It has long been reasoned that different clinical and histologic presentations of pulmonary fibrosis may share similar molecular mechanisms, but evidence supporting this notion has been lacking. Our detection of nuclear β-catenin staining in fibroblasts and alveolar epithelial cells in end-stage fibrotic lungs from patients with SSc-ILD () demonstrates that β-catenin may be activated in another form of pulmonary fibrosis. These findings corroborate previous observations in samples from patients with IPF (19
) and suggest that up-regulation of nuclear β-catenin may be a common feature of end-stage lung fibrosis. The observed increase in β-catenin nuclear staining in human lungs likely reflects bona fide
Wnt/β-catenin signaling because microarray data from SSc-affected tissues reveals alterations in multiple Wnts, their regulators, receptors, and targets (16
). Although Wnt/β-catenin signaling can be induced in a murine model of lung fibrosis using intratracheal bleomycin (17
), the temporal sequence and relative contributions of Wnt activation in epithelial cells, pulmonary interstitial cells, and progenitor cells are not fully established and are under active investigation by our lab. Ongoing studies using whole animal and cell type–specific knockout approaches are required to determine in which cell type β-catenin signaling casually contributes to lung fibrosis.
Since fibroblast proliferation and migration are thought to be critical steps in lung fibrogenesis (42
), we addressed the contribution of Wnt/β-catenin signaling to these phenotypes using cultures of normal human lung fibroblast and adenoviruses that can force activate (Ad-Wnt3a, Ad-S37A–β-catenin) or inhibit (Ad-ICAT) Wnt/β-catenin signaling. We found that activation of Wnt/β-catenin signaling enhances lung fibroblast proliferation in vitro
(Figures 5 and E2), which may contribute to the increase in fibroblast number and activity during fibrogenesis in vivo
). Although C-MYC and cyclinD1 are established β-catenin/TCF targets in epithelial cell types (46
), these genes were not obviously up-regulated in NHLFs (Figure E2), suggesting that other targets are responsible for fibroproliferation. Recently, Vugua and colleagues found that Wnt5a can increase fibroblast proliferation through a “noncanonical” or β-catenin/TCF-independent signaling mechanism (49
), indicating that canonical and noncanonical Wnts may contribute to fibroproliferation. We also found that Wnt/β-catenin signaling activation can enhance lung fibroblast motility and migration through a type 1 collagen matrix ( and data not shown), but the specific targets that promote these activities remain to be determined. For example, although MMP-1, -2, and -9 are known targets of β-catenin/TCF signaling in epithelial and immune cells (35
) and contribute to cell migrations in these cell types, these genes do not appear to be responsible for the enhanced motility observed our Wnt/β-catenin signaling activated lung fibroblasts (Figure E3). Gene expression analysis from Wnt3a-treated NIH 3T3 embryonic fibroblasts revealed an increase in genes associated with the cell cycle, motility, and adhesion (50
), consistent with the Wnt-activated phenotypes characterized in this study. Given that Wnt/β-catenin target genes are cell-type and cell-context dependent, defining the Wnt/β-catenin–regulated target genes in human lung fibroblasts requires similar unbiased microarray approaches.
The transdifferentiation of fibroblasts into myofibroblasts and consequent up-regulation of extracellular matrix components is typically viewed as a hallmark of fibrogenesis (2
). Since some of the classic markers of fibroblast “activation” and myofibroblast differentiation contain potential TCF-binding sites in their proximal promoter regions (51
), we reasoned that these markers would be co-regulated by β-catenin signaling. However, in striking contrast to the effects of the well established profibrotic factor TGF-β on these fibroblasts (, and and Figure E4), β-catenin signaling failed to increase mRNA expression of CTGF, COL1, α-SMA, and TGF-β upon either short-term (24 h) () or longer-term (6 d) () Wnt/β-catenin signaling activation. These data indicate that β-catenin signaling in normal human lung fibroblast cultures is not sufficient to activate many of the classic (TGF-β–activated) targets of myofibroblast differentiation. Consistent with these results, Chen and colleagues did not detect increased expression of a fibrotic gene cluster in their analyses of NIH 3T3 cells treated with recombinant Wnt3a (50
Two recent studies indicate that a systemic elevation of Wnt proteins or injury-induced reductions in a Wnt inhibitor can contribute to fibrosis in other tissues (7
). Specifically, age-related up-regulation of canonical Wnts can drive muscle stem cell differentiation along a fibroblastic, as opposed to a myogenic, lineage, resulting in fibrosis at the expense of muscle maintenance (7
). Moreover, an injury-induced model of kidney fibrosis was associated with decreased levels of sFRP4, a decoy receptor for Wnt, which correlated with increased levels of the signaling form of β-catenin as well as fibrotic markers such α-SMA (21
). Administration of recombinant sFRP4 reduced the levels of active β-catenin and α-SMA expression and the number of myofibroblasts. Since Wnt/β-catenin signaling appears insufficient to up-regulate the expression and number α-SMA–positive lung fibroblasts in culture (), β-catenin signaling may contribute to α-SMA expression and myofibroblast abundance in vivo
by promoting myofibroblast proliferation or by cell fate decisions that generate the myofibroblastic lineage.
Altogether, the results from our study demonstrate that sustained activation of Wnt/β-catenin signaling in normal adult lung fibroblasts, as might occur in SSc-associated ILD, can enhance activities associated with fibrosis, such as proliferation and motility. Whether these Wnt/β-catenin signaling–dependent fibroblast activities substantially contribute to disease progression needs to be determined using targeted knock-out models. However, given that Wnt/β-catenin signaling controls cell-fate decisions throughout development, often controlling the balance between progenitor cells and their descendants (53
), we speculate that cells serving as progenitors to fibroblasts and myofibroblasts may be a key target of Wnt/β-catenin signaling in pulmonary fibrosis. The identification of such fibroblast progenitors in lung and their response to Wnt/β-catenin signaling will be the subject of future investigations.