Augmented Wnt Signaling in a Mammalian Model of Accelerated Aging
Hongjun Liu1,
Maria M Fergusson1*,
Rogerio M. Castilho2*,
Jie Liu1,
Liu Cao1,
Jichun Chen3,
Daniela Malide4,
Ilsa I. Rovira1,
Daniel Schimel5,
Calvin J. Kuo6,
J. Silvio Gutkind2,
Paul M. Hwang1 and
Toren Finkel1
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.
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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.
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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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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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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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