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First published online February 6, 2008
doi: 10.1242/10.1242/jcs.016980

Journal of Cell Science 121, 487-495 (2008)
Published by The Company of Biologists 2008
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A role for membrane-bound CD147 in NOD2-mediated recognition of bacterial cytoinvasion

Andreas Till1,*, Philip Rosenstiel1,2,*,{ddagger}, Karen Bräutigam1, Christian Sina3, Gunnar Jacobs1, Hans-Heinrich Oberg4, Dirk Seegert5, Trinad Chakraborty6 and Stefan Schreiber1,3,{ddagger}

1 Institute for Clinical Molecular Biology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 12, 24105 Kiel, Germany
2 Max-Planck Institute for Molecular Genetics, Berlin, Germany
3 1st Department for General Internal Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 12, 24105 Kiel, Germany
4 Institute of Immunology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 12, 24105 Kiel, Germany
5 CONARIS Research Institute AG, Kiel, Germany
6 Institute for Medical Microbiology and Hygiene, University of Giessen, Germany


Figure 1
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  		Fig. 1. The CARD domains of NOD2 interact with the intracellular stalk of CD147. (A) HEK293 cells stably transfected with NOD2 expression construct (HEKNOD2) or empty vector (HEKmock) were used for co-immunoprecipitation of CD147 and NOD2. CD147 immunocomplexes and whole cell lysates were assayed for endogenous and precipitated NOD2 and CD147. Precipitates from irrelevant antibody were used as specificity control (ctrl). (B) HEK293 cells were transfected with constructs containing Xpress-tagged NOD2 or NOD2 domains. After precipitation of endogenous CD147, co-precipitated NOD2 or NOD2 domains were visualized using anti-Xpress antibody. Only full-length NOD2 and a fusion protein containing both CARD domains were found in CD147 immmunocomplexes (black arrowheads). (C) HEK293 cells were transfected with constructs containing Xpress-tagged NOD2 or NOD2 domains in combination with a construct containing the intracellular domain of CD147 (IC) fused to EGFP. After precipitation of Xpress-tagged NOD2 domains, EGFP-CD147-IC was identified in the immunocomplexes using anti-GFP antibody. Only full-length NOD2 and both CARD domains were able to precipitate with GFP-CD147-IC (indicated by black arrowheads). Antibody alone (Ab) was used as control. (D) CD147 immunocomplexes derived from THP1 monocytes were used to verify the interaction of CD147 with endogenous NOD2. Detection of two specific bands by use of NOD2 antibody points to the presence of more than one NOD2 isoform.

 

Figure 2
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  		Fig. 2. Expression and regulation of CD147. (A) CD147 mRNA levels in monocytic THP1 cells are upregulated by stimulation with MDP, TNF-{alpha} plus IFN-{gamma} and infection with L. monocytogenes. (B) THP1 monocytes were stimulated for 24 hours with different agents (thick line) or left untreated (thin line). Cell surface expression of CD147 protein was detected by FACS. Irrelevant antibody was used as specificity control (gray curve). A variety of proinflammatory stimuli (MDP, LPS, TNF-{alpha}/IFN-{gamma}) lead to an increased expression of CD147. Note that TNF-{alpha} alone showed only a minor effect.

 

Figure 3
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  		Fig. 3. Co-localization of CD147 and NOD2 at the membrane of epithelial and myelomonocytic cells. (A) HeLaS3 cells were transiently transfected with GFP-NOD2 (green) and stained for CD147 (red) and DAPI as a DNA counterstain (blue). (B) Transfected cells were infected with L. monocytogenes (MOI=100) and fixed after 1 hour. In the basal as well as in the infected state, NOD2 and CD147 colocalize in the membrane compartment. An accumulation of both NOD2 and CD147 in areas of early bacterial invasion is detectable (open arrowheads and inset in B). (C) Indirect immunofluorescence micrographs of PMA-differentiated THP1 myelomonocytic cells. Staining with specific antibodies against endogenous NOD2 (red channel) and CD147 (green channel) shows a partial colocalization at the cell membrane (arrowheads in merged image). Images are representative of at least three independent experiments. Original magnification: 100x.

 

Figure 4
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  		Fig. 4. CD147 negatively regulates NOD2-mediated NF-{kappa}B activation and IL8 release. (A) NOD2-dependent MDP-induced activation of NF-{kappa}B was determined by dual-luciferase assay. Overexpression of full-length CD147 (FL) or its intracellular domain (IC), but not of the extracellular domain (EC) lead to a decrease of NOD2-dependent NF-{kappa}B activation. (RLU, relative light units; vc, vector control; **P<0.01 for transfection of CD147 constructs versus vc). (B) Overexpression of full-length CD147 impairs NOD2-dependent IL8 release in a dose-dependent manner. HEK293 cells were transiently transfected with pcDNA4-Xpress-NOD2 and increasing amounts of pEGFP-N1-CD147 and were left untreated or were treated with MDP. Supernatants were assayed for IL8 by enzyme-linked immunosorbent assay. Transfection with empty vector pEGFP-N1 served as control (indicated by `–'). Data represent the mean ± s.d. of three independent experiments (**P<0.01 for transfection of CD147 construct versus control).

 

Figure 5
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  		Fig. 5. Gene silencing of CD147 induces upregulation of NOD2-mediated NF-{kappa}B activation and IL8 release. HEKNOD2 cells and SW480 cells expressing endogenous NOD2 were transfected with pSUPER.neo+GFP vectors containing three different target sequences for CD147 (CD147-si1 to si3) or irrelevant control sequence (ctrl-si). (A) After selection and sorting, HEKNOD2 cells were assayed for CD147 expression by immunoblotting. Equal loading was monitored by concomitant detection of β-actin (lower panel). (B) Stably transfected shRNA HEKNOD2 cells were stimulated with MDP or left untreated. Induction of NF-{kappa}B activity was determined as described in Materials and Methods. Data are expressed as relative luciferase activity (RLU, mean ± s.d.; n=3 independent experiments). (C) Transiently transfected SW480 cells were stimulated with MDP (50 µg/ml) overnight and assayed by ELISA for IL8 release. Gene silencing of CD147 augmented NF-{kappa}B activation and IL8 secretion in both transfected HEK293 cells and SW480 cells expressing endogenous NOD2 (++P<0.01 for MDP-stimulated versus unstimulated cells; **P<0.01 for CD147-si versus ctrl-si).

 

Figure 6
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  		Fig. 6. Surface expression of CD147 modifies invasion of L. monocytogenes into epithelial cells. (A) HEK293 cells were transiently transfected with pcDNA4-Xpress-NOD2 and/or pEGFP-N1-CD147 and infected with L. monocytogenes for 1 hour. After killing of extracellular bacteria by gentamicin, cells were lysed and plated on solid medium. CFU of Listeria were expressed as relative invasion adjusted to the cell number (measured in parallel by MTT test). Transient overexpression of NOD2 led to a protection of HEK293 against infection, whereas this effect was abolished by parallel overexpression of CD147. CD147 overexpression alone enhanced the relative infection rate of Listeria. (B) HEKNOD2 cells stably transfected with siRNA constructs targeting CD147 were used for gentamicin protection assays as described. Gene-silencing of CD147 strongly reduced the infection by Listeria monocytogenes. Antibodies directed against the extracellular part of CD147 inhibited invasion [relative invasion as a percentage of control; mean ± s.d. (n=3); *P<0.05, **P<0.01 for experiment versus control].

 

Figure 7
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  		Fig. 7. CD147 does not influence expression of host cell entry factors. (A) Gentamicin protection assays with Listeria monocytogenes wild-type (L.m.) or mutant strains deficient in either internalin A or internalin B ({Delta}InlA, {Delta}InlB) were performed with HEK293NOD2 cells as described in Materials and Methods. Deletion of InlB markedly reduced the ability of Listeria to invade into HEK293 cells, whereas deletion of InlA only had a marginal effect. Relative invasion is shown as percentage of CFUs compared to Listeria wild-type strain; data represent the mean ± s.d. (n=3); **P<0.01. (B) Cell surface expression of c-Met and E-cadherin. Cells stably transfected with pSUPER constructs silencing CD147 expression (red) or transiently transfected with EGFP-CD147 (green) were assayed for cell surface expression of Met (upper panel) or E-cadherin (lower panel) as described. Untransfected cells were used as positive control (black), cells treated with secondary antibody alone were used as specificity control (gray curve). Neither overexpression nor knock-down of CD147 influenced Met or E-cadherin expression level.

 

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