ABSTRACT
Thrombospondin-1 and 2 have each been implicated in collagen fibrillogenesis. We addressed the possibility that deficits in lysyl oxidase (LOX) contribute to the extracellular matrix (ECM) phenotype of TSP-deficient bone. We examined detergent insoluble (mature cross-linked) and soluble (newly secreted) ECM fractions prepared from diaphyseal cortical bone. Detergent-insoluble hydroxyproline content, an indicator of cross-linked collagen content and LOX function, was reduced in female TSP-deficient bones. In male diaphyses, only TSP2 deficiency affected insoluble hydroxyproline content. Western blot suggested that removal of the LOX-pro-peptide (LOPP), an indication of LOX activation, was not affected by TSP status. Instead, the distribution of pro-LOX and mature LOX between immature and mature ECM was altered by TSP-status. LOX was also examined in primary marrow-derived mesenchymal stem cells (MSC) treated with ascorbate. Relative LOPP levels were elevated compared to WT in MSC conditioned medium from female TSP-deficient mice. When cells were serum starved to limit LOX pro-peptide removal, pro-LOX levels were elevated in TSP2-/- cells compared to wild-type. This phenotype was associated with a transient increase in BMP1 levels in TSP2-/- conditioned medium. TSP2 was detected in bone tissue and osteoblast cell culture. TSP1 was only detected in insoluble ECM prepared from WT diaphyseal bone samples. Our data suggest that the trimeric thrombospondins contribute to bone matrix quality by regulating the distribution of pro and mature LOX between newly secreted, immature ECM and mature, cross-linked ECM.
