Immunoproteasome subunit ß5i/LMP7-deficiency in atherosclerosis

Abstract

Management of protein homeostasis by the ubiquitin-proteasome system is critical for atherosclerosis development. Recent studies showed controversial results on the role of immunoproteasome (IP) subunit β5i/LMP7 in maintenance of protein homeostasis under cytokine induced oxidative stress. The present study aimed to investigate the effect of β5i/LMP7-deficiency on the initiation and progression of atherosclerosis as a chronic inflammatory, immune cell driven disease. LDLR^-/-LMP7^-/- and LDLR^-/- mice were fed a Western-type diet for either 6 or 24 weeks to induce early and advanced stage atherosclerosis, respectively. Lesion burden was similar between genotypes in both stages. Macrophage content and abundance of polyubiquitin conjugates in aortic root plaques were unaltered by β5i/LMP7-deficiency. In vitro experiments using bone marrow-derived macrophages (BMDM) showed that β5i/LMP7-deficiency did not influence macrophage polarization or accumulation of polyubiquitinated proteins and cell survival upon hydrogen peroxide and interferon-γ treatment. Analyses of proteasome core particle composition by Western blot revealed incorporation of standard proteasome subunits in β5i/LMP7-deficient BMDM and spleen. Chymotrypsin-, trypsin- and caspase-like activities assessed by using short fluorogenic peptides in BMDM whole cell lysates were similar in both genotypes. Taken together, deficiency of IP subunit β5i/LMP7 does not disturb protein homeostasis and does not aggravate atherogenesis in LDLR^-/- mice.

Figure 1

β5i/LMP7-deficiency does not aggravate atherosclerotic lesion progression in LDLR^−/− mice. LDLR^−/−LMP7^−/− and LDLR^−/− littermate mice were fed a high-fat Western-type diet (WD) for either 6 or 24 weeks to induce early and advanced stage atherosclerosis, respectively. (A) Representative en face stainings of aortic arch/whole aorta (upper rows) and Oil Red O stainings with hematoxylin counterstain of aortic root cross-sections (bottom rows) for early (upper panels) and advanced (lower panels) stage atherosclerosis with quantification of lesion sizes. (B) Representative stainings and quantifications of (upper row) macrophages (MAC-2), (middle row) necrotic core (NC; Movat pentachrome staining), and (bottom row) ubiquitinated proteins (FK2; magnification: 10x) of aortic root cross-sections of advanced stage atherosclerosis. Data in graphs are presented as individual values with median and interquartile ranges indicated. n.s. = statistically non-significant. Unpaired t-test (Welch test for unequal variances) or Mann-Whitney U test.

Figure 2

β5i/LMP7-deficiency does not influence macrophage polarization and stress response in BMDM of LDLR^−/− mice. BMDM isolated from LDLR^−/−LMP7^−/− and LDLR^−/− littermate mice: (A) M1/M2 polarization – mRNA expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and arginase-1 determined by quantitative real-time RT-PCR after treatment of BMDM with IFN-γ (100 U/ml) and IL-4 (4 U/ml) over 6 hours for M1 and M2 macrophage phenotype polarization, respectively; C = untreated cells; graphs show fold-change in mRNA expression levels relative to untreated BMDM of LDLR^−/− mice; n = 5 experiments. Unpaired t-test (Welch test for unequal variances) or Mann-Whitney U test. (B) Superoxide production of IFN-γ-treated (100 U/ml) BMDM determined by electron paramagnetic resonance (EPR) technique; n = 5–7 per group. Two-way ANOVA followed by Sidak’s multiple comparisons test. (C) Representative Western blot with anti-ubiquitin antibody of lysates of IFN-γ (100 U/ml) treated BMDM; Bortezomib (5 ng/ml) treated BMDM served as positive controls; n = 4 experiments; LC indicates amidoblack staining as loading control. (D) Representative Western blot with anti-ubiquitin antibody of lysates of BMDM after treatment with hydrogen peroxide (H₂O₂) over 1 hour; n = 3 experiments. LC indicates GAPDH staining as loading control. (E) Survival of BMDM treated with H₂O₂ over 1 hour; n = 4 experiments. Kruskal-Wallis test with post hoc comparison by Dunn’s multiple comparison test. Data in graphs are presented as mean ± SEM. n.s. = statistically non-significant.

Figure 3

Impact of β5i/LMP7-deficiency on proteasome composition and proteolytic activity in cells and tissues of LDLR^−/− mice. (A) Western blot analysis of standard proteasome (β1, β2, and β5) and immunoproteasome (β1i, β2i, and β5i) subunit expression in BMDM of LDLR^−/−LMP7^−/− and LDLR^−/− at baseline and after treatment with IFN-γ (100 U/ml) over 8 hours (left panel). Right panel shows Western blot analyses of pooled spleen protein samples from LDLR^−/−LMP7^−/− and LDLR^−/− mice at baseline and after feeding a high-fat Western-type diet (WD) over 6 weeks (n = 11 mice per group). LC indicates actin staining (for BMDM) or amidoblack staining (for spleen) as loading controls. (B) Representative Western blots of standard proteasome and immunoproteasome subunits of isolated 26 S proteasome derived from murine LDLR^−/−LMP7^−/− and LDLR^−/− BMDM (left panel) and spleen (right panel) lysates. (C) Chymotrypsin-, trypsin- and caspase-like proteasome activities (expressed as relative fluorescence units, RFU) of murine LDLR^−/−LMP7^−/− and LDLR^−/− BMDM; n = 4 experiments. Unpaired t-test or Mann-Whitney U test. Data in graphs are presented as mean ± SEM.