Figure 1.
HO-1 expression in response to MALP-2 is dependent on TLR2 and TLR6.
A. THP-1 cells were preincubated with anti-TLR2 (10 µg/ml) or anti-TLR6 (1 µg/ml) for 1 h, and then stimulated with MALP-2 (5 ng/ml) for 16 h. HO-1 protein expression was analyzed by Western blot, and then densitometric analysis was performed after normalization with β-actin protein levels. B. Cells were transiently transfected with a dominant negative (DN)-TLR2 or DN-TLR6. After 20 h of transfection, cells were incubated with 5 ng/ml of MALP-2 for another 16 h. The cell lysates were prepared and separated by SDS-PAGE for Western blot analysis of HO-1. C. Cells were cotransfected with a DN-TLR2 or DN-TLR6 and HO-1-luc reporter gene, and then stimulated with MALP-2 (5 ng/ml) for 8 h. The luciferase activity derived from HO-1 activation was normalized the transfection efficiency with β-gal, and pZERO was used as a control vector. Data shown represent results from three independent experiments (means ± SEM). *, P<0.05 and **, P<0.01 for significant difference between compared groups.
Figure 2.
MALP-2-stimulation induces c-Src phosphorylation and HO-1 expression.
A. THP-1 cells were stimulated with MALP-2 (5 ng/ml) for the indicated time points and lysed. The lysates were analyzed by Western blotting with a phosphorylated (p)-c-Src antibody (upper panel) or total c-Src antibody (lower panel). The results of a representative experiment are shown, and densitometric quantification of relative protein levels of p-c-Src from three experiments are presented under the panels. B. Cells were preincubated with PP1 for 1 h prior to stimulation with MALP-2. Cell lysates were prepared and HO-1 proteins were detected by Western blotting. C. Cells were transfected with c-Src specific siRNA or control (con) siRNA prior to 5.0 ng/ml MALP-2 treatment. Total c-Src and HO-1 protein expression levels were detected by Western blotting. Results shown are representative of at least three separate experiments. **, P<0.01 for significant difference between compared groups.
Figure 3.
MALP-2 induces HO-1 expression in a Btk-dependent manner.
A. THP-1 cells were stimulated with MALP-2 for the indicated time intervals, the cell lysates were subjected to Western blotting using an anti-phospho-Btk at Tyr223. *, P<0.05 and **, P<0.01 as compared with the basal level. B. Cells were pretreated with or without PP1 for 1 h, and then incubated in the absence or presence of 5 ng/ml MALP-2 for 10 min. Cell fractions were prepared and subjected to Western blotting analysis with an anti- phospho-Btk antibody. Data shown represents three experiments (means ± SEM). C. Cells were transfected with siRNA of c-Src, and then treated with MALP-2 for 10 min. Phosphorylated Btk was detected by Western blotting. Results shown are representative of at least three separate experiments. D. THP-1 cells were pretreated with a Btk inhibitor FLM-A13 (30 µM and 100 µM) for 1 h, and incubation was continued with MALP-2 (5 ng/ml) for another 16 h. Samples were subjected to Western blotting for the detection of HO-1 expression. Densitometry analysis was performed on at least three Western blots quantified by scanning densitometry. E. Cells were transiently transfected with HO-1-luc reporter gene, and then pretreated with LFM-A13. After incubation for 1 h, cells were stimulated with MALP-2 (5 ng/ml) for 8 h. The luciferase activity derived from HO-1 activation was normalized to the transfection efficiency with β-gal. Data represent means ± SEM from at least three independent experiments. *, P<0.05 and **, P<0.01 for significant difference between compared groups.
Figure 4.
MyD88 does not play a central role in MALP-2-induced HO-1 expression.
A. THP-1 cells were transiently transfected with a dominant negative plasmid encoding MyD88 (DN-MyD88) or empty vector (pDeNy, lane 2). Cell lysates were prepared, and samples were immunoblotted with an anti-HO-1 antibody. B. Cells were transfected with MyD88 siNRA or control siRNA, and then treated with MALP-2 (5 ng/ml) for 16 h. Expression of MyD88 and HO-1 were analyzed by Western blotting. All values are expressed as means ± SEM obtained from three independent experiments. **, P<0.01 for significant difference between compared groups.
Figure 5.
Mal is essential for the expression of HO-1 induced by MALP-2.
A. Cells were transiently transfected with a dominant negative plasmid encoding Mal (DN-Mal) or empty vector (pDeNy, lane 2). Cell lysates were prepared, and samples were immunoblotted with an anti-HO-1 antibody. B. Cells were transfected with Mal siNRA or control siRNA, and then treated with MALP-2 (5 ng/ml) for 16 h. Expression of Mal and HO-1 were analyzed by Western blot. C. Cells were cotransfected with a DN-MyD88 or DN-Mal and HO-1-luc reporter gene, and then treated with MALP-2 for 8 h. The luciferase activity derived from HO-1 activation was normalized to the transfection efficiency with β-gal, and pDeNy was used as a control vector. All values are expressed as means ± SEM obtained from three independent experiments. *, P<0.05 and **, P<0.01 for significant difference between compared groups.
Figure 6.
Involvement of PI3K in the up-regulation of HO-1 by MALP-2.
A. Cells were stimulated with MALP-2 for the indicated time intervals, the cell lysates were subjected to Western blotting using an anti-p-Akt antibody. B. THP-1 cells were pretreated with LY294002 (25 µM) for 1 h, and then incubated with 5 ng/ml MALP-2 for another 16 h. Densitometric analysis was performed after normalization with total Akt or β-actin. All of the values are expressed as means ± SEM of three independent experiments. *, P<0.05 for comparisons within groups.
Figure 7.
MALP-2 induces PI3K activation is regulated by the formation of a Btk, Mal, c-Src and p85α complex.
A. Cells were pretreated with or without LFM-A13 (100 µM), or PP1 (50 µM) for 1 h, and then incubated in the absence or presence of 5 ng/ml MALP-2 for 15 min. Cell fractions were prepared and subjected to Western blotting analysis with an anti-p-Akt antibody. Data shown represents three experiments (means ± SEM). *, P<0.05 and **, P<0.01 for significant difference between compared groups. B, C. Cells were transfected with siRNA of Mal or MyD88, and then treated with MALP-2 for 15, 60 or 120 min. Phosphorylated Akt was detected by Western blotting. *, P<0.05 for significant difference between compared groups. D. Cells were treated with MALP-2 for the indicated times, the cell lysates were subjected to immunoprecipitation using an anti-c-Src or anti-p85α antibody, and followed by immunoblotting with anti-Btk, anti-Mal, anti-MyD88, anti-c-Src or anti p85α antibody. All figures are representative of three independent experiments.
Figure 8.
MALP-2 stimulates translocation of Nrf2 to the nucleus and binding to HO-1 ARE in THP-1 cells.
A. Immunofluorescence images of THP-1 cells pretreated with or without LY294002 (25 µM) and MyD88 siRNA in the presence of MALP-2 (5 ng/ml) for 2 h. Cells were fixed and then stained with 4′-6-diamidino-2-phenylindole or anti-Nrf2 antibody, followed by incubation with cy3-conjugated anti-rabbit antibody. Images were obtained using a confocal microscope. B. EMSA was performed using MALP-2-stimulated nuclear THP-1 extracts and biotinylated HO-1-ARE probe. The specificity of Nrf2 was verified by competition analysis with an excess of nonlabeled specific (cold probe) or nonspecific (NF-κB probe) oligonucleotide probe. For supershift analysis, nuclear extracts from MALP-2-treated THP-1 cells were preincubated with 1 µg anti-Nrf2 supershift antibody before EMSA.
Figure 9.
Schematic model depicting the potential signaling pathway involved in MALP-2-induced HO-1 expression in THP-1 cells.
The cytoplasmic adaptor Mal recruits the TLR2/6 heterodimer in response to MALP-2 stimulation. Mal binds to p85α, the regulatory subunit of PI3K, in the presence of Btk and c-Src. PI3K activates downstream events like Akt phosphorylation and Nrf2 nuclear translocation, which in turn initiates HO-1 expression.