The JI PBL Intereron Source
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xiao, Y. Q.
Right arrow Articles by Henson, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xiao, Y. Q.
Right arrow Articles by Henson, P. M.
The Journal of Immunology, 2006, 176: 1209-1217.
Copyright © 2006 by The American Association of Immunologists

Oxidants Selectively Reverse TGF-{beta} Suppression of Proinflammatory Mediator Production1

Yi Qun Xiao*, Celio G. Freire-de-Lima{dagger}, William J. Janssen*, Konosuke Morimoto*, Dennis Lyu*, Donna L. Bratton* and Peter M. Henson2,*

* Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; and {dagger} Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Although TGF-{beta} inhibits the production of proinflammatory mediators in vitro and in vivo, its anti-inflammatory activities may be ineffective in early or severe acute inflammatory circumstances. In this study, we suggest a role for oxidative stress on TGF-{beta} signaling, leading to prevention of its normal anti-inflammatory effects but leaving its Smad-driven effects on cellular differentiation or matrix production unaffected. Stimulation of the RAW 264.7 macrophage cells, human or mouse alveolar macrophages with LPS led to NF-{kappa}B-driven production of proinflammatory mediators, which were inhibited by TGF-{beta}. This inhibition was prevented in the presence of hydrogen peroxide. We found that hydrogen peroxide acted by inducing p38 MAPK activation, which then prevented the ERK activation and MAPK phosphatase-1 up-regulation normally induced by TGF-{beta}. This was mediated through Src tyrosine kinases and protein phosphatase-1/2A. By contrast, hydrogen peroxide had no effects on TGF-{beta}-induced Smad2 phosphorylation and SBE-luc reporter gene transcription.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Transforming growth factor-{beta} is a pleiotropic cytokine that regulates numerous physiological process, including cell proliferation, differentiation, apoptosis, early embryonic development, and extracellular matrix protein synthesis (1, 2, 3, 4). Upon apoptotic cell recognition and removal, the phagocytes produce active TGF-{beta}, which plays a pivotal role in the resolution of inflammation (5, 6, 7, 8). However, in pulmonary fibrotic diseases, TGF-{beta} causes the accumulation of extracellular matrix and induces transdifferentiation of fibroblasts to myofibroblasts (9, 10).

TGF-{beta} initiates signaling by inducing the association of type I and type II transmembrane serine/threonine kinase receptors. Upon ligand binding, the type II receptor transphosphorylates type I receptor kinase domain, which then propagates the signal through phosphorylation of Smad proteins (2, 11, 12). TGF-{beta} also activates MAPK pathways that participate in both the Smad-dependent as well as Smad-independent transcriptional responses to TGF-{beta} (4, 13, 14). Previously, we have demonstrated that TGF-{beta} inhibits proinflammatory cytokine production through cross talk between MAPKs, specifically, ERK-dependent inhibition of p38 MAPK caused by up-regulation of MAPK phosphatase 1 (MKP-1)3 (15). These anti-inflammatory effects appeared independent of the Smad signaling pathway.

Despite the observations that TGF-{beta} produced in response to apoptotic cells can lead to resolution of inflammation, these anti-inflammatory effects appear to be overwhelmed in severe inflammatory circumstances, such as acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS) (16). In contrast, the elevated TGF-{beta} levels formed within 24 h of the diagnosis of ARDS were nevertheless implicated in the early increase in collagen turnover and the enhancement of epithelial permeability in humans (17, 18, 19, 20). Enhancement of pulmonary edema was demonstrated in a murine model of ALI as well (19). In seeking an explanation for the apparent selective ineffectiveness of TGF-{beta} as an anti-inflammatory cytokine in these circumstances, we questioned whether the presence of oxidants might alter TGF-{beta} signaling. There is an extensive body of experimental evidence supporting the role of oxidants and oxidative injury in the pathogenesis of ALI/ARDS (21, 22, 23, 24). One of the downstream effects of oxidative stress is the activation of transcription factor NF-{kappa}B, potent inducer of proinflammatory mediators. Expression of proinflammatory cytokines is rapidly increased in experimental models of ARDS, in patients at risk for ARDS, and in patients with established ARDS (21). In the present study, we determined whether inclusion of oxidant stress reversed the normally anti-inflammatory effects of TGF-{beta} signaling in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Abs and reagents

TGF-{beta}1 was from R&D Systems. LPS (Escherichia coli 0111:B4) was from List Biological Laboratories. Hydrogen peroxide and catalase were from Sigma-Aldrich. SB 203580, okadaic acid, PP2, and Protease Inhibitor Cocktail Set I were from Calbiochem. Lipofectamine Plus reagent was from Invitrogen Life Technologies. Anti-p38 MAPK phosphospecific Ab was from Calbiochem. Phospho-ERK (E-4), ERK-2 (K 23), phospho-MEK-1, p38 (c-20), and MKP-1 (v-15) Abs were from Santa Cruz Biotechnology. Phospho-Smad-2 and {beta}-actin Abs were from Cell Signaling Technology. Unless otherwise specified in the text, all reagents have been tested as inhibitor-alone controls in the experiments without showing any effects.

Cell culture, stimulation, and measurement of proinflammatory mediators by ELISA

RAW 264.7 cells (obtained from the American Type Culture Collection), human alveolar macrophages obtained from the bronchial alveolar lavage fluid of normal volunteers, and 3T3-L1 cells (kindly provided by Dr. W. P. Schiemann, National Jewish Medical and Research Center, Denver, CO) were cultured in DMEM supplemented with 10% heat-inactivated endotoxin-free FBS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin under a humidified 5% CO2 atmosphere at 37°C. The cells (0.5 x 106 cells/well) were plated in each well of a 24-well tissue culture plate and incubated overnight.

Female ICR mice, 10 wk of age, were obtained from Taconic Farms. LPS was used to elicit alveolar macrophages. Briefly, mice were anesthetized with Avertin (Sigma-Aldrich) and then treated with 200 mg of LPS instilled directly into the trachea using a modified feeding needle (Popper and Sons). Twelve days later, the mice were euthanized with i.p. pentobarbital (Abbott Laboratories), and whole-lung lavage was performed using ice-cold PBS with 1 mM EDTA (Sigma-Aldrich). Approximately 1 x 106 alveolar macrophages were obtained from each mouse. Samples were pooled, washed twice in ice-cold PBS, and then used in in vitro studies. Mice were housed and studied under Institutional Animal Care and Use Committee-approved protocols in the animal facility of the National Jewish Medical and Research Center. All of the human studies were reviewed and approved by the institutional review committee at the National Jewish Medical and Research Center.

Cells were washed twice in serum-free DMEM and were either left unstimulated or treated for 18 h with various agents. The supernatants were collected and measured for MIP-2, IL-6, and IL-1{beta} by ELISA according to the manufacturer’s instructions (ELISA Tech).

NO production assay

NO levels produced by RAW 264.7 cells were measured by reducing the nitrate accumulated over 18 h to nitrite with nitrate reductase (23) and measuring the nitrite concentration by the method of Green et al. (24). The nitrite concentrations were quantified by using a double three-point standard curve of NaNO2 concentrations (in a linear range between 1 and 80 µM).

Transient cell transfection and reporter gene assays

pNF-{kappa}B-Luc ({kappa}B4, 6x; Clontech Laboratories) and pSBE-Luc (kindly provided by Dr. W. P. Schiemann) luciferase reporter gene constructs were transfected into RAW 264.7 cells by using Lipofectamine Plus reagent according to the manufacturer’s instructions. pSV-{beta}-galactosidase vector (Promega) was cotransfected as internal control to measure differences in transfection efficiency. Luciferase and {beta}-galactosidase activities were measured 18 h after LPS stimulation using the Luciferase Assay System (Promega) and Galacto-Light (Tropix), respectively.

Immunoblotting analysis

Immunoblotting analysis was conducted as described previously with some modification (25). Briefly, cells (3.0 x 105 cells/well) were plated in each well of a 12-well tissue culture plate and incubated overnight. The cells were washed twice in serum-free DMEM and were serum-starved for 2 h before stimulation. Afterward, the cells were lysed in lysis buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM DTT, 0.5% Triton X-100, and 1x Protease Inhibitor Cocktail Set I), resolved on 10% SDS-PAGE, and blotted to nitrocellulose membranes. The membranes were probed with primary Abs at 4°C overnight and incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary Abs for 1 h at room temperature. Proteins were visualized by ECL (Amersham Biosciences) according to the manufacturer’s instructions.

Equal loading of proteins in each lane was confirmed either by Ponceau S staining or reprobed with corresponding Abs against the native proteins (15) or {beta}-actin. The results shown are representative of at least three separate experiments.

Statistical analysis

All data are presented as means ± SEM from three or more separate experiments. The means were analyzed using ANOVA for multiple comparisons. When ANOVA indicated significance, the Tukey-Kramer honestly significant difference test for all pairs was used to compare groups. All data were analyzed using JMP statistical software (version 3; SAS Institute) for the Macintosh computer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Hydrogen peroxide overrides the inhibitory effects of TGF-{beta} on LPS-induced inflammatory mediator production and NF-{kappa}B-driven reporter gene transcription

To examine whether oxidative stress alters the inhibition by TGF-{beta} of proinflammatory mediator production, RAW 264.7 cells, mouse or human alveolar macrophages (only human IL-8 and IL-6 were measured because of the limited normal volunteers) were pretreated with 100 µM hydrogen peroxide, followed by stimulation with LPS (100 ng/ml), TGF-{beta} (10 ng/ml), or the combination of TGF-{beta} and LPS. As shown in Figs. 1, A and B, and 5D, at this concentration, hydrogen peroxide alone had little effect on MIP-2, IL-8, IL-6, IL-1{beta}, NO production, or inducible NO synthase (iNOS) synthesis but reversed the TGF-{beta} inhibition of LPS-induced generation of these mediators (p < 0.05), and this was recovered by the removal of the H2O2 with catalase (p < 0.05) (Fig. 1B). Because the proinflammatory mediators are driven by NF-{kappa}B, we next tested whether hydrogen peroxide reversed TGF-{beta} suppression of NF-{kappa}B reporter gene transcription. As shown in Fig. 1C, hydrogen peroxide alone slightly increased NF-{kappa}B activation and additively increased LPS-induced NF-{kappa}B activation. Importantly, hydrogen peroxide markedly reversed the TGF-{beta} suppression of NF-{kappa}B activation (p < 0.05), and this was recovered by catalase (p < 0.05) (26). Collectively, these findings suggest that oxidative stress inhibits TGF-{beta} suppression of proinflammatory mediator production at the level of NF-{kappa}B. By contrast, hydrogen peroxide had no effect on the production of an AP-1-dependent chemokine, MCP-1, or on TGF-{beta}-increased AP-1 reporter gene transcription (p, NS) (Fig. 2).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 1. TGF-{beta}-mediated inhibition of LPS-induced MIP-2, IL-8, IL-1{beta}, IL-6, and NO production and NF-{kappa}B activation are reversed by hydrogen peroxide. RAW 264.7 cells (A) or mouse or human alveolar macrophages (B) were initially cultured in the presence or absence of catalase (500 U), hydrogen peroxide (100 µM), and TGF-{beta} (10 ng/ml) for 1 h and subsequently cultured in the presence or absence of LPS (100 ng/ml) for 18 h. Culture supernatants were analyzed for MIP-2, IL-8, IL-1{beta}, IL-6 by ELISA, and NO as described in Materials and Methods. #, Significantly different from LPS (p < 0.05). *, Significantly different from TGF-{beta} plus LPS (p < 0.05). {dagger}, Significantly different from H2O2, TGF-{beta} plus LPS. C, RAW 264.7 cells were transiently cotransfected with pNF-{kappa}B-luc and pSV-{beta} galactosidase constructs. After 48 h, the cells were incubated in the presence or absence of catalase (500 U), hydrogen peroxide (100 µM), and TGF-{beta} (10 ng/ml) for 1 h, followed by incubation with LPS (100 ng/ml) for 18 h. The luciferase assays, which were normalized to {beta}-galactosidase, are expressed as relative luciferase units. #, Significantly different from LPS (p < 0.05). *, Significantly different from TGF-{beta} plus LPS (p < 0.05). {dagger}, Significantly different from H2O2, TGF-{beta} plus LPS.

 


View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 5. Hydrogen peroxide-mediated inhibition of LPS and TGF-{beta}-induced MKP-1 and iNOS expression is reversed by okadaic acid and PP2. A, RAW 264.7 cells were incubated in the presence or absence of SB 203580 (10 µM) or okadaic acid (10 nM) for 1 h and then stimulated with LPS (100 ng/ml) for 15 min. The total cell lysates were immunoblotted with phospho-MEK-1 and phospho-ERK Abs, respectively. B, RAW 264.7 cells were incubated in the presence or absence of PP2 (10 µM) for 60 min, followed by incubation with hydrogen peroxide (100 µM) and LPS (100 ng/ml) for 15 min. The total cell lysates were immunoblotted with phospho-ERK. Ratios of p-ERK and ERK-2 were analyzed using densitometric analysis. #, (p-44) significantly different from LPS (p < 0.05). *, (p-44) significantly different from H2O2 plus LPS (p < 0.05). C, RAW 264.7 cells were initially cultured in the presence or absence of PP2 (10 µM) or okadaic acid (10 nM) for 1 h, followed by incubation with hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 60 min and then stimulated with LPS (100 ng/ml) for 15 min. Total cell lysates were immunoblotted with MKP-1 Ab. D, RAW 264.7 cells were initially cultured in the presence or absence of SB 203580 (10 µM), PP2 (10 µM), or okadaic acid (10 nM) for 1 h and subsequently cultured in the presence or absence of hydrogen peroxide (100 µM), TGF-{beta} (10 ng/ml), and LPS (100 ng/ml) for 18 h. The total cell lysates were immunoblotted with iNOS Ab.

 


View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2. TGF-{beta}-mediated increase of LPS-induced MCP-1 production and AP-1 activation are not affected by hydrogen peroxide in RAW 264.7 cells. A, RAW 264.7 cells were initially cultured in the presence or absence of hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 1 h and subsequently cultured in the presence or absence of LPS (100 ng/ml) for 18 h. Culture supernatants were analyzed for MCP-1 by ELISA. B, RAW 264.7 cells were transiently cotransfected with AP-1-luc and pSV-{beta} galactosidase constructs. After 48 h, the cells were incubated in the presence or absence of hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 1 h, followed by incubation with LPS (100 ng/ml) for 18 h. The luciferase assays, which were normalized to {beta}-galactosidase, are expressed as relative luciferase units.

 
Hydrogen peroxide-induced p38 MAPK phosphorylation suppresses TGF-{beta}-mediated ERK phosphorylation

We have reported previously that, when bioactive TGF-{beta} is added to macrophages, ERK is activated, reaching a maximum at 60 min and resulting in up-regulation of the MKP-1 phosphatase. This ultimately results in the deactivation of p38 MAPK and decreased NF-{kappa}B transactivation (15). In this study, we show that hydrogen peroxide itself led to p38 MAPK phosphorylation, which reached a maximum at 15 min. Meanwhile, basal ERK phosphorylation was not affected (Fig. 3A). Based on the time course of TGF-{beta}-stimulated ERK (15) and hydrogen peroxide-stimulated p38 MAPK, we used the time point of maximum stimulation by TGF-{beta} and hydrogen peroxide in the following studies. As expected, hydrogen peroxide-induced p38 MAPK phosphorylation was inhibited by catalase (500 U) (Fig. 3B). Importantly, pretreatment of RAW 264.7 cells with hydrogen peroxide inhibited TGF-{beta}-induced ERK phosphorylation (Fig. 3C). This inhibition was reversed by catalase and by the p38 MAPK inhibitor, SB 203580 (10 µM) (27), indicating that hydrogen peroxide inhibits TGF-{beta}-induced ERK phosphorylation through p38 MAPK. Notably, catalase and SB 203580 alone slightly increased baseline ERK phosphorylation (Fig. 3C); these results confirm the cross-inhibition from p38 MAPK to ERK. Moreover, pretreatment of the cells with SB 203580 inhibited hydrogen peroxide-induced NF-{kappa}B activation (p < 0.05) (Fig. 3D). Collectively, these findings suggested that hydrogen peroxide increases p38 MAPK phosphorylation and activation of NF-{kappa}B. Meanwhile, hydrogen peroxide-induced p38 MAPK phosphorylation suppresses TGF-{beta}-induced ERK phosphorylation and therefore overrides TGF-{beta} inhibition of NF-{kappa}B.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 3. Blockade of hydrogen peroxide-induced p38 MAPK phosphorylation by catalase or SB 203580 reverses its inhibition on TGF-{beta}-induced ERK phosphorylation. A, Time course for phosphorylation of p38 MAPK and ERK by hydrogen peroxide. RAW 264.7 cells were stimulated with hydrogen peroxide (100 µM) for the time indicated. The total cell lysates were immunoblotted with phospho-p38 and phospho-ERK Abs, respectively. B, Inhibition by catalase on hydrogen peroxide-induced p38 MAPK phosphorylation. RAW 264.7 cells were pretreated with catalase (500 U) for 60 min and then stimulated with hydrogen peroxide (100 µM) for 15 min. Total cell lysates were immunoblotted with phospho-p38 MAPK Ab. C, Hydrogen peroxide-mediated inhibition of TGF-{beta}-induced ERK phosphorylation was reversed by catalase or SB 203580. RAW 264.7 cells were incubated in the presence or absence of catalase (500 U) or SB 203580 (10 µM) for 60 min followed by incubation with hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 60 min. Total cell lysates were immunoblotted with phospho-ERK Abs. Ratios of p-ERK and ERK-2 were analyzed using densitometric analysis. *, (p-42 and p-44) significantly different from H2O2, plus TGF-{beta} (p < 0.05). D, Hydrogen peroxide-induced NF-{kappa}B activation was inhibited by SB 203580. RAW 264.7 cells were transiently cotransfected with pNF-{kappa}B-luc and pSV-{beta} galactosidase constructs. After 48 h, the cells were incubated in the presence of the indicated concentrations (micromolar) of SB 203580 for 1 h, followed by incubation with hydrogen peroxide (100 µM) for 18 h. The luciferase assays, which were normalized to {beta}-galactosidase, are expressed as relative luciferase units. #, Significantly different from control (p < 0.05). *, Significantly different from H2O2 (p < 0.05).

 
Hydrogen peroxide blocks LPS- and TGF-{beta}-induced up-regulation of MKP-1

As shown in Fig. 4, LPS induced a small increase in MKP-1 expression that was substantially enhanced with the addition of TGF-{beta}. Hydrogen peroxide blocked both of these effects. As reported previously (15), MKP-1 increases in these systems are dependent on ERK activation, and, in keeping with this, ERK phosphorylation and MPK-1 expression were decreased in the presence of hydrogen peroxide and restored in the presence of SB 203580. A reciprocal effect was seen for p38 MAPK activation. The experiment suggests that oxidants activate p38 MAPK, which inhibits ERK and thereby reduces MKP-1 expression. Noticeably, there is a difference in the amount of phospho-ERK-42 and -44 between RAW 264.7 macrophages and mouse alveolar macrophages, although the same trend was shown under the same experimental conditions. We assume that is due to the difference in the abundance of ERK subtypes in these two cells.



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 4. Hydrogen peroxide suppresses LPS and TGF-{beta}-induced MKP-1 expression by activation of p38 MAPK and inhibition of ERK. RAW 264.7 cells (A) or mouse alveolar macrophages (B) were incubated in the presence or absence of SB 203580 (10 µM) for 60 min, followed by incubation with hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 60 min and then stimulated with LPS (100 ng/ml) for 15 min. Total cell lysates were immunoblotted with MKP-1, phospho-ERK, and phospho-p38 MAPK Abs, respectively.

 
Inhibition of LPS- and TGF-{beta}-induced MKP-1 expression and iNOS synthesis by hydrogen peroxide was reversed by okadaic acid and PP2

Our previous studies indicated that, after LPS stimulation, p38 MAPK maintained suppression of ERK activation (15). Further investigation in this study showed that the suppression was upstream of MEK-1 and reversed by the p38 inhibitor SB 203580 and the protein phosphatase-1/2A inhibitor okadaic acid (28) (Fig. 5A). It has been shown that hydrogen peroxide increases p38 MAPK activation through Src-family tyrosine kinases (29, 30, 31). Consistent with this, the Src-family tyrosine kinase inhibitor PP2 (32) reversed the hydrogen peroxide suppression of ERK (Fig. 5B). To determine whether hydrogen peroxide overrides the inhibitory effect of TGF-{beta} through the signal pathway outlined above, RAW 264.7 cells were pretreated with okadaic acid or PP2 before incubation with hydrogen peroxide, TGF-{beta}, and LPS. As shown in Fig. 5C, hydrogen peroxide-mediated inhibition of LPS- or TGF-{beta} plus LPS-induced MKP-1 expression was completely reversed by okadaic acid and PP2, respectively. Consistent with this result, hydrogen peroxide reversal of TGF-{beta} inhibition of LPS-induced generation of iNOS was restored by okadaic acid, PP2, and SB 203580 (Fig. 5D).

Hydrogen peroxide abrogates the inhibitory effect of TGF-{beta} independent of Smad signaling

TGF-{beta} appears to activate ERK through Ras/MEK-1/ERK pathways (12, 33). The other well-defined signaling pathway of TGF-{beta} is through the phosphorylation of a Smad complex and its translocation into the nucleus. To examine whether oxidative stress could affect the Smad signaling pathway, RAW 264.7 cells or mouse alveolar macrophages were stimulated with hydrogen peroxide, TGF-{beta}, and the combination of hydrogen peroxide and TGF-{beta} followed by blotting with phospho-Smad-2. As shown in Fig. 6A, hydrogen peroxide did not affect TGF-{beta}-induced Smad-2 phosphorylation in both RAW 264.7 and mouse alveolar macrophages. Furthermore, RAW 264.7 cells transfected with a SBE-luc reporter gene construct showed no effect of hydrogen peroxide on the SBE-luc reporter gene transcription (p, NS) (Fig. 6B). These findings suggest that oxidants override the TGF-{beta}-mediated inhibitory effect on proinflammatory mediator production independent of Smad signaling.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 6. Hydrogen peroxide does not affect TGF-{beta}-induced Smad-2 phosphorylation and SBE-luc reporter gene activation. A, RAW 264.7 cells (upper panels) or mouse alveolar macrophages (lower panels) were incubated with hydrogen peroxide (100 µM), TGF-{beta} (10 ng/ml), or hydrogen peroxide plus TGF-{beta} for 60 min. The total cell lysates were immunoblotted with phospho-Smad-2 Ab. B, RAW 264.7 cells were transiently cotransfected with SBE-luc and pSV-{beta} galactosidase constructs. After 48 h, the cells were incubated in the presence or absence of hydrogen peroxide (100 µM), TGF-{beta} (10 ng/ml), or hydrogen peroxide plus TGF-{beta} for 18 h. The luciferase assays, which were normalized to {beta}-galactosidase, are expressed as relative luciferase units.

 
Dysregulation of proinflammatory mediator production by hydrogen peroxide is stimulus and cell type independent

The fibroblast cell line 3T3-L1 was preincubated with hydrogen peroxide and TGF-{beta} for 1 h, followed by stimulation with TNF-{alpha} for 18 h. As shown in Fig. 7, A and B, 100 µM hydrogen peroxide alone had little effect on IL-6 production and iNOS induction, but the TGF-{beta} inhibition of TNF-{alpha}-induced generation of IL-6 and iNOS were totally reversed. Consistent with these results, TNF-{alpha}-induced p38 MAPK phosphorylation was inhibited by TGF-{beta}. However, the combination of hydrogen peroxide and TNF-{alpha} prevented the inhibition. Moreover, hydrogen peroxide blocked TNF-{alpha}- or TGF-{beta}-plus-TNF-{alpha}-mediated MKP-1 up-regulation (Fig. 7C). The same results were obtained in RAW 264.7 cells stimulated with TNF-{alpha} as well (data not shown).



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 7. Dysregulation of proinflammatory mediator production by hydrogen peroxide is stimulus and cell type independent. A, 3T3-L1 cells were incubated in the presence or absence of hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 1 h and subsequently cultured in the presence or absence of TNF-{alpha} (10 ng/ml) for 18 h. Culture supernatants were analyzed by ELISA for IL-6. #, Significantly different from TNF-{alpha} (p < 0.05). *, Significantly different from TGF-{beta} plus TNF-{alpha} (p < 0.05). B, The total cell lysates were analyzed by Western blot for iNOS. C, 3T3-L1 cells were incubated in the presence or absence of hydrogen peroxide (100 µM) and TGF-{beta} (10 ng/ml) for 60 min and then stimulated with TNF-{alpha} (10 ng/ml) for 15 min. Total cell lysates were immunoblotted with phospho-p38 MAPK and MKP-1 Abs, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TGF-{beta} production in the inflamed lung has been suggested to suppress the production of proinflammatory mediators and promote the resolution of the inflammation (8). This effect of activated TGF-{beta} would be expected from both soluble and tissue-bond material. However, the inhibitory effect of TGF-{beta} appears to be blocked in ARDS (21, 22, 23, 24). In the present study, we provide evidence that hydrogen peroxide prevented TGF-{beta}-mediated inhibition of proinflammatory mediator production and NF-{kappa}B activation. It is suggested that blockade of these effects by oxidants may explain the presence of both TGF-{beta} and production of proinflammatory mediators early in acute inflammatory reactions (i.e., leading to reversal of TGF-{beta} anti-inflammatory effects). Meanwhile, the effects of TGF-{beta} that are driven by Smad pathway appear unaffected by the oxidants and could proceed throughout the inflammatory reaction. Later, as the oxidative environment decreases, TGF-{beta}’s anti-inflammatory effects could recover, leading eventually to resolution of the inflammation.

ARDS is a syndrome resulting from uncontrolled acute inflammatory response in the lung and remains a major cause of morbidity and mortality (20). Although the anti-inflammatory cytokine TGF-{beta} was found dramatically increased within 24 h of ALI, overproduced oxidants may prevent its normal inhibitory effect, permitting oxidative cell damage and inflammatory cytokine production. The suggested selectivity of this effect would not affect TGF-{beta} Smad pathway-dependent processes, e.g., collagen turnover and fibroliferation (19).

In the present study, TGF-{beta} blocked LPS-induced increases in MIP-2, IL-8, IL-1{beta}, IL-6, NO production, iNOS synthesis, and NF-{kappa}B activation. Hydrogen peroxide also induced some proinflammatory mediator production. However, TGF-{beta} failed to suppress LPS-induced inflammatory mediators and NF-{kappa}B activation in the presence of hydrogen peroxide (Fig. 1). Our previous studies have suggested that the suppression by TGF-{beta} occurs at the level of MAPKs by ERK-dependent inhibition of p38 MAPK (15). Therefore, the effect of oxidative stress on the balance between ERK and p38 MAPK activation was a major focus of this study. Hydrogen peroxide alone activated p38 MAPK without affecting the basal level of ERK activation. Importantly, the catalase as well as the p38 MAPK-specific inhibitor SB 203580, which was shown to inhibit hydrogen peroxide-induced p38 MAPK activation, also reversed hydrogen peroxide-mediated inhibition of TGF-{beta}-induced ERK activation, implying that TGF-{beta} inhibition of p38 MAPK through ERK was no longer functional in the presence of hydrogen peroxide. This was further confirmed by the results from p38 MAPK phosphorylation and MKP-1 expression (Fig. 4); namely, oxidative stress overrode TGF-{beta} signaling through p38-mediated ERK and MKP-1 inhibition. These results further support the concept that considerable cross talk occurs between the different MAPKs. In these studies, hydrogen peroxide was added to the cells before or simultaneously with TGF-{beta}. This experimental design presumptively mimicked the situation in vivo during oxidant-mediated injury. By contrast, preincubation with TGF-{beta} before hydrogen peroxide addition prevented the p38 MAPK activation (data not shown). Presumably, this resulted from prior up-regulation of MKP-1. Therefore, it seems likely that potential effects also are temporally regulated. Notably, LPS and TGF-{beta} have been reported to induce reactive oxygen species (ROS) (34, 35, 36, 37, 38, 39). We suggest that the signaling events observed for LPS and TGF-{beta} already integrated the ones from ROS; therefore, the effects of ROS induced by themselves are masked in both proinflammatory and anti-inflammatory conditions. When another source of oxidant exists, the proinflammatory or anti-inflammatory balance is dysregulated.

It has been reported that c-Src can be directly activated by hydrogen peroxide treatment and can contribute to NF-{kappa}B activation (40). Thus, inhibition of hydrogen peroxide-induced p38 MAPK by the Src family inhibitor PP2 supports a role for Src family tyrosine kinases acting as upstream effectors to mediate hydrogen peroxide signaling in our system. It was reported that oxidants might prime macrophages for altered responsiveness to LPS through PI3K-mediated NF-{kappa}B nuclear translocation (32). However, TGF-{beta} caused inhibition of NF-{kappa}B transactivation mainly through p38 MAPK and the basal transcriptional factor TATA-binding protein (15, 41). Therefore, it seems unlikely that hydrogen peroxide overrode TGF-{beta}’s anti-inflammatory effect through PI3K. Members of a family of dual-specificity phosphatases principally inhibit MAPK’s activity. This ERK-mediated up-regulation of MKP-1 appeared to be involved in deactivation of p38 MAPK (15). By contrast, in our previous study (28) and this study, we provided evidence that the protein phosphatases contribute to inhibition of ERK by hydrogen peroxide-induced p38 MAPK activation, further confirming that phosphatases play key roles in regulation the balance of MAPKs in vivo. Hydrogen peroxide increased p38 MAPK activation, and by inactivating the MEK-ERK pathway, led to prevention of the MKP-1 up-regulation.

In the present study, it is suggested that hydrogen peroxide blocks the TGF-{beta}-induced Ras/MEK-1/ERK pathway. However, the TGF-{beta}-induced Smad-2 phosphorylation, as well as activity of the SBE-luc reporter gene, was shown to be independent of hydrogen peroxide treatment (Fig. 5). Therefore, it seems that oxidative stress selectively suppressed the non-Smad signaling of TGF-{beta} in this system. The canonical proinflammatory pathways initiate NF-{kappa}B activation via serine phosphorylation of I{kappa}B by the I{kappa}B kinase complex, which causes degradation of I{kappa}B (42). In contrast, oxidative stress can activate NF-{kappa}B independent of I{kappa}B kinase activation and I{kappa}B degradation (40); instead, I{kappa}B is phosphorylated on tyrosine. Although the mechanism is unknown at this moment, it has been suggested that tyrosine phosphorylation of I{kappa}B is capable of activating NF-{kappa}B in the absence of ubiquitin-dependent degradation of I{kappa}B. Therefore, we do not exclude the possibility that hydrogen peroxide activates NF-{kappa}B via this pathway as well.

In the present study, we demonstrate that oxidative stress overrides the TGF-{beta}-mediated inhibitory effect on proinflammatory mediator production independent of Smad signaling (Fig. 8). It is noteworthy that the inhibitory effect of TGF-{beta} and its antagonistic regulation by hydrogen peroxide appear to be cell-type and stimulus independent. More importantly, we suggest that the TGF-{beta} signal pathways leading to the suppression of inflammation are separately regulated from those driven through Smad, such as collagen synthesis, fibrotic response, inhibition of cell proliferation, and differentiation (43, 44, 45, 46, 47, 48, 49, 50, 51). Because of TGF-{beta}’s numerous homeostatic functions, blocking TGF-{beta} signaling may have serious adverse consequences. Alternatively, specific inhibition of Smad signaling might prevent fibrotic responses while leaving the anti-inflammatory effects of TGF-{beta} unaltered. Moreover, recognition of oxidative stress-mediated signal pathways may open up a new field of cell regulation via specific and targeted control of its effects on inflammatory diseases.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 8. Schematic diagram of the mechanism by which oxidative stress overrides the inhibitory effect of TGF-{beta} on NF-{kappa}B activation. TGF-{beta} inhibits LPS or TNF-{alpha}-induced proinflammatory mediator production through suppression of p38 MAPK and NF-{kappa}B. The pre-existed oxidant prevents the inhibitory by TGF-{beta} via activation of p38 MAPK and stimulation of protein phosphatase, which blocks ERK activity and MKP-1 expression and, therefore, further increases p38 MAPK and NF-{kappa}B activation.

 

   Acknowledgments
 
We acknowledge Lindsay Guthrie for performing the ELISA. We thank Dr. R. William Vandivier for the suggestions and help with the statistical analysis. We thank Dr. Gregory P. Cosgrove for organizing the collection of human bronchial alveolar lavage fluid. We thank Drs. Jennifer A. Kench, Valeria de Matos Borges, and Caroline M. Cole for collecting mouse bronchial alveolar lavage fluid.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by National Institutes of Health Grants HL67671, AI058228, and HL34303. C.G.F.-L. is supported by Human Frontier Science Program Long-Term Fellowship LT00608/2002-C. Back

2 Address correspondence and reprint requests to Dr. Peter M. Henson, Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: hensonp{at}njc.org Back

3 Abbreviations used in this paper: MKP-1, MAPK phosphatase 1; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; iNOS, inducible NO synthase; ROS, reactive oxygen species. Back

Received for publication April 1, 2005. Accepted for publication November 15, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Chin, B. Y., I. Petrache, A. M. Choi, M. E. Choi. 1999. Transforming growth factor {beta}1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages. J. Biol. Chem. 274: 11362-11368. [Abstract/Free Full Text]
  2. Dennler, S., M. J. Goumans, P. ten Dijke. 2002. Transforming growth factor {beta} signal transduction. J. Leukocyte Biol. 71: 731-740. [Abstract/Free Full Text]
  3. Huang, Y., D. Hutter, Y. Liu, X. Wang, M. S. Sheikh, A. M. Chan, N. J. Holbrook. 2000. Transforming growth factor-{beta}1 suppresses serum deprivation-induced death of A549 cells through differential effects on c-Jun and JNK activities. J. Biol. Chem. 275: 18234-18242. [Abstract/Free Full Text]
  4. Hartsough, M. T., K. M. Mulder. 1997. Transforming growth factor-{beta} signaling in epithelial cells. Pharmacol. Ther. 75: 21-41. [Medline]
  5. Fadok, V. A., D. L. Bratton, A. Konowal, P. W. Freed, J. Y. Westcott, P. M. Henson. 1998. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-{beta}, PGE2, and PAF. J. Clin. Invest. 101: 890-898. [Medline]
  6. Fadok, V. A., D. L. Bratton, D. M. Rose, A. Pearson, R. A. Ezekewitz, P. M. Henson. 2000. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405: 85-90. [Medline]
  7. McDonald, P. P., V. A. Fadok, D. Bratton, P. M. Henson. 1999. Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-{beta} in macrophages that have ingested apoptotic cells. J. Immunol. 163: 6164-6172. [Abstract/Free Full Text]
  8. Huynh, M. L., V. A. Fadok, P. M. Henson. 2002. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-{beta}1 secretion and the resolution of inflammation. J. Clin. Invest. 109: 41-50. [Medline]
  9. Krein, P. M., B. W. Winston. 2002. Roles for insulin-like growth factor I and transforming growth factor-{beta} in fibrotic lung disease. Chest 122: 289S-293S. [Abstract/Free Full Text]
  10. Desmouliere, A., A. Geinoz, F. Gabbiani, G. Gabbiani. 1993. Transforming growth factor-{beta} 1 induces {alpha}-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J. Cell Biol. 122: 103-111. [Abstract/Free Full Text]
  11. Shi, Y., J. Massague. 2003. Mechanisms of TGF-{beta} signaling from cell membrane to the nucleus. Cell 113: 685-700. [Medline]
  12. Derynck, R., Y. E. Zhang. 2003. Smad-dependent and Smad-independent pathways in TGF-{beta} family signalling. Nature 425: 577-584. [Medline]
  13. Hartsough, M. T., R. S. Frey, P. A. Zipfel, A. Buard, S. J. Cook, F. McCormick, K. M. Mulder. 1996. Altered transforming growth factor signaling in epithelial cells when ras activation is blocked. J. Biol. Chem. 271: 22368-22375. [Abstract/Free Full Text]
  14. Mulder, K. M.. 2000. Role of Ras and Mapks in TGF{beta} signaling. Cytokine Growth Factor Rev. 11: 23-35. [Medline]
  15. Xiao, Y. Q., K. Malcolm, G. S. Worthen, S. Gardai, W. P. Schiemann, V. A. Fadok, D. L. Bratton, P. M. Henson. 2002. Cross-talk between ERK and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by transforming growth factor-{beta}. J. Biol. Chem. 277: 14884-14893. [Abstract/Free Full Text]
  16. Haddad, J. J.. 2002. Science review: redox and oxygen-sensitive transcription factors in the regulation of oxidant-mediated lung injury: role for nuclear factor-{kappa}B. Crit. Care 6: 481-490. [Medline]
  17. Ware, L. B., M. A. Matthay. 2000. The acute respiratory distress syndrome. N. Engl. J. Med. 342: 1334-1349. [Free Full Text]
  18. Bellingan, G. J.. 2002. The pulmonary physician in critical care * 6: the pathogenesis of ALI/ARDS. Thorax 57: 540-546. [Abstract/Free Full Text]
  19. Pittet, J. F., M. J. Griffiths, T. Geiser, N. Kaminski, S. L. Dalton, X. Huang, L. A. Brown, P. J. Gotwals, V. E. Koteliansky, M. A. Matthay, D. Sheppard. 2001. TGF-{beta} is a critical mediator of acute lung injury. J. Clin. Invest. 107: 1537-1544. [Medline]
  20. Fahy, R. J., F. Lichtenberger, C. B. McKeegan, G. J. Nuovo, C. B. Marsh, M. D. Wewers. 2003. The acute respiratory distress syndrome: a role for transforming growth factor-{beta} 1. Am. J. Respir. Cell Mol. Biol. 28: 499-503. [Abstract/Free Full Text]
  21. Park, W. Y., R. B. Goodman, K. P. Steinberg, J. T. Ruzinski, F. Radella, II, D. R. Park, J. Pugin, S. J. Skerrett, L. D. Hudson, T. R. Martin. 2001. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 164: 1896-1903. [Abstract/Free Full Text]
  22. Schwartz, M. D., E. E. Moore, F. A. Moore, R. Shenkar, P. Moine, J. B. Haenel, E. Abraham. 1996. Nuclear factor-{kappa}B is activated in alveolar macrophages from patients with acute respiratory distress syndrome. Crit. Care Med. 24: 1285-1292. [Medline]
  23. Schmidt, H. H., P. Wilke, B. Evers, E. Bohme. 1989. Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol. Biochem. Biophys. Res. Commun. 165: 284-291. [Medline]
  24. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, S. R. Tannenbaum. 1982. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126: 131-138. [Medline]
  25. Frasch, S. C., J. A. Nick, V. A. Fadok, D. L. Bratton, G. S. Worthen, P. M. Henson. 1998. p38 mitogen-activated protein kinase-dependent and -independent intracellular signal transduction pathways leading to apoptosis in human neutrophils. J. Biol. Chem. 273: 8389-8397. [Abstract/Free Full Text]
  26. Pawate, S., Q. Shen, F. Fan, N. R. Bhat. 2004. Redox regulation of glial inflammatory response to lipopolysaccharide and interferon-{gamma}. J. Neurosci. Res. 77: 540-551. [Medline]
  27. Xiao, Y. Q., K. Someya, H. Morita, K. Takahashi, K. Ohuchi. 1999. Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils. Biochim. Biophys. Acta 1450: 155-163. [Medline]
  28. Gardai, S. J., B. B. Whitlock, Y. Q. Xiao, D. B. Bratton, P. M. Henson. 2004. Oxidants inhibit ERK/MAPK and prevent its ability to delay neutrophil apoptosis downstream of mitochondrial changes and at the level of XIAP. J. Biol. Chem. 279: 44695-44703. [Abstract/Free Full Text]
  29. Khadaroo, R. G., J. Parodo, K. A. Powers, G. Papia, J. C. Marshall, A. Kapus, O. D. Rotstein. 2003. Oxidant-induced priming of the macrophage involves activation of p38 mitogen-activated protein kinase through an Src-dependent pathway. Surgery 134: 242-246. [Medline]
  30. Takano, T., K. Sada, H. Yamamura. 2002. Role of protein-tyrosine kinase syk in oxidative stress signaling in B cells. Antioxid. Redox. Signal 4: 533-541. [Medline]
  31. He, J., T. Takano, J. Ding, S. Gao, C. Noda, K. Sada, S. Yanagi, H. Yamamura. 2002. Syk is required for p38 activation and G2/M arrest in B cells exposed to oxidative stress. Antioxid. Redox Signal. 4: 509-515. [Medline]
  32. Khadaroo, R. G., A. Kapus, K. A. Powers, M. I. Cybulsky, J. C. Marshall, O. D. Rotstein. 2003. Oxidative stress reprograms lipopolysaccharide signaling via Src kinase-dependent pathway in RAW 264.7 macrophage cell line. J. Biol. Chem. 278: 47834-47841. [Abstract/Free Full Text]
  33. Hu, P. P., X. Shen, D. Huang, Y. Liu, C. Counter, X. F. Wang. 1999. The MEK pathway is required for stimulation of p21WAF1/CIP1 by transforming growth factor-{beta}. J. Biol. Chem. 274: 35381-35387. [Abstract/Free Full Text]
  34. Shibanuma, M., T. Kuroki, K. Nose. 1991. Release of H2O2 and phosphorylation of 30-kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor {beta}1. Cell Growth Differ. 2: 583-591. [Abstract]
  35. Ohba, M., M. Shibanuma, T. Kuroki, K. Nose. 1994. Production of hydrogen peroxide by transforming growth factor-{beta} 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J. Cell Biol. 126: 1079-1088. [Abstract/Free Full Text]
  36. Thannickal, V. J., B. L. Fanburg. 1995. Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor {beta}1. J. Biol. Chem. 270: 30334-30338. [Abstract/Free Full Text]
  37. Seekamp, A., C. Lalonde, D. G. Zhu, R. Demling. 1988. Catalase prevents prostanoid release and lung lipid peroxidation after endotoxemia in sheep. J. Appl. Physiol. 65: 1210-1216. [Abstract/Free Full Text]
  38. Milligan, S. A., J. M. Hoeffel, I. M. Goldstein, M. R. Flick. 1988. Effect of catalase on endotoxin-induced acute lung injury in unanesthetized sheep. Am. Rev. Respir. Dis. 137: 420-428. [Medline]
  39. Amari, T., K. Kubo, T. Kobayashi, M. Sekiguchi. 1993. Effects of recombinant human superoxide dismutase on tumor necrosis factor-induced lung injury in awake sheep. J. Appl. Physiol. 74: 2641-2648. [Abstract/Free Full Text]
  40. Fan, C., Q. Li, D. Ross, J. F. Engelhardt. 2003. Tyrosine phosphorylation of I{kappa}B{alpha} activates NF-{kappa}B through a redox-regulated and c-Src-dependent mechanism following hypoxia/reoxygenation. J. Biol. Chem. 278: 2072-2080. [Abstract/Free Full Text]
  41. Carter, A. B., K. L. Knudtson, M. M. Monick, G. W. Hunninghake. 1999. The p38 mitogen-activated protein kinase is required for NF-{kappa}B-dependent gene expression: the role of TATA-binding protein (TBP). J. Biol. Chem. 274: 30858-30863. [Abstract/Free Full Text]
  42. Li, Q., I. M. Verma. 2002. NF-{kappa}B regulation in the immune system. Nat. Rev. Immunol. 2: 725-734. [Medline]
  43. Ito, T., J. D. Williams, D. Fraser, A. O. Phillips. 2004. Hyaluronan attenuates transforming growth factor-{beta}1-mediated signaling in renal proximal tubular epithelial cells. Am. J. Pathol. 164: 1979-1988. [Abstract/Free Full Text]
  44. Asano, Y., H. Ihn, K. Yamane, M. Jinnin, Y. Mimura, K. Tamaki. 2004. Phosphatidylinositol 3-kinase is involved in {alpha}2(I) collagen gene expression in normal and scleroderma fibroblasts. J. Immunol. 172: 7123-7135. [Abstract/Free Full Text]
  45. Verrecchia, F., A. Mauviel. 2004. TGF-{beta} and TNF-{alpha}: antagonistic cytokines controlling type I collagen gene expression. Cell. Signal. 16: 873-880. [Medline]
  46. Flanders, K. C.. 2004. Smad3 as a mediator of the fibrotic response. Int. J. Exp. Pathol. 85: 47-64. [Medline]
  47. Tokumasa, A., T. Katsuno, T. S. Tanaga, K. Yokote, Y. Saito, Y. Suzuki. 2004. Reduction of Smad3 accelerates re-epithelialization in a murine model of colitis. Biochem. Biophys. Res. Commun. 317: 377-383. [Medline]
  48. Hu, M. G., G. F. Hu, Y. Kim, T. Tsuji, J. McBride, P. Hinds, D. T. Wong. 2004. Role of p12CDK2-AP1 in transforming growth factor-{beta}1-mediated growth suppression. Cancer Res. 64: 490-499. [Abstract/Free Full Text]
  49. Seoane, J., H. V. Le, L. Shen, S. A. Anderson, J. Massague. 2004. Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation. Cell 117: 211-223. [Medline]
  50. Ohnishi, H., T. Miyata, H. Yasuda, Y. Satoh, K. Hanatsuka, H. Kita, A. Ohashi, K. Tamada, N. Makita, T. Iiri, et al 2004. Distinct roles of Smad2-, Smad3-, and ERK-dependent pathways in transforming growth factor-{beta}1 regulation of pancreatic stellate cellular functions. J. Biol. Chem. 279: 8873-8878. [Abstract/Free Full Text]
  51. Miyazono, K., S. Maeda, T. Imamura. 2004. Coordinate regulation of cell growth and differentiation by TGF-{beta} superfamily and Runx proteins. Oncogene 23: 4232-4237. [Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
C. G. Freire-de-Lima, Y. Q. Xiao, S. J. Gardai, D. L. Bratton, W. P. Schiemann, and P. M. Henson
Apoptotic Cells, through Transforming Growth Factor-beta, Coordinately Induce Anti-inflammatory and Suppress Pro-inflammatory Eicosanoid and NO Synthesis in Murine Macrophages
J. Biol. Chem., December 15, 2006; 281(50): 38376 - 38384.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xiao, Y. Q.
Right arrow Articles by Henson, P. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xiao, Y. Q.
Right arrow Articles by Henson, P. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS