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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Shock. 2014 Dec;42(6):562–569. doi: 10.1097/SHK.0000000000000242

Regulation of Endothelial Cell Inflammation and Lung PMN Infiltration by Transglutaminase 2

Kaiser M Bijli 1,#, Bryce G Kanter 1, Mohammad Minhajuddin 1,€, Antony Leonard 1, Lei Xu 2, Fabeha Fazal 1, Arshad Rahman 1,*
PMCID: PMC4236274  NIHMSID: NIHMS615191  PMID: 25057925

Abstract

We addressed the role of transglutaminase2 (TG2), a calcium-dependent enzyme that catalyzes crosslinking of proteins, in the mechanism of endothelial cell (EC) inflammation and lung PMN infiltration. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor NF-κB and its target genes, VCAM-1, MCP-1, and IL-6. RNAi knockdown of TG2 inhibited these responses. Analysis of NF-κB activation pathway showed that TG2 knockdown was associated with inhibition of thrombin-induced DNA binding as well as serine phosphorylation of RelA/p65, a crucial event that controls transcriptional capacity of the DNA-bound RelA/p65. These results implicate an important role for TG2 in mediating EC inflammation by promoting DNA binding and transcriptional activity of RelA/p65. Because thrombin is released in high amounts during sepsis and its concentration is elevated in plasma and lavage fluids of patients with Acute Respiratory Distress Syndrome (ARDS), we determined the in vivo relevance of TG2 in a mouse model of sepsis-induced lung PMN recruitment. A marked reduction in NF-κB activation, adhesion molecule expression, and lung PMN sequestration was observed in TG2 knockout mice compared to wild type mice exposed to endotoxemia. Together, these results identify TG2 as an important mediator of EC inflammation and lung PMN sequestration associated with intravascular coagulation and sepsis.

Keywords: Endothelium, signal transduction, transcription factors, adhesion molecules, sepsis

INTRODUCTION

Acute lung Injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in critically ill patients. A characteristic feature of ALI/ARDS is an exuberant inflammatory response involving massive infiltration of polymorphonuclear lymphocytes (PMN) into the lung that ultimately causes capillary-alveolar barrier dysfunction, subsequent pulmonary edema with severe consequences for pulmonary gas exchange (1). Despite the use of state-of-art treatment, mortality associated with ALI and ARDS remains high, ~25-40% in the reported cases (2). In addition to inflammation, activation of intravascular coagulation is emerging as an important component of ALI (3), particularly in the settings of sepsis, a prominent extrapulmonary cause responsible for ~ 40% of ALI in humans (4). Increasing evidence indicates a close interaction and bidirectional cooperation between these two systems, whereby inflammation not only leads to activation of coagulation, but coagulation also promotes inflammation (5). One of the main interfaces linking coagulation and inflammation is thrombin, a procoagulant serine protease whose concentrations are elevated in plasma and lavage fluids of patients with ALI/ARDS (6). Consistent with this, studies in animal models have demonstrated that impairing thrombin/PAR1 (protease-activated receptor 1) signaling via activated protein C (APC) reduces lung inflammation and injury in mice (7, 8) whereas infusion of thrombin induces lung vascular injury and tissue edema primarily by promoting PMN sequestration in microvessels (9).

Recruitment of PMN from blood into the lung is initiated by adhesion of PMN to the vascular endothelium, which in turn is mediated by the interaction of adhesive proteins, E-selectin, intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cell (EC) surface, with their counter receptors, P-selectin glycoprotein ligand-1 (PSGL-1), β2-integrins (LFA-1 [CD11a/CD18] and Mac-1 [CD11b/CD18]) and very late antigen-4 (VLA-4) respectively, on PMN (10, 11). Other proteins that act in concert with adhesion molecules to promote EC-PMN interactions and to enable the adherent PMN to migrate across endothelial barrier into tissue parenchyma and subsequently into the alveolar space include cytokines (TNFα, IL-6) and chemokines (IL-8, MCP-1) (10, 11). Expression of these proteins (adhesion molecules, cytokines, and chemokines) in turn critically depends on activation of the transcription factor nuclear factor-kappa B (NF-κB) (11, 12). Among the central events controlling NF-κB activation involves serine phosphorylation of IκBα (Ser32and Ser36) and RelA/p65 (Ser276 and Ser536) (11, 12). Phosphorylation targets IκBα for ubiquitination and proteasome-mediated degradation, causing the release of NF-κB (predominantly RelA/p65 homodimer in EC) for its nuclear translocation and subsequent binding to the promoter of target genes, including adhesion molecules, cytokines, and chemokines (11, 12). Phosphorylation of RelA/p65 at Ser276 or Ser536 serves to confer transcriptional competency to NF-κB bound to the promoter (11, 12).

Transglutaminase 2 (TG2) is a multifunctional enzyme and the most ubiquitous among the transglutaminase family of proteins (13). Among its several functions include calcium-dependent cross-linking of proteins, GTP binding and hydrolysis, and protein scaffolding (13). Under normal physiological conditions, TG2 exists as a catalytically inactive protein due to low Ca2+ concentrations (14). However, under stress, loss in Ca2+ homeostasis (increase in calcium concentration) can activate intracellular TG2 resulting in cross-linking of cellular proteins (14). TG2 has been shown to play a role in the activation of NF-κB in cancer cells (15), and conversely, TG2 expression is induced by NF-κB in liver cells (16). TG2 has been implicated in a number of pathological processes including myocardial hypertrophy, tissue fibrosis, celiac disease, cancer, wound healing and inflammation (13, 17). In a recent study, Oh et al. (18) have identified epithelial TG2 as an important mediator of bleomycin-induced lung inflammation and fibrosis in mice. However, the role of TG2 in mediating EC inflammation and lung PMN recruitment is unclear. Because thrombin can elicit Ca2+ transients (19), a requirement for TG2 as well as NF-κB activation in EC (20), we addressed the possibility that TG2 plays a critical role in activating NF-κB to promote EC inflammation in vitro and lung PMN sequestration in vivo in mice. Our data establish that TG2 is an important mediator of NF-κB activation and EC inflammation associated with intravascular coagulation, and show the relevance of this molecule in the mechanism of lung PMN sequestration associated with sepsis.

MATERIALS AND METHODS

Reagents

Human thrombin was purchased from Enzyme Research Laboratories (South Bend, IN). Lipopolysaccharide (LPS) of E. coli origin and DEAE Dextran were purchased from Sigma Chemical Company (St. Louis, MO), and the recombinant TNFα was obtained from Promega (Madison, WI). Antibodies to RelA/p65, IκBα, ICAM-1, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). An antibody to TG2 was obtained from Lab Vision (Fremont, CA). Antibodies to phospho-(Ser276)-RelA/p65 and phospho-(Ser536)-RelA/p65 were obtained from Cell Signaling (Beverly, MA) or from Imgenex (San Diego, CA). Plasmid maxi kit from QIAGEN Inc. (Valencia, CA); RelA/p65 transcription factor assay kit from Cayman Chemical (Ann Arbor, MI); protein assay kit and nitrocellulose membrane were from Bio-Rad Laboratories (Hercules, CA). All other materials were from Fisher Scientific (Pittsburg, PA) or VWR Scientific Products Corporation (Gaithersburg, MD).

Endothelial cells

Human pulmonary artery endothelial cells (HPAEC) were purchased from Lonza (Walkersville, MD). HPAEC were cultured as described (21) in gelatin-coated flasks using endothelial basal medium 2 (EBM2) with bullet kittm additives (BioWhittaker, Walkersville, MD). HPAEC used in the experiments were between3 and 6 passages.

Mice

TG2 knockout (KO) mice were obtained from Dr. Gerry Melino (22) and bred at the University of Rochester. Age-matched C57BL/6 mice were used as controls (Jackson Laboratory, Bar Harbor, ME). WT and KO mice were subjected to intraperitoneal (i.p.) injection of LPS (10 mg/kg body weight) to induce lung inflammation. Lungs from these mice were then harvested at the indicated times after LPS challenge for determination of NF-κB activation, proinflammatory gene expression, and lung PMN infiltration. All mice care and treatment procedures were approved by the University of Rochester Committee on Animal Resources and performed in adherence to the National Institute of Health guidelines.

RNAi knockdown

SMARTpool short interfering RNA (siRNA) specific for human TG2 (siRNA-TG2) and a non-targeting siRNA control (siRNA-Con) were obtained from Dharmacon (Lafayette, CO). siRNA-TG2 or siRNA-Con were transfected into EC using DharmaFect1 siRNA Transfection Reagent (Dharmacon) essentially as described (21). Briefly, 50-100 nM siRNA was mixed with the DharmaFect1 and added to cells that were 50-60% confluent. At 24-36 h after transfection, cells were stimulated with thrombin or TNFα and lysed after the indicated time periods for various analyses.

Immunoblot analysis

After appropriate treatments, the cells were subjected to lysis using radioimmune precipitation (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25 mM EDTA, pH 8.0, 1% deoxycholic acid, 1% Triton-X, 5 mM NaF, 1 mM sodium orthovanadate supplemented with protease inhibitor cocktail [Sigma]) or phosphorylation lysis buffer (50 mM HEPES, 150 mM NaCl, 200 μM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 1.5 mM magnesium chloride, 10% glycerol, 0.5 to 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride [PMSF], and protease inhibitor cocktail [Sigma]). Lung homogenates were prepared using RIPA buffer supplemented with protease inhibitor cocktail. Briefly, lung tissue (100 mg) was mechanically homogenized in 1.0 ml of RIPA buffer, and the homogenates were incubated on ice for 1 h to achieve total cell lysis. Equal amounts of protein from the cell lysates or lung homogenates as determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) were electrophoresed and subsequently transferred onto nitrocellulose membranes for immmunoblotting as described (21). Representative blots presented in the results section come from the same membrane which may have more samples in various groups.

Nuclear extract preparation and assessment of RelA/p65 DNA binding

Nuclear extracts were prepared as described (23). Briefly, cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 400 μl of buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM [DTT], and 0.5 mM PMSF). After 15 min, NP-40 was added to a final concentration of 0.6%, and the samples were centrifuged to collect the supernatants containing the cytoplasmic proteins. The pelleted nuclei were resuspended in 50 μl of buffer B (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After 0.5 h at 4°C, lysates were centrifuged and supernatants containing the nuclear proteins were collected. The DNA binding activity of RelA/p65 was determined using an ELISA-based DNA binding assay kit (Cayman Chemical, Ann Arbor, MI) as described (24).

NF-κB transcriptional activity

The transcriptional activity of NF-κB was determined as described (21). The plasmid pNF-κBLUC containing 5 copies of consensus NF-κB sequences linked to a minimal E1B promoter-luciferase gene was purchased from Stratagene (La Jolla, CA). Briefly, 5 μg pNF-κB-LUC was mixed with 50 μg/ml DEAE-dextran in serum-free EBM2 and the mixture was added onto cells which were 60-80% confluent. The transfection mixture also included 0.125 μg pTKRLUC plasmid (Promega, Madison, WI) containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter, which was used to normalize the transfection efficiency. After 1 h, cells were incubated for 4 min with 10% dimethyl sulfoxide (DMSO) in serum-free EBM2. The cells were then washed 2× with PBS and grown to confluency using EBM2-10% FBS. After appropriate treatments, cell extracts were prepared and assayed for Firefly and Renilla luciferase activities using Promega Biotech Dual Luciferase Reporter Assay System. The data were expressed as a ratio of Firefly to Renilla luciferase activity. In experiments evaluating the effect of TG2 knockdown on NF-κB transcriptional activity, cells were first transfected with TG2 siRNA as described above. After 24 h, cells were again transfected with pNF-κB-LUC and pTKRLUC using DEAE-dextran as above.

ELISA

The levels of IL-6 and MCP-1 in HPAEC culture supernatants and the levels of VCAM-1 in mouse lung homogenates were determined using ELISA kits from R&D Systems (Minneapolis, MN) as described (24). The levels of IL-1β, IL-6, MIP-1α, MIP-1β, MCP-1, and GM-CSF in mouse lung homogenates were determined using multiplex immune assay system from Millipore Corp. (Bradford, MA) according to manufacturers’ recommendations.

Lung tissue myeloperoxidase (MPO) activity

Lung tissue MPO activity was determined as a measure of PMN infiltration as described (24). Briefly, 100 mg lung tissue in 1 ml HTAB (hexadecyltrimethylammonium bromide) buffer (pH 6.0) was subjected to sonication at 30 cycles, twice, for 30 s on ice, followed by centrifugation at 12,000 rpm for at 4°C. The clear supernatant was collected and the protein concentration was determined by bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). To assay MPO activity, the reaction was performed in a 96-well plate as described (25). Briefly, 10 μl of supernatant was added to a reaction mixture containing 290 μl of 50 mM phosphate buffer, 3 μl of substrate solution (containing 20 mg/ml o-dianisidine hydrochloride), and 3 μl of 20 mM H2O2 to start the reaction. After 5 min, the reaction was stopped by addition of 3 μl of 30% sodium azide and the absorbance was measured at 460 nm. MPO activity was determined by using the curve obtained from the standard MPO (Sigma Chemical Company) and expressed as units/mg protein.

Statistical Analysis

Results are presented as mean ± SE and were analyzed by using standard one-way ANOVA. The significance between the groups was determined using Tukey's test (Prism 5.0, GraphPad Software, San Diego). A p value < 0.05 between two groups was considered statistically significant. The number of mice used for each experimental group ranged from 3-7.

RESULTS

RNAi knockdown of TG2 attenuates thrombin-induced EC Inflammation by inhibiting NF-κB activity

We used RNAi knockdown approach to determine the role of TG2 in mediating EC inflammation. Cells were transfected with short-interfering RNA (siRNA) targeting TG2 (siRNA-TG2) or control siRNA (siRNA-Con) and the total cell lysates were analyzed for TG2 levels. We observed a marked depletion of TG2 in cells transfected with siRNA-TG2 compared to the cells transfected with siRNA-Con (Figure 1, A and B). TG2 levels in cells transfected with siRNA-TG2 or siRNA-Con remained unaffected upon stimulation with thrombin or TNFα (Figure 1, A and B). Depletion of TG2 by this approach impaired thrombin-induced VCAM-1 expression (Figure 1C). Interestingly, TG2 knockdown had no effect on VCAM-1 expression induced by TNFα(Figure 1D), a prototype mediator of EC inflammation. We also determined if TG2 knockdown inhibited other proinflammatory genes in response to thrombin. Results showed that TG2 knockdown was effective in preventing the basal as well as thrombin-induced MCP-1 and IL-6 release (Figure 1, E and F). Because these genes are primarily regulated by NF-κB (11, 12), we assessed the possibility that TG2 regulates EC inflammation by mediating NF-κB activation. A marked reduction in thrombin-induced transcriptional activity of NF-κB was noted in TG2-depleted cells (Figure 2A), consistent with the effect of TG2 knockdown on proinflammatory gene expression. We also determined whether TG2 knockdown influences NF-κB activity in response to TNFα. In contrast to its effect on thrombin response, TG2 knockdown showed no effect on TNFα-induced NF-κB activity (Figure 2B), suggesting a stimulus-specific regulation of NF-κB by TG2. To further examine the stimulus-specific effect of TG2 on NF-κB activity, we evaluated the effect of TG2 knockdown of LPS-and PMA-induced NF-κB activity. Results showed that like thrombin, LPS- and PMA-induced NF-κB activity was inhibited upon TG2 depletion (Figure 2C). Thus, TG2 appears to be an important mediator of NF-κB activity induced by a variety of proinflammatory mediators except TNFα.

Figure 1.

Figure 1

Figure 1

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Involvement of TG2 in thrombin-induced EC inflammation. (A&B) RNAi knockdown of TG2 in EC. HPAEC were transfected with control siRNA (siRNA-Con) or TG2-specific siRNA (siRNA-TG2) for 24-36 h. Cells were challenged for 6 h with thrombin (5 U/ml) or TNFα (100 U/ml). Total cell lysates were immunoblotted with an antibody to TG2 or actin. The bar graphs represent the effect of siRNA on TG2 level normalized to actin level. Data are means ± S.E. (n = 4 for each condition). ***p < 0.001 versus siRNA-Con controls. (C&D) Effect of RNAi knockdown of TG2 on thrombin- and TNFα-induced VCAM-1 protein expression. Total cell lysates from (A&B) were immunoblotted with an antibody to VCAM-1 or actin. The bar graphs represent the effect of TG2 knockdown on thrombin- and TNFα-induced VCAM-1 expression normalized to actin level. Data are means ± S.E. (n = 3-4 for each condition). *p < 0.05 or **p < 0.01 versus siRNA-Con untreated control; #p < 0.05 versus siRNA-Con thrombin-treated control. (E&F) Effect of TG2 knockdown on thrombin-induced MCP-1 and IL-6 release. Culture supernatants from cells transfected and treated similarly as in (A) were analyzed by ELISA for MCP-1 or IL-6 levels. Data are means ± S.E. (n = 5-6 for each condition). **p < 0.01 or ***p < 0.001 versus siRNA-Con untreated controls; ###p < 0.001 versus siRNA-Con thrombin-treated controls.

Figure 2.

Figure 2

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Involvement of TG2 in thrombin-induced NF-κB activation in EC. (A&B) Effect of RNAi knockdown of TG2 on thrombin- and TNFα-induced NF-κB transcriptional activity. HPAEC were transfected with siRNA-TG2 or siRNA-Con using DharmaFect1. After 12 h, cells were again transfected with NF-κBLUC construct using DEAE-Dextran as described in Materials and Methods. Twelve hours later, cells were challenged with (A) thrombin [5 U/ml] or (B) TNFα [100 U/ml] for 6 h. Cells were lysed and the extracts were assayed for Firefly and Renilla (F/R) luciferase activities. Data are means ± S.E. (n = 5-6 for each condition). **p < 0.01 or ***p < 0.001 versus siRNA-Con untreated controls; ##p < 0.01 versus siRNA-Con thrombin-stimulated controls. (C) Effect of RNAi knockdown of TG2 on LPS-and PMA-induced NF-κB transcriptional activity. HPAEC were transfected with siRNA-TG2 and NF-κBLUC construct essentially as in (A&B). Cells were challenged with LPS (0.1 μg/ml) or PMA (50 nM) for 7 h and the cell extracts were assayed for Firefly and Renilla (F/R) luciferase activities. Data are means ± S.E. (n = 6 for each condition). **p < 0.01 or ***p < 0.001 versus siRNA-Con untreated controls; #p < 0.05 versus siRNA-Con LPS-stimulated controls; €€€p < 0.001 versus siRNA-Con PMA-stimulated controls. (D) Effect of RNAi knockdown of TG2 on thrombin-induced RelA/p65 DNA binding. HPAEC were transfected with siRNA-TG2 or siRNA-Con using DharmaFect1. After 24-36 h, cells were challenged for 1 h with thrombin (5 U/ml). Nuclear extracts were prepared and assayed for RelA/p65 DNA binding activity by ELISA as described in Materials and Methods. The bar graphs represent the effect of TG2 knockdown on thrombin-induced RelA/p65 DNA binding. Data are means ± S.E. (n = 3-4 for each condition). ***p < 0.001 versus siRNA-Con untreated control; ##p < 0.01 versus siRNA-Con thrombin-stimulated control. (E&F) Effect of RNAi knockdown of TG2 on thrombin- and TNFα-induced phosphorylation of RelA/p65. HPAEC were transfected with siRNA-TG2 or siRNA-Con using DharmaFect1. After 24-36 h, cells were challenged with (E) thrombin (5 U/ml) for 1 h or (F) TNFα (100 U/ml) for 30 min. Total cell lysates were immunoblotted with an anti-phospho-RelA/p65 (Ser536) antibody. RelA/p65 levels were used to monitor loading. The bar graphs represent the effect of TG2 knockdown on (E) thrombin- and (F) TNFα-induced phosphorylation of RelA/p65. Data are means ± S.E. (n = 3 for each condition). ***p < 0.001 versus siRNA-Con untreated controls; ###p < 0.001 versus siRNA-Con thrombin-treated control.

RNAi knockdown of TG2 inhibits thrombin-induced NF-κB activity by impairing DNA binding and phosphorylation of RelA/p65

To determine if TG2 mediates thrombin-induced NF-κB activity by promoting its DNA binding activity in the nucleus, we assessed the effect of TG2 knockdown on this response. Analysis of nuclear extracts from TG2-depleted cells showed a significant impairment in DNA binding of RelA/p65 induced by thrombin (Figure 2D). Because phosphorylation of RelA/p65 enhances its transcriptional capacity (11, 12, 21) , we next addressed the possible contribution of this event in the mechanism of TG2-mediated NF-κB activity. To this end, TG2-depleted cells were treated with thrombin and total cell lysates were analyzed for Ser536 phosphorylation of RelA/p65. Depletion of TG2 markedly inhibited phosphorylation of RelA/p65 upon thrombin challenge of EC (Figure 2E). By contrast, TNFα-induced Ser536 phosphorylation of RelA/p65 was insensitive to TG2 depletion (Figure 2F), consistent with its effect on TNFα-induced NF-κB transcriptional activity (Figure 2B). These results show that TG2 controls thrombin-induced NF-κB signaling by promoting DNA binding and increasing the transcriptional capacity of bound RelA/p65.

Genetic ablation of TG2 reduces sepsis-induced NF-κB activation and adhesion molecule expression in mouse lung

To determine if the above effects of TG2 knockdown in cultured EC can be recapitulated in vivo in the intact lungs of mice, we used the well-established mouse model of sepsis-induced lung inflammation. The rationale for this was that activation of intravascular coagulation and generation of thrombin has been shown to play an important role in the evolution of lung inflammatory responses in this model (26). Wild type (WT) and TG2 knockout (KO) mice were challenged with i.p. LPS and the lungs were analyzed for NF-κB activation by measuring the degradation of IκBα and phosphorylation of RelA/p65. Figure 3 shows the immunoblots exhibiting the tissue levels of IκBα and phosphorylation status of RelA/p65 in the lungs of WT and KO m ice challenged with LPS. IκBα levels were significantly decreased in the lungs of WT mice exposed to LPS compared to WT mice treated with saline (Figure 3A). The LPS-induced decrease in IκBα levels was partly inhibited in the lungs of KO mice challenged with LPS (Figure 3A). We also determined if TG2 deficiency affects RelA/p65 phosphorylation. We specifically chose to look at Ser276 phosphorylation of RelA/p65 since this event is known to be triggered both by LPS and thrombin to stimulate NF-κB transcriptional activity (11, 12, 27). LPS caused a substantial increase in RelA/p65 phosphorylation in the lungs of WT mice and this response was reduced in KO mice (Figure 3B). We next addressed the consequence of reduced NF-κB activation in KO mice on the levels of VCAM-1 and ICAM-1 in the lungs of these mice. Analyses by ELISA and immunoblotting of lung homogenates from LPS-challenged WT mice showed a significant increase in the levels of these adhesion molecules compared to saline-exposed WT mice, and this increase was attenuated in LPS-challenged KO mice (Figure 4, A and B), consistent with the effect of TG2 deficiency on NF-κB activation in these mice. We also measured the effect of TG2 deficiency on other proinflammatory genes (IL-1β, IL-6, MIP-1α, MIP-1β, MCP-1, and GM-CSF) in the lungs of mice challenged with LPS. As expected, a marked increase in the levels of these proinflammatory mediators was noted in lungs of LPS-challenged WT mice compared to saline-challenged WT mice. However, the levels of these proteins remain unaffected in LPS-challenged KO mice (Figure 5).

Figure 3.

Figure 3

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Involvement of TG2 in LPS-induced NF-κB activation in mouse lungs. (A) Effect of TG2 deficiency on IκBα degradation in the lungs of mice challenged with LPS. Wild type (WT) or TG2-knockout (KO) mice were challenged with LPS (i.p.) for 1 h. Lungs were isolated and analyzed for IκBα levels by immunoblotting. Actin levels were used to monitor loading. The bar graph represents the effect of TG2 deficiency on LPS-induced IκBα degradation normalized to actin levels. Data are mean ± SEM (n= 3-5 for each condition). ***p < 0.001 versus saline-treated WT; ###p < 0.001 versus LPS-treated WT. (B) Effect of TG2 deficiency on RelA/p65 phosphorylation in the lungs of mice challenged with LPS. WT and KO mice were challenged with LPS (i.p.) for 1 h. Lung homogenates were immunoblotted with an anti-phospho-RelA/p65 (Ser276) antibody. p70S6 kinase (p70S6K) levels were used to monitor loading. The bar graph represents the effect of TG2 deficiency on LPS-induced RelA/p65 phosphorylation normalized to p70S6K level. Data are mean ± SEM (n= 3-5 for each condition). *p < 0.05 versus saline-treated WT; #p < 0.05 versus LPS-treated WT.

Figure 4.

Figure 4

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Involvement of TG2 in LPS-induced adhesion molecule expression in mouse lungs. (A&B) Effect of TG2 deficiency on adhesion molecule expression in the lungs of mice challenged with LPS. WT and KO mice were challenged with LPS (i.p.) for 4 h. (A) Lung homogenates were analyzed for VCAM-1 levels by ELISA. The bar graph represents the effect of TG2 deficiency on LPS-induced VCAM-1 levels. Data are means ± S.E. (n = 3-6 for each condition). ***p < 0.001 versus saline-treated WT; #p < 0.05 versus LPS-treated WT. (B) Lung homogenates were analyzed for ICAM-1 levels by immunoblotting. Actin levels were used to monitor loading. The bar graph represents the effect of TG2 deficiency on LPS-induced ICAM-1 expression normalized to actin level. Data are means ± S.E. (n = 4-7 for each condition). **p < 0.01 versus saline-treated WT; #p < 0.05 versus LPS-treated WT.

Figure 5.

Figure 5

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TG2 deficiency fails to inhibit LPS-induced expression of cytokines and chemokines in mouse lungs. WT and KO mice were challenged with LPS (i.p.) for 4 h. Lung homogenates were analyzed for (A) IL-1β, (B) IL-6, (C) MIP-1α, (D) MIP-1β, (E) MCP-1, and (F) GM-CSF levels by multiplex immune assay system. Data are means ± S.E. (n = 3-6 for each condition). *p < 0.05, **p < 0.01 or ***p < 0.001 versus saline-treated WT.

Genetic ablation of TG2 reduces sepsis-induced lung PMN infiltration

Given the role of adhesion molecules in mediating the adhesion of PMN to facilitate their transmigration across the endothelial barrier, we evaluated whether levels of adhesion molecules correlate with PMN infiltration into the lungs of saline- and LPS-treated WT and KO mice. We used tissue myeloperoxidase (MPO) activity as a marker to monitor PMN infiltration into the lung. LPS-treated WT mice showed ~ 4-fold increase in lung tissue MPO activity compared to saline-treated WT and KO mice (Figure 6). The lung tissue MPO activity in LPS-treated KO mice was markedly lower than that in LPS-treated WT mice (Figure 6), consistent with levels of adhesion molecules in these mice (Figure 4). Together, these data show that TG2 deficiency dampens NF-κB activation, adhesion molecule expression, and lung PMN infiltration associated with sepsis.

Figure 6.

Figure 6

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Effect of TG2 deficiency on LPS-induced lung PMN sequestration. WT and KO mice were challenged with LPS (i.p.) for 6.5 h and the lungs were analyzed for PMN infiltration by measuring tissue MPO activity. Data are means ± S.E. (n = 4-6 for each condition). ***p < 0.001 compared with saline-treated control; ## p < 0.01 compared with LPS-treated control.

DISCUSSION

The present study demonstrates an important role of TG2 in the mechanism of EC inflammation associated with intravascular coagulation and lung PMN infiltration in the setting of sepsis. Our in vitro data show that TG2 mediates thrombin-induced EC inflammation by promoting NF-κB signaling via phosphorylation and DNA binding of RelA/p65. Our in vivo data recapitulates our in vitro findings and implicates a role for TG2 in the mechanism of adhesion molecule expression and lung PMN infiltration in mice exposed to endotoxemia.

Studies have shown that activation of endothelial NF-κB plays an essential role in septic multiple organ inflammation and injury (28). Conditional blockade of NF-κB pathway in the endothelium is sufficient to reduce multiple organ (including lung) inflammation, prevent vascular leak, and improve survival in murine models of sepsis (28). Interestingly, deficiency of TG2 also confers protection from sepsis-induced myocardium and renal tissue damage and lethality in mice (29). Our finding that TG2 knockdown attenuates thrombin-induced NF-κB activity identifies TG2 as a critical mediator of EC inflammation. Indeed, TG2 knockdown was associated with a significant decrease in the levels of VCAM-1 as well as IL-6 and MCP-1 following thrombin challenge of EC. Intriguingly, however, TNFα-induced NF-κB activity and expression of VCAM-1 was insensitive to TG2 depletion, indicating a stimulus-specific regulation of endothelial NF-κB by TG2. Together, these data reveal the importance of endothelial TG2 in regulating NF-κB and EC inflammation associated with intravascular coagulation.

Although, the relationship between TG2 and NF-κB in other cell types has been previously reported (15, 30, 31), the mechanism by which TG2 regulates NF-κB in EC appears to be different from that in other cell types. Studies have shown that TG2 regulates NF-κB in other cell types primarily at the level of IκBα but via a non-canonical pathway, whereby it catalyzes the polymerization of IκBα, which in turn leads to depletion of free IκBα and thereby nuclear translocation and DNA binding of NF-κB (15, 30, 31). By contrast, TG2 regulation of thrombin-induced NF-κB activity in EC relies on RelA/p65 phosphorylation in addition to its DNA binding activity. Furthermore, this mechanism of NF-κB regulation by TG2 in EC appears to be stimulus-specific as TNFα-induced phosphorylation of RelA/p65 was insensitive to TG2 knockdown. These data explain the lack of inhibition of TNFα-induced NF-κB transcriptional activity and further underscore the importance TG2 in EC inflammation associated with intravascular coagulation.

The above in vitro studies identifying TG2 as a critical determinant of thrombin-induced EC inflammation prompted us to investigate the in vivo relevance of TG2 in sepsis-induced lung PMN infiltration in mice. The rationale for using this model is that intravascular coagulation has emerged as an important component of lung inflammation and injury associated with sepsis (3, 8, 26). Indeed, elevated levels of thrombin have been found in BAL fluids of patients with sepsis and interstitial lung diseases (32). Furthermore, recombinant activated protein C (APC) has shown beneficial effects on sepsis-induced vascular injury and lethality in mice primarily by impairing thrombin/PAR-1 signaling (7). Studies have also shown that activation of endothelial NF-κB impairs the protein C anticoagulation mechanism (33), which is an important anti-inflammatory and cytoprotective mechanism during sepsis (34), causing lung inflammation and injury. Consistent with our in vitro data, we observed increased RelA/p65 phosphorylation in the lungs of WT mice challenged with LPS, which was significantly inhibited in the lungs of LPS-challenged TG2 KO mice. Similarly, we also noted enhanced degradation of IκBα in the lungs of LPS-challenged WT mice. In view of these observations, we determined the effect of TG2 deficiency on LPS-induced VCAM-1 and ICAM-1 (markers of EC inflammation and activation) as well as IL-1β, IL-6, MIP-1α, MIP-1β, MCP-1, and GM-CSF (markers of systemic inflammation) in the lung. Analysis of lungs revealed that VCAM-1 tissue levels were markedly reduced in the lungs of LPS-challenged KO mice compared to LPS-challenged WT mice. Similarly, LPS-induced ICAM-1 levels were also markedly decreased in the lungs of KO mice compared to WT mice. Because expression of these adhesion molecules, particularly that of VCAM-1 is predominantly restricted to EC, attenuated expression of these adhesion molecules in LPS-challenged KO mice is consistent with our in vitro data, and points to an important role of EC-derived TG2 in the mechanism of sepsis-induced NF-κB activation and adhesion molecule expression in the lung.

Unlike adhesion molecules, the levels of IL-1β, IL-6, MIP-1α, MIP-1β, MCP-1, and GM-CSF induced by LPS were not affected in the lungs of KO mice compared to WT mice. It should be noted that similar effects of EC-selective blockade of NF-κB on sepsis-induced tissue levels of adhesion molecules and IL-1β, IL-6, TNFα, and MCP-1 in the lungs of mice have also been reported (28, 35). Intriguingly, however, these results are not consistent with our in vitro data showing inhibition of IL-6 and MCP-1 release from EC upon TG2 depletion. Considering that endothelium is a major source of adhesion molecules but not that of cytokines and chemokines (35), it is likely that EC-derived IL-6, MCP-1, TNFα, and GM-CSF in the lungs of LPS-challenged KO mice are inhibited; however, this inhibition is masked by overwhelming production of these molecules by other lung cells including epithelial cells, fibroblasts, and the infiltrating leukocytes. To further test this possibility, we assessed the consequence of reduced adhesion molecule expression (in the face of unaltered cytokine/chemokine levels) on lung PMN infiltration in TG2 KO mice challenged with LPS. We found that TG2 deficiency was associated with reduced lung PMN infiltration induced by LPS challenge. These data are consistent with an earlier study which reported reduced PMN infiltration in the kidneys and peritoneum of TG2 KO mice subjected to endotoxemia (29).

In summary, this study reveals that TG2 is a critical mediator of EC inflammation and lung PMN infiltration associated with intravascular coagulation and sepsis. It also provides several pieces of evidence supporting the contention that EC-derived TG2 is critically involved in lung inflammation during sepsis. These include 1) in vitro evidence of the importance of TG2 in EC inflammation, 2) lack of effect of TG2 deficiency on LPS-induced IL-1β, IL-6, MIP-1α, MIP-1β, MCP-1, and GM-CSF levels which may derive predominantly from cells other than EC such as epithelial and inflammatory cells, and 3) the reduced expression of adhesion molecules in TG2 KO mice, particularly that of VCAM-1 whose expression is largely restricted to the endothelium. However, it should be emphasized that the present study does not exclude the involvement of TG2 derived from other cells, particularly epithelial TG2, in this model of lung inflammation. In view of a recent report implicating a role for epithelial TG2 in lung inflammation and fibrosis in mice challenged with bleomycin (18), it is likely that epithelial TG2 also contribute to lung PMN infiltration attributed to sepsis; however, determining the precise contribution of endothelial vs. epithelial TG2 in this model of lung inflammation requires additional comprehensive studies using mice with cell-specific deletion of TG2.

Acknowledgments

This work was supported by National Heart, Lung, and Blood InstituteGrants HL67424, HL096907, and HL116632. This study was also supported in part by National Institute of EnvironmentalHealth Sciences Center (EHSC) Grant ES-01247, and an SDG Grant from American Heart Association (KMB).

Footnotes

All authors declare that they do not have any financial conflict of interest with any entity whatsoever.

REFERENCES

  • 1.Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000;342(18):1334–1349. doi: 10.1056/NEJM200005043421806. [DOI] [PubMed] [Google Scholar]
  • 2.Erickson SE, Martin GS, Davis JL, Matthay MA, Eisner MD, Network NNA. Recent trends in acute lung injury mortality: 1996-2005. Crit Care Med. 2009;37(5):1574–1579. doi: 10.1097/CCM.0b013e31819fefdf. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Idell S. Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Crit Care Med. 2003;31(4 Suppl):S213–220. doi: 10.1097/01.CCM.0000057846.21303.AB. [DOI] [PubMed] [Google Scholar]
  • 4.Wheeler AP, Bernard GR. Acute lung injury and the acute respiratory distress syndrome: A clinical review. Lancet. 2007;369(9572):1553–1564. doi: 10.1016/S0140-6736(07)60604-7. [DOI] [PubMed] [Google Scholar]
  • 5.Levi M, van der Poll T. Two-way interactions between inflammation and coagulation. Trends Cardiovasc Med. 2005;15(7):254–259. doi: 10.1016/j.tcm.2005.07.004. [DOI] [PubMed] [Google Scholar]
  • 6.Gando S, Nanzaki S, Morimoto Y, Kobayashi S, Kemmotsu O. Systemic activation of tissue-factor dependent coagulation pathway in evolving acute respiratory distress syndrome in patients with trauma and sepsis. J Trauma. 1999;47(4):719–723. doi: 10.1097/00005373-199910000-00017. [DOI] [PubMed] [Google Scholar]
  • 7.Niessen F, Furlan-Freguia C, Fernández JA, Mosnier LO, Castellino FJ, Weiler H, Rosen H, Griffin JH, Ruf W. Endogenous epcr/apc-par1 signaling prevents inflammation-induced vascular leakage and lethality. Blood. 2009;113(12):2859–2866. doi: 10.1182/blood-2008-12-192385. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 8.Conway EM, Van de Wouwer M, Pollefeyt S, Jurk K, Van Aken H, De Vriese A, Weitz JI, Weiler H, Hellings PW, Schaeffer P, et al. The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappab and mitogen-activated protein kinase pathways. J Exp Med. 2002;196(5):565–577. doi: 10.1084/jem.20020077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Malik AB, Horgan MJ. Mechanisms of thrombin-induced lung vascular injury and edema. Am Rev Respir Dis. 1987;136(2):467–470. doi: 10.1164/ajrccm/136.2.467. [DOI] [PubMed] [Google Scholar]
  • 10.Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell. 1994;76(2):301–314. doi: 10.1016/0092-8674(94)90337-9. [DOI] [PubMed] [Google Scholar]
  • 11.Rahman A, Fazal F. Blocking nf-κb: An inflammatory issue. Proc Am Thorac Soc. 2011;8(6):497–503. doi: 10.1513/pats.201101-009MW. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hayden MS, Ghosh S. Signaling to nf-kappab. Genes Dev. 2004;18(18):2195–2224. doi: 10.1101/gad.1228704. [DOI] [PubMed] [Google Scholar]
  • 13.Lorand L, Graham RM. Transglutaminases: Crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol. 2003;4(2):140–156. doi: 10.1038/nrm1014. [DOI] [PubMed] [Google Scholar]
  • 14.Siegel M, Strnad P, Watts RE, Choi K, Jabri B, Omary MB, Khosla C. Extracellular transglutaminase 2 is catalytically inactive, but is transiently activated upon tissue injury. PLoS One. 2008;3(3):e1861. doi: 10.1371/journal.pone.0001861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Mann AP, Verma A, Sethi G, Manavathi B, Wang H, Fok JY, Kunnumakkara AB, Kumar R, Aggarwal BB, Mehta K. Overexpression of tissue transglutaminase leads to constitutive activation of nuclear factor-kappab in cancer cells: Delineation of a novel pathway. Cancer Res. 2006;66(17):8788–8795. doi: 10.1158/0008-5472.CAN-06-1457. [DOI] [PubMed] [Google Scholar]
  • 16.Kuncio GS, Tsyganskaya M, Zhu J, Liu SL, Nagy L, Thomazy V, Davies PJ, Zern MA. Tnf-alpha modulates expression of the tissue transglutaminase gene in liver cells. Am J Physiol. 1998;274(2 Pt 1):G240–245. doi: 10.1152/ajpgi.1998.274.2.G240. [DOI] [PubMed] [Google Scholar]
  • 17.Iismaa SE, Mearns BM, Lorand L, Graham RM. Transglutaminases and disease: Lessons from genetically engineered mouse models and inherited disorders. Physiol Rev. 2009;89(3):991–1023. doi: 10.1152/physrev.00044.2008. [DOI] [PubMed] [Google Scholar]
  • 18.Oh K, Park HB, Byoun OJ, Shin DM, Jeong EM, Kim YW, Kim YS, Melino G, Kim IG, Lee DS. Epithelial transglutaminase 2 is needed for t cell interleukin-17 production and subsequent pulmonary inflammation and fibrosis in bleomycin-treated mice. J Exp Med. 2011;208(8):1707–1719. doi: 10.1084/jem.20101457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Tiruppathi C, Ahmmed GU, Vogel SM, Malik AB. Ca2+ signaling, trp channels, and endothelial permeability. Microcirculation. 2006;13(8):693–708. doi: 10.1080/10739680600930347. [DOI] [PubMed] [Google Scholar]
  • 20.Paria BC, Bair AM, Xue J, Yu Y, Malik AB, Tiruppathi C. Ca2+ influx induced by protease-activated receptor-1 activates a feed-forward mechanism of trpc1 expression via nuclear factor-kappab activation in endothelial cells. J Biol Chem. 2006;281(30):20715–20727. doi: 10.1074/jbc.M600722200. [DOI] [PubMed] [Google Scholar]
  • 21.Bijli KM, Fazal F, Rahman A. Regulation of rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2. Am J Respir Cell Mol Biol. 2012 doi: 10.1165/rcmb.2012-0047OC. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.De Laurenzi V, Melino G. Gene disruption of tissue transglutaminase. Mol Cell Biol. 2001;21(1):148–155. doi: 10.1128/MCB.21.1.148-155.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kefer J, Rahman A, Anwar KN, Malik AB. Decreased oxidant buffering impairs nf-kappab activation and icam-1 transcription in endothelial cells. Shock. 2001;15(1):11–15. doi: 10.1097/00024382-200115010-00002. [DOI] [PubMed] [Google Scholar]
  • 24.Fazal F, Bijli KM, Murrill M, Leonard A, Minhajuddin M, Anwar KN, Finkelstein JN, Watterson DM, Rahman A. Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung pmn infiltration. PLoS One. 2013;8(3):e59965. doi: 10.1371/journal.pone.0059965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Li X, Schwacha MG, Chaudry IH, Choudhry MA. Acute alcohol intoxication potentiates neutrophil-mediated intestinal tissue damage after burn injury. Shock. 2008;29(3):377–383. doi: 10.1097/shk.0b013e31815abe80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Murakami K, Okajima K, Uchiba M, Johno M, Nakagaki T, Okabe H, Takatsuki K. Activated protein c prevents lps-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol. 1997;272(2 Pt 1):L197–202. doi: 10.1152/ajplung.1997.272.2.L197. [DOI] [PubMed] [Google Scholar]
  • 27.Lin CC, Shih CH, Yang YL, Bien MY, Lin CH, Yu MC, Sureshbabu M, Chen BC. Thrombin induces inducible nitric oxide synthase expression via the mapk, msk1, and nf-κb signaling pathways in alveolar macrophages. Eur J Pharmacol. 2011;672(1-3):180–187. doi: 10.1016/j.ejphar.2011.10.005. [DOI] [PubMed] [Google Scholar]
  • 28.Ye X, Ding J, Zhou X, Chen G, Liu SF. Divergent roles of endothelial nf-kappab in multiple organ injury and bacterial clearance in mouse models of sepsis. J Exp Med. 2008;205(6):1303–1315. doi: 10.1084/jem.20071393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Falasca L, Farrace MG, Rinaldi A, Tuosto L, Melino G, Piacentini M. Transglutaminase type ii is involved in the pathogenesis of endotoxic shock. J Immunol. 2008;180(4):2616–2624. doi: 10.4049/jimmunol.180.4.2616. [DOI] [PubMed] [Google Scholar]
  • 30.Lee J, Kim YS, Choi DH, Bang MS, Han TR, Joh TH, Kim SY. Transglutaminase 2 induces nuclear factor-kappab activation via a novel pathway in bv-2 microglia. J Biol Chem. 2004;279(51):53725–53735. doi: 10.1074/jbc.M407627200. [DOI] [PubMed] [Google Scholar]
  • 31.Kumar S, Mehta K. Tissue transglutaminase constitutively activates hif-1α promoter and nuclear factor-κb via a non-canonical pathway. PLoS One. 2012;7(11):e49321. doi: 10.1371/journal.pone.0049321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Günther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P, Grimminger F, Walmrath D, Temmesfeld-Wollbrück B, Seeger W. Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome. Am J Respir Crit Care Med. 2000;161(2 Pt 1):454–462. doi: 10.1164/ajrccm.161.2.9712038. [DOI] [PubMed] [Google Scholar]
  • 33.Song D, Ye X, Xu H, Liu SF. Activation of endothelial intrinsic nf-{kappa}b pathway impairs protein c anticoagulation mechanism and promotes coagulation in endotoxemic mice. Blood. 2009;114(12):2521–2529. doi: 10.1182/blood-2009-02-205914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Taylor FB, Chang A, Esmon CT, D'Angelo A, Vigano-D'Angelo S, Blick KE. Protein c prevents the coagulopathic and lethal effects of escherichia coli infusion in the baboon. J Clin Invest. 1987;79(3):918–925. doi: 10.1172/JCI112902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Xu H, Ye X, Steinberg H, Liu SF. Selective blockade of endothelial nf-kappab pathway differentially affects systemic inflammation and multiple organ dysfunction and injury in septic mice. J Pathol. 2010;220(4):490–498. doi: 10.1002/path.2666. [DOI] [PubMed] [Google Scholar]

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