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Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.
Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.
Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.
Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.
Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.
Lung fibrosis including human idiopathic pulmonary fibrosis (IPF) is caused by accumulation of myofibroblasts in injured lung. Myofibroblasts are derived from three sources: (1) proliferation and differentiation of fibroblasts, (2) epithelial mesenchymal transition, and (3) fibrocytes from circulation. However, the mechanism of accumulation of myofibroblasts in injured lung is unknown.
We found that a transcription factor, Yin Yang 1, plays an important role in fibroblast differentiation in lung fibrosis. Decreasing YY1 expression can inhibit differentiation of fibroblasts mediated by nuclear factor-κB and can decrease lung fibrosis in silica- and bleomycin-treated mice.
Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic interstitial lung disease associated with high mortality (median survival of newly diagnosed patients is ~ 3 yr) and a uniformly poor prognosis (1). IPF is the pathologic hallmark of interstitial lung diseases and is characterized by increased deposition of extracellular matrix proteins including collagen. This lethal lung disorder presents major clinical challenges, because effective therapeutic agents for reversing lung fibrosis have not yet been discovered (2). One current hypothesis is that IPF represents a chronic injury and repair response to specific environmental insults, such as silica or asbestos. However, the precise molecular mechanisms underlying persistent fibroblast activation remain poorly understood.
After inhalation of profibrotic stimuli (e.g., asbestos, silica, or bleomycin), alveolar macrophages and neutrophils produce cytokines including transforming growth factor (TGF)-β1 and tumor necrosis factor (TNF)-α that contribute to lung inflammation and fibrosis by different mechanisms (3, 4). TGF-β plays an essential role in wound healing and matrix molecule deposition. It induces myofibroblast differentiation and alveolar remodeling in vivo (5), and overexpression of this potent profibrotic mediator leads to progressive fibrosis in mice, with minimal inflammation (6). TNF-α also contributes to lung fibrosis (7), and its effects may be mediated through activation of other growth factors. For example, TNF-α–deficient mice are protected against bleomycin-induced lung inflammation via reduced apoptosis of inflammatory cells (8). Inhibition of TNF-αwith infliximab may stabilize the progression of pulmonary fibrosis associated with collagen vascular disease (9).
Myofibroblasts are derived from epithelial to mesenchymal transition (EMT) (10), circulating fibrocytes (11), or resident fibroblasts, in response to lung injury or chronic inflammation induced by stimuli, such as silica and bleomycin, and are now recognized as major effector cells in pulmonary fibrosis. They are characterized by the expression of α-smooth muscle actin (α-SMA), enhanced proliferation, and synthesis of extracellular matrix proteins (12), and are thought to be derived from fibroblasts via the activity of TGF-β and other stimuli (13). In pulmonary fibrosis, myofibroblasts acquire resistance to apoptosis, which may account for the increased number of these cells present in fibroblastic foci (14). These myofibroblasts accumulate in the injured lung and block alveolar gas exchange. Preventing the formation of myofibroblasts would provide protection against lung fibrosis, and this could be achieved by inhibition of α-SMA expression in lung fibroblasts (15).
Yin Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor that can either activate or repress gene transcription, and plays an important role in cellular proliferation, differentiation, and apoptosis. Growing evidence indicates that YY1 contributes to the pathogenesis of cancer and inflammation (16). TNF-α–induced YY1 represses Fas expression, providing a mechanism whereby YY1 contributes to TNF-α–induced cell survival (17). In fibroblasts, TNF-α induces YY1 in a nuclear factor (NF)-κB–dependent manner (18), supporting a link between the NF-κB pathway and YY1 expression (17). YY1 can bind to and activate the type I and type II collagen and α-SMA gene promoters in fibroblasts (19–21). Although these reports suggest that YY1 may play a role in fibrotic responses, very little is known about the expression or function of YY1 in fibrotic conditions in vivo.
Embryonic fibroblasts from YY1-deficient mice demonstrated reduced proliferation in vitro in proportion to the levels of YY1 protein expression. This finding indicates that YY1 controls fibroblast proliferation in a gene dosage-dependent manner (22). When designing the present study, we considered that YY1 overexpression is associated with hyperproliferation and increased differentiation of cells, and that YY1 can regulate collagen gene expression (23). Therefore, we investigated the role of YY1 in fibrotic lung diseases using tissues and cells from human subjects. Here we report that YY1 expression is markedly increased in lung tissue obtained from human subjects with IPF and in two mouse models of lung fibrosis. Using loss-of-function strategies, we show that YY1 plays an important and previously underappreciated role in lung fibrosis by regulating fibroblast activation and differentiation. Some of the results of these studies have been previously reported in the form of an abstract (24).
The WI-38 and LL97A human fibroblast lines were purchased from ATCC (Manassas, VA). 293FT cells were obtained from Invitrogen (Carlsbad, CA). YY1 heterozygotes (YY1+/−) and conditional knockout YY1 (YY1f/f) mice were obtained from Y. Shi (Harvard University, Cambridge, MA). All animals were grouped according to age (6–12 wk) or weight (20–35 g). YY1+/− mice were on a C57BL/6 background (≥ 6 generations). YY1f/f mice were on a C57BL/6 background (four generations). These mice received 200–300 μg of silica or three units of bleomycin per kg of body weight by intratracheal injection. All animals were housed under specific pathogen-free conditions at the National Institutes of Health (NIH) in an American Association for the Accreditation of Laboratory Animal Care-approved facility. The University of Rochester Medical Center animal care and use committee approved all experimental procedures.
Lungs were fixed with 10% buffered formalin and embedded in paraffin. Hematoxylin and eosin and Masson's trichrome stains were used for analysis of pathologic changes. Immunohistochemical staining for α-SMA and YY1 tissues was performed according to a previous paper (25). The severity of fibrosis was evaluated in stained sections by an individual who was blinded to the genotypes of the mice. For area analysis of fibrotic changes, a quantitative fibrotic scale (Ashcroft scale) was used (26) based on hematoxylin and eosin staining. A numerical fibrotic score (Ashcroft scale) was obtained as follows: the severity of the fibrotic changes in each lung section was given as the mean score from the observed microscopic fields. Twenty fields within each lung section were observed at a magnification of ×10, and each field was assessed individually for fibrotic severity and allotted a score from 0 (normal) to 8 (total fibrosis). The severity score for each field was averaged and presented as the average for each lung section. To avoid bias, all histologic specimens were evaluated in a blinded fashion. Each specimen was scored independently by three observers and the mean of their individual scores was taken as the fibrotic score.
Cells or lung tissues were solubilized in lysis buffer. Western blot, mRNA extract, and quantitative polymerase chain reaction (PCR) were performed according to previous protocols (27). Data were analyzed using NIH Image J software or by the 2−Δ(ΔCT) method and normalized to the expression of the GAPDH housekeeping gene. The sequences of the primers are listed in Table E1 (see online supplement).
Plasmids containing the wild-type YY1 promoter (or a YY1 promoter with a mutant NF-κB binding site) driving a luciferase reporter gene were kindly presented by Dr. Denis C. Guttridge (Ohio State University). Transiently transfection protocol was the same as a previous paper (28). We transiently transfected the reporter constructs into WI38 cells, which were then stimulated with TGF-β for 24 hours or with TNF-α for 6 hours. Using the same procedure, we cotransfected a p65 (NF-κB) plasmid with wild-type or mutated YY1 promoter constructs. Construction of α-SMA luciferase reporter is shown in the online supplement.
LL97A cells, derived from human fibroblasts obtained from patients with IPF, were transduced with YY1 shRNA, and were plated in four-well chamber slides. Cells were supplemented with Dulbecco's modified Eagle medium and 10% fetal bovine serum at 37°C for 4 days. Anti–α-SMA antibody (1A4; DAKO Cytomation, Carpinteria, CA), anti-collagen (LF-67; Larry W. Fisher, Ph.D., Matrix Biochemistry Section, Craniofacial and Skeletal Diseases Branch of NIH), and anti-YY1 antibody (H-414; Santa Cruz, Santa Cruz, CA) were stained according to a previous paper (25). The cells were examined with a Zeiss fluorescence microscope. Package and transduction of lentiviral shRNA and adenoviral-cre are listed in the online supplement.
Quantitative PCR, YY1 promoter reporter assay, and histologic score of lung fibrosis were analyzed for statistical significance by using the paired Student t test using Microsoft Excel software. A P value of less than 0.05 was considered statistically significant.
Lung fibrosis results from proliferation and differentiation of fibroblasts in response to profibrotic cytokines, such as TGF-β and TNF-α. To test the possibility that these cytokines induce YY1 expression, we grew a human lung fibroblast line (WI38) in vitro and exposed cells to TGF-β for different times. We found that YY1 expression was up-regulated by TGF-β, as demonstrated by Western blot (Figure 1A) and quantitative PCR (Figure 1B). Using the same protocol as TGF-β, we determined that YY1 protein and mRNA expression are also increased by TNF-α stimulation in WI38 cells (Figures 1C and 1D). Taken together, these data indicate that YY1 is up-regulated by the profibrotic cytokines TGF-β and TNF-α in human lung fibroblasts. Next we investigated the mechanisms by which YY1 is up-regulated by these cytokines.
Recent studies have found that TNF-α can up-regulate YY1 expression through NF-κB activation in tumor cells (17). To determine whether TGF-β induces YY1 expression through NF-κB in lung fibroblasts, first we evaluated if NF-κB was activated by TGF-β. Fibroblasts (WI38) were stimulated with TGF-β after being serum starved for 24 hours. NF-κB and phospho I-κB expression were determined by immunoblot. We found that NF-κB and phospho I-κB were up-regulated by TGF-β in fibroblasts, beginning at 4 hours (Figure 2A). NF-κB can up-regulate YY1 expression by directly binding to the YY1 promoter in cancer cells (18). Second, we used an I-κB inhibitor (Bay11–7085), which blocks I-κB degradation and quenches NF-κB activity. We found that incubation with Bay11–7085 abolished TGF-β–induced YY1 expression (Figure 2B). The quantitative data of Figure 2B is shown in Figure E5A. Next, we studied TGF-β–induced YY1 promoter activity with a wild-type YY1 promoter reporter construct and a construct in which a putative NF-κB binding site was mutated (18). Transfected cells were stimulated with TGF-β, and reporter gene activity was analyzed by luminometry. Interestingly, YY1 promoter activity was significantly increased in lung fibroblasts in response to both TGF-β stimulation and cotransfection with the NF-κB subunit p65 (RelA; Figure 2C). We found that disruption of a potential NF-κB binding site on the YY1 promoter inhibited the enhancing effect of NF-κB, suggesting that this involves direct binding to the YY1 promoter (Figure 2D). Together, these findings suggest that the pathway leading to YY1 up-regulation by TGF-β is mediated through NF-κB in lung fibroblasts.
We next wanted to determine whether YY1 is induced in human IPF and in mouse models of lung fibrosis. To address these possibilities, we used immunohistochemistry (IHC) and immunoblot assays separately performed with anti-YY1 and anti–fibroblast-specific protein (FSP) 1 antibodies. In lung sections from humans with IPF, we conducted IHC staining with anti-YY1 or anti-FSP1 antibodies, and found that YY1 was overexpressed together with the fibroblast marker FSP1 (Figure 3A). Double staining of the IPF lung in Figure 3A with anti-YY1 and anti-FSP1 antibodies is shown in Figure E3C and confirms YY1 overexpression in FSP1-positive fibroblasts. Furthermore, lung tissues from patients with IPF were homogenized and YY1 protein and mRNA were determined by Western blot and quantitative PCR, respectively. Both YY1 protein (Figure 3B) and mRNA (Figure 3C) from lungs of patients with IPF were increased. These data show that YY1 is increased in lung tissue and lung fibroblasts from human subjects with IPF.
Silica and bleomycin are widely used to induce lung fibrosis in mice, and act via different mechanisms. We next investigated whether YY1 expression was also up-regulated in these fibrotic models. Wild-type mice were exposed to silica, bleomycin, or saline control using standard approaches (see Methods), and lungs were excised and analyzed 21 days later using IHC. Interestingly, YY1 overexpression was localized to FSP1- and α-SMA–positive cells in both silica- (Figure 3D) and bleomycin-induced lung fibrosis (Figure 3G). Magnification ×10 of the lung in Figures 3D and 3G is shown as supplementary data (Figures E2A and E2B). Immunoblot analysis confirmed that total YY1 expression was up-regulated in both silica (Figure 3E) and bleomycin mouse models (Figure 3H). Using quantitative PCR, we also found that YY1 transcript levels were increased both in silica- (Figure 3F) and bleomycin-exposed mice (Figure 3I). Taken together, these findings indicate that lung fibrotic responses are associated with enhanced expression of YY1 mRNA and protein in both human subjects and two widely used mouse models.
We further explored the mechanism by which YY1 may regulate fibroblast differentiation and activation. α-SMA is a hallmark of myofibroblasts and is well-accepted as a marker of pulmonary fibrosis. Although YY1 can bind to the α-SMA promoter, it is unknown whether it up-regulates or down-regulates α-SMA promoter activity (21, 29). To investigate the ability of YY1 to regulate α-SMA promoter activity, we created a murine 740-bp construct driving the firefly luciferase gene (α-SMA–Luc). This construct was transfected with or without a YY1 expression vector into WI38 cells. Using gel shift assays or CHIP assays, we confirmed that nuclear YY1 can directly bind to the α-SMA promoter in vitro and in vivo (Figures E1A–E1C). Overexpressed YY1 alone significantly increased α-SMA reporter activity, and this effect was further enhanced by TGF-β stimulation (Figure 4A). The enhancing effects of YY1 on α-SMA–Luc activity were further augmented by cotransfection with NF-κB. This finding suggests that TGF-β induces α-SMA promoter activity mediated by NF-κB and YY1. Therefore, in addition to type I collagen (19), the α-SMA gene represents another potential YY1 target in lung fibroblasts.
We then tested whether reducing the expression of YY1 would inhibit α-SMA or collagen expression in human lung fibroblasts. A fibroblast cell line (LL97A) was derived from a human subject with IPF and infected using different YY1 lentiviral shRNA constructs. After transduction with two controls and two YY1 lentiviral shRNA constructs, LL97A cells were allowed to recover for 3 days and then selected in puromycin to enrich for transduced cells. Figure 4B shows that decreased YY1 expression inhibited both α-SMA and collagen expression in human lung fibroblasts, as determined by immunofluorescence staining. These data suggest that knockdown of YY1 expression in lung fibroblasts inhibits fibroblast activation. Taken together, these data provide support for the idea that α-SMA is a previously unrecognized and important target of YY1 in lung fibroblasts and suggest that YY1 may be a potential therapeutic target for fibrotic lung disease through decreasing α-SMA and collagen.
We next investigated whether decreasing YY1 expression is able to protect against lung fibrosis in mice. To test this hypothesis, we exposed partially YY1-deficient mice (YY1+/−), or littermate controls on the C57BL/6 background, to silica particles by intratracheal injection. Three weeks later, mice were killed and the lungs stained using IHC, and connective tissue was analyzed using Masson trichrome straining. We found that lung fibrosis was markedly decreased in YY1+/− compared with littermate mice. Expression of the myofibroblast marker α-SMA, collagen, FSP1-positive cells, and inflammation were markedly greater in wild-type mice compared with YY1+/− littermates (Figure 5A). Magnification ×10 of the lung in Figure 5A is shown in Figure E2C. Lung fibrosis scores from heterozygote YY1+/− and wild-type mice were evaluated by the Ashcroft scale. Whole lung was scored using a double-blind method. Using this approach, lung fibrosis was significantly diminished in YY1+/− mice compared with wild-type littermates (Figure 5B). In addition, collagen I protein expression was greatly reduced in YY1+/− mice (Figure E5C). This suggests that lung fibrosis was significantly inhibited in YY1+/− mice, and that decreased YY1 expression protects against lung fibrosis.
To determine if inducible deletion of YY1 could be used in a therapeutic regimen, we used floxed-YY1 mice (YY1f/f) in which lox P sites were stably integrated into the introns between YY1 exon 1. Both wild-type and YY1f/f mice were exposed to silica particles using our standard protocol, except that both groups then received an intratracheal adenoviral vector expressing cre recombinase on Day 12, and lungs were harvested 21 days later. This strategy likely targets multiple lung cell types including lung epithelial cells, which also express YY1 (Figure E3A), and could potentially differentiate into fibroblasts in mice exposed to silica (30). Figure 6 shows that when compared with wild-type mice that received silica followed by adenoviral-cre, the induction of lung fibrosis, and α-SMA, collagen, FSP1, and YY1 expression, were all markedly attenuated in YY1f/f mice (Figure 6A). Arrows indicate that YY1 was decreased in epithelial cells of YY1f/f mice. Magnification ×10 of Figure 6A is shown in Figure E2D. Lung fibrosis was attenuated in YY1f/f mice as determined by Ashcroft scored using a double-blind method (Figure 6B). YY1 mRNA and protein expression were similarly reduced in YY1f/f compared with wild-type mice exposed to adenovirus-cre, as expected (Figures 6C and 6D). These suggest that partially decreasing YY1 expression by adeno-cre in type 1 epithelial cells (Figure E3A) and potentially other cell types can protect against lung fibrosis by reducing accumulation of fibroblasts.
YY1 is a versatile transcription factor with pivotal roles in normal biologic processes, such as development, differentiation, replication, and cell proliferation, and is increasingly linked with pathologic conditions. For example, YY1 overexpression is strongly linked with cancer development and progression (31). In this study, we demonstrate that YY1 is overexpressed by fibrotic cytokines TGF-β and TNF-α in lung fibroblasts, and that this involved activation of NF-κB. In addition, we found that YY1 expression is up-regulated in or near FSP1- and α-SMA–positive cells in human IPF and two murine models of lung fibrosis. In addition YY1 expression in lung fibroblasts isolated from mice instilled with bleomycin was dramatically increased, compared with fibroblasts from mice instilled with phosphate-buffered saline (Figure E5B). Furthermore, we found that YY1 can directly up-regulate α-SMA expression in pulmonary fibroblasts leading to fibroblast differentiation. Using gene-targeted mice and a conditional deletion strategy, we found that reducing YY1 expression protects against lung fibrosis in vivo. These novel data will be considered in turn.
Pulmonary fibrosis results from activation and differentiation of fibroblasts, which overexpress extracellular matrix proteins and α-SMA in the injured lung. In this study, we found that fibrotic cytokines induced YY1 expression, and that YY1 mRNA and protein were up-regulated in mouse model of lung fibrosis, and in lungs from human subjects with IPF (Figure 3A). We focused on lung fibroblasts because lung fibroblasts ultimately control deposition of extracellular matrix and their differentiation into myofibroblasts characterizes the injury and repair response. Furthermore, we found that both α-SMA and collagen expression were reduced in YY1-deficient mice. We did not find that YY1 was overexpressed in type II alveolar epithelial cells (Figure E3B), and macrophages (Figure E3C, middle panel) in the human IPF and in the lung of bleomycin-instilled mouse (Figure E3C, top panel). We concluded that YY1 expression by fibrotic cytokines seems to contribute to differentiation of fibroblasts into α-SMA expressing myofibroblasts in lung fibrosis. The precise molecular mechanisms for this effect requires further study, including the potential for YY1 directly to regulate genes involved in fibroblast activation.
In addition to TNF-α and TGF-β, other cytokines and stimuli may induce YY1 expression in lung fibroblasts. Silica and asbestos particles were recently shown to activate the Nalp3 containing inflammasome and induce caspase-1–driven processing of mature IL-1β (32), which we also found up-regulated fibroblast YY1 expression in vitro (Figure E4). Additionally, we found that cytokine-dependent induction of YY1 required the transcription factor NF-κB, in keeping with prior studies in other cell types (17, 33). This implied that blockade of the NF-κB pathway might protect against lung fibrosis by decreasing YY1 expression. Interestingly, a recent report demonstrated that Bay11–7085 decreased silica-induced inflammation and collagen deposition (34), although potential effects of NF-κB inhibition on YY1 expression were not investigated in that report. Thus, in addition to promoting the expression of cytokines, chemokines, and other inflammatory mediators, these data suggest that by inducing YY1 expression, NF-κB promotes the expression of genes involved in tissue repair responses. In other models, YY1 expression mediated by NF-κB can down-regulate Fas and death receptor 5 (17, 35) expression, suggesting a mechanism by which lung myofibroblasts may be protected from apoptosis. Smads are also activated by TGF-β during lung fibrosis, and interestingly Smad3 can bind to different domains of YY1 (36). It will be interesting in future studies to investigate interactions between YY1 and other transcription factors in lung fibrosis and fibrosis in other organs. Because TGF-β and TNF-α are universally implicated in injury and repair and remodeling, we predict that YY1 will play a role in fibroblast differentiation and activation in diverse tissues, and that the paradigms we established in this report should be generalizable.
In this study, we demonstrate that YY1 directly binds to the proximal α-SMA promoter (in keeping with a recent report ), and that overexpressed YY1 enhances α-SMA gene expression. In contrast, using shRNA-mediated knock-down of YY1 expression in human lung fibroblasts (Figure 4C), we found that loss of YY1 markedly reduces α-SMA expression. Because YY1 can directly bind to the promoter regions of both the collagen (19) and α-SMA genes and is required for their expression, we concluded that YY1 directly regulates two crucial genes (α-SMA and collagen) involved in fibroblast activation and myofibroblast differentiation.
Lung myofibroblasts derive from multiple sources including resident cells, circulating fibrocytes (11), or via EMT (10, 38). Our observation that intratracheally administered adeno-cre attenuates fibrotic reactions supports a potential role for YY1 in EMT, because our strategy likely targeted epithelial cells. This approach can be potentially used for a therapeutic strategy. Future studies are needed in this area. In addition to regulating myofibroblast gene expression, YY1 may also confer resistance to apoptosis (39), which is known to be induced during lung fibrotic responses after exposure to silica (40), TGF-β (41), and TNF-α (17, 42). Thus, decreasing YY1 expression may attenuate fibrotic responses by multiple mechanisms, in keeping with a growing role for YY1 in cell survival in response to injury (43, 44).
In addition to direct effects in fibroblasts, decreasing YY1 expression may inhibit early inflammation during injury and repair responses, and may help to protect against lung fibrosis by this mechanism. In support of this idea, we detected lower levels of inflammatory cytokines in bronchoalveolar lavage fluids from silica-challenged YY1+/− compared with wild-type controls (Figure E6). Because complete YY1 deficiency in mice results in early embryonic lethality, heterozygous mice (YY1+/−) were used in this report. YY1 heterozygous mice express about 50% of normal YY1 levels (22, 45). Thus, even partial reduction of YY1 levels can protect against lung fibrosis in mice. Future studies should expose the possibility that YY1 may be a therapeutic target in lung fibrosis, where new therapies are urgently needed.
YY1 is generally considered a constitutively expressed nuclear phosphoprotein, but there is growing evidence that YY1 expression can be dynamically regulated, including by compounds in therapeutic use. For example, YY1 and NF-κB expression are diminished in B cells by rituximab, which is an anti-CD20 antibody used for treating B-cell non-Hodgkin lymphoma (39). Nitric oxide can also inhibit YY1 expression in human tumor cells (46) and in lung fibroblasts, which may explain some of the therapeutic benefits of inhaled nitric oxide in lung fibrosis (47). In addition to regulating gene expression directly at the transcriptional level, YY1 is intimately involved in chromatin remodeling and epigenetic imprinting. For example, YY1 associates with histone H3–specific methyltransferases to its target promoters (48), and together with Polycomb group family members generates high levels of H3-K27 trimethylation that are needed to maintain a repressed chromatin state (49–51). It will be interesting in future studies to determine whether YY1 regulates fibroblast activation or differentiation in vivo via epigenetic mechanisms.
The authors thank Yang Shi (Harvard University) for kindly providing YY1+/− and YY1f/f mice; Denis C. Guttridge (Ohio State University) for providing YY1 promoter driven luciferase reporter constructs; and Weihua Wang (University of Rochester) for some of the fluorescent stainings in the online supplement.
Supported by a grant from American Lung Associate (RG-10327-N to J.G.); National Institutes of Health (R01 HL073952 to S.N.G. and HL75432 and HL095402 to P.J.S.); and the University of Rochester Medical School Department of Medicine.
Conception and design: J.G. and P.S.; analysis and interpretation: X.L., H.X., L.R., and M.W.; drafting the manuscript for important intellectual content: J.G., P.S., and S.G.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201002-0232OC on December 17, 2010
Author Disclosure: X.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.J.S. has received grants from the NIH and consultancy fees from Intermune Pharmaceuticals. H.X. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.N.G. has received grants from the NIH; he and his institution have received consultancy fees from Merck; and he holds stock in Pfizer. J.G. and his institution have received grants from ALA; he has received travel support from the University of Rochester.