Severe osteopenia in CFTR-null mice

Bone 35 (2004) 595 – 603 www.elsevier.com/locate/bone

Severe osteopenia in CFTR-null mice Fariel Dif, a Caroline Marty, b Claude Baudoin, b Marie-Christine de Vernejoul, b and Giovanni Levi a,* a

UMR5166 CNRS-MNHN, Evolution des Re´gulations Endocriniennes, 75231 Paris Cedex 5, France b INSERM U606, Centre Viggo Petersen, Hoˆpital Lariboisie`re, 75475 Paris Cedex 10, France Received 2 February 2004; revised 11 May 2004; accepted 12 May 2004 Available online 17 July 2004

Abstract Osteoporosis is a common complication in cystic fibrosis (CF) patients. In this study, we performed a histomorphometric analysis of the bones of a mouse genetic model of human CF in which both copies of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are inactivated. We find that, even in the absence of obvious nutritional and therapeutic differences, the CFTR mutation is associated with severe osteopenia. Bone mineral density (BMD) of total body and of individual bones is significantly diminished. CFTR mutants display a striking significant (50%) reduction of cortical bone width and thinner trabeculae. Analysis of dynamic parameters indicates a significant reduction of bone formation and a concomitant strong increase in bone resorption. Active osteoclasts where found mostly associated with cortical bone. Our data support the concept that CF-associated osteoporosis is part of the syndromic symptoms associated with the CFTR mutation. D 2004 Elsevier Inc. All rights reserved. Keywords: Cystic fibrosis; CFTR; Osteoporosis; Bone; Mutant mice

Introduction Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disease present in the Caucasians population (1 in every 2500 newborns) [1]. The CF gene [2,3] codes for the CF transmembrane conductance regulator protein CFTR, an ATP-gated chloride channel that is regulated by cyclic-AMP-dependent protein kinase phosphorylation [4,5]. The CFTR gene is expressed in epithelial cells where it regulates the luminal secretion of chloride and the active ion and water transport in the airway epithelial cells [6,7]. Mutations of CFTR lead to dysfunction of chloride and sodium channels and to airway mucus dehydration. Primarily a disease of children and young adults, CF has pleiotropic manifestations that include failure of the mucociliary clearance apparatus in the lung, meconium ileus, pancreatic enzyme insufficiency, and sweat duct dysfunction [8,9]. * Corresponding author. UMR5166 CNRS-MNHN, Evolution des Re´gulations Endocriniennes, 7, rue Cuvier, 75231 Paris Cedex 5, France. Fax: +33-1-40793621. E-mail address: [email protected] (G. Levi). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.05.021

Over the last 50 years, dramatic advances in CF research and treatment have helped to extend the median age of survival for CF patients from 5 years to about 35 years. With this increased survival, further complications of the disease have emerged, an example of which is low bone mineral density (BMD) [10 –16]. Deficient hard tissue mineralization is a recognized but poorly understood complication of cystic fibrosis. Reports on CF patients include evidence of reduced bone mineral density, kyphosis, and pathological fractures; both children and adults are affected by these complications [17 –19]. In CF patients, osteoporosis begins in early adolescence [18,20,21]. Both before and after lung transplantation, CF patients have lower BMD than any other patient group with chronic lung disease [22 – 24]. Low BMD is clinically important in CF, as it has resulted in an increased rate of fracture compared to the general population. Vertebral and rib fractures are particularly detrimental, as septum clearance can be compromised, resulting in exacerbation of pulmonary symptoms. High risk of fractures may also affect quality of life. Further, some centers now consider symptomatic osteopenia to be a relative contraindication to lung transplantation [17,18]. Whether CFTR dysfunction directly leads to hard tissue hypomineralization in CF disease has been difficult to

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establish as demineralization could also depend on a number of accompanying disease factors, including pancreatic insufficiency, calcium or vitamin D nutritional deficiency, reduced exercise capacity, glucocorticoid therapy, delayed puberty, and chronic lung infection [14,25,26]. To evaluate whether osteopenia is directly linked to the CFTR mutation, we analyzed bone histomorphometric parameters in a genetic mouse model of CF in which most of these disease factors do not contribute to the phenotype.

Materials and methods Animal model This study was performed on an established mouse model of cystic fibrosis in which part of the CFTR exon 10 is replaced by two neomycin resistance (Neo) genes driven by different promoters [27]. This replacement introduces a chain termination codon at amino acid position 489 in the CFTR sequence. This mouse model of cystic fibrosis was maintained on a mixed genetic background by the ‘‘Centre de Distribution, de Typage et d’Archivage Animal’’ UPS44 CNRS in Orleans. All animals used in this study were female. Normal and mutant littermates were fed together by the mother until 3 weeks of age. Twenty-four hours after weaning, the mice were sacrificed and their bones were analyzed. Throughout this study, we performed experiments on groups composing homozygous mutants (Cftr / ), heterozygous mutants (Cft+/ ), and normal littermates (Cftr+/+). Each mouse was individually weighed and the length between the tip of the nose to the beginning of the tail was measured with a caliber. For each animal, one femur was removed and multiple sagittal sections were prepared and observed. When longer survival times were needed (for example for tetracycline or calcein double labeling), particular care was taken to assure that normal and mutant littermates were exposed to the same feeding and environmental conditions. Dual-energy X-ray absorptiometry (DEXA) Three-week-old CF mice and their normal littermates were anesthetized by intraperitoneal injection of pentobarbital. The bone mineral density (BMD, mg/cm2) and the bone mineral content (BMC) of total body, femoral, and caudal vertebrae (second and third caudal vertebrae) were measured on groups of 10 –12 mice using a PIXImus mouse densitometer (Lunar GE Medical Systems, France; Software version 1.44). We choose to use this procedure as its accuracy, sensitivity, and reproducibility have been previously demonstrated comparing this instrument to Hologic QDR 2000 devices [50]. Each animal was weighed and its length was measured. The surface area of individual bones was determined.

Bone histomorphometry Femurs were removed from mice immediately after sacrifice and the surrounding soft tissue was discarded. These bone specimens were stored in 70% ethanol and dehydrated in graded ethanol, defatted in xylene, and embedded in methyl methacrylate. Frontal sections (5 Am) of the central region of the distal femur were cut using an SM2500S microtome (Leica, Germany). Two nonconsecutive sections were stained for tartrate-resistant acid phosphatase (TRAP) detection using naphthol ASTR phosphate (Sigma, France) as substrate and then counterstained with toluidine blue (pH 4.3). Other sections were stained with toluidine blue for evaluation of the formation parameters or with aniline blue for the analysis of static parameters. Double labeling of tetracycline and calcein was performed as described [28]. A first tetracycline injection (20 mg/kg) was followed by a second injection of calcein (10 mg/kg) 2 days later. Mice were killed 24 h after the second injection and their femurs dissected and analyzed. Histomorphometric parameters were recorded at this standard sampling site in compliance with the recommendations of the American Society for Bone and Mineral Research (ASBMR) Histomorphometry Nomenclature Committee [29]. Measurements were performed in an area located at about 200 Am from the growth plate. The bone surface (BS), the trabecular bone volume (BV/TV), the trabecular bone width (Tb.Wi), the trabecular separation (Tb.Sp), the cortical width (Ct.Wi), and the osteoid thickness (O.Th) were measured using a software package developed for bone histomorphometry (Morphome´trie Osseuse, Biocom, Les Ulis, France). Osteoclast numbers expressed as N.Oc/T.Am (per mm2) were evaluated on TRAP-stained sections. Osteoid and osteoblast surfaces (expressed as a % of bone surface) were evaluated on toluidine blue stained sections. Observations were performed using an objective eyepiece Leitz intergrateplate II at 128 magnification. Unstained sections of 10 Am were examined under fluorescent light to determine the rate of bone formation using the same image analyzer (Biocom) by a semiautomatic method. The mineralizing surfaces were measured in the same area using the objective eyepiece Leitz intergrateplate II. The mineral apposition rate (MAR) was calculated according to the ASBMR nomenclature. Biochemical analyses Urine samples were collected in the morning to measure the titers of deoxypyridinoline (Metra DPD EIA kit, Quidel Sandiego, CA, USA) and creatinine with the creatinine procedure nj355 (Sigma diagnostic). Statistical analysis Statistical differences between the means in the different groups tested were evaluated by Fisher’s test. Results are

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expressed as means F SEM. A value of P < 0.05 was considered significant. Covariance analysis of BMD versus body weight was performed on groups of at least 15 Cftr / , Cftr+/ , and Cftr+/+ 3-week-old gender-matched mice.

Results Dual-energy X-ray absorptiometry Bone mineral density (BMD) and bone mineral content (BMC) were measured by DEXA in groups of Cftr / , Cftr+/ , and Cftr+/+ 3-week-old gender-matched mice. In Cftr / mice, the whole body BMC and BMD were significantly reduced (46% and 16%, respectively) compared to age- and gender-matched Cftr+/ and Cftr+/+ littermates (Fig. 1A). No significant difference was observed between Cftr+/ and Cftr+/+ mice. The reduction in BMC and BMD observed in Cftr / mice was more pronounced when individual bones were analyzed separately: 55% and 26% reduction of BMC and BMD, respectively, in caudal vertebrae (Fig. 1B) and 48% and 20% reduction of BMC and BMD in the femurs (Fig. 1C). The total area is

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reduced by 35%, the femur area is reduced by 36%, and the vertebrae area is reduced by 39% in the Cftr / mice compared to the wild-type mice. The bones of mutant mice appeared less radio opaque; no evidence of skeletal malformation was seen in Cftr / mice at 3 weeks of age (Fig. 1D). It has been previously described that CF mice have significantly reduced body weights as compared to gender-matched littermates [27]. To exclude the possibility that the reduced bone parameters where only the consequence of the reduced body mass of mutant mice, we have analyzed the correlation between weight and BMD in a group of 15 mutant female mice compared to an equivalent group of normal female littermates. Covariance analyses performed on total body and on femurs (Figs. 1E and F) show an obvious difference in the slope (Figs. 1E and F), supporting the notion that factors different from reduced body weight alone affect the BMD of these mice. To confirm these results, we also compared the BMD of selected Cftr / and Cftr+/+ littermates of very similar weight and body size. Table 1 shows that invariably Cftr / animals had a lower BMD than weight-matched littermates.

Fig. 1. Osteopenia in Cftr / mice. (A) BMC and BMD measured from total body, (B) vertebrae, and (C) femur of Cftr+/+, Cftr / , and Cftr+/ 3-week-old mice. Microradiographs of Cftr+/+, Cftr / , and Cftr+/ hind limbs (D). Covariance analysis of body weight and BMD in total body (E) and femora (F) of Cftr+/+() and Cftr / (.) mice, dotted lines correspond to the mean values of each group. Bars in histograms represent the means F SEM (n = 10 – 12 mouse per group). Asterisks indicate statistically significant differences (**P < 0.001, ***P < 0.0001) evaluated by Fisher’s test.

0.033 0.036 0.037 0.038 0.035 F 0.003 0.029 F 0.001 6.5 7.0 7.0 7.5 6.8 F 0.4 6.3 F 0.2 8.75 F 1.3

Six CFTR mice were paired by weight with six normal littermates; the difference in weight did not exceed 3% in each case. This small difference in weight was associated to a strong reduction of the values of BMD of the CFTR / animals (in average, 18% for femoral BMD and 11% for total body BMD). Body weight and femur size are not significantly different.

9.5 F 0.5

0.027 0.031 0.031 0.030 0.029 F 0.002 0.025 F 0.001

0.026 0.034 6.5 8.5

8.9 10.9 11.2 11.2 Mean F SD 9.76 F 1.4

/

9.5 10.5 10.5 10.5 9.9 F 0.6

9.25

9 9.25 9.75 9.25 9.5 10.5 9.1

10 16 5 3 8 18 18 13 0.025 0.024 0.024 0.025 0.025 0.026 0.028

13 11 16 10 8 24 29 33 0.028 0.028 0.029 0.031 0.030 0.029 0.031

7 5 5 0 5 6 6 14 6.1 6.2 6.2 6.5 6.2 6.6 6.5

7.7 7.7 8.5 8.5 8.9 11.2 7.9

3 2 1 0 0 2 0 0

/ Relative WT difference (%) / Relative WT difference (%) / Relative WT difference (%) / Relative WT difference (%) / WT

Femur size (mm) Total body BMD (g/cm2) Femoral BMD (g/cm2) Body size (cm) Body weight (g)

Table 1 Comparison of BMDs of littermates paired by weight

1.09 1.6 5.4 0 0 0 0 0

F. Dif et al. / Bone 35 (2004) 595–603 Relative difference (%)

598

Bone histomorphometry Histomorphometric measurements were made on femur sagittal sections stained with toluidine blue (Fig. 2A). Bones from Cftr / mice were characterized by a severe reduction in bone surface (Fig. 2B) and trabecular bone volume (Fig. 2C). The trabecular bone loss of Cftr / mice was also manifested by a decrease in the number (data not shown) and thickness of the trabeculae (Fig. 2D) and by an increased trabecular separation when compared to Cftr+/ and Cftr+/+ littermates (Fig. 2E); these last two groups were nod significantly different. The cortical thickness of the femurs from Cftr / mice was about half of that found in normal and heterozygous animals, suggesting that the osteopenic phenotype of these mice is more severe in cortical than in trabecular bone (Fig. 3). Osteoblast surface (Fig. 4A) and osteoid thickness (Fig. 4B) were considerably decreased in Cftr / mice (about 70% and 40%, respectively) demonstrating a reduced rate of bone apposition. To further analyze bone metabolism, the rate of new bone apposition was measured after double labeling with tetracycline and calcein. We first measured the mineralizing surface [MS/BS (%)], which was significantly reduced in Cftr / mice when compared both to Cftr+/+ and Cftr+/ mice; no significant difference was observed between these last two genotypes (Fig. 4C). Dual tetracycline and calcein labeling in Cftr+/+ mice feature two fluorescent bands. In contrast, in Cftr / mice, there was clearly less separation of the two fluorescent bands compared with Cftr+/+ and Cftr+/ mice. In fact, when animals were examined at a 2day interval, bone formation was so slow that often no obvious separation could be seen between the red and green bands, but rather only a single yellow trait band could be observed (Fig. 4E). The trabecular mineral apposition rate of Cftr / mice was consistently 50% slower than in normal littermates (Fig. 4D). In the cortical bone of the Cftr / mice, the rate of bone apposition is so low that it was not possible to include these measurements. Trabecular bone formation rate showed a reduction of 92% between Cftr+/+ and Cftr / mice; means were 0.25 (Am2/Am/day) for Cftr+/+, 0.26 (Am2/Am/day) for Cftr+/ , and 0.02 (Am2/ Am/day) for Cftr / . The ratio between D-pyridinoline and creatinine in urine reflects the global rate of bone resorption. When this value was measured in Cftr+/+, Cftr+/ , and Cftr / mice, it appeared that bones of mutant mice were degraded more rapidly than those of control littermates (Fig. 5A). To better quantify this process, we visualized the presence of active osteclasts by TRAP staining of normal and mutant bones. Surprisingly, in the trabecular region, the density of osteoclasts was reduced in Cftr / mice (Figs. 5B – D); however, the increased rate of bone resorption could be accounted for a dramatic increase of the number of osteoclasts found near to cortical bone (Figs. 5E –G). The N.Oc/BS on cancellous bone was significantly reduced (51%) in Cftr / mice

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Fig. 2. Static histomorphometric parameters in Cftr / mice. (A) Representative sagittal sections of 3-week-old wild-type and Cftr / femora, stained by aniline blue. The black square indicates the metaphyseal region selected for measuring the histomorphometric parameters shown in the following panels. (B) Average bone surface (BS/TV), (C) trabecular bone volume (BV/TV), (D) trabecular width (Tb.Wi), and (E) trabecular separation (Tb.Sp). Bars represent the means F SEM (n = 20 – 24 sections per group). Asterisks indicate statistically significant differences (*P < 0.01; **P < 0.001) evaluated by Fisher’s test. Magnification bar: 0.75 mm.

compared to their wild type (means: 19/mm2 for Cftr+/+, 15/ mm2 for Cftr+/ , and 9/mm2 for Cftr / ).

Discussion More than 170 different mutations in the CFTR gene have been described in CF patients [1,30], about 70% of these mutations share the minimal deletion of three bases in exon 10 (D F508) [2].

Murine models of CF have been developed by disruption or alteration (D F508 mutation) of exon 10 of the murine homologue of CFTR for the purpose of investigating the pathogenesis of the disease [27,31,32]. These mutants reproduce several aspects of the human disease such as lung [33] and severe bowel disease [8,31]. Death, resulting from intestinal obstruction, usually occurs around 3 weeks of age; this is why we had to perform our analysis on 3-week-old mice. However, the severity of individual symptoms of the disease developed by CF mice is different

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Fig. 3. Reduced cortical bone width in Cftr / mice. (A – B) Cortical regions from sections of femora of 3-week-old wild-type and Cftr / mice stained by aniline bleu. Arrows indicate the cortical width. (C) Histogram of cortical width (Ct.Wi) in 3-week-old Cftr+/ + , Cftr / , and Cftr+/ mice. Values represented are the means F SEM (n = 20 – 24 sections per group). Asterisks indicate a statistically significant difference (**P < 0.001, ***P < 0.0001). Magnification bar: 50 Am.

from that observed in humans. The most obvious effect of the Cftr / genotype is shortened life span due primarily to complications arising from obstruction of the intestinal tract. Pathological changes in the respiratory tract of Cftr / mice include increased number of goblet cells, dilation of gland ducts in the nasal, and proximal trachea and destructive change in the epithelia of the upper airways. In contrast to human patients, CF mice do not require specific pharmacological treatments such as antibiotics and glucocorticoids. CF mice therefore represent an ideal model to evaluate the association between the CFTR mutation and other complications of the disease, such as reduced bone tissue mineralization, in the absence of many of the confounding disease factors that are present in human CF patients. For example, it has been recently suggested that malnutrition and chronic use of intravenous and oral corticosteroids might be at the origin of reduced bone mineral density in CF patients [34] and thus would not be directly linked to the CFTR mutation. In the present study, we investigated the bone mineral status and established the histomorphometric profile of

bones of Cftr homozygous mutant compared to normal littermates reared under identical conditions. We show that the presence of the Cftr mutation is invariably associated with severe osteopenia. To take in account the fact that Cftr mutant mice are, in average, smaller that their control littermates, we performed a regression analysis of BMD versus body weight: We show that the observed osteopenia cannot be only ascribed to the reduced size of the animals. Our findings suggest that the osteopenia found in CF patients [13 – 15,26,35,36] might be one of the syndromic aspects of this disease. We find that bones from Cftr / mice are characterized by severe osteopenia in both trabecular and cortical bone. This phenotype is associated to a drastic reduction in bone formation accompanied by an increase in the rate of bone resorption. Our data suggest therefore that in Cftr / mice, osteopenia derives from an imbalance in bone modeling and/or remodeling. The situation might be similar in CF patients. Indeed it has previously been suggested that an imbalance between bone formation and degradation occurs in this disease [37].

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Fig. 4. Reduced bone formation in Cftr / mice. Histomorphometric indicators of bone formation are reduced in Cftr / mice. (A) Osteoblast surface (Ob.S/BS), (B) osteoid thickness indicator of mineral apposition rate in femora (O.Th), (C) mineralizing surface (MS/BS), and (D) mineral apposition rate (MAR) for 3-week-old Cftr+/ + , Cftr / , and Cftr+/ mice. (E) In vivo analysis of bone formation in Cftr+/ + and Cftr / mice, fluorescent micrographs of two representative sections of trabecular surface demonstrating tetracycline or calcein double fluorochrome labeling (see Materials and methods for details). Note the decrease in the distance between the two labeled lines in Cftr / mice. Bars represent the means F SEM (n = 20 – 24 sections per group). Asterisks indicate statistically significant differences (**P < 0.001, ***P < 0.0001) evaluated by Fisher’s test. Magnification bar: 7 Am.

In vertebrates, bone is constantly renewed through resorption of preexisting bone by osteoclasts followed by de novo bone formation by the osteoblasts. Physiologically, these two processes are balanced to maintain a stable bone mass. It has been assumed that bone formation and bone resorption are linked during bone remodeling; implying that the altered functionality of one cell type affects the function of the other cell type to maintain a constant bone mass. We have demonstrated evidence of accelerated bone resorption without a compensatory increase in bone formation in Cftr / mice. Actually, the rate of bone resorption is greatly increased in Cftr / animals, which could explain the dramatic reduction in bone mass. Our observations indicate that the Cftr mutation is associated with increased number and activity of osteoclasts and reduced osteoblastic function. These cellular functions are under complex multifactorial endocrine, paracrine, and

genetic regulation. In particular, most circulating hormones (e.g., estrogens, TSH, PTH, prolactin, GH, leptin [38 – 47]) have major effects in the control of bone homeostasis and their imbalance is at the origin of many forms of human osteoporosis. CFTR is present in most secretory epithelia and in the hypothalamus [48]. It has been shown that CF patients present multiple endocrine defects including hypothyroidism, altered levels of circulating TSH, and prolactin and sex hormones [49]. Preliminary observations (data not shown) indicate a defective endocrine status of the Cftr / mice with an increased level of PTH that can be the initial cause of the increased in cortical bone resorption. It is therefore conceivable that a complex primary endocrine lesion present in CF patients is at the origin of their osteopenic status. However, we cannot exclude the possibility that CFTR itself is present in bone cells and its mutation directly results in the phenomena described here.

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Fig. 5. Increased bone resorption in Cftr / mice. (A) Bone resorption was evaluated by the [D-pyrimidinoline]/[creatinine] ratio in the urine of 3-week-old Cftr+/+, Cftr / , and Cftr+/ mice (n = 11 – 18 sections per group). (B) Trabecular osteoclast number (N.Oc/T.Am) and (E) cortical osteoclast number (N.Oc/ T.Am) in a proximal region of femora (n = 10 – 12 mice per group). Femoral sections of 3-week-old wild-type and Cftr / mice stained for tartrate-resistant acid phosphatase (TRAP) and counterstained with methyl-green and toluidin blue. The regions between primary and secondary spongiosa indicate red osteoclasts (C and D) or cortical region (F and G). The arrows indicate red osteoclasts in cortical bone of Cftr / mice. Bars represent the means F SEM. Asterisks indicate statistically significant differences (***P < 0.0001) evaluated by Fisher’s test. Magnification bar: 50 Am.

Our data strongly suggest that osteopenia is one of the symptoms directly associated with the CFTR mutation. The complete elucidation of the mechanism leading to low bone density in CF patients should help in refining appropriate therapeutic strategies in the future.

the colony of mutant CFTR mice. We wish to thank Christine Forgeron, Dr. Valerie Geoffroy, and Geremie Odillard for excellent suggestions and technical help. We are grateful to Prof. Barbara Demeneix and Dr. Paul Kelly for critical reading of the manuscript.

Acknowledgments

References

This work was supported by a grant from ‘‘Vaincre la Mucoviscidose’’ to G.L. and by the European Consortiums GENOSPORA (QLK6-1999-02108) and ANABONOS ‘‘Molecular Mechanisms of Bone Formation and Anabolism’’ to G.L. and M-C. D. Fariel Dif is recipient of a doctoral fellowship of ‘‘Vaincre la Mucoviscidose.’’ We are grateful to the personnel of the ‘‘Centre de Distribution, de Typage et d’Archivage Animal’’ UPS44 CNRS in Orleans and in particular with Mme. M.F. Bertrand for maintaining

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