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Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process Mohammed S. Razzaque,* Despina Sitara,* Takashi Taguchi,† Rene´ St-Arnaud,‡ and Beate Lanske*,1 *Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts, USA; †Department of Pathology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; ‡Genetics Unit, Shriners Hospital, Montreal, Que´bec, Canada To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5432fje; doi: 10.1096/fj.05-5432fje SPECIFIC AIMS We sought to determine the effects of in vivo genetic ablation of fibroblast growth factor 23 (Fgf-23–/–) on premature aging and survival. Upon finding that Fgf-23–/– mice exhibit numerous biochemical and morphological features consistent with premature aging-like phenotypes, along with severe atherosclerosis, widespread soft tissue calcifications, and increased vitamin D activities, we investigated the role of vitamin D in Fgf-23–/– mice by ablating its activation pathway by generating Fgf-23/1␣ hydroxylase double mutant animals (Fgf-23–/–/ 1␣(OH)ase–/–). Fgf-23–/–/1␣(OH)ase–/– compound mutants, not only eliminated atherosclerosis, ectopic calcifications, and other premature aging-like features documented in Fgf-23–/– mice, but also prolonged their survival.
PRINCIPAL FINDINGS 1. Premature aging-like phenotypes of the Fgf-23–/– mouse We recently generated two independent Fgf-23–/– mouse lines by replacing the entire coding region of the Fgf-23 gene with lacZ and the neomycin resistance gene. In contrast to the normal phenotype of heterozygous mice, Fgf-23–/– mice exhibit multiple features resembling human aging. Fgf-23–/– mice develop normally until 2 wk of age, and are macroscopically indistinguishable from their wild-type littermates; however, visible growth retardation is apparent from 3 wk onwards, associated with uncoordinated sluggish movements, followed by premature lethality between 4 –13 wk. In addition to short life span, Fgf-23–/– mice show numerous biochemical and morphological features consistent with premature aging-like features, including kyphosis, hypogonadism, osteopenia, emphysema, amyloid deposition, increased apoptosis, sparse hair, barely detectable subcutaneous fat, severe muscle wasting, uncoordinated movement, T cell dysregulation, 720
and atrophy of the intestinal villi, skin, thymus, and spleen (Fig. 1). Fgf-23–/– mice also exhibit widespread soft tissue calcifications in lung, kidney, skeletal muscle, skin, urinary bladder, testes, heart, and arterial walls, as detected by von-Kossa staining. Soft tissue calcifications appear as early as 6 wk postnatally and progress with age. In general, the distribution of ectopic calcification in Fgf-23–/– mice is compatible with that in natural human aging. 2. Expression of klotho and 1␣(OH)ase in Fgf-23–/– mice Mice homozygous for hypomorphic alleles of the klotho gene exhibit a number of premature aging features, similar to those we described above for Fgf-23–/– mice; these include kyphosis, ectopic calcifications, atherosclerosis, hypogonadism, emphysema, osteopenia, generalized organ atrophy, and short life span. Therefore, we studied the level of klotho expression in Fgf-23–/– mice. Quantitative real-time PCR revealed a significant decrease in the expression of klotho in kidney of Fgf23–/– mice. In contrast to the reduced renal expression of klotho, the renal expression of the 1␣(OH)ase gene was significantly increased in Fgf-23–/– mice, which is also reflected by significantly increased serum levels of 1,25 dihydroxyvitamin D3. To study the role of increased vitamin D activities in Fgf-23–/– mice, we generated a new mouse line, deficient in both 1␣(OH)ase and Fgf-23 (Fgf-23–/–/1␣(OH)ase–/–). 3. Genetic rescue of premature aging-like phenotypes of Fgf-23–/– mice To determine whether some of the premature aginglike features in Fgf-23–/– mice are mediated through the increased vitamin D activities, we examined the phenotype of Fgf-23–/–/1␣(OH)ase–/– double knockout mice; 1
Correspondence: Department of Developmental Biology, Harvard School of Dental Medicine, Research and Education Building, Room 303, 188 Longwood Ave., Boston, MA 02115, USA. E-mail: beate_lanske@hsdm.harvard.edu 0892-6638/06/0020-0720 © FASEB
CONCLUSIONS AND SIGNIFICANCE We present here a novel role of Fgf-23 in aging and longevity. Our results show for the first time that Fgf-23–/– mice develop multiple premature aging-like phenotypes, including reduced life span, infertility, osteopenia, atherosclerosis, atrophy of the skin, and other internal organs; all resembling features observed during human aging. These extensive premature aginglike phenotypes were caused by the disruption of a single gene, Fgf-23, a secreted protein that appears to exert its effects through binding with cell surface receptors; Fgf-23–/– mice thus are very different from described previously models of premature-aging syndromes, which are mostly due to dysregulation of nuclear proteins. Fgf-23–/– mice show increased vitamin D activities, and abnormal mineral ion homeostasis leading to widespread soft tissue calcifications. Aboli-
Figure 1. Macroscopic and microscopic features of various organs at 6 wk of age. H&E stained sections of A) wild-type and B) Fgf-23⫺/⫺ testes, and of C) wild-type and D) Fgf-23⫺/⫺ lungs. The mutant testis shows severe atrophy, while mutant lung exhibits typical emphysematous changes as observed during aging. Toluidine blue staining of an Fgf-23⫺/⫺ kidney (E); arrows depict renal stones found only in the mutants. Congo red staining indicates amyloid deposition found in small and medium sized arteries of the mutant kidney (F ), and in the heart muscle (H). von Kossa staining demonstrates severe calcification of the aortic wall in Fgf-23⫺/⫺ animals (G). Macroscopic image of small intestine of wild-type (I ) and Fgf-23⫺/⫺ animal (J ) (n⫽6), showing ballooning of mutant intestine. Hematoxilin/eosin staining of cross sections of intestine obtained from wild-type (K) and Fgf-23⫺/⫺ (L) littermate; reduced height of intestinal villi, and atrophy of intestinal mucosa are noted in Fgf-23⫺/⫺ mice (L). Immunohistochemical evaluation by ␣-smooth muscle actin antibody staining presents the dramatic reduction in vascularization and thinning of smooth muscle layer in the intestine of mutant animals (N), when compared with wild-type littermates (M). BrdU staining demonstrates the striking diminution of proliferative cells in mutant intestine (P) vs. wild-type mice (O). Increased apoptosis in the mutant skin (R) is visible by positive TUNEL staining mostly located in hair follicles when compared with wild-type littermates (Q). BrdU labeling of the skin shows a dramatic decrease in proliferative cells in Fgf-23⫺/⫺ mice (T) vs. wild-type littermate (S) (⫻10).
these newly generated mice were indistinguishable from wild-type mice, in terms of their appearances and body weight (Fig. 2). The thymus, intestine and genital organs in Fgf-23–/–/1␣(OH)ase–/– mice were of similar size and shape, as those of wild-type littermates. Histological analysis showed that atrophic changes in the skin, and intestine, widely observed in Fgf-23–/– mice, are considerably ameliorated and rescued in Fgf-23–/–/ 1␣(OH)ase–/– mice. Ectopic calcification completely disappeared in double knockout mice. These in vivo genetic studies suggest that some of the premature aging-like phenotypes observed in Fgf-23–/– mice are due to enhanced vitamin D activities. ROLE OF Fgf-23 AND VITAMIN D IN PREMATURE AGING
Figure 2. A) Macroscopic image of a wild-type (WT), Fgf23⫺/⫺, and Fgf-23⫺/⫺/1␣(OH)ase⫺/⫺ double mutant at ⬃6 wk of age. B) Body weight curve and C) survival curve of control, Fgf-23⫺/⫺ and Fgf-23⫺/⫺/1␣(OH)ase⫺/⫺ mice (n⬎15) showing weight gain and prolonged life span in compound mutants. Hematoxilin/eosin staining of intestinal sections of D) wildtype, E) Fgf-23⫺/⫺, and F ) Fgf-23⫺/⫺/1␣(OH)ase⫺/⫺ double knockout animals. Single Fgf-23⫺/⫺ mice exhibit severe atrophy of the intestine, which is significantly improved by the deletion of the 1␣(OH)ase gene. Similar improvement was also noted in the skin section of Fgf-23⫺/⫺/1␣(OH)ase⫺/⫺ double knockout animals, compared with Fgf-23⫺/⫺ mice; G) wildtype; H ) Fgf-23⫺/⫺; and I) Fgf-23⫺/⫺/1␣(OH)ase⫺/⫺ double knockout animals (D–I: ⫻10). 721
tion of vitamin D activities from Fgf-23–/– mice by genetically ablating the 1␣(OH)lase gene rescues a number of systemic aging-like phenotypes of the Fgf23–/– mice, suggesting that "premature aging-like phenotypes” in these genetically ablated mice are partly exerted through abnormal regulation of vitamin D. Fgf-23–/– mice show most of the pathophysiological features found in human premature aging syndromes and some of these premature aging-like phenotypes are visible already at 6 wk of age, which is relatively earlier than other existing models of accelerated aging. This novel function of Fgf-23 in premature aging might help us to determine its role in human progeroid syndromes. The fact that Fgf-23–/– mice show systemic aging-like phenotypes despite its restricted expression mainly in adult bone cells (osteoblasts and osteocytes) would suggest that these aging-like phenotypes are likely to be non-cell-autonomous phenomena, and exerted through the pleiotropic hormonal functions of Fgf-23. Based on the strikingly similar biochemical and morphological phenotypes of Fgf-23–/– and klotho mutant mice, and the reduced expression of klotho in Fgf-23–/– mice, we presume that part of the aging process may be regulated through a common humoral signaling pathway in these two strains of mice. The fact that lowering serum levels of vitamin D by dietary restriction results in alleviation of some of the aging-like phenotypes of klotho mutant mice is in accord with our results; the premature aging-like phenotypes of Fgf-23–/– mice are rescued by genetic ablation of the 1␣(OH)ase gene, suggesting these are downstream events resulting from increased vitamin D activities. Based on our current results and taking into account earlier studies, we propose a new model of interactions among Fgf-23, vitamin D, and klotho (Fig. 3). Both Fgf-23 and klotho are negative regulators of vitamin D metabolism by suppressing the expression of the 1␣(OH)ase gene. It has been shown that 1,25 dihydroxyvitamin D3 can induce klotho, as demonstrated by up-regulation of renal expression of klotho after 1,25 dihydroxyvitamin D3 injection into wild-type mice. However, despite significantly increased levels of 1,25 dihydroxyvitamin D3 , we found decreased renal expression of klotho in Fgf-23–/– mice, suggesting that interactions of vitamin D and klotho are partly mediated through Fgf-23. More important, in Fgf-23–/–/1␣(OH)ase–/– compound mutants, where synthesis of the active form of vitamin D was completely abolished, the expression of klotho was still low, similar to that found in Fgf-23–/– mice, again emphasizing that vitamin D-mediated expression of
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Figure 3. Schematic diagram showing the possible interactions among Fgf-23, vitamin D, and klotho.
klotho is regulated through Fgf-23. When we injected control mice with recombinant FGF-23, we did not find any change of renal expression of klotho compared with the vehicle-treated mice, although these FGF-23-treated mice did show low serum phosphate, compared with vehicle-treated control mice. However, when Fgf-23–/– mice were treated with FGF-23, we found induction of renal expression of klotho. Although the premature aging features observed in Fgf-23–/– mice are unlikely to be due to reduce expression of klotho, to clarify this issue we plan other studies to determine whether pharmacological or genetic restoration of klotho affects the aging phenotypes in Fgf-23–/–. We presume that part of the premature aging process may be regulated through a common humoral signaling pathway. Our proposed model (Fig. 3) should provide a basis for further molecular studies to establish the exact mechanisms by which Fgf-23 interacts with klotho to develop premature aging-like phenotypes. In conclusion, our results provide a novel causative link between Fgf-23 and multiple aging-like phenotypes and are expected to shed new insights into mechanism of the aging process. The results obtained from the genetic ablation of Fgf-23–/– also support the novel concept that a humoral factor(s) can be responsible for regulating the aging process. Finally, Fgf-23–/– mice provide an important tool to study the effects of genetic or pharmacological interventions to delay premature aging-like features. Our knowledge of intrinsic factors influencing aging process will facilitate understanding the role of extrinsic factors that have differential affect on individuals.
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RAZZAQUE ET AL.