Novel non-collagenous modulators of biomineralization in bone and dentin

The formation of mineralized tissues such as bones and teeth is governed on many levels by molecular, genetic, and cellular events to culminate in terminally differentiated cells that secrete a specialized organic matrix, which is later mineralized through the coordinated deposition of calcium phosphate crystals. The formation of this hard tissue phase is tightly regulated by components of the extracellular matrix (ECM), since deviations from this equilibrium result in prevalent, debilitating health problems such as arthritis, vascular calcification, periodontal disease and osteoporosis. The development of a more causative therapy for diseases that are accompanied by deviant hard tissue mineralization necessitates a profound understanding of the molecular mechanisms that control the normal mineralization process. Numerous minor protein constituents of the ECM in hard tissues have been identified and play crucial roles in the process of biomineralization. The topic of this thesis was the identification and functional analysis of such minor constituents of the ECM in mineralized tissues, bone and dentin. Our studies on calreticulin (CRT) have demonstrated, for the first time that this Ca 2+binding protein is indeed a component of the ECM of hard tissues. Functional studies have shown that CRT promotes certain aspects of collagen fibrillogenesis in vitro and that it binds to hydroxyapatite (HAP), the major mineral component of bone and dentin, in a dose dependent manner. Deregulation of normal CRT expression levels by either overexpression or repression in cell culture had profound effects on the speed and extent of mineral formation. These data collectively indicate that CRT is not only a component of the ECM, but that it actively participates in the regulation of mineral formation. In the second part of this thesis, we showed that nucleobindin (NUC) mRNA is expressed by ameloblast and odontoblasts and that the corresponding protein is located in odontoblasts, ameloblasts and the dentin matrix. Based on its previously identified role as a major calcium-binding protein in the ER, these findings suggest that NUC might participate in Ca2+ homeostasis and ion transport to the ECM. Lastly, the presence of prothrombin (PT) protein was confirmed in the osteoid. However, it was shown that PT is not produced by osteoblasts, and most likely is delivered to the bone matrix from the serum. PT associates with type I collagen, but not with type II collagen fibrils, and inhibits collagen fibrillogenesis and HAP crystal growth in a dose dependent manner in vitro, suggesting that the serum protein PT may act as a negative regulator of mineralization