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attract migration of embryonic stem cells via activation of p38 and c-Jun amino-terminal kinase
Stem Cells Research Laboratory, The Charles A. Dana Research Institute and Harvard-Thorndike Laboratory, Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA
1Correspondence: Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215, USA. E-mail: yxiao{at}caregroup.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) plays an important role in the pathogenesis of myocardial infarction. Stem cells are able to regenerate infarcted myocardium. This study investigated whether TNF- was able to induce migration of embryonic stem cells (ESCs) in vitro. We used a Transwell assay in which neonatal rat cardiomyocytes, with or without transfection of TNF- cDNA, were plated in the lower compartments and mouse ESCs tagged with green fluorescent protein were added to the upper compartments. TNF- level was significantly increased in the medium of the lower compartments seeded with TNF--transfected cardiomyocytes. Compared with the controls, overexpression of TNF- significantly enhanced migration of ESCs to the lower compartments. This enhancement was attenuated by preincubation of ESCs with the antibody against the type II TNF- receptor (TNF-RII), but not by the antibody against the type I TNF- receptor (TNF-RI). Western blot analysis showed that the phosphorylated protein levels of p38 and c-Jun amino-terminal kinase (JNK) were significantly increased in TNF--treated ESCs. Inhibition of the activity of p38 or JNK significantly attenuated TNF--induced ESC migration. Our data demonstrate that excessive TNF- stimulates TNF-RII and enhances migration of ESCs in vitro. Activation of p38 and JNK is required for TNF--enhanced ESC migration.—Chen, Y., Ke, Q., Yang, Y., Rana, J. S., Tang, J., Morgan, J. P., Xiao, Y.-F. Cardiomyocytes overexpressing TNF- attract migration of embryonic stem cells via activation of p38 and c-Jun amino-terminal kinase.
Key Words: myocardial infarction • transfection • Transwell assay
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
and animal studies (3
4
5
6)
. The engrafted stem cells improved cardiac performance of injured hearts via regeneration of infarcted myocardium and neovascularization (7
8
9
10)
. Embryonic stem cells (ESCs) are pluripotent cells that retain the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and other types of cells (11
, 12)
. Our recent study demonstrated that intravenously infused mouse ESCs were able to migrate to virally injured myocardium and significantly increase the survival rate in myocarditic mice via the reduction of myocardial necrosis and inflammatory infiltration (13)
. However, the process of the migration of infused ESCs and repair of damaged myocarditic hearts remains to be determined.
Clinical data show that TNF- generation and release occur in patients with acute MI (14)
. This release contributes to myocardial injury and dysfunction (15)
. There is a correlation between infarct size and TNF- concentration in patients with MI (16)
. High concentrations of TNF- appear in the circulation of patients with heart failure and the levels are directly proportional to a patient’s functional class and prognosis. In rat experiments, TNF- production of infarcted myocardium started on day 1 and was sustained for 35 days (17)
. Expression of TNF- also occurred in the myocardium contralateral to the infarcted site. It has been shown that TNF- affects the growth and function of myocytes and fibroblast cells (18)
.
Several studies show that TNF- activates and enhances migration of vascular smooth muscle cells (VSMC; refs 19
, 20
). TNF- also stimulates the migration of neutrophils in vitro in a dose- and time-dependent manner (21)
. Additional evidence demonstrates that TNF- increases the proliferation and migration of intraepithelial lymphocytes (22)
. Recent experimental results demonstrate that each step of transendothelial migration of flowing neutrophils is regulated by TNF- stimulation (23)
. Local production of TNF- provides an important signal for Langerhans cell (LC) migration in humans during cutaneous immune and inflammatory reactions (24
, 25)
. These results indicate that TNF- enhances the migration of different cell types in vitro and in vivo. However, any possible effect from TNF- on migration of stem cells may have important implications for stem cell therapy and needs to be delineated. Therefore, this study was to test whether TNF- was able to enhance the migration of ESCs in vitro and, if so, to define the mechanism of the enhancement.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cells were cultured in flasks (TI-25) at 37°C in humidified atmosphere with 5% CO2. To maintain ESC self-renewal and pluripotency, leukemia inhibitory factor (LIF, 1000 U/mL) was added to the culture medium to prevent ESC differentiation. ESCs after 3 days in culture were transfected with a GFP gene for identification of migrated cells (9)
. Two days after GFP transfection and selection with neomycin, ESCs with stable expression of GFP were trypsinized and suspended in culture medium. Collected cells were centrifuged at 500 x g for 5 min at room temperature and resuspended in medium for cell migration experiments.
Transfection of plasmid TNF- cDNA
The plasmid of TNF- cDNA was a generous gift from Dr. Paul Robbins (Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA). The TNF- cDNA was originally cloned into the plasmid pAdLox, then transfected into the E1 and E3 deleted Psi-5 adenovirus. Expression was driven by the cytomegalovirus promoter. Adenovirus-mediated gene transfer was used for transfection of TNF- cDNA (5 µL, 9.9x1011 particles/mL) in the cultured neonatal rat cardiomyocytes (80% confluence) plated in a TI-75 flask. Transfected cells were incubated at 37°C in air with 5% CO2 added and 98% relative humidity for 1 day. These cardiac cells were trypsinized and plated to each lower compartment (2x105 cells/well) of a 6.5 mm diameter Transwell dish with or without a gelatin-coated glass coverslip (3 mm diameter) placed on the bottom. After another day of culture these cells were ready for migration experiments. The content of TNF- released by cardiomyocytes into the culture medium was measured with a commercial ELISA kit specifically designed for detection of mouse TNF- (R&D Systems, Inc., MN, USA). Expression of TNF- in cultured neonatal rat cardiomyocytes was also detected by immunostaining. Two days after TNF- transfection, cultured rat cardiomyocytes were washed with phosphate-buffered saline (PBS) twice, then fixed in 4% paraformaldehyde. A rat (monoclonal) anti-TNF biotin-conjugate antibody (Biosource International Inc., Camarillo, CA, USA) was used for incubation of cardiomyocytes. Streptavidin-R-phycoerythrin (R-PE) conjugates (Biosource International Inc., CA, USA) were used as the second antibody for fluorescence.
Migration assay
Migration of ESCs was assessed in Transwell plates of 6.5 mm diameter with 5 µm pore filters. Mouse embryonic fibroblast feeder cells (STO, ATCC) were plated at 3 x 104 cells/well on gelatin-coated filters. Nonadherent cells were removed after 18 h. The adherent cells were cultured for 2 days to obtain confluent STO monolayers. Each lower compartment with or without one glass coverslip (3 mm diameter) on the bottom was plated with neonatal rat cardiomyocytes. Myocytes in the treated group were transfected with TNF- cDNA. Before adding ESCs to the upper compartment, STO monolayers were treated with mitomycin-C (10 µg/mL) for 90 min and washed three times with the culture medium. GFP-tagged ESCs (6x104 cells) were added to each upper compartment. After a 5 or 24 h incubation period, the number of GFP-positive cells in the lower compartments was measured. ESC migration was also examined in cardiomyocytes transfected with the virus vector (without TNF- cDNA) as an additional control. The number of migrated GFP-positive cells was counted under fluorescent microscopy or quantified with fluorescence-activated cell sorter (FACS).
Flow cytometry
To determine the number of migrated ESCs (GFP-tagged), cells in the lower compartment of Transwell dishes were trypsinized, then incubated with goat anti-rat sarcomeric actin (1:100) for 1 h at room temperature. PE-conjugated rabbit anti-rat IgG (1:100, 1 h at 21–23°C) served as the second antibody. Primary culture of neonatal rat cardiomyocytes showed that >95% were stained positive to sarcomeric actin. After washing, the cells were resuspended in 2% paraformaldehyde solution and the samples (10,000 events per sample) were acquired on FACScan flow cytometry (BDIS) equipped with a standard filter setup. Multiparameter data acquisition and analyses were performed with CELLUQEST software (BDIS). Cells were separated based on different fluorescence staining.
Western blot analysis
Stem cells were either unstimulated or incubated with TNF- (2 or 20 ng/mL) for different periods at 37°C. Cells were harvested in RIPA buffer (1xPBS, 1% NP-40, 0.15% SDS, 0.5% sodium deoxycholate, 1.25 mM sodium fluoride, 1 mM sodium pyrophosphate). The insoluble cell debris was removed by centrifugation at 4°C for 10 min at 12,000 x g. After addition of an equal volume of loading buffer (0.25 M TRIS (pH 6.8), 10% glycerol, 1% SDS, 0.5% ß-mercaptoethanol), samples were boiled and separated by 4–15% SDS-PAGE ready gel and transferred to 0.2 µm polyvinylidene difluoride membranes (BioRad, Hercules, CA, USA) for staining. The nonspecific binding sites on the membrane were blocked after treatment with PBS containing 7.5% w/v nonfat dry milk, 1% normal horse serum, and 0.1% Tween 20 for 1 h. The membrane was then incubated overnight at 4°C with anti-phosphotyrosine mAbs (1:1000) against p42/44, JNK, and p38 (Cell Signaling Technology, Inc., Beverly, MA, USA). Proteins were visualized with horseradish peroxidase-conjugated second antibody (1:2000) and an enhanced chemiluminescence (ECL) Luminol reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA). To ensure similar amounts of proteins in each sample, the same membrane was stripped off, reprobed with mAbs against p42/44, JNK, or p38, and developed with HRP-conjugated secondary Abs by ECL.
Data analysis
Values are presented as mean ± SE. Results between two individual groups were compared by the unpaired Student’s t test. Data delivered from more than two experimental groups were tested by one-way ANOVA. Differences between control and treated groups were considered statistically significant when P values were <0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced ESC migration in vitro on ESC migration, the lower compartments of the Transwell chambers were plated with neonatal rat cardiomyocytes with or without transfection of the TNF- cDNA. TNF- production by control cardiomyocytes in the culture medium of the lower compartments was 49 ± 21 pg/mL. However, cardiomyocytes transfected with TNF- cDNA produced significantly more TNF- in the medium: 1301 ± 53 pg/mL (n=8, P<0.001). Compared with the control cardiomyocytes (Fig. 1
A, B), cardiomyocytes with overexpression of TNF- attracted more GFP-positive ESCs migrated to the lower compartments within 24 h after coculture (Fig. 1C, D
). Under fluorescence microscopy, more GFP-positive cells were detected on the coverslips seeded with TNF--transfected cardiomyocytes than those plated with control cardiac cells. The averaged numbers of migrated GFP-positive cells were 108 ± 9 cells/mm2 for the TNF--transfected group (n=5 runs, P<0.001) and 26 ± 7 cells/mm2 for the control (n=5 runs). Immunostaining showed that compared with control cardiomyocytes (Fig. 2
A, upper panel), TNF- level was much higher in the cardiomyocytes transfected with TNF- cDNA (Fig. 2A
, lower panel). Cardiomyocytes with overexpression of TNF- attracted more GFP-positive ESCs (Fig. 2B
, lower panel) from the upper compartment.
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In another experiment, we tested whether ESCs preferred to migrate toward cardiomyocytes with overexpression of TNF-. Coverslips (3 mm diameter) seeded with control (one side) or TNF--transfected (another side) cardiomyocytes were placed separately on the bottom of 6-well Transwell dishes with separation in the middle of each lower compartment. GFP-positive ESCs were added to the upper compartments as described above. Twenty-four hours after coculture, migrated GFP-positive cells on each coverslip were counted under fluorescence microscopy. The number of migrated ESCs was significantly increased from 41 ± 6 cells/mm2 for the control to 91 ± 5 cells/mm2 (n=3, P<0.05 vs. control) for the TNF--transfected cardiomyocytes. These results demonstrate that TNF- excessively expressed in cardiomyocytes also served as a chemoattractant in the process of ESC migration in vitro.
Role of TNF- receptor II in TNF--induced ESC migration
The effects of TNF- on migration of ESCs were assessed with flow cytometry. GFP-tagged ESCs were added to the upper compartments and cocultured with cardiomyocytes plated in the lower compartments for 5 h. After these treatments, the cardiomyocytes and migrated ESCs in the lower compartments were trypsinized and stained. TNF--transfected cardiomyocytes attracted significantly more ESCs to the lower compartments. Figure 3
shows that 10.9% (control, R2) of total cells in the lower compartments seeded with control cardiomyocytes were migrated GFP-positive ESCs. The percentage of GFP-positive cells in the lower compartments was increased to 19.8% when the cardiomyocytes were transfected with TNF- cDNA (Fig. 3
, TNF-, R2). However, cardiomyocytes transfected with the blank viral vector (no TNF- cDNA) did not enhance ESC migration (Fig. 3
, virus control). The averaged data show that in the control group, 1127 ± 38 GFP-positive ESCs were detected in each 10,000 cells. However, in the TNF- transfected myocytes, the number of GFP-positive ESCs was increased to 1989 ± 52 cells/10,000 cells (n=18, P<0.001), 76.5% greater for TNF--transfected cardiomyocytes (Fig. 4
, TNF-) than for nontransfected heart cells (Fig. 4
, control). These results suggest that TNF- enhanced migration of ESCs in vitro.
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The activities of TNF- are mediated through two functionally distinct cell surface receptors, the TNF-RI and TNF-RII. To determine which receptor type was more responsible for TNF--induced ESC migration, GFP-tagged ESCs were preincubated with the neutralizing antibody against TNF-RI or TNF-RII (3 µg/mL, BD Biosciences, San Jose, CA, USA). After incubation for 60 min, antibody-treated ESCs were added to the upper compartments of Transwell dishes and cocultured with TNF--overexpressing cardiomyocytes seeded in the lower compartments for 5 h. The total number of cells in the lower compartments was counted and the percentage (%) of GFP-positive cells was calculated. Compared with 19.8% GFP-positive cells in the TNF--treated group (Fig. 3
, TNF-), the antibody against the type II receptor of TNF- markedly reduced the number of migrated GFP-positive ESCs to 12.4% (Fig. 3
, TNF+anti-RII). In contrast, the antibody against TNF-RI did not significantly alter the TNF--induced ESC migration (19.1%, Fig. 3
, TNF+anti-RI). Figure 4
shows the averaged data for the control and TNF--transfected cardiomyocytes. Obviously, the cardiomyocytes transfected with TNF- attracted more ESCs and pretreatment of ESCs with the anti-TNF-RII antibody, but not the anti-TNF-RI antibody, significantly reduced the attraction. However, in the non-TNF--transfected cardiomyocytes, preincubation of ESCs with the anti-TNF-RII antibody did not significantly alter ESC migration (121% of control, n=3 runs, P>0.05). An immunohistologic staining assay showed similar results of TNF--enhanced ESC migration and inhibition of the anti-TNF-RII antibody (Fig. 5
). Our data indicate that TNF--induced ESC migration resulted from stimulation of TNF-RII of ESCs.
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Signal transduction pathways in TNF--induced ESC migration
TNF- binds to its receptors and activates different intracellular signaling components. Extracellular signal-regulated kinases (ERKs) are critical for the responses to cytokine stimuli. To test which kinase(s) played a critical role in TNF--induced ESC migration, we investigated the effects of TNF- on p42/44 (ERK1/2), p38 MAPK, and JNK during TNF--induced ESC migration. Western blot analysis showed that total protein levels of p38 and JNK were not significantly altered at different time points in ESCs treated with 20 ng/mL TNF- (Fig. 6
A). However, the levels of both phosphorylated p38 and JNK were markedly increased in TNF--treated ESCs (Fig. 6B
). Increases in phosphorylated p38 and JNK reached a peak from 5 to 30 min after incubation of ESCs with 20 ng/mL TNF-, then began to subside in the presence of TNF- (Fig. 6B
). Similar results were obtained in three additional trials. Relative quantification of phosphorylated JNK by measurement of the intensities of the bands shows that TNF- stimulation for 5 to 30 min caused 2.1- to 3.8-fold increases (P<0.05) in phosphorylated 44 kDa and 56 kDa JNK proteins in cultured ESCs. Phosphorylated protein levels returned toward the control 60 min after application of TNF-. In contrast, levels of total or phosphorylated ERK1/2 (p42/44) did not change in ESCs treated with 20 ng/mL TNF- (data not shown). A lower concentration (2 ng/mL) of TNF- produced an effect on p38 and JNK phosphorylation similar to that of 20 ng/mL (data not shown). These results indicate that the effects of TNF- on ESC migration may result from enhanced activities of p38 and JNK.
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To further assess the roles of p38 and JNK in TNF--induced ESC migration, two specific inhibitors widely used for signaling studies were tested: SB203580 (Sigma), an agent that selectively inhibits p38, and SP600125 (Calbiochem, CA), a selective inhibitor of JNK. The lower compartments of Transwell dishes were plated with neonatal rat cardiomyocytes transfected with TNF- cDNA. ESCs were preincubated with the specific inhibitors of either SB203580 (10 µM) or SP600125 (10 µM) or both together (10 µM each) for 30 min. Inhibitor-incubated ESCs with either one or both of the inhibitors were added to the upper compartments and cocultured for 5 h with TNF--transfected cardiomyocytes. Figure 7
shows that compared with the nontransfected myocytes, overexpression of TNF- in TNF--transfected cardiomyocytes significantly increased migrated GFP-positive cells to 208 ± 2.4% of the control (n=5, P<0.001). Application of either inhibitor at 10 µM significantly attenuated the effects of TNF- on ESC migration: 132 ± 2.4% and 146 ± 2.4% of the control for the p38 inhibitor SB203580 (n=5) and for the JNK inhibitor SP600125 (n=5), respectively. Incubation of ESCs with the two inhibitors together (SB203580 plus SP600125) further reduced TNF--induced ESC migration to 115 ± 1.6% of the control (n=5, Fig. 7
). Our results demonstrate that p38 MAPK and JNK play important roles in TNF--induced ESC migration. However, the number of migrated ESCs in the presence of either one or both of the inhibitors remains significantly greater than the control group, which suggests that other signaling pathways may also be involved in TNF--induced ESC migration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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attract the migration of ESCs from one culture compartment to another. It has been shown that TNF- enhances migration of VSMCs (19
, 20)
, neutrophils (21)
, dendritic cells in the skin (27)
, and Langerhans cells (LCs) during cutaneous immune and inflammatory reactions (24
, 25)
. TNF- stimulates and regulates every step of the migration of flowing neutrophils (23)
. Generation and release of TNF- occur during the pathophysiological response to myocardial injury (14)
. Treatment with the monoclonal antibody against TNF- can be cardioprotective, particularly in the setting of heart failure with acute myocardial infarction (15)
. TNF- also affects fibroblast cell growth (18)
. These results indicate that TNF- production is a part of the important intrinsic response to myocardial injury.
We recently reported that intravenously infused ESCs migrated to myocarditic hearts and differentiated into myocytes in injured myocardium of encephalomyocarditic mice (13)
. The survival rate in the ESC-treated myocarditis group was significantly higher than the untreated myocarditis group. However, the mechanism of ESC migration to myocarditic hearts is unclear. Recent studies have shown that expression of TNF- (28)
and TNF ligand superfamily costimulatory molecules (29)
is significantly greater in the myocarditic group than in the control group in both patients and murine animal models. Therefore, our present data suggest that TNF- may play an important role in migration of ESCs to virally injured hearts after an intravenous infusion of stem cells (13)
. TNF- production during acute myocardial injury may help the heart to recruit extracardiac stem cells and regenerate damaged myocardium. Our previous data showed that stem cell transplantation produced a greater improvement of cardiac function in pigs with acute myocardial infarction than with chronic infarction (10
, 30)
, which might result from more TNF- production during the acute stage of infarction. Moreover, in TNF- gene-deficient mice [TNF-(-/-)] with acute myocarditis via the encephalomyocarditis virus (EMCV), the survival rate of TNF-(-/-) mice after EMCV infection was significantly lower than that of TNF-(+/+) mice. Injection of recombinant human TNF- improved the survival of TNF-(-/-) mice in a dose-dependent manner, indicating that TNF- is important for protection against viral myocarditis (31)
. TNF- is necessary for adhesion molecule expression and recruitment of leukocytes to inflammatory sites; thus, a lack of this cytokine resulted in failure to eliminate infectious agents and an increase in mortality (31)
. Extracardiac progenitor cells in adult human beings are capable of migrating to transplanted hearts (32
, 33)
, but it remains undetermined whether TNF- or other cytokines play a crucial role in this process. Our data suggest that TNF- may attract the migration of stem cells to injured myocardium.
TNF- activity is regulated by two distinct receptors, TNF-RI and TNF-RII, which mediate discrete cellular responses (34)
. The expression of TNF-RI and TNF-RII in ESCs was confirmed by RT-PCR and Northern blot analysis (35)
. In the present study, we found that the enhanced migration of ESCs toward cultured rat cardiomyocytes overexpressing TNF- was significantly attenuated by preincubation of ESCs with the antibody against TNF-RII, but not against TNF-RI. This result is consistent with the report that TNF--induced LC migration was decreased in gene-targeted knockout mice lacking TNF-RII (36)
. However, another study showed that the signaling pathways of both TNF receptors are important in mediating corneal LC migration in TNF receptor knockout mice (37)
. In addition, TNF-RI played a critical role in TNF- promotion of cutaneous inflammation (38)
and in the TNF--induced VSMC migration, which is MAPK dependent (39)
. These results demonstrate that TNF- induces migration of different cell types in vitro and in vivo. The findings also indicate that either TNF-RI or TNF-RII or both regulate TNF--induced cell migration. These various observations may result from using different experimental conditions and different cell types from various preparations or animals. Indeed, it has been reported that a broad spectrum of biological effects of TNF- depends on the type and growth state of the target cell (40)
. In the present study, our data demonstrate that TNF-RII, not TNF-RI, plays a crucial role in TNF--induced migration of ESCs. This is consistent with the finding that TNF- promotes migration of cutaneous dendritic cells primarily through the selective activation of TNF-RII (27
, 36)
.
It has been found that TNF- enhances cell migration in different types of cells (19
20
21
22
23
24
25)
. Binding of TNF- to its receptors activates different intracellular signaling components. The family of MAPKs plays a key role in cellular responses to cytokine stimulation. The three main families of MAPKs are the extracellular signal-regulated kinase (ERK)1/2 (p42/44), p38, and JNK (41)
. Mammalian ERK families are rapidly and transiently activated in response to stimulation of mouse macrophages with TNF-. The JNK and p38 pathways have been reported to be critical in cytokine-induced signaling of cell migration, whereas ERKs respond to mitogen and growth factors that regulate cell proliferation and differentiation (41)
. In this study, our results show that p42/44 ERKs were not altered in ESCs after treatment with TNF-. However, stimulation of ESCs with TNF- increased the levels of phosphorylated proteins of p38 and JNK whereas the total protein levels of p38 and JNK did not change. The specific inhibitors SB203580 and SP600125 for p38 and JNK, respectively, significantly attenuated TNF--induced migration of ESCs. These results suggest that p38 and JNK play critical roles in TNF--induced ESC migration, which is consistent with the findings that a variety of extracellular stimuli, such as cytokines and growth factors, activate the p38 signaling pathway, thereby causing migration of various cell types (19
20
21
22
23
24
25)
. For example, stem cell factor-induced migration of mast cells requires activation of p38 MAPK whereas mitogen-induced extracellular kinase (MEK) is much less involved (42)
. Migration of endothelial cells induced by platelet-derived growth factor (43)
and vascular endothelial growth factor (44)
also involves activation of p38 MAPK. A recent study showed that TNF--induced eotaxin release of human eosinophils occurred via phosphorylation of p38, but not ERK (45)
. Two recent studies show that JNK, and to some extent p38, but not ERK, play a predominant role in TNF--induced CD44 expression in human monocytic cells (46)
. Moreover, TNF--induced JNK phosphorylation is one of the major signaling pathways for TNF--induced cardiac hypertrophy (47)
. TNF- has been shown to activate all three types of MAPKs (ERK1/2, p38, and JNK) in different cell types (38)
, but our data show that enhanced migration of ESCs by TNF- resulted from activation of p38 and JNK, not ERK1/2. These results are consistent with the reports that growth factors enhanced migration of ESCs through activation of JNK (47
, 48)
. In engineered HeLa cells with an overexpression of TNF-RII, TNF- activates JNK but not p38 (49)
. However, our observations show that TNF--induced ESC migration resulted from stimulation of TNF-II and activation of p38 and JNK. A possible explanation for activation of both p38 and JNK in ESCs after stimulation of TNF-RII is that a "cross-talk" may occur, possibly via a TNF-R-associated factor (TRAF) 2 that binds both TNF-RI and TNF-RII and has been shown to be capable of activating p38 MAPK (50). Elucidation of the mechanism(s) of mobilization and homing of stem cells is of great importance to the potential of using a less invasive way (i.e., intravenous infusion) in stem cell therapy.
In summary, our data show that TNF- enhances migration of ESCs in vitro and that the enhanced migration is mediated via stimulation of TNF-RII and activation of p38 and JNK. This conclusion is based on the observed attenuation of TNF--enhanced ESC migration by the antibody against TNF-RII, the increase in phosphorylated protein levels of p38 and JNK, and the inhibition of TNF--enhanced ESC migration by specific inhibitors of p38 and JNK. Taken together, our results suggest a predominant role of p38 and JNK in TNF--induced ESC migration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 17, 2003. Accepted for publication August 5, 2003.
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