Yasuji Mori

Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-β involves functional cooperation with p300/CBP transcriptional coactivators

Transforming growth factor-β (TGF-β) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-β- or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected fibroblasts, and reduced the basal level of collagen gene expression. This effect was due to specific interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-β to stimulate COL1A2 promoter activity in the presence of E1A. The effect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferase-deficient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-deficient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-β. These results indicate, for the first time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-β stimulation of collagen gene expression in fibroblasts.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-β (TGF-β) stimulates collagen synthesis and accumulation, and aberrant TGF-β signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-β involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-β induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-β stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-β enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-β. In contrast, the TGF-β response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-β responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-β target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-β and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

Peroxisome proliferator-activated receptor-γ abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator

Ligands of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) abrogate the stimulation of collagen gene transcription induced by transforming growth factor‐beta (TGF‐β). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR‐γ in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF‐β activity by PPAR‐γ ligands involves cellular PPAR‐γ, since 15‐deoxy‐A12,14‐prostaglandin J2 (15d‐PGJ2) failed to block TGF‐β‐induced responses in either primary cultures of PPAR‐γ‐null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi‐mediated knockdown of PPAR‐γ. Next, we examined the molecular basis underlying the abrogation of TGF‐β signaling by PPAR‐γ in normal human fibroblasts in culture. The results demonstrated that Smad‐dependent transcriptional responses were blocked by PPAR‐γ without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF‐β, and the accumulation of p300 on consensus Smad‐binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR‐γ. Wild‐type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF‐β‐induced stimulation of COL1A2 in the presence of PPAR‐γ ligands. Collectively, these results indicate that PPAR‐γ blocked Smad‐mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF‐β‐induced collagen gene expression. Pharmacological activation of PPAR‐γ thus may represent a novel therapeutic approach to target p300‐dependent TGF‐β profibrotic responses such as stimulation of collagen gene expression.—Ghosh, A. K., Bhattacharyya, S., Wei, J., Kim, S., Barak, Y., Mori, Y., and Varga, J. Peroxisome proliferator‐activated receptor‐γ abrogates Smad‐dependent collagen stimulation by targeting the p300 transcriptional coactivator. FASEB J. 23, 2968–2977 (2009). www.fasebj.org

A non-Smad mechanism of fibroblast activation by transforming growth factor-β via c-Abl and Egr-1: selective modulation by imatinib mesylate

The nonreceptor protein tyrosine kinase c-Abl regulates cell proliferation and survival. Recent studies provide evidence that implicate c-Abl as a mediator for fibrotic responses induced by transforming growth factor-β (TGF-β), but the precise mechanisms underlying this novel oncogene function are unknown. Here, we report that when expressed in normal fibroblasts, a constitutively active mutant of Abl that causes chronic myelogenous leukemia (CML) stimulated the expression and transcriptional activity of the early growth response factor 1 (Egr-1). Mouse embryonic fibroblasts (MEFs), lacking c-Abl, were resistant to TGF-β stimulation. Responsiveness of these MEFs to TGF-β could be rescued by wild-type c-Abl, but not by a kinase-deficient mutant form of c-Abl. Furthermore, Abl kinase activity was necessary for the induction of Egr-1 by TGF-β in normal fibroblasts, and Egr-1 was required for stimulation of collagen by Bcr-Abl. Lesional skin fibroblasts in mice with bleomycin-induced fibrosis of skin displayed evidence of c-Abl activation in situ, and elevated phospho-c-Abl correlated with increased local expression of Egr-1. Collectively, these results position Egr-1 downstream of c-Abl in the fibrotic response, delineate a novel Egr-1-dependent intracellular signaling mechanism that underlies the involvement of c-Abl in certain TGF-β responses, and identify Egr-1 as a target of inhibition by imatinib. Furthermore, the findings show in situ activation of c-Abl paralleling the upregulated tissue expression of Egr-1 that accompanies fibrosis. Pharmacological targeting of c-Abl and its downstream effector pathways may, therefore, represent a novel therapeutic approach to blocking TGF-β-dependent fibrotic processes.

Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis: Implications for Scleroderma

Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

Negative modulation of α1(I) procollagen gene expression in human skin fibroblasts: Transcriptional inhibition by interferon-γ

Interferon‐γ (IFN‐γ), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of α1(I) procollagen gene (COL1A1) expression by recombinant IFN‐γ. The results showed that IFN‐γ stimulated the rapid accumulation of interferon regulated factor (IRF)‐1 mRNA, followed by a delayed and dose‐dependent inhibition of α1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF‐1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre‐mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter–chloramphenicol acetyltransferase reporter gene plasmids, IFN‐γ selectively inhibited promoter activity and abrogated its stimulation induced by TGF‐β. The inhibition by IFN‐γ was not due to downregulation of TGF‐β receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN‐α and IFN‐β by themselves had little effect on promoter activity, but IFN‐α augmented the inhibitory effect of IFN‐γ. Using a series of 5′ deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN‐γ response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF‐1 but no homologs of previously characterized IFN‐γ response elements. The putative IFN‐γ response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN‐γ. Gel mobility shift analysis showed that two distinct and specific DNA‐protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN‐γ response region. IFN‐γ did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN‐γ inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF‐1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN‐γ regulation of fibroblast function. J. Cell. Physiol. 179:97–108, 1999.

Selective inhibition of collagen gene expression in fibroblasts by an interferon-gamma transgene.

Because interferon-gamma (IFN-gamma) selectively inhibits collagen gene expression, we hypothesized that expression of IFN-gamma cDNA in fibroblasts might be a useful strategy to inhibit the development of fibrosis. A replication-deficient E1-, E3- adenovirus vector encoding murine IFN-gamma (AdCM VmIFN gamma) was constructed. Infection of murine fibroblasts with AdCM VmIFN gamma in vitro was well tolerated. The results showed that IN F-gamma mRNA was expressed in infected cells, and as much as 17.7 ng of mIFN-gamma/10(6) cells was secreted into culture supernatants. Steady-state levels of alpha 1 (I) procollagen mRNA were decreased by 90% in infected cells compared to uninfected cells. The inhibition of collagen mRNA expression was partially abrogated with a neutralizing anti-mIFN-gamma antibody. Secretion of total collagen by AdCM VmIFN gamma-infected fibroblasts was decreased by 60% compared to uninfected cells. Induction cells. Induction of cytokine responsive gene-2 expression in AdCM VmIFN gamma-infected cells demonstrated that suppression of collagen production was a selective response. The results suggest a novel strategy of cytokine gene transfer and expression for the treatment of fibrotic lung diseases.

Inhibition of collagen gene expression by interferon-γ: Novel role of the CCAAT/enhancer binding protein β (C/EBPβ)

By inhibiting collagen synthesis, interferon‐γ (IFN‐γ) plays a key role in maintaining connective tissue homeostasis, but the mechanisms are not well‐understood. In addition to intracellular signaling through the canonical JAK–STAT transduction pathway, IFN‐γ was recently shown to regulate gene expression via the CCAAT/enhancer‐binding protein β (C/EBPβ) as well. Because C/EBPβ is a crucial mediator of immune and inflammatory responses, and has been implicated in regulation of collagen synthesis by tumor necrosis factor‐α, we examined its role in the inhibitory effects of IFN‐γ. The results demonstrated that IFN‐γ caused increased C/EBPβ expression in dermal fibroblasts and enhanced its binding to cognate DNA sequences in the α2(I) procollagen gene (COL1A2) promoter in vitro and in vivo. Disruption of C/EBP binding by deletion or site‐directed mutagenesis abrogated the inhibition of collagen promoter activity in transient transfection assays, as did cotransfection with dominant negative C/EBPβ, indicating a functional role of cellular C/EBPβ in mediating the IFN‐γ response. Rapid phosphorylation of the ERK1/2 MAP kinases induced by IFN‐γ was accompanied by phosphorylation and nuclear translocation of cellular C/EBPβ, and pretreatment of fibroblasts with ERK1/2 kinase inhibitor blocked C/EBPβ phosphorylation, as well as inhibition of COL1A2 promoter activity, elicited by IFN‐γ. These results provide compelling evidence for a novel C/EBPβ‐dependent IFN‐γ signaling pathway responsible for inhibition of collagen gene transcription. Taken together with recent reports, the findings indicate that intracellular pathways mediating negative regulation of collagen synthesis in response to distinct inflammatory signals that converge on C/EBPβ. J. Cell. Physiol. 207: 251–260, 2006.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

BACKGROUND The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-beta (TGF-beta) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-beta, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-beta. To explore this notion, we characterized TGF-beta-induced activation of fibroblasts from CCN2-null (CCN2(-/-)) mouse embryos. METHODS The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-beta signal transduction and regulation of collagen gene expression were examined in CCN2(-/-) MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. RESULTS Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2(-/-) MEFs was markedly reduced compared to wild type MEFs, TGF-beta-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2(-/-) MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. CONCLUSION Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-beta-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.