Figure 1.
Cebpd plays an important role in CIA mice.
A, Age- and sex-matched C57BL/6 mice were immunized with chicken CII emulsified in CFA at day 0. The mice were scored every 2–3 days without knowledge of their genotypes. Clinical score of arthritis: the scores for four paws were obtained for each mouse, and the total severity score for the group was divided by the number of animals in the group to obtain an average severity score. B, The rear paws and joints from Cebpd+/+ and Cebpd−/− mice were stained with hematoxylin and eosin after collagen-induced arthritis. C, Histopathological scores: the score from two ankle joints in each mouse was assessed by two orthopedic surgeons blind to the genotypes of the mice. The average histopathological score in the Cebpd −/− group was significantly lower than that of the wild-type group (p = 0.016). *p<0.05; n = 10 for control and n = 6 for the experimental group.
Figure 2.
Arthritic pannus and blood vessels are decreased in CIA Cebpd−/− mice.
The rear paws and joints from Cebpd+/+ and Cebpd−/− mice were immunohistochemically stained after collagen-induced arthritis. A, Ki-67 staining was used as a proliferating cell marker. B, CD31 staining was used as an endothelial cell marker. C, F4/80 staining was used as a macrophage/monocyte marker. ND: no difference. **p<0.01, Student’s t test; n = 6−10 per group.
Figure 3.
CEBPD promotes rFLS proliferation and migration in vitro.
A, IL-1β and TNFα induce CEBPD expression in THP-1 cells, THP-1-derived macrophages and mouse primary macrophages. THP-1 cells, THP-1-derived macrophages and mouse primary macrophages were starved in serum-free medium for 6 h and then treated with TNFα or IL-1β for 3 h. Samples of total RNA and cell lysates were harvested for RT-PCR and Western blotting, respectively. B, The effect of lentiviral shCEBPD in THP-1 cells. CEBPD expression upon TNFα treatment and inactivation by lentiviral shCEBPD (sh-D) and shluciferase (sh-L) was examined by RT-PCR and Western blot. GAPDH mRNA and β-actin protein were used as the respective internal controls. C-D, Macrophage CEBPD affects the proliferation and migration of rFLS. The conditioned media were harvested as described in the Materials and Methods for further experiments. C, rFLS were treated with conditioned media for 24 h, and the cell viability was measured using a Cell Counting Kit-8. The data are presented as the means ± SE of three independent experiments performed in triplicate. The asterisks represent significant differences (*p<0.05, **p<0.01, ***p<0.001; Student’s t test). D, CM-CEBPD promotes the migration of rFLS cells. The data are presented as the means ± SE of three independent experiments performed in triplicate. The asterisks represent significant differences (*p<0.05, **p<0.01; Student’s t test).
Figure 4.
CEBPD promotes endothelial cell tube formation.
A, The angiogenic effect of CM-CEBPD on HUVECs. The positive control is HUVECs that were treated with EGM-2. The negative control is HUVECs that were treated with serum-free EBM-2 medium. B, The angiogenic effects of exposing HUVECs for 24 h to conditioned medium from THP-1 cells that were infected with lentivirus shCEBPD (CM-shD) or shLuciferase (CM-shL) with or without TNFα. The number of intersections between branches of assembled endothelial cell networks was counted in the whole field. The data are presented as the means ± SE of three independent experiments performed in triplicate. The asterisks represent significant differences (*p<0.05, **p<0.01; Student’s t test).
Figure 5.
CCL20, CXCL1, IL23A and TNFAIP6 genes are responsive to CEBPD activation in THP-1 cells.
A, CEBPD participates in TNFα-induced CCL20, CXCL1, IL23A and TNFAIP6 transcripts in THP-1 cells. Total RNA was extracted from TNFα-treated THP-1 cells treated with lentiviral shCEBPD (sh-D) or shluciferase (sh-L) for RT-PCR analysis. B, Ccl20, Cxcl1, Il23a and Tnfaip6 transcripts in TNFα-treated primary macrophages of WT or Cebpd-deficient mice. Total RNA was extracted from TNFα-treated macrophages of WT or Cebpd-deficient mice for RT-PCR analysis. C, Putative CEBPD-binding motifs in the CCL20, CXCL1, IL23A and TNFAIP6 reporters. The 5′-flanking regions of human CCL20, CXCL1, IL23A and TNFAIP6 genes were subcloned into pGL3 basic reporters. (D) CEBPD activates CCL20, CXCL1, IL23A and TNFAIP6 reporter activities in THP-1 cells. pcDNA3/HA/CEBPD (pcHA/CEBPD) or pcDNA3/HA (pcHA) was cotransfected with the CCL20 (−1000 to +50), CXCL1 (−970 to +51), IL23A (−1200 to +60) or TNFAIP6 (−600 to +76) reporter. The data are presented as the means ± SE of relative luciferase activity from three independent experiments in duplicate. E, CEBPD contributes to TNFα-induced CCL20, CXCL1, IL23A and TNFAIP6 reporter activities in THP-1 cells. Oligonucleotides of siCEBPD (si-D) or siLacZ (si-C) were cotransfected with various indicated reporters. The lysates of transfectants were harvested after treating with TNFα for 6 h. The data are presented as the means ± SE of relative luciferase activity. The asterisks represent significant differences (*p<0.05; Student’s t test). F, CEBPD binds to the 5′-flanking regions of the CCL20, CXCL1, IL23A and TNFAIP6 genes in vivo. Chromatin was isolated for ChIP analysis from THP-1 cells with or without TNFα treatment, as described in the Materials and Methods. The locations of the PCR primers in the 5′-flanking regions of CCL20 (−344 to −7), CXCL1 (−736 to −406), IL23A (−379 to −45) and TNFAIP6 (−354 to −6) are presented in the left panel. Chromatin was separately immunoprecipitated with control IgG and CEBPD antibodies and then amplified by PCR with the indicated primers, as shown in the right panel.
Table 1.
Immune factors responding to the induction of CEBPD in macrophages.
Figure 6.
CCL20, CXCL1, IL23A and TNFAIP6 contribute to the proliferation and migration of rFLS cells, but only CXCL1 and TNFAIP6 increase endothelial cell tube formation.
A, CCL20, CXCL1, IL23A and TNFAIP6 contribute to the proliferation and migration of rFLS cells. The purified rFLS were cultured with the conditioned medium harvested from TNFα-treated THP-1 cells containing the indicated lentivirus, namely shLuciferase (Sh-L), shCCL20 (Sh-CC), shCXCL1 (Sh-CX), shIL23A (Sh-IL) or shTNFAIP6 (Sh-TA). The migration and proliferation assays were conducted using a Boyden chamber assay and a CCK-8 kit, respectively. The results are shown as the means ± SE. The asterisks represent significant differences (*p<0.05; Student’s t test). B, The endothelial tube formation assay was conducted using conditioned media harvested from THP-1 cells as described in the Materials and Methods. The results are shown in the upper panel and compared statistically in the lower panel. The number of intersections between branches of assembled endothelial cell networks was counted in the whole field. The data are presented as the means ± SE. The asterisks represent significant differences (*p<0.05, **p<0.01; Student’s t test).
Figure 7.
Inotilone and rosmanol inhibit the proliferation and migration of rFLS and the endothelial tube formation of HUVECs.
A, Inotilone and rosmanol inhibit CEBPD, CCL20, CXCL1, IL23A and TNFAIP6 transcription in THP-1 cells. Total RNA was harvested from THP-1 cells pretreated with inotilone (Ino) or rosmanol (Ros) then treated with 20 ng/ml TNFαas described in the Materials and Methods. B-D, Inotilone and rosmanol inhibit the proliferation and migration of rFLS and endothelial cell tube formation. The conditioned media were harvested to assess their effects on the proliferation and migration of rFLS and on the endothelial tube formation of HUVECs. The data are presented as the means ± SE of three independent experiments performed in triplicate. The asterisks represent significant differences (*p<0.05, **p<0.01; Student’s t test).