Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs.

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Inflammation in arthritis induces expression of BMP3, an inhibitor of bone formation.

OBJECTIVES: Inflammation in diseases such as rheumatoid arthritis (RA) stimulates osteoclast-mediated articular bone erosion and inhibits osteoblast-mediated bone formation, leading to a net loss of bone. Pro-inflammatory cytokines and antagonists of the Wnt signalling pathway have been implicated in the inhibition of osteoblast differentiation and activity in RA, contributing to the erosive process and impairing erosion healing. Importantly, osteoblast differentiation and function are also regulated by the osteogenic bone morphogenetic protein (BMP) signalling pathway, which is antagonized by BMP3. We therefore examined the potential role of BMP3 in inflammatory arthritis. METHOD: Two murine models of RA, K/BxN serum transfer arthritis (STA) and antigen-induced arthritis (AIA), were used to establish the temporal expression of BMP3 and the cellular sources of BMP3 mRNA and protein in inflammatory arthritis. To determine the effects of inflammation on the expression of BMP3 in osteoblasts, murine calvarial osteoblasts were treated with pro-inflammatory cytokines and BMP3 expression was assessed. RESULTS: In both murine models of RA, BMP3 mRNA and protein are highly expressed by osteoblasts lining inflammation-bone interfaces late in the course of arthritis. Synovial tissues are not a significant source of BMP3. BMP3 expression is induced in osteocalcin-expressing osteoblasts in vitro following stimulation by tumour necrosis factor (TNF). CONCLUSIONS: These data implicate BMP3 as a novel factor that may act locally to contribute to the erosive process and inhibit the repair of articular bone in RA through inhibition of osteoblast differentiation and function.

Impaired muscle function in a mouse surgical model of post-traumatic osteoarthritis.

OBJECTIVES: Using a mouse surgical model of post-traumatic osteoarthritis (OA), we sought to determine if muscle function is altered following acute joint injury and whether this relates to OA progression. DESIGN: Male C57BL/6 mice underwent surgical transection of the medio-meniscal tibial ligament destabilisation of the medial meniscus (DMM) or sham surgery on one knee. Tibialis anterior (TA) muscle function was assessed in situ at 1, 4 and 8 weeks post-surgery. Cartilage damage and joint inflammation were assessed by histologic scoring. Muscle mRNA expression was quantified by qRT-PCR. RESULTS: Tetanic and twitch force production between DMM and sham muscle did not differ at 1 week post-surgery. Muscle function improved in both groups with time, but specific force production in DMM muscles was 18% and 22% lower than sham muscles at 4 and 8 weeks post-surgery respectively. At 8 weeks post-surgery, DMM muscles had a 40% slower relaxation rate and reduced expression of sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (Serca) pump mRNA compared to sham muscles; both observations indicate likely alterations in muscle Ca(2+) handling. There were no histologic signs of muscle atrophy or inflammation in DMM TA muscles. Specific force production in both sham and DMM mice showed a negative correlation with the severity of joint inflammation. CONCLUSIONS: Acute knee injury in the DMM model of post-traumatic OA leads to a persistent deficit in TA muscle function that occurs in the absence of muscle atrophy. This study highlights that the impact of acute knee injury is unlikely to be limited to the muscles controlling knee movement.

Activated human T cells express alternative mRNA transcripts encoding a secreted form of RANKL.

Receptor activator of nuclear factor-kappaB-ligand (RANKL), encoded by the gene TNFSF11, is required for osteoclastogenesis, and its expression is upregulated in pathologic bone loss. Transcript variants of TNFSF11 messenger RNA (mRNA) have been described that encode a membrane-bound and a putative secreted form of RANKL. We identify a TNFSF11 transcript variant that extends the originally identified transcript encoding secreted RANKL. We demonstrate that this TNFSF11 transcript variant is expressed by the human osteosarcoma cell line, Saos-2, and by both primary human T cells and Jurkat T cells. Of relevance to the production of RANKL in pathologic bone loss, expression of this secreted TNFSF11 transcript is upregulated in Jurkat T cells and primary human T cells upon activation. Furthermore, this transcript can be translated and secreted in Jurkat T cells in vitro and is able to support osteoclast differentiation. Our data highlight the complexity of the TNFSF11 genomic locus, and demonstrate the potential for the expression of alternate mRNA transcripts encoding membrane-bound and secreted forms of RANKL. Implications of alternate mRNA transcripts encoding different RANKL protein isoforms should be carefully considered and specifically examined in future studies, particularly those implicating RANKL in pathologic bone loss.

Calcitonin impairs the anabolic effect of PTH in young rats and stimulates expression of sclerostin by osteocytes.

The therapeutic goal of increasing bone mass by co-treatment of parathyroid hormone (PTH) and an osteoclast inhibitor has been complicated by the undefined contribution of osteoclasts to the anabolic activity of PTH. To determine whether active osteoclasts are required at the time of PTH administration, we administered a low dose of the transient osteoclast inhibitor salmon calcitonin (sCT) to young rats receiving an anabolic PTH regimen. Co-administration of sCT significantly blunted the anabolic effect of PTH as measured by peripheral quantitative computer tomography (pQCT) and histomorphometry in the femur and tibia, respectively. To determine gene targets of sCT, we carried out quantitative real time PCR and microarray analysis of metaphyseal samples 1.5, 4 and 6.5h after administration of a single injection of PTH, sCT or PTH+sCT. Known targets of PTH action, IL-6, ephrinB2 and RANKL, were not modified by co-administration with sCT. Surprisingly, at all time points, we noted a significant upregulation of sclerostin mRNA by sCT treatment, as well as down-regulation of two other osteocyte gene products, MEPE and DMP1. Immunohistochemistry confirmed that sCT administration increased the percentage of osteocytes expressing sclerostin, suggesting a mechanism by which sCT reduced the anabolic effect of PTH. Neither mRNA for CT receptor (Calcr) nor labeled CT binding could be detected in sclerostin-enriched cells differentiated from primary calvarial osteoblasts. In contrast, osteocytes freshly isolated from calvariae expressed a high level of Calcr mRNA. Furthermore immunohistochemistry revealed co-localization of CT receptor (CTR) and sclerostin in some osteocytes in calvarial sections. Taken together these data indicate that co-treatment with sCT can blunt the anabolic effect of PTH and this may involve direct stimulation of sclerostin production by osteocytes. These data directly implicate calcitonin as a negative regulator of bone formation through a previously unsuspected mechanism.

RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis.

OBJECTIVES: Receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) have been demonstrated to be critical regulators of osteoclast generation and activity. In addition, RANKL has been implicated as an important mediator of bone erosion in rheumatoid arthritis (RA). However, the expression of RANKL and OPG at sites of pannus invasion into bone has not been examined. The present study was undertaken to further elucidate the contribution of this cytokine system to osteoclastogenesis and subsequent bone erosion in RA by examining the pattern of protein expression for RANKL, OPG and the receptor activator of NF-kappaB (RANK) in RA at sites of articular bone erosion. METHODS: Tissues from 20 surgical procedures from 17 patients with RA were collected as discarded materials. Six samples contained only synovium or tenosynovium remote from bone, four samples contained pannus-bone interface with adjacent synovium and 10 samples contained both synovium remote from bone and pannus-bone interface with adjacent synovium. Immunohistochemistry was used to characterize the cellular pattern of RANKL, RANK and OPG protein expression immediately adjacent to and remote from sites of bone erosion. RESULTS: Cellular expression of RANKL protein was relatively restricted in the bone microenvironment; staining was focal and confined largely to sites of osteoclast-mediated erosion at the pannus-bone interface and at sites of subchondral bone erosion. RANK-expressing osteoclast precursor cells were also present in these sites. OPG protein expression was observed in numerous cells in synovium remote from bone but was more limited at sites of bone erosion, especially in regions associated with RANKL expression. CONCLUSIONS: The pattern of RANKL and OPG expression and the presence of RANK-expressing osteoclast precursor cells at sites of bone erosion in RA contributes to the generation of a local microenvironment that favours osteoclast differentiation and activity. These data provide further evidence implicating RANKL in the pathogenesis of arthritis-induced joint destruction.