Identification of osteopontin as a novel ligand for the integrin α8β1 and potential roles for this integrin-ligand interaction in kidney morphogenesis

Epithelio-mesenchymal interactions during kidney organogenesis are disrupted in integrin alpha 8 beta 1-deficient mice. However, the known ligands for integrin alpha 8 beta 1-fibronectin, vitronectin, and tenascin-C-are not appropriately localized to mediate all alpha 8 beta 1 functions in the kidney. Using a method of general utility for determining the distribution of unknown Fntegrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for alpha 8 beta 1. We have coexpressed the extracellular domains of the mouse alpha 8 and beta 1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the alpha 8 beta 1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with alpha 8 beta 1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In "far Western" blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to alpha 8 beta 1-AP. Cell adhesion assays using K562 cells expressing alpha 8 beta 1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin alpha 8 beta 1 may help regulate kidney development and other morphogenetic processes.