Figure 2.

Toll-like receptor 7-mediated mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) upregulation is required for matrix metalloproteinase-9 (MMP-9) production in alveolar macrophages. (A) MH-S cells were stimulated with or without R848 (10 µM). RNA was extracted at 24 h after stimulation and applied to microarray chip. Relative levels of mRNA expression were compared to unstimulated cells. Data were compiled from one experiment. (B,C) MH-S cells were stimulated with or without (B) R848 (10 µM) and (C) imiquimod (Imiq, 1 µg/ml). The level of Malt1 mRNA was quantified by qPCR and normalized against Actb at indicated time points (n = 4, four independent experiments). (D) MH-S cells were stimulated with or without Imiq (1 µg/ml) for indicated periods of time. Cell lysates were analyzed for MALT1 and β-actin protein by Western blotting. Bar graphs show the levels of MALT1 protein normalized against β-actin (n = 4, four independent experiments. One representative experiment is shown). The MALT1-to-β-actin ratio at each time point without stimulation (Ctrl) was taken as 1.0. (E) MH-S cells were pretreated with z-VRPR-fmk (zVRPR, MALT1 inhibitor, 100 µM) for 6 h before stimulation with or without Imiq (1 µg/ml) for another 18 and 24 h. MMP-9 concentration was quantified by ELISA (n = 3, three independent experiments). (F) Primary alveolar macrophages from wild-type and MALT1-deficient mice were stimulated with or without Imiq (1 µg/ml) for 48 h. The concentration of MMP-9 in culture supernatants was quantified by ELISA. Each dot represents cells from one mouse and data are a compilation of three independent experiments. n.s., not significant, *p < 0.05, **p < 0.01, and ***p < 0.001 compared to control. (B–D) were analyzed by Mann–Whitney U-test, and (E,F) were analyzed by one-way ANOVA followed by (E) Tukey’s and (F) Sidak’s post hoc tests.