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. 2016 Oct 7;8(10):2392-2406.
doi: 10.18632/aging.101060.

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice

Joohwee Kim  1   2 , Vivek Vaish  1   2 , Mingxiao Feng  1   2 , Kevin Field  1 , Ioulia Chatzistamou  3 , Minsub Shim  1   2
Affiliations

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice

Joohwee Kim et al. Aging (Albany NY). .

Abstract

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated β-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders.

Keywords: COX2; TP53; p16; premature aging; prostaglandin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Generation of tamoxifen-inducible COX2 transgenic mouse. Diagram of CAG/CAT/hCOX2 construct is shown. (B) Expression of COX2 in various tissues of tamoxifen-inducible COX2 transgenic mouse. COX2 expression was induced in 5-week-old (CATflCOX2:Rosa-Cre ERT2) mice by intra-peritoneal injections of tamoxifen. Paraffin-embedded sections of 20-week-old COX2 transgenic mice were stained for COX2 using a human COX2-specific antibody. The tissues shown are heart (a), kidney (b), skeletal muscle (c), pancreas (d), small intestine (e), and skin (f). Scale bar=100 μm (C) General appearance of 20-week-old control (CT) and COX2 transgenic (TG) mice.
Figure 2
Figure 2
Cross-sections of dorsal skin from 20-week-old control (A, C, and F) and COX2 transgenic (B, D, and G) female littermates. Epidermis (E), dermis (D), adipose under the dermis (A), and muscle (M) are indicated. The COX2 transgenic skin exhibits sebaceous gland hyperplasia and a decrease in subcutaneous fat and muscle. Scale bar=100 μm (E) Quantification of the thickness of subcutaneous fat in control and COX2 transgenic mice (***: p<0.001). (H) A representative photo of 20- week-old COX2 transgenic and age-matched control male mice at 20 days after hair removal on dorsal area. (I) Quantification of hair re-growth in control and COX2 transgenic mice (***: p<0.001).
Figure 3
Figure 3
(A) Longevity in control (n=23) and COX2 transgenic (n=25) mice. (B) H&E stained sections of hind leg skeletal muscle from 22-week-old control (CT) and COX2 transgenic (TG) female littermates. Cross sections of the extensor digitorum longus (EDL) muscle are shown. Scale bar=100 μm (C) Frequency distribution of skeletal muscle fiber cross-sectional area (CSA) between control (CT) and COX2 transgenic (TG) mice. (D) Heart weight/body weight (HW/BW) ratios in tamoxifen-induced control (CT) and COX2 transgenic (TG) male littermates at the age of 22 weeks. HW/BW ratios in COX2 transgenic mice were significantly higher than those in control mice (paired t-test, p<0.001). (E) Quantitative assessment of fat content by X-ray densitometry of control (CT) and transgenic (TG) mice (n=5). (F) White blood cell (WBC) counts in tamoxifen-induced control (CT) and COX2 transgenic littermates (paired t-test, *: p<0.05, **: p<0.01, n=4). (G) The opacity of cornea developed in COX2 transgenic (TG) mice.
Figure 4
Figure 4
(A) A representative photo of testis from 23-week-old control (CT) and COX2 transgenic (TG) male littermates. The size of the testis is reduced in COX2 transgenic mice (n=4). (B) Cross-sections of testis from control (CT) and COX2 transgenic (TG) littermates at 25 weeks. While control testis contains sperms (arrows), sperms were not observed in COX2 transgenic testis. Scale bar=50 μm
Figure 5
Figure 5
H&E staining of ovarian sections from 40-week-old (A) control (CT) and (B) COX2 transgenic mice (magnification x5). (C) Higher magnification (x10) view of the ovary shown in (B). (D) The ovary from 22-week-old COX2 transgenic mouse showing a cystic structure filled with a clear fluid (arrow). (E) H&E stained bursal cyst in COX2 transgenic mouse. The cyst contains pale eosinophilic proteinaceous material. Scale bar=100 μm.
Figure 6
Figure 6
H&E stained sections of pancreas from COX2 transgenic mice (TG) showing the loss of the pancreatic acini, the increase of the metaplastic pancreatic ducts with dysplastic changes, peri-ductal fibrosis, and mononuclear inflammatory cell infiltration in the highly vascularized fibrotic stroma. Hyperplasia of the Langerhans islets was also noticed. Scale bar=100 μm.
Figure 7
Figure 7
(A) and (B) Immunohistochemical staining of p16 in the pancreata from 25-week-old control and COX2 transgenic littermates (magnification x20). (C) and (D) Immunohistochemical staining of phospho-H2AX in the pancreata from 23-week-old control and COX2 transgenic littermates (magnification x20). (E) and (F) Immunohistochemical staining of phospho-H2AX in the skin from 25-week-old control and COX2 transgenic littermates (magnification x20). Scale bar=100 μm.
Figure 8
Figure 8
(A) and (B) Adult mouse lung fibroblasts from control and COX2 transgenic mice were incubated with 4-hydroxytamoxifen for 24 hours to induce COX2 expression in transgenic fibroblasts. The cells were cultured in the regular culture medium for 48 hours before SA-β-Gal staining. Representative pictures from 3 independent experiments are shown. Quantification results are shown in (C). (D-F) Western blot analysis of COX2 and p53 in pancreas (D), skin (E), and muscle (F) tissues of control and COX2 transgenic littermates.
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