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.

Exclusion of all three calbindins from a calcium‐ferry role in rat enamel cells

“…Accordingly, these data added further molecular support to previous evidence that calbindin-9 kDa protein is expressed in rat enamel cells (23)(24)(25)(26)(27).…”

“…a product of the S100G gene), which, like calbindin-28 kDa, has classically been considered a calcium ferry; and calretinin (a CALB2 product), a 30-kDa homolog of calbindin-28 kDa, which has been rather loosely linked to calcium transport (9,17,18,(20)(21)(22). Diverse evidence obtained from immunolocalization, radioimmunoassay, and mRNA analyses (23)(24)(25)(26)(27) collectively indicate that calbindin-9 kDa is expressed in calcium-transporting enamel cells, but the reported quantification [0.01% of soluble protein in incisor enamel organ (24)] implies insufficient abundance to serve as a calcium ferry (1,11). The level of calbindin-9 kDa during enamel maturation [where its expression appears maximal (23)] remains to be quantified, and it has not yet been established whether molars express similarly low amounts of calbindin-9 kDa as incisors.…”

Ca2+ transport and signalling in enamel cells

“…A number of studies reported the expression of calbindin 9kDa and calbindin 28kDa in ameloblasts of rat molars and incisors (Berdal et al . , ; Hubbard, , ; Turnbull et al . ; Kutuzova et al .…”

Physiological implications of DLX homeoproteins in enamel formation

“…Notably, the transition from secretion to post‐secretion stage is coincident with a dramatic down‐regulation of enamel matrix protein production (Papagerakis et al, 1999). Conversely, expression levels of the tissue non‐specific alkaline phosphatase and calcium‐binding proteins are up‐regulated (Berdal et al, 1996; Hotton et al, 1999). This transition is accompanied by an abrupt up‐regulation of the Dlx2 transcription factor in the ameloblasts (Lézot et al, 2000a).…”