Analysis of the Actions of RARγ Agonists on Growing Osteochondromas in a Mouse Model.

The actions of the retinoic acid nuclear receptor gamma (RARγ) agonist, palovarotene, on pre-existing osteochondromas were investigated using a mouse multiple osteochondroma model. This approach was based on the knowledge that patients often present to the clinic after realizing the existence of osteochondroma masses, and the findings from preclinical investigations are the effects of drugs on the initial formation of osteochondromas. Systemic administration of palovarotene, with increased doses (from 1.76 to 4.0 mg/kg) over time, fully inhibited tumor growth, keeping the tumor size (0.31 ± 0.049 mm3) similar to the initial size (0.27 ± 0.031 mm3, p = 0.66) while the control group tumor grew (1.03 ± 0.23 mm3, p = 0.023 to the drug-treated group). Nanoparticle (NP)-based local delivery of the RARγ agonist also inhibited the growth of osteochondromas at an early stage (Control: 0.52 ± 0.11 mm3; NP: 0.26 ± 0.10, p = 0.008). Transcriptome analysis revealed that the osteoarthritis pathway was activated in cultured chondrocytes treated with palovarotene (Z-score = 2.29), with the upregulation of matrix catabolic genes and the downregulation of matrix anabolic genes, consistent with the histology of palovarotene-treated osteochondromas. A reporter assay performed in cultured chondrocytes demonstrated that the Stat3 pathway, but not the Stat1/2 pathway, was stimulated by RARγ agonists. The activation of Stat3 by palovarotene was confirmed using immunoblotting and immunohistochemistry. These findings suggest that palovarotene treatment is effective against pre-existing osteochondromas and that the Stat3 pathway is involved in the antitumor actions of palovarotene.

Wnt signaling in cartilage development and diseases: lessons from animal studies.

Cartilage not only plays essential roles in skeletal development and growth during pre- and postnatal stages but also serves to provide smooth movement of skeletons throughout life. Thus, dysfunction of cartilage causes a variety of skeletal disorders. Results from animal studies reveal that ß-catenin-dependent canonical and independent non-canonical Wnt signaling pathways have multiple roles in regulation of cartilage development, growth, and maintenance. ß-Catenin-dependent signaling is required for progression of endochondral ossification and growth of axial and appendicular skeletons, while excessive activation of this signaling can cause severe inhibition of initial cartilage formation and growth plate organization and function in mice. In contrast, non-canonical Wnt signaling is important in columnar organization of growth plate chondrocytes. Manipulation of Wnt signaling causes or ameliorates articular cartilage degeneration in rodent osteoarthritis models. Human genetic studies indicate that Wnt/ß-catenin signaling is a risk factor for osteoarthritis. Accumulative findings from analysis of expression of Wnt signaling molecules and in vivo and in vitro functional experiments suggest that Wnt signaling is a therapeutic target for osteoarthritis. The target tissues of Wnt signaling may be not only articular cartilage but also synovium and subchondral bone.

Possible Contribution of Wnt-Responsive Chondroprogenitors to the Postnatal Murine Growth Plate.

Active cell proliferation and turnover in the growth plate is essential for embryonic and postnatal bone growth. We performed a lineage tracing of Wnt/ß-catenin signaling responsive cells (Wnt-responsive cells) using Axin2CreERT2 ;Rosa26ZsGreen mice and found a novel cell population that resides in the outermost layer of the growth plate facing the Ranvier's groove (RG; the perichondrium adjacent to growth plate). These Wnt-responsive cells rapidly expanded and contributed to formation of the outer growth plate from the neonatal to the growing stage but stopped expanding at the young adult stage when bone longitudinal growth ceases. In addition, a second Wnt-responsive sporadic cell population was localized within the resting zone of the central part of the growth plate during the postnatal growth phase. While it induced ectopic chondrogenesis in the RG, ablation of ß-catenin in the Wnt-responsive cells strongly inhibited expansion of their descendants toward the growth plate. These findings indicate that the Wnt-responsive cell population in the outermost layer of the growth plate is a unique cell source of chondroprogenitors involving lateral growth of the growth plate and suggest that Wnt/ß-catenin signaling regulates function of skeletal progenitors in a site- and stage-specific manner. © 2019 American Society for Bone and Mineral Research.

Alpha 5 Integrin Mediates Osteoarthritic Changes in Mouse Knee Joints.

Osteoarthritis (OA) is one of most common skeletal disorders and can affect synovial joints such as knee and ankle joints. α5 integrin, a major fibronectin receptor, is expressed in articular cartilage and has been demonstrated to play roles in synovial joint development and in the regulation of chondrocyte survival and matrix degradation in articular cartilage. We hypothesized that α5 integrin signaling is involved in pathogenesis of OA. To test this, we generated compound mice that conditionally ablate α5 integrin in the synovial joints using the Gdf5Cre system. The compound mice were born normally and had an overall appearance similar to the control mice. However, when the mutant mice received the OA surgery, they showed stronger resistance to osteoarthritic changes than the control. Specifically the mutant knee joints presented lower levels of cartilage matrix and structure loss and synovial changes and showed stronger biomechanical properties than the control knee joints. These findings indicate that α5 integrin may not be essential for synovial joint development but play a causative role in induction of osteoarthritic changes.

The type 2 diabetes associated rs7903146 T allele within TCF7L2 is significantly under-represented in Hereditary Multiple Exostoses: insights into pathogenesis.

Hereditary Multiple Exostoses (HME) is an autosomal-dominant disorder characterized by benign cartilage tumors (exostoses) forming near the growth plates, leading to severe health problems. EXT1 and EXT2 are the two genes known to harbor heterozygous loss-of-function mutations that account for the vast majority of the primary genetic component of HME. However, patients present with wide clinical heterogeneity, suggesting that modifier genes play a role in determining severity. Our previous work has pointed to an imbalance of ß-catenin signaling being involved in the pathogenesis of osteochondroma formation. TCF7L2 is one of the key 'gate-keeper' TCF family members for Wnt/ß-catenin signaling pathway, and TCF7L2 and EXT2 are among the earliest associated loci reported in genome wide appraisals of type 2 diabetes (T2D). Thus we investigated if the key T allele of single nucleotide polymorphism (SNP) rs7903146 within the TCF7L2 locus, which is strongly over-represented among T2D cases, was also associated with HME. We leveraged genotype data available from ongoing GWAS efforts from genomics and orthopedic centers in the US, Canada and Italy. Collectively 213 cases and 1890 controls were analyzed and, surprisingly, the T allele was in fact significantly under-represented in the HME patient group [P = 0.009; odds ratio = 0.737 (95% C.I. 0.587-0.926)]; in addition, the direction of effect was consistent within each individual cohort. Immunohistochemical analyses revealed that TCF7L2 is differentially expressed and distributed in normal human growth plate zones, and exhibits substantial variability in human exostoses in terms of staining intensity and distribution. In summary, the data indicate that there is a putative genetic connection between TCF7L2 and EXT in the context of HME. Given this observation, we suggest that these loci could possibly modulate shared pathways, in particular with respect to ß-catenin, and their respective variants interplay to influence HME pathogenesis as well as T2D.