Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

Takeo Iwasaki,Kenji Miyado,Ichiro Kawase,Yoshito Takeda,Takashi Kijima,Mayumi Suzuki,Hideshi Kaneko,Toshihisa Komori,Bo Zhou,Tadashi Osaki,Seigo Minami,Eisuke Mekada,Ping He,Sho Goya,Mitsuhiro Yoshida,Isao Tachibana,Toru Kumagai

doi:10.1074/jbc.m801902200

Takeo Iwasaki, Kenji Miyado + Show 15 more

Open Access

https://doi.org/10.1074/jbc.m801902200

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Abstract

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.

Highlights

The tetraspanin proteins include at least 33 members, including CD9, CD63, CD81, CD82, and CD151 in mammals
CD9 and CD81 Are Down-regulated by Smoking-related Inflammatory Stimuli in RAW264.7 Macrophages—Cigarette smoke reduces the expression and activity of histone deacetylases (HDACs) in macrophages of chronic obstructive pulmonary disease (COPD) patients, resulting in amplification of proinflammatory gene transcription [5]
They affect tumor cell motility in vitro and tumor metastasis in vivo most likely by regulating function of integrins and production of matrix metalloproteinase (MMP) [7,8,9, 29]. It has not been investigated if tetraspanins play a role in motility and MMP production of macrophages

Summary

EXPERIMENTAL PROCEDURES

Immunoblotting—A mouse macrophage line, RAW264.7, and a human alveolar epithelial cell line, A549, were serumstarved for 24 h and treated with 10 ng/ml trichostatin A (TSA; Wako Pure Chemical Industries, Osaka, Japan) or 0.1% cigarette smoke extract (CSE) as described previously [10] for 48 h. Culture supernatants were studied for MMP-9 activity in gelatin zymography. After 4 h, cells migrating to the lower surface were counted with Diff-Quick stain (International Reagents, Hyogo, Japan). The cells were cultured for 24 h in serum-free DMEM, and the culture supernatants were studied for MMP-9 activity by gelatin zymography. Mice—The generation of CD9/CD81 DKO mice was described previously [13]. Bronchoalveolar Lavage and Gelatin Zymography—Lungs of anesthetized mice were subjected to lavage with 3 volumes of 1 ml of phosphate-buffered saline containing 0.1% bovine serum albumin. The total mineral content and strength strain index were determined as described previously [16]

RESULTS

Are Less Motile and Produce More

DISCUSSION