Figure 1.
Infection of TIGK epithelial cells with P. gingivalis expressing SerB diminishes IL8 promoter activity and phosphorylation of NF-κB p65.
(A) TIGKs were transiently transfected with the pIL-8 κB-Luc luciferase reporter plasmid for IL8 promoter activity, or pRL-CMV (CMV) in which Renilla luciferase is driven by the cytomegalovirus promoter, and infected with P. gingivalis WT or ΔserB mutant at a MOI of 10. At 16 h after infection, TIGKs were stimulated with TNF-α (10 ng/ml) for 3 h or left unstimulated and luciferase activity was measured and normalized to Renilla luciferase. Results are presented as fold relative to the activity of the non-stimulated control. Values are mean ± SD; n = 3; *, p<0.05. Data shown are representative of 2 biological replicates. (B) Immunoblots (IB) of lysates of TIGKs infected with P. gingivalis WT or ΔserB at a MOI of 10. At 16 h after infection, TIGKs were stimulated with TNF-α (10 ng/ml) or left unstimulated for the indicated time periods prior to cell lysis. Blots were probed with the antibodies indicated. Actin was used as a loading control. Result is representative of 2 biological replicates. (C) Densitometry of immunoblot in B) showing ratio of phospho-NF-κB p65 (S536) relative to total immunodetectable NF-κB p65. (D) Confocal microscopy showing expression of SerB by intracellular P. gingivalis. TIGKs were infected with P. gingivalis expressing FLAG-SerB at a MOI of 10 for 2 h, and stained with DAPI (cyan), fluorescein isothiocyanate (FITC)-phalloidin (green) or anti-FLAG (magenta). Bar = 5 µm. (E) Higher magnification of P. gingivalis indicated by arrow in D). Bar = 1 µm.
Figure 2.
Ectopically expressed SerB binds to and dephosphorylates NF-κB p65.
(A) TIGKs transfected with empty vector or Myc-SerB were stimulated with TNF-α (10 ng/ml) for 5 min or left unstimulated. Cells were cross-linked with DSP and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoblots (IB) show cell lysates prior to immunoprecipitation (5% input) and immunoprecipitate (IP) with Myc antibodies. Result is representative of 3 biological replicates. (B) Densitometry of immunoblot in A) showing ratio of immunoprecipitated NF-κB p65 relative to immunoprecipitated Myc-SerB. (C) Confocal microscopic images of TIGKs expressing GFP-NF-κB p65 (green) and either Myc (Vector) or Myc-SerB. At 36 h after transfection, cells were stimulated with TNF-α (10 ng/ml) for 5 min or left unstimulated. Cells were fixed and stained with anti-Myc (red). Bars = 5 µm. Data shown are representative of 2 biological replicates. (D) The intensity (Olympus FluoView software) of the fluorescence signals of GFP-NF-κB p65 (green) and either of Myc (red, Vector) or Myc-SerB (red) on the x-y lines indicated in C) are shown. (E) TIGKs transfected with empty vector or Myc-SerB, and GFP-NF-κB p65 or GFP-NF-κB p65 S536D, were cross-linked with DSP and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoblots (IB) show cell lysates prior to immunoprecipitation (5% input) and immunoprecipitate (IP) with Myc antibodies. Result is representative of 2 biological replicates. (F) Densitometry of immunoblot in E) showing ratio of immunoprecipitated GFP-NF-κB p65 or p65 S536D relative to immunoprecipitated Myc-SerB.
Figure 3.
SerB dephosphorylates NF-κB p65 but not p105.
(A) TIGKs were transfected with empty vector or Myc-SerB. At 36 h after transfection, cells were stimulated with TNF-α (5 ng/ml), and at the time periods shown cell extracts were prepared and immunoblotted (IB) with the antibodies indicated. Actin was used as a loading control. Result is representative of 3 biological replicates. (B) Densitometry of immunoblot in A) showing ratio of phospho-NF-κB p65 (S536) relative to total immunodetectable NF-κB p65. (C) TIGKs were transiently transfected with empty vector or Myc-SerB. At 36 h after transfection, cells were stimulated with TNF-α (5 ng/ml), and at the time periods shown cell extracts were prepared immunoblotted (IB) with the antibodies indicated. Result is representative of 3 biological replicates. (D) Densitometry of immunoblot in C) showing ratio of phospho-NF-κB p105 (S933) relative to total immunodetectable NF-κB p105. (E) Densitometry of immunoblot in C) showing ratio of phospho-NF-κB p50 relative to actin.
Figure 4.
SerB inhibits nuclear translocation of NF-κB p65 but not p105/p50.
(A) Confocal microscopy of TIGKs transiently co-transfected with Myc (Vector) or Myc-SerB along with GFP-NF-κB p65 or GFP-NF-κB p65 S536D and left unstimulated or stimulated with TNF-α (5 ng/ml) for 30 min. Cells were fixed and stained with DAPI and anti-Myc. Bars = 5 µm. Result is representative of 3 biological replicates. (B) Quantification of nuclear translocation in cells in A). Results are expressed as percentage of Myc positive cells with nuclear GFP- NF-κB p65 and are the mean with SEM of three independent experiments. At least 90 Myc/GFP positive cells were counted per test. *, p<0.05. (C) Confocal microscopy of TIGKs transiently co-transfected with Myc (Vector) or Myc-SerB along with GFP-NF-κB p105 and left unstimulated or stimulated with TNF-α (5 ng/ml) for 30 min. Cells were fixed and stained with DAPI and anti-Myc. Bars = 5 µm. Result is representative of 3 biological replicates. (D) Confocal microscopy of TIGKs transiently co-transfected with Myc (Vector) or Myc-SerB along with GFP-NF-κB p50 and left unstimulated or stimulated with TNF-α (5 ng/ml) for 30 min. Cells were fixed and stained with DAPI and anti-Myc. Bars = 5 µm. Result is representative of 3 biological replicates.
Figure 5.
SerB dephosphorylation of p65 inhibits IL8 promoter activity and IL-8 production.
(A) TIGKs were transiently co-transfected with Myc (Vector) or Myc-SerB, and either of GFP, GFP-NF-κB p65 or GFP-NF-κB p50, along with pIL-8 κB-Luc luciferase reporter for IL8 promoter activity or pRL-CMV Renilla luciferase control. Cells were stimulated with TNF-α (40 ng/ml) as indicated, and after 3 h TNF-α-induced IL8 κB luciferase activity was measured and normalized to Renilla luciferase. Results are presented as fold relative to the activity of the non-stimulated control and are means ± SD of 6 biological replicates. *, p<0.05. (B) TIGKs were transiently transfected with Myc (Vector) or Myc-SerB and at 36 h after transfection stimulated with TNF-α (5 ng/ml) as indicated. At the indicated time periods, the level of IL-8 in culture supernatants was measured by ELISA. Values are mean ± SD of 6 biological replicates. *, p<0.05. (C) TIGKs were transiently co-transfected Myc (Vector) or Myc-SerB, and either of GFP, GFP-NF-κB p65 or GFP-NF-κB p50. Cells were stimulated with TNF-α (5 ng/ml) as indicated, and after 4 h the level of IL-8 in culture supernatants was measured by ELISA. Values are mean ± SD of 6 biological replicates. *, p<0.05.
Figure 6.
SerB HAD superfamily motifs are required for dephosphorylation of NF-κB p65.
(A) Schematic view of the SerB structure and derivatives. ACT domains are indicated by white boxes and HAD superfamily motifs are shown with black boxes. (B) TIGKs transiently expressing SerB mutants were stimulated with TNF-α (5 ng/ml) for 5 min as indicated, and cell lysates immunoblotted (IB). Actin was used as a loading control. Result is representative of 2 biological replicates. (C) Densitometry of immunoblot in B) showing ratio of phospho-NF-κB p65 (S536) relative to total immunodetectable NF-κB p65. (D) TIGKs transiently expressing Myc-SerB or Myc-SerB Δ198–358 were stimulated with TNF-α (10 ng/ml) for 5 min or left unstimulated. Cells were cross-linked with DSP and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoblots (IB) show cell lysates prior to immunoprecipitation (10% input) and immunoprecipitates (IP) with Myc antibodies. Result is representative of 3 biological replicates. (E) TIGKs were transiently co-transfected with Myc (Vector), Myc-SerB or Myc-SerB Δ198–358, and pIL-8 κB-Luc luciferase reporter plasmid for IL8 promoter activity or pRL-CMV control. Cells were stimulated with TNF-α (10 ng/ml) or left unstimulated, and after 3 h IL8 κB luciferase activity was measured and normalized to Renilla luciferase. Results are presented as fold relative to the activity of the non-stimulated control. Values are mean ± SD of 8 biological replicates. *, p<0.05. (F) TIGKs transiently expressing Myc (Vector), Myc-SerB or Myc-SerB Δ198-358 were stimulated with TNF-α (5 ng/ml) as indicated. At 4 h after stimulation, the level of IL-8 in culture supernatants was measured by ELISA (upper panel). Results are mean ± SD of 6 biological replicates. *, p<0.05. Cells were lysed and immunoblotted (IB) with Myc antibodies (lower panel) to confirm expression of Myc-SerB or Myc-SerB Δ198–358. Actin was used as a loading control.
Figure 7.
SerB HAD superfamily motifs are required for inhibition of nuclear translocation of NF-κB p65.
(A) Confocal microscopy images of TIGKs transiently co-transfected with Myc (Vector), Myc-SerB or Myc-SerB Δ198-358 along with GFP-NF-κB p65 and left unstimulated or stimulated with TNF-α (5 ng/ml) for 30 min. Cells were fixed and stained with DAPI and anti-Myc. Bars = 5 µm. Result is representative of 3 biological replicates. (B) Quantification of nuclear translocation in cells transfected with Myc (Vector), Myc-SerB or Myc-SerB Δ198–358 along with GFP-NF-κB p65 and stimulated with TNF-α or left unstimulated. Results are expressed as percentage of Myc positive cells with nuclear GFP-NF-κB p65 and are the mean ± SD of 6 biological replicates. At least 90 Myc/GFP positive cells were counted per test. *, p<0.05.