Role of osteocyte CFTR in skeletal aging

Abstract

Essential to human health, skeletal homeostasis is maintained by different types of bone cells, namely, osteocytes, osteoblasts and osteoclasts with a variety of genes and signaling pathways involved. Bone mass and strength decline with age, as osteoporosis is commonly seen in postmenopausal women and older men. Osteocytes, the most abundant type of cells constituting the main structure of bones, are long-living cells and recently revealed to be significantly affected by aging. However, the cellular and molecular mechanisms underlying skeletal aging remain largely unknown. CFTR (cystic fibrosis transmembrane conductance regulator) is an anion channel, mutations in which result in cystic fibrosis (CF), the most common genetic diseases in Caucasian. Though primarily regarded as a lung disease, CF presents problems in other organ-systems including the bones. Direct or indirect role of CFTR in bone cells, though proposed, remain unclear. Recent studies have suggested that CFTR expression is decreased with age in the prostate and sperm in humans. In airway cells, CFTR deficiency shares common phenotypes with cell senescence. Therefore, we hypothesized that CFTR expression in bone cells might decrease with age, contributing to age-related bone impairment; targeting CFTR might be a novel therapeutic strategy for the treatment of age-related skeletal diseases. In the present study, by using the CFTR deficient (ΔF508 mutation) mouse model, transcriptome analysis, an osteocyte line with CRISPR/Cas9-based CFTR knockout, old-aged mice as well as human bone biopsy samples, we explored the role of CFTR in osteocyte function and aging, and tested the potential effect of recently developed pharmaceuticals targeting CFTR on age-related bone disorder. The present results showed that CFTR deficiency (ΔF508) in mice resulted in low bone mineral density (BMD), abnormality of osteocytes and altered transcriptome profile in bone tissues. CFTR mRNA and protein expression was confirmed in osteocytes in mouse and human bone tissues as well as in MLO-Y4, a commonly used osteocyte cell line. Knockdown of CFTR in MLO-Y4 impaired the mineralization in vitro. Knockout of CFTR by CRISPR-Cas9 technology in MLO-Y4 significantly retarded cell growth and survival with overwhelmed inflammation. A direct protective role of CFTR in osteocytes for maintaining bone integrity is therefore suggested. In following investigation into the role of CFTR in osteocyte aging, we measured BMD of wild-type and ΔF508 mice of different ages. Results showed that BMD in wild-type mice age-dependently dropped by ~21% from 6- to 18-month-old, while in ΔF508 mice, BMD sustained low but only 5% decrease was seen, suggesting possible premature bone loss/deficiency in ΔF508 mice. In addition, a series of genes/proteins associated with cellular senescence were significantly upregulated in bone tissues in ΔF508 mice at an early age (3-month-old), as compared to the aged-matched wild-type bones, suggesting an activation of cellular senescence in bones bearing CFTR deficiency. CFTR expression in bones, especially in osteocytes, were found to be reduced with age in wild-type mice. Ovariectomized rats exhibited diminished CFTR expression in osteocytes. Human bone biopsy samples from female subjective of different ages show similar age-dependent downregulation of CFTR in osteocytes. Consistently, the expression of CFTR decreased in MLO-Y4 cells with a high passage number, as compared to that of lower ones. Further study on the MLO-Y4 cells with CFTR knockout showed that the KO line exhibited surge in senescence activation genes and phenotypes (reactive oxygen species, senescence-associated secretory phenotype). CFTR expression decline with age contributing to osteocyte aging is therefore suggested by these results. We next explored the potential of enhancing CFTR in rescuing age-related bone disorder. Overexpression of CFTR by local injection of CFTR-containing virus into femur regions increased expression of genes promoting bone formation and osteocyte maturation. Two newly developed CFTR modulators VX809 and VX770 reversed the impaired mineralization in MLO-Y4 cells of a high passage number, although no additive effect of the two drugs were observed. Importantly, in old-aged wild-type mice, periodic local injection of VX809 into lumbar vertebrae and femur for 4 weeks enhanced bone mass. We also investigated the effect of magnesium implant, which show beneficial effect in CFTR mutant mice. Therefore, targeting/enhancing CFTR in bones is suggested as a new strategy for age-related bone disorder. Taken together, the present study has shown a direct role of CFTR pivotal to osteocyte function and survival. Age-dependent decline in CFTR may underlie the molecular mechanism of skeletal aging. Such a previously undefined role of osteocyte CFTR in skeletal aging may facilitate the development of new strategies for the prevention and treatment of age-related bone diseases.