Articular cartilage is classified into permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in growth plate. In the process of synovial joint development, articular cartilage is originated from the interzone, developing at the edge of the cartilaginous anlagen, it establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators such as Wnts, GDF5, Erg, and PTHLH coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracerllular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier’s groove, the intra-articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Further, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site.

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies. Clinically relevant antibodies for this target have not yet been developed. Here, we describe the development of an ALK-ADC, CDX-0125-TEI, which selectively targets both wild-type and mutated ALK-expressing neuroblastomas. CDX-0125-TEI exhibited efficient antigen binding and internalization, and cytotoxicity at picomolar concentrations in cells with different expression of ALK on the cell surface. In vivo studies showed that CDX-0125-TEI is effective against ALK wild-type and mutant patient-derived xenograft models. These data demonstrate that ALK is a bona fide immunotherapeutic target and provide a rationale for clinical development of an ALK-ADC approach for neuroblastomas and other ALK-expressing childhood cancers such as rhabdomyosarcomas.

Objective To determine whether and how transcription factor Erg participates in the genesis, establishment and maintenance of articular cartilage. Methods Floxed Erg mice were mated with Gdf5-Cre mice to create conditional mutants lacking Erg in their joints. Mutant and control joints were subjected to morphological and molecular characterization and also experimental osteoarthritis (OA) surgery. Gene expression, promoter reporter assays and gain- and loss-of-function in vitro tests were used to characterize molecular mechanisms of Erg action. Results Conditional Erg ablation did not elicit obvious changes in limb joint development and overall phenotype in juvenile mice. Over aging, however, mutant joints became spontaneously deranged and exhibited clear OA-like phenotypic defects. Mutant joints in juvenile mice were more sensitive to surgically induced OA and became defective sooner than operated control joints. Global gene expression data and other studies identified PTHrP and lubricin as possible downstream effectors and mediators of Erg action in articular chondrocytes. Reporter assays using control and mutated promoter/enhancer constructs did indicate that Erg acted on ets DNA binding sites to stimulate PTHrP expression. ERG was up-regulated in severely affected areas in human OA articular cartilage, but remained barely appreciable in less affected cartilage areas. Conclusion The study shows for the first time that Erg is a critical molecular regulator of articular cartilage’s endurance over postnatal life and ability to mitigate spontaneous and experimental OA. Erg appears to do so through its regulation of PTHrP and lubricin expression, factors known for their protective roles in joints.

The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ-null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ-null mice. The antagonist-induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle-treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ-null mice, and increases in pSmad1/5/8 levels and Id1-luc activity were observed in RARγ antagonist-treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096-1105, 2017.

Retinoic acid signaling regulates several biological events, including myogenesis. We previously found that retinoic acid receptor γ (RARγ) agonist blocks heterotopic ossification, a pathological bone formation that mostly occurs in the skeletal muscle. Interestingly, RARγ agonist also weakened deterioration of muscle architecture adjacent to the heterotopic ossification lesion, suggesting that RARγ agonist may oppose skeletal muscle damage. To test this hypothesis, we generated a critical defect in the tibialis anterior muscle of 7-week-old mice with a cautery, treated them with RARγ agonist or vehicle corn oil, and examined the effects of RARγ agonist on muscle repair. The muscle defects were partially repaired with newly regenerating muscle cells, but also filled with adipose and fibrous scar tissue in both RARγ-treated and control groups. The fibrous or adipose area was smaller in RARγ agonist-treated mice than in the control. In addition, muscle repair was remarkably delayed in RARγ-null mice in both critical defect and cardiotoxin injury models. Furthermore, we found a rapid increase in retinoid signaling in lacerated muscle, as monitored by retinoid signaling reporter mice. Together, our results indicate that endogenous RARγ signaling is involved in muscle repair and that selective RARγ agonists may be beneficial to promote repair in various types of muscle injuries.