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The distribution and subcellular localization of the 1,25-dihydroxyvitamin D3 receptor (VDR) in the epiphyseal cartilage of normal weaning rats were examined immunocytochemically at the light and electron microscope level using a monoclonal anti-VDR antibody (9A7 gamma). VDR immunoreactivity was detected in the nuclei of chondrocytes in all zones of the epiphyseal plate cartilage from the resting to calcifying chondrocytes, and at much lower concentrations, in the cytoplasms. Perichondrial mesenchymal cells contained no VDR immunoreactivity. VDR immunoreactivity developed in the nuclei of cells in the lateral margin area as they acquired the chondroblast phenotype. VDR immunoreactivity was also found over the nucleoli of chondrocytes in all cells zones of the epiphyseal plate and appeared in the nucleoli of the cells in the lateral margin area before immunostaining of the nuclei, as the mesenchymal cells differentiated into chondroblasts. Electron microscopy showed that the immunoreactivity for 1,25(OH)2D3 receptor, indicated by gold particles, was associated with scattered clumps of compact chromatin and small clumps of dispersed chromatin. But the nuclei immunostaining patterns before and after mitosis were different in proliferative chondrocytes. The heterochromatin along the nuclear envelope was immunonegative in interphase chondrocytes, but there was VDR immunostaining over the rim of the perinuclear chromatin just after mitosis. In the nucleoli, the dense fibrillar component was immunostained, but the fibrillar centers and the perinuclear chromatin were not. This distribution of VDR immunoreactivity suggests that the hormone is directly involved in differentiation, proliferation and maturation of cartilage cells, and also with extracellular calcification in epiphyseal cartilage. The presence of immunoreactive VDR receptors in nucleoli of chondrocytes, particularly the fibrillar component, suggests that 1,25(OH)2D3 may be involved in regulation of ribosomal genes.

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Calbindin-D 28 kDa (CaBP 28 kDa), a vitamin D-dependent calcium-binding protein, has been associated with calcium handling by cells. We have investigated the expression of this protein in the rat incisor enamel organ, an epithelium interposed between a mineralizing matrix and connective tissue rich in blood vessels, by radioimmunoassay (RIA), Western blotting, and quantitative protein A-gold immunocytochemistry with antibodies to rat kidney CaBP 28 kDa. RIA of cytosolic extracts showed that enamel organs contained relatively high concentrations of CaBP 28 kDa (compared to kidney; see review by Christakos S., C. Gabrielides, and W.B. Rhoten 1989 Endocr. Rev., 10:3-25). Immunoblotting of proteins extracted from enamel organ strips revealed an intensely-stained band near 28 kDa throughout amelogenesis following ameloblast differentiation. Immunocytochemically, CaBP 28 kDa was localized exclusively within ameloblasts. The density of labelling increased from the presecretory stage to the secretory stage and fluctuated across the maturation stage in relation to ameloblast modulation. Ruffle-ended ameloblasts consistently showed the most intense immunoreaction. Gold particles were present throughout the cytoplasm and nuclei of ameloblasts but regions rich in rough endoplasmic reticulum or cell webs showed a higher immunolabelling. Some gold particles were also associated with the external face of the rough endoplasmic reticulum. Multivesicular bodies in maturation stage ameloblasts were occasionally immunoreactive. These data suggest that the intracellular concentration of CaBP 28 kDa is regulated throughout amelogenesis reflecting a stage-specific control of calcium homeostasis in ameloblasts.

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Experimental and clinical data indicate that dentin mineralization is vitamin D-dependent. This calcium-regulating steroid controls protein synthesis, for instance that of osteocalcin in osteoblasts. This protein also elaborated by odontoblasts was used as a molecular marker for vitamin D action on odontoblasts. Since the most characteristic protein synthesized by odontoblasts is the dentin phosphoprotein which is thought to regulate hydroxyapatite growth, its cellular and extracellular distribution was also studied. Tooth formation in the molars and incisors of successive generations of vitamin D-deficient animals (-D) and in controls (+D) was compared by microadiography, toluidine blue histochemistry, and immunocytochemistry. In -D samples, the presence of dentin phosphoprotein in odontoblasts indicated that their differentiation occurred despite major morphological disturbances at the cusp tips. In contralateral teeth, osteocalcin was depleted in odontoblasts and dentin, suggesting an inhibition of protein synthesis induced by vitamin D-deficiency. In the extracellular matrix of +D animals, phosphoprotein distribution was associated with dentin, especially within actively forming calcospherites at the mineralization front. In contrast, in -D dentin, the mineralization defects corresponded to irregular absence of histochemically detectable phosphoprotein. This protein indeed appeared either absent or uniformly sparse in -D dentin by immunocytochemistry. These data suggest that vitamin D acts directly on odontogenic cells at various synthetic (osteocalcin) or secretory (phosphoprotein) levels indicating that odontoblasts are target-cells for vitamin D. Therefore, this hormone could contribute to the regulation of extracellular mineralization during dentinogenesis, via different mechanisms in the processing of matrix protein.

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This electron microscope study describes the subcellular occurrence and distribution of immunoreactive calbindin-D9K in the trabecular metaphyseal and compact cortical bone of normal rats, rachitic vitamin-D-deficient rats, and rachitic rats given 1,25-(OH)2D3. Undecalcified bones were embedded in Lowicryl K4M and calbindin-D9K antigenicity was detected by the protein A-gold method. Immunoreactive calbindin-D9K was localized in the cytoplasm and cell processes of osteoblasts and osteocytes. Immunoreactive calbindin-D9K was also found within matrix vesicles and calcifying matrix vesicles, where it lay over the needle-shaped crystallites, at the apparent site of initial crystal formation, but not along the whole crystallites. In fully mineralized bone it occurred at the same site, over the crystallites. Calibindin-D9K was vitamin-D-dependent in the osteoblasts and matrix vesicles, where its presence was correlated with the reappearance of crystallites in 1,25-(OH)2D3-treated vitamin-D-deficient rats. This suggests that immunoreactive calbindin-D9K is involved in mineral deposition in bone matrix vesicles. Abnormal intracellular calcification associated with calbindin-D9K antigenicity in the osteoblasts of 1,25-(OH)2D3-treated vitamin-D-deficient rats indicates that immunoreactive calbindin-D9K may also play a part in abnormal intracellular mineral deposition.

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The immunocytochemical patterns of calbindin-D9k (CaBP 9k) and calbindin-D28k (CaBP 28k) were compared by light and electron microscopy throughout amelogenesis. Labelling on serial sections and co-localization of CaBPs confirmed that the two proteins were restricted to a single cell type, the ameloblasts. Their quantity increased during presecretion, was stable during secretion and alternately high and low during the cyclic modulation of ameloblasts which occurs during maturation. Ruffle-ended ameloblasts contained the highest apparent concentration. Investigations with several fixatives indicated that the CaBPs were present in the cytosol and the nucleus, although there were slight differences with various fixatives by light microscopy. Their concentrations in these compartments varied in parallel throughout amelogenesis. However, mitochondria contained only immunoreactive CaBP 9k. While the distribution of CaBP 9k in zones containing Golgi apparatus and rough endoplasmic reticulum was similar, CaBP 28k concentration has, in another paper, been shown to be higher near the rough endoplasmic reticulum.

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Calbindin-D9K immunoreactivity was localized by electron microscopy in rat calcifying epiphyseal plate cartilage. Antigen-antibody reaction sites were visualized by the presence of protein A-gold complex particles on undecalcified material embedded in Lowicryl K4M. Immunoreactive calbindin-D9K was found in the hyaloplasm of hypertrophic chondrocytes and inside and at the ends of their cell processes. It was localized outside the cells, inside matrix vesicles (MVs), often against the inner face of the delimiting membrane, and inside the trilaminar membrane. Immunoreactive calbindin-D9K appeared to be extruded from the chondrocytes into the matrix vesicles when the latter were formed during the budding of cell processes. In calcifying MVs, gold particles were detected over the needle-shaped crystallites and often over the crystallites lying against the inner leaflet of the vesicular membrane. At a later stage of matrix vesicle calcification after MV membrane disruption, the number of gold particles remained unchanged over the clusters of crystallites at the loci from which the crystallites appeared to have grown and radiated. At a yet more advanced stage of calcification, they remained in the same areas, which were limited to the lateral edges of calcified cartilage longitudinal septa. These results suggest that immunoreactive calbindin-D9K plays a role in calcium input to matrix vesicles and may be involved in matrix vesicle calcification, perhaps in the initial event of matrix vesicle crystal nucleation.

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It has been suggested that vitamin D is involved in the process of cell differentiation and extracellular mineralization during tooth development. One of the best-defined molecular markers of the action of vitamin D is a calcium-binding protein of Mr 28,000 called calbindin D-28 K (CaBP 28 K). Since this protein is present in growing teeth, we have examined its synthesis in teeth from vitamin D-replete and -deplete rats by Western blotting and immunocytochemistry with an antiserum to CaBP 28 K purified from rat kidney. The CaBP 28 K present in the enamel organ is a single molecular species migrating near 30 k Da, similarly to the kidney protein. The differentiation and maturation of odontogenic cells were followed during early postnatal development (2-12 days) in rat molars. At the light-microscope level, CaBP 28 K was only found in a single cell-type, the ameloblasts. The expression of this protein appeared to be developmentally controlled, since its distribution varied with the cell stage and the functional steps of amelogenesis. The protein was localized in the basal compartment of ameloblasts from the presecretory stage. During the early secretory stage, the concentration of cytoplasmic CaBP 28 K formed a gradient from the apical to the basal pole of the ameloblasts. Staining appeared homogeneous in the cytoplasm of later secretory ameloblasts. CaBP 28 K was discontinuously distributed during the maturation stage. This discontinuity might be related to cyclical changes in mature ameloblasts. In all stages, ameloblasts from vitamin-D-deficient rats appeared depleted of CaBP 28 K.

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The present study focuses on the ultrastructure of enamel organ cells and the immunolocalization of calbindins D-9kDa and -28kDa during enamel secretion in Vitamin D-deficient rats. Vitamin D-deficiency disturbed the deposition of the layer of inner enamel and depleted the calbindins-content of ameloblasts. These data raise the possibility of a direct action of Vitamin D on the physiology of ameloblasts through ionic calcium homeostasis.

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This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae, and in the pericanalicular walls containing the cell processes. Calbindin-D9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)2-VitD3. Thus, the synthesis of immunoreactive calbindin-D9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its localization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)

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Clinical observation of patients with disordered phosphocalcium metabolism has demonstrated that dyschromia and/or dental dysplasias systematically accompany such disorders. A certain action of this steroid on dental buds has been demonstrated after analysis of the effects of experimental vitamin D deficiency in the rat: vitamin D would seem to control the behaviour of cells undergoing differentiation and also after this process is complete. Dentinogenesis and amelogenesis would appear principally to be affected. Two proteins, calbindins D-9K and -28K, may constitute the molecular mediators of this ameloblastic regulation.

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Regulation of the expression of vitamin D-dependent calcium-binding protein (Mr 9000 CaBP) gene by 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was studied in the rat duodenum. In vivo stimulation of Mr 9000 CaBP synthesis was analyzed using a complementary DNA probe and by measuring the rate of Mr 9000 CaBP gene transcription in isolated nuclei (run-on assay). A single 1,25-(OH)2D3 injection (650 pmol/100 g of body weight) induced a 2-fold increase in Mr 9000 CaBP gene transcription within 15 min in the duodenum of vitamin D-deficient rats. RNA synthesis was maximal at 1 h, then decreased until 16 h of postinjection. There was an initial transient accumulation of Mr 9000 CaBP mRNA (from 7 to 15 min), which was followed by a second, significant increase, by 3 h which remained elevated until 16 h. The magnitude and time course of the Mr 9000 CaBP increase was similar to that of its mRNA as early as 1 h after 1,25-(OH)2D3 administration. Mr 9000 CaBP gene transcription was not significantly induced by 1,25-(OH)2D3 in vitamin D-replete rats and no transient accumulation of Mr 9000 CaBP mRNA was observed. Thus, 1,25-(OH)2D3 modulates Mr 9000 CaBP gene expression in at least two ways, a rapid transcriptional stimulation and a post-transcriptional effect preventing degradation of Mr 9000 CaBP transcripts and accounting for their accumulation several hours after the hormone treatment.

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The role of vitamin D on tooth-germ development was studied. The molars of vitamin D-deficient rats were compared with those of vitamin D-replete controls. The deficiency disturbed enamel and dentine mineralization and decreased their matrix secretion. Morphogenesis was affected; teeth were flattened and the whole of the epithelio-mesenchymal junction rippled. Where this irregularity was maximal, the inner dental epithelium and stratum intermedium were intermingled and the adjoining sub-odontoblast cells were mixed with poorly polarized odontoblasts. The cytodifferentiation of both central and sub-odontoblastic cells was inhibited. Thus vitamin D has a role in the early events of tooth development: morphogenesis, histodifferentiation and cytodifferentiation of pulp cells as well as in enamel and dentine mineralization.

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The presence of vitamin-D-dependent calcium-binding protein (CaBP-9K) in tibial growth-plate cartilage was immunohistochemically demonstrated using a specific antibody to rat duodenal CaBP-9K. The protein was found to be mainly localized in the cytoplasm of maturing chondrocytes. In hypertrophic chondrocytes, CaBP-9K concentrations decreased, and the protein was found in the cytoplasmic processes. No CaBP-specific immunoreactivity was seen in the hypertrophic chondrocytes of the lower calcified hypertrophic zone; in contrast, the protein was found in the extracellular lateral edges of longitudinal septa, i.e. where matrix vesicles are preferentially localized and where cartilage mineralization is initiated. These findings suggest that vitamin D has a direct function in this tissue. It also seems likely that CaBP-9K is an indicator of chondrocyte maturation, and that it is involved in the matrix vesicle-associated process of cartilage calcification.

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The distribution of the vitamin-D dependent calcium-binding protein (Calbindin-D 28K) (CaBP-28K) in the tibial growth plate cartilage of the rat has been studied immunohistochemically using an antibody raised against rat renal CaBP-28K. The protein was detected mainly in the nuclei of chondrocytes and occasionally in the juxtanuclear cytoplasm. The distribution was not uniform throughout the growth plate, but concentrated in the proliferatively active chondrocytes of the resting and proliferative zones. These findings raise the possibility that CaBP-28K may be involved in the mitotic activity of the chondrocytes, acting as a regulator of the proliferative process, perhaps via intranuclear calcium.

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Some applications to man of specific markers of the molecular action of vitamin D (1.25(OH)2D3 receptors and antibodies to hormone-dependent proteins (CaBP and cDNA] are reported in this study. On case of type II vitamin-dependent rickets was characterized by 1.25(OH)2D3 plasma level greater than 250 pg/ml and a ten-fold decrease of the number of binding sites of the hormone in cultured skin fibroblasts. We propose that CaBP 28K and/or 9K-containing cells, such as Purkinje's cells and chondroblasts may be targets for vitamin D action. Detection in fetuses, from the 20th week of gestation, of CaBP 9K messenger RNA in the duodenum and sternum and presence of CaBP 28K and 9K in the chondroblasts of the upper extremity of tibia, suggest that vitamin D acts on the nucleus of its target-cells during fetal development. Finally, discovery of the gene of CaBP 9K in man opens the prospect of studies which will improve the understanding of the mechanism of action of vitamin D.

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Lung mechanics was studied at 50 days of age in 7 rachitic rats born from mothers deprived of vitamin D. They were compared with 7 control rats raised in the same conditions but fed a diet supplemented with vitamin D. The animals were anesthetized, tracheotomized, and paralyzed. Quasi-static pressure-volume curves of the respiratory system and of the lungs were obtained. Body weight of the rachitic rats was within the range of the control rats, but dry lung weight (LW) was significantly lower (p less than 0.01). Lung volumes in absolute terms and when normalized for LW were significantly lower than in the control rats. Chest wall compliance (Ccw) of the rachitic rats was within the range of values of the control rats, except for 2 animals with an infinite Ccw. Analysis of the pressure-volume curves of the lungs of the rachitic rats compared with those in the control animals showed a significant decrease in lung compliance (CL) and in CL/LW (p less than 0.01), indicating a decrease in lung distensibility. The more severe the rickets (according to microradiographic criteria of the tibia), the lower the CL/LW. It is speculated that decrease in lung distensibility may be related to abnormal lung growth caused by disturbed alveolar formation and lung connective tissue development. These abnormalities could be due to vitamin D deficiency acting on the growing lung, as on the growing bones, by a mechanism involving proteoglycans.

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Microradiographic examination of metaphyses in long and short tubular growing bones allowed detection of a repetitive, clearly defined pattern of three adjacent zones; the latter are successively formed by the mineralization of cartilaginous longitudinal intercolumnar septa and by the subsequent apposition of other mineralized tissues concurrently with resorption. Consequently, each zone of the metaphysis includes mineralized tissues of various compositions and ages, identifiable by their different mineralization densities. Microradiography of pieces of the growing skeleton in several animal species shows that the same organization is not only present in long and short tubular bones but also in many other such as the pelvis and scapula, cuboid bones like the calcaneum and talus, and cartilaginous bones at the base of the skull. This suggests that there is no difference between the osteogenesis pattern of these bones and tubular ones. The problem of identifying the factors generating such metaphyseal organization is raised.

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Mineralized ring, an elongated tubular structure ensheathing bone metaphysis, is described at successive stages of long bone growth. It is shown that the mineralized ring is characterized by different morphological aspects during its development, corresponding to successive growth stages of one and the same anatomical formation. Its origin is different from periosteal bone and its development is parallel to that of the metaphysis.

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