Osteoblast CFTR inactivation reduces differentiation and osteoprotegerin expression in a mouse model of cystic fibrosis-related bone disease.

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover--impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr-/-) mouse model. In the murine calvarial organ culture assay, Cftr-/- calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr-/- compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr-/- compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr-/- murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr-/- murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD

Reduced bone length, growth plate thickness, bone content, and IGF-I as a model for poor growth in the CFTR-deficient rat

<div><p>Background</p><p>Reduced growth and osteopenia are common in individuals with cystic fibrosis (CF). Additionally, improved weight and height are associated with better lung function and overall health in the disease. Mechanisms for this reduction in growth are not understood. We utilized a new CFTR knockout rat to evaluate growth in young CF animals, via femur length, microarchitecture of bone and growth plate, as well as serum IGF-I concentrations.</p><p>Methods</p><p>Femur length was measured in wild-type (WT) and SD-<i>CFTR</i>^{<i>tm1sage</i>} (<i>Cftr-/-</i>) rats, as a surrogate marker for growth. Quantitative bone parameters in <i>Cftr-/-</i> and WT rats were measured by micro computed tomography (micro-CT). Bone histomorphometry and cartilaginous growth plates were analyzed. Serum IGF-I concentrations were also compared.</p><p>Results</p><p>Femur length was reduced in both <i>Cftr-/-</i> male and female rats compared to WT. Multiple parameters of bone microarchitecture (of both trabecular and cortical bone) were adversely affected in <i>Cftr-/-</i> rats. There was a reduction in overall growth plate thichkness in both male and female <i>Cftr-/-</i> rats, as well as hypertrophic zone thickness and mean hypertrophic cell volume in male rats, indicating abnormal growth characteristics at the plate. Serum IGF-I concentrations were severely reduced in <i>Cftr-/-</i> rats compared to WT littermates.</p><p>Conclusions</p><p>Despite absence of overt lung or pancreatic disease, reduced growth and bone content were readily detected in young <i>Cftr-/-</i> rats. Reduced size of the growth plate and decreased IGF-I concentrations suggest the mechanistic basis for this phenotype. These findings appear to be intrinsic to the CFTR deficient state and independent of significant clinical confounders, providing substantive evidence for the importance of CFTR on maintinaing normal bone growth.</p></div

Association of Cystic Fibrosis Genetic Modifiers with Congenital Bilateral Absence of the Vas Deferens

Objective: To investigate whether genetic modifiers of cystic fibrosis (CF) lung disease also predispose to congenital bilateral absence of the vas deferens (CBAVD) in association with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. We tested the hypothesis that polymorphisms of transforming growth factor (TGF)-beta 1 (rs 1982073, rs 1800471) and endothelin receptor type A (EDNRA) (rs 5335, rs 1801708) are associated with the CBAVD phenotype. Design: Genotyping of subjects with clinical CBAVD. Setting: Outpatient and hospital-based clinical evaluation. Patient(s): DNA samples from 80 subjects with CBAVD and 51 healthy male controls from various regions of Europe. This is one of the largest genetic studies of this disease to date. Intervention(s): None. Main Outcome Measure(s): Genotype analysis. Result(s): For single nucleotide polymorphism (SNP) rs 5335, we found increased frequency of the CC genotype among subjects with CBAVD. The difference was significant among Turkish patients versus controls (45.2% vs. 19.4%), and between all cases versus controls (36% vs. 15.7%). No associations between CBAVD penetrance and polymorphisms rs 1982073, rs 1800471, or rs 1801708 were observed. Conclusion(s): Our findings indicate that endothelin receptor type A polymorphism rs 5335 may be associated with CBAVD penetrance. To our knowledge, this is the first study to investigate genetic modifiers relevant to CBAVD. (Fertil Steril (R) 2010; 94: 2122-7. (C) 2010 by American Society for Reproductive Medicine.)Wo

Micro CT of femurs.

<p>Micro CT of femurs.</p

<i>Cftr</i>-/- rats have reduced size and femur length.

<p><b>(</b>A) <i>Cftr-/-</i> rats are notably smaller in size compared to their wild type (WT) littermates. Animal total body weights are demonstrated versus age of the animals in <i>Cftr-/-</i> rats (•) compared to WT rats (▯). These findings are consistent with previously published data by Tuggle et al [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.ref013" target="_blank">13</a>]. (B) Excised femurs obtained at sacrifice were measured by digital calipers. Femur length is reduced in <i>Cftr-/-</i> rats (•) compared to WT rats (▯) regardless of age or gender (p<0.0001).</p

Cartilaginous growth plate analysis.

<p>Cartilaginous growth plates were evaluated based on overall growth plate thickness, average proliferating zone thickness, average number of proliferating cells/column, average hypertropic zone thickness, mean volume of hypertrophic cells, and mean hypertrophic cell volume standard deviation. Pictured growth plates are from the rats included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g003" target="_blank">Fig 3</a> (as well as microCT images in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g002" target="_blank">Fig 2</a>). They include 38 day old WT (A) and 42 day old <i>Cftr-/-</i> (C) male rats. Female bone images are from 42 day old <i>Cftr-/-</i> (B) and WT (D) rats. Each larger image is at 2X, with inserts demonstrating areas of measurement at 20X. Green outlined cells represent the proliferative zone and blue outlined cells are the hypertrophic zone. Both male and female <i>Cftr-/-</i> rats demonstrated a reduction in overall growth plate thickness (43% reduction in males and 35% in females). However, in males there was also a reduction in the hypertrophic zone thickness, mean volume of hypertrophic cells and hypertrophic cell volume standard deviation in the <i>Cftr-/-</i> rats, but not in the proliferative zone thickness or average number of proliferating cells/column. These findings are suggestive of a difference in the maturation from the proliferative zone into the hypertrophic zone, or differences in cellular activity of the hypertrophic zone, between the <i>Cftr-/-</i> and the WT rats.</p

Histomorphometric analysis.

<p>Representative histology images demonstrate histomorphologic findings of reduced bone content in <i>Cftr-/-</i> rats (more predominantly in females and detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.t002" target="_blank">Table 2</a>). Each figure demonstrates a blue line of 1000 microns in length for size consistency and photographed at 1.25X magnification. Pictured images obtained from 38 day old WT (A) and 42 day old <i>Cftr-/-</i> (C) male rats. Female bone images are from 42 day old <i>Cftr-/-</i> (B) and WT (D) rats. Histology images are from the male and female rat femurs pictured in microCT images (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g002" target="_blank">Fig 2</a>).</p

Micro CT imaging demonstrates reduced bone content.

<p>Representative 3D micro CT images of cortical and trabecular bone structures from young <i>Cftr-/-</i> and WT rats. MicroCT images demonstrate reduced bone content in <i>Cftr-/-</i> rats (more predominantly in females and detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.t001" target="_blank">Table 1</a>). MicroCT images obtained from 38 day old WT and 42 day old <i>Cftr-/-</i> male rats. Female rat microCT images are from 42 day old <i>Cftr-/-</i> and WT rats. MicroCT images are from the male and female rat femurs pictured in histology images (Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.g004" target="_blank">4</a>).</p

IGF-I is reduced in <i>Cftr-/-</i> rats.

<p>Serum IGF-I concentrations were reduced in <i>Cftr-/-</i> (•) male (261.5 ± 65.7 vs. 850.8 ± 61.1 ng/mL, p<0.0001, (A) and female (512.6 ± 70.1 vs. 1139 ± 149.7 ng/mL, p = 0.001, (B) rats compared to WT (▯). Age and weight adjusted IGF-I concentrations are found in the supplemental figure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188497#pone.0188497.s001" target="_blank">S1 Fig</a>).</p