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Differential Roles of JNK, ERK1/2, and p38 Mitogen-Activated Protein Kinases on Endothelial Cell Tissue Repair Functions in Response to Tumor Necrosis Factor-αKanaji N.a · Nelson A.d · Wang X.d · Sato T.b · Nakanishi M.c · Gunji Y.d · Basma H.d · Michalski J.d · Farid M.d · Rennard S.I.d · Liu X.d
aDivision of Endocrinology and Metabolism, Kagawa University, Kagawa, bDepartment of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, and cThird Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan; dDivision of Pulmonary, Critical Care, Sleep and Allergy Medicine, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebr., USA
Dr. Xiangde Liu
Division of Pulmonary, Critical Care, Sleep and Allergy Medicine
Department of Internal Medicine, University of Nebraska Medical Center
Omaha, NE 68198-5910 (USA)
Tumor necrosis factor (TNF)-α can alter tissue repair functions in a variety of cells including endothelial cells. However, the mechanism by which TNF-α mediates these functional changes has not fully been studied. We investigated the role of mitogen-activated protein kinases (MAPKs) on mediating the regulatory effect of TNF-α on the tissue repair functions of human pulmonary artery endothelial cells (HPAECs). TNF-α protected HPAECs from undergoing apoptosis induced by serum and growth factor deprivation, augmented collagen gel contraction, and stimulated wound closure. TNF-α activated c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38. Inhibitors of JNK (SP600125, 5 µ
© 2012 S. Karger AG, Basel
Pulmonary vascular endothelial cells play an important role in the pathogenesis of inflammatory lung diseases including chronic obstructive pulmonary disease and pulmonary hypertension . Tumor necrosis factor (TNF)-α is an inflammatory cytokine, and has been implicated in a diverse range of inflammatory, infectious, and malignant diseases. In response to TNF-α, endothelial cells display adhesion molecules such as E-selectin and intracellular adhesion molecule-1 . In combination with the release of chemokines, these responses lead to recruitment of leukocytes, which can promote inflammation . TNF-α can also alter endothelial cell functions such as migration and survival, and by these mechanisms TNF-α may contribute to the development of fibrosis by modulating tissue repair/remodeling [3,4,5,6].
Mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs including embryogenesis, proliferation, differentiation, migration, and apoptosis . Among more than a dozen MAPKs in mammals, the best known are c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and p38 families . These MAPKs are known to respond to a wide range of extracellular cues including cellular stressors, growth factors, and inflammatory cytokines such as TNF-α [2,8,9]. MAPKs play a role in the pathogenesis of pulmonary diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease [10,11,12]. MAPKs are also involved in regulating several critical functions of endothelial cells including vasodilatation, angiogenesis, and chemoattraction [13,14,15]. Recently, it was reported that MAPKs are involved in corneal endothelium defect repair . In addition, we recently reported that TNF-α promotes tissue repair/remodeling via NF-ĸB signaling in human pulmonary artery endothelial cells (HPAECs) , and that p38 MAPK modulates endothelial cell survival and tissue repair in response to IL-1β stimulation .
The current study was designed to investigate the role of MAPKs in mediating TNF-α-induced pulmonary endothelial cell tissue repair functions. We here show that TNF-α can stimulate JNK, ERK1/2, and the p38α isoform in pulmonary endothelial cells and that suppression of either JNK or ERK1/2 blocks while suppression of p38α augments endothelial cell tissue repair functions induced by TNF-α including cell survival, contraction of collagen gels, and cell migration.
Recombinant human TNF-α was purchased from R&D Systems (Minneapolis, Minn., USA). Anti-p38α, anti-p38β, anti-p38γ, anti-p38δ and anti-phosphorylated p38, anti-phosphorylated JNK, and anti-phosphorylated ERK1/2 antibodies (the above antibodies were used at a 1:1,000 or 1:2,000 dilution following the manufacturer’s instructions) were purchased from Cell Signaling Technology, Inc. (Danvers, Mass., USA). Anti-β-actin (used at a 1:10,000 dilution) was purchased from Sigma-Aldrich (St. Louis, Mo., USA). HRP-conjugated anti-mouse and rabbit IgG antibodies were purchased from Rockland (Gilbertsville, Pa., USA). Selective MAPK inhibitors (JNK inhibitor: SP600125, ERK1/2 inhibitor: PD98059, and p38 inhibitor: SB203580) were purchased from Calbiochem (San Diego, Calif., USA) and dissolved in dimethyl sulfoxide (DMSO). The small interference RNAs (siRNAs) targeting p38 MAPK isoforms (α, β, γ, and δ) and nontargeting control-siRNA were purchased from Dharmacon (Lafayette, Colo., USA).
HPAEC were purchased from Clonetics, Lonza (Walkersville, Md., USA). Cells were maintained in endothelial basal medium (EBM-2; Lonza) supplemented with growth factors and 2% fetal bovine serum according to the manufacturer’s recommendations (complete EBM). Cells were treated with TNF-α (0.5 ng/ml) and/or MAPK inhibitors (5 µm) in a 1:1 mixture of complete EBM to EBM-2 resulting in a final concentration of 1% serum (1:1 EBM) for 2 days. Concentrations of TNF-α (0.5 ng/ml) and MAPK inhibitors (5 µm) were chosen based on preliminary experiments with TNF-α (0.5–2 ng/ml) and MAPK inhibitors (5–10 µm) assessing their effect on HPAEC-mediated collagen gel contraction. DMSO was used as a solvent control for the MAPK inhibitors. To induce apoptosis, cells were incubated in EBM-2 basal medium, which is without any growth factors or serum, for 4 days in the presence or absence of TNF-α (0.5 ng/ml) and/or MAPK inhibitors (5 µm). Cells were then fixed and stained with a commercially available cell staining solution, i.e. PROTOCOL HEMA3 (Fisher Scientific Company L.L.C., Kalamazoo, Mich., USA). A Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) equipped with a DP71 digital camera and DP Controller software (Olympus, Tokyo, Japan) was used to take photomicrographs.
Following treatment, cells were washed with ice-cold PBS and total protein was extracted with lysis buffer (35 mm Tris-HCl, pH 7.4, 0.1% Triton X-100, 0.4 mm EGTA, 10 mm MgCl2) containing a protease inhibitor cocktail (Sigma-Aldrich). Lysates were centrifuged at 13,000 g for 10 min and the protein concentration was measured using a BIO-RAD Protein Assay Kit (Bio-Rad, Hercules, Calif., USA). Lysates were diluted with 2× sample buffer (125 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue, 10% β-mercaptoethanol) and heated at 95°C for 5 min. Five micrograms of proteins were loaded, separated by electrophoresis in 7.5% SDS-polyacrylamide gels, and transferred to a PVDF membrane (Bio-Rad). The membrane was blocked in 5% skim milk for 1 h at room temperature and incubated with proper concentrations of first antibody at 4°C overnight. Targeting proteins were subsequently detected using horseradish peroxidase-conjugated IgG with an enhanced chemiluminescence plus detection system (ECL plus) and Typhoon Scanner (Amersham Pharmacia Biotech, Little Chalfont, UK). To quantify phosphorylation of MAPKs, densitometric analysis was performed using ImageQuant software (Amersham Biosciences Corp., Piscataway, N.J., USA).
To selectively silence each isoform of p38 MAPK, RNA interference was performed. Briefly, cells were seeded at a cell density of 1 × 105 cells/ml. The next day, cells were transfected with siRNA targeting individual p38 MAPK isoforms or nontargeting control-siRNA (the final concentration of siRNA was 50 nm) in OptiMEM I (Invitrogen, Carlsbad, Calif., USA) using Lipofectamine2000 (Invitrogen). After 8 h of incubation, media were changed to complete EBM. Cell lysates were extracted on day 4 and the efficacy of RNA interference was assessed by immunoblot.
Native type I collagen gels were prepared by mixing a solution of rat tail tendon collagen (approximately 3 mg/ml), complete EBM, and cell suspension so that the final mixture resulted in 0.75 mg/ml collagen and 3 × 105 cells/ml. A 500-µl aliquot of the resulting solution was then cast into each well of a 24-well culture plate and allowed to polymerize. After gelation was completed, in about 20 min at room temperature, the gels were gently released into 60-mm dishes (3 gels in each dish) containing 5 ml of 1:1 EBM with or without TNF-α (0.5 ng/ml) and/or MAPK inhibitors (5 µm) on day 0 and incubated at 37°C for 4 days. Gel size was measured with an Optomax V image analyzer (Optomax, Burlington, Mass., USA). Data are expressed as the percentage of gel area compared with the original gel size.
Cells were pretreated with or without TNF-α (0.5 ng/ml) and/or MAPK inhibitors (5 µm) in a 1:1 EBM for 2 days. Fixed width linear wounds were then created using a cell scraper (Corning Inc., Corning, N.Y., USA), and the remaining cells were incubated in a 1:4 mixture of complete EBM to EBM-2 for 16 h in the presence or absence of freshly added TNF-α (0.5 ng/ml) and/or p38 inhibitors (5 µm). Cells were then fixed, stained, and photographed. To quantify cell migration, a grid was overlaid on the wounded area (line markers in the figures) on each image at 0 and 16 h after wounding using Photoshop Elements 2.0 software (Adobe Systems Inc., San Jose, Calif., USA) to make 1,064 tiny squares. The number of squares that contained migrated cells was counted. Three fields for each condition were quantified. Data are presented as the percentage of squares occupied by migrated cells after wounding.
Nearly confluent cells were treated with or without TNF-α (0.5 ng/ml) and/or MAPK inhibitors (5 µm) in EBM-2 only (serum- and growth factor-free medium) for 2 days. Both attached and floating cells were then collected and fixed with cold 70% ethanol in PBS at 4°C for 30 min. Cells were then pelleted by centrifugation and resuspended in the staining solution (50 µg propidium iodide, 100 µg RNase A in 1 ml PBS). After 1 h of staining at 4°C, flow cytometric DNA content profiling was performed.
Data are expressed as means ± SD. An unpaired two-tailed Student t test was used for single comparisons. Two-way ANOVA followed by Bonferroni’s correction was used to compare variances of multiple groups using PRISM 4 software. p < 0.05 was considered statistically significant.
We recently reported that TNF-α can promote HPAEC survival and tissue repair functions including wound closure and collagen gel contraction . The mechanism by which the effect of TNF-α is mediated, however, has not been defined. In the current study, therefore, we investigated the role of MAPKs in mediating TNF-α prevention of apoptosis and augmentation of tissue repair functions in HPAEC. First, we demonstrated that JNK, ERK1/2, and p38 MAPKs were activated in response to TNF-α as evidenced by phosphorylation of the MAPKs. This was readily demonstrated 20 min after stimulation (fig. 1a). At 40 and 60 min after stimulation, however, phosphorylation of the MAPKs was gradually diminished. At each time point, there was no significant difference in terms of phosphorylation intensity among JNK, ERK, and p38 (data not shown).
The p38 complex includes 4 different isoforms: p38α, β, γ, and δ . To identify the p38 isoforms that are activated by TNF-α, we performed RNA interference targeting individual isoforms. The siRNA targeting each p38 isoform could significantly and specifically suppress the respective target . Phosphorylation of p38 in response to TNF-α was significantly blocked in the cells transfected with siRNA targeting p38α (p < 0.05 compared to control-siRNA or siRNAs targeting p38β, γ, or δ), but not the other 3 isoforms (siRNAs targeting p38β, γ, or δ, p > 0.05), indicating that TNF-α induced phosphorylation of the p38α isoform (fig. 1b).
Since cell viability is important for tissue repair/remodeling, particularly in the face of injury, we investigated if inhibition of MAPKs affects cell survival/apoptosis. Cells were cultured to subconfluence (before treatment, fig. 2a). When the cells were subsequently cultured in basal medium without serum and growth factors, the majority of the cells detached and underwent cell death within 4 days (control + DMSO, fig. 2a). An average of 22.5 ± 5.3% of the cells survived after 4 days under this condition. In the presence of TNF-α (0.5 ng/ml), a higher number of cells survived (37.3 ± 8.8%, p < 0.05). Either SP600125 (JNK inhibitor) or PD98059 (inhibitor of MEK; upstream regulator for ERK1/2) reduced cell survival when added alone (SP600125: 10.8 ± 1.0% survived, PD98059: 12.8 ± 0.5%, p < 0.05, fig. 2a) and almost inhibited the ability of TNF-α to prevent cell death (SP600125: 20.7 ± 4.2% survived, PD98059; 19.5 ± 0.9%, p < 0.05, fig. 2a). In contrast, SB203580 (inhibitor of p38α and β) protected against cell death both when added alone (53.4 ± 14.3% survived, p < 0.05 compared to DMSO alone 22.5 ± 5.3%) and when added in the presence of TNF-α (76.1 ± 19.7% survived, p < 0.05 compared to TNF-α alone, 37.3 ± 8.8%; or p < 0.05 compared to SB203580 alone, 53.4 ± 14.3%, fig. 2a). The effect of SB203580 on cell survival was very significantly different from that of either SP600125 or PD98509 regardless of TNF-α presence or lack thereof (p < 0.01), while there was no difference between SP600125 and PD 98509 (p > 0.05).
Similar to the effect of the pharmacologic p38 inhibitor SB203580, siRNA targeting p38α also reduced cell death following withdrawal of serum and growth factors (surviving cells: 1.0 ± 0.2% with control-siRNA vs. 21.2 ± 5.1% with p38α-siRNA, p < 0.05, fig. 2b). In addition, inhibition of p38α by siRNA further augmented survival in the presence of TNF-α (4.7 ± 0.9% with control-siRNA vs. 41.5 ± 6.4% with p38α-siRNA-transfected cells, p < 0.05)
To determine if the cell death induced by deprivation of serum and growth factors is through induction of apoptosis, DNA content profiling by FACS analysis was conducted. Consistent with the microscopic observation and cell number count, DNA content profiling assay demonstrated that an average of 28.7 ± 1.9% of the cells contained less than the diploid amount of DNA after 2-day culture in the basal medium only (DMSO, fig. 3), indicating that these cells had undergone apoptosis. As expected, TNF-α alone significantly decreased the number of apoptotic cells, that is, cells containing less than the diploid amount of DNA (11.6 ± 3.2%, p < 0.05 compared to DMSO only). Interestingly, in the presence of either SP600125 or PD98059, however, TNF-α significantly increased the percentage of apoptotic cells (47.4 ± 6.2% or 49.5 ± 6.2%, respectively, p < 0.01 compared to TNF-α alone), and these numbers were even higher than that of DMSO alone (28.7 ± 1.9%, p < 0.05) or of the inhibitors alone (36.8 ± 3.7% for SP600125 alone or 35.6 ± 3.0% for PD98059 alone, p < 0.05). In contrast, SB203580 significantly decreased the percentage of apoptotic cells in the presence (2.6 ± 0.4%, p < 0.05 compared to DMSO + TNF-α only, fig. 3) or absence of TNF-α (18.77 ± 4.0%, p < 0.05 compared to DMSO only, fig. 3). Further, the effect of SB203580 on apoptosis was not only opposite to but also very significantly different from that of either SP600125 or PD98509 (p < 0.01) regardless of TNF-α presence or not, while there was no significant difference between the effect of SP600125 and PD98509 (p > 0.05).
The collagen gel contraction assay is an in vitro model of remodeling of extracellular matrix and is thought to reflect tissue repair functions. Thus, we investigated if inhibition of MAPK signaling affects the contraction of collagen gels in the presence and absence of TNF-α. Under control conditions, HPAECs were able to contract native type I collagen gels. Gel size decreased gradually and reached 75.0 ± 1.4% of the initial size on day 4 (fig. 4a). TNF-α augmented collagen gel contraction mediated by HPAEC (64.1 ± 3.4%, p < 0.05 compared to DMSO alone). SP600125 and PD98059 each reduced gel contraction, although the effect did not achieve statistical significance (fig. 4a). In contrast, the TNF-α augmentation of collagen gel contraction was significantly blocked by either SP600125 or PD98059 (72.6 ± 2.6 or 71.9 ± 3.9%, respectively, p < 0.05 compared to TNF-α alone). SB203580 enhanced gel contraction when added alone and further enhanced contraction in the presence of TNF-α (56.0 ± 4.4%, p < 0.05 compared to TNF-α alone). Similar to the effect on apoptosis, the effect of SB203580 on collagen gel contraction mediated by HPAEC was not only opposite to but also significantly different from that of SP600125 or PD98509 (p < 0.05), while there was no difference between SP600125 and PD98509 (p > 0.05).
Augmentation of collagen gel contraction by the p38 inhibitor was confirmed using RNA interference. While the cells transfected with control-siRNA contracted to 74.2 ± 2.5% of the initial size on day 4, cells transfected with p38α-siRNA contracted significantly more robustly (59.1 ± 3.9%, p < 0.05, fig. 4b). More importantly, TNF-α-augmented gel contraction was further enhanced by p38α-siRNA (64.4 ± 4.1% in control-siRNA vs. 46.4 ± 6.0% in p38α-siRNA, p < 0.05).
Next, we evaluated the role of MAPKs in regulating TNF-α-mediated wound closure by HPAECs. Under control conditions, endothelial cells migrated to cover 42.8 ± 5.9% of the wound (DMSO alone, fig. 5a). TNF-α stimulated wound closure significantly (69.9 ± 3.6%, p < 0.05 compared to DMSO alone). The JNK inhibitor alone (PD98059) had no effect on wound closure, while the MEK inhibitor inhibited wound closure slightly, but significantly (SP600125, 26.0 ± 7.7%, p < 0.05 compared to DMSO alone, fig. 5a). Both inhibitors significantly blocked TNF-α-stimulated wound closure (SP600125: 29.6 ± 5.4% and PD98059: 38.2 ± 11.6%, respectively, p < 0.01 compared to TNF-α alone). In contrast to SP600125 and PD98059, SB203580 stimulated HPAEC-mediated wound closure (66.5 ± 2.6%) and it was as potent as TNF-α (69.9 ± 3.6% for TNF-α alone). In addition, when SB203580 and TNF-α were added together, HAPEC-mediated wound closure was even further enhanced (89.2 ± 4.6%, p < 0.05 compared to either SB203580 or TNF-α alone, fig. 5a). Again, the effect of SB203580 on cell migration was not only opposite to but also very significantly different from that of SP600125 and PD98509 (p < 0.01), while there was no difference between SP600125 and PD98509 (p > 0.05).
Similar to the effect of p38 inhibitor on cell migration, suppression of p38α by siRNA resulted in significant augmentation of wound closure (62.4 ± 6.8% for p38α-siRNA vs. 43.0 ± 4.7% for control-siRNA-transfected cells, p < 0.05). TNF-α-augmented wound closure was further enhanced significantly in cells transfected with p38α-siRNA (60.4 ± 4.8% for control-siRNA vs. 92.0 ± 3.4% for p38α-siRNA, p < 0.01, fig. 5b).
The current study explores the mechanism by which TNF-α increases the survival of HPAECs in the absence of serum and growth factors and augments collagen gel contraction and promotes wound closure. TNF-α induces phosphorylation of JNK, ERK1/2, and p38. Suppression of JNK or ERK1/2 blocks, while suppression of p38α augments, the effects TNF-α on HPAEC survival, gel contraction, and wound closure. This suggests that the JNK and ERK1/2 pathways mediate TNF-α-augmented tissue repair functions in HPAECs positively and that p38α regulates them negatively in addition to the NF-ĸB pathway as illustrated in figure 6. These findings provide a new mechanistic insight into TNF-α modulation of endothelial cell tissue repair functions.
Cell viability is a key feature of tissue repair following injury. TNF-α is known to initiate an apoptotic process in many types of cells [5,6,18]. In the current study, however, we demonstrated that TNF-α prolonged HPAEC survival following withdrawal of serum and growth factors. This seems to be due to a reduction in apoptotic cell death in that fewer cells had hypodiploid DNA content in the presence of TNF-α.
We have previously reported that inflammatory cytokines IL-1β and TNF-α modulate endothelial cell survival through p65 activation  and that p38 modulates endothelial cell survival and tissue repair function in response to IL-1β stimulation . In the current study, we further report that TNF-α also induced MAPK (ERK, JNK, and p38) activation; furthermore, MAPK kinase inhibitors can also modulate endothelial cell survival. These findings suggested that both NF-ĸB and MAPK signaling pathways are involved in regulating endothelial cell survival in response to inflammatory cytokine stimulation. Interestingly, cross talk or interplay between these two signal pathways had been previously reported [19,20,21,22]. In this regard, Gorina et al.  reported that lipopolysaccharide induced early activation of NF-ĸB and MAPK kinases as well as late activation of STAT1 signaling in astrocytes, while Bladh et al.  reported that inhibition of ERK signaling participated in repression of NF-ĸB activity by glucocorticoids. Whether there is an interplay between NF-ĸB and MAPK signaling in our system, however, remains to be determined.
The current study demonstrated that TNF-α induced phosphorylation of MAPK kinases (p38, JNK, and ERK1/2) after 20 min of stimulation, and inhibition of JNK and ERK1/2 activation by the pharmacologic inhibitors resulted in blockade of TNF-α protection against cell death. In contrast, the p38 signaling pathway played an opposite role in regulating HPAEC survival. Inhibition of p38 by the pharmacologic inhibitor not only prolonged cell survival under the basal conditions but also significantly potentiated the effect of TNF-α on cell survival. Furthermore, p38α, but not other isoforms of p38, was responsible for regulating HPAEC survival in that only p38α-siRNA but not the siRNAs targeting other p38 isoforms protected cells in the presence or absence of TNF-α. Consistent with our findings, there is increasing evidence showing that inhibition of p38 MAPK protects endothelial cells from apoptosis [23,24]. In particular, inhibition of the p38α isoform leads the cells resistant to apoptosis [17,25,26]. These results suggest that p38α plays a pro-apoptotic role while JNK or ERK1/2 plays an anti-apoptotic role in HPAECs.
Consistent with previous reports [18,24,26,27], we have also demonstrated that suppression of the activated p38α by either a pharmacological inhibitor (SB203580) or siRNA protects endothelial cells from apoptosis in response to growth factor withdrawal. The effect of TNF-α on cell survival in the presence or absence of p38 inhibitor, however, has been controversial. In this regard, it has been reported that TNF-α could induce apoptosis in various kinds of cells including endothelial cells [18,28], and p38 inhibitor further enhanced TNF-α -induced apoptosis. In contrast, we found that TNF-α alone or TNF-α plus p38 inhibitor could further enhance cell survival. Consistent with our findings, Porras et al.  reported that cardiomyocytes and fibroblasts lacking p38α are more resistant to apoptosis induced by different stimuli. Interestingly, we also found that TNF-α, in the presence of inhibitors of JNK or ERK1/2, increased apoptosis, consistent with TNF-α activating both pro- and anti-apoptotic pathways.
Collagen gel contraction is an in vitro model of tissue repair/remodeling. It has been reported that MAPK inhibitors (JNK, ERK, or p38 inhibitors) inhibit gel contraction mediated by fibroblasts [29,30,31]. In the current study, we investigated the role of MAPKs in regulating endothelial cell-mediated collagen gel contraction in response to TNF-α stimulation. TNF-α augmented collagen gel contraction by HAPECs and, similar to previous reports using fibroblasts, inhibition of JNK and ERK1/2 partially blocked TNF-α-augmented collagen gel contraction, suggesting that collagen gel contraction augmented by TNF-α is mediated through JNK and ERK1/2 activation, at least in part. Unlike JNK and ERK1/2, however, p38 MAPK had the opposite effect on collagen gel contraction. Specifically, both a pharmacologic p38 inhibitor and an siRNA targeting p38α significantly augmented collagen gel contraction mediated by HPAECs. These results suggest that p38, specifically p38α, acts opposite to other MAPK components (JNK and ERK) in regulating extracellular matrix remodeling by endothelial cells in response to TNF-α stimulation.
Similar to collagen gel contraction, wound closure is also an in vitro measure of tissue repair functions following injury. In the current study, we also assessed the effect of TNF-α on endothelial cell wound closure and the role of MAPKs in mediating the effect of TNF-α. Inhibition of JNK and ERK1/2 blocked TNF-α-augmented wound closure, suggesting that TNF-α-augmented wound closure is mediated via JNK and ERK1/2 pathways, which is similar to previous reports [32,33]. Interestingly, we also demonstrated that p38 MAPK had an opposite effect, that is, blockade of p38 signaling by a pharmacologic p38 inhibitor or an siRNA targeting p38α accelerated wound closure under control conditions as well as in the presence of TNF-α. Thus, consistent with the effect on collagen gel contraction, activation of JNK and ERK1/2 is required for TNF-α-augmented wound closure while p38α activation acts opposite to JNK or ERK1/2 in modulating the effect of TNF-α on endothelial cell migration.
The findings of the current study may have clinical implications for inflammatory lung diseases including COPD [12,34]. In this regard, excessive apoptosis of pulmonary endothelial cells has been suggested to contribute to the development of COPD . It has also been reported that phospho-p38-positive cells in alveolar walls were increased in COPD patients compared with smoking and nonsmoking control subjects . Consistent with our findings, over activation of the p38 MAPK in the airway walls may result in apoptosis of endothelial cells and by this mechanism p38 may contribute to the development of COPD. Therefore, p38 inhibition has potential therapeutic benefit for inflammatory lung diseases including COPD .
There are several limitations to the current study. First, we did not investigate any downstream signaling from the MAPKs that may be responsible for cell survival (for instance, caspases and Bcl-2 family proteins) and tissue repair functions altered by TNF-α. Second, we did not assess cross talk among MAPKs, although the activity of one MAPK can be influenced by another  or interplay between NF-ĸB and MAPK signaling pathways although we have reported that NF-ĸB also play a role in modulating endothelial cell survival and functions . Third, we used only one type of endothelial cells. It remains unclear if other endothelial cells would function in the same way as the HPAECs used in the current study.
In summary, TNF-α increases the survival of HPAECs and augments collagen gel contraction and wound closure. TNF-α activates JNK, ERK1/2, and p38α MAPKs. Inhibition of either JNK or ERK1/2 blocks while suppression of p38α augments the effects of TNF-α. These findings suggest that different MAPKs can positively or negatively modulate tissue repair/remodeling functions of endothelial cells, especially in an inflammatory milieu where the inflammatory cytokine TNF-α is present.
The authors thank Ms. Lillian Richards for the excellent secretarial support of the manuscript.
Dr. Xiangde Liu
Division of Pulmonary, Critical Care, Sleep and Allergy Medicine
Department of Internal Medicine, University of Nebraska Medical Center
Omaha, NE 68198-5910 (USA)
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