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

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.

Figure 1. 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.

Figure 2. PIM2 induced upregulation of COX-2 and MMP-9 requires Notch1 mediated activation of PI3 Kinase pathway.

(A). COX-2 and MMP-9 transcript levels were analyzed in mouse macrophages pretreated with LY294002 (50 µM), AKT inhibitor II (10 µM) or 0.1% DMSO as vehicle control and then cultured in the presence of PIM2 (4.0 µg/ml) for 12 h. (B). MMP-9 at the cell surface was analyzed by flow cytometry on PIM2 treated macrophages cultured with or without AKT inhibitor II (10 µM) or 0.1% DMSO as vehicle control. (C). Kinetics of phosphorylation of AKT and 4EBP1 upon treatment of PIM2. The blots were stripped and probed for total levels of AKT and 4EBP1 as control. (D). RAW 264.7 macrophages were transiently transfected with either control siRNA or AKT siRNA. After three days of transfection, cells were treated with PIM2 and protein levels of AKT as well as COX-2 and MMP-9 were analyzed. The blots from three independent experiments were densitometrically quantitated and represented as fold change over medium. (E). RAW 264.7 macrophages were transfected with either empty vector or dominant-negative AKT (AKT-DN) followed by treatment with PIM2. Transcript levels of COX-2 were analyzed by quantitative real-time PCR and (F). Surface expression of MMP-9 was analyzed by flow cytometry using rabbit anti-MMP-9 antibody followed by probing with Cy-2 labeled anti-rabbit secondary antibody. (G). Mouse macrophages were pretreated with LY294002 (50 µM) or GSI-I (10 µM) and activation of 4EBP1 at 30 and 60 min post treatment of PIM2 was analyzed. Histogram represents densitometric analysis of phospho-4EBP1 normalized against total 4EBP1 derived from three independent experiments. The results shown represent three independent experiments. Med, Medium.

Figure 3. 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.

Figure 4. Soluble Jagged 1 significantly inhibits PIM2 triggered cleaved Notch1 (NICD), COX-2 or MMP-9 expression.

(A). Cleaved Notch1 levels were assessed in RAW 264.7 macrophages transfected either with soluble Jagged1 construct or Vector construct followed by treatment with PIM2. (B). As described in (A), in soluble Jagged1 construct transfected cells, transcript levels of COX-2 were analyzed by quantitative real-time PCR and (C). surface expression of MMP-9 by flow cytometry. The results presented are representative of three independent experiments. Med, Medium.

Figure 5. TLR2-MyD88 axis plays important role in PIM2 triggered Notch1 activation.

(A). TLR2 dominant-negative construct significantly reduced PIM2 triggered MMP-9 expression as assayed by MMP-9 luciferase reporter activity as described in Materials and Methods. (B). siRNA mediated downregulation of MyD88 in PIM2 triggered nuclear localization of NICD as well as (C). in expression of Notch1 target gene, Hes1, as analyzed by confocal microscopy. (D). RAW 264.7 macrophages were transfected with either vector or dominant-negative TLR2 (TLR2-DN) and Hes1-Luc, followed by treatment with PIM2. The Hes1 promoter activity was evaluated by Luciferase assay. Data presented in the figure is representative of three independent experiments. Med, Medium.

Figure 6. PIM2 induced activation of ERK1/2 and NF-κB is dependent on Notch1 mediated activation of PI3 kinase pathway.

(A). Mouse macrophages were treated with U0126 (10 µM), SB203580 (20 µM), SP600125 (50 µM), Manumycin (20 µM) or DMSO as vehicle control, prior to treatment with PIM2 for 12 h and COX-2 and MMP-9 mRNA expression levels were analyzed by real time PCR. (B). MMP-9 levels on the cell surface were assessed by flow cytometry on PIM2 treated macrophages pretreated with Manumycin (20 µM), U0126 (10 µM) or DMSO. (C). Time dependent activation of Raf-1 and ERK1/2 upon treatment macrophages with 4.0 µg/ml of PIM2. (D). Mouse peritoneal macrophages were pretreated with LY294002 (50 µM) or GSI-I (10 µM) and phosphorylation status of Raf-1 and ERK1/2 at indicated time points after treatment with PIM2 was assessed. (E). Mouse macrophages were treated with PIM2 for indicated time points and were fractionated into cytosolic, nuclear fractions and probed for NF-κB by immunoblotting. (F). Nuclear translocation of NF-κB upon treatment of macrophages with PIM2. Macrophages were treated with U0126 (10 µM), LY294002 (50 µM), GSI-I (10 µM) prior to PIM2 treatment. The graphs show the average of three independent experiments±SEM. Representative blots from three independent experiments are shown. Med, Medium.

Figure 7. Association of PIM2 triggered cell surface MMP-9 associates with CD44.

(A). Mouse macrophages were treated with PIM2 for 12 h and cell surface levels of CD44 were analyzed by probing with anti-CD44 or isotype matched control antibody followed by Cy5-conjugated goat anti- rat IgG. (B). CD44 levels on the cell surface were assessed by flow cytometry on PIM2 treated macrophages pretreated with LY294002 (50 µM) or GSI-I (10 µM). (C). Surface expression of MMP-9 and CD44 were evaluated by immunofluorescent staining using confocal microscopy. After 12 h treatment with PIM2, macrophages were probed with anti MMP-9 followed by anti rabbit IgG-Cy-2(green) as well as anti CD44 followed by anti- rat IgG-Cy5 (red). Merged image shows the areas of colocalization in yellow. The results are representative of three independent experiments. Med, Medium.

Figure 8. Model depicting PIM2 triggered signaling cascades regulating COX-2 and MMP-9 gene expression.