Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging

Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging, but the mechanism remains unclear. Tetraspanins have emerged as key players in malignancy and inflammatory diseases. Here, we found that CD9/CD81 double knockout (DKO) mice with a COPD-like phenotype progressively developed a syndrome resembling human aging, including cataracts, hair loss, and atrophy of various organs, including thymus, muscle, and testis, resulting in shorter survival than wild-type (WT) mice. Consistent with this, DNA microarray analysis of DKO mouse lungs revealed differential expression of genes involved in cell death, inflammation, and the sirtuin-1 (SIRT1) pathway. Accordingly, expression of SIRT1 was reduced in DKO mouse lungs. Importantly, siRNA knockdown of CD9 and CD81 in lung epithelial cells additively decreased SIRT1 and Foxo3a expression, but reciprocally upregulated the expression of p21 and p53, leading to reduced cell proliferation and elevated apoptosis. Furthermore, deletion of these tetraspanins increased the expression of pro-inflammatory genes and IL-8. Hence, CD9 and CD81 might coordinately prevent senescence and inflammation, partly by maintaining SIRT1 expression. Altogether, CD9/CD81 DKO mice represent a novel model for both COPD and accelerated senescence.


Transmission electron microscopy
Tissue samples were fixed at 4 ºC in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) and post-fixed at room temperature in 2% osmium tetroxide in the same buffer. After dehydration in ethanol, the specimens were embedded in Epon 812 resin (TAAB, Berkshire, UK). Ultrathin sections (60 nm) were stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H-7100; Hitachi, Tokyo, Japan)

Lung function test
Lung volume and compliance were measured with a whole-body plethysmograph as part of the Pulmonary Maneuvers System (Buxco Electronics, Wilmington, NC). Anesthetized mice were attached via a tracheostomy tube to a mechanical ventilator in a sealed plethysmograph. A quasi-static pressure-volume maneuver was used to measure lung compliance. Data for all maneuvers were analyzed with Biosystem XA software (Buxco Electronics).

Grip test and spontaneous locomotor activity
Forearm grip strength was assessed 3 times in 8-wk-old mice using a grip strength meter (GPM-100; Melquest, Toyama, Japan) according to the manufacturer's instructions (1). Spontaneous locomotor activities of test and control mice were measured simultaneously in separate transparent-plastic cages using a LOCOMO sensor system consisting of LOCOMO sensor units (LS-5), a counter interface (CIF-mini4), a control unit (LCU-20), and a personal computer with WinCIF II mini software (Melquest). The counter interface counted the interruptions of latticed infrared-beams by a mouse moving in the cage inside the sensor.

Micro-computed tomography (CT)
The mice were anesthetized with isoflurane and placed in the chamber of a CT scanner for small animals (Latheta LCT-200; Hitachi, Tokyo, Japan) (2). Bone mineral density and the muscle mass were assessed by analyzing the volume using the tibia. Visceral adipose tissue was analyzed by assessing fat content within the peritoneal cavity. Three-dimensional CT pictures for whole body were reconstructed with VGStudio MAX2.0 software (Volume Graphics, Tokyo, Japan).

Immunohistochemistry
Mice paraffin sections (WT and DKO) were stained using indirect immunohistochemistry as described previously with minor modifications (3). Briefly, deparaffinized and rehydrated tissue sections were heated in citrate buffer (15 min, at 121°C, 10 mM, pH 6.0), and blocked with 10% normal serum. Bioinformatics IPA(Ingenuity Systems; www.ingenuity.com) is a knowledge base that contains approximately 3,000,000 biological and chemical interactions and functional annotations with definite scientific evidence. By uploading the list of Gene IDs, the network-generation algorithm identifies genes integrated in a global molecular network. IPA calculates a P-value score that reflects the statistical significance of the association between the genes and the networks by Fisher's exact test. Array data were analyzed using IPA, comparing the gene expression lists of 15W CD9/CD81 DKO lungs and WT lungs; significantly differentially regulated genes were classified into subcategories by gene functions.
TargetMine(http://targetmine.nibio.go.jp) is an integrative data analysis platform for gene set analysis and knowledge discovery within the framework of a data warehouse. TargetMine models biological entities such as genes and proteins as "objects" and their relationships as "references" to other objects.
TargetMine features a variety of biological data types and interactive tools to analyse them. In this study, biological pathway data from KEGG and Gene Ontology (GO) Slim associations from the GO consortium were used to assign functional annotations to the differentially expressed genes (DEGs) in    Table 1. Histological analysis of aging phenotype in CD9/CD81 DKO mice.
Shown are tables regarding histological analysis of aged mice from WT type (left) and DKO mice (right) (n=2-3, 18 months of age). Pathological scoring is reported as negative (-), mild (), moderate (+) and severe (2+) by experts in animal pathology. Multiple organ atrophy was confirmed in aged DKO mice. However, no significant difference was observed in several organs such as heart, kidney, liver, gastrointestinal tract and aorta. By hematological and serological test, level of creatinine and phosphate was indistinguishable, whereas that of cholesterol and calcium was minimally decreased, and that of platelet was increased. ** P < 0.01 versus WT.