ABSTRACT
Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4 ß1 and α9 ß1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl(-/-) mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl(-/-) mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN.
Subject(s)
Bone Marrow/metabolism , Hematopoietic Stem Cells/metabolism , Megakaryocytes/metabolism , Osteopontin/metabolism , Thrombin/metabolism , Animals , Cell Differentiation , Cell Movement , Megakaryocytes/cytology , Mice , Stem Cell Niche , Tumor MicroenvironmentABSTRACT
Mature megakaryocytes (MM) can be up to 65 µM in diameter and due to their size, viable and pure MM populations have been difficult to isolate in large numbers. Here in, we report a fluorescence activated cell sorting (FACS) method by which viable and pure populations of 8 N, 16 N, 32 N, and 64 N MM can be isolated from murine bone marrow (BM).
Subject(s)
Bone Marrow Cells/physiology , Megakaryocytes/physiology , Animals , Cell Survival , Cells, Cultured , Flow Cytometry , Immunomagnetic Separation , Mice , Mice, Inbred C57BL , Mice, Transgenic , Platelet Membrane Glycoprotein IIb/metabolism , PolyploidyABSTRACT
Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin. The N-terminal thrombin cleavage fragment of OPN through its binding to the alpha(9)beta(1) and alpha(4)beta(1) integrins plays a key role in the attraction, retention, regulation, and release of hemopoietic stem and progenitor cells to, in, and from their BM niche. Thrombin-cleaved OPN (trOPN) acts as a chemoattractant for stem and progenitor cells, mediating their migration in a manner that involves interaction with alpha(9)beta(1) and alpha(4)beta(1) integrins. In addition, in the absence of OPN, there is an increased number of white blood cells and, specifically, stem and progenitor cells in the peripheral circulation.
Subject(s)
Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Integrin alpha4beta1/metabolism , Integrins/metabolism , Osteopontin/physiology , Animals , Base Sequence , CHO Cells , Cell Line , Chemotaxis/drug effects , Chemotaxis/physiology , Cricetinae , Cricetulus , DNA Primers/genetics , Fetal Blood/cytology , Gene Expression , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoiesis/drug effects , Hematopoiesis/genetics , Hematopoiesis/physiology , Hematopoietic Stem Cells/drug effects , Humans , In Vitro Techniques , Integrin alpha4beta1/genetics , Integrins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Osteopontin/deficiency , Osteopontin/genetics , Osteopontin/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thrombin/metabolismABSTRACT
Primary olfactory neurons situated in the nasal septum project axons within fascicles along a highly stereotypical trajectory en route to the olfactory bulb. The ventral fascicles make a distinct dorsovental turn at the rear of the septum so as to reach the olfactory bulb. In the present study we have used a brain and nasal septum coculture system to examine the role of target tissue on the peripheral trajectory of olfactory sensory axons. In cultures of isolated embryonic nasal septa, olfactory axons form numerous parallel fascicles that project caudally in the submucosa, as they do in vivo. The ventral axon fascicles in the septum, however, often fail to turn, and do not project dorsally towards the roof of the nasal cavity. The presence of olfactory bulb, cortical, or tectal tissue apposed to the caudal end of the septum rescued this phenotype, causing the ventral fascicles to follow a normal in vivo-like trajectory. Ectopic placements of the explants revealed that brain tissue is not tropic for olfactory axons but appears to maintain the peripheral trajectory of growing axons in the nasal septum. Although primary olfactory axons are able to penetrate into olfactory bulb in vitro, they only superficially enter cortical tissue, whereas they do not grow into tectal explants. The ability of axons to differentially grow into different brain regions was shown to be unrelated to the migratory behavior of olfactory ensheathing cells, indicating that olfactory axons are directly responsive to guidance cues in the brain.
