Thrombospondins 1 and 2 affect lysyl oxidase protein and collagen matrix maturation in cortical bone of growing male and female mice via non-redundant pathways

Abstract

Thrombospondin-2-deficiency is associated with impaired matrix maturation in osteoblasts and cortical bone of growing mice. Here we addressed the possibility that lysyl oxidase (LOX) contributes to this phenotype. After overnight serum starvation, pro-LOX levels were elevated compared to wild-type in marrow-derived osteoblasts from male and female TSP2-/- mice. The liberated LOX pro-peptide (LOPP) was faintly visible in serum-starved cultures. When serum was maintained, pro-LOX content was not affected by TSP2 status, but relative LOPP levels were elevated in cultures from female TSP2-/- mice. Two isoforms of pro-LOX at 75 kDa and 50 kDa were detected in detergent soluble protein extracts of diaphyseal tissue from growing mice. In female mice, TSP2 status did not affect detergent soluble pro-LOX content or the relative contribution of each band to the total signal. Instead, levels of the 50 kDa band were reduced in female TSP1-/- samples. In male diaphyseal tissue, total pro-LOX content and the contribution each isoform made to the total signal was not affected by TSP1 or TSP2 status. We did not detect 32 kDa mature LOX in detergent soluble preparations of cells or whole bone tissue. Detergent insoluble hydroxyproline content was reduced in diaphyseal tissue obtained from female TSP1-/- and TSP2-/- mice. In male diaphyseal cortical samples, TSP2 but not TSP1 deficiency was associated with reduced insoluble hydroxyproline content. Our data suggest that the trimeric thrombospondins contribute to bone matrix quality via non-redundant mechanisms that are dependent on the unique tissue milieu of the male and female skeleton.