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Augmented Wnt Signaling in a Mammalian Model of Accelerated Aging
Hongjun Liu,1
Maria M Fergusson,1*
Rogerio M. Castilho,2*
Jie Liu,1
Liu Cao,1
Jichun Chen,3
Daniela Malide,4
Ilsa I. Rovira,1
Daniel Schimel,5
Calvin J. Kuo,6
J. Silvio Gutkind,2
Paul M. Hwang,1
Toren Finkel1
Abstract:
The contribution of stem and progenitor cell dysfunction anddepletion in normal aging remains incompletely understood. Weexplored this concept in the Klotho mouse model of acceleratedaging. Analysis of various tissues and organs from young Klothomice revealed a decrease in stem cell number and an increasein progenitor cell senescence. Because klotho is a secretedprotein, we postulated that klotho might interact with othersoluble mediators of stem cells. We found that klotho boundto various Wnt family members. In a cell culture model, theWnt-klotho interaction resulted in the suppression of Wnt biologicalactivity. Tissues and organs from klotho-deficient animals showedevidence of increased Wnt signaling, and ectopic expressionof klotho antagonized the activity of endogenous and exogenousWnt. Both in vitro and in vivo, continuous Wnt exposure triggeredaccelerated cellular senescence. Thus, klotho appears to bea secreted Wnt antagonist and Wnt proteins have an unexpectedrole in mammalian aging.
1 Cardiology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA. 2 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892, USA. 3 Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA. 4 Light Microscopy Core Facility, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA. 5 Mouse Imaging Facility, NIH, Bethesda, MD 20892, USA. 6 Division of Hematology, Stanford University School of Medicine, Stanford, CA 94305, USA.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: finkelt{at}nih.gov
Resident and circulating stem and progenitor cells are criticalfor ongoing tissue maintenance and repair, and it is often postulatedthat stem and progenitor cell depletion or dysfunction mightcontribute to aging (1). We therefore examined stem cell dynamicsin a genetic model of accelerated aging. Mice lacking klothoexpression, henceforth termed Klotho mice, have a shortenedlife span and exhibit a number of early-onset age-related changes,including arteriosclerosis, decreased fertility, and skin atrophy(2). Klotho is a transmembrane protein with a large extracellulardomain composed of two repeats (KL1 and KL2 domains) that sharesimilarity to Family I glycosidases. In addition to being cell-associated,the extracellular portion of klotho is secreted and can be detectedin the circulation of animals and humans (3). It is generallybelieved that secreted klotho is the form most likely mediatingthe protein's longevity effects.
One alteration in Klotho animals is the early appearance ofage-related changes in the skin. To assess whether these phenotypicchanges were accompanied by alterations in stem cell number,we identified the number of long-term 5-bromo-2'-deoxyuridine(BrdU)–retaining cells in the skin of either wild-typeor age-matched Klotho animals (4). These label-retaining cells(LRCs) are a convenient method to identify stem cells withintheir niche (5). At an age of 2.5 months, Klotho mice had significantlyfewer LRCs than their wild-type littermates [wild type: 80 ±5 LRCs (±SD) per set of three follicles versus Klotho:35 ± 3 LRCs, n = 30, P <0.05 paired t test]. SkinLRCs are confined to a specialized region of the follicle knownas the bulge region, and the stem cells contained within thisniche are enriched for CD34 expression (6). The bulge regionin Klotho animals was consistently smaller with reduced CD34expression (Fig. 1A). Hair follicle epidermal stem cells arealso a source of transient amplifying (TA) cells induced byacute wounding (7). Consistent with a defect in the number ofLRCs, epidermal wounding resulted in a diminished number ofTA cells in the Klotho animals (Fig. 1B) and a deficit in woundclosure (Fig. 1C).
Fig. 1.. Altered stem and progenitor cells in Klotho mice. (A) LRCs within the skin follicles of 80-day-old wild-type (WT) or Klotho (Klo) mice (top). Higher magnification of representative bulge regions stained for CD34 (bottom). (B) TA cells, identified by positive brown nuclear BrdU staining, after skin wounding. (C) Assessment of skin closure 4 days after creating a 1-cm wound (n = 4 pairs, *P < 0.05 paired t test). (D) Evidence for senescence within the hair follicle of Klotho animals as assessed by ß-gal staining (SAß-gal) and nuclear foci of -H2AX and 53BP1 (red) in 4',6'-diamidino-2-phenylindole (DAPI)–stained nuclei (blue). Percentage of positive nuclear staining in either wild-type or Klotho follicles is shown ± SD. (E) SAß-gal staining of small intestine. (F) Determination of the absolute number of c-kit+sca-1+ Lin– HSCs in bilateral femur and tibias of either wild-type or Klotho animals (n = 3 pairs, *P < 0.05 paired t test). (G) Representative cell cycle analysis of HSCs from wild-type and Klotho animals demonstrating a decrease in HSC quiescence (% G0) and increased proliferation (% G1). DNA content is displayed along the x axis; RNA content determined by Pyronin Y (PY) staining is displayed along the y axis.
[View Larger Version of this Image (57K GIF file)]
Age-matched wild-type and Klotho skin sections also exhibiteddifferences in senescence-associated endogenous ß-galactosidase(SAß-gal) activity (Fig. 1D). The observed SAß-galstaining occurred in the outermost epidermal layer includingthe acellular stratum corneum. The specificity and physiologicalsignificance of this staining is unclear. Examination of numerousrandom follicles revealed that the Klotho animals also had intenseß-galactosidase staining within the follicles, especiallywithin regions known to contain rapidly dividing progenitorcells (8). We observed little to no SAß-gal stainingin the intrafollicular regions. Senescent cells often activatethe DNA damage response (DDR) pathway, as evidenced by the developmentof nuclear foci of proteins such as phosphorylated histone (H2AX),ataxia telangiectasia mutated (ATM), and binding protein 1 (53BP1)(9). The DDR pathway was activated in multiple random Klothofollicles but not in age-matched wild-type mice (Fig. 1D).
Klotho animals also demonstrated increased SAß-galstaining in the small intestine, especially within intestinalcrypts, an area enriched for stem and progenitor cells (Fig. 1E).Similar analysis of the testis in male animals also demonstratedevidence of increased progenitor cell senescence (fig. S1).In the bone marrow of Klotho mice, there was also a reductionin the population of cells bearing the cell surface phenotypeof c-kit+ sca-1+ lineage negative that encompasses the hematopoieticstem cell (HSC) (Fig. 1F). This reduction of HSC in Klotho animalswas accompanied by a marked increase in the percentage of stemcells that were actively dividing (Klotho HSCs: 28.4 ±3.7% in G1 versus wild-type HSCs: 10.2 ± 1.1% in G1,n = 3 animal pairs per group, P <0.05 paired t test) (Fig. 1G).
Given that stem cell biology is regulated by a number of secretedfactors, we wondered whether there might be a functional interactionbetween klotho and one of these known stem cell regulators.In the course of our experiments, we noted that the subcellulardistribution of klotho and Wnt proteins within transfected cellsoverlapped (fig. S2). Thus, we sought to determine whether klothoand Wnt3 could form a direct molecular complex. Epitope-taggedWnt3 and myc-tagged klotho were readily detectable in transfectedcell lysates (Fig. 2A). Klotho associated with immunoprecipitatedWnt3, and the reciprocal immunoprecipitation of klotho containedWnt3 (Fig. 2A). A single extracellular KL domain was sufficientto mediate the observed interaction with Wnt3 (Fig. 2B). TheWnt binding domain was contained within the amino-terminal portionof klotho's KL1 domain (amino acids 1 to 285) (fig. S3). Full-lengthklotho also immunoprecipitated with a number of other Wnt isoformsincluding Wnt1, Wnt4, and Wnt5a (fig. S4).
Fig. 2.. Interaction of klotho with Wnt and inhibition of Wnt signaling. (A) HEK-293 cells were transiently transfected with myc- and hemagglutinin (HA)–tagged expression constructs encoding Wnt3 and murine klotho (Klo). Reciprocal coimmunoprecipitation (IP) of klotho and Wnt3 from cell lysates is also demonstrated. WB, Western blot. (B) Schematic diagram of klotho demonstrating the two extracellular klotho repeats (KL1 and KL2) followed by the single-pass transmembrane domain (–). Flag-tagged full-length klotho (Klo) and truncation mutants were assessed for Wnt binding. (C) HEK-293 cells were transfected with a Wnt3 expression construct (100 ng DNA) and the indicated amount of a full-length klotho expression vector along with either an active (TOPFlash) or inactive (FOPFlash) Wnt luciferase reporter. (D) Conditioned medium mixes were incubated for 24 hours with HEK-293 cells previously transfected with either the active or inactive Wnt luciferase reporter. All Wnt activity measurements represent the ratio of TOP/FOP activity obtained from a single experiment performed in triplicate and are representative of at least three similar experiments.
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We further analyzed the biological effects of the observed klotho/Wntinteraction. We coexpressed Wnt3 and klotho in human embryonickidney (HEK) 293 cells along with a Wnt-responsive reporter.The presence of Wnt3 increased reporter activity by approximately7 times, whereas the addition of increasing amounts of klothoreduced Wnt activity in a dose-dependent fashion (Fig. 2C).Various structural mutants containing a single KL1 domain oraltering conserved amino acids believed to be important in klotho'sß-glucuronidase activity failed to abrogate klotho-mediatedWnt inhibition (fig. S5). In contrast, klotho constructs thatfailed to physically interact with Wnt3 also failed to inhibitWnt activity (fig. S6).
We next asked whether klotho could inhibit Wnt activity in acell-free system. Conditioned media with and without eitherklotho or Wnt3a were prepared and mixed in various combinationsbefore being placed on HEK-293 cells transfected with a Wntreporter construct. These results indicated that secreted klothocould inhibit soluble Wnt activity (Fig. 2D). In contrast, secretedklotho-conditioned medium was ineffective in inhibiting Wntsignaling directly stimulated by intracellular ß-cateninexpression (fig. S7).
We next tested whether animals lacking klotho expression hadincreased Wnt activity. We crossed Klotho animals with the TOPGALreporter strain in which the activity of a ß-galactosidasereporter is under the control of Wnt-responsive elements (10).Analysis of the skin of Klotho/TOPGAL animals demonstrated anincrease in ß-galactosidase reporter activity comparedwith that of age-matched wild-type/TOPGAL controls (Fig. 3A).Quantitative analysis confirmed this augmented Wnt activityin the skin and small intestine of Klotho mice and demonstratedthat the increased expression of the Wnt-dependent ß-galactosidasereporter was similar to that of Axin2, a known Wnt target gene(fig. S8). Other potential transcriptional targets of the Wntpathway also showed increased transcription in Klotho animals(fig. S9).
Fig. 3.. Inhibition of Wnt signaling by klotho in vivo. (A) Endogenous Wnt activity in skin from 14-day-old wild-type or Klotho mice crossed with the TOPGAL reporter strain. (B) Wnt activity in tibias of 14-day-old Klotho/TOPGAL or wild-type/TOPGAL mice. (C) Longitudinal microcomputerized CT sections from the tibia of 3-month-old Klotho (top) or wild-type (bottom) animals. (D) Horizontal three-dimensional reconstruction of cortical bone (yellow) and trabecular bone (green). (E) Calculation of the trabecular to total bone volume from the tibia of three pairs of 3-month-old wild-type or Klotho animals. (F) Quantification of the percentage of ß-galactosidase positive follicles from 12-day-old TOPGAL mice injected 4 days earlier with either a control virus (Ad.FC), an adenovirus encoding the Wnt inhibitor (Ad.DKK1), or an adenovirus encoding klotho (Ad.Klo). Approximately 500 random follicles were assessed per condition. (G) Hyperplastic skin phenotype of K5rtTA/tet-Wnt1 transgenic mice treated with doxycycline is blocked by prior injection of an adenovirus encoding for either DKK-1 or klotho. Mice were killed on postnatal day 21.
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Whereas normal aging is associated with bone loss, studies inmice and humans have established that augmented Wnt signalingleads to increased bone mass (11). We therefore monitored Wntactivity in bones of aged-matched wild-type or Klotho littermates.Using the TOPGAL reporter, we detected augmented reporter activityin sections obtained from the proximal tibia of 2-week-old Klothomice (Fig. 3B and fig. S10). At this time point, Klotho micelack any discernible phenotype, suggesting that the increasein Wnt activity precedes the apparent onset of accelerated aging.We also analyzed by microcomputerized tomography (µCT)the tibias of 3-month-old wild-type or Klotho animals. Klothomice demonstrated nearly 5 times as much tibial trabecular bonemass (Fig. 3, C to E). An increase in overall tibial and vertebralcolumn trabecular bone density has actually been previouslynoted but unexplained in Klotho mice (12). In contrast, otherbones in the Klotho animals exhibit decreased bone density (2).The basis for this regional difference in bone density is unclearbut may relate to the complex interplay of klotho's abilityboth to alter Wnt signaling and to regulate other pathways involvedin calcium and vitamin D homeostasis (13–16).
To test whether augmented klotho expression could inhibit Wntsignaling in vivo, we analyzed TOPGAL reporter mice in the firstfew weeks of life, when the kinetics of the hair follicle cycleare synchronized (17). On postnatal day 8, mice were injectedsubcutaneously with one of three different recombinant adenovirusesencoding either klotho (Ad.Klo), the Wnt inhibitor DKK-1 (Ad.DKK1),or an adenovirus encoding the immunoglobulin G Fc fragment (Ad.FC)that served as a control. Four days after injection (postnatalday 12), we harvested the injected area and assessed Wnt activityin the hair follicle. In control-injected skin, Wnt activitywas clearly visible around numerous growing hair follicles,whereas this activity was reduced in both the DKK-1 and klothoinjected skin (fig. S11). Assessment of more than 500 randomfollicles in each condition revealed that klotho was roughlyequivalent to DKK-1 in the ability to quantitatively suppressWnt's biological activity (Fig. 3F).
To assess whether the expression of klotho could also blockthe effects of pathological Wnt expression, we crossed two strainsof mice, one that expressed the tetracycline-inducible transcriptionalactivator (rtTA) under the control of the cytokeratin 5 promoter(K5rtTA) and another that expressed Wnt1 under the control ofmultiple tet-responsive elements (tet-Wnt1) (18, 19). The resultantcross generated the experimental line K5rtTA/tet-Wnt1, whichprovided tetracycline-inducible expression of Wnt1 in the basallayer of stratified epithelium. In our transgenic animals, ectopicexpression of Wnt1 from postnatal day 12 to 21 resulted in increasedepidermal thickness and marked follicular hyperplasia (Fig. 3G).The local injection of either DKK-1 or klotho, but not the controladenovirus, blocked these pathological Wnt-induced skin changes(Fig. 3G).
To begin to directly assess the role of Wnt proteins in aging,we tested whether continuous Wnt exposure induced cellular senescence.We grew primary mouse embryonic fibroblasts (MEFs) in the presenceor absence of Wnt3a-conditioned medium. Consistent with Wntproteins having mitogenic effects, analysis of BrdU incorporationrevealed that Wnt-conditioned medium initially acted to increaseMEF cell proliferation (Fig. 4A). However, over time, continuousWnt exposure resulted in a marked decrease in proliferation(Fig. 4A and fig. S12). Wnt exposure did not increase the levelof apoptosis (fig. S13); rather, assessment of cells grown inthe continuous presence of Wnt3a demonstrated a flattened morphologywith evidence of increased SAß-gal activity (Fig. 4Band fig. S14). Similarly, continuous Wnt3a exposure triggeredthe DDR pathway as well as nuclear foci of HP1-, a marker ofsenescence-associated heterochromatin formation (Fig. 4C). Theinhibitory effects of long-term Wnt3a-conditioned media on MEFproliferation (fig. S15) and BrdU incorporation (Fig. 4D) wereattenuated by the addition of soluble klotho. Similar resultswere obtained with purified Wnt3a protein rather than Wnt3a-conditionedmedium (Fig. 4E and fig. S16) and when using human rather thanmouse cells (fig. S17).
Fig. 4.. Senescence induced by increased Wnt activity. (A) BrdU incorporation for MEFs grown in the presence or absence of Wnt3a conditioned media. Cells were assayed from passage 1 (P1) through passage 4 (P4). (B) Senescence associated ß-galactosidase staining (SAß-gal) for MEFs (P4) grown in standard media (–) or mixed with the indicated L-cell conditioned medium. (C) MEFs (P4) with or without continuous Wnt3a exposure were assessed for senescence-associated heterochromatin (HP1-) and the activation of the DNA damage response. (D) Level of BrdU incorporation (P4) for MEF cells continuously treated with L-Wnt3a conditioned media (CM) mixed with either vector-transfected or klotho-transfected conditioned medium. (E) Growth of MEFs instandard medium (diamonds), or supplemented with Wnt3a at 10 ng/ml (squares) or 30 ng/ml (triangles). (Inset) P7 cells were stained for activation of H2AX. (F) Skin sections obtained from K5rtTA/tet-Wnt1 transgenic mice either treated or untreated with doxycycline (Dox) from postnatal days 2 through 21. The senescence-associated marker -H2AX is induced in the setting of continuous in vivo Wnt1 (+Dox) exposure.
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Finally, we tested whether Wnt could induce senescence in vivoby analyzing the skin of K5rtTA/tet-Wnt1 transgenic animals.Beginning on postnatal day 2, littermate transgenic animalswere placed on a diet with or without doxycycline (Dox) andskin samples were collected for study on day 21. Analysis ofmultiple random follicles (n >100) demonstrated an increasein senescence markers in those animals exposed to continuousWnt1 expression. On day 21, 70% of the follicles in Wnt1-expressinganimals exhibited staining for -H2AX, whereas less than 5% ofuninduced follicles demonstrated this phenotype (Fig. 4F). Similarly,whereas follicles from young animals without Dox treatment showedlittle to no evidence of senescence, their Wnt1-induced littermatesdemonstrated multiple areas of discrete SAß-gal staining(fig. S18).
We demonstrate that klotho acts as a Wnt antagonist and thatchronic Wnt stimulation may contribute to stem cell depletionand aging. During the course of our studies, two other studiesappeared demonstrating that forced constitutive Wnt signalingwithin HSC led to a rapid exhaustion of long-term repopulatingstem cells (20, 21). These results, as well as the observationsin Brack et al. (22), are broadly consistent with our observationsof Klotho mice. Previous attempts to understand the basis ofthe observed Klotho aging phenotype have implicated alterationsin insulin signaling as well as, more recently, the fibroblastgrowth factor 23 pathway (14–16, 23). Further analysisis therefore required to fully understand how these variousklotho-regulated pathways potentially intersect and whetherany biological hierarchy exists. Nonetheless, our results providean unexpected connection between aging and the well-studiedWnt signaling pathway and suggest that strategies targetingsoluble mediators of stem cell function may provide new therapeuticstrategies to combat aging and potentially age-related diseases.
We thank R. Moon for the Wnt (TOP/FOP) reporter constructs, M. Kuro-o for generating the klotho mice, and H. Dietz for providing the klotho mice. The TOPGAL mouse was generated by E. Fuchs and kindly provided by Y. Yang. We also thank J. G. Kang for technical help. This work was supported by Intramural NIH funds (T.F.), a grant from the Ellison Medical Foundation (T.F.), and an NIH 1 R01 DK069989-01 to C.J.K.
Received for publication 9 April 2007. Accepted for publication 9 July 2007.
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To whom correspondence should be addressed. E-mail: 
-H2AX and 53BP1 (red) in 4',6'-diamidino-2-phenylindole (DAPI)–stained nuclei (blue). Percentage of positive nuclear staining in either wild-type or Klotho follicles is shown ± SD. (E) SAß-gal staining of small intestine. (F) Determination of the absolute number of c-kit+sca-1+ Lin– HSCs in bilateral femur and tibias of either wild-type or Klotho animals (n = 3 pairs, *P < 0.05 paired t test). (G) Representative cell cycle analysis of HSCs from wild-type and Klotho animals demonstrating a decrease in HSC quiescence (% G0) and increased proliferation (% G1). DNA content is displayed along the x axis; RNA content determined by Pyronin Y (PY) staining is displayed along the y axis.
, a marker of senescence-associated heterochromatin formation (
