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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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PIM2 induced expression and activation of Notch1 is involved in induced expression of COX-2 and MMP-9 in macrophages.(A). Real-time PCR quantification of Notch1 mRNA expression levels in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h (B). Kinetics of protein expression of Cleaved Notch1 was analyzed in mouse macrophages treated with 4.0 µg/ml of PIM2 for the indicated time points. (C). Hes1 transcript levels were assessed by real time PCR in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h. (D). RAW 264.7 macrophages stably transfected with pCMV-NICD, Notch intracellular domain, (RAW-NICD) or vector pCMV (RAW-Vec) were analyzed for the expression of NICD, COX-2, MMP-9 and β-actin as loading control. (E). PIM2 (4.0 µg/ml) treated macrophages were cultured with or with out γ-secretase inhibitor GSI-I (10 µM) and the levels of COX-2 and MMP-9 transcript (at 12 h) were analyzed by quantitative real time PCR. (F). Macrophages were treated with 4.0 µg/ml of PIM2 for 12 h cultured in the presence of LY294002 (50 µM), GSI-I (10 µM) or 0.1% DMSO and cell surface expression of MMP-9 was analyzed by flow cytometry. (G). RAW 264.7 macrophages were transfected with 100 nM of small interfering RNA (siRNA) directed to either Notch1 or RBP-Jk siRNA or control siRNA. Three days post transfection, cells were treated with PIM2 for 12 h and protein levels of COX-2 as well as MMP-9 were analyzed. The blots from three independent experiments were quantitated by densitometry and represented as fold change over medium. The results are expressed as mean±SEM of three independent experiments and the blots are representative of three independent experiments. Med, Medium.
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pone-0004911-g003: PIM2 induced expression and activation of Notch1 is involved in induced expression of COX-2 and MMP-9 in macrophages.(A). Real-time PCR quantification of Notch1 mRNA expression levels in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h (B). Kinetics of protein expression of Cleaved Notch1 was analyzed in mouse macrophages treated with 4.0 µg/ml of PIM2 for the indicated time points. (C). Hes1 transcript levels were assessed by real time PCR in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h. (D). RAW 264.7 macrophages stably transfected with pCMV-NICD, Notch intracellular domain, (RAW-NICD) or vector pCMV (RAW-Vec) were analyzed for the expression of NICD, COX-2, MMP-9 and β-actin as loading control. (E). PIM2 (4.0 µg/ml) treated macrophages were cultured with or with out γ-secretase inhibitor GSI-I (10 µM) and the levels of COX-2 and MMP-9 transcript (at 12 h) were analyzed by quantitative real time PCR. (F). Macrophages were treated with 4.0 µg/ml of PIM2 for 12 h cultured in the presence of LY294002 (50 µM), GSI-I (10 µM) or 0.1% DMSO and cell surface expression of MMP-9 was analyzed by flow cytometry. (G). RAW 264.7 macrophages were transfected with 100 nM of small interfering RNA (siRNA) directed to either Notch1 or RBP-Jk siRNA or control siRNA. Three days post transfection, cells were treated with PIM2 for 12 h and protein levels of COX-2 as well as MMP-9 were analyzed. The blots from three independent experiments were quantitated by densitometry and represented as fold change over medium. The results are expressed as mean±SEM of three independent experiments and the blots are representative of three independent experiments. Med, Medium.

Mentions: In order to further delineate the signaling events, peritoneal macrophages were treated with LY294002, a PI3K inhibitor before PIM2 treatment. The inhibitor LY294002 significantly reduced PIM2 activated 4EBP1 phosphorylation (Figure 2G). During this stage of current investigation, experimental results obtained in parallel suggested the involvement of Notch1 signaling in PIM2 induced COX-2 and MMP-9 expression. These results are presented in detail in Figure 3 of this manuscript. Since it has been suggested that PI3K-AKT pathway can operate in concert as a gain control for Notch signal responses in many cell types, attempts were carried out to assess whether Notch activation inhibitor, γ-secretase inhibitor-I (GSI-I) could abrogate PIM2 triggered phopshorylation of AKT, mTOR, and 4EBP1. GSI-I, when tested, significantly blocked PIM2 triggered AKT, mTOR and 4EBP1 (Figure S6 and Figure 2G). These findings implicate possible cross-talks between PI3K and Notch signaling cascades during PIM2 induced expression of COX-2 and MMP-9.


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 induced expression and activation of Notch1 is involved in induced expression of COX-2 and MMP-9 in macrophages.(A). Real-time PCR quantification of Notch1 mRNA expression levels in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h (B). Kinetics of protein expression of Cleaved Notch1 was analyzed in mouse macrophages treated with 4.0 µg/ml of PIM2 for the indicated time points. (C). Hes1 transcript levels were assessed by real time PCR in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h. (D). RAW 264.7 macrophages stably transfected with pCMV-NICD, Notch intracellular domain, (RAW-NICD) or vector pCMV (RAW-Vec) were analyzed for the expression of NICD, COX-2, MMP-9 and β-actin as loading control. (E). PIM2 (4.0 µg/ml) treated macrophages were cultured with or with out γ-secretase inhibitor GSI-I (10 µM) and the levels of COX-2 and MMP-9 transcript (at 12 h) were analyzed by quantitative real time PCR. (F). Macrophages were treated with 4.0 µg/ml of PIM2 for 12 h cultured in the presence of LY294002 (50 µM), GSI-I (10 µM) or 0.1% DMSO and cell surface expression of MMP-9 was analyzed by flow cytometry. (G). RAW 264.7 macrophages were transfected with 100 nM of small interfering RNA (siRNA) directed to either Notch1 or RBP-Jk siRNA or control siRNA. Three days post transfection, cells were treated with PIM2 for 12 h and protein levels of COX-2 as well as MMP-9 were analyzed. The blots from three independent experiments were quantitated by densitometry and represented as fold change over medium. The results are expressed as mean±SEM of three independent experiments and the blots are representative of three independent experiments. Med, Medium.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654112&req=5

pone-0004911-g003: PIM2 induced expression and activation of Notch1 is involved in induced expression of COX-2 and MMP-9 in macrophages.(A). Real-time PCR quantification of Notch1 mRNA expression levels in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h (B). Kinetics of protein expression of Cleaved Notch1 was analyzed in mouse macrophages treated with 4.0 µg/ml of PIM2 for the indicated time points. (C). Hes1 transcript levels were assessed by real time PCR in mouse macrophages after treatment with 4.0 µg/ml of PIM2 for 12 h. (D). RAW 264.7 macrophages stably transfected with pCMV-NICD, Notch intracellular domain, (RAW-NICD) or vector pCMV (RAW-Vec) were analyzed for the expression of NICD, COX-2, MMP-9 and β-actin as loading control. (E). PIM2 (4.0 µg/ml) treated macrophages were cultured with or with out γ-secretase inhibitor GSI-I (10 µM) and the levels of COX-2 and MMP-9 transcript (at 12 h) were analyzed by quantitative real time PCR. (F). Macrophages were treated with 4.0 µg/ml of PIM2 for 12 h cultured in the presence of LY294002 (50 µM), GSI-I (10 µM) or 0.1% DMSO and cell surface expression of MMP-9 was analyzed by flow cytometry. (G). RAW 264.7 macrophages were transfected with 100 nM of small interfering RNA (siRNA) directed to either Notch1 or RBP-Jk siRNA or control siRNA. Three days post transfection, cells were treated with PIM2 for 12 h and protein levels of COX-2 as well as MMP-9 were analyzed. The blots from three independent experiments were quantitated by densitometry and represented as fold change over medium. The results are expressed as mean±SEM of three independent experiments and the blots are representative of three independent experiments. Med, Medium.
Mentions: In order to further delineate the signaling events, peritoneal macrophages were treated with LY294002, a PI3K inhibitor before PIM2 treatment. The inhibitor LY294002 significantly reduced PIM2 activated 4EBP1 phosphorylation (Figure 2G). During this stage of current investigation, experimental results obtained in parallel suggested the involvement of Notch1 signaling in PIM2 induced COX-2 and MMP-9 expression. These results are presented in detail in Figure 3 of this manuscript. Since it has been suggested that PI3K-AKT pathway can operate in concert as a gain control for Notch signal responses in many cell types, attempts were carried out to assess whether Notch activation inhibitor, γ-secretase inhibitor-I (GSI-I) could abrogate PIM2 triggered phopshorylation of AKT, mTOR, and 4EBP1. GSI-I, when tested, significantly blocked PIM2 triggered AKT, mTOR and 4EBP1 (Figure S6 and Figure 2G). These findings implicate possible cross-talks between PI3K and Notch signaling cascades during PIM2 induced expression of COX-2 and MMP-9.

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