Calbindin-D9k and calbindin-D28k expression in rat mineralized tissues in vivo

Abstract

Following their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium-binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin-D9k and calbindin-D28k in rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin-D28k accounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100-fold more concentrated than calbindin-D9k in both tissue types. Western blot analysis confirmed that the anticalbindin-D28k reactive species corresponded to the well characterized renal calbindin-D28k in the ectomesenchyme. In this tissue, calbindin-D28k was ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin-D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin-D28k appeared to be associated with rough endoplasmic reticulum. Calbindin-D9k antisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (approximately 10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins-D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D-dependent calcium-binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tissues, enamel, dentine, and bone.