CALBINDIN-D-9K AND CALBINDIN-D-28K EXPRESSION IN RAT MINERALIZED TISSUES IN-VIVO

Following their terminal differentiation, highly specialized cells, am eloblasts, odontoblasts, and osteoblasts sequentially elaborate minera lized tissues, While the developmental expression pattern of matrix pr oteins has been studied extensively, less attention has been paid to t he molecules involved ire calcium handling, such as calcium-binding pr oteins,This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D-9k and calbindin-D-28k in rat mandib ular bone and incisor based on several methods established on rat amel oblasts in vivo, Radioimmunoassays showed that calbindin-D-28k account s for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 1 00-fold more concentrated than calbindin-D-9k in both tissue types, We stern blot analysis confirmed that the anticalbindin-(28k) reactive sp ecies corresponded to the well characterized renal calbindin-D-28k in the ectomesenchyme, In this tissue, calbindin-D-28k was ultrastructura lly immunolocalized in the odontoblasts, Quantitative immunocytochemis try showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D pr oteins and mRNAs suggests that their expression is regulated at the su bcellular level, In particular, immunoreactive calbindinD(28k) appeare d to be associated with rough endoplasmic reticulum, Calbindin-D-9k an tisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (similar to 10 ng/mg of total pro tein), Finally, in situ hybridization showed transcripts for both calb indins-D in ameloblasts nd also in osteoblasts, In summary, the presen t results support the concept that an elevated expression of these vit amin D-depndent calcium-binding proteins-may characterize the phenotyp e of cells directly involved in the elaboration of mineralized tissues , enamel, dentine, and bone.