Figure 1.
CFTR expression in osteoblasts.
(A) Total RNA was harvested from mouse tissues. Cftr mRNA expression in murine primary osteoblast cultures, pancreas, liver and lung was assessed by real-time RT PCR. (B) Calvariae were harvested from Cftr+/+ and Cftr−/− 4-day-old pups, fixed, sectioned immunostained for CFTR using the 3G11 rat anti-CFTR monoclonal antibody and counterstained with hematoxylin. (C) Osteoblasts were prepared by digestion from murine calvariae and cultured on collagen-coated glass slides. CFTR (red) was identified by immunofluorescence using the 3G11 antibody. Nuclei (blue) were identified by DAPI staining. Two adjacent osteoblasts are shown.
Figure 2.
Lack of CFTR expression in osteoclasts.
(A) Cftr mRNA expression analysis of calvarial osteoblasts, undifferentiated and osteoclast-differentiated (M-CSF/RANKL-treated) RAW264.7 murine monocyte cells. Trap mRNA expression served as a control for osteoclast formation. (B) Immunofluorescence did not reveal CFTR (red) expression in murine bone marrow-derived multinucleated osteoclasts. Membrane-localized protein Na+/H+ exchange regulatory cofactor-1 (NHERF1) (green) served as a positive control for membrane staining. Nuclei (blue) was identified by DAPI staining.
Figure 3.
CFTR inactivation reduced osteoblast differentiation and bone formation.
(A) The murine calvarial organ culture assay was performed on Cftr+/+ and Cftr−/− mouse pups. After two weeks in culture, new bone formation and osteoblast number was significantly less with Cftr inactivation. Yellow arrows indicate the area of actively mineralizing bone indicated by orange G staining that is distinct from the red staining of eosin. (B) Murine calvarial osteoblasts harvested from Cftr+/+ and Cftr−/− calvariae were grown in culture. Seven days after reaching confluence, osteoblast differentiation was assessed by alkaline phosphatase staining. (C) Proliferation, as assessed by BrdU staining, was modestly increased in Cftr+/+ compared to Cftr−/− calvarial osteoblasts. (D) The proportion of calvarial osteoblasts that were viable, undergoing early apoptosis or late apoptosis was unchanged with CFTR inactivation. (* = p<0.05; ** = p<0.01)
Figure 4.
CFTR inactivation increased osteoclastogenesis and Rankl:Opg osteoblast expression.
(A) Bone marrow was flushed from Cftr+/+ and Cftr−/− mice and osteoclast formation assays performed. Osteoclasts were identified by TRAP staining as cells containing ≥3 or more nuclei. More osteoclasts were found in Cftr−/− bone marrow. (B) Rankl and Opg mRNA expression was determined by real-time RT PCR in Cftr+/+ and Cftr−/− calvarial osteoblasts with and without PTH (1–34) 10 nM treatment for 24 hours. Overall, CFTR inactivation increased the Rankl:Opg ratio in both PTH (1–34)-treated and untreated osteoblasts. (C) In long-term osteoblast cultures, decreased Opg expression persisted post-confluence despite no significant difference in Rankl. Opg expression remained significantly lower at 7 days in Cftr−/− osteoblasts, but did not reach significance at 14 days post-confluence. (* = p<0.05; ** = p<0.01; *** = p<0.001; NS = not significant)
Figure 5.
CFTR inactivation blocked Wnt3a and PTH-activated osteoblast canonical Wnt signaling.
(A) Calvarial osteoblasts harvested from Cftr+/+ and Cftr−/− mouse pups were cultured. Cyclic AMP accumulation, a marker of PTH receptor activation, was not significantly different between Cftr+/+ and Cftr−/− osteoblasts after 10 minutes of PTH (1–34) 10 nM treatment. (B) Activation of canonical Wnt signaling was assessed utilizing the TOP-Flash and FOP-Flash Wnt luciferase reporter vectors. Canonical Wnt activity increased after 48 hours of PTH (1–34) 10 nM and Wnt3a 10 ng/ml treatment in Cftr+/+ but not in Cftr−/− calvarial osteoblasts. (* = p<0.05; NS = not significant)