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Mutations within the CFTR gene are central to the pathophysiology of cystic fibrosis. CFTR encodes a chloride channel that is located primarily on epithelial cell membranes and is responsible for the regulation of transmembrane chloride and other ion transport. Recent studies indicate a potential association between mutation of the CFTR gene and osteoporosis in patients with CF. Dif et al1 reported an abnormal skeletal phenotype in CFTR-null mice with striking osteopenia, reduced cortical width and thinning of the trabeculae, while in a study of adults with CF, the ΔF508 mutation was shown to be an independent risk factor for low bone mineral density.2 An association between CFTR mutations and bone disease might be mediated either indirectly by effects of the mutations on other systems (for example, the endocrine system), or it could be due to abnormally functioning CFTR in bone cells. However, to date, CFTR has not been reported in bone cells. We have therefore investigated the expression of CFTR in human bone using both in situ and in vitro approaches.
Immunolocalisation of CFTR was performed in human neonatal bone sections, primary human osteoblasts, an osteoblastic cell line (MG63) and osteoclasts cultured from peripheral blood mononuclear cells.3 T84 colonic carcinoma cells were used as a positive control. The primary antibody was mouse IgG2A anti-human CFTR (C-terminus specific) mono-clonal antibody (clone 24-1) (R&D Systems, Abingdon, UK).4 CFTR in bone sections was visualised using a HRP-DAB staining technique. For cell cultures, an FITC-conjugated goat anti-mouse IgG secondary antibody was added for visualisation. Neonatal rib bone was collected post-mortem from six full-term infants (3 boys, 2 girls and 1 of unknown sex) who had no evidence of growth retardation or skeletal abnormalities. These samples were obtained with informed parental consent after approval by the local research ethics committee.
In neonatal bone sections, strong CFTR expression was detected in osteoblasts on forming surfaces (fig 1A) and in osteocytes newly incorporated into bone, whereas more deeply embedded osteocytes showed no CFTR expression (data not shown). Osteoclasts, defined by their multinucleate appearance and associated resorption pits, also expressed CFTR (fig 1B). In addition, CFTR expression was seen in an uncharacterised population of haematopoietic cells within the bone marrow. No CFTR expression was seen in resting, proliferating or hypertrophic chondrocytes in the growth plate.
In primary osteoblast cultures, immunofluorescence was confined to the cytoplasm of osteoblastic cells and was localised to small areas, giving a speckled appearance (fig 1C). This speckled appearance was also seen in the MG63 osteoblastic cell line (data not shown). In cultured osteoclastic cells, immunofluorescence was also confined to the cytoplasm (fig 1D). T84 cells showed positivity for CFTR within the cytoplasm, with strong perinuclear staining (data not shown). In addition, Western blot analysis using the mouse IgG2A anti-human CFTR (C-terminus specific) monoclonal antibody (clone 24-1) confirmed the presence of CFTR in the osteoblastic cell line and in primary human osteoblasts (data not shown).
Our results show, for the first time, the presence of CFTR in human osteoblasts, osteocytes and osteoclasts. Although there are known limitations regarding the sensitivity and specificity of the anti-human CFTR (clone 24-1) antibody, the inclusion of appropriate positive and negative controls and confirmation of our findings by Western blot analysis in osteoblastic cells support this conclusion. The functional significance of these findings and their implications for bone disease associated with cystic fibrosis require further study. Other chloride channels are known to be essential for bone cell function,5 and our finding that CFTR is expressed by normal bone cells suggests that it might also have a physiological role.
Dr Elizabeth Shead was funded though a clinical scientist training programme supported by the Workforce Development Confederation.
Competing interests: None declared.
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