P0833 - Investigation of RARg Signaling in Human Growth-Plate Chondrocytes

Background
Pediatric fractures involving the growth-plate (GP) require careful follow-up, as these fractures can lead to inhibition of bone growth, resulting in progressive growth imbalance, deformity and/or painful conditions. Currently, surgical intervention is the only means to correct substantial imbalance and deformity due to a physeal injury. However, these procedures are invasive and cause substantial burden to the child and their family. Developing novel drug-based therapy for correcting physeal injuries will be beneficial for reducing the need of invasive surgery and correcting relatively minor imbalances that may occur.

Our previous studies show that retinoic acid receptor (RAR) isoform RAR gamma (RARg) is a critical regulator of bone growth. RARg is dominantly expressed in GP chondrocytes, regulates matrix synthesis and turn over, and transition of cartilage to bone transition. Pharmacological activation of RARg causes early closure of the GP and inhibits further bone growth while inactivation delays maturation of growth-plate chondrocytes. These findings indicate that RARg agonists/antagonists may have remarkable therapeutic potential for the treatment of abnormal endochondral ossification and bone growth. To translate these previous research findings towards human therapy, we investigated the role of RARg signaling in human GP.

Methods
We analyzed expression of RARg and its signaling-related molecules in damaged or normal growth plates collected at the time of epiphyseiodesis surgery via immunohistochemistry. Surgical specimens were immediately fixed in 4% PFA, decalcified and sectioned.

Results
RARg and Aldh1a2, a rate-limiting enzyme of retinoic acid synthesis were detected in pre-and hypertrophic chondrocytes in the intact growth-plate. In damaged growth-plate, the typical pattern of columnar growth-plate was completely gone. There was no sign of progression of active endochondral ossification as the remnant of cartilage (resting or arrested cartilage) is completely sealed by bone tissue. In a subsequent experiment, we compared the response to RARg agonist in human and mouse chondrocytes. Human GP-chondrocytes were isolated from the GP of polydactyly samples and mouse chondrocytes were isolated from new bone mouse epiphysis. Cells were maintained in culture until confluent and then treated with 100nM of selective RARg agonist for 47h. Total RNA was purified and subjected to Clariom Gene Array. In ether cells, pharmacological activation of RARg down-regulated cartilage-matrix genes and up-regulated expression of genes related to MMPs, angiogenesis and osteoclastgenesis.

Conclusion
These findings clearly indicate that RARg signaling is present and functioning in human cartilage, and (2) selective RARg agonists might be an effective therapeutic drug utilized for growth restriction/epiphyseiodesis.