CoPP IX for 16 h. The expression of Nrf2 or HO-1 was examined. D, E: Cells were incubated with CoPP IX for 16 h or 24 h, and then (D) the expression of Nrf2 was examined or (E) the cytosolic (CE) and nuclear (NE) extracts were prepared and subjected to Western blot using an anti-Nrf2 Ab. Lamin A and GAPDH were used as a marker protein for nuclear and cytosolic extracts, respectively. F: Cells were treated with CoPP IX for 16 h. Cells were fixed, and then labeled using an anti-Nrf2 Ab and FITC-conjugated secondary Ab. Individual cells were imaged.
Figure 2. Overexpression of Nrf2-dependent HO-1 inhibits VCAM-1 and ICAM-1 expression in HTSMCs. Cells were pretreated with CoPP IX for 16 h in the presence or absence of ZnPP IX, and then treated with TNF- for (A) 24 h or (B) 5 h. The protein levels and mRNA expression of VCAM-1 and ICAM-1 were determined. C:
Cells were transiently transfected with an ICAM-1- or VCAM-1-luc reporter gene, pretreated with CoPP IX for 16 h in the presence or absence of ZnPP IX, and then treated with TNF- for 4 h. The luciferase activity of ICAM-1 or VCAM-1 was measured. D, E: HTSMCs were pretreated with CoPP IX for 16 h in the presence or absence of ZnPP IX, and then treated with TNF- for 24 h. The (D) THP-1 cells adherence and (E) IL-6 generation were measured. F, G: Cells were pretreated with CoPP IX for 16 h, transfected with siRNA of HO-1, Nrf2, or scrambled, and then incubated with TNF- for 24 h. The (F) levels of VCAM-1 and ICAM-1 expression and (G) IL-6 generation were measured. Data are expressed as mean ± S.E.M. of three independent experiments. Significant differences between the compared groups are indicated: *P < 0.05; #P < 0.01.
Figure 3. Overexpression of HO-1 inhibits VCAM-1 and ICAM-1 expression in vivo.
Mice were intra-tracheally injected with CoPP IX for 16 h in the presence or absence of ZnPP IX, and then injected with TNF- for 24 h. A: Airway tissues were homogenized to extract protein. The levels of ICAM-1 and VCAM-1 expression were determined. B: BAL fluid was acquired and leukocyte count was determined by a hemocytometer. Significant differences between the compared groups are indicated:
*P < 0.05; #P < 0.01. C: Immunohistochemical staining for VCAM-1, ICAM-1, or -actin in serial sections of the airways from BSA-treated mice (Sham), TNF--injected mice (TNF-), CoPP pretreated mice (CoPP IX + TNF-), and CoPP
IX-pretreated in the presence of ZnPP IX mice (CoPP IX + ZnPP IX + TNF-). The airway smooth muscle (ASM) is indicated by an arrowhead. The arrow indicates smooth muscle cells overlapping with VCAM-1 and ICAM-1 expression. H&E stain for the airway from untreated mice was observed (top).
Figure 4. TNF--enhanced ICAM-1 and VCAM-1 expression are mediated by the formation of a TNFR1/c-Src complex. A: Cells were pretreated with an anti-TNFR1 neutralizing Ab or PP1, and then incubated with TNF- for 24 h. The levels of VCAM-1 and ICAM-1 expression were determined. B: Cells were pretreated with or without PP1 (10 M), and then stimulated with TNF- for the indicated time intervals. The cell lysates were subjected to Western blot using an anti-phospho-c-Src (Tyr416) Ab. C: Cells were treated with TNF- for the indicated time intervals and the cell lysates were subjected to immunoprecipitation using an anti-TNFR1 Ab, and then the immunoprecipitates were analyzed by Western blot using an c-Src or anti-TNFR1 Ab.
Figure 5. c-Src involves in TNF--mediated NADPH oxidase activation. A: DHE and DCF fluorescence intensities after stimulation with TNF- for 1 h in the presence or absence of PP1 (10 M) were measured by a flow cytometer. B, C: Cells were pretreated with PP1 for 1 h, and then treated with TNF- for 1 h. B: NADPH oxidase activity was measured. Data are expressed as mean ± S.E.M. of three independent experiments. Significant differences between the compared groups are indicated: *P <
0.05; #P < 0.01. C: The membrane and cytosolic extracts were prepared and subjected to Western blot using an anti-p47phox Ab. D: Cells were pretreated without or with PP1 for 1 h, and then stimulated with TNF- for 10 min. Total cell lysates were subjected to immunoprecipitation with anti-p47phox Ab and the immunoprecipitates were analyzed by Western blot using an anti-phosphotyrosine Ab. E: Cells were pretreated without or with PP1, and then stimulated with TNF- for 10 min. The cell lysates were subjected to immunoprecipitation using an anti-c-Src Ab, and then the immunoprecipitates were analyzed by Western blot using an anti-p47phox or anti-c-Src Ab.
Figure 6. Overexpression of HO-1 inhibits TNF--induced ROS generation by
down-regulation of the association of TNFR1/c-Src/p47phox. A: DHE and DCF fluorescence intensities of HTSMCs after stimulation with TNF- for 1 h in the presence or absence of CoPP IX (1 M) or ZnPP IX (1 M) were measured. B: DHE or DCF fluorescence image after 1 h of stimulation with TNF- in the presence or absence of CoPP IX or ZnPP IX. Images shown are representative of three independent experiments with similar results. C: Cells were pretreated with CoPP IX for 16 h in the presence or absence of ZnPP IX or pretreated with DPI or APO, and then treated with TNF- for 1 h. NADPH oxidase activity was determined. Data are expressed as mean ± S.E.M. of three independent experiments. Significant differences between the compared groups are indicated: *P < 0.05. D: Cells were pretreated with CoPP IX for 16 h in the presence or absence of ZnPP IX or pretreated with APO, and then treated with TNF- for 1 h. The membrane and cytosolic extracts were prepared and subjected to Western blot using an anti-p47phox Ab. E, F, G: Cells were pretreated with CoPP IX in the presence or absence of ZnPP IX, and then stimulated with
TNF- for 10 min. E: Total cell lysates were subjected to immunoprecipitation with anti-p47phox Ab and the immunoprecipitates were analyzed by Western blot using an anti-phosphotyrosine or anti-p47phox Ab. F: The cell lysates were subjected to Western blot using an anti-phospho-c-Src (Tyr416) or anti-c-Src Ab. G: The cell lysates were subjected to immunoprecipitation using an anti-TNFR1 or anti-c-Src Ab, and then the immunoprecipitates were analyzed by Western blot using an anti-TNFR1, anti-p47phox, or anti-c-Src Ab.
Figure 7. Overexpression of Nrf2-dependent HO-1 inhibits NF-B activation and translocation. A: Cells were stimulated with TNF- for the indicated time intervals.
The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser536) or anti-phospho-IB (Ser32) Ab. The cytosolic and nuclear extracts were prepared and subjected to Western blot using an anti-p65 or anti-IB Ab (left). Cells were pretreated with HLN (1 M), PP1 (10 M), NAC (10 mM), DPI (10 M), or APO (100 M), and then treated with TNF- for 1 h. The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser536) Ab (right). B, C: Cells were pretreated with CoPP IX for 16 h in the presence or absence of ZnPP IX, and then treated with TNF- for 1 h. B: The cell lysates were subjected to Western blot using an anti-phospho-p65 (Ser536) or anti-phospho-IB (Ser32) Ab. The cytosolic and
nuclear extracts were prepared and subjected to Western blot using an anti-p65 or anti-IB Ab. C: Cells were fixed, and then labeled with anti-p65 Ab and FITC-conjugated secondary Ab. Individual cells were imaged as described in Methods. D:
Cells were transiently transfected with a NF-B-luc reporter gene, and then treated with TNF- for the indicated time intervals (left) or for 1 h in the presence or absence of CoPP IX or ZnPP IX (right). The luciferase activity of NF-B was measured. Data represent the mean ± S.E.M. from at least three independent experiments. *P < 0.05;
#P < 0.01 as compared with the basal level (left). Significant differences between the compared groups are indicated: *P < 0.05; #P < 0.01 (right).
Figure 8. Adenovirus-regulated HO-1 transduction reduces TNF--induced inflammatory responses. A: HTSMCs were infected with an indicated multiplicity of infection (MOI) of either Adv or Adv-HO-1 for the indicated time intervals. HTSMCs were infected with 10 MOI of Adv or Adv-HO-1 for 48 h in the presence or absence of ZnPP IX, followed by treatment with TNF- for (B, C, D) 24 h or (E, F) 1 h. The (B) levels of VCAM-1 and ICAM-1 proteins, (C) THP-1 cells adherence, (D) IL-6 generation, (E) DCF fluorescence intensity, and (F) p65 phosphorylation at Ser536 were measured. Data are expressed as mean ± S.E.M. of three independent experiments. Significant differences between the compared groups are indicated: *P <
0.05; #P < 0.01.
Figure 9. Endogenous CO production in CoPP IX-treated HTSMCs and its role on the attenuation of TNF--mediated adhesion molecules expression and ROS generation. Cells were pretreated with 50 M CO-RM2, and then incubated with TNF- for (A, C) 24 h or (B) 1 h. A: The levels of VCAM-1 and ICAM-1 expression were determined. B: The fluorescence intensity (relative DCF fluorescence) was measured. C: IL-6 generation was measured. Cells were pretreated with CoPP IX in the presence or absence of 20 g/ml hemoglobin (Hb), and then incubated with
TNF- for (D, G) 24 h or (E, F) 1 h. D: The levels of VCAM-1 and ICAM-1 expression were determined. E: The membrane and cytosolic extracts were prepared and subjected to Western blot using an anti-p47phox Ab. F: The fluorescence intensity (relative DCF fluorescence) was measured. G: IL-6 generation was measured. Data are expressed as mean ± S.E.M. of three independent experiments. Significant
differences between the compared groups are indicated: *P < 0.05; #P < 0.01.
Figure 10. A proposed pathway for overexpression of Nrf2-dependent HO-1 protects against TNF--induced oxidative stress and airway inflammation. TNF- stimulates ROS production through TNFR1/c-Src/NADPH oxidase, in turn initiates the activation of NF-B. Activated NF-B was recruited to the promoter regions of ICAM-1 and VCAM-1 leading to an increase of adhesion molecules expression.
Moreover, CoPP IX is capable of inducing HO-1 expression, activating Nrf2-dependent pathway, resulting in inhibition of c-Src and p47phox activation, ROS generation, IL-6 production, and NF-B activation, and suppressing adhesion molecules expression and THP-1 cells adherence on HTSMCs.