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PIM2 Induced COX-2 and MMP-9 expression in macrophages requires PI3K and Notch1 signaling.

Bansal K, Kapoor N, Narayana Y, Puzo G, Gilleron M, Balaji KN - PLoS ONE (2009)

Bottom Line: PIM2 triggered significant p65 nuclear factor -kappaB (NF-kappaB) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling.Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NF-kappaB to respective promoters.Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.

ABSTRACT
Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor -kappaB (NF-kappaB) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NF-kappaB to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

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Related in: MedlinePlus

PIM2 induces expression of COX-2 and MMP-9 in mouse peritoneal macrophages.(A). Mouse peritoneal macrophages were treated with 0.5 and 4.0 µg/ml of PIM2 and mRNA levels of COX-2 and MMP-9 were analyzed by Quantitative real time PCR. (B). The levels of COX-2 and MMP-9 protein expression were evaluated by immunoblotting in total cell lysates prepared from macrophages treated with 4.0 µg/ml of PIM2 for different time points. (C). Flow cytometric analysis of MMP-9 expression on the surface of PIM2 treated macrophages. Cells were probed with anti-MMP-9 or isotype matched control antibody followed by anti- rabbit FITC. (D). Immunoflourescent staining of MMP-9 on macrophages treated with 4.0 µg/ml of PIM2 as analyzed by confocal microscopy. Cells were fixed and MMP-9 expression was detected by binding of specific or isotype matched antibodies followed by probing with Cy-2 (green) labeled anti-rabbit secondary antibody. Nucleus of macrophages were stained with nuclear staining dye, Hoechst 33342 (blue) and plasma membrane with anti-MHC Class I antibody-Cy5 (red). (E). Mouse macrophages were cultured with or with out NS-398 (10 µM) and treated with 4.0 µg/ml of PIM2 for 12 h. The protein levels of MMP-9 were analyzed by immunoflourescent staining of MMP-9 followed by flow cytometry. The data represented in the figure are representative of three independent experiments. Med, Medium.
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pone-0004911-g001: PIM2 induces expression of COX-2 and MMP-9 in mouse peritoneal macrophages.(A). Mouse peritoneal macrophages were treated with 0.5 and 4.0 µg/ml of PIM2 and mRNA levels of COX-2 and MMP-9 were analyzed by Quantitative real time PCR. (B). The levels of COX-2 and MMP-9 protein expression were evaluated by immunoblotting in total cell lysates prepared from macrophages treated with 4.0 µg/ml of PIM2 for different time points. (C). Flow cytometric analysis of MMP-9 expression on the surface of PIM2 treated macrophages. Cells were probed with anti-MMP-9 or isotype matched control antibody followed by anti- rabbit FITC. (D). Immunoflourescent staining of MMP-9 on macrophages treated with 4.0 µg/ml of PIM2 as analyzed by confocal microscopy. Cells were fixed and MMP-9 expression was detected by binding of specific or isotype matched antibodies followed by probing with Cy-2 (green) labeled anti-rabbit secondary antibody. Nucleus of macrophages were stained with nuclear staining dye, Hoechst 33342 (blue) and plasma membrane with anti-MHC Class I antibody-Cy5 (red). (E). Mouse macrophages were cultured with or with out NS-398 (10 µM) and treated with 4.0 µg/ml of PIM2 for 12 h. The protein levels of MMP-9 were analyzed by immunoflourescent staining of MMP-9 followed by flow cytometry. The data represented in the figure are representative of three independent experiments. Med, Medium.

Mentions: In this study, we examined the molecular details of PIM2 induced signaling events that are leading to the expression of COX-2 and MMP-9. PIM2 treatment of mouse peritoneal macrophages triggers the expression of COX-2 and MMP-9 both at mRNA and protein levels (Figure 1A–B). PIM2 triggered expression of both COX-2 and MMP-9 transcripts as early as 2 hours, clearly peaking in their expression at 12 hrs (Figure S1A). Accordingly, PIM2 induced COX-2 and MMP-9 protein expression could be detected as early as 2 hours (faint band) up to 16 hours of the post-PIM2 treatment (Figure 1B). Further, in addition to the release of MMP-9 in the medium, experiments were carried out to explore whether PIM2 increases the cell membrane associated MMP-9 by flow cytometry. PIM2 treatment increased the percentage of cells that were stained positive for membrane MMP-9 compared to untreated cells (Figure 1C). Confocal microscopy studies further validated PIM2 triggered increased cell-surface association of MMP-9, where in macrophages were stained for nucleus (with nuclear staining dye, Hoechst 33342, blue), plasma membrane (with anti-MHC Class I antibody, red) and for MMP-9 (with anti-MMP-9 antibody, green). The merged images from confocal microscopic analysis revealed that MMP-9 or MHC class I do not colocalize clearly demonstrating a distinct cell-surface association of MMP-9 (Figure 1D and Figure S1B). COX-2 is known to regulate mycobacteria triggered MMP-9 expression and accordingly, when tested, COX-2 inhibitor NS-398 markedly blocked MMP-9 expression both at RNA (Figure S2A) and protein levels clearly implicating the role COX-2 dependent signaling events in PIM2 induced MMP-9 expression (Figure S2B). In order to further ascertain the specific effect of NS-398, we analyzed the cell-surface expression of MMP-9 upon PIM2 treatment in the presence or absence of NS-398. Flow cytometry results presented in Figure 1E demonstrate that blocking COX-2 activity by NS-398 significantly reduced PIM2 triggered surface MMP-9 expression. Further, purified PGE2 increased MMP-9 expression in a dose dependent manner in mouse macrophages (Figure S3). In order to exclude the autocrine or secondary effect of macrophage produced cytokines on PIM2 triggered COX-2 and MMP-9 expression, mouse macrophages were treated with PIM2 in the presence or absence of cycloheximide. The data presented clearly demonstrates that PIM2 triggered COX-2 and MMP-9 mRNA expression was not affected by cycloheximide clearly ruling out the autocrine effect of PIM2 induced cytokines on COX-2 and MMP-9 expression (Figure S4A–B). However, interestingly, cycloheximide augmented PIM2 triggered COX-2 mRNA expression. Extensive literature survey suggests that the regulation of COX-2 gene is complex and transcription of COX-2 is reported to vary across the cell types as well as among responses of a given cell type to variety of stimuli [54]–[56]. This effect has been attributed to the presence of AU-rich elements in 3′-untranslated region of COX-2 mRNA, which is suggested to be an important determinant in COX-2 mRNA stability in human macrophages [55] as well as in diverse cell types [54].


PIM2 Induced COX-2 and MMP-9 expression in macrophages requires PI3K and Notch1 signaling.

Bansal K, Kapoor N, Narayana Y, Puzo G, Gilleron M, Balaji KN - PLoS ONE (2009)

PIM2 induces expression of COX-2 and MMP-9 in mouse peritoneal macrophages.(A). Mouse peritoneal macrophages were treated with 0.5 and 4.0 µg/ml of PIM2 and mRNA levels of COX-2 and MMP-9 were analyzed by Quantitative real time PCR. (B). The levels of COX-2 and MMP-9 protein expression were evaluated by immunoblotting in total cell lysates prepared from macrophages treated with 4.0 µg/ml of PIM2 for different time points. (C). Flow cytometric analysis of MMP-9 expression on the surface of PIM2 treated macrophages. Cells were probed with anti-MMP-9 or isotype matched control antibody followed by anti- rabbit FITC. (D). Immunoflourescent staining of MMP-9 on macrophages treated with 4.0 µg/ml of PIM2 as analyzed by confocal microscopy. Cells were fixed and MMP-9 expression was detected by binding of specific or isotype matched antibodies followed by probing with Cy-2 (green) labeled anti-rabbit secondary antibody. Nucleus of macrophages were stained with nuclear staining dye, Hoechst 33342 (blue) and plasma membrane with anti-MHC Class I antibody-Cy5 (red). (E). Mouse macrophages were cultured with or with out NS-398 (10 µM) and treated with 4.0 µg/ml of PIM2 for 12 h. The protein levels of MMP-9 were analyzed by immunoflourescent staining of MMP-9 followed by flow cytometry. The data represented in the figure are representative of three independent experiments. Med, Medium.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654112&req=5

pone-0004911-g001: PIM2 induces expression of COX-2 and MMP-9 in mouse peritoneal macrophages.(A). Mouse peritoneal macrophages were treated with 0.5 and 4.0 µg/ml of PIM2 and mRNA levels of COX-2 and MMP-9 were analyzed by Quantitative real time PCR. (B). The levels of COX-2 and MMP-9 protein expression were evaluated by immunoblotting in total cell lysates prepared from macrophages treated with 4.0 µg/ml of PIM2 for different time points. (C). Flow cytometric analysis of MMP-9 expression on the surface of PIM2 treated macrophages. Cells were probed with anti-MMP-9 or isotype matched control antibody followed by anti- rabbit FITC. (D). Immunoflourescent staining of MMP-9 on macrophages treated with 4.0 µg/ml of PIM2 as analyzed by confocal microscopy. Cells were fixed and MMP-9 expression was detected by binding of specific or isotype matched antibodies followed by probing with Cy-2 (green) labeled anti-rabbit secondary antibody. Nucleus of macrophages were stained with nuclear staining dye, Hoechst 33342 (blue) and plasma membrane with anti-MHC Class I antibody-Cy5 (red). (E). Mouse macrophages were cultured with or with out NS-398 (10 µM) and treated with 4.0 µg/ml of PIM2 for 12 h. The protein levels of MMP-9 were analyzed by immunoflourescent staining of MMP-9 followed by flow cytometry. The data represented in the figure are representative of three independent experiments. Med, Medium.
Mentions: In this study, we examined the molecular details of PIM2 induced signaling events that are leading to the expression of COX-2 and MMP-9. PIM2 treatment of mouse peritoneal macrophages triggers the expression of COX-2 and MMP-9 both at mRNA and protein levels (Figure 1A–B). PIM2 triggered expression of both COX-2 and MMP-9 transcripts as early as 2 hours, clearly peaking in their expression at 12 hrs (Figure S1A). Accordingly, PIM2 induced COX-2 and MMP-9 protein expression could be detected as early as 2 hours (faint band) up to 16 hours of the post-PIM2 treatment (Figure 1B). Further, in addition to the release of MMP-9 in the medium, experiments were carried out to explore whether PIM2 increases the cell membrane associated MMP-9 by flow cytometry. PIM2 treatment increased the percentage of cells that were stained positive for membrane MMP-9 compared to untreated cells (Figure 1C). Confocal microscopy studies further validated PIM2 triggered increased cell-surface association of MMP-9, where in macrophages were stained for nucleus (with nuclear staining dye, Hoechst 33342, blue), plasma membrane (with anti-MHC Class I antibody, red) and for MMP-9 (with anti-MMP-9 antibody, green). The merged images from confocal microscopic analysis revealed that MMP-9 or MHC class I do not colocalize clearly demonstrating a distinct cell-surface association of MMP-9 (Figure 1D and Figure S1B). COX-2 is known to regulate mycobacteria triggered MMP-9 expression and accordingly, when tested, COX-2 inhibitor NS-398 markedly blocked MMP-9 expression both at RNA (Figure S2A) and protein levels clearly implicating the role COX-2 dependent signaling events in PIM2 induced MMP-9 expression (Figure S2B). In order to further ascertain the specific effect of NS-398, we analyzed the cell-surface expression of MMP-9 upon PIM2 treatment in the presence or absence of NS-398. Flow cytometry results presented in Figure 1E demonstrate that blocking COX-2 activity by NS-398 significantly reduced PIM2 triggered surface MMP-9 expression. Further, purified PGE2 increased MMP-9 expression in a dose dependent manner in mouse macrophages (Figure S3). In order to exclude the autocrine or secondary effect of macrophage produced cytokines on PIM2 triggered COX-2 and MMP-9 expression, mouse macrophages were treated with PIM2 in the presence or absence of cycloheximide. The data presented clearly demonstrates that PIM2 triggered COX-2 and MMP-9 mRNA expression was not affected by cycloheximide clearly ruling out the autocrine effect of PIM2 induced cytokines on COX-2 and MMP-9 expression (Figure S4A–B). However, interestingly, cycloheximide augmented PIM2 triggered COX-2 mRNA expression. Extensive literature survey suggests that the regulation of COX-2 gene is complex and transcription of COX-2 is reported to vary across the cell types as well as among responses of a given cell type to variety of stimuli [54]–[56]. This effect has been attributed to the presence of AU-rich elements in 3′-untranslated region of COX-2 mRNA, which is suggested to be an important determinant in COX-2 mRNA stability in human macrophages [55] as well as in diverse cell types [54].

Bottom Line: PIM2 triggered significant p65 nuclear factor -kappaB (NF-kappaB) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling.Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NF-kappaB to respective promoters.Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.

ABSTRACT
Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor -kappaB (NF-kappaB) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NF-kappaB to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

Show MeSH
Related in: MedlinePlus