Cross Talk between Id1 and Its Interactive Protein Dril1 Mediate Fibroblast Responses to Transforming Growth Factor-β in Pulmonary Fibrosis
2008; Elsevier BV; Volume: 173; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.070915
ISSN1525-2191
AutoresLing Lin, Zhihong Zhou, Liang Zheng, Sean Alber, Simon C. Watkins, Prabir Ray, Naftali Kaminski, Yingze Zhang, Danielle Morse,
Tópico(s)Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
ResumoThe presence of activated fibroblasts or myofibroblasts represents a hallmark of progressive lung fibrosis. Because the transcriptional response of fibroblasts to transforming growth factor-β1 (TGF-β1) is a determinant of disease progression, we investigated the role of the transcriptional regulator inhibitor of differentiation-1 (Id1) in the setting of lung fibrosis. Mice lacking the gene for Id1 had increased susceptibility to bleomycin-induced lung fibrosis, and fibroblasts lacking Id1 exhibited enhanced responses to TGF-β1. Because the effect of Id1 on fibrosis could not be explained by known mechanisms, we performed protein interaction screening and identified a novel binding partner for Id1, known as dead ringer-like-1 (Dril1). Dril1 shares structural similarities with Id1 and was recently implicated in TGF-β1 signaling during embryogenesis. To date, little is known about the function of Dril1 in humans. Although it has not been previously implicated in fibrotic disease, we found that Dril1 was highly expressed in lungs from patients with idiopathic pulmonary fibrosis and was regulated by TGF-β1 in human fibroblasts. Dril1 enhanced activation of TGF-β1 target genes, whereas Id1 decreased expression of these same molecules. Id1 inhibited DNA binding by Dril1, and the two proteins co-localized in vitro and in vivo, providing a potential mechanism for suppression of fibrosis by Id1 through inhibition of the profibrotic function of Dril1. The presence of activated fibroblasts or myofibroblasts represents a hallmark of progressive lung fibrosis. Because the transcriptional response of fibroblasts to transforming growth factor-β1 (TGF-β1) is a determinant of disease progression, we investigated the role of the transcriptional regulator inhibitor of differentiation-1 (Id1) in the setting of lung fibrosis. Mice lacking the gene for Id1 had increased susceptibility to bleomycin-induced lung fibrosis, and fibroblasts lacking Id1 exhibited enhanced responses to TGF-β1. Because the effect of Id1 on fibrosis could not be explained by known mechanisms, we performed protein interaction screening and identified a novel binding partner for Id1, known as dead ringer-like-1 (Dril1). Dril1 shares structural similarities with Id1 and was recently implicated in TGF-β1 signaling during embryogenesis. To date, little is known about the function of Dril1 in humans. Although it has not been previously implicated in fibrotic disease, we found that Dril1 was highly expressed in lungs from patients with idiopathic pulmonary fibrosis and was regulated by TGF-β1 in human fibroblasts. Dril1 enhanced activation of TGF-β1 target genes, whereas Id1 decreased expression of these same molecules. Id1 inhibited DNA binding by Dril1, and the two proteins co-localized in vitro and in vivo, providing a potential mechanism for suppression of fibrosis by Id1 through inhibition of the profibrotic function of Dril1. Fibrosis is a feature of many chronic diseases, and the unfortunate consequence of losing normal organ tissue to scar formation is functional impairment or outright organ failure. One determinant of the progression of lung fibrosis is the presence and extent of fibroblastic foci.1Kuhn C McDonald JA The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis.Am J Pathol. 1991; 138: 1257-1265PubMed Google Scholar Most cells in the fibroblast foci are myofibroblasts, defined by the presence of α-smooth muscle actin (α-SMA) and increased contractile properties.1Kuhn C McDonald JA The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis.Am J Pathol. 1991; 138: 1257-1265PubMed Google Scholar, 2Phan SH The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (414) Google Scholar Myofibroblasts exhibit elevated matrix protein synthesis and increased production of growth factors and cytokines such as transforming growth factor-β1 (TGF-β1).2Phan SH The myofibroblast in pulmonary fibrosis.Chest. 2002; 122: 286S-289SCrossref PubMed Scopus (414) Google Scholar Inhibitor of differentiation-1 (Id1) is the first discovered member of an evolutionarily conserved family of four Id proteins (designated Id1 to Id4) that act as dominant-negative inhibitors of basic helix-loop-helix (bHLH) transcription factors.3Hara E Yamaguchi T Nojima H Ide T Campisi J Okayama H Oda K Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.J Biol Chem. 1994; 269: 2139-2145Abstract Full Text PDF PubMed Google Scholar Id1 has been identified as a TGF-β-responsive gene, but its response appears to be cell-type-specific: Id1 was reported to be robustly, although transiently, induced in fibroblasts by TGF-β14Chambers RC Leoni P Kaminski N Laurent GJ Heller RA Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching.Am J Pathol. 2003; 162: 533-546Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar but suppressed in human epithelial cells.5Kang Y Chen CR Massague J A self-enabling TGFbeta response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells.Mol Cell. 2003; 11: 915-926Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar The suppression of Id1 in epithelial cells is thought to play an important role in the TGF-β1 cytostatic program,5Kang Y Chen CR Massague J A self-enabling TGFbeta response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells.Mol Cell. 2003; 11: 915-926Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar just as the increase of Id1 in fibroblasts is thought to play a role in cell-cycle entry.3Hara E Yamaguchi T Nojima H Ide T Campisi J Okayama H Oda K Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.J Biol Chem. 1994; 269: 2139-2145Abstract Full Text PDF PubMed Google Scholar Id proteins have been implicated in a variety of biological processes such as cellular growth, senescence, differentiation, apoptosis, angiogenesis, neoplastic transformation, and T-cell receptor signaling,4Chambers RC Leoni P Kaminski N Laurent GJ Heller RA Global expression profiling of fibroblast responses to transforming growth factor-beta1 reveals the induction of inhibitor of differentiation-1 and provides evidence of smooth muscle cell phenotypic switching.Am J Pathol. 2003; 162: 533-546Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 6Qi Z Sun XH Hyperresponse to T-cell receptor signaling and apoptosis of Id1 transgenic thymocytes.Mol Cell Biol. 2004; 24: 7313-7323Crossref PubMed Scopus (18) Google Scholar, 7Lyden D Young AZ Zagzag D Yan W Gerald W O'Reilly R Bader BL Hynes RO Zhuang Y Manova K Benezra R Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts.Nature. 1999; 401: 670-677Crossref PubMed Scopus (781) Google Scholar, 8Benezra R Davis RL Lockshon D Turner DL Weintraub H The protein Id: a negative regulator of helix-loop-helix DNA binding proteins.Cell. 1990; 61: 49-59Abstract Full Text PDF PubMed Scopus (1804) Google Scholar, 9Kreider BL Benezra R Rovera G Kadesch T Inhibition of myeloid differentiation by the helix-loop-helix protein Id.Science. 1992; 255: 1700-1702Crossref PubMed Scopus (223) Google Scholar, 10Schindl M Oberhuber G Obermair A Schoppmann SF Karner B Birner P Overexpression of Id-1 protein is a marker for unfavorable prognosis in early-stage cervical cancer.Cancer Res. 2001; 61: 5703-5706PubMed Google Scholar although the role that each Id family member plays in the regulation of these functions and the mechanisms by which they act are still areas of active investigation. Biological responses that have so far been described for Id1 invite questions about its role in fibrosis, but to date very little is known. Global expression profiling of fibroblast responses to TGF-β1 has shown that Id1 is highly induced by TGF-β1, raising the possibility that Id1 plays a part in determining fibroblast behavior in the setting of fibrosis. We hypothesized that Id1 induced by TGF-β1 would act in a negative regulatory manner to inhibit fibrosis in vivo and delay or halt myofibroblast differentiation in vitro. To test this hypothesis, we examined the effect of Id1 in a murine bleomycin model of lung fibrosis and in fibroblast responses to TGF-β1 stimulation. We present evidence here that Id1 does in fact inhibit the progression of fibrosis in these models. Surprisingly, the inhibition of α-SMA promoter activation by Id1 did not rely on the presence of E-boxes, the known recognition sequence for bHLH family proteins. This finding suggested that Id1 was not acting via inhibition of traditional bHLH partners. As a result of yeast two-hybrid protein interaction screening, we identified a novel binding partner for Id1, known as dead ringer-like-1 (Dril1). Dril1 shares structural similarities with Id1 and other bHLH proteins, and Dril1 was also recently implicated in TGF-β1 signaling during embryogenesis.11Callery EM Smith JC Thomsen GH The ARID domain protein dril1 is necessary for TGF(beta) signaling in Xenopus embryos.Dev Biol. 2005; 278: 542-559Crossref PubMed Scopus (16) Google Scholar To date, little is known about the function of Dril1 in humans. Although it has not been implicated in fibrotic disease, our studies show that Dril1 is highly expressed in lungs of patients with idiopathic pulmonary fibrosis (IPF) and is regulated by TGF-β1 in human fibroblasts. Our studies define the function of Id1 and Dril1 in fibroblast responses to TGF-β1 and explore the relationship between these two molecules, providing evidence for a regulatory role of Id1 and Dril1 in the progression of fibrosis. Id1−/− mice were provided by Dr. Robert Benezra (Sloan Kettering Institute, New York, NY) and were generated as previously described.12Yan W Young AZ Soares VC Kelley R Benezra R Zhuang Y High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice.Mol Cell Biol. 1997; 17: 7317-7327Crossref PubMed Scopus (208) Google Scholar Mice were backcrossed with C57BL/6 for seven additional generations at the University of Pittsburgh; wild-type littermates were used as controls. All animals were housed in accordance with guidelines from the American Association for Laboratory Animal Care and Research Protocols and were approved by the Animal Care and Use Committees of the Pittsburgh University School of Medicine. Gender- and age-matched mice were assigned to different treatment groups. Bleomycin was administered as previously described.13Zhou Z Song R Fattman CL Greenhill S Alber S Oury TD Choi AM Morse D Carbon monoxide suppresses bleomycin-induced lung fibrosis.Am J Pathol. 2005; 166: 27-37Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Human fetal lung fibroblasts (MRC-5; American Tissue Cell Culture, Rockville, MD), primary adult human lung fibroblasts, and mouse embryonic fibroblasts from Id1+/+ and Id−/− mice (kind gift of Dr. Robert Benezra) were maintained and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.1% gentamicin. For siRNA inhibition, MRC-5 cells were transfected with control siRNA or target siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) at a final concentration of 20 nmol/L using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. For adenovirus infection, cells were seeded and grown to 50% confluence, then incubated with adenovirus in serum-free medium. Four hours later, adenovirus was replaced by complete medium and cells were harvested for Western blot analysis after 48 hours of incubation. Lung tissues were processed as previously described.13Zhou Z Song R Fattman CL Greenhill S Alber S Oury TD Choi AM Morse D Carbon monoxide suppresses bleomycin-induced lung fibrosis.Am J Pathol. 2005; 166: 27-37Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar After blocking, incubation with primary antibody and three washes with bovine serum albumin, the sections were incubated with cy3-conjugated AffinPure (Jackson ImmunoResearch, West Grove, PA) goat anti-rabbit or anti-mouse IgG (H+L) (1:3000) for 1 hour, stained with Hoechst dye for 30 seconds, and mounted in Gelvatol (Sigma-Aldrich, Milwaukee, WI) after washing. Images were collected with an Olympus Provis microscope (Olympus, Tokyo, Japan) as well as an Olympus Floview scanning confocal microscope. The Hybrid Hunter yeast two-hybrid system (Invitrogen) was used to identify Id11-interacting proteins. The full length Id1 was cloned into the yeast expression plasmid LexZeo and sequenced to confirm that the gene was cloned in frame with LexA. A human adult lung cDNA expression library (cloned into pYESTrp2) was purchased from Invitrogen. The bait plasmid was transformed into L40 yeast and expression was confirmed by immunoblot analysis. Bait plasmid was also tested for nonspecific activation. To identify Id1-interacting proteins, L40 was transformed with bait strain and library and Leu+ transformants were tested for β-galactosidase activity. A total of 88 colonies were screened by polymerase chain reaction (PCR). Oligonucleotides were labeled with biotin for chemiluminescence using a biotin 3′ end DNA labeling kit (Pierce, Rockford, IL). After labeling, equal amounts of complementary strands were mixed together and allowed to anneal for 1 hour at 37°C to form double-stranded probe. Gel mobility shift assays were performed using the Lightshift chemiluminescent electrophoretic mobility shift assay kit (Pierce) according to the manufacturer's instructions. Briefly, nuclear protein was extracted using a nuclear extract kit (Active Motif, Carlsbad, CA). Four to eight μg of nuclear proteins were incubated in binding buffer with 50 fmol biotin end-labeled target DNA for 20 minutes at room temperature. Protein-DNA complexes were then separated from the free oligonucleotide by electrophoresis through 4% native polyacrylamide gels containing 0.5× Tris-borate-ethylenediaminetetraacetic acid and transferred to a nylon membrane at 380 mA for 60 minutes. After cross-linking the transferred DNA to the membrane at 120 mJ/cm2 using a UV-light cross-linker instrument equipped with 254-nm bulbs, we incubated the membrane in blocking buffer for 15 minutes and then conjugated/blocking buffer for 15 minutes. After four washes, the membrane was incubated in substrate equilibration buffer for 5 minutes, then substrate working solution for 5 minutes, and then exposed to Biomax MR film (Kodak, Rochester, NY). Streptavidin beads were incubated with biotin-labeled double-stranded target DNA oligonucleotide and negative control DNA oligonucleotide at 4°C for 3 hours, respectively. Cells were lysed by ice-cold phosphate-buffered saline (PBS) containing 0.5% Triton X-100, 5 mmol/L ethylenediaminetetraacetic acid, 125 μmol/L phenylmethyl sulfonyl fluoride, protease inhibitor cocktail, and phosphatase inhibitor cocktail (1:100 dilution; Sigma, St. Louis, MO). Cell lysates were then centrifuged at 10,000 × g for 30 minutes. The supernatant was divided equally and added to the mixture of DNA oligonucleotide and streptavidin beads and further incubated overnight at 4°C. Beads were then washed with ice-cold PBS containing 0.5% Triton X-100 and 5 mmol/L ethylenediaminetetraacetic acid for four times. Proteins were eluted by adding 2× sodium dodecyl sulfate loading buffer and boiling for 5 minutes, and then were analyzed by Western blotting using rabbit anti-Dril1 antibody. The α-SMA constructs were kindly provided by Dr. Gary K. Owens (Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA). The E-box mutant constructs (E1 and E2) contained the following mutated sequences in bold: E1, CAAGCT; E2, GTACTGA. Details of the generation of the mutants has previously been published.14Kumar MS Hendrix JA Johnson AD Owens GK Smooth muscle alpha-actin gene requires two E-boxes for proper expression in vivo and is a target of class I basic helix-loop-helix proteins.Circ Res. 2003; 92: 840-847Crossref PubMed Scopus (66) Google Scholar Cells were seeded at 3 × 104 cells/cm2 into 6-well or 12-well plates the day before transfection and were transiently transfected (in triplicate) using Lipofectamine 2000 according to the manufacturer's instructions. Cell extracts were prepared 48 hours after transfection using a passive lysis buffer (Promega, Madison, WI), and β-galactosidase activity was measured using the Galacto-Star system (Applied Biosystems, Foster City, CA). β-Galactosidase activity was normalized to activity of Renilla luciferase measured using the dual luciferase assay system from Promega. Each construct was tested in triplicate per experiment, and the data shown represent three independent experiments. Lung tissues were homogenized in 0.5 mol/L acetic acid and incubated with shaking for 24 hours at 4°C. Homogenate was spun at 15,000 × g, and supernatant was analyzed for soluble collagen by the Sircol collagen assay kit following the instructions of the manufacturer (Biocolor Ltd., Newtownabbey, UK). Lung tissue or cells was homogenized and RNA was extracted using the Trizol method (Invitrogen). Standard Northern blotting and quantitative RT-PCR were performed as described elsewhere.15Morse D Pischke SE Zhou Z Davis RJ Flavell RA Loop T Otterbein SL Otterbein LE Choi AM Suppression of inflammatory cytokine production by carbon monoxide involves the JNK pathway and AP-1.J Biol Chem. 2003; 278: 36993-36998Crossref PubMed Scopus (355) Google Scholar Probes and primers for the Id1, TIMP1, MMP9, procollagen, and fibronectin genes were obtained from Applied Biosystems. Real-time quantitative PCR was conducted by the TaqMan core facility of the Genomics and Proteomics Core Laboratories of the University of Pittsburgh. Gene expression was analyzed by the δδCt method, with 18s rRNA as the endogenous control, and one of the control samples was chosen as a calibrator sample. Cell extracts were prepared from 100-mm dishes in 800 μl of RIPA (radio-immunoprecipitation assay) lysis buffer (Sigma) containing protease inhibitors. The protein extract was incubated with either Id1- or Dril1-specific rabbit polyclonal antibody at 4°C overnight with rotation. After the addition of protein A beads (Santa Cruz Biotechnology Inc.) for another 4 hours. The complexes were then washed with RIPA buffer, and the proteins were extracted in reducing sampling buffer and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis. To characterize Id1 expression in fibrosis, we began by examining lungs of mice after bleomycin treatment for Id1 mRNA and protein expression. Fourteen days after bleomycin treatment, whole lung mRNA levels for Id1 were increased; this corresponded with a time-dependent increase in Id1 protein levels as assessed by Western blotting of whole lung homogenate (Figure 1, A and B). Similarly, Id1 expression increased at early time points after TGF-β1 treatment of both embryonic human lung fibroblasts (MRC-5) and primary adult human lung fibroblasts (Figure 1C and data not shown). The increase in protein levels correlated with an early increased expression of Id1 mRNA after TGF-β1 treatment (Figure 1D). The rise in Id1 expression occurred predominantly in the nucleus, although a modest increase in cytoplasmic expression was also seen by immunostaining (Figure 1F). The image shown in Figure 1 was generated using mouse wild-type fibroblasts; Id1−/− controls were used to confirm antibody specificity (not shown). Identical results were obtained using human fibroblasts (not shown). In both Western and Northern blots for Id1, the expression level appeared to fall below the baseline level after an initial early rise. We confirmed this observation using quantitative RT-PCR. After an initial sharp rise in Id1 mRNA expression, there is late suppression of Id1 mRNA levels (Figure 1E), indicating a biphasic response of Id1 to TGF-β1 stimulation. To determine whether Id1 plays a functional role in experimental fibrosis, mice lacking the gene for Id1 were treated with intratracheal bleomycin. At a dose of 0.1 U per age- and weight-matched mouse, the Id1−/− mice exhibited increased mortality when compared with WT mice (Figure 2A). The Id1−/− mice displayed evidence of increased respiratory distress 10 days after bleomycin treatment and succumbed to death from the 12th day onwards. A lower dose of bleomycin (0.06 U per mouse) allowed for improved survival of the Id1−/− mice, but lung collagen content was increased in the Id1−/− mice relative to wild-type 14 days after bleomycin treatment (Figure 2B). The expression of fibronectin and procollagen mRNA (assessed by real-time PCR) was also increased in the lungs of Id1−/− mice treated with bleomycin relative to control mice (Figure 2, C and D). Lungs were also harvested for α-SMA staining 1 week after bleomycin treatment. Lungs of mice lacking the gene for Id1 did not have noticeably increased α-SMA expression at baseline, and histologically were indistinguishable from those of wild-type mice. After bleomycin treatment, Id1−/− mice exhibited appreciably more parenchymal α-SMA staining than wild-type mice; representative images are shown in Figure 2E. Quantitation of α-SMA immunofluorescence for each group is shown in Figure 2F. In wild-type mice, there was a threefold induction over baseline, whereas in Id1−/− mice the α-SMA staining increased fivefold over baseline. Treatment of fibroblasts with TGF-β1 results in a characteristic increase in the expression of proteins involved in fibrosis, including matrix proteins, α-SMA, and plasminogen activator inhibitor-1 (PAI-1). To determine whether the presence or absence of Id1 would affect this characteristic response in vitro, fibroblasts from Id1−/− and control mice were compared. As expected, TGF-β1 stimulation led to an increase in expression of fibronectin, α-SMA, and PAI-1 within 48 hours. Fibroblasts lacking the gene for Id1 had an even greater response to TGF-β1 than did the wild-type fibroblasts (Figure 3A). This finding was confirmed in human lung fibroblasts using siRNA to inhibit the expression of Id1 (Figure 3, B–D). It should be mentioned that the level of α-SMA expression in Figure 3D is identical to the level of expression in the unstimulated lanes in Figure 3C; the same cellular protein was used to generate both figures. The apparently higher expression of α-SMA in Figure 3D is attributable to a longer film exposure time. This indicates that even in the absence of exogenous TGF-β1 stimulation, Id1 may be acting to maintain a low baseline level of α-SMA expression. Taken together, these in vitro observations are consistent with our in vivo finding that Id1 acts to suppress fibrosis. Id1 is best known for its ability to bind bHLH proteins and prevent their association with DNA. The DNA binding recognition sequence of bHLH proteins is the E-box (CANNTG); this element is present in the promoter regions of a number of TGF-β1-regulated genes, including PAI-1, α-SMA, Smad7, and collagen-1.14Kumar MS Hendrix JA Johnson AD Owens GK Smooth muscle alpha-actin gene requires two E-boxes for proper expression in vivo and is a target of class I basic helix-loop-helix proteins.Circ Res. 2003; 92: 840-847Crossref PubMed Scopus (66) Google Scholar, 16Kato M Zhang J Wang M Lanting L Yuan H Rossi JJ Natarajan R MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors.Proc Natl Acad Sci USA. 2007; 104: 3432-3437Crossref PubMed Scopus (615) Google Scholar, 17Allen RR Qi L Higgins PJ Upstream stimulatory factor regulates E box-dependent PAI-1 transcription in human epidermal keratinocytes.J Cell Physiol. 2005; 203: 156-165Crossref PubMed Scopus (34) Google Scholar, 18Stopa M Anhuf D Terstegen L Gatsios P Gressner AM Dooley S Participation of Smad2. Smad3, and Smad4 in transforming growth factor beta (TGF-beta)-induced activation of Smad7. The TGF-beta response element of the promoter requires functional Smad binding element and E-box sequences for transcriptional regulation.J Biol Chem. 2000; 275: 29308-29317Crossref PubMed Scopus (123) Google Scholar We postulated that Id1 might modulate the expression of these genes by a mechanism involving E-box binding. To test this hypothesis, we transfected Id1−/− and wild-type fibroblasts with control and E-box mutant α-SMA promoter constructs fused with a β-galactosidase reporter (kindly provided by Dr. Gary K. Owens). The α-SMA promoter contains two E-boxes at −214 bp (E1) and −252 bp (E2); the constructs tested were mutated at one or both of these sites. After transfection, fibroblasts were stimulated with TGF-β1, and β-galactosidase activity was assessed. As shown in Figure 4A, mutation of either or both E-box elements did not alter the enhanced promoter responsiveness in the Id1−/− fibroblasts, indicating that Id1 is likely acting through an E-box-independent mechanism. Because the effect of Id1 on α-SMA promoter activation did not require the presence of intact E-boxes and therefore might not require traditional bHLH binding partners, we sought novel Id1-interacting proteins by yeast two-hybrid screening. Of the 12 individual interacting proteins identified (from a total of 73 independent clones sequenced), 3 were known Id1-interacting proteins (HEB, E2–2, and E2A), 3 were unknown proteins, and the remainder were novel proteins not previously known to interact with Id1. Of these, the most promising candidate for a fibrosis-related molecule was Dril1. Interaction between Dril1 and Id1 was confirmed by co-immunoprecipitation (Figure 4D). Dril1 is a DNA-binding protein containing an A/T rich interaction domain (ARID). ARID domain proteins are members of a highly conserved family involved in chromatin remodeling and cell-fate determination. Very little is known about the role of Dril1 in humans, but it shares some similarities with Id1, including a helix-loop-helix structure.19Suzuki M Okuyama S Okamoto S Shirasuna K Nakajima T Hachiya T Nojima H Sekiya S Oda K A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer.Oncogene. 1998; 17: 853-865Crossref PubMed Scopus (37) Google Scholar Little is known about control of Dril1 expression, and it is not known whether TGF-β1 has any role in regulating its expression. To determine whether Dril1 is responsive to TGF-β1, human fibroblasts were treated with TGF-β1 and analyzed by Western blotting for expression of Dril1. As shown in Figure 4, C and D, a time- and dose-dependent increase in Dril1 expression occurs after TGF-β1 treatment, with maximal expression at the 6-hour time point. The protein for dose response was collected at the 6-hour time point. Densitometry of Western blots showing the time course of Dril1 response to TGF-β1 is shown in Figure 4E. Whole tissue homogenate of lungs explanted from patients with IPF and control lungs were analyzed by Western blotting for Dril1 expression. Expression of Dril1 was uniformly increased in IPF lungs as compared with controls (Figure 5A), suggesting that Dril1 may play a role in human disease. This finding was confirmed by Dril1 immunostaining of control and IPF lungs (Figure 5B). Co-staining of human IPF lung with antibodies against Dril1 and the fibroblast marker vimentin indicates that Dril1 is likely expressed in fibroblasts in IPF. Some of the co-stained cells have the typical morphology of a fibroblast (Figure 5C, left), whereas others have a more cuboidal morphology (Figure 5C, right). Given that we had identified Dril1 as an Id1-binding protein, we tested whether Id1 and Dril1 co-localize in fibroblasts in vitro in response to TGF-β1 stimulation. Human fibroblasts were stained for Id1 and Dril1 before and 2 hours after TGF-β1 treatment. This time point corresponds with declining levels of Id1 expression and rising levels of Dril1 expression, providing the highest levels of both proteins according to our Western blot kinetics. Expression of both proteins increased as expected after TGF-β1 treatment. Dril1 and Id1 both stained primarily in the nucleus, although cytoplasmic Id1 also increased after TGF-β1 stimulation (Figure 5D). Co-localization of the two proteins occurred mainly in the nucleus. Co-staining was also performed in the IPF lung to demonstrate co-localization in vivo. Figure 5E shows co-expression of Id1 and Dril1 in end-stage IPF/UIP lung. The areas of greatest co-localization (yellow) again occur in the nucleus. Because Id1 and Dril1 interact physically and co-localize in vitro and in vivo, we hypothesized that Id1 might inhibit Dril1 DNA binding in the same way that Id1 is known to inhibit DNA binding of classical bHLH family members. Dril1 is known to bind AT-rich regions of DNA, and using a published consensus sequence for Dril120Gregory SL Kortschak RD Kalionis B Saint R Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.Mol Cell Biol. 1996; 16: 792-799Crossref PubMed Scopus (136) Google Scholar (5′-AATAAATTAAGTTTAAAATATTTT-3′), we tested the effect of increasing intracellular Id1 levels on binding of Dril1 to DNA probe by electrophoretic mobility shift assay. Intracellular Id1 expression levels were manipulated using Id1 adenovirus and Id1 siRNA to increase or decrea
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