F-spondin (spondin-1) null mice exhibit increased bone formation, decreased osteoclast function and accelerated osteoarthritis

Abstract

Purpose: We have previously reported that F-spondin (spondin-1), a neuroregulatory protein, is upregulated by chondrocytes in osteoarthritis. These studies showed that spondin-1, a member of the TSR (thrombospondin) type I class super family, activated latent TGF-b1, which appeared to account for selected in vitro effects, including induction of the hypertrophic chondrocyte phenotype. In this study we generated Spon1 knockout mice to investigate the effect of F-spondin in vivo in i) skeletal development and ii) OA progression following surgical destabilization of the medial meniscus (DMM). Methods: Knockout mice were generated in collaboration with the Texas Institute of Genomic Medicine by targeted deletion of exon 1 in C57BL/6 mice. Exon deletion was confirmed by Southern blot analysis and PCR using probes specific for wild type (WT) and mutant loci. Total TGFb-1 was detected using R D p<0.002). Similarly, cultured chondrocytes isolated from the rib cages of 5 day old Spon1-/mice also produced significantly less TGF-b1 (30%) compared to WT controls (p<0.01). To determine whether Spon1 deletion affected bone phenotype, we performed microCT of tibia and femurs in mutant and WT mice aged 1-6 months Relative to WT mice, Spon1-/exhibited increased bone formation at 6 months (Figure 1), evidenced by, a) increased trabecular and cortical bone volume fraction (Bone volume/Total volume: 0.26 0.03 versus 0.16 0.05, p<0.0002; Fig 1), b) decreased trabecular spacing (0.14 0.02 versus 0.19 0.03, p<0.0003); and c) increased trabecular number (7.7 1.2, versus 5.5 0.9, p<0.0005). Interestingly, no significant changes were observed at 1 or 3 months, suggesting that Spon1 effects are age-dependent. Histologically, tibia from Spon1-/mice displayed increased bone ingrowth in the bone marrow cavity, particularly within the spongy bone of the ephysiseal regions. This was accompanied by decreased TRAP staining compared to WT mice, suggesting decreased numbers of osteoclasts. Supporting this observation, osteoclast differentiation, performed by RANKL, M-CSF induction of nonadherent bone-marrow cells, revealed impaired differentiation in Spon1-/mice compared to WT. Preliminary analyses suggest that Spon1 deletion also accelerated cartilage degradation in the DMM model of osteoarthritis. The increased severity of osteoarthritis-like cartilage destruction 7 weeks post surgery was by accompanied by increased thickening of subchondral bone (Figure 2). Conclusions: Our studies indicate that spondin-1 (F-spondin), a latent TGF-b1 activating ECM protein, over-expressed in OA bone and cartilage, regulates bone metabolism in aging mice. Spon1-/mice exhibit increased trabecular and cortical bone formation, decreased osteoclast function and increased susceptibility to surgical induced OA. Together these data suggest that a primary function of spondin-1 in skeletal tissue is the regulation of bone mass via latent TGF-b1 activation. Further studies are in progress regarding the potential of spondin-1 as a drugable target in future therapy of osteoporosis or osteoarthritis. 103 GENE ARRAY PROFILING OF ARTICULAR CHONDROCYTES IN MICE WITH DIFFERENT SUSCEPTIBILITY TO NATURAL DISEASE REVEALS SPECIFIC GENE SIGNATURES LINKED TO HEALTHY AGEING AND SPONTANEOUS OA.