|Title:||Channel function of CFTR in bone cells for microenvironment homeostasis|
|Advisors:||Ruan, Yechun (BME)|
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Biomedical Engineering|
|Pages:||74 pages : color illustrations|
|Abstract:||The physiological microenvironment for bone cells (e.g., osteocytes and osteoblasts), including the chemical composition, pH and volume of the surrounding interstitial fluid as well as the mechanical forces loaded upon them, is believed to be important for bone cells to function. However, how bone cells interact with these environmental factors remains largely unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel best known for its role in regulating electrolytes and water transport in epithelial tissues. Studies have also demonstrated the channel function of CFTR to be mechanosensitive. Given the reported expression of CFTR in bone cells and the correlation of CFTR mutation with bone defects, we hypothesized that CFTR might play a role in regulating the bone cell microenvironment via its channel functions.|
Using patch-clamp analysis in MLO-Y4 cells, a mouse osteocyte-like cell line, whole-currents promoted by forskolin (a cAMP activator) and blocked by CFTRinh-172 (a CFTR selective inhibitor) were observed, suggesting CFTR channel function in bone cells. Fluorometric imaging measurement of intracellular Cl- concentration ([Cl-]i) and membrane potential (Vm) in MLO-Y4 cells showed that CFTR inhibitors induced increases in [Cl-]i and Vm, suggesting CFTR in mediating Cl- efflux in bone cells. Further studies using siRNA-based knockdown and CRISPR-Cas9-based knockout of CFTR in MLO-Y4 cells confirmed CFTR channel function mediating Cl-efflux in bone cells. In addition, fluorometric pH dye monitoring intracellular pH of MLO-Y4 cells showed that CFTRinh-172 impeded pH change recovery from alkalization induced by the removal of extracellular CO2/HCO3-, suggesting the role of CFTR in mediating the exclusion of HCO3- and thus in regulating extracellular pH in bone cells. Consistent with this, we found in RNA sequencing analysis of bone tissues from CFTR mutant (DF508) mice that key genes encoding V-ATPase, a proton pump critically involved in pH regulation, were significantly upregulated in DF508 bones as compared to the wild type. To demonstrate possible mechano-sensitivity of CFTR in bone cells, we used both ultrasound stimulation and stretching forces. Ultrasound elicited intracellular Ca2+ elevation and [Cl-]i responses which were largely inhibited by CFTRinh-172. Stretching of MLO-Y4 cells upregulated COX-2, a key osteogenic gene, which was also inhibited by CFTRinh-172. These results suggested a possible role of CFTR in mediating mechanical stimulation in bone cells. We also tested a mouse osteoblast cell line, MC3T3, which showed similar function of CFTR as Cl- channel. Together, these results have demonstrated previously undefined Cl-/HCO3- channel function of CFTR in bone cells and suggested its regulatory roles in maintaining the homeostasis of the microenvironment such as mediating electrolyte/water transport, adjusting pH and responding to mechanical stimulation.
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