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FGF-23–Klotho signaling stimulates proliferation and prevents vitamin D–induced apoptosis
Correspondence to Beate Lanske: beate_lanske{at}hsdm.harvard.edu
Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23–/– or Klotho–/– knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23–Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1
-hydroxylase expression and phosphoinositide-3 kinase–dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho.
B, inhibitor B; PI3K, phosphoinositide-3 kinase; PTEC, proximal tubule epithelial cells. © 2008 Medici et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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The phenotypes observed from both Fgf-23–/– and Klotho–/– mice are consistent with premature aging. These include shortened life span, severe atrophy of most tissues/organs, soft tissue calcifications, infertility, pulmonary emphysema, osteoporosis, and arteriosclerosis (Kuro-o et al., 1997; Razzaque et al., 2005, 2006; Torres et al., 2007). Fgf-23–/– and Klotho–/– mice also have increased serum phosphate levels. FGF-23–Klotho has been found to inhibit phosphate transport in renal proximal tubule epithelial cells (PTEC) by direct regulation of the sodium phosphate cotransporters (Yu and White, 2005).
Both Fgf-23–/– and Klotho–/– mice have unusually high serum levels of 1,25(OH)2D3 (Tsujikawa et al., 2003; Shimada et al., 2004; Sitara et al., 2004). Although vitamin D precursor molecules are steroid derived (Johnson et al., 2002), an enzyme called 1-hydroxylase (CYP27B1) is essential for the final synthesis of active vitamin D (Dardenne et al., 2001). In fact, targeted inactivation of 1-hydroxylase in mouse models (1-hydroxylase–/–) results in undetectable serum levels of 1,25(OH)2D3 (Dardenne et al., 2001). Therefore, it is reasonable to suggest that elevated serum levels of 1,25(OH)2D3 in Fgf-23–/– or Klotho–/– mice might be caused by the ability of these molecules to regulate expression of the 1-hydroxylase gene. The correlation between elevated levels of active vitamin D and the premature aging–like phenotype in Fgf-23–/– and Klotho–/– mice makes it tempting to hypothesize that excessive active vitamin D might have cytotoxic effects on various tissues, resulting in the observed atrophy. Studies in prostate and breast cancer cells have shown that exposure of cells to high levels of active vitamin D can have apoptotic effects (Narvaez et al., 2001; Johnson et al., 2002, 2006). Excessive activation of the vitamin D receptor causes transcription of genes associated with mitochondrial export of cytochrome c and subsequent cleavage of caspase-9. This action induces cleavage of caspase-3, which promotes DNA fragmentation causing apoptosis (Demay, 2006).
In this study, we show that FGF-23–Klotho stimulates mitogenic and cell survival pathways to prevent atrophy of tissues caused by excessive production of active vitamin D. High levels of 1,25(OH)2D3 are suppressed by FGF-23–Klotho signaling through inhibited expression of renal 1-hydroxylase, an enzyme responsible for synthesis of active vitamin D. Furthermore, we show that vitamin D–induced caspase activity is inhibited by signaling pathways initiated by FGF-23–Klotho, thus directly preventing apoptosis.
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B (IB), and GSK-3β (glycogen synthase kinase-3β) compared with control (vehicle treated) cells. However, exposure to both FGF-23 and Klotho caused significant up-regulations in phosphorylation of all of these proteins (Fig. 1).
These data were confirmed by multiplex ELISA, which also showed increases in CREB (cAMP response element binding), p70S6K, and STAT3 (signal transducer and activator of transcription 3) phosphorylation by FGF-23–Klotho. Addition of a small molecule Ras inhibitor before FGF-23 and Klotho treatment prevented increased phosphorylation of CREB, ERK1/2, JNK, p38, p70S6K, and STAT3. Addition of a small molecule inhibitor against phosphoinositide-3 kinase (PI3K) prevented increased phosphorylation of IB, p70S6K, AKT, and GSK-3β. Combined effects of both Ras and PI3K inhibitors lowered all FGF-23–Klotho-induced phosphorylations to background levels (Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200803024/DC1).
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FGF-23–Klotho signaling stimulates proliferation
Because the observed phosphorylation patterns are known to be associated with proliferation signaling pathways (Bennasroune et al., 2004), we performed ELISA to detect potential effects of FGF-23–Klotho on expression of the cell cycle proteins Cyclin D1 and c-myc. Consistent with the phosphorylation patterning, we found that levels of Cyclin D1 and c-myc were significantly increased with the addition of FGF-23 and Klotho to cell cultures compared with control cells. As expected, FGF-23 or Klotho alone had no significant effect on Cyclin D1 or c-myc expression. Ras inhibitor was sufficient to abolish most of the increased expression of these proteins under the influence of FGF-23–Klotho, whereas PI3K inhibitor had only a minimal effect (Fig. 2, A and B).
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Fgf-23–/– and FGF-23 transgenic mice were generated as previously described (Larsson et al., 2004; Sitara et al., 2004). Kidneys and intestines from these mice at 6 wk of age were sectioned and stained for Ki67 expression to measure proliferation in vivo. Tissues from wild-type mice at 6 wk of age were used as a comparative control. In the intestine, we found that the number of Ki67-positive nuclei was significantly reduced in Fgf-23–/– mice, whereas in FGF-23 transgenic mice it was greatly increased compared with the wild type (Fig. 2, D and E). Similar results were observed in skin and spleen tissues (unpublished data). Interestingly, in the kidney, there were dramatic changes in levels of cytoplasmic Ki67 but no positive staining for nuclear Ki67. We also attempted BrdU incorporation, but there was no positive staining in the nuclei from kidney sections (unpublished data). These data suggest that the role of FGF-23–Klotho is not to control proliferation in the kidney in vivo but rather to regulate mineral-ion homeostasis as previously described (Yu and White, 2005).
FGF-23–Klotho signaling prevents vitamin D–induced apoptosis
Because active vitamin D has recently been described to have anti-cancer effects (Johnson et al., 2002, 2006) and serum levels of 1,25(OH)2D3 are excessively high in Fgf-23–/– or Klotho–/– mice (Tsujikawa et al., 2003; Shimada et al., 2004; Sitara et al., 2004), we hypothesized that these elevated levels might promote apoptosis, thus causing the observed atrophy of tissues. We first showed the direct effect of FGF-23–Klotho on expression of 1-hydroxylase, the enzyme responsible for final synthesis of active vitamin D metabolite. Protein levels of 1-hydroxylase were observed by ELISA, showing that FGF-23 and Klotho together were able to greatly reduce expression of this enzyme in PTEC cells compared with the control, whereas FGF-23 or Klotho alone had little effect. Addition of Ras and PI3K inhibitors showed significant rescue of 1-hydroxylase expression, however full restoration was not achieved. These data suggest that other signaling pathways may also be involved in regulation of 1-hydroxylase expression. Interestingly, no statistically significant changes in expression of 1-hydroxylase were found with treatment of FHs74Int cells (Fig. 3 A).
This is consistent with the fact that PTEC of the kidney are the primary site of 1-hydroxylase activity for synthesis of active vitamin D (Dardenne et al., 2001) not small intestinal epithelium.
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To directly observe whether kidneys of Fgf-23–/– mice express higher levels of 1-hydroxylase, thus resulting in the previously described high levels of serum vitamin D, we performed real-time quantitative PCR using RNA isolated from these tissues compared with those of wild-type mice. As expected, we found levels of 1-hydroxylase to be far higher in kidney tissue of knockout mice compared with that of wild-type mice. We also found that there was no significant change in 1-hydroxylase mRNA in intestinal tissue (Fig. 4 A).
Histology of kidney and intestinal tissues from Fgf-23–/– mice show high levels of apoptosis compared with those of wild-type mice, as observed by TUNEL staining. Most significantly, tissues from Fgf-23–/–/1-hydroxylase–/– mice show a complete rescue of the apoptosis seen in Fgf-23–/– mice (Fig. 4, B and C). Similar results were found in other organs including skin, spleen, liver, lung, ovaries, and testes (unpublished data). These data suggest that systemically elevated active vitamin D is the cause of tissue atrophy observed in Fgf-23–/– mice.
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Perhaps the most interesting aspect of our study is that although virtually all internal organs showed evidence of vitamin D–induced apoptosis in Fgf-23–/– mice, only select tissues (intestine, spleen, and skin) showed effects of FGF-23–Klotho on proliferation. Although kidneys of Fgf-23–/– mice showed no evident change in proliferation, in vitro studies of kidney PTEC demonstrated greatly increased proliferation as a result of exposure to exogenous FGF-23 and Klotho. These data suggest that there are distinct regulatory factors controlling in vivo proliferation for each organ that may render FGF-23–Klotho ineffective for this purpose. However, it is clear that FGF-23–Klotho does inhibit expression of 1-hydroxylase in kidney cells (in vitro and in vivo) and subsequent production of active vitamin D. Although the concentrations of active vitamin D used for our in vitro studies may not be the same as those reached in vivo in Fgf-23–/– mice, evidence of apoptotic rescue by genetic knockout of 1-hydroxylase suggests that active vitamin D is the cause of the observed tissue atrophy.
The potential use of FGF-23–Klotho as an antiaging therapy is tempting; however, there is great risk. Although excessive serum levels of active vitamin D are clearly responsible for several aging-like phenotypes, including tissue atrophy, moderate production of active vitamin D is still essential for normal bone mineralization (Christakos et al., 2006). Also, lack of systemic vitamin D has been linked to increased risk in development of various forms of cancer including colorectal, breast, lung, ovarian and prostate carcinomas, as well as non-Hodgkin's lymphoma. Furthermore, there is strong evidence that vitamin D deficiency increases the risk of developing other pathological conditions such as multiple sclerosis, type 1 and type 2 diabetes mellitus, rheumatoid arthritis, osteoarthritis, hypertension, and stroke (Grant, 2006; Peterlik and Cross, 2006). Pharmacological doses of vitamin D analogues have been used as potential therapies against these conditions (Hayes, 2000; Holick, 2006; Deeb et al., 2007). The ability of FGF-23–Klotho signaling to inhibit synthesis of active vitamin D through inhibitory control of 1-hydroxylase gene expression, as well as to induce signaling pathways that counter-regulate vitamin D–induced apoptotic signaling, could potentially slow the aging process. However, it would also likely increase the risk of developing diseases associated with vitamin D deficiency. Therefore, it is evident that a balance of moderate levels of systemic active vitamin D is essential for maintaining overall health and longevity.
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Immunoblotting
Western blotting was performed using the following antibodies at concentrations (and using protocols) recommended by the respective manufacturers: P-ERK1/2, ERK1/2, P-p38, p38, P-JNK, JNK, P-AKT, AKT, P-GSK-3β, GSK-3β, and IB (Cell Signaling Technology); P-IB (Santa Cruz Biotechnology, Inc.); Klotho (provided by M. Kuro-o); and -tubulin (EMD). HRP-conjugated secondary antibodies (EMD) were used at a dilution of 1:5,000. Adjustments of total image size, brightness, and contrast were made using Photoshop CS (Adobe).
Multiplex ELISA
Signal transduction was assessed using the Beadlyte 8-plex Multi-Pathway Signaling kit (Millipore). Active caspase-3 beadmates (Upstate Biotechnology) were used to assess apoptosis. P-AKT, P-GSK-3β (Cell Signaling Technology), Cyclin D1, c-myc, and CYP27B1 (Santa Cruz Biotechnology, Inc.) antibodies were conjugated to various Bio-Plex carboxylated beads (with unique optical codes) using the Bio-Plex Amine Coupling kit (Bio-Rad Laboratories). -Tubulin antibody (EMD) was also conjugated to Bio-Plex carboxylated beads to be used as an internal control. Samples were run on a Luminex 200 multiplex testing system using the Universal Cell Signaling Assay kit and corresponding protocol (Millipore). Experimental values were divided by the -tubulin control values to provide normalized data.
Flow cytometry
PTEC and FHs74Int cells were stained in suspension for BrdU or TUNEL incorporation with the 5-Bromo-2'-deoxy-uridine Labeling and Detection kit I and In Situ Cell Death Detection kit, Fluorescein (Roche), respectively, using the protocols provided by the manufacturer (Roche). Flow cytometry was performed at the Harvard Medical School Department of Pathology flow cytometry core facility using a FACSDCalibur (BD Biosciences) cell sorter isolating 30,000 cells per sample.
Real-time quantitative PCR
Quantitative RT-PCR was performed to quantify the relative expression of 1-hydroxylase mRNA in the kidney and intestine of 6-wk-old wild-type or Fgf-23–/– mice. Tissues were snap frozen in liquid nitrogen and total RNA was isolated by phenol chloroform extraction using Trizol Reagent. Subsequently, 1 µg of RNA was reverse transcribed using the QuantiTectR Reverse Transcription kit (Qiagen). RT-PCR was performed using a real-time PCR system (ABI 7300; Applied Biosystems) with the following conditions: initial denaturing at 95°C for 2 min, followed by 45 three-step cycles of denaturing at 95°C for 10 s, annealing at 55°C for 20 s, and extension at 68°C for 1 min. The following primers were used: 1a-hydroxylase forward, 5'-CAGATGTTTGCCTTTGCCC-3' and reverse, 5'-TGGTTCCTCATCGCAGCTTC-3'; and m36B4 forward, 5'-AGATGCAGCAGATCCGCAT-3' and reverse, 5'-GTTCTTGCCCATCAGCACC-3'. All samples were run in triplicate using SYBR green (Eppendorf) and compared with levels of endogenous m36B4 as an internal control.
Mice
Fgf-23–/–, 1-hydroxylase–/–, and FGF-23 transgenic mice were generated as previously described (Dardenne et al., 2001; Larsson et al., 2004; Sitara et al., 2004). Fgf-23–/–/1-hydroxylase–/– mice were created by crossbreeding FGF-23–/– with 1-hydroxylase–/– mice (Razzaque et al., 2006). Standard PCR genotyping was performed using the following primers: Fgf-23, forward, 5'-GGATCCCCACCTCAGTTCTCA-3' and reverse, 5'-TAGCCGTGTACAGGTGGGTCA-3'; 1-hydroxylase forward, 5'-GCACCTGGCTCAGGTAGCTCTTC-3' and reverse, 5'-GTCCCAGACAGAGACATCCGT-3'; and FGF-23 Transgenic forward, 5'-GGCAACATTTTTGGATCA-3' and reverse, 5'-CCGGGGCTTCAGCACGTT-3'.
Immunohistochemistry
Immunohistochemistry was performed using Ki67 antibody (Dako) according to the manufacturer's guidelines with a 1:100 dilution. Nova Red (Vector Laboratories) POD substrate was used for Ki67 staining. TUNEL staining was conducted using the In Situ Cell Death Detection kit, Fluorescein and protocol. All sections (provided by T. Taguchi, Nagasaki University, Nagasaki, Japan) for Ki67 were counterstained with hematoxylin, whereas sections for TUNEL were counterstained with DAPI in fluorescent mounting medium (Vector Laboratories). Data were quantified by counting the number of Ki67- or TUNEL-positive nuclei per mm2. Images were acquired using a fluorescence microscope (80i; Nikon) at 25°C with 10 or 20x magnifications. Fluorescein filters were used to detect TUNEL. Images were captured using a charge-coupled device camera (Orca 100; Hamamatsu Photonics) and MetaMorph software (MDS Analytical Technologies). Adjustments of total image size, brightness, and contrast were made using Photoshop CS.
Statistics
One-way analysis of variance was performed and confirmed with two-tailed paired Student's t test using Prism 4 software (GraphPad Software, Inc.). P-values <0.05 were considered significant.
Online supplemental material
Fig. S1 shows phosphorylation targets of FGF-23–Klotho signaling by ELISA and control immunoblotting for Klotho expression. Fig. S2 shows dose-dependent effects of active vitamin D on apoptosis (TUNEL) of PTEC or FHs74Int cells by flow cytometry. Fig. S3 is a schematic diagram of FGF-23–Klotho signal transduction. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200803024/DC1.
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Acknowledgments |
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This work was supported by grants R01-073944 (to B. Lanske), R01-077276 (to M.S. Razzaque), and P01-AR048564 (to B.R. Olsen) from the National Institutes of Health.
Submitted: 5 March 2008
Accepted: 7 July 2008
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