Interaction and Regulation of HSC with Osteopontin Is Mediated through α₄β₁ and α₉β₁Integrins, Which Are Differentially Expressed in the HSC Pool.

Osteoblasts are a key cellular component of the hemopoietic stem cell (HSC) niche and directly regulate the HSC pool. Molecules synthesised by osteoblasts both promote or inhibit HSC proliferation. Osteopontin (Opn) is an osteoblast produced, RGD containing protein with roles in cell adhesion and migration. Until recently, the role of Opn in hemopoiesis was seen as restricted to the regulation of bone turnover. However, from analysis of hemopoiesis in the Opn null mouse, we have demonstrated that Opn plays a critical role in regulating the HSC pool. Furthermore Opn is critical in trans-marrow migration and lodgement of HSC within the BM after transplantation. When added to in vitro HSC cultures, exogenous thrombin-cleaved Opn also inhibits cell proliferation and potently suppresses HSC differentiation. We have now demonstrated that this interaction occurs in an RGD-independent manner via the cryptic SVVYGLR epitope revealed on the N-terminal fragment of Opn following thrombin cleavage. This epitope has previously been shown to bind to α₄β₁ and α₉β₁. HSC are known to express α₄β₁, but we have now shown that within the HSC pool this occurs in a differential manner, mimicking that of CD38, with more committed CD34⁺CD38⁺ cord blood progenitors having the highest levels of expression. In addition, we have shown the previously unrecognised characteristic of human marrow and cord blood HSC, the expression of α₉β₁, which also occurs in a differential manner, but mimicking CD34. Expression of α₉β₁ is highest on cord blood CD34⁺CD38⁻ cells, a population highly enriched for HSC. Using the synthetic SVVYGLR peptide in culture, we re-capitulated the thrombin-cleaved Opn induced suppression of HSC differentiation in a dose dependent manner. Antibody blocking experiments demonstrated that binding to this peptide was occurring through both α₄β₁ and α₉β₁. In contrast, suppression of HSC proliferation and differentiation did not occur through the upstream alternate α₄β₁ binding site. Furthermore, we have now demonstrated endogenous binding of Opn to α₄β₁ and α₉β₁ to cord blood HSC in vivo. Together, these data provide strong evidence that Opn is an important component of the HSC niche which acts as a physiological negative regulator. Furthermore, our studies identify the previously unrecognised characteristic of HSC, the expression of α₉β₁, which together with α₄β₁ provides two receptors on HSC with differing expression signatures and potentially a mechanism for fine tunning the physiological effects of Opn.