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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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P. gingivalis 381-induced proteolysis of RIPK1 and RIPK2 in HAEC.HAEC were treated with medium (M) or with P. gingivalis strain 381 (MO1 100) for 0.25, 0.5, 1, 2, 6, 12, 24 or 48 h. Whole cell lysates were analyzed for the detection of A) RIPK1, B) RIPK2 with an anti N′-terminal RIPK2 antibody (left panel) or an anti C′-terminal RIPK2 antibody (right panel), or C) NOD1 (left panel) and NOD2 (right panel). Full-length RIPK1 (74-kDa) and RIPK2 (61-kDa) are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. Molecular weight (MW) ladder is indicated on the left in kDa. GAPDH was detected as a loading control. (−) protein levels in medium-treated cells were similar at all time points. Densitometric analysis is presented below respective blots as the mean (+/− SEM) ratio of NOD1 (or NOD2) to GAPDH protein levels (arbitrary densitometric units (A.D.U.) from at least 3 independent membranes. Means are displayed within the bar charts.
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ppat-1002723-g001: P. gingivalis 381-induced proteolysis of RIPK1 and RIPK2 in HAEC.HAEC were treated with medium (M) or with P. gingivalis strain 381 (MO1 100) for 0.25, 0.5, 1, 2, 6, 12, 24 or 48 h. Whole cell lysates were analyzed for the detection of A) RIPK1, B) RIPK2 with an anti N′-terminal RIPK2 antibody (left panel) or an anti C′-terminal RIPK2 antibody (right panel), or C) NOD1 (left panel) and NOD2 (right panel). Full-length RIPK1 (74-kDa) and RIPK2 (61-kDa) are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. Molecular weight (MW) ladder is indicated on the left in kDa. GAPDH was detected as a loading control. (−) protein levels in medium-treated cells were similar at all time points. Densitometric analysis is presented below respective blots as the mean (+/− SEM) ratio of NOD1 (or NOD2) to GAPDH protein levels (arbitrary densitometric units (A.D.U.) from at least 3 independent membranes. Means are displayed within the bar charts.

Mentions: We previously demonstrated that P. gingivalis induces the activation (defined as an up regulation of cell surface CAM, TLR, activation of NF-κB and secretion of chemokines, including IL-8 and MCP-1) of primary HAEC and human umbilical vein endothelial cells (HUVEC) in tissue culture [33], [35], [37], [39], [47] and acceleration of aortic lesion development in mouse models of P. gingivalis infection via TLR mediated signaling [31], [34], [38]. To begin to address the role of P. gingivalis-mediated intracellular signaling responses in HAEC, we first examined the expression level of the intracellular kinases RIPK1 and RIPK2. HAEC were treated with live organism at a multiplicity of infection (MOI) of 100 and protein levels were monitored over time. Target sites of antibodies to RIPK1 and RIPK2 used in this study are described in Figure S1. Treatment of HAEC with P. gingivalis strain 381 induced an immediate (15 min) and significant reduction of full-length RIPK1 and RIPK2 protein levels as determined by Western blot analysis of whole cell lysates (Figure 1A and B). The reduction of full-length RIPK1 and RIPK2 in HAEC was transient since full-length protein levels were observed at later time points. We also observed prominent low molecular weight (LMW) immunoreactive bands in samples treated with P. gingivalis with an antibody to RIPK1 (14-, 28- and 32-kDa) (Figure 1A and Figure 2), and with antibodies directed to the N′-terminal and C′-terminal region of RIPK2 (20- and 42-kDa), respectively (Figure 1B). Noteworthy, the sum of the immunoreactive bands observed for RIPK1 and RIPK2 was approximate to the molecular weight of RIPK1 (74-kDa) and RIPK2 (61-kDa), respectively. The detection of multiple bands with the RIPK2 C′-terminal antibody (Figure 1B-right panel) may represent fragments of additional proteolytic processing of RIPK2 following an initial cleavage event. Similar findings were observed in HUVEC (data not shown). In contrast to RIPK1 and RIPK2, we did not observe LMW immunoreactive bands with antibodies to NOD1 or NOD2, the cytosolic PRR of RIPK2, in HAEC treated with P. gingivalis. However, NOD1 levels were decreased in response to P. gingivalis with an initial decrease in protein levels and a partial recovery later in the infection. NOD2 levels were relatively stable in response to P. gingivalis as to untreated HAEC. (Figure 1C).


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)

P. gingivalis 381-induced proteolysis of RIPK1 and RIPK2 in HAEC.HAEC were treated with medium (M) or with P. gingivalis strain 381 (MO1 100) for 0.25, 0.5, 1, 2, 6, 12, 24 or 48 h. Whole cell lysates were analyzed for the detection of A) RIPK1, B) RIPK2 with an anti N′-terminal RIPK2 antibody (left panel) or an anti C′-terminal RIPK2 antibody (right panel), or C) NOD1 (left panel) and NOD2 (right panel). Full-length RIPK1 (74-kDa) and RIPK2 (61-kDa) are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. Molecular weight (MW) ladder is indicated on the left in kDa. GAPDH was detected as a loading control. (−) protein levels in medium-treated cells were similar at all time points. Densitometric analysis is presented below respective blots as the mean (+/− SEM) ratio of NOD1 (or NOD2) to GAPDH protein levels (arbitrary densitometric units (A.D.U.) from at least 3 independent membranes. Means are displayed within the bar charts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369954&req=5

ppat-1002723-g001: P. gingivalis 381-induced proteolysis of RIPK1 and RIPK2 in HAEC.HAEC were treated with medium (M) or with P. gingivalis strain 381 (MO1 100) for 0.25, 0.5, 1, 2, 6, 12, 24 or 48 h. Whole cell lysates were analyzed for the detection of A) RIPK1, B) RIPK2 with an anti N′-terminal RIPK2 antibody (left panel) or an anti C′-terminal RIPK2 antibody (right panel), or C) NOD1 (left panel) and NOD2 (right panel). Full-length RIPK1 (74-kDa) and RIPK2 (61-kDa) are indicated with arrows. Prominent P. gingivalis-induced LMW bands are indicated with asterisks. Molecular weight (MW) ladder is indicated on the left in kDa. GAPDH was detected as a loading control. (−) protein levels in medium-treated cells were similar at all time points. Densitometric analysis is presented below respective blots as the mean (+/− SEM) ratio of NOD1 (or NOD2) to GAPDH protein levels (arbitrary densitometric units (A.D.U.) from at least 3 independent membranes. Means are displayed within the bar charts.
Mentions: We previously demonstrated that P. gingivalis induces the activation (defined as an up regulation of cell surface CAM, TLR, activation of NF-κB and secretion of chemokines, including IL-8 and MCP-1) of primary HAEC and human umbilical vein endothelial cells (HUVEC) in tissue culture [33], [35], [37], [39], [47] and acceleration of aortic lesion development in mouse models of P. gingivalis infection via TLR mediated signaling [31], [34], [38]. To begin to address the role of P. gingivalis-mediated intracellular signaling responses in HAEC, we first examined the expression level of the intracellular kinases RIPK1 and RIPK2. HAEC were treated with live organism at a multiplicity of infection (MOI) of 100 and protein levels were monitored over time. Target sites of antibodies to RIPK1 and RIPK2 used in this study are described in Figure S1. Treatment of HAEC with P. gingivalis strain 381 induced an immediate (15 min) and significant reduction of full-length RIPK1 and RIPK2 protein levels as determined by Western blot analysis of whole cell lysates (Figure 1A and B). The reduction of full-length RIPK1 and RIPK2 in HAEC was transient since full-length protein levels were observed at later time points. We also observed prominent low molecular weight (LMW) immunoreactive bands in samples treated with P. gingivalis with an antibody to RIPK1 (14-, 28- and 32-kDa) (Figure 1A and Figure 2), and with antibodies directed to the N′-terminal and C′-terminal region of RIPK2 (20- and 42-kDa), respectively (Figure 1B). Noteworthy, the sum of the immunoreactive bands observed for RIPK1 and RIPK2 was approximate to the molecular weight of RIPK1 (74-kDa) and RIPK2 (61-kDa), respectively. The detection of multiple bands with the RIPK2 C′-terminal antibody (Figure 1B-right panel) may represent fragments of additional proteolytic processing of RIPK2 following an initial cleavage event. Similar findings were observed in HUVEC (data not shown). In contrast to RIPK1 and RIPK2, we did not observe LMW immunoreactive bands with antibodies to NOD1 or NOD2, the cytosolic PRR of RIPK2, in HAEC treated with P. gingivalis. However, NOD1 levels were decreased in response to P. gingivalis with an initial decrease in protein levels and a partial recovery later in the infection. NOD2 levels were relatively stable in response to P. gingivalis as to untreated HAEC. (Figure 1C).

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