Published online before print January 29, 2004, doi: 10.1161/01.RES.0000119170.70818.4F
© 2004 American Heart Association, Inc.
Molecular Medicine |
Essential Role for Smad3 in Regulating MCP-1 Expression and Vascular Inflammation
From the Division of Cardiovascular Medicine (M.W.F., K.S., M.L., K.T., Z.C., D.I.S., P.L., M.K.J.) and Department of Pathology (R.H., R.N.M.), Brigham and Women’s Hospital, Boston, Mass; Duke University (J.P.F., X.-F.W.), Durham, NC.
Correspondence to Mark W. Feinberg and Mukesh K. Jain, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115. E-mail mfeinberg{at}rics.bwh.harvard.edu and mjain@rics.bwh.harvard.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Transforming growth factor (TGF)-ß1 is a pleiotropic growth factor with known inhibitory effects on immune cell activation. However, the specific mechanism(s) and in vivo significance of the effectors of TGF-ß1 modulation in the context of vascular inflammation are not well characterized. The chemokine monocyte chemoattractant protein (MCP)-1 is critical for the recruitment of macrophages in inflammatory disease states. In this study, we provide definitive evidence that the ability of TGF-ß1 to inhibit MCP-1 expression is mediated via its effector Smad3. Adenoviral overexpression of Smad3 potently repressed inducible expression of endogenous MCP-1. Conversely, TGF-ß1 inhibition of cytokine-mediated induction of MCP-1 expression was completely blocked in Smad3-deficient macrophages. Consistent with this impaired response, cardiac allografts in Smad3-deficient mice developed accelerated intimal hyperplasia with increased infiltration of adventitial macrophages expressing MCP-1. Previous studies show that MCP-1 inducibility is regulated by an AP-1 complex composed of c-Jun/c-Fos heterodimers. We demonstrate that the inhibitory effect of Smad3 occurs via a novel antagonistic effect of Smad3 on AP-1 DNA-protein binding and activity. Thus, Smad3 plays an essential role in modulating vascular inflammation characteristic of transplant-associated arteriopathy, is important in regulating MCP-1 expression, and plays a critical role in the ability of TGF-ß1 to repress stimuli from a major inflammatory signaling pathway.
Key Words: transforming growth factor-ß • Smads • vascular inflammation • AP-1 • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Accumulating clinical and experimental studies suggest that chronic inflammation contributes to the development of atherosclerosis, transplant arteriopathy, and restenosis after mechanical injury. A pathophysiological event common to these vascular pathologies is the recruitment and infiltration of monocytes/macrophages into the blood vessel wall.1–4
MCP-1, a member of the C-C chemokine ß subfamily, was originally identified for its potent chemotactic activity toward monocytes.5 Several lines of evidence suggest a critical role for MCP-1 in vascular disease states.6 Recent experimental studies demonstrated a reduction in atherosclerotic lesion formation in mice deficient in MCP-1 or its receptor CCR2 in atherosclerotic-prone mice.7,8 Conversely, macrophage-specific overexpression of MCP-1 resulted in the acceleration of vascular lesion size and infiltration of macrophages in atherosclerosis-prone mice.9 MCP-1 also contributes to the development of restenosis after mechanical balloon injury. In several animal models, blockade of MCP-1 or deficiency of CCR2 decreased neointimal hyperplasia after arterial injury.10–12 Although MCP-1 is an important mediator for the development of native atherosclerosis and restenosis, its role in transplant-associated arteriopathy (TxAA) has not yet been defined. The observation that elevated serum levels of MCP-1 in heart transplant patients are associated with the presence of TxAA suggests that MCP-1 may be an important mediator in this process.13 Furthermore, induction of MCP-1 expression correlated with the development of TxAA in rat cardiac allografts and its expression colocalized with macrophages.14 Thus, identification of mechanisms or signaling pathways to inhibit MCP-1 expression may offer novel strategies to diminish the inflammatory response for a broad range of vascular disease states.
Members of the TGF-ß superfamily consist of a large group of secreted polypeptides that play essential roles in diverse cellular processes including development, cell growth and differentiation, extracellular matrix deposition, and immune modulation.15–17 With respect to immune modulation, the prototypic ligand TGF-ß1 is a particularly potent regulator of inflammatory responses in a number of cell types within the vascular system.18 The role of TGF-ß1 in inflammation is highlighted in mice bearing a targeted deletion for this gene. These mice die perinatally and exhibit a systemic inflammatory reaction characterized by multiorgan infiltration of leukocytes, increased circulating monocytes, and tissue necrosis.19,20 Whereas previous studies support a role for TGF-ß1 in inhibiting the expression of MCP-1 and other macrophage activation markers,21–23 the specific mechanism(s) and in vivo significance of the effectors of TGF-ß1 modulation are not well understood.
Cellular signaling through the TGF-ß superfamily occurs via intracellular mediators, termed Smads, which translocate to the nucleus where they direct transcriptional responses. Three classes of Smads—pathway-restricted, common, and inhibitory—are responsible for coordinating the downstream signaling effects.15,17,24 TGF-ß/activin receptors phosphorylate the pathway restricted Smads, Smad2, and Smad3, whereas bone morphogenic protein receptors activate Smads 1, 5, and 8. On phosphorylation, these pathway-restricted Smads may hetero-oligomerize with Smad4, the only common Smad, and translocate to the nucleus where they may participate in regulating transcriptional events. Smad6 and Smad7, known as inhibitory Smads, are structurally divergent from other Smads and function to block TGF-ß signaling by preventing activation of pathway restricted Smads.15,17,24
The importance of Smad proteins in regulating inflammatory events by TGF-ß1 is highlighted by the phenotype of Smad3-deficient mice.25–27 These mice exhibit spontaneously activated T cells, impaired mucosal immunity, and abnormal wound healing; however, the contribution of Smad3 in regulating macrophage activation in these mice and its role in vascular inflammation in vivo remains unknown. In the work reported in this study, we demonstrate by both gain and loss of function experiments that the ability of TGF-ß1 to inhibit LPS-mediated induction of MCP-1 in macrophages is Smad3 dependent and involves inhibitory effects on AP-1 activity and DNA-protein binding. Moreover, hearts transplanted into Smad3-/- mice develop accelerated intimal hyperplasia with increased infiltration of adventitial macrophages that express MCP-1. These data indicate a novel mechanism by which TGF-ß1 via Smad3 can suppress inflammatory responses in the vascular system.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
An expanded Materials and Methods section can be found in the online data supplement at http://circres.ahajournals.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
TGF-ß1 and Smad3 Can Inhibit Cytokine-Induced Expression of MCP-1
In macrophages, stimuli such as LPS can activate MCP-1 expression by stimulating members of the AP-1 family.21,28 To examine whether TGF-ß1 can inhibit inducible expression of MCP-1 mRNA and protein in a macrophage cell line, J774a cells were stimulated with the phorbol ester PMA (100 nmol/L) or LPS (10 ng/mL) in the presence or absence of pretreatment with active TGF-ß1 (10 ng/mL) for 30 minutes. As demonstrated in Figure 1, MCP-1 mRNA and protein are expressed at low levels in unstimulated cells and induced markedly by both LPS and PMA. However, pretreatment with TGF-ß1 potently attenuated this induction (Figures 1A and 1B). In contrast, TGF-ß1 increased PAI-1 expression, a well-established target for TGF-ß1.17 Previous work has demonstrated that cytokine activation of MCP-1 occurs primarily at the level of transcription.29 Induction of MCP-1 expression is mediated in part through two tandem AP-1 sites and an NF-
B site in the proximal promoter of this gene depending on the cell type and stimulus used.29–31 Consistent with these results, transient transfection assays using an MCP-1 promoter construct showed an increase in transcriptional activity induced by LPS that could be attenuated by pretreatment with TGF-ß1 in RAW264.7 cells (Figure 1C, left panel). We also examined whether pre-, co-, or postincubation with TGF-ß1 would have similar effects. We found that TGF-ß1 inhibition of the MCP-1 promoter can occur, but is attenuated, if LPS stimulation is allowed to proceed first for 1 to 6 hours in comparison to co- or preincubation with TGF-ß1 (online Figure 1A, in the online data supplement at http://circres.ahajournals.org).
|
Smad proteins constitute a family of intracellular effectors that mediate TGF-ß signal transduction and gene expression.17 To assess whether the TGF-ß1 inhibition of MCP-1 gene expression was Smad dependent, we performed cotransfection assays of the MCP-1 promoter construct with several Smad expression constructs followed by stimulation with LPS. As shown in Figure 1C, only Smad3 was able to repress the MCP-1 promoter in a manner similar to TGF-ß1, an effect enhanced by treatment with exogenous TGF-ß1. In contrast, the inhibitory Smads, Smad6, and Smad7, transactivated the MCP-1 promoter to levels even higher than that achieved by LPS stimulation. The authenticity of the Smad3 expression construct was verified by its ability to induce the PAI-1 promoter (Figure 1C). Taken together, these data indicate that TGF-ß1 inhibits inducible expression of MCP-1, an effect that may be mediated via Smad3.
Adenoviral Overexpression of Smad3 Inhibits MCP-1 Expression and AP-1 DNA-Protein Binding
To address whether constitutive overexpression of Smad3 can inhibit inducible expression of endogenous MCP-1, we adenovirally overexpressed Ad-Smad3 or Ad-CMV-ß-galactosidase (ß-gal) in J774a cells followed by stimulation with PMA or LPS. As demonstrated in Figure 2A, whereas TGF-ß1 alone can inhibit PMA- or LPS-induced MCP-1 mRNA, constitutive overexpression of Smad3 markedly augmented this inhibitory effect. As a positive control of TGF-ß/Smad responsiveness, we assessed PAI-1 expression under the same conditions. In contrast to the inhibitory effect on MCP-1, overexpression of Smad3 in the presence of TGF-ß1 induced PAI-1 mRNA (Figure 2A).
|
In theory, the inhibitory effect of Smad3 may occur by direct DNA binding or through interaction with coactivators (eg, p300/CBP), corepressors (eg, TGIF, SnoN, and c-Ski), or sequence-specific transcription factors (eg, AP-1, TFE3, Sp1, Evi-1, and GATA-3).17,32 Examination of the -486 bp MCP-1 promoter failed to reveal the presence of a consensus Smad3 binding site. Because transcriptional activation of the MCP-1 promoter is critically regulated by the proximal AP-1 sites,29–31 we assessed whether Smad3 can affect AP-1 DNA-protein binding in J774a cells. As demonstrated in Figure 2B, a dominant gel-shift complex is induced after treatment with PMA and competed away by a wild-type, but not a mutated, competitor oligonucleotide. In addition, supershift studies demonstrate that c-Jun, JunB, and c-Fos are the major AP-1 proteins present. To address whether constitutive overexpression of Smad3 can alter AP-1 DNA-protein binding, we infected J774a cells with adenoviral constructs Ad-Smad3 or Ad-CMV-ß-gal (ctrl). As shown in Figure 2B, although TGF-ß1 alone can inhibit PMA-induced AP-1 DNA-protein binding, constitutive overexpression of Smad3 markedly enhanced this inhibitory effect. In contrast, there was no inhibitory effect observed of Ad-Smad3 plus TGF-ß1 on NF-B DNA-protein binding (online Figure 1E). Taken together, these data indicate that Smad3 may act as a transcriptional repressor by preventing AP-1 DNA-protein binding in J774a cells and raises the possibility that TGF-ß1 via Smad3 may regulate the macrophage response to inflammatory stimuli.
TGF-ß1 Inhibition of MCP-1 Expression Is Mediated by a Smad3-c-Jun Interaction
The activation of AP-1 by inflammatory stimuli involves the participation of various members of the AP-1 family of bZIP transcription factors and results in their binding to specific DNA sequences.33 These proteins may dimerize with other family members to form transcriptionally active AP-1 complexes. Whereas the Jun family members (c-Jun, JunB, and JunD) can both homo- and heterodimerize with Fos members (c-Fos, FosB, Fra-1, and Fra-2), the Fos family members can only heterodimerize with Jun members.33 AP-1 proteins may also interact with other transcriptional regulators to modulate different signaling pathways. Indeed, a recent investigation verified that AP-1 proteins, c-Jun and c-Fos, can interact with Smad3 to control transcriptional responses to TGF-ß1.34 Although this report suggested a synergistic cooperativity of AP-1 and Smad3 in gene activation, the mechanisms for Smad3-mediated gene repression via AP-1 are not well characterized. Because the regulation of AP-1 transcription factors by TGF-ß1 may vary with the specific family member, we first examined the effect of TGF-ß1 pretreatment on the protein expression levels of c-Jun and c-Fos after stimulating with PMA. As demonstrated in Figure 3A, TGF-ß1 inhibited PMA-induced expression of c-Fos; however, there was no inhibitory effect on c-Jun expression after 6 hours of stimulation (Figure 3A). Although decreased c-Fos expression will prevent heterodimerization, c-Jun homodimerization and AP-1 DNA-binding should still occur. Indeed, reporter gene transfection experiments using c-Jun alone demonstrated transactivation of both the -486 bp MCP-1 promoter construct and an AP-1 concatamer (data not shown). Thus, our observation of decreased AP-1 DNA-binding in the presence of TGF-ß1 and Smad3 overexpression (Figure 2B) suggests that in response to TGF-ß1, Smad3 may also interact with c-Jun to prevent its DNA-binding.
|
To verify that Smad3 functionally interacts with c-Jun, we performed coimmunoprecipitation studies in 293T cells. An 
Flag antibody immunoprecipitated c-Jun protein from lysates of cells transfected with Flag-Smad3, c-Jun, and a constitutively active TGF-ßtype I receptor (TGFßRI), but not with Flag-Smad3+empty vector or c-Jun+empty vector (Figure 3B, left panel). Recent characterization of the Smad3/c-Jun interaction using GST adsorption assays also showed that the N-terminal domain of Smad3 and the basic DNA binding domain and leucine zipper (bZIP) of c-Jun mediate this physical interaction.34 Furthermore, substitution of four critical lysines residues with alanines (at positions 40, 41, 43, and 44) within the N-terminal domain of Smad3 significantly blocked this interaction.34 To assess the degree to which the full-length Smad3 mutation construct, Smad3(4A), may interact with c-Jun, we cotransfected 293T cells with c-Jun+TGFßRI and either wild-type Flag-Smad3 or Flag-Smad3(4A). As shown in Figure 3C, an Flag antibody immunoprecipitated c-Jun from lysates transfected with the wild-type Flag-Smad3 construct. In contrast, this immunoprecipitated band is markedly attenuated from lysates transfected with the Flag-Smad3(4A). These findings are consistent with the hypothesis that Smad3 may inhibit the transactivation of AP-1–dependent promoters through a direct interaction of Smad3 with c-Jun. To examine this, we first performed transient transfection experiments with an AP-1-luciferase construct containing seven concatamerized consensus AP-1 sites and verified that TGF-ß1 could inhibit induction of AP-1 by LPS or PMA (online Figure 1B). To assess this effect directly, we cotransfected Smad3, c-Jun, and c-Fos. Increasing molar amounts of Smad3 in the presence or absence of TGF-ß1 effectively inhibited c-Jun+c-Fos induction of the AP-1-luciferase construct (Figure 3D). However, when the Smad3(4A) construct is cotransfected in a similar manner, no inhibition was detected (Figure 3E). Similar results were observed using the MCP-1 promoter (Figure 3F). Taken together, these data indicate that in response to TGF-ß1, Smad3 mediates its inhibitory effect by interacting with c-Jun and interfering with the ability of c-Jun to transactivate AP-1–bearing promoters.
TGF-ß1 Inhibition of MCP-1 Expression and AP-1 DNA-Protein Binding Are Blocked in Smad3-/- Macrophages
The studies above support an inhibitory role for Smad3 on MCP-1 expression. To determine whether Smad3 is requisite for TGF-ß1 mediated inhibition, we performed loss-of-function studies. Smad3-/- and Smad3+/+ bone marrow–derived macrophages were harvested and examined for their responsiveness to LPS in the presence or absence of TGF-ß1. As shown in Figure 4A, although TGF-ß1 can inhibit LPS-mediated induction of MCP-1 protein in Smad3+/+ cells, the inhibitory effect of TGF-ß1 is completely blocked in Smad3-/- macrophages. Similarly, although TGF-ß1 can inhibit PMA-induced AP-1 DNA-protein binding in Smad3+/+ macrophages, there is no inhibitory effect observed in response to TGF-ß1 in Smad3-/- macrophages (Figure 4B). These data indicate that Smad3 is required to mediate the inhibitory effect of TGF-ß1 on MCP-1 expression and suggest that Smad3 may be critical for regulating inflammatory responses in vivo.
|
Cardiac Allografts of Smad3-/- Recipient Mice Develop an Accelerated Inflammatory Arteriopathy
Recruitment of activated macrophages is critical to the development of transplant arteriopathy, a process characterized by a diffuse and concentric intimal narrowing in blood vessels of the transplanted organ.2 To determine the role of Smad3 in an in vivo model of vascular inflammation, we examined the development of TxAA in hearts transplanted into Smad3-/- or Smad3+/+ mice. In light of our observation that Smad3 inhibits MCP-1, we hypothesized that Smad3 deficiency would accelerate TxAA. We transplanted bm12 (major histocompatibility complex (MHC) class II-mismatched) donor hearts heterotopically into C57BL/6 (B6, H-2b) Smad3-/- or Smad3+/+ recipients without immunosuppression. Grafts were harvested at 6 weeks after transplantation as we typically detect well-developed neointimal expansion of the coronary arteries after about 8 weeks.35 As shown in Figure 4C, coronary arteries of allografts from Smad3+/+ recipient mice exhibit minimal neointima formation at this 6-week time point; however, there is accelerated intimal hyperplasia within allografts from the Smad3-/- mice. Furthermore, there is marked induction of MCP-1 expression in the Smad3-/- recipient allografts in comparison to the Smad3+/+ recipient allografts. Indeed, MCP-1 expression colocalized to CD11b-positive macrophages, which accumulated in the adventitia of the coronary arteries of Smad3-/- allografts (Figure 4C). In contrast, there was no difference of low-level immunostaining for CD3+ cells (T lymphocytes) within allografts from either Smad3-/- or Smad3+/+ mice (data not shown). In addition, although low-level immunostaining for smooth muscle -actin (vascular smooth muscle cells) was observed within the neointima of allografts from Smad3-/- mice, there was no immunostaining detected within the minimal neointima of Smad3+/+ mice (data not shown). Finally, to rule out the possibility that the accelerated arteriopathy and enhanced MCP-1 expression observed in Smad3-/- mice was not due to a decrease in TGF-ß1 expression, we immunostained for TGF-ß1. We found that by comparison to Smad3+/+, TGF-ß1 expression of Smad3-/- donor hearts is not decreased and is present in both the perivascular and neointimal areas (online Figure 2). Taken together, these findings suggest that Smad3 is critical in modulating the transplant-associated inflammatory process and in regulating the expression of MCP-1 in vivo.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The chemokine MCP-1 is critical for the recruitment of macrophages in a variety of inflammatory vascular disease states. As such, identification of mechanisms limiting MCP-1 expression may offer novel strategies to diminish the inflammatory response. One of the most important properties of TGF-ß1 is its ability to modulate inflammatory stimuli. Our previous observations demonstrate that TGF-ß1 inhibits cytokine-mediated induction of proinflammatory gene expression in macrophages.22,23 However, the mechanism by which TGF-ß1 is able to mediate this function and in vivo significance of such effects are not well understood. In this report, we describe a definitive role for the TGF-ß downstream effector Smad3 in suppressing inflammatory events within the vascular system in vivo and in macrophages ex vivo in response to TGF-ß1. In support, we found that of all the Smads examined, only Smad3 could inhibit induction of MCP-1 transcriptional activity in an analogous manner as TGF-ß1 (Figure 1C). In addition, the inhibitory effect of Smad3 was enhanced with the addition of exogenous TGF-ß1. Furthermore, adenoviral overexpression of Smad3 significantly repressed inducible expression of endogenous MCP-1 (Figure 2A). Conversely, TGF-ß1 inhibition of LPS-mediated induction of MCP-1 expression was completely blocked in Smad3-deficient macrophages (Figure 4A). Consistent with this impaired response, we found abundantly more perivascular CD11b-positive macrophages within allografts from Smad3-/- mice than Smad3+/+ mice (Figure 4C). Moreover, nearly all the MCP-1–positive cells colocalized with these adventitial macrophages (Figure 4C). Perivascular inflammation is an important hallmark in the development of transplant arteriopathy by contributing to adventitial scarring and arterial lumen narrowing.2 Collectively, these findings are consistent with lesions found early in the development of transplant arteriopathy, typified by a preponderance of inflammatory cells rather than smooth muscle cells.2,36 Thus, expression of chemokines such as MCP-1 by adventitial macrophages may allow for potentiation of macrophage infiltration resulting in an accelerated inflammatory arteriopathy as found in the allografts from Smad3-/- mice.
Previous studies highlight the critical role of AP-1 proteins in the induction of MCP-1.29–31 Our studies demonstrate by both gain- and loss-of-function strategies that the TGF-ß1 inhibition of cytokine-induced AP-1 DNA binding is Smad3 dependent (Figures 2 and 3
). Our results also suggest that in response to TGF-ß1, the inhibitory effect of Smad3 on AP-1 is mediated through at least two potential mechanisms: by decreasing c-Fos expression and by a direct interaction with c-Jun (Figure 4). The net result of these effects is a reduction in AP-1 DNA binding and transcriptional activity. Although we have demonstrated an inhibitory effect of TGF-ß1 and Smad3 on the induction of MCP-1, two recent studies suggest opposite effects.37,38 We believe the discrepancy of their observed effects is due to either the failure to coincubate cells with TGF-ß1 for the total time that the stimulus was used37 or differences in cell-type examined (eg, astrocytic cells).38
Our data do not exclude the possibility of additional mechanisms by which Smad3 may inhibit AP-1 function. In light of our observations that JunB is also present in the AP-1 complex (Figure 2B), we cannot exclude the possibility that JunB may also participate in the inhibitory effect of Smad3 on AP-1. Indeed, Smad3 has also been found to interact with JunB in vitro.39 Another possible inhibitory mechanism is coactivator competition. For example, Smad3 can interact with p300, a coactivator that is required for optimal AP-1 mediated transactivation.40 However, because we have previously demonstrated that overexpression of p300 could only partially rescue the inhibitory effects of TGF-ß1, alternative inhibitory mechanisms are likely to be involved.23 Thus, our current observations and previously published data support dual inhibitory effects of Smad3 on AP-1 dependent promoters through the direct effect on AP-1 DNA binding and transcriptional activity as well as an indirect effect that may be conferred by coactivator competition. These inhibitory effects may underlie the more potent repression achieved by TGF-ß/Smad3 on cytokine-mediated induction of AP-1 dependent promoters (eg, MCP-1 and MMP-12) in comparison to non–AP-1–dependent promoters (eg, iNOS) (Figure 1 and Feinberg et al22 and Werner et al23).
Several experimental studies implicate a salutary effect of TGF-ß1 on vascular inflammation. For example, blockade of TGF-ß1 ligand or the TGF-ß type II receptor accelerates the development of atherosclerotic lesion formation in ApoE-/- atherosclerotic-prone mice.41,42 In patients, the serum concentration of active TGF-ß1 is inversely correlated with the severity of atherosclerotic disease.43 In the context of TxAA, cardiac allografts from recipient mice heterozygous for TGF-ß1 displayed a marked increase in TxAA in comparison to wild-type controls.44 The observations in this study that robust macrophage infiltration and accelerated vascular arteriopathy occur in allografts of Smad3-deficient mice coupled with impairment of TGF-ß1 inhibition in Smad3-deficient macrophages indicate that Smad3 is the downstream effector mediating the immunosuppressive effects of TGF-ß1.
Taken together, these observations indicate that Smad3 plays an essential role in modulating vascular inflammation by regulating MCP-1 expression. Furthermore, we provide evidence that the Smad3 inhibition occurs through a novel antagonistic effect of Smad3 on the AP-1 signaling pathway in macrophages. To our knowledge, this is the first report to demonstrate an essential role of this Smad protein in vascular inflammation in vivo. Targeting the Smad3 pathway may provide a novel antiinflammatory strategy for transplant arteriopathy and atherosclerosis.
|
Acknowledgments |
|---|
This work was supported by NIH grants HL67755 (to M.W.F.), HL03747 (to M.K.J.), GM-67049 (to R.M. and K.S.), and HL-43364 (to P.L.). This work was also supported by American Heart Association grant 0250030N and American Diabetes Association grant 1-03-JF-00 (to M.K.J.), and a Roche Organ Transplantation Research Foundation grant (to K.S.).
|
Footnotes |
|---|
Original received July 8, 2003; resubmission received December 5, 2003; revised resubmission received January 15, 2004; accepted January 16, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.[CrossRef][Medline] [Order article via Infotrieve]
2. Libby P, Pober JS. Chronic rejection. Immunity. 2001; 14: 387–397.[CrossRef][Medline] [Order article via Infotrieve]
3. Schwartz RS, Henry TD. Pathophysiology of coronary artery restenosis. Rev Cardiovasc Med. 2002; 3 (suppl 5): S4–S9.[Medline] [Order article via Infotrieve]
4. Simon DI, Dhen Z, Seifert P, Edelman ER, Ballantyne CM, Rogers C. Decreased neointimal formation in Mac-1-/- mice reveals a role for inflammation in vascular repair after angioplasty. J Clin Invest. 2000; 105: 293–300.[Medline] [Order article via Infotrieve]
5. Rollins BJ, Yoshimura T, Leonard EJ, Pober JS. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol. 1990; 136: 1229–1233.[Abstract]
6. Gerard C, Rollins BJ. Chemokines and disease. Nat Immun. 2001; 2: 108–115.[CrossRef][Medline] [Order article via Infotrieve]
7. Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998; 2: 275–281.[CrossRef][Medline] [Order article via Infotrieve]
8. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998; 394: 894–897.[CrossRef][Medline] [Order article via Infotrieve]
9. Aiello RJ, Bourassa PA, Lindsey S, Weng W, Natoli E, Rollins BJ, Milos PM. Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 1999; 19: 1518–1525.
10. Usui M, Egashira K, Ohtani K, Kataoka C, Ishibashi M, Hiasa K, Katoh M, Zhao Q, Kitamoto S, Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy inhibits restenotic changes (neointimal hyperplasia) after balloon injury in rats and monkeys. FASEB J. 2002; 16: 1838–1840.
11. Mori E, Komori K, Yamaoka T, Tanii M, Kataoka C, Takeshita A, Usui M, Egashira K, Sugimachi K. Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits. Circulation. 2002; 105: 2905–2910.
12. Roque M, Kim W, Gazdoin M, Malik A, Reis ED, Fallon JT, Badimon JJ, Charo IF, Taubman M. CCR2 deficiency decreases intimal hyperplasia after arterial injury. Arterioscler Thromb Vasc Biol. 2002; 22: 554–559.
13. Gullestad L, Simonsen S, Ueland T, Holm T, Aass H, Andreassen AK, Madsen S, Geiran O, Froland SS, Aukrust P. Possible role of proinflammatory cytokines in heart allograft coronary artery disease. Am J Cardiol. 1999; 84: 999–1003.[CrossRef][Medline] [Order article via Infotrieve]
14. Russell ME, Adams DH, Wyner LR, Yamashita Y, Halnon NJ, Karnovsky MJ. Early and persistent induction of monocyte chemoattractant protein-1 In rat cardiac allografts. Proc Natl Acad Sci U S A. 1993; 90: 6086–6090.
15. Mehra A, Wrana JL. TGF-ß and the Smad signal transduction pathway. Biochem Cell Biol. 2002; 80: 605–622.[CrossRef][Medline] [Order article via Infotrieve]
16. Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF-ß signal transduction. J Cell Sci. 2001; 114: 4359–4369.[Medline] [Order article via Infotrieve]
17. Massague J, Wotton D. Transcriptional control by the TGF-ß/Smad signaling system. EMBO J. 2000; 19: 1745–1754.[CrossRef][Medline] [Order article via Infotrieve]
18. Metcalfe JC, Grainger DJ. TGF-ß: implications for human vascular disease. J Hum Hypertens. 1995; 9: 679.[Medline] [Order article via Infotrieve]
19. Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB, Sporn MB, Ward JM, Karlsson S. Transforming growth factor ß1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A. 1993; 90: 770–774.
20. Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D. Targeted disruption of the mouse transforming growth factor-ß1 gene results in multifocal inflammatory disease. Nature. 1992; 359: 693–699.[CrossRef][Medline] [Order article via Infotrieve]
21. Kitamura M. Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-ß1. J Immunol. 1997; 159: 1404–1411.[Abstract]
22. Feinberg MW, Jain MK, Werner F, Sibinga NE, Wiesel P, Wang H, Topper JN, Perrella MA, Lee ME. Transforming growth factor-ß1 inhibits cytokine-mediated induction of human metalloelastase in macrophages. J Biol Chem. 2000; 275: 25766–25773.
23. Werner F, Jain MK, Feinberg MW, Sibinga NE, Pellacani A, Wiesel P, Chin MT, Topper JN, Perrella MA, Lee ME. Transforming growth factor-ß1 inhibition of macrophage activation is mediated via Smad3. J Biol Chem. 2000; 275: 36653–36658.
24. Derynck R, Zhang Y, Feng XH. Smads: transcriptional activators of TGF-ß responses. Cell. 1998; 95: 737–740.[CrossRef][Medline] [Order article via Infotrieve]
25. Datto MB, Frederick JP, Pan L, Borton AJ, Zhuang Y, Wang XF. Targeted disruption of Smad3 reveals an essential role in transforming growth factor ß-mediated signal transduction. Mol Cell Biol. 1999; 19: 2495–2504.
26. Yang X, Letterio JJ, Lechleider RJ, Chen L, Hayman R, Gu H, Roberts AB, Deng C. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-ß. EMBO J. 1999; 18: 1280–1291.[CrossRef][Medline] [Order article via Infotrieve]
27. Zhu Y, Richardson JA, Parada LF, Graff JM. Smad3 mutant mice develop metastatic colorectal cancer. Cell. 1998; 94: 703–714.[CrossRef][Medline] [Order article via Infotrieve]
28. Introna M, Bast RC Jr, Tannenbaum CS, Hamilton TA, Adams DO. The effect of LPS on expression of the early "competence" genes JE and KC in murine peritoneal macrophages. J Immunol. 1987; 138: 3891–3896.[Abstract]
29. Martin T, Cardarelli PM, Parry GC, Felts KA, Cobb RR. Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-B and AP-1. Eur J Immunol. 1997; 27: 1091–1097.[Medline]
[Order article via Infotrieve]
30. Shyy JY, Lin MC, Han J, Lu Y, Petrime M, Chien S. The cis-acting phorbol ester "12-O-tetradecanoylphorbol 13-acetate"-responsive element is involved in shear stress-induced monocyte chemotactic protein 1 gene expression. Proc Natl Acad Sci U S A. 1995; 92: 8069–8073.
31. Lim SP, Garzino-Demo A. The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the ß-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-B consensus sites. J Virol. 2000; 74: 1632–1640.
32. Dennler S, Goumans MJ, ten Dijke P. Transforming growth factor ß signal transduction. J Leukoc Biol. 2002; 71: 731–740.
33. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991; 1072: 129–157.[Medline] [Order article via Infotrieve]
34. Qing J, Zhang Y, Derynck R. Structural and functional characterization of the transforming growth factor-ß-induced Smad3/c-Jun transcriptional cooperativity. J Biol Chem. 2000; 275: 38802–38812.
35. Shimizu K, Schonbeck U, Mach F, Libby P, Mitchell RN. Host CD40 ligand deficiency induces long-term allograft survival and donor-specific tolerance in mouse cardiac transplantation but does not prevent graft arteriosclerosis. J Immunol. 2000; 165: 3506–3518.
36. Shimizu K, Aikawa M, Takayama K, Libby P, Mitchell RN. Direct anti-inflammatory mechanisms contribute to attenuation of experimental allograft arteriosclerosis by statins. Circulation. 2003; 108: 2113–2120.
37. Xiao Y, Malcolm K, Worthen GS, Gardai S, Scheimann WP, Fadok VA, Bratton DL, Henson PM. Cross-talk between ERK and p38 MAPK mediates selective suppression of pro-inflammatory cytokines by transforming growth factor-ß. J Biol Chem. 2002; 277: 14884–14893.
38. Abraham S, Sawaya B, Safak M, Batuman O, Khalili K, Amini S. Regulation of MCP-1 gene transcription by Smads and HIV-1 Tat in human glial cells. Virology. 2003; 309: 196–202.[CrossRef][Medline] [Order article via Infotrieve]
39. Liberati NT, Datto MB, Frederick JP, Shen X, Wong C, Rougier-Chapman EM, Wang XF. Smads bind directly to the Jun family of AP-1 transcription factors. Proc Natl Acad Sci U S A. 1999; 96: 4844–4849.
40. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell. 1996; 85: 403–414.[CrossRef][Medline] [Order article via Infotrieve]
41. Mallat Z, Gojova A, Marchiol-Fournigault C, Esposito B, Kamate C, Merval R, Fradelizi D, Tedgui A. Inhibition of transforming growth factor-ß signaling accelerates atherosclerosis and induces an unstable plaque phenotype in mice. Circ Res. 2001; 89: 930–934.
42. Lutgens E, Gijbels M, Smook M, Heeringa P, Gotwals P, Koteliansky V, Daemen M. Transforming growth factor-ß mediates balance between inflammation and fibrosis during plaque progression. Arterioscler Thromb Vasc Biol. 2002; 22: 975–982.
43. Grainger DJ, Kemp PR, Metcalfe JC, Liu AC, Lawn RM, Williams NR, Grace AA, Schofield PM, Chauhan A. The serum concentration of active transforming growth factor-ß is severely depressed in advanced atherosclerosis. Nat Med. 1995; 1: 74–79.[CrossRef][Medline] [Order article via Infotrieve]
44. Koglin J, Glysingjensen T, Raisanensokolowski A, Russell ME. Immune sources of transforming growth factor-ß1 reduce transplant arteriosclerosis: insight derived from a knockout mouse model. Circ Res. 1998; 83: 652–660.
This article has been cited by other articles:
 |
|
 |
|
  K. Shimizu, M. Minami, R. Shubiki, M. Lopez-Ilasaca, L. MacFarlane, Y. Asami, Y. Li, R. N. Mitchell, and P. Libby CC Chemokine Receptor-1 Activates Intimal Smooth Muscle-Like Cells in Graft Arterial Disease Circulation, November 3, 2009; 120(18): 1800 - 1813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Cao, A. K. Wara, B. Icli, X. Sun, R. R. S. Packard, F. Esen, C. J. Stapleton, M. Subramaniam, K. Kretschmer, I. Apostolou, et al. Kruppel-like Factor KLF10 Targets Transforming Growth Factor-{beta}1 to Regulate CD4+CD25- T Cells and T Regulatory Cells J. Biol. Chem., September 11, 2009; 284(37): 24914 - 24924. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Tsai, S. T. Hollenbeck, E. J. Ryer, R. Edlin, D. Yamanouchi, R. Kundi, C. Wang, B. Liu, and K. C. Kent TGF-{beta} through Smad3 signaling stimulates vascular smooth muscle cell proliferation and neointimal formation Am J Physiol Heart Circ Physiol, August 1, 2009; 297(2): H540 - H549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Zhang, S. Tsai, K. Kato, D. Yamanouchi, C. Wang, S. Rafii, B. Liu, and K. C. Kent Transforming Growth Factor-{beta} Promotes Recruitment of Bone Marrow Cells and Bone Marrow-derived Mesenchymal Stem Cells through Stimulation of MCP-1 Production in Vascular Smooth Muscle Cells J. Biol. Chem., June 26, 2009; 284(26): 17564 - 17574. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Shimizu and R. N. Mitchell The Role of Chemokines in Transplant Graft Arterial Disease Arterioscler Thromb Vasc Biol, November 1, 2008; 28(11): 1937 - 1949. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Shimizu, P. Libby, R. Shubiki, M. Sakuma, Y. Wang, K. Asano, R. N. Mitchell, and D. I. Simon Leukocyte Integrin Mac-1 Promotes Acute Cardiac Allograft Rejection Circulation, April 15, 2008; 117(15): 1997 - 2008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Minami, K. Shimizu, Y. Okamoto, E. Folco, M.-L. Ilasaca, M. W. Feinberg, M. Aikawa, and P. Libby Prostaglandin E Receptor Type 4-associated Protein Interacts Directly with NF-{kappa}B1 and Attenuates Macrophage Activation J. Biol. Chem., April 11, 2008; 283(15): 9692 - 9703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Kim, S. A. Ham, S. U. Kim, J.-Y. Hwang, J.-H. Kim, K. C. Chang, C. Yabe-Nishimura, J.-H. Kim, and H. G. Seo Transforming Growth Factor-{beta}1 Is a Molecular Target for the Peroxisome Proliferator-Activated Receptor {delta} Circ. Res., February 1, 2008; 102(2): 193 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bujak, G. Ren, H. J. Kweon, M. Dobaczewski, A. Reddy, G. Taffet, X.-F. Wang, and N. G. Frangogiannis Essential Role of Smad3 in Infarct Healing and in the Pathogenesis of Cardiac Remodeling Circulation, November 6, 2007; 116(19): 2127 - 2138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Gourdy, A. Schambourg, C. Filipe, V. Douin-Echinard, B. Garmy-Susini, B. Calippe, F. Terce, F. Bayard, and J.-F. Arnal Transforming Growth Factor Activity Is a Key Determinant for the Effect of Estradiol on Fatty Streak Deposit in Hypercholesterolemic Mice Arterioscler Thromb Vasc Biol, October 1, 2007; 27(10): 2214 - 2221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-M. Ka, X.-R. Huang, H.-Y. Lan, P.-Y. Tsai, S.-M. Yang, H.-A. Shui, and A. Chen Smad7 Gene Therapy Ameliorates an Autoimmune Crescentic Glomerulonephritis in Mice J. Am. Soc. Nephrol., June 1, 2007; 18(6): 1777 - 1788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bujak and N. G. Frangogiannis The role of TGF-{beta} signaling in myocardial infarction and cardiac remodeling Cardiovasc Res, May 1, 2007; 74(2): 184 - 195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Ma, Q. Wang, T. Fei, J.-D. J. Han, and Y.-G. Chen MCP-1 mediates TGF-{beta}-induced angiogenesis by stimulating vascular smooth muscle cell migration Blood, February 1, 2007; 109(3): 987 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fujimi, M. P. MacConmara, A. A. Maung, Y. Zang, J. A. Mannick, J. A. Lederer, and P. H. Lapchak Platelet depletion in mice increases mortality after thermal injury Blood, June 1, 2006; 107(11): 4399 - 4406. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. da Cunha, B. Martin-McNulty, J. Vincelette, L. Zhang, J. C. Rutledge, D. W. Wilson, R. Vergona, M. E. Sullivan, and Y.-X. Wang Interaction between mild hypercholesterolemia, HDL-cholesterol levels, and angiotensin II in intimal hyperplasia in mice J. Lipid Res., March 1, 2006; 47(3): 476 - 483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Feinberg, Z. Cao, A. K. Wara, M. A. Lebedeva, S. SenBanerjee, and M. K. Jain Kruppel-like Factor 4 Is a Mediator of Proinflammatory Signaling in Macrophages J. Biol. Chem., November 18, 2005; 280(46): 38247 - 38258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Smith, A. Yndestad, B. Halvorsen, T. Ueland, T. Waehre, K. Otterdal, H. Scholz, K. Endresen, L. Gullestad, S. S. Froland, et al. Potential anti-inflammatory role of activin A in acute coronary syndromes J. Am. Coll. Cardiol., July 21, 2004; 44(2): 369 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. W. Feinberg, M. Watanabe, M. A. Lebedeva, A. S. Depina, J.-i. Hanai, T. Mammoto, J. P. Frederick, X.-F. Wang, V. P. Sukhatme, and M. K. Jain Transforming Growth Factor-{beta}1 Inhibition of Vascular Smooth Muscle Cell Activation Is Mediated via Smad3 J. Biol. Chem., April 16, 2004; 279(16): 16388 - 16393. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||