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The F508del Mutation in Cystic Fibrosis Transmembrane Conductance Regulator Gene Impacts Bone Formation

      The F508del mutation in the cystic fibrosis transmembrane conductance regulator (Cftr) gene is believed to be an independent risk factor for cystic fibrosis–related bone disease. In this study, we evaluated the bone mineral density as well as the histomorphometric parameters of bone formation and bone mass in both F508del-Cftr homozygous mice (F508del Cftrtm1Eur) and Cftr+/+ littermate controls at 6 (prepubertal), 10 (pubertal), and 14 (young adult) weeks of age in both sexes. The bone architecture of F508del Cftrtm1Eur and wild-type (WT) littermate mice was evaluated by bone densitometry, microcomputed tomography, and analysis of the dynamic parameters of bone formation. Serum levels of both insulin-like growth factor 1 and osteocalcin also were determined. Reduced bone mineral density, lower femoral bone mass, and altered trabecular bone architecture were observed in F508del Cftrtm1Eur mice compared with controls at 6, 10, and 14 weeks of age. A decrease in the bone formation rate in F508del Cftrtm1Eur mice was shown compared with control mice, independently of age and sex. In addition, we found lower insulin-like growth factor 1 levels in F508del Cftrtm1Eur mice compared with age-matched controls, whereas osteocalcin levels were normal. Severe osteopenia and altered bone architecture were found in young and mature adult F508del Cftrtm1Eur mice. Our findings show that the F508del mutation in CFTR impacts trabecular bone mass by reducing bone formation.
      Cystic fibrosis (CF) is an autosomal-recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent anion channel leading to progressive pulmonary damage and, ultimately, to death.
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      Cystic fibrosis since 1938.
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      Identification of the cystic fibrosis gene: genetic analysis.
      • Riordan J.R.
      CFTR function and prospects for therapy.
      Over the past 3 decades, reduced bone mineral density (BMD) and low bone mass has been reported in children, adolescents, and adults with CF, independently of sex, compared with the general population.
      • Aris R.M.
      • Merkel P.A.
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      • Watts N.B.
      • White T.B.
      Guide to bone health and disease in cystic fibrosis.
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      Impact of cystic fibrosis on bone health.
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      BMD and body composition in children and young patients affected by cystic fibrosis.
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      A roadmap to the brittle bones of cystic fibrosis.
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      European cystic fibrosis bone mineralisation guidelines.
      Brittle bones in CF disease were confirmed by densitometric data, fracture incidence, and impaired quality of life in young patients.
      • Gore A.P.
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      A roadmap to the brittle bones of cystic fibrosis.
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      The prevalence of osteoporosis, osteopenia, and fractures among adults with cystic fibrosis: a systematic literature review with meta-analysis.
      The cause of the CF-related low BMD is multifactorial including vitamin K and D insufficiencies, calcium malabsorption, malnutrition, glucocorticoid use, delayed puberty, and pulmonary infection/systemic inflammation.
      • Sermet-Gaudelus I.
      • Bianchi M.L.
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      • Morton A.
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      • Compston J.E.
      • Conway S.P.
      • Castanet M.
      • Wolfe S.
      • Haworth C.S.
      European cystic fibrosis bone mineralisation guidelines.
      • Javier R.M.
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      Bone disease in cystic fibrosis: what's new.
      • Sermet-Gaudelus I.
      • Castanet M.
      • Retsch-Bogart G.
      • Aris R.M.
      Update on cystic fibrosis-related bone disease: a special focus on children.
      • Ionescu A.A.
      • Nixon L.S.
      • Evans W.D.
      • Stone M.D.
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      • Chatham K.
      • Shale D.J.
      Bone density, body composition, and inflammatory status in cystic fibrosis.
      The bone disease occurs around puberty owing to a poor acquisition of peak bone mass and worsens with age.
      • Bianchi M.L.
      • Romano G.
      • Saraifoger S.
      • Costantini D.
      • Limonta C.
      • Colombo C.
      BMD and body composition in children and young patients affected by cystic fibrosis.
      • Sparks A.A.
      • McGee S.J.
      • Boone C.E.
      • Neuringer I.P.
      • Jones S.K.
      • Aris R.M.
      ‘Old’ bones in young bodies: the tale of cystic fibrosis.
      However, low BMD has been observed in CF children younger than the age of 6 years with mild disease and normal nutritional status, suggesting that CF-related low BMD also may be owing to the intrinsic defect (ie, CFTR dysfunction).
      • Bianchi M.L.
      Osteoporosis in children and adolescents.
      • Sermet-Gaudelus I.
      • Souberbielle J.C.
      • Ruiz J.C.
      • Vrielynck S.
      • Heuillon B.
      • Azhar I.
      • Cazenave A.
      • Lawson-Body E.
      • Chedevergne F.
      • Lenoir G.
      Low bone mineral density in young children with cystic fibrosis.
      Whether CFTR activity plays a direct role in the low bone mass in children with CF is unknown, but can be hypothesized on the basis of animal studies in the CFTR-null mouse. Smaller bones with decreased BMD, cortical bone thinning, and altered trabecular architecture were found in Cftr−/− mice despite no difference in osteoblast and osteoclast numbers as compared with control mice, suggesting that CFTR protein may influence bone cell activity rather than number.
      • Haston C.K.
      • Li W.
      • Li A.
      • Lafleur M.
      • Henderson J.E.
      Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice.
      • Dif F.
      • Marty C.
      • Baudoin C.
      • de Vernejoul M.C.
      • Levi G.
      Severe osteopenia in CFTR-null mice.
      Another study
      • Pashuck T.D.
      • Franz S.E.
      • Altman M.K.
      • Wasserfall C.H.
      • Atkinson M.A.
      • Wronski T.J.
      • Flotte T.R.
      • Stalvey M.S.
      Murine model for cystic fibrosis bone disease demonstrates osteopenia and sex-related differences in bone formation.
      reported sex-related differences in the bone formation rate of gut-corrected Cftr−/− mice, suggesting inadequate bone formation in females but increased formation in males. The expression of CFTR protein has been identified by immunohistochemistry in human bone cells.
      • Shead E.F.
      • Haworth C.S.
      • Condliffe A.M.
      • McKeon D.J.
      • Scott M.A.
      • Compston J.E.
      Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human bone.
      We previously reported the expression of CFTR mRNA and protein in primary human osteoblasts (cells that form bone) and showed that inhibition of CFTR-mediated Cl channel activity affects the release of osteoprotegerin and prostaglandin E2, two key regulators of bone formation.
      • Le Heron L.
      • Guillaume C.
      • Velard F.
      • Braux J.
      • Touqui L.
      • Moriceau S.
      • Sermet-Gaudelus I.
      • Laurent-Maquin D.
      • Jacquot J.
      Cystic fibrosis transmembrane conductance regulator (CFTR) regulates the production of osteoprotegerin (OPG) and prostaglandin (PG) E(2) in human bone.
      We recently discovered a defective CFTR Cl channel activity and a deficit of osteoprotegerin production by primary osteoblasts from a 25-year-old CF patient with the F508del/G542X mutation in CFTR.
      • Gimenez A.
      • Le Henaff C.
      • Norez C.
      • Guillaume C.
      • Ravoninjatovo B.
      • Laurent-Maquin D.
      • Becq F.
      • Jacquot J.
      Deficit of osteoprotegerin release by osteoblasts from a patient with cystic fibrosis.
      One study in patients with CF with at least one F508del allele showed a direct association between the F508del mutation and low BMD in both sexes.
      • King S.J.
      • Topliss D.J.
      • Kotsimbos T.
      • Nyulasi I.B.
      • Bailey M.
      • Ebeling P.R.
      • Wilson J.W.
      Reduced bone density in cystic fibrosis: deltaF508 mutation is an independent risk factor.
      However, the impact of the F508del allele mutation in CFTR on bone formation and bone mass remains unknown. In this study, we evaluated the impact of the F508del mutation in CFTR on the BMD status and bone formation in F508del-CFTR homozygous mice in relation to age (prepubertal, pubertal, and young adult) and sex compared with normal Cftr+/+ littermates.

      Materials and Methods

      Mice

      Rotterdam homozygous F508del-CFTR mice (F508del Cftrtm1Eur) that express the clinically common F508del mutation in Cftr at wild-type (WT) protein level and their normal Cftr+/+ homozygous littermates (in the FVB background)
      • van Doorninck J.H.
      • French P.J.
      • Verbeek E.
      • Peters R.H.
      • Morreau H.
      • Bijman J.
      • Scholte B.J.
      A mouse model for the cystic fibrosis delta F508 mutation.
      were obtained from the Centre de Distribution, Typage et Archivage Animal, Centre National de la Recherche Scientifique (Orléans, France). At 6, 10, and 14 weeks of age, F508del Cftrtm1Eur and Cftr+/+ littermate (WT-CFTR) male and female mice were anesthetized by intraperitoneal injection of ketamine (80 mg/kg; Virbac, Carros, France) and xylazine (20 mg/kg Rompun; Bayer, Leverkusen, Germany) as previously described.
      • Bonvin E.
      • Le Rouzic P.
      • Bernaudin J.F.
      • Cottart C.H.
      • Vandebrouck C.
      • Crie A.
      • Leal T.
      • Clement A.
      • Bonora M.
      Congenital tracheal malformation in cystic fibrosis transmembrane conductance regulator-deficient mice.

      Dual-Energy X-Ray Absorptiometry, Scanning Electron Microscopy, and X-Ray Spectra

      The BMD (mg/cm2) of total body and femoral bones free of soft tissue were measured from groups of 8 to 10 F508del Cftrtm1Eur and WT-CFTR mice using a PIXImus mouse densitometer (software version 1.44; Lunar GE Medical Systems, Paris, France). The femoral bones were stored in 70% ethanol and dehydrated in graded ethanol, defatted in xylene, and embedded in methyl methacrylate. Measurements of total calcium (Ca2+) and phosphate (P) atomic concentrations in undecalcified femoral sections from X-ray spectra were performed to calculate a mean value of the Ca/P atomic ratio, indicative of calcium phosphate levels in the bone,
      • Dorozhkin S.V.
      • Epple M.
      Biological and medical significance of calcium phosphates.
      as reported in our laboratory.
      • Drevet R.
      • Velard F.
      • Potiron S.
      • Laurent-Maquin D.
      • Benhayoune H.
      In vitro dissolution and corrosion study of calcium phosphate coatings elaborated by pulsed electrodeposition current on Ti6Al4V substrate.

      Bone Histomorphometry

      Two nonconsecutive sections of femoral bones were stained for tartrate-resistant acid phosphatase detection using naphthol AS-TR phosphate (Sigma, St. Quentin Fallavier, France) as substrate and then counterstained with toluidine blue (pH 4.3). The bone surface, trabecular bone volume, trabecular bone width, and trabecular separation were measured using a software package developed for bone histomorphometry (Microvision, Evry, France). Osteoclast numbers expressed as N.Oc/T.Am (per mm2) were evaluated on tartrate-resistant acid phosphatase–stained sections. Histomorphometric parameters were recorded at this standard sampling site in compliance with the recommendations of the American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee.
      • Parfitt A.M.
      • Drezner M.K.
      • Glorieux F.H.
      • Kanis J.A.
      • Malluche H.
      • Meunier P.J.
      • Ott S.M.
      • Recker R.R.
      Bone histomorphometry: standardization of nomenclature, symbols, and units Report of the ASBMR Histomorphometry Nomenclature Committee.
      Double labeling of tetracycline and calcein in vivo was performed as described.
      • Vignery A.
      • Baron R.
      Dynamic histomorphometry of alveolar bone remodeling in the adult rat.
      The mineral apposition rate was calculated according to the American Society for Bone and Mineral Research nomenclature. Femoral bones of 6-week-old F508del Cftrtm1Eur and WT-CFTR mice were scanned with microcomputed tomography (μCT-40; Scanco Medical AG, Brüttiselen, Switzerland) to assess three-dimensional bone morphology.

      Serum Analysis

      Sera were obtained from F508del Cftrtm1Eur and WT-CFTR mice and stored at −85°C until analysis of duplicate samples. In the active insulin-like growth factor 1 (IGF-1) enzyme-linked immunosorbent assay, IGF-1 was separated from its binding proteins in serum by using an ethanol and HCl-based extraction solution provided by the manufacturer (Millipore, Molsheim, France). Analysis of bone turnover biomarkers was undertaken with a commercial assay (enzyme-linked immunosorbent assays for osteocalcin, IL-6, and tumor necrosis factor-α; Millipore).

      Statistical Analysis

      Data are presented as mean ± SEM for 6 to 10 animals per group. Significance of the in vivo response was determined using one-way analysis of variance with Dunnett post hoc analysis. Significance between groups was determined using two-way analysis of variance with Bonferroni post hoc analysis. A P value <0.05 was considered significant.

      Results

      F508del CFTR Mutation Results in a Decreased Bone Mass

      In a recent study it was reported that femurs of adult F508del Cftrtm1Eur mice of 11 to 15 weeks of age presented with a significantly lower bone volume compared with Cftr+/+ controls whereas weight, length, and BMD were similar in the two mouse groups.
      • Paradis J.
      • Wilke M.
      • Haston C.K.
      Osteopenia in Cftr-deltaF508 mice.
      In our study, measures of body weight, femur length, and BMD were performed in growing F508del Cftrtm1Eur mice at 6 weeks (prepubertal), 10 weeks (pubertal), and 14 weeks (young adult) of age in both sexes. No significant difference in femur length was observed between F508del Cftrtm1Eur mice and sex-matched controls in all ages. As expected, the average body weight of F508del Cftrtm1Eur mice was 24%, 20%, and 14% less than control mice at 6, 10, and 14 weeks of age, respectively (Figure 1, A–C).
      Figure thumbnail gr1
      Figure 1F508del Cftrtm1Eur mice had lower body weight compared with WT control mice at 6, 10, and 14 weeks of age. A: Six-week-old WT control and F508del Cftrtm1Eur male mice. B: Femur length was not significantly different between F508del Cftrtm1Eur and WT control mice between both sexes and at all ages. C: A lower weight in F508del Cftrtm1Eur mice was found compared with WT control mice in both sexes and at all ages. Data are mean ± SEM for 8 to 10 animals per group. Statistical differences are indicated by analysis of variance. *P < 0.05, **P < 0.01, and ***P < 0.001.
      We then examined bone microarchitecture in 6-week-old mice of both genotypes by microcomputed tomography. Representative three-dimensional images of the distal femur are shown in Figure 2A. The plate-like structure of the trabecular bone was reduced markedly, and the connecting rods of trabeculae were disrupted in F508del Cftrtm1Eur mice compared with sex-matched WT controls. We further assessed BMD of F508del Cftrtm1Eur mice and their littermate controls by dual-energy X-ray absorptiometry at 6, 10, and 14 weeks of age. At 6 weeks, the whole-body BMD in F508del Cftrtm1Eur mice was reduced significantly (6% and 15% in males and females, respectively) compared with sex-matched controls (Figure 2B). The reduction in BMD was more pronounced in F508del Cftrtm1Eur mice when femurs were analyzed separately in both sexes, namely a 14% and 22% reduction of BMD in femurs of male and female 6-week-old F508del Cftrtm1Eur mice, respectively, compared with sex-matched controls (Figure 2C). The reduction in BMD persisted in both whole body and femurs of 10-week-old F508del Cftrtm1Eur mice in both sexes. However, there was a trend toward BMD normalization in both whole body and femoral bone in 14-week-old F508del Cftrtm1Eur mice, which was more pronounced in females (Figure 2C).
      Figure thumbnail gr2
      Figure 2F508del Cftrtm1Eur mice had lower bone mass and BMD compared with WT control mice at 6, 10, and 14 weeks of age. A: Morphology study by microcomputed tomography scanning of trabecular bone in the femurs of 6-week-old WT control and F508del Cftrtm1Eur mice. Note that the plate-like architecture of the trabecular bone is reduced markedly and the connecting rods of trabeculae are disrupted in F508del Cftrtm1Eur mice compared with WT control mice, in both sexes. B and C: BMD values measured from total body and femur, respectively, were lower in F508del Cftrtm1Eur mice compared with WT control mice at 6, 10, and 14 weeks of age. A trend toward BMD normalization in both whole body and femoral bone was observed in 14-week-old F508del Cftrtm1Eur mice, which was more pronounced in females. Data are mean ± standard error of the mean for 8 to 10 animals per group. Statistical differences are indicated by analysis of variance. *P < 0.05, **P < 0.01, and ***P < 0.001.

      F508del CFTR Mutation Results in Altered Bone Architecture

      We next assessed architectural parameters in femoral sagittal sections from F508del Cftrtm1Eur mice by microcomputed tomography and histologic measurements (Figure 3). In the femur of 6-week-old F508del Cftrtm1Eur mice, two-dimensional coronal sections adjacent to the growth plate (Figure 3A) showed reduced trabecular bone compared with sex-matched controls in both sexes. Figure 3B shows a reduction of trabecular bone in distal femoral sections of 6-week-old F508del Cftrtm1Eur mice in both sexes. Quantification of structural parameters revealed that bone volume/tissue volume (percentage of trabecular bone volume) was decreased by 40% and 36% in 6-week-old F508del Cftrtm1Eur male and female mice, respectively, compared with sex-matched controls. Trabecular thickness was decreased by 17% and 9% in 6-week-old F508del Cftrtm1Eur male and female mice, respectively, confirming the microcomputed tomography findings. Trabecular separation was increased by 53% and 69% in 6-week-old F508del Cftrtm1Eur mice, respectively, in both sexes (Figure 3, C and D). As shown in Figure 3E, the decrease of trabecular bone volume in femurs of F508del Cftrtm1Eur mice persisted at both 6 and 14 weeks whereas trabecular bone volume was normalized in F508del Cftrtm1Eur mice at 14 weeks of age, in females but not in males.
      Figure thumbnail gr3
      Figure 3Static histomorphometric parameters of bone structure in WT control and F508del Cftrtm1Eur mice. A: Representative two-dimensional sectional scans showed thinner trabeculae of femoral bone in 6-week-old F508del Cftrtm1Eur mice compared with WT control mice, in both sexes. B: Representative sagittal sections of femoral bones in 6-week-old F508del Cftrtm1Eur and WT control mice. Scale bar = 0.75 mm. C and D: Significant increases in thickness of trabeculae and in space between trabeculae, respectively, were observed in 6-week-old F508del Cftrtm1Eur mice compared with WT control mice, in both sexes. E: The femur of F508del Cftrtm1Eur mice had decreased trabecular bone volume at both 6 and 10 weeks in both sexes whereas trabecular bone volume was normalized in F508del Cftrtm1Eur mice at 14 weeks of age, in females but not in males. Data are mean ± standard error of the mean for 8 to 10 animals per group. Statistical differences are indicated by analysis of variance. *P < 0.05 and ***P < 0.001.

      F508del CFTR Mutation Results in Reduced Bone Formation

      Bone remodeling is a process that includes the tight coupling between bone formation and bone resorption. Because the F508del Cftrtm1Eur mice showed decreased bone mass, we examined whether these changes were related to the alterations in tartrate-resistant acid phosphatase–positive osteoclasts, a specific marker indicative of the rate of bone resorption (Figure 4A). Quantitative analysis of osteoclasts (N.Oc/T.Am) lining the femoral trabecular bone at 6, 10, and 14 weeks of age is shown in Figure 4B. No significant difference in osteoclast number was observed in 6- and 14-week-old F508del Cftrtm1Eur mice compared with sex-matched control mice. In contrast, a significant increase in osteoclast number was found in F508del Cftrtm1Eur mice at 10 weeks in both sexes (Figure 4B).
      Figure thumbnail gr4
      Figure 4Osteoclast number in femoral bones of WT control and F508del Cftrtm1Eur mice. A: Representative images of tartrate-resistant acid phosphatase–stained sections showing osteoclasts (arrows) from 6-week-old WT control and F508del Cftrtm1Eur female mice. Original magnification, ×50. Scale bar = 10 µm. B: No significant difference in the osteoclast number/bone surface was observed in 6- and 14-week-old F508del Cftrtm1Eur mice compared with sex-matched control mice. In contrast, a significant increase in osteoclast number was found in F508del Cftrtm1Eur mice at 10 weeks in both sexes. Data are mean ± standard error of the mean for 8 to 10 animals per group. Statistical difference is indicated by analysis of variance. **P < 0.01.
      To analyze bone formation dynamics, the rate of new bone apposition (ie, the trabecular mineral appositional rate) was evaluated in vivo after double labeling with tetracycline and calcein in F508del Cftrtm1Eur mice (Figure 5A). We found that the mineral apposition rate in F508del Cftrtm1Eur mice was consistently lower than in sex-matched controls, at all ages (Figure 5B). To investigate whether bone defects observed in F508del Cftrtm1Eur mice are related to nutritional deficiency and altered mineral homeostasis, we measured the total calcium and phosphate atomic ratio in undecalcified bone trabecular sections by X-ray analysis. No significant change in the Ca/P atomic ratio in bones from X-ray spectra was found between the F508del Cftrtm1Eur and control mice at 14 weeks of age, in both sexes (Table 1). To further investigate whether bone defects observed in F508del Cftrtm1Eur mice may be related to alteration in changes of circulating levels of bone-growth factors, we measured levels of serum IGF-1 and osteocalcin in F508del Cftrtm1Eur mice compared with control mice at 6, 10, and 14 weeks of age (Figure 6). We found lower levels of serum IGF-1 in F508del Cftrtm1Eur mice compared with age-matched control mice (Figure 6A). Control WT mice showed an expected increase in IGF-1 with time, which was less pronounced in F508del Cftrtm1Eur mice. No significant difference was observed in the serum level of osteocalcin between F508del Cftrtm1Eur and control WT mice (Figure 6B).
      Figure thumbnail gr5
      Figure 5F508del Cftrtm1Eur mice had a lower rate of bone formation compared with WT control mice at all ages, in both sexes. A: In vivo analysis of bone formation in 6-week-old WT control and F508del Cftrtm1Eur female mice, fluorescent images of two representative sections of trabecular surface showing tetracycline and calcein double-fluorochrome labeling. White crockets specify the distance between labeled bone surfaces, labeling for calcein was reduced in F508del Cftrtm1Eur compared with WT control mice. Scale bar = 10 μm. B: The femur from F508del Cftrtm1Eur mice had decreased mineral apposition rate compared with sex-matched WT controls at 6, 10, and 14 weeks of age. Statistical differences are indicated by analysis of variance. *P < 0.05, **P < 0.01, and ***P < 0.001.
      Table 1Ca/P Atomic Ratio of Femoral Bones in 14-Week-Old F508del Cftrtm1eur and Sex-Matched WT Mice
      GenotypeSexNCa/P atomic ratioP value
      WTMale31.62 ± 0.01NS
      F508delMale31.63 ± 0.03NS
      WTFemale31.57 ± 0.02NS
      F508delFemale31.58 ± 0.02NS
      Undecalcified femoral sections coated with a conductive layer (carbon) of 14-week-old WT control and F508del Cftrtm1Eur mice were observed using a LaB6 electron microscope (JEOL JSM-5400LV) operating at 15 kV, which is equipped with an ultra-thin window Si (Li) detector for X-ray measurements. The Ca/P values represent the means ± standard error of the mean of three distinct areas analyzed in three femoral sections for three animals per group.
      Ca/P, the atomic ratio of total calcium/phosphates in trabecular bone measured on the basis of high resolution X-ray spectra; NS, not significant.
      Figure thumbnail gr6
      Figure 6Serum IGF-1 and osteocalcin levels in F508del Cftrtm1Eur mice. A: Serum IGF-1 levels were lower in F508del Cftrtm1Eur compared with WT control mice, in all ages. B: No significant difference in serum levels of osteocalcin was observed between F508del Cftrtm1Eur and WT control mice. Blood collected by cardiac puncture in anesthetized animals was analyzed at 6 weeks (WT, 5 females and 5 males; F508del, 5 females and 5 males), at 10 weeks (WT, 2 females and 2 males; F508del, 3 females and 3 males), and at 14 weeks of age (WT, 5 females and 6 males; F508del, 5 females and 6 males). Statistical difference is indicated by analysis of variance. **P < 0.01.

      Discussion

      Our data clearly indicate that F508del mutation in CFTR results in decreased bone mass and bone formation, independently of sex. A severe osteopenic phenotype was seen in 6-week-old F508del Cftrtm1Eur mice, with reduced BMD, lower bone mass, and reduction in the number and thickness of trabeculae compared with control littermates. This phenotype was associated with a decreased rate of new bone formation at all ages. In addition, at 10 weeks (pubertal age), we found that the number of osteoclasts was increased in F508del Cftrtm1Eur female and male mice compared with sex-matched controls, supporting the idea that high bone turnover was increased in F508del Cftrtm1Eur mice at this pubertal period. We found a persistent deficit in both BMD and bone mass in F508del Cftrtm1Eur mice despite significant, quantitative improvements in females at 14 weeks of age (young adult age). Although F508del Cftrtm1Eur mice are, on average, smaller than their control littermates, we show that the observed osteopenic phenotype cannot be ascribed only to the reduced size of CF mice. Compared with sex-matched controls, a lower trabecular bone volume consistently was found in F508del Cftrtm1Eur mice, which was more pronounced in males. Our data therefore do not support the sex-related differences in bone loss described in the gut-corrected Cftr−/− mouse model, reporting inadequate bone formation in females but increased bone formation in males.
      • Pashuck T.D.
      • Franz S.E.
      • Altman M.K.
      • Wasserfall C.H.
      • Atkinson M.A.
      • Wronski T.J.
      • Flotte T.R.
      • Stalvey M.S.
      Murine model for cystic fibrosis bone disease demonstrates osteopenia and sex-related differences in bone formation.
      By using quantitative microcomputed tomography imaging of femurs, Paradis et al
      • Paradis J.
      • Wilke M.
      • Haston C.K.
      Osteopenia in Cftr-deltaF508 mice.
      reported reduced bone volume in adult F508del Cftrtm1Eur mice although measures of weight, length, BMD, and osteoclast numbers were similar to those of control littermates. Our data support some of these observations by showing a strong reduction (49%) in bone volume and no significant change in BMD of femoral bone in adult F508del Cftrtm1Eur mice in both sexes. Also in agreement with the latter, we noted that osteoclast number in femurs of 14-week-old F508del Cftrtm1Eur mice did not differ from those in control mice. We also found a slower rate in bone formation in F508del Cftrtm1Eur male and female mice compared with sex-matched controls. Given the lower bone volume now reported in two CF animal models (B6 Cfttm1Kth and Cftrtm1Eur mice) with the F508del mutation (this study and the study by Paradis et al
      • Paradis J.
      • Wilke M.
      • Haston C.K.
      Osteopenia in Cftr-deltaF508 mice.
      ), all data suggest that the F508del mutation in CFTR does not increase the osteoclast number, but rather may modify the activity of bone cells. It is therefore reasonable to hypothesize that the effects of F508del CFTR on bone cells may depend on the stage of development (ie, growing versus adult F508del Cftrtm1Eur mice). This may very well reflect a different regulation of the osteoblast/osteoclast balance in growing individuals. Indeed, in growing individuals, bone formation must exceed bone resorption to gain bone mass, allowing skeletal growth. On the other hand, during the growth phase, balanced bone resorption is essential for mineralized cartilage and woven bone erosion, two processes that contribute physiologically to longitudinal development. In our growing 10-week-old F508del Cftrtm1Eur mice, we show a higher osteoclast number compared with 6- and 14-week-old F508del Cftrtm1Eur mice. Therefore, the regulation of osteoblast and osteoclast activities must be different in growing mice compared with young adult mice. Whatever they might be, our results show that F508del mutation in CFTR in growing mice can physiologically dissociate osteoclast function from osteoblast function, with a higher bone resorption activity in the stage of bone trabecular development, contributing to an osteopenic phenotype. These findings are consistent with clinical studies reporting that levels of bone turnover markers were higher in F508del homozygous CF patients compared with healthy controls
      • Haworth C.S.
      • Selby P.L.
      • Webb A.K.
      • Dodd M.E.
      • Musson H.
      • Mc L.N.R.
      • Economou G.
      • Horrocks A.W.
      • Freemont A.J.
      • Mawer E.B.
      • Adams J.E.
      Low bone mineral density in adults with cystic fibrosis.
      and were more evident in male compared with female patients with CF.
      • King S.J.
      • Topliss D.J.
      • Kotsimbos T.
      • Nyulasi I.B.
      • Bailey M.
      • Ebeling P.R.
      • Wilson J.W.
      Reduced bone density in cystic fibrosis: deltaF508 mutation is an independent risk factor.
      Our finding that F508del Cftrtm1Eur mice are characterized by decreased new bone formation, independently of age and sex, may be relevant to patients with CF with the F508del mutation. Consistently, three clinical reports through histomorphometric data in young adult CF patients revealed both a reduced BMD and lower trabecular bone volume compared with age- and sex-matched healthy control subjects, which was ascribed to low bone formation.
      • Elkin S.L.
      • Vedi S.
      • Bord S.
      • Garrahan N.J.
      • Hodson M.E.
      • Compston J.E.
      Histomorphometric analysis of bone biopsies from the iliac crest of adults with cystic fibrosis.
      • Haworth C.S.
      • Freemont A.J.
      • Webb A.K.
      • Dodd M.E.
      • Selby P.L.
      • Mawer E.B.
      • Adams J.E.
      Hip fracture and bone histomorphometry in a young adult with cystic fibrosis.
      • Grey V.
      • Atkinson S.
      • Drury D.
      • Casey L.
      • Ferland G.
      • Gundberg C.
      • Lands L.C.
      Prevalence of low bone mass and deficiencies of vitamins D and K in pediatric patients with cystic fibrosis from 3 Canadian centers.
      A link between reduced BMD, bone resorption, and alteration in bone turnover markers has been described accompanying exacerbations of lung infection with systemic inflammation in CF patients.
      • Ionescu A.A.
      • Nixon L.S.
      • Evans W.D.
      • Stone M.D.
      • Lewis-Jenkins V.
      • Chatham K.
      • Shale D.J.
      Bone density, body composition, and inflammatory status in cystic fibrosis.
      • Aris R.M.
      • Stephens A.R.
      • Ontjes D.A.
      • Denene Blackwood A.
      • Lark R.K.
      • Hensler M.B.
      • Neuringer I.P.
      • Lester G.E.
      Adverse alterations in bone metabolism are associated with lung infection in adults with cystic fibrosis.
      A recent study showed a positive correlation between osteoclast number and serum levels of tumor necrosis factor-α and osteocalcin in CF patients.
      • Shead E.F.
      • Haworth C.S.
      • Barker H.
      • Bilton D.
      • Compston J.E.
      Osteoclast function, bone turnover and inflammatory cytokines during infective exacerbations of cystic fibrosis.
      The F508del Cftrtm1Eur mouse strain studied here has not been reported previously to develop lung inflammatory disease,
      • van Doorninck J.H.
      • French P.J.
      • Verbeek E.
      • Peters R.H.
      • Morreau H.
      • Bijman J.
      • Scholte B.J.
      A mouse model for the cystic fibrosis delta F508 mutation.
      although recent data based on quantitative lung histology showed mild inflammation in mature adult (16 to 19 weeks) F508del Cftrtm1Eur mice compared with normal mice, under pathogen-free conditions.
      • Wilke M.
      • Buijs-Offerman R.M.
      • Aarbiou J.
      • Colledge W.H.
      • Sheppard D.N.
      • Touqui L.
      • Bot A.
      • Jorna H.
      • de Jonge H.R.
      • Scholte B.J.
      Mouse models of cystic fibrosis: phenotypic analysis and research applications.
      In the present study, we found no significant difference in the serum osteocalcin level between F508del Cftrtm1Eur and control mice, as well as in the serum levels of tumor necrosis factor-α and IL-6 cytokines (data not shown).
      Although much attention has been focused on growth deficits in children, adolescents, and adults, evidence suggests that individuals with CF show abnormalities at birth. Circulating levels of IGF-1 have been shown to directly regulate bone growth and density.
      • Yakar S.
      • Rosen C.J.
      • Beamer W.G.
      • Ackert-Bicknell C.L.
      • Wu Y.
      • Liu J.L.
      • Ooi G.T.
      • Setser J.
      • Frystyk J.
      • Boisclair Y.R.
      • LeRoith D.
      Circulating levels of IGF-1 directly regulate bone growth and density.
      Studies have indicated that CF newborns are shorter and have a lower body weight that non-CF newborns.
      • Festini F.
      • Taccetti G.
      • Repetto T.
      • Reali M.F.
      • Campana S.
      • Mergni G.
      • Marianelli L.
      • de Martino M.
      Gestational and neonatal characteristics of children with cystic fibrosis: a cohort study.
      Additional studies have suggested that the reduced levels of serum IGF-1 might be responsible, at least in part, for the growth defect reported in patients with CF,
      • Switzer M.
      • Rice J.
      • Rice M.
      • Hardin D.S.
      Insulin-like growth factor-I levels predict weight, height and protein catabolism in children and adolescents with cystic fibrosis.
      which also was observed in both Cftr−/− mouse and newborn Cftr−/F508 pig models.
      • Rogan M.P.
      • Reznikov L.R.
      • Pezzulo A.A.
      • Gansemer N.D.
      • Samuel M.
      • Prather R.S.
      • Zabner J.
      • Fredericks D.C.
      • McCray Jr, P.B.
      • Welsh M.J.
      • Stoltz D.A.
      Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth.
      • Rosenberg L.A.
      • Schluchter M.D.
      • Parlow A.F.
      • Drumm M.L.
      Mouse as a model of growth retardation in cystic fibrosis.
      IGF-1 has been identified as an independent predictor of low BMD in CF patients
      • Gordon C.M.
      • Binello E.
      • LeBoff M.S.
      • Wohl M.E.
      • Rosen C.J.
      • Colin A.A.
      Relationship between insulin-like growth factor I, dehydroepiandrosterone sulfate and proresorptive cytokines and bone density in cystic fibrosis.
      and in Cftr−/− mice.
      • Rosenberg L.A.
      • Schluchter M.D.
      • Parlow A.F.
      • Drumm M.L.
      Mouse as a model of growth retardation in cystic fibrosis.
      In the present study, serum IGF-1 level was lower in F508del Cftrtm1Eur mice compared with age-matched controls, as previously reported in the Cftr−/− mice.
      • Haston C.K.
      • Li W.
      • Li A.
      • Lafleur M.
      • Henderson J.E.
      Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice.
      However, in the study by Haston et al,
      • Haston C.K.
      • Li W.
      • Li A.
      • Lafleur M.
      • Henderson J.E.
      Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulator-deficient mice.
      the median value of serum IGF-1 in 12-week-old Cftr−/− mice was lower (210 ng/mL) compared with the median value of serum IGF-1 of 500 ng/mL that we observed in our 10- and 14-week-old F508del Cftrtm1Eur mice. An alternate explanation for this difference is that no CFTR protein is expressed in the Cftr−/− mouse model, whereas in the F508del Cftrtm1Eur mouse model the F508del mutation in Cftr expresses the F508del CFTR protein similar to the WT protein level. Rogan et al
      • Rogan M.P.
      • Reznikov L.R.
      • Pezzulo A.A.
      • Gansemer N.D.
      • Samuel M.
      • Prather R.S.
      • Zabner J.
      • Fredericks D.C.
      • McCray Jr, P.B.
      • Welsh M.J.
      • Stoltz D.A.
      Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth.
      also reported a significant reduction in serum IGF-1 levels in both newborn Cftr−/F508 pigs and humans with CF and suggested that the decrease is not only a consequence of malnutrition or pulmonary inflammation, but that loss of CFTR function may have an additional, more direct effect in reducing IGF-1 levels. These data and the present study make it likely that F508del CFTR expression associated with low circulating IGF-1 level might be a causal factor in reduced bone formation. How the F508del mutation in CFTR decreases the serum IGF-1 level remains, to date, to be investigated.
      In humans, vitamin D deficiency also results in impaired bone density owing to decreased calcium absorption. Although lower serum 25-hydroxyvitamin D levels commonly are seen in patients with CF compared with the non-CF population, cross-sectional studies have shown no clear association between the vitamin D status and BMD of CF patients.
      • Haworth C.S.
      Impact of cystic fibrosis on bone health.
      In addition, vitamin D supplementation shows no evidence of benefits on BMD, fractures, and bone turnover markers in CF individuals.
      • Sermet-Gaudelus I.
      • Bianchi M.L.
      • Garabedian M.
      • Aris R.M.
      • Morton A.
      • Hardin D.S.
      • Elkin S.L.
      • Compston J.E.
      • Conway S.P.
      • Castanet M.
      • Wolfe S.
      • Haworth C.S.
      European cystic fibrosis bone mineralisation guidelines.
      • Haworth C.S.
      • Jones A.M.
      • Adams J.E.
      • Selby P.L.
      • Webb A.K.
      Randomised double blind placebo controlled trial investigating the effect of calcium and vitamin D supplementation on bone mineral density and bone metabolism in adult patients with cystic fibrosis.
      Adequate calcium intake is essential to achieve an optimal peak bone mass. In the present study, we reported a comparable total calcium/phosphate atomic ratio in femoral bone between adult F508del Cftrtm1Eur and control mice, suggesting that bone calcium and phosphate content was not affected in F508del Cftrtm1Eur mice. In support of this contention, Hillman et al
      • Hillman L.S.
      • Cassidy J.T.
      • Popescu M.F.
      • Hewett J.E.
      • Kyger J.
      • Robertson J.D.
      Percent true calcium absorption, mineral metabolism, and bone mineralization in children with cystic fibrosis: effect of supplementation with vitamin D and calcium.
      showed that in patients with CF the percentage of true calcium absorption was in the normal range based on age and pubertal state compared with the control population.
      Our data generate the hypothesis that the diminished bone mass observed in F508del-CFTR mice may imply an inherent defect of the F508del mutation, leading to a reduction of new bone formation in vivo that is not sex- and age-specific. These data are the first to show an important role for CFTR in maintaining bone mass, as well as new bone formation. Most experts in the European CF bone mineralization working group
      • Sermet-Gaudelus I.
      • Bianchi M.L.
      • Garabedian M.
      • Aris R.M.
      • Morton A.
      • Hardin D.S.
      • Elkin S.L.
      • Compston J.E.
      • Conway S.P.
      • Castanet M.
      • Wolfe S.
      • Haworth C.S.
      European cystic fibrosis bone mineralisation guidelines.
      found that bone loss is observed most often in the peripubertal age range (8 to 10 years) in CF patients. In accordance with these clinical data, we show a marked bone mass loss and altered trabecular bone architecture in F508del Cftrtm1Eur mice at the prepubertal state. Our data support the conclusion that the F508del-CFTR mutation may contribute to bone disease by slowing the new bone formation in infants and young children with cystic fibrosis. Additional molecular and cellular studies are required to elucidate how the F508del mutation in CFTR could disrupt the process of osteogenic differentiation, which could take place in bone marrow stromal cells, to affect both the osteoblastogenesis and osteoclastogenesis processes. The murine F508del Cftrtm1Eur model detailed earlier may become a valuable tool to identify new anabolic targets for the treatment of CF-related bone disease.

      Acknowledgments

      We thank Marie Françoise Bertrand (CDTA, Orleans, France) for providing Rotterdam FVB Cftrtm1Eur mice (which were developed with the support of EUROCARECF) and Christine Guillaume and Sylvie Bouthors (Equipe EA 4691, Biomateriaux et Inflammation en Site Osseux, SFR CAP-Santé, FED 4231, Université de Reims Champagne-Ardenne, Reims, France) for their excellent technical assistance.

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      • This Month in AJP
        The American Journal of PathologyVol. 180Issue 5
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          The following highlights summarize research articles that are published in the current issue of The American Journal of Pathology.
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