Figure 1.
NF-κB signaling regulates pro-inflammatory gene expression in senescent endothelial cells.
(A–C) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding cyclin-dependent kinase inhibitor 1A (p21) or cyclin-dependent kinase inhibitor 2A (p16) to induce senescence. Expression of pro-inflammatory genes, such as the chemokine (C–C motif) ligand 2 (CCL2), E-selectin (SELE), and vascular cell adhesion molecule 1 (VCAM1), in senescent endothelial cells was examined by real-time PCR at 6 days after infection with the retroviral vector for p16 (A) or p21 (B) or after mock infection. Expression of pro-inflammatory genes was also examined in human endothelial cells undergoing replicative senescence (C). We defined cells with replicative senescence as cultures that did not show an increase in cell numbers and remained subconfluent for 2 weeks. n = 5. (D) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding p21 to induce senescence (p21-senescent). Six days after infection, the cells were transduced with 3 sets of siRNAs for RELA (1–3), siRNAs for IKKs (α, β, γ subunits), or control siRNA (siCont). Expression of pro-inflammatory genes was examined by real-time PCR after 72 hours. n = 3. (E) Expression of RELA in the nuclear and cytoplasmic fractions and phospho-RELA in whole cell lysates was examined by western blotting at 6 days after retroviral infection. Samples were prepared as in Figure 1B. Histone H3 expression served as the internal control for nuclear extracts and the level of nuclear RELA relative to Histone H3 was quantified. n = 3. Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 2.
CDC42 signaling regulates pro-inflammatory gene expression in senescent endothelial cells.
(A) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding p21 to induce senescence (p21-senescent). Six days after infection, the cells were transduced with 3 sets of siRNAs for CDC42, siRNAs for PAK2, or control siRNA (siCont). Expression of pro-inflammatory genes was examined by real-time PCR after 72 hours. n = 3. (B) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding p21 to induce senescence (p21) and were harvested at 6 days after infection. The pull-down assay for active CDC42 (GTP-CDC42) was performed as described in Methods. The graph indicates the relative level of active CDC42. n = 3. (C) Expression of phopho-PAK2 and total PAK2 was examined by western blotting in human endothelial cells prepared as in Figure 2B. (D) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding active CDC42 (CDC42 V12), and expression of pro-inflammatory genes was examined by real-time PCR at 6 days after infection. n = 3. (E) Proliferation of endothelial cells infected with a retroviral vector encoding active CDC42 (CDC42 V12) or an empty vector (Mock). n = 3. (F) Human endothelial cells were infected with an empty vector (Mock) or a retroviral vector encoding active CDC42 (CDC42 V12). Six days after infection, the cells were transduced with 3 sets of siRNAs for RELA, siRNA for IKKs, or control siRNA (siCont). Expression of pro-inflammatory genes was examined by real-time PCR after 72 hours. n = 3. (G) Human endothelial cells were infected with a retroviral vector encoding p21 to induce senescence (p21-senescent). Six days after retroviral infection, the cells were infected with an adenoviral vector encoding a dominant-negative form of myc-tagged CDC42 (CDC42 N17) or LacZ. Expression of phospho-RELA (Ser536) and total RELA were examined by western blotting at 48 hours after adenoviral infection. (H) Expression of pro-inflammatory genes in endothelial cells prepared as in Figure 2G was examined by real-time PCR. n = 3. Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 3.
CDC42 signaling has a weaker influence on acute inflammation.
(A and B) Human endothelial cells were transduced with siRNA for CDC42, PAK2, RELA, IKKs, or control siRNA (siCont). Cells were treated with TNF-α (A) or LPS (B) at 60 hours after siRNA transduction and were harvested 24 hours later to examine the expression of pro-inflammatory genes by real-time PCR. (C) Knockdown efficiency of siRNAs for CDC42, PAK2, and NFκB signaling in endothelial cells at 60 hours after transduction. The graph shows expression of each gene in siRNA-treated cells relative to that in siCont-treated cells. Each experiment in Figure 3A–C was repeated three times, and the results were consistent. Representative results are shown.
Figure 4.
CDC42 signaling regulates chronic inflammation associated with senescence.
(A) Immunostaining for p53, p21, and RELA in sections of the lungs and the renal glomeruli from endothelial cell-specific Mdm2 conditional knockout mice (Mdm2 CKO, Pdgfb-Cre-ER; Mdm2loxP/loxP) and their littermate controls (Cont, Mdm2loxP/loxP). Arrowheads indicate positive staining of capillary endothelial cells. Scale bar = 100 µm. (B) Expression of p21 and pro-inflammatory genes in the lungs of Mdm2 CKO mice (Pdgfb-Cre-ER; Mdm2loxP/loxP), their littermate controls (Cont, Mdm2loxP/loxP), Cdc42 CKO mice (Pdgfb-Cre-ER; Cdc42loxP/loxP), and Mdm2 & Cdc42 CKO mice (Pdgfb-Cre-ER; Mdm2loxP/loxP; Cdc42loxP/loxP) was examined by real-time PCR. n = 4–6. (C) Expression of Cdc42 was examined by real-time PCR in the lungs of Cdc42 CKO mice (Pdgfb-Cre-ER; Cdc42loxP/loxP), their littermate controls (Cont 1, Cdc42loxP/loxP), Mdm2 & Cdc42 CKO mice (Pdgfb-Cre-ER; Mdm2loxP/loxP; Cdc42loxP/loxP), and their littermate controls (Cont 2, Mdm2loxP/loxP; Cdc42loxP/loxP). n = 5–6. (D) Immunostaining for p21 in paraffin-embedded sections of the aorta from Apoe knockout mice (Apoe KO, Apoe−/−) and wild-type littermates (WT, Apoe+/+). Black arrowheads indicate negative staining of aortic endothelial cells. Red arrowheads indicate positive staining. Scale bar = 100 µm. (E) Expression of Cdkn1a (p21), Cd68, and pro-inflammatory genes in the aortas of Cdc42 CKO mice (Pdgfb-Cre-ER; Cdc42loxP/loxP), their littermate controls (Cont, Cdc42loxP/loxP), Apoe KO mice (Apoe−/−;Cdc42loxP/loxP), and Apoe KO & Cdc42 CKO mice (Apoe−/−; Pdgfb-Cre-ER; Cdc42loxP/loxP) was examined by real-time PCR. n = 4–6. (F) Immunostaining for p21 in frozen sections of the aorta from Apoe KO mice (Apoe−/−), wild-type littermates (WT, Apoe+/+), Apoe KO littermates (Apoe−/−;Cdc42loxP/loxP), and Apoe KO & Cdc42 CKO mice (Apoe−/−; Pdgfb-Cre-ER; Cdc42loxP/loxP). Black arrowheads indicate negative staining of aortic endothelial cells for p21. Red arrowheads indicate positive staining for p21. Scale bar = 20 µm. (G) Oil red O staining of aortic sinus sections from Apoe KO mice (Apoe−/−;Cdc42loxP/loxP) and Apoe KO & Cdc42 CKO mice (Apoe−/−; Pdgfb-Cre-ER; Cdc42loxP/loxP). Scale bar = 100 µm. (H) Quantification of the atherosclerotic lesion area relative to the total area at the level of the aortic sinus in Apoe KO mice and Apoe KO & Cdc42 CKO mice. n = 5. Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 5.
CDC42 promotes senescence-associated inflammation in worms.
(A) Expression of sym-1 in wild-type (WT) and nol-6 mutant worms was examined by real-time PCR on day 9. n = 3. (B) Survival curves of wild-type (WT) and nol-6 mutant worms maintained at 22°C. n = 50. (C) Expression of sym-1 by wild-type (WT) and nol-6 mutants treated with RNAi for cdc-42 or control RNAi (Cont) was examined by real-time PCR. mRNA was analyzed 48 hours after starting feeding RNAi. n = 4. (D) Survival curves of nol-6 mutant worms treated with RNAi for cdc-42, max-2, or control RNAi (Cont). n = 50. Data are shown as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.