4GalT1 in the regulation of thrombopoiesis. We provide unequivocal evidence that ?4GalT1-dependent type-2 LacNAc synthesis is crucial for thrombopoiesis: 1) ?4GalT1-deficient mice are thrombocytopenic due to impaired megakaryocyte (MK) migration towards the bone marrow vascular niche in the absence of ?4GalT1. ?4GalT1 deficiency in MKs leads to increased ?1 integrin signaling and activity, thereby impairing MK migration. Deletion of the ?1 integrin subunit in ?4GalT1-deficient MKs restores thrombopoiesis, clearly showing that posttranslational modifications, specifically LacNAc decorations of the ?1 integrin, regulate its function and ultimately thrombopoiesis; 2) The chemokine CXCL12 signals via JAK2 and FAK activation to regulate integrin function to promote cell migration. Our results show that CXCL12 activates JAK2 signaling in MKs to upregulate type-2 LacNAc synthesis and ?1 integrin expression to promote MK migration; 3) Platelets isolated from patients with JAK2V617 activating mutation have increased expression of type-2 LacNAc and ?1 integrin subunit, and decreased phosphorylation of FAK. We therefore hypothesize that ?1 integrin signaling and function is regulated via CXCR4, JAK2 and type-2 LacNAc synthesis to promote MK migration and thrombopoiesis. We propose the following aims to investigate our guiding hypothesis:
In Aim 1 we will investigate the role of ?1 integrin in MK and platelet function in the absence of ?4GalT1- dependent type-2 LacNAc synthesis.
In Aim 2 we will determine the molecular mechanism of CXCL12- dependent type-2 LacNAc upregulation mediated by ?4GalT1 in MKs, with focus on JAK2 signaling.
In Aim 3 we will define the role of ?4GalT1 dependent type-2 LacNAc in MKs with activating JAK2V617F mutation. This study will address the gap of knowledge on the role of glycans in thrombopoiesis and will lead to the development of approaches to treat patients with dysregulated platelet production, including patients with myeloporliferative proliferative disease.
The proposed project will improve our understanding of the mechanisms how glycans regulate thrombopoiesis. The knowledge gained with this study may lead to the development of new strategies to treat patients with thrombocytopenia or to contain platelet production in the settings of thrombocytosis.
| Fischer, K; Ohori, S; Meral, F C et al. (2017) Testing the Efficacy of Contrast-Enhanced Ultrasound in Detecting Transplant Rejection Using a Murine Model of Heart Transplantation. Am J Transplant 17:1791-1801 |
