Compound Deletion of Thrombospondin-1 and-2 Results in a Skeletal Phenotype not Predicted by Single Gene Knockouts

The trimeric thrombospondin homologs, TSP1 and TSP2, are both components of bone tissue and contribute in redundant and distinct ways to skeletal physiology. TSP1-null mice display increased femoral cross-sectional area and thickness due to periosteal expansion, as well as diminished matrix quality and impaired osteoclast function. TSP2-null mice display increased femoral cross-sectional thickness and reduced marrow area due to increased endosteal osteoblast activity, with very little periosteal expansion. Osteoblast lineage cells are reduced in TSP2null mice, but not in TSP1-null. The functional effects of combined TSP1 and TSP2 deficiency remain to be elucidated. Here, we examined the spectrum of detergent soluble proteins in diaphyseal cortical bone of growing (6-week old) male and female mice deficient in both thrombospondins (double knockout (DKO)). Of 3429 detected proteins, 195 were differentially abundant in both male and female DKO bones. Physiologically relevant annotation terms identified by Ingenuity Pathway Analysis included "ECM degradation" and "Quantity of Monocytes." Manual inspection revealed that a number of proteins with shared expression among osteoclasts and osteocytes were reduced in DKO bones. To associate changes in protein content with phenotype, we examined 12-week old male and female DKO and WT mice. DKO mice were smaller than WT and in male DKO, femoral cross section area was reduced. Some of the male DKO femora also had a flattened, less circular cross-section. Male DKO bones were less stiff in bending and they displayed reduced ultimate load. Displacements at yield load and at max load were both elevated in male DKO. However, the ratios of post-yield to pre-yield displacements significantly diminished in DKO suggesting proportionally reduced post-yield behavior. Male DKO mice also exhibited reductions in trabecular bone mass, which were surprisingly associated with equivalent osteoblast numbers and accordingly increased osteoblast surface. Marrow-derived colony forming unit-fibroblastic was reduced in male and female DKO mice. Together our data suggest that when both TSP1 and TSP2 are absent, a unique, sex-specific bone phenotype not predicted by the single knockouts, is manifested.