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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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Classical apoptotic stimuli do not induce the proteolysis of RIPK1 or RIPK2 in HUVEC.HUVEC were treated with medium, P. gingivalis 381 (MOI 100), 2 µM staurosporine (STS) 25 µg/ml cycloheximide (CHX), 10 ng/ml TNFα, or co-treated with 25 µg/ml CHX and 10 ng/ml TNFα for 6 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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ppat-1002723-g004: Classical apoptotic stimuli do not induce the proteolysis of RIPK1 or RIPK2 in HUVEC.HUVEC were treated with medium, P. gingivalis 381 (MOI 100), 2 µM staurosporine (STS) 25 µg/ml cycloheximide (CHX), 10 ng/ml TNFα, or co-treated with 25 µg/ml CHX and 10 ng/ml TNFα for 6 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.

Mentions: To determine if P. gingivalis-induced proteolysis of RIPK1 and RIPK2 was a product of apoptosis, we treated cells with apoptotic stimuli and monitored apoptosis by flow cytomery detection of annexin V/propidium iodide staining and by Western blot detection of cleaved caspase 3 and its substrate PARP. Treatment of HUVEC with staurosproine or co-treatment with CHX and TNFα (CHX+TNFα) induced significant apoptotic cell populations as compared to HUVEC treated with CHX alone, TNFα alone or medium (Figure S5A). We also observed dose-dependent apoptotic cell populations treated with P. gingivalis 381, suggesting that a proportion of the cells undergo apoptosis in response to infection. Induction of apoptosis of HUVEC by staurosporine or CHX+TNFα treatment was also confirmed through the cleavage of caspase 3 and PARP (Figure S5B). Importantly, we did not observe the induction of RIPK1 or RIPK2 cleavage in HUVEC in response to staurosporine or CHX+TNFα treatment (Figure 4). Furthermore, P. gingivalis itself did not induce the cleavage of caspase 3, but appeared to induce the complete proteolysis of PARP. These findings demonstrate that apoptotic stimuli alone do not induce the cleavage of RIPK1 or RIPK2 in HUVEC.


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)

Classical apoptotic stimuli do not induce the proteolysis of RIPK1 or RIPK2 in HUVEC.HUVEC were treated with medium, P. gingivalis 381 (MOI 100), 2 µM staurosporine (STS) 25 µg/ml cycloheximide (CHX), 10 ng/ml TNFα, or co-treated with 25 µg/ml CHX and 10 ng/ml TNFα for 6 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g004: Classical apoptotic stimuli do not induce the proteolysis of RIPK1 or RIPK2 in HUVEC.HUVEC were treated with medium, P. gingivalis 381 (MOI 100), 2 µM staurosporine (STS) 25 µg/ml cycloheximide (CHX), 10 ng/ml TNFα, or co-treated with 25 µg/ml CHX and 10 ng/ml TNFα for 6 h. Whole cell lysates were analyzed for the detection of RIPK1 (left panel) or RIPK2 (right panel). Full-length RIPK1 and RIPK2 are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. MW ladder is indicated on the left in kDa. GAPDH was detected as a loading control.
Mentions: To determine if P. gingivalis-induced proteolysis of RIPK1 and RIPK2 was a product of apoptosis, we treated cells with apoptotic stimuli and monitored apoptosis by flow cytomery detection of annexin V/propidium iodide staining and by Western blot detection of cleaved caspase 3 and its substrate PARP. Treatment of HUVEC with staurosproine or co-treatment with CHX and TNFα (CHX+TNFα) induced significant apoptotic cell populations as compared to HUVEC treated with CHX alone, TNFα alone or medium (Figure S5A). We also observed dose-dependent apoptotic cell populations treated with P. gingivalis 381, suggesting that a proportion of the cells undergo apoptosis in response to infection. Induction of apoptosis of HUVEC by staurosporine or CHX+TNFα treatment was also confirmed through the cleavage of caspase 3 and PARP (Figure S5B). Importantly, we did not observe the induction of RIPK1 or RIPK2 cleavage in HUVEC in response to staurosporine or CHX+TNFα treatment (Figure 4). Furthermore, P. gingivalis itself did not induce the cleavage of caspase 3, but appeared to induce the complete proteolysis of PARP. These findings demonstrate that apoptotic stimuli alone do not induce the cleavage of RIPK1 or RIPK2 in HUVEC.

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