Transglutaminase-Mediated Bone Formation in Zebrafish

Abstract

The integrity of the human skeleton is maintained by a delicate balance of bone deposition and resorption. Disruption of this balance results in diseases such as sclerosteosis or osteoporosis, which are characterized by high or low bone mass respectively. Further, the skeleton is prone to injuries such as fractures and breaks throughout the human life-span. It has therefore become critical to understand the underlying mechanisms of bone formation and homeostasis in order to better target and treat such ailments. Two mammalian enzyme transglutaminases, TG2b and FXIIIa, which catalyze the formation of protein-protein cross-links, have been associated with bone mineralization in vitro. However, mouse single knockouts of these enzymes show no skeletal phenotype. In this study we demonstrate functional and transcriptional compensation for the loss of TG2 in various tissues of TG2 knockout mice; specifically, we demonstrate a compensation mechanism in the skeleton. To overcome this complication we utilize the zebrafish (danio rerio) model system to examine the role of transglutaminases in vivo. Zebrafish have become an invaluable model to the study of developmental processes due to several unique characteristics such as, transparency during early development, short gestation time and a remarkable regeneration capability. We characterized the zebrafish transglutaminase gene family and identified thirteen TG genes. Of these thirteen genes, eleven were homologous to one of three mammalian transglutaminases, TG1, TG2, or FXIIIa, and two were specific to zebrafish. We show that transglutaminase activity promotes proper bone mineralization in both developing vertebrae and regenerating fin bones. Further, we show evidence for transglutaminases functioning in bone mineralization by promoting collagen type I deposition and activating canonical beta-catenin signaling during bone regeneration. Importantly, these studies settled the previous discrepancy between in vitro studies and in vivo mouse studies on the role of TGs in osteo-chondrogenic differentiation by demonstrating a complex compensation mechanism in mammalian tissue. This identification of TGs in bone mineralization identifies a novel therapeutic target for various bone pathologies, such as bone-like tissue transformation in heterotropic ossification seen in musculoskeletal trauma, spinal cord injury and combat wounds.