Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins.

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.

Hemopoietic stem cells with higher hemopoietic potential reside at the bone marrow endosteum.

It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions. This methodology resulted in up to a sixfold greater recovery of primitive hemopoietic cells (lineage(-)Sca(+)Kit(+) [LSK] cells) and HSC as shown by transplant studies. HSC from different anatomical regions of the marrow exhibited important functional differences. Compared with their central marrow counterparts, HSC isolated from the endosteal region (a) had 1.8-fold greater proliferative potential, (b) exhibited almost twofold greater ability to home to the BM following tail vein injection and to lodge in the endosteal region, and (c) demonstrated significantly greater long-term hemopoietic reconstitution potential as shown using limiting dilution competitive transplant assays.

Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.

Although recent data suggests that osteoblasts play a key role within the hematopoietic stem cell (HSC) niche, the mechanisms underpinning this remain to be fully defined. The studies described herein examine the role in hematopoiesis of Osteopontin (Opn), a multidomain, phosphorylated glycoprotein, synthesized by osteoblasts, with well-described roles in cell adhesion, inflammatory responses, angiogenesis, and tumor metastasis. We demonstrate a previously unrecognized critical role for Opn in regulation of the physical location and proliferation of HSCs. Within marrow, Opn expression is restricted to the endosteal bone surface and contributes to HSC transmarrow migration toward the endosteal region, as demonstrated by the markedly aberrant distribution of HSCs in Opn-/- mice after transplantation. Primitive hematopoietic cells demonstrate specific adhesion to Opn in vitro via beta1 integrin. Furthermore, exogenous Opn potently suppresses the proliferation of primitive HPCs in vitro, the physiologic relevance of which is demonstrated by the markedly enhanced cycling of HSC in Opn-/- mice. These data therefore provide strong evidence that Opn is an important component of the HSC niche which participates in HSC location and as a physiologic-negative regulator of HSC proliferation.

A novel 110 kDa form of myosin XVIIIA (MysPDZ) is tyrosine-phosphorylated after colony-stimulating factor-1 receptor signalling.

Macrophage colony-stimulating factor (M-CSF or CSF-1) controls the development of macrophage lineage cells via activation of its tyrosine kinase receptor, c-Fms. After adding CSF-1 to M1 myeloid cells expressing CSF-1R (CSF-1 receptor), tyrosine phosphorylation of many cellular proteins occurs, which might be linked to subsequent macrophage differentiation. The biological significance and characterization of such proteins were explored by a dual strategy comprising two-dimensional SDS/PAGE analysis of cell lysates of CSF-1-treated M1 cells expressing the wild-type or a mutated receptor, together with an enrichment strategy involving a tyrosine-phosphorylated receptor construct. In the present study, we report the identification by MS of a novel, low-abundance, 110 kDa form of myosin XVIIIA (MysPDZ, myosin containing PDZ domain), which appears to be preferentially tyrosine-phosphorylated after CSF-1R activation when compared with other known isoforms. Receptor mutation studies indicate that CSF-1R-dependent tyrosine phosphorylation of p110myosin XVIIIA requires Tyr-559 in the cytoplasmic domain of the receptor and is therefore Src-family kinase-dependent. Gelsolin, Erp61 protein disulphide-isomerase and possibly non-muscle myosin IIA were also tyrosine-phosphorylated under similar conditions. Similar to the more abundant p190 isoform, p110 myosin XVIIIA lacks a PDZ domain and, in addition, it may lack motor activity. The phosphorylation of p110 myosin XVIIIA by CSF-1 may alter its cellular localization or target its association with other proteins.