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 (). 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
). 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
) 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 [37
]), and that overexpressed YY1 enhances α-SMA gene expression. In contrast, using shRNA-mediated knock-down of YY1 expression in human lung fibroblasts (), 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
). 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
). 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
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
). 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
). It will be interesting in future studies to determine whether YY1 regulates fibroblast activation or differentiation in vivo
via epigenetic mechanisms.