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TRENDS in Molecular Medicine
Vol.12 No.7 July 2006
Hypervitaminosis D and premature aging: lessons learned from Fgf23 and Klotho mutant mice Mohammed S. Razzaque and Beate Lanske Department of Developmental Biology, Harvard School of Dental Medicine, Research and Educational Building, 190 Longwood Ave, Boston, MA 02115, USA
The essential role of low levels of vitamin D during aging is well documented. However, possible effects of high levels of vitamin D on the aging process are not yet clear. Recent in vivo genetic-manipulation studies have shown increased serum level of vitamin D and altered mineralion homeostasis in mice that lack either fibroblast growth factor 23 (Fgf23) or klotho (Kl) genes. These mice develop identical phenotypes consistent with premature aging. Elimination or reduction of vitamin-D activity from Fgf23 and Kl mutant mice, either by dietary restriction or genetic manipulation could rescue premature aging-like features and ectopic calcifications, resulting in prolonged survival of both mutants. Such in vivo experimental studies indicated that excessive vitamin-D activity and altered mineral-ion homeostasis could accelerate the aging process.
Humoral factors and aging Aging is a progressive biological process that is regulated by complex genetic interactions and influenced by environmental factors. It is difficult to study the molecular events of the aging process because of its slow progressive nature. Determining the mechanism of normal aging is important to understand the molecular pathology of the diseases associated with either premature aging (see Glossary) or with advanced aging. In vivo genetic-manipulation studies combined with molecular profiling have greatly broadened the understanding and scope of aging research. The generation of mice with specific deletions of targeted genes has offered a useful tool to study the possible role of a particular gene during aging. Two mouse models that lack either fibroblast growth factor 23 (Fgf23) or klotho (Kl) genes have provided the opportunity to study the roles and regulation of humoral factors on progression of aging. Studies in model organisms have demonstrated the importance of endocrine control on aging and longevity, particularly of the insulin–insulin-like-growth factor-1 (IGF-I) system [1]. Moreover, roles of DNA damage, oxidative stress, genomic instability and apoptosis have been extensively studied and they are known to affect the Corresponding authors: Razzaque, M.S. (mrazzaque@hms.harvard.edu), Razzaque, M.S. (razzaquems@yahoo.com). Available online 30 May 2006
progression of the aging process [2–4]. However, the potential role of humoral factor(s), such as Fgf23 and klotho, that regulate the aging process has not been studied in similar depth and details. A comprehensive review of all aspects of aging is beyond the scope of this article; rather, we will briefly summarize the lessons learned from the study of mice that lack either Fgf23 or Kl in regulating the aging process. Notably, in vivo genetic manipulation could induce unexpected compensatory or redundancy effects, and, therefore, might complicate interpretation of discrete effects of targeted molecules on aging. However, experimental evidences suggest that abnormal mineral-ion homeostasis or premature-agingGlossary Autosomal dominant hypophosphatemic rickets (ADHR): it is an inherited disease accompanied by renal phosphate wasting due to gain-of-function mutations in the FGF23 gene that prevent cleavage and inactivation of FGF23. Ectopic calcification: pathological accumulation of Ca2C salts in the tissues that alters the functionality of the affected organs. Familial tumoral calcinosis (FTC): it is an autosomal recessive disease that is associated with hyperphosphatemia and ectopic calcifications, due to lossof-function mutations in either FGF23 or GALNT3 gene. Fibroblast growth factor 23 (FGF23): it is a circulating factor that is mainly produced by bone cells. It helps maintain physiological balance of phosphate. Hypervitaminosis D: vitamin-D toxicity that is due to increased activity of vitamin D (usually harmful), leading to abnormal soft tissue and vascular calcifications. Klotho: the klotho gene (Kl) encodes a type-I membrane glycoprotein (130 kDa) with a short cytoplasmic domain (ten amino acids). Klotho is expressed in renal tubular epithelial cells, parathyroid secretory cells and epithelial cells of the choroid plexus. Klotho has many functions, including regulation of mineral-ion homeostasis. Membrane protein: most of the cellular functions and cell-matrix interactions are performed through membrane proteins, which consist of transmembrane and anchored proteins. Membrane proteins are classified into five types: (i) type I membrane proteins; (ii) type II membrane proteins; (iii) multipass transmembrane proteins; (iv) lipid-chain-anchored membrane proteins; and (v) glycosyl phosphatidylinositol-anchored membrane proteins. Type I membrane proteins have a single transmembrane stretch of hydrophobic amino acid sequence, with the N-terminus exposed to the extracellular side and the C-terminus exposed on the cytoplasmic side of the membrane. Premature aging: early appearance of aging features before actual age. Premature aging is a pathological condition that is present in humans in various forms; Werner’s syndrome is one of such human diseases. Animal models of premature aging are useful in studying the molecular mechanisms of aging and age-related diseases. Tumor-induced osteomalacia (TIO): it is a paraneoplastic disease characterized by hypophosphatemia. It is caused by renal phosphate wasting due to excessive production of FGF23 by the tumor. X-linked hypophosphatemia (XLH): it is an X-linked dominant disorder characterized by renal phosphate wasting, rickets and growth retardation. It is caused by inactivating mutations of PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X-chromosome.
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like phenotypes of mice that lack Fgf23 or Kl could be reversed by either exogenous or endogenous restoration of FGF23 and klotho, respectively [5,6]. Fibroblast growth factor 23 FGF23 is a 30-kDa secreted protein that is processed by a pro-convertase enzyme into two smaller fragments of w18 kDa (amino fragment) and 12 kDa (carboxy fragment). The biological significance of these fragments is not clearly defined, and is an area of intense research. The human FGF23 gene was cloned from sequence homology of the mouse Fgf23 gene [7]. The in vivo biological activity of FGF23 was initially studied using tumor tissues that were obtained from patients with tumor-induced osteomalacia (TIO), where high levels of FGF23 produce clinical symptoms such as hypophosphatemia and osteomalacia. When tumor cells that secrete FGF23 were experimentally transplanted into nude mice, hypophosphatemia or osteomalacia was reproduced into the host animals, emphasizing the notion that high levels of FGF23 in patients with TIO are responsible for producing the clinical symptoms [8]. Another rare human disease – autosomal dominant hypophosphatemic rickets (ADHR) – is associated with missense mutations in the human FGF23 gene. Mutations in the 176RXXR179 site (Arg176Gln, Arg179Trp and Arg179Gln) prevent cleavage and inactivation of FGF23 in ADHR, leading to massive urinary phosphate wasting [9]. FGF23 is also involved in producing clinical symptoms in patients with X-linked hypophosphatemia (XLH), a disease that has the highest incidence rate among patients with hereditary hypophosphatemia. XLH is present in about one out of 20 000 individuals, and results from mutations in the phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), which contains 22 exons [10,11]. The Hyp mouse spontaneously lacks the 3 0 region of Phex and is the animal model for XLH. This mouse develops hypophosphatemia and has shown skeletal features consistent with rickets and/or osteomalacia [10,11]. Increased levels of Fgf23 have been detected in Hyp mice, and genomic deletion of Fgf23 reverted hypophosphatemia in these mice [12,13]. It was initially proposed that PHEX could cleave FGF23 and, thereby, inactivate its bioactivity; however, subsequent studies suggested that FGF23 is actually a downstream factor of functional PHEX, which seems to be involved in the expression of FGF23 instead of cleaving it [14,15]. Recently, missense mutations in the UDP-N-acetyl-aD-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) have been associated with hyperostosis–hyperphosphatemia syndrome (HHS) and familial tumoral calcinosis (FTC) with hyperphosphatemia [16,17]. Interestingly, mutations in FGF23 have also been reported in patients with FTC [18]. FTC and HHS are autosomal recessive disorders characterized by unusually normal or elevated serum levels of vitamin D, hyperphosphatemia and ectopic calcifications. The fact that two different genes can cause the same human disease suggests a possible role for GALNT3-mediated glycosylation in controlling FGF23 activity. Similar to patients with FTC, Fgf23 knockout mice also develop www.sciencedirect.com
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hyperphosphatemia, hypervitaminosis D and widespread ectopic calcifications, involving lung, kidney, heart, blood vessels and skin. These mice, however, develop also other features, including severe growth retardation and infertility, that are not usually observed in patients with FTC, emphasizing the species variation and the complexity to reconcile every aspect of mouse phenotypes with corresponding human diseases. Genetically modified-animal studies have provided further insights into the role of FGF23 in phosphate homeostasis and skeletogenesis. For example, transgenic mice that overexpress Fgf23 under the control of the a1(I)collagen promoter exhibited hypophosphatemia due to excessive urinary loss of phosphate [3]. Similar observations were also reported in transgenic mice that overexpress Fgf23 under the control of other promoters [19,20]. The opposite effect was documented in Fgf23 knockout mice, which develop severe hyperphosphatemia and increased serum levels of vitamin D [13,21]. The phenotype of transgenic mice that overexpress Fgf23 is similar to that of patients with ADHR, XLH and TIO, whereas the phenotype of Fgf23 knockout mice mimics that of patients with FTC. These in vivo geneticmanipulation studies reinforce the notion that Fgf23 is essential to maintain phosphate homeostasis. Another important aspect that emerges from in vivo mouse genetic studies is that FGF23 is a negative regulator of vitamin-D synthesis, as it appears from the high serum level of vitamin D in Fgf23 knockout mice [13,21] and the low level of vitamin D in Fgf23 transgenic mice [20]. However, other aspects of FGF23 need to be further investigated to have a comprehensive understanding of its biology (Box 1). In addition to the roles of FGF23 in maintaining mineral-ion and vitamin-D homeostasis, recent studies have suggested that FGF23 has also a role in the aging process, possibly by interacting with another known age-associated protein, Klotho [5]. Klotho Klotho, a type-I membrane protein, contains a putative signal sequence at its N-terminus and a single transmembrane domain near its C-terminus [6]. The extracellular domain of klotho consists of two internal repeats that share sequence homology with b-glucosidases (of both bacteria and plants) [6,22,23]. Klotho is a member of the glycosidase superfamily 1 [24]. Cellular expression of klotho is detected predominantly in the kidney (distal convoluted tubules) and brain (choroid plexus) by immunohistochemistry [25]. Knock-in into mice of the lacZ gene Box 1. Unknown facts on FGF23 † How does FGF23 bind with the receptor to exert its bio-activity? † Is there any FGF23-specific receptor in addition to classic FGF receptors? † What are the roles of amino and carboxy fragments of FGF23 in receptor binding? † What are the effector cells to which circulating FGF23 binds to exert its bio-activity? † Is there any autocrine or paracrine effect of FGF23 in addition to its endocrine functions? † Which are the downstream molecules directly induced by FGF23?
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just downstream of the translational initiation codon of Kl showed expression of klotho in the parathyroid gland and sinoatrial cells of the heart [26,27]. Besides these tissues, low expression of klotho was also reported in the pituitary gland, placenta, skeletal muscle, urinary bladder, aorta, pancreas, testis, ovary and colon [6]. In addition to the membrane protein, Kl gene also encodes a splice variant that lacks the transmembrane domain to generate a secreted form of the protein [28,29]. Studies have detected secreted klotho in serum and cerebrospinal fluid [30]. Klotho not only regulates mineral-ion homeostasis [31,32] but is also involved in numerous other important functions that include (but are not limited to): (i) antiapoptotic function [33]; (ii) suppression of oxidative stress [34]; (iii) protection of the kidney from ischemic renal damages [35]; (iv) regulation of vasculogenesis [26]; (v) protection against endothelial dysfunction [36,37]; and (vi) involvement in bone growth, development and maintenance [38–40]. Klotho gene polymorphism has also suggested that Kl gene might be a genetic risk factor for atherosclerotic coronary-artery diseases but not for vasospastic angina in Japanese patients [41]. In a human study, a link between a functional variant of the klotho gene (KL-VS) and high-density lipoprotein cholesterol, blood pressure and longevity has been reported. Individuals with homozygous KL-VS seem to be more likely to develop cardiovascular diseases and have an increased mortality risk that individuals without KL-VS [42]. Short lifespan that is due to accelerated aging is also observed in mice lacking klotho activities [6]. Because klotho exerts protective effects on a wide range of cells and tissues, it is not surprising that lack of its activity might induce features consistent with premature aging in mammals. Mammalian aging Mammalian aging might be defined as an overall decline in the functionality of the vital systems; decreased reproductive capacity and increased mortality are consequences of aging. Therefore, aging results in an increased vulnerability of the tissues and organs that is due to decreased cellular viability. Human and experimental studies suggest that few selective genes and pathways might be coordinately regulating the biological clock of aging. Oxidative stress, DNA damage and mitochondrial dysfunction might affect mammalian aging, possibly by altering the normal cellular and subcellular activities [43,44]. Furthermore, loss of genomic stability has an impact on cellular viability and survival [45,46]. Also, numerous endocrine and humoral factors, including insulin, IGF-1, klotho and FGF-23 might influence mammalian aging and overall survival by regulating metabolic balance or by exerting effects on mineral-ion homeostasis [1,5,6,47]. In Box 2, some of the key factors that are involved in mammalian aging are listed. The relevance of some of these factors in mammalian aging is briefly presented below. (i) Telomeres are composed of a repetitive DNA sequence and associated proteins that enable cells to distinguish chromosome ends from DNA double-strand breaks, and help protecting chromosome from www.sciencedirect.com
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Box 2. Potential factors involved in mammalian aging † Factors that regulate DNA damage, repair and nuclear function. † Factors that regulate genetic maintenance of telomeres. † Factors that regulate oxidative stress and mitochondrial dysfunction. † Factors that regulate the effects of caloric restriction. † Factors that regulate insulin and IGF-1 signaling. † Factors that regulate mineral-ion homeostasis.
degradation and recombination. Telomeres also maintain genomic integrity by preventing end-to-end fusion of chromosomes. Telomeres shorten with each somatic cell division and, therefore, elderly individuals have shorter telomeres than younger adults [43]. Short or dysfunctional telomeres are associated with cellular senescence, a key component of mammalian aging. Mice that lack the telomerase RNA component show selective signs of premature aging, including gray hair, alopecia, skin ulcerations, impaired wound healing, cancer and shortened lifespan. Such features of premature aging were evident between the third and the fourth generation in these mice [44]. (ii) Mitochondrial DNA encodes mitochondrial enzymes that are involved in cellular respiration, and its dysfunction might influence mammalian aging. Ageinduced mutations in mitochondrial DNA accumulate in post-mitotic tissues such as CNS neurons, and cardiac and skeletal muscles, and disrupt normal functionality of the involved tissues, possibly by inducing senescent changes [46]. In senescence, endogenous cellular protection system is attenuated; this makes cells and organs more vulnerable to extrinsic and intrinsic stress. Particularly mitochondria, which are actively involved in maintaining cellular energetic and ionic homeostasis, are susceptible to injury in cells that undergo senescent changes. Proof-reading-deficient version of the mitochondrial DNA polymerase g (POLG) expressed in mice has been shown to induce features of accelerated aging by promoting apoptosis [45] and, thus, providing an in vivo experimental evidence of altered mitochondrial DNA activity on premature aging. (iii) The role of insulin–IGF-1 signaling in aging is one of the most extensively studied pathways. Reduced insulin–IGF-1 activity has been shown to be associated with prolonged lifespan in various species [47]. Mice with heterozygous deletions of the IGF-1 receptor have longer lifespan, although survival is gender specific [48]. Recently, extended lifespan in klotho transgenic mice has been associated with suppressed insulin and IGF-1 activities [49]. Although several important factors that affect mammalian aging have been identified, the way these factors cross-talk and coordinately regulate aging needs further elucidation. To date, it seems that chronic dysregulation of cellular homeostasis that is influenced by numerous intrinsic and extrinsic factors leads to an overall decline of functionality of the organ systems, inducing irreversible age-associated changes. Recent generation of Fgf23- and Kl-deficient mice has provided an additional evidence
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for a close association between hypervitaminosis D and accelerated aging. Klotho mutant mice and premature aging The mutation generated by the insertion of ectopic DNA into the regulatory region of the mouse klotho gene induces multiple phenotypes resembling human aging [6]. Klotho mutants develop arteriosclerosis, osteoporosis, skin atrophy, pulmonary emphysema, infertility and have short lifespan [6]. Even if the expression of klotho is limited to certain organs, Kl-deficient mice manifest systemic-aging features in organs such as the stomach, lung, submandibular gland and skin that do not express klotho, suggesting that klotho has pleiotropic humoral effects. Moreover, endogenous restoration of klotho into Kl-deficient mice that carry klotho transgene has been shown to rescue premature aging-like features [6]. In contrast to Kl-deficient mice, klotho transgenic mice have an extended lifespan, possibly by repressing intracellular signals of insulin and IGF-1 [49]. In a human study, an association between aging and a functional variant of klotho has been reported [50]. These human and experimental studies suggest a potential role of klotho in the regulation of mammalian aging. Fgf23 mutant mice and premature aging Recently, we have shown a novel role of Fgf23 in aging [5]. Fgf23K/K mice develop extensive premature aging-like features that include, but are not limited to, reduced lifespan, infertility, osteoporosis, arteriosclerosis, atrophy of the skin and emphysema. All these systemic premature aging-like features of Fgf23K/K mice are caused by
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inactivation of the Fgf23 gene, which has a restricted expression, mainly in bone cells (osteoblasts and osteocytes). The extensive extra-skeletal involvement of soft tissues and organ systems in Fgf23K/K mice suggests that premature aging-like features are likely to be mediated by pleiotropic humoral effects of Fgf23. Indeed, abnormal mineral-ion homeostasis in Fgf23K/K mice could be reversed by providing exogenous FGF23 to these mice, indicating that the phenotype of these mice is a consequence of the inactivation of its functions [5]. Fgf23K/K mice show high biochemical and morphological similarities with mice that are homozygous for hypomorphic alleles of the klotho gene [5,6]. Both Fgf23and Kl-deficient mice have shortened lifespan, growth retardation, infertility, emphysema, muscle atrophy, hypoglycemia, ectopic calcification and elevated serum levels of phosphate and vitamin D. Vitamin-D receptors (VDRs) are widely distributed in various organs and tissues [51–53], and a continuous local stimulation of VDRs by excessive level of circulating vitamin D in both Fgf23- and Kl-deficient mice might exacerbate premature aging-like phenotypes with or without development of abnormal calcification. The biochemical and morphological phenotypes of both these mice are summarized in Table 1. The strikingly similar phenotypes of Fgf23- and Kldeficient mice imply that the premature aging process is a consequence of disruption of a common humoral signaling pathway. In a recent in vitro study, klotho has been shown to be a cofactor in FGF23 signaling [54]. FGF-23 was also able to bind to multiple FGF receptors in the presence of other cofactors such as heparin or glycosaminoglycan to
Table 1. Similarities in the phenotypes of mice lacking either Fgf23 or Kla Phenotype
Fgf23K/K Mice
Kl-deficient mice
Lifespan Body weight Growth retardation Kyphosis Skin atrophy Body hair Muscle wasting Thymus atrophy Spleen atrophy Intestinal atrophy Ectopic calcifications Hypogonadism Infertility Gait walk Atherosclerosis and/or arteriosclerosis Emphysema Physical activity Serum 1,25(OH)2D3 Serum phosphate Serum calcium Serum PTH Osteopenia Altered skeletal mineralization
Short Reduced Present Present Present Sparse Present Present Present Present Present Present Present Present Present Present Sluggish High High High Low Present Present
Short Reduced Present Present Present Sparse Present Present Present Not known Present Present Present Present Present Present Sluggish High High High Low Present Present
a
Mice that lack both genes encoding Fgf23 and 1a-hydroxylase Prolonged Rescued Rescued Rescued Rescued Normal Rescued Rescued Rescued Rescued Rescued Rescued Rescued Rescued Rescued Rescued Normal Not measured Low Low High Yes (rickets) Reversed
Kl-deficient mice on a vitamin-D-deficient diet Prolonged Rescued Rescued Rescued Rescued Not known Not known Not known Not known Not known Rescued Rescued Rescued Not known Rescued Rescued Normal Low Normal to low Normal to low Not known Partly rescued Reversed
Comparison of phenotype among Fgf23K/K mice [5,13,21], Kl-deficient mice [6,31], mice that lack both genes encoding Fgf23 and 1a-hydroxylase [5,57] and Kl-deficient mice fed a vitamin-D-deficient diet [31,32]. Note that there are remarkable similarities in the phenotypes of Fgf23 and klotho mutant mice. Suppression of vitamin-D activities, either by genomic deletion of 1a-hydroxylase from Fgf23K/K mice, or providing a vitamin-D-deficient diet to Kl-deficient mice rescued most of the premature aging-like phenotypes in both Fgf23 and klotho mutant mice [5,31,57].
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FGF23 Klotho GAG
FGFR
p
p38 MAPK
p
ERK
Genes regulating mineral-ion homeostasis Nucleus Figure 1. FGF23 and receptor interaction. FGF23 can bind with known FGF receptors (FGFRs) with various affinity, and can phosphorylate ERK and p38 MAPK [55] to induce the expression of molecules that regulate mineral-ion homeostasis. Note that FGF23–FGFR binding uses sulfated glycosaminoglycan (GAG) [56] and klotho [54] as co-factors.
activate and generate downstream signaling events [55,56] (Figure 1). Role of vitamin D in premature aging of Fgf23 and klotho mutant mice Both Fgf23- and Kl-deficient mice have altered mineralion homeostasis and increased renal expression of the gene that encodes 1a-hydroxylase, in addition to elevated serum levels of vitamin D [13,20,31]. Manipulation of vitamin-D activities by dietary restriction rescues the aging phenotypes of Kl-deficient mice (Table 1). Lowering vitamin-D levels in mice that lack klotho by providing a vitamin-D-deficient diet has resulted in no ectopic calcification. These mice also regain body weight and fertility and, most importantly, have prolonged lifespan when fed a vitamin-D-deficient diet, suggesting that the premature aging-like features in mice that lack klotho are downstream events resulting from increased activity of vitamin D [31]. Similarly, when vitamin-D activities were genetically suppressed from Fgf23K/K mice by generating mice that lack both genes encoding Fgf23 and 1ahydroxylase, several premature-aging-like features of Fgf23 K/K mice were rescued [5,57]. In contrast to Fgf23K/K mice, mice that lack both genes encoding Fgf23 and 1a-hydroxylase have similar appearance and body weight to wild-type littermates. Suppression of vitamin-D activities from Fgf23K/K mice led to no ectopic calcifications in the kidney, heart and lung. The generalized atrophic changes in skin, intestine and other organs of Fgf23K/K mice were rescued in mice that lack both genes encoding Fgf23 and 1a-hydroxylase, and the overall effect was a dramatically increased lifespan in vitamin-Ddeficient Fgf23K/K mice. It is, therefore, possible that the premature-aging-like features in Fgf23- and Kl-deficient mice are due to hypervitaminosis D and altered mineralion homeostasis (Table 1). www.sciencedirect.com
Fgf23K/K mice have hypervitaminosis D, hyperphosphatemia and premature-aging-like features, whereas Fgf23 transgenic mice have opposite features such as low serum level of vitamin D, hypophosphatemia and no sign of premature-aging-like features, apart from the development of rickets that are due to chronic vitamin-D deficiency [3,20]. Similarly, in contrast to the extensive premature aging and short lifespan of Kl-deficient mice [6], klotho transgenic mice have prolonged lifespan [49]. Because reduction of vitamin-D activity from Kl-deficient mice rescued premature aging-like features (Table 1), it will be interesting to know whether prolonged survival of klotho transgenic mice and altered insulin signaling have any association with reduced vitamin-D activity. Another aspect that deserves further investigation is to determine whether excessive levels of FGF23 might change insulin signaling, as shown in klotho transgenic mice [49]. Also, longevity studies need to be performed in Fgf23 transgenic mice to determine the potential role of FGF23 as a survival factor. Vitamin D and aging FGF23 has an important role in premature aging, and such premature aging is a vitamin-D-mediated process [5]. The normal aging process is associated with vitamin-D deficiency, which leads to senile osteoporosis. However, using Fgf23K/K mice and klotho mutant mice, it seems that excessive levels of vitamin D are associated with accelerated aging, possibly by inducing extensive softtissue calcification. The notion that vitamin-D deficiency is associated with a normal aging process, whereas excessive levels of vitamin D are associated with an accelerated aging process is clinically important because a careful and balanced approach to treat senile osteoporosis with vitamin-D supplements might be needed. Careful re-assessment of both beneficial and adverse effects of
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vitamin-D supplements that are routinely and widely prescribed to elderly individuals is needed. This re-evaluation will be useful in determining potential undesirable effects of vitamin D, and will help generate public awareness about adverse effects of uncontrolled use or consumption of arbitrary amounts of vitaminD supplements. It is important to note that the aging process varies among species; the premature-aging features that are observed in genetically altered mice might not exactly mimic those in humans. However, the understanding of premature aging in Fgf23 and klotho mutant mice will be useful to determine the effects of excessive vitamin-D activity and altered mineral-ion homeostasis in human aging. Such understanding will help envision clinical situations beyond aging; for example, in patients with end-stage renal disease (ESRD) and hyperphosphatemia, long-term use of active vitamin-D metabolites is one of the common treatment options. Such treatment is not always effective and might increase the risk of cardiovascular diseases of these patients because of abnormal calcification. Although it is essential to treat ESRD patients with secondary hyperparathyroidism effectively, a balanced approach to minimize soft-tissue calcification of these patients is required. Abnormal tissue calcification causes w50% of the cardiovascular deaths among patients that undergo dialysis. Uremic tumoral calcinosis is a severe complication that is encountered in ESRD patients [58]. The most commonly affected sites of uremic tumoral calcinosis are the elbow, wrist, shoulder and hip, which restrict joint mobility leading to various neurovascular and musculoskeletal
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symptoms that are due to compression of the adjoining structures. Continuous use of high-Ca2C-level dialysate and prolonged administration of vitamin D are the main underlying cause of tumoral calcinosis in patients with chronic renal failure [59]. Milliner et al. [60] showed a strong association between vitamin-D therapy and softtissue calcifications in pediatric patients. They also showed that the probability of calcinosis was greater in patients receiving calcitriol therapy than those treated without calcitriol. Resolution of tumoral calcinosis in hemodialysis patient was achieved by using low-Ca2Clevel dialysate [61]. Furthermore, withdrawal of vitaminD therapy from patients with tumoral calcinosis could markedly improve the lesion [62], emphasizing the fact that manipulation of vitamin-D–Ca2C axis might greatly ameliorate ectopic calcification. Concluding remarks Recent studies using genetic model systems, ranging from yeast and nematodes to mice, have greatly enhanced the understanding of the aging process; such studies have highlighted that certain genetically conserved pathways might regulate this process. Here, we have presented our view on how understanding the mechanisms of hyperphosphatemia- and hypervitaminosis-D-induced premature aging in Fgf23- and Kl-deficient mice might help determine their roles in human aging. In vivo genetic manipulation studies have suggested a novel link between Fgf23 and klotho during aging. In Figure 2, possible downstream events that might be responsible for inducing premature-aging-like features in Fgf23- and Kl-deficient mice are presented. Fgf23–/– mice Klotho–/– mice
Local activation of VDR
Abnormality in VDRresponsive organs
Renal 1α (OH)ase
Renal NaPi
Serum vitamin D
Serum PO4
Abnormal calcifications
Serum Ca2+
Impaired vasculogenesis
Premature aging-like features TRENDS in Molecular Medicine
Figure 2. Possible downstream events due to loss of either Fgf23 or klotho functions. Loss of Fgf23 or klotho activity affects both mineral-ion homeostasis and vitamin-D homeostasis [13,21,31,32,64] to produce extensive calcifications and soft-tissue abnormalities in mice that lack Fgf23 or Kl [5,6,57]. Furthermore, impaired vasculogenesis seems to accentuate the process of premature aging in Fgf23- and klotho-deficient mice [5,26]. Abbreviations: NaPi, sodium-phosphate cotransporters; 1a(OH)ase, 1a hydroxylase; VDR, vitamin-D receptor. www.sciencedirect.com
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Box 3. Outstanding questions † Can vitamin-D overload accelerate the human aging process? † How FGF23, klotho and vitamin D coordinately regulate aging? † Can vitamin D be used selectively to treat senile osteoporosis without provoking calcification? † Can FGF23 act as an anti-aging factor? † Can FGF23 affect insulin signaling?
The inter-relationships among FGF23, klotho and vitamin D, and how these molecules coordinately regulate aging await further studies. Experiments have indicated that klotho is an anti-aging factor [49]; long-term studies will provide information on whether FGF23 might have similar anti-aging effects. In Box 3, issues that still need to be resolved have been summarized. The physical, behavioral, morphological and biochemical similarities of Fgf23 and klotho mutant mice suggest the existence of a common humoral signaling pathway that might be important in maintaining normal mineral-ion homeostasis. Disruption of such signaling cascade not only affects vitamin-D homeostasis but also alters mineral-ion homeostasis. This results in accelerated aging in Fgf23 and klotho mutant mice, which might be due to hypervitaminosis D. The results obtained from Fgf23- and Kl-deficient mice highlight two important issues that might be of interest to the clinicians dealing with ESRD patients: (i) widespread soft-tissue calcification is intensified by active vitamin-D metabolites in hyperphosphatemic microenvironment; and (ii) such abnormal calcification can be prevented by reducing vitamin-D activities. The challenge will be to find the right treatment, and to determine optimum amount of vitamin-D metabolites or alternative therapies that might be beneficial to the patients, without compromising their cardiovascular functions. Bone fractures that are due to osteoporosis are one of the most common problems of elderly individuals, particularly of women. Although vitamin D is traditionally used for treating senile osteoporosis to reduce the risk of fracture in the elderly population, recent studies have recommended very high daily doses of vitamin D to diminish the risk of fracture [63]. Since vitamin-D overload has the potential to accelerate the aging process, developing a system to treat senile osteoporosis with vitamin D without provoking calcification or other features of aging might be an ideal approach to deal with age-associated skeletal diseases. In summary, based on recent studies, we have briefly presented experimental evidences that suggest that excessive levels of vitamin D has the potential to accelerate the aging process by abnormally increasing serum levels of Ca2C and phosphate. Acknowledgements The institutional supports and technical helps to M.S.R. from the Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, are acknowledged.
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