Excess BMP Signaling in Heterotopic Cartilage Forming in Prg4-null TMJ Discs

Abstract

Heterotopic cartilage develops in certain pathologic conditions, including those affecting the human temporomandibular joint (TMJ), but the underlying molecular mechanisms remain obscure. This is in part due to the fact that a reliable animal model of such TMJ diseases is not available. Here, we show that aberrant chondrocyte differentiation and ectopic cartilage formation occur spontaneously in proteoglycan 4 (Prg4) mutant TMJ discs without further invasive procedure. By 2 mo of age, mutant disc cells displayed chondrocyte transdifferentiation, accompanied by strong expression of cartilage master gene Sox9 and matrix genes aggrecan and type II collagen. By 6 mo, heterotopic cartilage had formed in the discs and expressed cartilage hypertrophic markers Runx2 and ColX. The ectopic tissue grew in size over time and exhibited regional mineralization by 12 mo. Bone morphogenetic protein (BMP) signaling was activated with the ectopic chondrogenic cells and chondrocytes, as indicated by phosphorylated Smad 1/5/8 nuclear staining and by elevated expression of Bmp2, Bmpr1b, Bmpr2, and BMP signaling target genes. Likewise, we found that upon treatment with recombinant human BMP 2 in high-density micromass culture, mutant disc cells differentiated into chondrocytes and synthesized cartilage matrix more robustly than control cells. Importantly, a specific kinase inhibitor of BMP receptors drastically attenuated chondrogenesis in recombinant human BMP 2-treated mutant disc cultures. Unexpectedly, we found that Prg4 was expressed at joint-associated sites, including disc/muscle insertion and muscle/bone interface, and all these structures were abnormal in Prg4 mutants. Our data indicate that Prg4 is needed for TMJ disc integrity and function and that its absence leads to ectopic chondrogenesis and cartilage formation in conjunction with abnormal BMP signaling. Our findings imply that the BMP signaling pathway could be a potential therapeutic target for prevention or inhibition of ectopic cartilage formation in TMJ disease.

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling

“…Cultures were prepared from glenoid fossa/eminence samples isolated from 2 month-old wild-type mice (n=32) as described (Bechtold et al, 2015) with some modifications. Isolated glenoid fossae were incubated with 2.5 U/ml dispase (MP Biomedicals, LLC, Solon, OH) and 600 U/ml (3mg/ml) type I collagenase (Worthington Biochemical Corporation, Lakewood, NJ) in HBSS with Ca 2+ and Mg 2+ for 10 min at 37°C with agitation, and then the surface was curetted.…”

“…(Hill et al, 2014; Koyama et al, 2014). We also reported that Prg4 -null mice displayed increased levels of chondrogenesis and ectopic cartilage formation within TMJ components (Bechtold et al, 2015; Koyama et al, 2014), but the cellular, signaling and molecular mechanisms underling such potentially important pathogenic transformation remains unclear.…”

Extracellular Matrix Mediates BMP-2 in a Model of Temporomandibular Joint Osteoarthritis

“…Results from this study illustrate that BMP-2 treatment potentiated the induction of Acan, Col2 , Col 10 , and Ihh in Bgn −/− Fmod −/− mice. Excess BMP-2 signaling has also been implicated in the pathogenesis of accelerated TMJ-OA in Pgr4-KO mice [Bechtold et al 2016]. In contrast, absence of BMP signaling in transgenic mice with Bmp1Ra conditionally deleted in neural crest cells led to failure of articular disc separation from the hypoplastic condyle and under development of the mandibular condylar cartilage [Gu et al 2014].…”

“…Abnormal activation of this signaling causes heterotopic cartilage and bone formation [Huegel et al 2015, Billings et al, 2008]. Specifically, excessive BMP-2 signaling has recently been shown to be involved in heterotopic cartilage formation and the pathogenesis of TMJ-OA [Bechtold et al 2016, Albilia et al, 2013]. However, the mechanism of excessive BMP-2 signaling on the mandibular condylar cartilage and its implications in TMJ-OA are unknown.…”

Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation

“…In seeking for effective treatment and functional repair strategies, murine model becomes a necessary and unique in vivo platform, as its availability for genetic modification and short lifespan enable pinpointing the TMJ development, biology and pathogenesis to specific molecules or signaling pathways (Suzuki and Iwata, 2016). Evaluation of the phenotype in genetically modified mice has provided new insights into the roles of individual matrix molecules in TMJ development and OA, such as lubricin (Bechtold et al, 2016; Hill et al, 2014; Koyama et al, 2014), biglycan and fibromodulin (Wadhwa et al, 2005), collagen types II (Ricks et al, 2013) and XI (Xu et al, 2003). In addition, aided by clinically relevant operations such as partial discectomy (Xu et al, 2009) and anterior crossbite prosthesis (Liu et al, 2014), TMJ OA can be induced in mice to study the disease initiation and progression in a well-defined timeframe in vivo.…”