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. 2008 Mar;212(3):275–285. doi: 10.1111/j.1469-7580.2008.00859.x

Histological and elemental analyses of impaired bone mineralization in klotho-deficient mice

Hironobu Suzuki 1, Norio Amizuka 2, Kimimitsu Oda 2,3, Masaki Noda 4, Hayato Ohshima 1, Takeyasu Maeda 2,5
PMCID: PMC2408997  PMID: 18248363

Abstract

The klotho gene-deficient mouse is known as an animal model for an accelerated gerontic state, mimicking osteoporosis, skin atrophy, ectopic calcification, and gonadal dysplasia. To elucidate the influence of klotho deficiency on bone mineralization, we examined the ultrastructures of osteoblasts and bone matrices in addition to performing the elemental mapping of calcium, phosphorus, and magnesium in the bone. Under anesthesia, 4- and 5-week-old klotho-deficient mice (klotho−/–mice) and their wild-type littermates were perfused with either 4% paraformaldehyde for light microscopic observation or 4% paraformaldehyde and 0.0125% glutaraldehyde for electron microscopic observation. The femurs and tibiae were processed for both observations. Paraffin sections were subject to alkaline phosphatase and tartrate resistant acid phosphatase histochemistry. Semithin and ultrathin sections obtained from epoxy resin-embedded specimens were used for detecting mineralization – according to von Kossa's staining method – and for elemental mapping by electron probe micro-analyzer, respectively. Alkaline phosphatase-positive plump osteoblasts adjacent to the growth plate normally developed cell organelles in the klotho−/–metaphyses. This, however, contrasted with the flattened osteoblasts covering the metaphyseal trabeculae and accompanied by small tartrate resistant acid phosphatase-positive osteoclasts. The wild-type mice displayed the mineralized matrix at the zone of hypertrophic chondrocyte of the growth plate and well-mineralized metaphyseal trabeculae parallel to the longitudinal axis of the bone. Alternatively, the klotho−/–mice demonstrated a thick mineralized matrix from the proliferative zone of the growth plate as well as the large non-mineralized area in the metaphyseal trabeculae. Consistently, electron probe micro-analysis verified sporadic distributions of higher or lower concentrations of calcium and phosphorus in each trabecule of the klotho−/–mice. The distribution of magnesium, however, was almost uniform. Under transmission electron microscopy, osteoblasts on the metaphyseal trabeculae displayed less-developed cell organelles in the klotho−/–mice. Thus, the klotho deficiency appears not only to reduce osteoblastic population, but also to disturb bone mineralization.

Keywords: bone, klotho, mineralization

Introduction

The klotho gene is involved in multiple aging phenotypes and age-related disorders. Klotho-deficient mice (klotho−/– mice) develop normally until 3 weeks of age and then become less active, ultimately dying by 8–9 weeks of age (Kuro-o et al. 1997). Conversely, the overexpression of the klotho gene in klotho−/– mice extends the life span (Kurosu et al. 2005). The defect in the klotho gene in mice gives rise to osteoporosis, skin atrophy, ectopic calcification, pulmonary emphysema, gonadal dysplasia, and defective hearing – all characteristics of human aging. The defect also induces various metabolic abnormalities which closely resemble those in conditions of food deprivation: lower levels of blood glucose, insulin and glycogen storage in the liver and lipid droplets in brown adipose tissue (Mori et al. 2000).

The klotho gene encodes secretory and membrane-bound forms of its protein, containing respectively one and two β-glucosidase-like domains in both humans (Matsumura et al. 1998) and mice (Shiraki-Iida et al. 1998). The membrane-bound isoform predominates in mice but is scanty in humans. The klotho gene is typically expressed in the kidney and the choroid plexus (Kuro-o et al. 1997). A human klotho protein indicates an 86% amino acid identity with the mouse protein, and its gene is encoded by a gene that spans over 50 kb on chromosome 13q12 (Matsumura et al. 1998), but no premature-aging syndromes have been linked to this region. It has been reported that klotho gene polymorphism in humans is associated with the pathophysiology of bone loss with aging (Kawano et al. 2002), spondylosis (Ogata et al. 2002), osteocalcin level (Mullin et al. 2005), bone mineral density (Yamada et al. 2005), osteonecrosis in patients with sickle cell anemia (Baldwin et al. 2005), and coronary artery disease (Arking et al. 2003; Imamura et al. 2006). Taken together, these findings indicate that klotho appears to have extreme influences on many organs that do not express this molecule, as supported by the fact that the klotho protein is a circulating factor related to human aging (Xiao et al. 2004).

The klotho protein directly binds the fibroblast growth factor receptor (FGFR) with the klotho-FGFR complex binding to the fibroblast growth factor (FGF)-23 with a higher affinity than FGFR or klotho protein alone (Kurosu et al. 2006; Urakawa et al. 2006). More recently, α-klotho has been reported to be involved in calcium homeostasis by mediating increased activity of Na+, K+-ATPase (Imura et al. 2007). FGF-23 regulates systemic phosphate homeostasis (Quarles, 2003). All the above imply the possibility that the klotho protein influences bone mineralization.

Klotho−/– mice have shown a low-turnover osteoporosis as a result of a markedly reduced bone formation that exceeds a decrease in bone resorption (Kawaguchi et al. 1999). On the other hand, it has been reported that klotho−/– mice exhibit elongated metaphyseal trabecular bone as well as an increase in the volume, number and thickness of this bone (Yamashita et al. 1998, 2000a). Although the mRNA levels of the receptor activator of NF-κB (RANK) and RANK ligand (RANKL) in mice homozygous for klotho gene deletion are the same as those of heterozygous and wild-type mice, the klotho−/– mice display high serum levels of calcium, phosphorus (Kuro-o et al. 1997; Kawaguchi et al. 1999), and osteoprotegerin (Yamashita et al. 2000b, 2001). In the physiological state, bone formation is coupled with bone resorption during the process of remodeling, which continuously takes place along the surface of bone, with resorption preceding formation. We should note here the hypothesis that osteoclastic bone resorption triggers the migration, differentiation and activation of osteoblasts, which is referred to as a ‘coupling phenomenon’ (Frost, 1964). However, the reduced function of osteoblasts may result from the specific effects of klotho deficiency rather than from decreased activities of osteoclasts, since not only osteoblastic bone formation but also the distribution of osteocytes and the synthesis of bone matrix proteins is disturbed (Suzuki et al. 2005). Therefore, the unique pathological state of klotho−/– bone cells may affect bone mineralization in a manner that must be different from osteoporotic bone mineralization. To date, little literature is available regarding bone mineralization in mice lacking the klotho gene, except for one study (Yamashita et al. 1998). Recently, a homozygous missense mutation in human klotho has caused severe tumoral calcinosis (Ichikawa et al. 2007), which is a rare autosomal recessive metabolic disorder characterized by extraosseous calcium phosphate deposition in the skin, muscles, joints, and visceral organs (Metzker et al. 1988). Hence, histological examination of the bone abnormality of klotho−/– mice should provide a cue to elucidate the influence on the mineralization with the loss of the klotho gene.

This study therefore aimed to examine morphological alterations in the bone mineralization of klotho−/– deficient mice at the histological, ultrastructural and elemental levels.

Materials and methods

Tissue preparation

Twelve male wild-type SPF/VAF (n = 6) and klotho−/– (n = 6) mice (Japan CLEA, Tokyo, Japan) at 4 and 5 weeks of age were used in this study. The care and use of animals followed the Guiding Principles for the Care and Use of Animals, as approved by Niigata University. Mice were anesthetized with an intraperitoneal injection of chloral hydrate (40 mg 100 g−1, b.w.). They were perfused with 4% paraformaldehyde in a 0.1 m phosphate buffer (pH 7.4), or 4% paraformaldehyde and 0.0125% glutaraldehyde in a 0.067 m phosphate buffer for light or electron microscopic observations, respectively. Femurs and tibiae were immediately removed and immersed with the same fixative for an additional 8 h at 4 °C. The samples were decalcified with 10% ethylenediamine tetraacetic disodium salt (EDTA-2Na) solution for light microscopy, or 5% EDTA-2Na solution for electron microscopy. For light microscopy, the decalcified specimens were dehydrated in an ascending series of alcohols prior to paraffin embedding, and the sections were mounted onto silanized glass slides (Dako Japan, Tokyo, Japan). For transmission electron microscopy (TEM), the decalcified specimens were post-fixed with 1% osmium tetraoxide with 1.5% potassium ferrocyanide in a 0.1 m cacodylate buffer for 3 h at 4 °C, dehydrated in ascending acetone solutions, and embedded in epoxy resin (Epon 812, Taab, Reading, UK). Ultrathin sections were prepared with an ultramicrotome and examined with an electron microscope (H-7000: Hitachi, Tokyo, Japan) following a brief staining with tannic acid, uranyl acetate, and lead citrate. Furthermore, some undecalcified specimens were dehydrated in ascending acetone solutions and embedded in Epon 812 for mineralization detection and elemental mapping. Sections of 1 µm were prepared with an ultramicrotome for mineralization detection, and femurs embedded in Epon 812 were ground for elemental mapping.

Immunohistochemistry for alkaline phosphatase (ALP) and enzyme histochemistry for tartrate-resistant acid phosphatase (TRAP)

After an inhibition of endogenous peroxidase activity with methanol containing 0.3% hydrogen peroxidase for 30 min, the dewaxed paraffin sections were pretreated with 1% bovine serum albumin (BSA; Seologicals Proteins Inc. Kankakee, IL, USA) in phosphate-buffered saline (1% BSA-PBS) for a further 30 min. They were then incubated for 2–3 h at room temperature with rabbit polyclonal antisera against alkaline phosphatase (ALP) (Oda et al. 1999) diluted at 1 : 250 with 1% BSA-PBS. Sections were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Amersham Bui., Tokyo, Japan). The immunoreaction was visualized by an incubation with 0.04% 3-3′-diaminobenzidine and 0.003% hydrogen peroxidase. The sections were faintly counterstained with methyl green. The detection of TRAP was performed as previously described (Amizuka et al. 1998); slides were rinsed with PBS and incubated in a mixture of 2.5 mg naphthol AS-BI phosphate (Sigma, St. Louis, MO, USA), 18 mg red violet LB (Sigma) salt, and 100 mm L (+) tartaric acid (0.76 g) diluted in 30 mL of a 0.1 m sodium acetate buffer (pH 5.0) for 15 min at 37 °C.

Von Kossa's staining for detection of mineralized matrix

Von Kossa's staining was performed as described previously (Sakagami et al. 2005). Briefly, undecalcified semithin epoxy resin sections were incubated with an aqueous solution of 5% silver nitrate (Wako Pure Chemical Industries, Osaka, Japan) for 60 min at room temperature under sunlight until they assumed a dark brown color. Following a distilled water rinse, the sections were incubated with a 5% sodium thiosulfate solution (Wako Pure Chemical Industries) for 5 min. The sections were faintly stained with toluidine blue.

Elemental mapping by EPMA

An electron probe microanalyzer (EPMA; 8705, Shimazu, Co. Ltd, Kyoto, Japan) was used for the elemental mapping of calcium (Ca), phosphorus (P), and magnesium (Mg) – the three major elements in the mineralized bone matrix. Undecalcified femurs at weeks 4 and 5 were embedded in epoxy resin, and trimmed with diamond disks until exposure of a sagittal plane containing the center of the implant. After polishing, the specimens were sputter-coated with carbon prior to elemental analysis. For each experiment, 256 × 256 pixel mapping was performed. The accelerating voltage and beam current were set to 15 kV and 0.03 µA, respectively, and integrating time was 0.05 s at each pixel.

Results

Histological observations of tibiae of 4- and 5-week-old wild-type mice and mice homozygous for klotho gene deletion

The tibiae of wild-type mice at 4 and 5 weeks of age showed well-defined metaphyseal trabeculae parallel to the longitudinal axis (Fig. 1A,G). ALP-immunopositive osteoblasts were evenly discernible on the metaphyseal trabeculae from their subchondral to the terminal regions (Fig. 1B,H). Many TRAP-positive osteoclasts were localized at the chondro-osseous junction and the termini of the metaphyseal trabeculae (Fig. 1C,I). In contrast, the 4-week-old klotho−/– tibia featured less-extended metaphyseal trabeculae compared with the age-matched wild-type counterparts (Fig. 1D). Unlike the wild-type mice, ALP-positive osteoblasts and TRAP-reactive osteoclasts could be observed at the chondro-osseous junction, but there were only a few osteoclasts on the metaphyseal trabeculae (Fig. 1E,F). Five-week-old klotho−/– mice displayed shorter but thicker metaphyseal trabeculae than did the wild-type mice (Fig. 1J). Consistent with 4-week-old klotho−/– mice, ALP-positive osteoblasts and TRAP-reactive osteoclasts accumulated at the chondro-osseous junction, with a few of them discernible on the metaphyseal trabeculae (Fig. 1K,L).

Fig. 1.

Fig. 1

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The tibiae of 4- (A–C) and 5- (G–I) week-old wild-type, and 4- (D–F) and 5- (J–L) week-old klotho−/– mice. The tibiae of 4- and 5-week wild-type mice have well-defined cortical bone and parallel metaphyseal trabeculae continuous with the growth plate each other (A,G). The metaphyseal trabeculae line parallel to the longitudinal axis of the tibia (A,G). ALP-immunopositive osteoblasts cover the surface of the metaphyseal trabeculae from beneath the growth plate (B,H). Many osteoclasts demonstrate a TRAP reaction on the trabecular bone (C,I). In contrast, the 4-week-old klotho−/– mouse shows the short metaphyseal trabeculae continuing to the thin layer of the hypertrophic chondrocytes (D), and the adjacent trabecular bones connect with each other in the 5-week-old mouse (J). Osteoblasts beneath the growth plate show strong ALP-immunoreactions, but the expression of ALP in the osteoblasts on the metaphyseal region is weak in both old klotho−/– mice (E,K). TRAP-positive osteoclasts are recognizable beneath the growth plate (F,L). Scale bars: (A,D,G,J): 250 µm; (A,D,G,J) insets: 100 µm; (B,C,E,F,H,I,K,L): 100 µm.

Mineralization of tibiae in wild-type mice and mice homozygous for klotho gene deletion

The mineralized cartilaginous matrix was found in the hypertrophic zone of the growth plate of the 4- and 5-week-old wild-type mice (Fig. 2A,E). In contrast, in the 4-, and 5-week-old klotho−/– mice, thick bands of the mineralized matrix were recognizable in the lower region of the proliferative chondrocytes (Fig. 1C,G). The cartilage mineralization was relatively restricted to the intercolumnar septum of the wild-type mice, while the klotho−/– mice featured broad mineralization, including a transverse partition of the cartilage column (Fig. 2A,C,E,G insets). On observing metaphyseal trabeculae, the 4- and 5-week-old wild-type mice exhibited well-mineralized trabeculae (Fig. 2B,F). However, 4-week-old klotho−/– mice displayed patchy unmineralized matrices (Fig. 2D) and, at 5 weeks, huge unmineralized trabeculae (Fig. 2H) were seen, despite the cartilaginous mineralization initiated from the proliferative zone in the growth plate. Thus, the matrix mineralization appeared to be accelerated at the klotho−/– growth plate cartilage but was extremely reduced in the metaphyseal trabeculae.

Fig. 2.

Fig. 2

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Observation of the mineralized matrix of tibias in the 4- (A–D) and 5- (E–H) week-old wild type (A,B,E,F) and klotho−/– (C,D,G,H) mice. Von Kossa's staining. Both wild-type mice have the mineralized matrix at the layer of the hypertrophic chondrocyte (A,E, and inset), and almost all trabeculae at the metaphyseal region indicate advanced mineralization (B,F). The thick mineralized matrix, meanwhile, is recognizable at the layer of cell proliferation in klotho−/– mice (C,G, and inset). In a view of the metaphyseal region, the non-mineralized matrix exists in 4-week-old klotho−/– mouse (D in arrows) and spreads in the 5-week-old klotho−/– mouse (H in arrows). Scale bars: 50 µm: (A,C,E,G) insets: 30 µm.

Elemental mapping of Ca, P and Mg in wild-type mice and mice homozygous for klotho gene deletion

The wild-type femur demonstrated a similar gradient distribution of Ca and P, both of which showed lower concentrations close to the growth plate, but higher concentrations when observing the metaphyseal trabeculae (Fig. 3A–D,G,H). Mg seemed to be much less concentrated than Ca and P, and evenly distributed in the trabeculae (Fig. 3E,F,I). These distribution patterns of Ca, P and Mg did not differ between 4- and 5-week-old wild-type mice (compare Figs 3 and 4). Surprisingly, the 4-week-old klotho−/– mice showed higher concentrations of Ca, P and Mg in closer proximity to the growth plate than did the wild-type mice (Fig. 3J–O). Furthermore, the focal deposition of highly concentrated Ca, P and Mg was present near the growth plate of the 5-week-old klotho−/–mice (Fig. 4J–O). The 5-week-old klotho−/–trabeculae featured a patchy area composed of high or low concentrations of Ca and P, indicating uneven distribution of calcium phosphate (Fig. 4P,Q). However, the Mg concentration was uniform in the klotho−/–trabeculae (Fig. 4R).

Fig. 3.

Fig. 3

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The elemental mapping of calcium, phosphorus and magnesium in femurs of the 4-week-old wild-type (A–I) and klotho−/– (J–R) mice. Red indicates a high, and dark blue, a low elemental concentration. The wild-type mouse reveals that the dose distributions of calcium and phosphorus gradually increase from the growth plate to the metaphyseal region (A–D), but that the magnesium concentration does not change at the low level (E,F). However, the klotho−/– mouse does not show a gradual distribution; the elements of calcium, phosphorus, and magnesium are highly distributed close to the growth plate compared with the wild-type mouse (J–O). Observing the metaphyseal trabeculae, the concentrations of calcium and phosphorus in the wild-type mouse are higher than klotho−/– mouse (G,H,P,Q), but no distinct difference in magnesium concentrations is indicated between both types of mice (i, r). Scale bars: (A,C,E,J,L,N): 500 µm; (B,D,F–I,K,M,O–R): 100 µm.

Fig. 4.

Fig. 4

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The elemental mapping of calcium, phosphorus, and magnesium in femurs of the 5-week-old wild-type (A–I) and klotho−/– (J–R) mice. Red indicates a high, and dark blue, a low elemental concentration. The dose distributions of calcium and phosphorus gradually increase from metaphysis to diaphysis in the wild-type mouse such as the 4-week-old mouse (A–D), but the magnesium concentration is roughly fixed (E,F). The klotho−/– mouse indicates focal distributions of highly concentrated calcium, phosphorus and magnesium beneath the growth plate (J–O). At the metaphyseal region, the klotho−/– mouse shows regions of high and low doses of calcium and phosphorus elements, but no distinct difference in magnesium concentration appears between both types of mice (G–I,P–R). Scale bars: (A,C,E,J,L,N): 500 µm; (B,D,F–I,K,M,O–R): 100 µm.

TEM observations on bone cells in wild-type mice and mice homozygous for klotho gene deletion

Metaphyseal trabeculae of the wild-type mice were covered with many plump osteoblasts with well-developed rough endoplasmic reticulum, indicative of an active synthesis of the bone matrix (Fig. 5A,B). Multinucleated osteoclasts developed ruffled borders adjacent to resorption lacunae (Fig. 5C). In contrast, flattened osteoblasts were scattered on klotho−/– primary trabeculae (Fig. 5D,E). Klotho−/– osteoclasts had a poorly developed ruffled border on the bone matrix (Fig. 5F). Thus, both osteoblasts and osteoclasts appeared to be morphologically less activated and poorly populated on the trabecular surfaces.

Fig. 5.

Fig. 5

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TEM observations of tibia in the wild-type (A–C) and klotho−/– (D–F) mice. Plump osteoblasts (OB) are localized on the mixed spicules composed of cartilage cores and the bone matrix (A). An OB shows well-developed rough endoplasmic reticulum, indicating abundant synthesis of the bone matrix (B). A multinucleated osteoclast (OC) has developed a ruffled border, adjacent to resorption lacunae (C). In contrast, a flat osteoblast is discernible on the bone matrix of the metaphyseal trabecula in the klotho−/– mouse (D,E). Note an osteocyte with fragmented pyknotic nuclei and a disrupted cell body (D, inset). An osteoblast-like cell shows less-developed cell organellae, including rough endoplasmic reticulum (E), but an osteoclast (OC) with a ruffled border is localized on the bone matrix similarly to the wild-type mouse (F). Scale bars: (A,C,D–F), 5 µm. (B,D) inset, (E): 2 µm.

Discussion

In this study, the osteoclastic activity and population of klotho−/– mice were markedly decreased, in accordance with the high serum levels of Ca, P (Kuro-o et al. 1997; Kawaguchi et al. 1999) and osteoprotegerin (Yamashita et al. 2000b, 2001). In the physiological state, the osteoclast appears to play an important role in the activation of osteoblasts, enabling normal bone remodeling (Frost, 1964; Nishino et al. 2001; Sakagami et al. 2005). Although a previous report on klotho−/– bone has demonstrated low bone turnover resembling senile osteoporosis (Kawaguchi et al. 1999), our own investigation indicates that the altered bone with klotho deficiency does not appear to be due merely to a less active coupling of osteoblasts and osteoclasts, but also to the unique pathological effect of the klotho−/– state. Patchy areas of unmineralized matrix in the metaphyseal trabeculae of 4- and 5-week-old klotho−/– mice appear to account for the reduced activity of osteoblasts and osteoclasts, but do not seem to reflect the senile osteoporosis. Rather, this resembles the bony abnormalities of op/op mice, which showed reduced populations of osteoblasts and patchy unmineralized areas in osteoblast-lacking diaphyses (Sakagami et al. 2005).

It is of paramount interest that, in the klotho−/– mice, cartilaginous mineralization occurs at a higher position of the growth plate, and many ALP-positive osteoblasts and TRAP-reactive osteoclasts accumulate at the chondro-osseous junction. The chondro-osseous junction is a site of actively collapsing cartilaginous matrices. Therefore, some other factors may compensatively stimulate osteoblastic activities, e.g. vascular endothelial growth factors (VEGF) (Gerber et al. 1999; Amizuka et al. 2004), transforming growth factors (TGF) (Thorp et al. 1992; Horner et al. 1998) or osteopontin (Ikeda et al. 1992), which could be expressed by chondrocytes. In the klotho−/– growth plate, von Kossa's staining demonstrated thick bands of the mineralized matrix from the height of the proliferative chondrocytes, which extended not only in the longitudinal intercolumnar septum, but also in the transverse partition. This may imply that the klotho deficiency provoked growth plate chondrocytes to mineralize the cartilaginous matrix prematurely. The stout, mineralized cartilaginous core may give rise to thick primary trabeculae which must be well mineralized, as evidenced by the increased contents of Ca and P close to the chondro-osseous junction (Fig 4). It seems therefore likely that ALP-positive osteoblasts could deposit well-mineralized bone matrices on the stout cartilaginous cores at this site, but then, metaphyseal osteoblasts are inhibited to synthesize a mineralized bone matrix, due to the klotho deficiency.

It is possible that abnormal bone mineralization is involved in the systemic phosphorus metabolism, which is regulated by many factors including FGF-23, secreted frizzled related protein-4, matrix extracellular phosphoglycoprotein, parathyroid hormone, and 1α,25(OH)2D3(Berndt et al. 2005). FGF-23, identified as a gene mutated in patients with autosomal dominant hypophosphatemic rickets (The ADHR Consortium, 2000), is secreted by osteoblasts and osteoclasts in addition to odontoblasts and cementoblasts (Yoshiko et al. 2007) and inhibits phosphate transport in renal proximal tubular cells (Baum et al. 2005). In addition, the klotho protein directly binds to FGFR, and the klotho-FGFR complex binds to FGF-23 with a higher affinity than FGFR or the klotho protein alone (Kurosu et al. 2006). The klotho protein also binds to FGF-23 (Urakawa et al. 2006). The increased serum level of phosphate, shortened life span, growth retardation, infertility, hypoglycemia, and vascular calcification seen in klotho−/– mice are consistent with that seen in FGF-23 deficient mice (Kuro-o et al. 1997; Shimada et al. 2004). Taken together, these findings show that klotho may evolve to implement the FGF-23 action, and in the physiological state, bone cells might have the klotho-FGFR complex, thus regulating the FGF-23 function in bone. Furthermore, α-klotho has been reported to be involved in calcium homeostasis by mediating increased activity of Na+, K+-ATPase (Imura et al. 2007). In contrast, in the klotho-deficient environment, increased serum levels of calcium and phosphorus might contribute to accelerated cartilaginous mineralization by prematurely differentiated chondrocytes, as well as an extraordinary accumulation of Ca and P in the subchondral bone. However, this does not seem to occur in the metaphyseal trabeculae since the osteoblasts have already been disturbed, as seen in ALP histochemistry and TEM observations.

Sera from aged donors inhibit osteoblast differentiation of mesenchymal stem cells, not only decreasing osteoblastic gene expression such as core binding factor/runt-related binding factor 2, ALP, type I collagen and osteocalcin, but also affecting mineralization (Abdallah et al. 2006). Bone marrow transplantation from the osteoporosis-prone mouse induced senile osteoporosis in the normal mouse (Ueda et al. 2007). It is therefore possible that some local factors work jointly or affect the klotho action for inhibiting premature differentiation and the short life span of bone cells. The klotho protein appears to interfere with intracellular signals of insulin/insulin-like growth factor (IGF-1), resulting in an insulin resistance and an extension of the life span (Kurosu et al. 2005). IGF-1, on the other hand, stimulates bone formation through osteoblasts without bone resorption (Sugimoto et al. 1997; Ogata et al. 2000; Akune et al. 2002), whose serum level decreased with aging. These findings seem to contradict previous findings on klotho−/– mice phenotypes – for example hypoglycaemia, a decreased serum level of insulin (Kuro-o et al. 1997), and an increased insulin sensitivity (Utsugi et al. 2000). Although it is possible that there are unknown factors interposed between klotho and IGF-1, both influence bone cells synergistically.

Acknowledgments

We thank Mr Masayoshi Kobayashi at the Center for Instrumental Analysis, Niigata University, for his invaluable technical assistance.

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