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. 2007 Jan 23;104(4):1248-53.
doi: 10.1073/pnas.0609149104. Epub 2007 Jan 16.

Cdc42 GTPase-activating Protein Deficiency Promotes Genomic Instability and Premature Aging-Like Phenotypes

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Free PMC article

Cdc42 GTPase-activating Protein Deficiency Promotes Genomic Instability and Premature Aging-Like Phenotypes

Lei Wang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Cdc42 is a member of the Rho GTPase family known to regulate cell actin cytoskeleton organization, polarity, and growth, but its function in mammalian organismal physiology remains unclear. We found that natural aging of WT mice is marked with increased Cdc42 activity in various tissues. Among the negative regulators of Cdc42, gene targeting of Cdc42 GTPase-activating protein (Cdc42GAP) results in constitutively elevated Cdc42-GTP level in diverse tissues of adult mice; significantly shortened life span of the animals; and multiple premature aging-like phenotypes, including a reduction in body mass, a loss of subdermal adipose tissue, severe lordokyphosis, muscle atrophy, osteoporosis, and reduction of reepithelialization ability in wound-healing. Cdc42GAP-/- mouse embryonic fibroblasts and/or tissues display reduced population doubling, significantly dampened DNA damage repair activity after DNA-damaging agent treatment, accumulated genomic abnormalities, and induction of p53, p16Ink4a, p21Cip1, and senescence-associated beta-galactosidase expressions. Furthermore, Cdc42 activation is sufficient to promote a premature cellular senescence phenotype that depends on p53. These results suggest a role of Cdc42 activity in regulating mammalian genomic stability and aging-related physiology.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Increased Cdc42 activity in the course of natural aging in mice and the effects of Cdc42GAP gene targeting on the growth, bone structure, life span, and fertility period of adult mice. (A) Normal aging in mice is associated with increased Cdc42 activity. (Left) The Cdc42-GTP levels of various tissues from young (2-month-old), middle-aged (12-month-old), and elderly (24-month-old) mice were examined by GST-PAK1 effector domain pull-down assays. A representative blot from two experiments is shown with densitometry quantifications. (Right) The Cdc42-GTP levels of different tissues from 1.5-month-old (Young) and 26-month-old (Old) WT mice were examined by the effector pull-down assay. The quantifications were obtained from three independent experiments. (B) Different organs from 8-month-old mice were lysed with sonication. The lysates were subjected to GST-PAK1 effector pull-down, and the bound proteins were immunoblotted by a monoclonal anti-Cdc42 antibody. One set of data from two repeated experiments is shown. The underlining numbers indicate relative Cdc42-GTP quantified by densitometry scanning. (C) Body weights of 20 mice in each group (Cdc42GAP+/+, Cdc42GAP+/−, and Cdc42GAP−/−) tracked with increasing age. (D) Survival curves of 16 control (Cdc42GAP+/+ and Cdc42GAP+/−) and 21 Cdc42GAP−/− mice. The median life spans were 12 and 27 months for Cdc42GAP−/− and control mice, respectively. (E) Photographs of representative 12-month-old live Cdc42GAP+/+ and Cdc42GAP−/− males. The Cdc42GAP−/− mouse shows reduced size and severe lordokyphosis. (F) X-rays of representative 15-month-old male mice showing skeletal changes with severe lordokyphosis in the Cdc42GAP−/− mouse. (G) Delayed, reduced, and shortened reproductive period in the Cdc42GAP−/− female mice.
Fig. 2.
Fig. 2.
Cdc42GAP−/− mice display premature aging-like phenotypes in various tissues. (A) H&E sections of 8-month-old female homozygous or WT mouse vertebral bodies. The Cdc42GAP−/− mouse shows a thin, bowed cortex and thin trabeculae in contrast with the WT mouse. (B) H&E sections of 12-month-old female mouse spleens. The Cdc42GAP-deficient mouse shows decreased volume in the white pulp and red pulp of the spleen and smaller lymphoid follicles and decreased red blood cells on the sinusoids of the red pulp. (C) H&E staining of 9-month-old female mouse dorsal skin sections. In the Cdc42GAP−/− mouse, the s.c. layer is completely collapsed due to the loss of adipocytes, and the muscle layer shows atrophy at the muscle fibers compared with the WT control. Ep, epidermis; de, dermis; sft, s.c. fat tissue; sm, skeletal muscle. (D) Wound healing ability compared in 8-month-old female mice. (E) H&E staining of a skin wound 4 days after wounding. The WT shows complete epithelialization of the wound, whereas there is no closure at the wound (indicated by asterisk) in the Cdc42GAP-deficient mouse. The experiments were repeated at least twice, and one set of representative data is shown.
Fig. 3.
Fig. 3.
Cdc42GAP−/− cells undergo early senescence and show increased genomic instability. (A and B) SA-β-gal activities in the spleen sections of 9-month-old female mice (A) and passage-6 MEF cells (B). The mutant MEF cells show a flattened and enlarged morphology that is associated with β-galactosidase staining. (C) MEF cells (passage 4) were replated at a density of 1 × 106 per 10-cm culture dish every 3 days, and the population doubling times were derived. (D) Metaphase spreading profiles of 50 splenocytes from 8-month-old female WT and homozygous mice. (E) Passage-6 MEF cells were subjected to karyotype analysis, and the chromosome abnormalities are summarized. (F) A set of representative pictures of chromosome structures are shown for both genotypes. Arrowheads point to the chromatid breaks, asterisks indicate chromosome fragments, and the plus sign indicates a fusion and complex rearrangement. (G) Passage-7 MEF cells were stained with anti-p-H2AX antibody and DAPI to reveal cell nucleus and DNA-damage foci. Three independent pairs of MEF cells were examined, and the pairs behaved similarly.
Fig. 4.
Fig. 4.
Impaired DNA damage repair ability of Cdc42GAP-deficient MEF cells after treatment with various DNA-damaging agents. MEF cells of early passages were treated with the indicated doses of IR, H2O2, camptothecin (CPT), methyl-methane sulfonate (MMS), or mitomycin C (MMC), and the surviving cell numbers under each condition were quantified after 7 days. The survival rates were normalized to those of the nontreated cells.
Fig. 5.
Fig. 5.
The early senescence of Cdc42GAP−/− cells depends on p53 activity. Protein lysates (100 μg) from MEF cells of passages 3 and 6 (A) or from different tissues of 8-month-old mice (B) were immunoblotted with the respective antibodies. (C) WT MEF cells transduced with EGFP or Cdc42F28L/EGFP were stained for β-galactosidase activity at an early passage (passage 6) to reveal the senescent cell populations. (D) The population doubling times of MEF cells of various genotypes at passages 4–9 were measured in cell culture. (E) The MEF cells at passage 7 were stained with the senescence marker SA-β-gal to determine the senescent cell populations. (F) A working model for a possible mechanism of Cdc42GAP deficiency-induced senescence. Cdc42GAP knockout causes a constitutively elevated Cdc42 activity, which in turn dampens the DNA damage repair ability. The accumulated genomic abnormalities resulting from the unrepaired damages stimulate a p53-mediated response that causes replicative senescence.

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