1,25-Dihydroxyvitamin D3 and fetal lung maturation: immunogold detection of VDR expression in pneumocytes type II cells and effect on fructose 1,6 bisphosphatase.

Lung maturation before birth includes type II pneumocyte differentiation with progressive disappearance of glycogen content and onset of surfactant synthesis. We have shown previously that 1,25-(OH)2D3 increases surfactant synthesis and secretion by type II cells and decreases their glycogen content in fetal rat lung explants. Recently, the gene coding fructose 1,6 bisphosphatase (F1,6BP), a regulatory enzyme of gluconeogenesis, has been identified in type II cells and its promoter bears a Vitamin D response element. Present results show:The coexistence of type II cells at different stages of maturation. in rat fetal lung on day 21 of gestation (electron microscopy), and the association between maturation of type II cells and disappearance of their glycogen content. The immunogold labeling of all type II cells when using the 9A7g VDR-antibody, with significantly more abundant gold particles in cells exhibiting an intermediate glycogen content. The expression of F1,6BP mRNA in a human type II cell line (NCI-H441) and the increase of this expression after 18h incubation with 1,25-(OH)2D3 (10(-8)M). These results bring further evidence for a physiological role of 1,25-(OH)2D3 during type II pneumocyte maturation. Activation of F1,6BP may participate to the 1,25-(OH)2D3 action on surfactant synthesis via the gluconeogenesis pathway.

PP60c-src expression in osteoclasts from osteopetrotic children and in giant tumor cells.

Malignant infantile osteopetrosis is a severe congenital disease characterized by impaired osteoclast activity. Among the multiple factors that influence bone resorption, the c-src protooncogene product pp60c-src plays an essential role, since mice which lack pp60c-src develop osteopetrosis. To gain insight into the possible role of pp60c-csrc in the pathogenesis of infantile osteopetrosis, we examined the osteoclasts of three children displaying the typical features of the disease, aged respectively one, four and seven months. pp60c-csrc expression and localization, together with the expression of a 80/85-kilodalton pp60c-src substrate, cortactin, were examined by immunoelectron microscopy. Osteoclasts from two giant cell tumors were used as controls. Bone and tumor samples were fixed in 4% paraformaldehyde, included in LR-White resin at -30 degrees C and the sections processed with mAb 327 or mAb anti p80/85 by an immunogold technique. pp60c-src was expressed in the cytoplasm, in nuclear membranes and in nuclei of the osteoclasts of the three osteopetrotic children. The subcellular localization of the kinase was not different from the localization in giant tumor cells. In both cases cortactin was abundant. In conclusion, in three children with malignant osteopetrosis, pp60c-src expression in osteoclasts does not appear to be involved in the pathogenesis of the disease. The presence of this protein, however, does not necessarily reflect normal c-src tyrosine kinase activity, nor normal c-src-dependent intracellular signaling pathways. Moreover the presence of the protein in nuclear membranes, and especially around nuclear pores supports the hypothesis that in osteoclasts, c-src may participate in the regulation of RNA processing.

Iron distribution in thalassemic bone by energy-loss spectroscopy and electron spectroscopic imaging.

Iron overload occurs frequently in thalassemia as a consequence of regular blood transfusions, and iron has been found to accumulate in bone, but skeletal toxicity of iron is not clearly established. In this study, bone biopsies of thalassemic patients were investigated by light (n = 6) and electron microscopy (n = 8) in order to analyze iron distribution and possible iron-associated cellular lesions. Sections (5 microns thick) were used for histomorphometry and iron histochemistry. Ultrathin sections were examined with an energy filtering transmission electron microscope. Iron was identified by electron energy loss spectroscopy (EELS), and iron distribution was visualized by electron spectroscopic imaging (ESI) associated with computer-assisted treatment (two-window method). This study shows that EELS allows the detection of 4500-9000 iron atoms, and that computer-assisted image processing is essential to eliminate background and to obtain the net distribution of an element by ESI. This study shows also that stainable iron was present along trabecular surfaces, mineralizing surfaces, and on cement lines in the biopsies of all patients. Moreover, iron was detected by EELS in small granules (diffusely distributed or condensed in large clusters), in osteoid tissue, and in the cytoplasm of bone cells, but not in the mineralized matrix. The shape and size (9-13 nm) of these granules were similar to those reported for ferritin. As for iron toxicity, all patients had osteoid volume and thickness and osteoblast surface in the normal range. Stainable iron surfaces did not correlate with osteoblast surfaces, plasma ferritin concentrations, or the duration of transfusion therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured circulating mononuclear cells from osteopetrotic infants express the osteoclast-associated vitronectin receptor and form multinucleated cells in response to 1,25-dihydroxyvitamin D3.

Malignant osteopetrosis is characterized by impaired osteoclast activity. Osteoclasts derive from hematopoietic stem cells. In osteopetrosis, marrow cavities fail to develop, resulting in extramedullary hematopoiesis and the presence of stem cells in the bloodstream. Resistance to 1,25-(OH)2D3 may be involved in the pathogenesis of the disease. Sensitivity to 1,25-(OH)2D3, calcitonin sensitivity, and expression of the osteoclast-associated vitronectin receptor (VR) was examined in cultures of circulating mononuclear cells of seven osteopetrotic infants (1.5-6 months old). Since peripheral blood from age-matched children contains few stem cells, umbilical cord blood was used as control. Mononucleated cells were isolated by the Ficoll-Hypaque method and cultured (10(6) cells per ml) in alpha-MEM containing 20% horse serum in presence or absence of added 1,25-(OH)2D3. VR was identified by immunochemical staining with MAb 23C6. 1,25-(OH)2D3 at 10(-8) M significantly stimulated the formation of multinucleated cells (MNC) in cultures from all osteopetrotic patients and cord blood samples. Cells from three of five patients responded to 10(-9) M 1,25-(OH)2D3, the minimal stimulatory concentration for cord blood. Salmon calcitonin (100 ng/ml) partially inhibited the 10(-8) M 1,25-(OH)2D3-induced MNC formation in cultures from three of six patients and in cultures of all cord blood samples. In both types of cultures mononuclear cells and MNC cross-reacted with MAb 23C6, and 1,25-(OH)2D3 concentration did not influence the number and percentage of these cells. This study does not support the hypothesis of 1,25-(OH)2D3 resistance in osteopetrotic infants and shows that mononuclear cells expressing VR, possibly osteoclast progenitors, develop in cultures of circulating mononuclear cells from these infants. 1,25-(OH)2D3 may not be closely involved in VR expression.

Bioactive parathyroid hormone in pregnant rats and fetuses.

Parathyroid function at the end of gestation (day 21) was investigated by measuring plasma calcium (PCa), immunoreactive parathyroid hormone (iPTH), bioactive parathyroid hormone (bioPTH; cytochemical bioassay), and bone histology in intact and thyroparathyroidectomized (TPTX; day 12, ether anesthesia) rats and their fetuses. In pregnant intact rats, PCa was significantly lower, and iPTH, bioPTH, and osteoclast number were higher than in nonpregnant rats. In fetuses, PCa was higher than maternal PCa and correlated with fetal bioPTH. TPTX suppressed maternal bioPTH and decreased iPTH and osteoclast number, whereas fetal iPTH and bioPTH were decreased with no change in osteoclast number. Fetal PCa was near normal and was correlated with maternal PCa but not with fetal bioPTH. The fetomaternal calcium gradient was maintained and even increased. This study shows that there is maternal physiological hyperparathyroidism and functional fetal parathyroid glands at the end of gestation in the rat. Parathyroid hormone does not seem to be responsible for maintaining the high fetomaternal calcium gradient in TPTX animals.