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Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Madrigal AG, Barth K, Papadopoulos G, Genco CA - PLoS Pathog. (2012)

Bottom Line: RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2.Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed.We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

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RIPK2 levels are stable in HAEC stimulated with TLR or NLR agonists.HAEC were treated with medium, P. gingivalis 381 (MOI 100), 10 µg/ml Pam3CSK4, 10 µg/ml FSL-1, 10 µg/ml P. gingivalis 381 LPS, 1.0 µg/ml E. coli 0111:B4 LPS, 100 ng/ml recombinant human TNF, 100 µg/ml iE-DAP, 100 µg/ml iE-DAP control, 1000 ng/ml C12-iE-DAP, 0.01% DMSO (C12-iE-DAP vehicle control), 100 µg/ml MDP, 100 µg/ml MDP control, or 1000 ng/ml L18-MDP for 2 h. Whole cell lysates were analyzed for the detection of RIPK2 and GAPDH. Full-length RIPK2 and RIPK2β are indicated with arrows. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.
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ppat-1002723-g003: RIPK2 levels are stable in HAEC stimulated with TLR or NLR agonists.HAEC were treated with medium, P. gingivalis 381 (MOI 100), 10 µg/ml Pam3CSK4, 10 µg/ml FSL-1, 10 µg/ml P. gingivalis 381 LPS, 1.0 µg/ml E. coli 0111:B4 LPS, 100 ng/ml recombinant human TNF, 100 µg/ml iE-DAP, 100 µg/ml iE-DAP control, 1000 ng/ml C12-iE-DAP, 0.01% DMSO (C12-iE-DAP vehicle control), 100 µg/ml MDP, 100 µg/ml MDP control, or 1000 ng/ml L18-MDP for 2 h. Whole cell lysates were analyzed for the detection of RIPK2 and GAPDH. Full-length RIPK2 and RIPK2β are indicated with arrows. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.

Mentions: To determine if P. gingivalis-induced proteolysis of RIPK2 was a result of cell activation via TLR or NLR-mediated signaling, HAEC were treated with a panel of synthetic or purified agonists to PRR that have been demonstrated to be activated by P. gingivalis or are direct activators of the NOD/RIPK2 signaling pathway. HAEC were untreated or treated with live P. gingivalis or with agonists to PRR, including Pam3CSK4, FSL-1, P. gingivalis LPS, E. coli LPS, iE-DAP, MDP or TNFα as a non-PRR activator of NF-κB or their vehicle controls for 2, 6, 12 and 24 h. To facilitate the passage of iE-DAP or MDP through the cell membrane, acylated derivatives of iE-DAP (C12-iE-DAP) and MDP (L18-MDP) were also used. Stimulation of HAEC with the panel of agonists did not result in the proteolysis of full-length RIPK2 or its mRNA splice variant, RIPK2β [49] (Figure 3). Similar results were obtained using a 100-fold range for all agonists examined over the full time course of the experiment (data not shown). Treatment of HAEC with the agonists resulted in the induction of IL-8 levels in a manner dependent on the agonist and concentration used (Figure S4). Noteworthy, NOD1 agonists (iE-DAP and C12-iE-DAP) induced significantly higher IL-8 levels in comparison to NOD2 agonists (MDP, L18-MDP), TLR2, and TLR4 agonists. Collectively, these findings suggest that the modification of RIPK2 observed in HAEC treated with P. gingivalis was not simply due to the activation of a single TLR, NOD or TNF-R1 canonical signaling pathway but a response driven by P. gingivalis.


Pathogen-mediated proteolysis of the cell death regulator RIPK1 and the host defense modulator RIPK2 in human aortic endothelial cells.

Madrigal AG, Barth K, Papadopoulos G, Genco CA - PLoS Pathog. (2012)

RIPK2 levels are stable in HAEC stimulated with TLR or NLR agonists.HAEC were treated with medium, P. gingivalis 381 (MOI 100), 10 µg/ml Pam3CSK4, 10 µg/ml FSL-1, 10 µg/ml P. gingivalis 381 LPS, 1.0 µg/ml E. coli 0111:B4 LPS, 100 ng/ml recombinant human TNF, 100 µg/ml iE-DAP, 100 µg/ml iE-DAP control, 1000 ng/ml C12-iE-DAP, 0.01% DMSO (C12-iE-DAP vehicle control), 100 µg/ml MDP, 100 µg/ml MDP control, or 1000 ng/ml L18-MDP for 2 h. Whole cell lysates were analyzed for the detection of RIPK2 and GAPDH. Full-length RIPK2 and RIPK2β are indicated with arrows. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.
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Related In: Results  -  Collection

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ppat-1002723-g003: RIPK2 levels are stable in HAEC stimulated with TLR or NLR agonists.HAEC were treated with medium, P. gingivalis 381 (MOI 100), 10 µg/ml Pam3CSK4, 10 µg/ml FSL-1, 10 µg/ml P. gingivalis 381 LPS, 1.0 µg/ml E. coli 0111:B4 LPS, 100 ng/ml recombinant human TNF, 100 µg/ml iE-DAP, 100 µg/ml iE-DAP control, 1000 ng/ml C12-iE-DAP, 0.01% DMSO (C12-iE-DAP vehicle control), 100 µg/ml MDP, 100 µg/ml MDP control, or 1000 ng/ml L18-MDP for 2 h. Whole cell lysates were analyzed for the detection of RIPK2 and GAPDH. Full-length RIPK2 and RIPK2β are indicated with arrows. A prominent P. gingivalis-induced LMW band is indicated with an asterisk. MW ladder is indicated on the left in kDa.
Mentions: To determine if P. gingivalis-induced proteolysis of RIPK2 was a result of cell activation via TLR or NLR-mediated signaling, HAEC were treated with a panel of synthetic or purified agonists to PRR that have been demonstrated to be activated by P. gingivalis or are direct activators of the NOD/RIPK2 signaling pathway. HAEC were untreated or treated with live P. gingivalis or with agonists to PRR, including Pam3CSK4, FSL-1, P. gingivalis LPS, E. coli LPS, iE-DAP, MDP or TNFα as a non-PRR activator of NF-κB or their vehicle controls for 2, 6, 12 and 24 h. To facilitate the passage of iE-DAP or MDP through the cell membrane, acylated derivatives of iE-DAP (C12-iE-DAP) and MDP (L18-MDP) were also used. Stimulation of HAEC with the panel of agonists did not result in the proteolysis of full-length RIPK2 or its mRNA splice variant, RIPK2β [49] (Figure 3). Similar results were obtained using a 100-fold range for all agonists examined over the full time course of the experiment (data not shown). Treatment of HAEC with the agonists resulted in the induction of IL-8 levels in a manner dependent on the agonist and concentration used (Figure S4). Noteworthy, NOD1 agonists (iE-DAP and C12-iE-DAP) induced significantly higher IL-8 levels in comparison to NOD2 agonists (MDP, L18-MDP), TLR2, and TLR4 agonists. Collectively, these findings suggest that the modification of RIPK2 observed in HAEC treated with P. gingivalis was not simply due to the activation of a single TLR, NOD or TNF-R1 canonical signaling pathway but a response driven by P. gingivalis.

Bottom Line: RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2.Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed.We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders.

Show MeSH
Related in: MedlinePlus