Citation: Selman M, Pardo A, Kaminski N (2008) Idiopathic Pulmonary Fibrosis: Aberrant Recapitulation of Developmental Programs? PLoS Med 5(3): e62. https://doi.org/10.1371/journal.pmed.0050062
Published: March 4, 2008
Copyright: © 2008 Selman et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: MS and AP are supported by Universidad Nacional Autónoma de México Grant SDI.PTID.05.6; NK is supported by National Institute of Health grants HL073745, HL0793941, HL0894932, and a generous donation by the Simmons family. The funders had no role in the decision to submit the article or in its preparation.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BMP, bone morphogenetic protein; EMT, epithelial–mesenchymal transition; FAP-α, fibroblast activation protein-α; IPF, idiopathic pulmonary fibrosis; LEF-1, lymphoid enhancer factor-1; PTEN, phosphatase and tensin homologue deleted on chromosome 10; Shh, Sonic hedgehog; TGF-β, transforming growth factor-β
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal disease of uncertain etiology . It has been proposed that IPF likely results from an aberrant activation of alveolar epithelial cells after injury that provoke the migration, proliferation, and activation of mesenchymal cells with the formation of fibroblastic/myofibroblastic foci, leading to the exaggerated accumulation of extracellular matrix with the irreversible destruction of the lung parenchyma [2–4]. The molecular mechanisms that determine the persistent nature of IPF are poorly understood. While aberrant activation of developmental pathways that are usually suppressed in adult tissues is often noticed in cancer, it is only rarely observed in non-malignant diseases. In the following pages we present evidence from gene expression studies and animal models of disease that suggest that IPF is characterized by aberrant activation of developmental pathways (Box 1).
Box 1. Developmental Pathways Likely Involved in Idiopathic Pulmonary Fibrosis
The Wnt signaling pathway: A signaling pathway that is involved in embryogenesis. Elimination of Wnt causes development of wingless fruit flies, hence the name Wnt (Wingless type). The central player in the signaling cascade of the canonical Wnt pathway is β-catenin. Nuclear β-catenin interacts with transcription factors such as LEF-1/T cell–specific transcription factor to affect transcription. Most Wnts work through specific receptors (Frizzled family of receptors and LRP5/6 co-receptors) and affect degradation and nuclear localization of β-catenin. In adults, dysregulation of the Wnt pathway leads to a variety of abnormalities and degenerative diseases, including cancer and fibrosis.
TGF-β signaling: The TGF-β superfamily of growth factors that includes TGF-β, activins, and BMPs regulates a plethora of biological processes during embryonic development as well as in adult life. A common mechanism through Smad activation applies to signaling mediated by all TGF-beta superfamily members. However, TGF-β ligands signal preferentially through Smad-2 and -3, whereas BMP signaling is mediated through Smad-1, -5, and -8. TGF-β and BMPs have opposite effects in a number of processes. Importantly, TGF-β has a profibrotic effect and induces epithelial–mesenchymal transition, while several BMPs display the contrary effect.
PTEN/PI3 kinase/Akt signaling pathway: The development and function of many tissues that feature tubular networks, including the lung, are regulated by intricate programs of epithelial cell morphogenesis. PTEN controls epithelial morphogenesis, integrating cellular polarity with tissue architecture. In the adult life, PTEN is an important tumor suppressor, and its dysfunction has been associated with cancer susceptibility. Importantly, PTEN becomes activated during wound healing, promoting fibroblast apoptosis. Decreased PTEN expression is found in IPF fibroblasts, which may enhance their survival, causing active fibroblastic foci persistence.
Hedgehog signaling pathway: A pathway critical in development. The pathway is named after its main ligand hedgehog. Sonic hedgehog is a morphogen that can specify multiple cell identities as a function of its concentration. In the lungs, Shh participates in branching morphogenesis, controlling bud size and shape. Lung-specific overexpression of Shh results in severe increase of interstitial tissue. Strong Shh expression has been identified in reactive alveolar epithelial cells in IPF lungs.
Cross-talk pathways: Emerging evidence supports the theory that the cell response to extrinsic signals relies not only on the effect of a single pathway, but on the integration of numerous signals from a plethora of cross-talking pathways. In this context, TGF-β/Smad, Wnt/β-catenin, BMP, PTEN, and members of the hedgehog and other families of secreted factors pathways operate through complex interconnections and feedback loops.
Transcriptional Signatures of IPF Lungs Are Enriched with Developmental Genes
Microarray analysis identified an IPF-specific gene expression signature characterized by the up-regulation of genes indicative of an active tissue remodeling program, including extracellular matrix and a large number of myofibroblast/smooth muscle cell–associated and epithelial cell–related genes [3,5]. Recently, we have reanalyzed previously published datasets [3,5,6] using analytical approaches that allow global and unbiased mapping of the functional themes that characterize IPF lungs in comparison to controls or to other interstitial lung disease. The analyses revealed that IPF lungs were significantly enriched with genes associated with lung development . The up-regulated development-relevant genes included several members of transcription factor families such as the Sry-related high mobility group box and forkhead box, and genes related to the Wnt/β-catenin pathway [6,7]. (For complete datasets see http://www.dom.pitt.edu/paccm/Genomics/data.htm).
While transcription factors active in morphogenesis and differentiation of the embryonic lung may be transiently expressed during adult lung repair, as in naphthalene injury , they are only rarely expressed in the normal lung. In fact, their sustained expression is often thought of as a marker for malignant transformation.
- Idiopathic pulmonary fibrosis is a devastating lung disorder of unknown etiology that inexorably leads to death in a relatively short time because of the lack of any effective therapy.
- IPF lungs are enriched with genes associated with lung development, indicating that embryonic signaling pathways involved in epithelium/mesenchymal communication and epithelial cell plasticity may be aberrantly activated in this disease.
- Developmental genes include members of transcription factor families such as the Sry-related high mobility group–box and forkhead box, and genes related to the Wnt/β-catenin pathway.
- Epithelial-to-mesenchymal transition, a key process in embryogenesis, may contribute to the fibroblast expansion in IPF lungs. Diverse molecular networks in IPF lungs seem to constitute an environment that drives epithelial cells to express mesenchymal cell properties.
- A better understanding of the dysfunctional activation of embryological pathways in IPF may result in novel, more effective therapeutic strategies.
Epithelial Cell Plasticity
Epithelial cells are motile and can migrate away from their nearest neighbors . However, under normal conditions they do not detach and move away from the epithelial layer. This arrangement can be disturbed by a process known as epithelial–mesenchymal transition (EMT). In the process of EMT, epithelial cells lose many of their epithelial characteristics and obtain properties that are distinctive of mesenchymal cells . They become migratory, down-regulate the expression of cell adhesion molecules, primarily E-cadherin, lose their apical–basal polarity, and express mesenchymal molecules such as fibronectin and N-cadherin [11,12]. EMT is a key process in embryogenesis, where it leads to the formation of a migratory mesenchyme that progresses along the primitive streak and populates new areas of the embryo that will develop into mesoderm and endoderm .
EMT May Contribute to the Fibroblast Expansion in IPF
An EMT-like process has been reported in cancer progression and metastasis, and in fibrotic disorders [13–23]. Recently, EMT was also observed in lung fibrosis by two groups that noticed numerous cells co-expressing epithelial and mesenchymal markers (thyroid transcription factor-1/α-smooth muscle actin  or surfactant protein C/N-cadherin ) within the expanded interstitium in IPF lungs. Moreover, using a triple transgenic mouse reporter system, Kim et al. demonstrated that EMT plays an important role during lung fibrogenesis and may be more widespread than previously thought .
While the mechanisms underlying EMT in IPF are still unclear, many EMT-related genes such as transforming growth factor (TGF)-β3 [26,27], lymphoid enhancer factor-1 (LEF-1) , and Slug, a TGF-β target gene required for EMT, as in the developing chicken heart , are up-regulated in IPF lungs (Table 1).
Bone Morphogenetic Proteins/TGF-β Balance and EMT
Bone morphogenetic proteins (BMPs) and TGF-β belong to a superfamily of multifunctional cytokines that includes different isoforms with highly specific functions including wound healing, extracellular matrix remodeling, and the control of epithelial– mesenchymal interactions during embryogenesis [29,30]. Importantly, BMPs antagonize the effects of TGF-β regarding EMT and induce the inverse process of mesenchymal-to-epithelial transition . In tubular epithelial cells, BMP-7 reverses EMT by directly counteracting TGF-β-induced Smad-dependent cell signaling . In kidney fibrosis, this antagonism may lead to regeneration of injured tissues, suggesting that reversal of EMT may have therapeutic advantages and that fibrosis may be reversible . BMP-2 is decreased and BMP-4 is increased in IPF lungs, compared with controls (Table 1). More importantly, gremlin, the main BMP antagonist that modulates early limb outgrowth and patterning in the mouse embryo , is increased in IPF lungs . Gremlin is also found in human diabetic nephropathy, where it colocalizes with TGF-β . TGF-β induces gremlin expression in association with EMT in lung epithelial cells . Taken together, these data suggest that increased TGF-β expression, decreased BMP-2 expression, and active BMP inhibition by gremlin create an EMT-favoring environment in IPF lungs (Figure 1).
In IPF lungs, TGF-β3, gremlin, and LEF-1 are up-regulated, while BMP-2 is down-regulated. Dysregulation of TGF-β/BMP pathway induces a fibrotic phenotype. EMT, so important in development, is likely to represent one possible response of the alveolar epithelial cell facing sustained injury. Increased expression of BMPs may not only antagonize TGF-β-induced EMT, enhancing re-epithelialization, but also may cause the opposite process, mesenchymal-to-epithelial transition (MET).
The occurrence of EMT in the lung represents a dramatic shift in cellular phenotype and requires reversal of early embryonic programs. Unlike kidney development, where the tubular epithelium originates from cells that undergo mesenchymal-to-epithelial transition [35,36], in the lung the formation of the various cell types lining the proximal and distal airways occurs through the differentiation of the epithelial precursor cells . In other words, it may be more “natural” for kidney epithelial cells to undergo EMT than for lung epithelial cells. In fact, Snail activation is sufficient to induce EMT and kidney fibrosis in adult transgenic mice . Thus, EMT in IPF probably represents a dramatic reprogramming of epithelial cells .
The Wnt Signaling Pathway
Wnts comprise a large family of secreted glycoproteins that activate multiple distinct types of intracellular signaling pathways through canonical and noncanonical Wnt pathways . Wnt signaling regulates a wide range of developmental processes, and its aberrant activation can lead to disease [41,42]. Canonical Wnt signaling inhibits the phosphorylation and degradation of β-catenin, allowing its translocation into the nucleus and its interaction with the high mobility group domain–containing, DNA-binding proteins (including the previously mentioned LEF-1) to regulate target gene expression [40–42]. β-catenin influences epithelial cell differentiation in the lung, and is required for the normal differentiation of the bronchiolar and alveolar epithelium .
Several Wnt genes are expressed during lung development. Wnt7b-deficient mice exhibit impaired alveolar type I cell differentiation, have hypoplastic lungs, and die at birth of respiratory failure . Similarly, Wnt5a-deficient mice also die shortly after birth from respiratory failure, but in contrast to Wnt7a-deficient mice, they exhibit increased proliferation of lung epithelial and mesenchymal compartments [45,46]. Interestingly, Wnt5a-null embryos showed increased expression of Sonic hedgehog (Shh), suggesting that Wnt5a signaling is required for the normal down-regulation of Shh, and that in the absence of Wnt5a, Shh-induced mesenchymal proliferation continues in late gestation. As will be discussed, epithelial expression of Shh has also been found in IPF lungs [47,48].
Five Key Papers in the Field
Armanios et al., 2007  Mutations of genes encoding telomerase reverse transcriptase and telomerase RNA support the idea that pathways leading to telomere shortening are involved in the pathogenesis of familial idiopathic pulmonary fibrosis.
Selman et al., 2006  This was the first paper to compare the gene expression profile of different interstitial lung diseases, providing evidence that idiopathic pulmonary fibrosis is characterized by a distinct transcriptional signature.
Thiery and Sleeman, 2006  In this detailed review on epithelial cell plasticity, the authors dissect the molecular events during which epithelial cells are transformed into mesenchymal cells and vice versa.
Koli et al., 2006  Findings suggest that overexpression of the BMP inhibitor gremlin may play a role in the pathogenesis of IPF, and may function to enhance the fibrotic response by modulating BMP-4 signaling in the lung.
Willis et al., 2005  The authors demonstrate for the first time the epithelial–mesenchymal transition in idiopathic pulmonary fibrosis.
The Wnt Signaling Pathway in IPF
Using gene expression microarrays, we demonstrated up-regulation of several members of the Wnt signaling pathway in IPF lungs, compared either with normal lungs or other interstitial lung diseases [3,5] (Table 1). For example, WISP-1 and the secreted frizzled-related protein 2 are increased in IPF compared with hypersensitivity pneumonitis . Several other Wnt pathway-related genes are also overexpressed in IPF lungs compared to normal controls (Table 1; dataset used in ; Gene Expression Omnibus database serial accession number GSE2052). The overall balance of the Wnt pathway genes overexpressed in IPF lungs seems to favor activation of the canonic pathway (Figure 2). To date, there is only one study that directly demonstrated aberrant nuclear localization of β-catenin in bronchiolar/alveolar epithelial cells and in fibroblasts from the fibroblastic foci in IPF lungs . Activation of β-catenin in epithelial cells is also indirectly corroborated by the overexpression of downstream genes such as MMP-7 and osteopontin [5,6], and may also be related to EMT.
The figure was generated using Ingenuity Pathways Analysis (Ingenuity Systems, http://www.ingenuity.com/). Increased genes are in increasing shades of red, decreased are in increasing shades of green. Combined red and green is when different members are changed in different directions.
Taken together, these data suggest a significant role for the Wnt pathway in IPF.
Fibroblast Expansion in IPF: Mirroring Aggressive Fibromatosis?
A key process in the development of IPF is the formation of the fibroblastic focus. It has been suggested that these foci represent discrete isolated foci of fibroblasts/myofibroblasts. However, using three-dimensional reconstruction of the IPF lungs, other studies have suggested that fibroblast foci are the leading edge of a complex reticulum that is highly interconnected extending from the pleura into the underlying parenchyma .
It is increasingly apparent that mesenchymal cells in fibroblastic foci in IPF exhibit a variety of abnormalities compared to normal lung fibroblasts or fibroblasts from other lung diseases. Some of these abnormalities are related to Wnt pathway signaling or to up-regulation of genes present in exaggerated fibroplasias. β-catenin stabilization (as observed in IPF) has been reported in hyperplastic cutaneous wounds, in keloid scars, and in aggressive fibromatosis, among other disorders [51–53]. Furthermore, transgenic mice that express stabilized β-catenin in mesenchymal cells develop aggressive fibromatosis suggesting a common role for β-catenin in abnormal fibrotic responses . These findings raise the possibility that unchecked activation of a process important in normal wound healing causes abnormal fibroproliferative processes.
Aggressive fibromatosis is a soft tissue tumor composed of a clonal population of mesenchymal, spindle-shaped cells. It is locally invasive, but rarely metastasizes. Microarray analysis identified four genes uniquely overexpressed in this disorder: ADAM12, WISP-1, SOX-11, and fibroblast activation protein-alpha (FAP-a) . WISP-1 and several members of the SOX family are up-regulated in IPF . FAP-α is selectively expressed by a subset of fibroblasts in areas of ongoing tissue injury in IPF lungs, but not in normal lung tissue or in tissue with centriacinar emphysema . FAP-α is a type II transmembrane serine protease expressed at sites of tissue remodeling in embryonic development [56,57]. FAP-α is highly expressed on reactive stromal fibroblasts in over 90% of human epithelial cancers, in healing wounds, and in sarcomas, but is not detected in fibroblasts of benign epithelial tumors or normal adult tissues [57,58].
An additional analogy between IPF and aggressive fibromatosis is the recent evidence that suggests involvement of pathways leading to telomere shortening in the pathogenesis of both disorders [59–61]. Telomeres are noncoding DNA sequences at the end of eukaryotic chromosomes that maintain chromosomal integrity and prevent replication of defective genes . When normal cells reach a critical telomere length, they exit the cell cycle and undergo senescence. The putative relationship between telomerase and human disease is demonstrated in two opposite situations. On one side of the spectrum, most human cancers are characterized by the expression of telomerase, which helps to maintain telomere length and enhance indefinite cell proliferation . On the other side of the spectrum, in pulmonary fibrosis and aggressive fibromatosis, telomerase activity seems to be diminished, with consequent premature telomere shortening. Two recent reports demonstrated that mutations in the genes encoding telomerase components are detected in patients with familial IPF, and occasionally in patients with sporadic IPF [59,60]. According to these studies, pulmonary fibrosis may be at least partially related to a telomerase deficiency and short dysfunctional telomeres, which after DNA damage leads to cell death or cell-cycle arrest. Interestingly, cigarette smoking, a strong environmental risk factor for IPF, causes telomere shortening in a dose-dependent manner . Since alveolar epithelial cells play a critical role in the pathogenesis of IPF, and telomerase expression is generally restricted to cells with the capacity to proliferate, it has been speculated that fibrotic response in patients with short telomeres is provoked by a loss of alveolar cells , which may be enhanced by exposure to cigarette smoke.
Naturally, there are many differences between aggressive fibromatosis and the fibroblastic foci of IPF, primarily the clonality of cells; however, some of the pathways that are activated in the former may also participate in the latter.
Phosphatase and Tensin Homologue Deleted on Chromosome 10
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a phosphatidylinositol phosphate phosphatase that is frequently deleted or inactivated in human cancers . PTEN is critical in development, and PTEN-null embryos die early during embryogenesis [66,67].
Significant to our discussion, a critical role for PTEN in the orchestrated death and removal of myofibroblasts has been suggested. In response to collagen matrix contraction, PTEN triggers fibroblast apoptosis by leading to decreased PI3 kinase activity and Akt phosphorylation . Myofibroblasts from IPF fibroblastic foci undergo decreased apoptosis compared to myofibroblasts from fibromyxoid lesions of cryptogenic organizing pneumonia, a potentially reversible lung disorder [69,70]. Impaired apoptosis may be at least partially associated with the decreased expression of PTEN observed in IPF lungs . Additionally, since PTEN suppresses fibroblast differentiation to myofibroblasts , its reduced expression in IPF fibroblastic foci may have a dual deleterious effect, inducing myofibroblast differentiation and increasing survival.
Importantly, the PTEN/PI3 kinase/Akt signaling pathway is a critical regulator of the interconnection between the TGF-β/Smad, Wnt/β-catenin, and BMP pathways [72–75].
Sonic (Shh), Indian (Ihh), and Desert (Dhh) are the hedgehog family members in mammals. Shh is expressed in the developing lung epithelium, and its primary receptor, Patched-1 (Ptc), is found in mesenchymal cells. In vitro and in vivo studies have shown that Shh increases the proliferation of lung mesenchymal cells, up-regulating the expression of smooth muscle actin and myosin .
In developing mice, Shh produced by the epithelium stimulates mesenchymal cell proliferation and differentiation, and importantly, its overexpression causes an aberrant increase in the ratio of interstitial mesenchyme to epithelial tubules [76,77].
In this context, a pattern of strong Shh expression has been identified in reactive alveolar epithelial cells as well as in epithelial cells lining the honeycomb cysts in IPF lungs [47,48], while microarrays indicated up-regulation of its primary receptor (Table 1), suggesting that the Shh pathway is activated on in this disease.
Identification and targeting of these abnormal mediators/pathways will eventually allow the development of therapeutic agents to control and hopefully achieve regression of IPF remodeling. Strategies designed to target the fibrogenic action of TGF-β with agents blocking its signaling pathway, such as the BMP-7, have led to repair of severely damaged renal tubular epithelial cells and to improvement of renal function and survival in mice with nephrotoxic serum nephritis . Along the same line of thought, hepatocyte growth factor, which among other functions inhibits TGF-β-induced EMT, may also be a useful therapeutic approach. It was recently shown that in vivo hepatocyte growth factor gene transfer reduced bleomycin-induced pulmonary fibrosis, improving alveolar epithelial repair, and importantly, decreasing TGF-β1 expression .
Aggressive fibromatosis: A disease characterized by the formation of tumors consisting of well-differentiated fibroblasts.
Bone morphogenetic proteins: A family of proteins that were originally described by their ability to cause bone and cartilage formation. Most of them belong to the TGF-β superfamily, and are implicated in various stages of embryonic development.
Epithelial–mesenchymal transition: The process in which an epithelial cell acquires characteristics often attributed to mesenchymal cells. It has been implicated in embryogenesis, cancer, and tissue fibrosis.
Gene expression microarrays: Methods that allow the parallel measurement of the expression of multiple transcripts even of complete genomes, usually by hybridization.
Idiopathic pulmonary fibrosis: A disease characterized by progressive scarring of the lungs, without any recognizable cause.
Myofibroblast: a fibroblast that has muscle cell–like properties such as contractility. These cells are important in wound healing and in fibrosis. In IPF, the cells form collections that are called myofibroblast foci.
Telomerase: An enzyme that adds DNA sequence repeats to the 3′ end of DNA in telomeric regions at the end of chromosomes. Telomerase mutations are associated with multiple inherited diseases, while abnormal telomerase activation has been implicated in cancer.
Many pathways that play an essential role during embryological development are inactivated later in life, although some of them may be transiently expressed during adult repair. Aberrant activation of these pathways during adult homeostasis leads to pathological events resulting in cancer, but may also be associated with the development of idiopathic pulmonary fibrosis. Dysfunctional activation of embryological pathways regularly repressed in the adult life may explain the persistent nature of the disease. Although some progress into unraveling the pathogenic mechanisms involved in IPF has been made, many open questions remain, and virtually no effective treatment is currently available. Designing and implementing interventions that target these embryological pathways may be required to develop novel anti-IPF therapies and to significantly improve the outcome of IPF patients.
- 1. Gross TJ, Hunninghake GW (2001) Idiopathic pulmonary fibrosis. N Engl J Med 345: 517–525.
- 2. Selman M, King TE, Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Ann Intern Med 134: 136–151.
- 3. Selman M, Pardo A, Barrera L, Estrada A, Watson SR, et al. (2006) Gene expression profiles distinguish idiopathic pulmonary fibrosis from hypersensitivity pneumonitis. Am J Respir Crit Care Med 173: 188–198.
- 4. Thannickal VJ, Toews GB, White ES, Lynch JP 3rd, Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annu Rev Med 55: 395–417.
- 5. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, et al. (2002) Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A 99: 6292–6297.
- 6. Pardo A, Gibson K, Cisneros J, Richards TJ, Yang Y, et al. (2005) Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. PLoS Med 2: e251.
- 7. Studer SM, Kaminski N (2007) Towards systems biology of human pulmonary fibrosis. Proc Am Thorac Soc 4: 85–91.
- 8. Park KS, Wells JM, Zorn AM, Wert SE, Laubach VE, et al. (2006) Transdifferentiation of ciliated cells during repair of the respiratory epithelium. Am J Respir Cell Mol Biol 34: 151–157.
- 9. Schock F, Perrimon N (2002) Molecular mechanisms of epithelial morphogenesis. Annu Rev Cell Dev Biol 18: 463–493.
- 10. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7: 131–142.
- 11. Moustakas A, Heldin CH (2007) Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression. Cancer Sci 98: 1512–1520.
- 12. Zavadil J, Bottinger EP (2005) TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24: 5764–5774.
- 13. Huber MA, Kraut N, Beug H (2005) Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 17: 548–558.
- 14. Christiansen JJ, Rajasekaran AK (2006) Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res 66: 8319–8326.
- 15. Lee JM, Dedhar S, Kalluri R, Thompson EW (2006) The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 172: 973–981.
- 16. Willis BC, Borok Z (2007) TGF-ß-induced EMT: mechanisms and implications for fibrotic lung disease. Am J Physiol Lung Cell Mol Physiol 293: L525–L534.
- 17. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, et al. (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110: 341–350.
- 18. Chaffer CL, Brennan JP, Slavin JL, Blick T, Thompson EW, et al. (2006) Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis: role of fibroblast growth factor receptor-2. Cancer Res 66: 11271–11278.
- 19. Zeisberg M, Yang C, Martino M, Duncan MB, Rieder F, et al. (2007) Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J Biol Chem 282: 23337–23347.
- 20. Alonso SR, Tracey L, Ortiz P, Pérez-Gómez B, Palacios J, et al. (2007) A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis. Cancer Res 67: 3450–3460.
- 21. Horikawa T, Yang J, Kondo S, Yoshizaki T, Joab I, et al. (2007) Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma. Cancer Res 67: 1970–1978.
- 22. Vasko V, Espinosa AV, Scouten W, He H, Auer H, et al. (2007) Gene expression and functional evidence of epithelial-to-mesenchymal transition in papillary thyroid carcinoma invasion. Proc Natl Acad Sci U S A 104: 2803–2808.
- 23. Lange-Sperandio B, Trautmann A, Eickelberg O, Jayachandran A, Oberle S, et al. (2007) Leukocytes induce epithelial to mesenchymal transition after unilateral ureteral obstruction in neonatal mice. Am J Pathol 171: 861–871.
- 24. Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, et al. (2005) Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1: potential role in idiopathic pulmonary fibrosis. Am J Pathol 166: 1321–1332.
- 25. Kim KK, Kugler MC, Wolters PJ, Robillard L, Galvez MG, et al. (2006) Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix. Proc Natl Acad Sci U S A 103: 13180–13185.
- 26. Nawshad A, Hay ED (2003) TGFbeta3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development. J Cell Biol 163: 1291–1301.
- 27. Romano LA, Runyan RB (2000) Slug is an essential target of TGFbeta2 signaling in the developing chicken heart. Dev Biol 223: 91–102.
- 28. LaGamba D, Nawshad A, Hay ED (2005) Microarray analysis of gene expression during epithelial-mesenchymal transformation. Dev Dyn 234: 132–142.
- 29. Lin SJ, Lerch TF, Cook RW, Jardetzky TS, Woodruff TK (2006) The structural basis of TGF-beta, bone morphogenetic protein, and activin ligand binding. Reproduction 132: 179–190.
- 30. Wahab NA, Mason RM (2006) A critical look at growth factors and epithelial-to-mesenchymal transition in the adult kidney. Interrelationships between growth factors that regulate EMT in the adult kidney. Nephron Exp Nephrol 104: e129–e134.
- 31. Zeisberg M, Shah AA, Kalluri R (2005) Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney. Biol Chem 280: 8094–8100.
- 32. Khokha MK, Hsu D, Brunet LJ, Dionne MS, Harland RM (2003) Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nat Genet 34: 303–307.
- 33. Koli K, Myllarniemi M, Vuorinen K, Salmenkivi K, Ryynanen MJ, et al. (2006) Bone morphogenetic protein-4 inhibitor gremlin is overexpressed in idiopathic pulmonary fibrosis. Am J Pathol 169: 61–71.
- 34. Dolan V, Murphy M, Sadlier D, Lappin D, Doran P, et al. (2005) Expression of gremlin, a bone morphogenetic protein antagonist, in human diabetic nephropathy. Am J Kidney Dis 45: 1034–1039.
- 35. Davies JA, Perera AD, Walker CL (1999) Mechanisms of epithelial development and neoplasia in the metanephric kidney. Int J Dev Biol 43: 473–478.
- 36. Romio L, Fry AM, Winyard PJ, Malcolm S, Woolf AS, et al. (2004) OFD1 is a centrosomal/basal body protein expressed during mesenchymal-epithelial transition in human nephrogenesis. J Am Soc Nephrol 15: 2556–2568.
- 37. Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD, et al. (2000) The molecular basis of lung morphogenesis. Mech Dev 92: 55–81.
- 38. Boutet A, De Frutos CA, Maxwell PH, Mayol MJ, Romero J, et al. (2006) Snail activation disrupts tissue homeostasis and induces fibrosis in the adult kidney. EMBO J 25: 5603–5613.
- 39. Selman M, Pardo A (2006) Role of epithelial cells in idiopathic pulmonary fibrosis: from innocent targets to serial killers. Proc Am Thorac Soc 3: 364–372.
- 40. Karner C, Wharton KA, Carroll TJ (2006) Apical-basal polarity, Wnt signaling and vertebrate organogenesis. Semin Cell Dev Biol 17: 214–222.
- 41. Cadigan KM, Liu YI (2006) Wnt signaling: complexity at the surface. J Cell Sci 119: 395–402.
- 42. Veeman MT, Axelrod JD, Moon RT (2003) A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev Cell 5: 367–377.
- 43. Mucenski ML, Nation JM, Thitoff AR, Besnard V, Xu Y, et al. (2005) Beta-catenin regulates differentiation of respiratory epithelial cells in vivo. Am J Physiol Lung Cell Mol Physiol 289: L971–L979.
- 44. Shu W, Jiang YQ, Lu MM, Morrisey EE (2002) Wnt7b regulates mesenchymal proliferation vascular development in the lung. Development 129: 4831–4842.
- 45. Shannon JM, Hyatt BA (2004) Epithelial-mesenchymal interactions in the developing lung. Annu Rev Physiol 66: 625–645.
- 46. Li C, Xiao J, Hormi K, Borok Z, Minoo P (2002) Wnt5a participates in distal lung morphogenesis. Dev Biol 248: 68–81.
- 47. Stewart GA, Hoyne GF, Ahmad SA, Jarman E, Wallace WA, et al. (2003) Expression of the developmental Sonic hedgehog (Shh) signalling pathway is up-regulated in chronic lung fibrosis and the Shh receptor patched 1 is present in circulating T lymphocytes. J Pathol 199: 488–495.
- 48. Coon DR, Roberts DJ, Loscertales M, Kradin R (2006) Differential epithelial expression of SHH and FOXF1 in usual and nonspecific interstitial pneumonia. Exp Mol Pathol 80: 119–123.
- 49. Chilosi M, Poletti V, Zamo A, Lestani M, Montagna L, et al. (2003) Aberrant Wnt/beta-catenin pathway activation in idiopathic pulmonary fibrosis. Am J Pathol 162: 1495–1502.
- 50. Cool CD, Groshong SD, Rai PR, Henson PM, Stewart JS, et al. (2006) Fibroblast foci are not discrete sites of lung injury or repair: the fibroblast reticulum. Am J Respir Crit Care Med 174: 654–658.
- 51. Cheon S, Poon R, Yu C, Khoury M, Shenker R, et al. (2005) Prolonged beta-catenin stabilization and tcf-dependent transcriptional activation in hyperplastic cutaneous wounds. Lab Invest 85: 416–425.
- 52. Cheon SS, Cheah AY, Turley S, Nadesan P, Poon R, et al. (2002) Beta-catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds. Proc Natl Acad Sci U S A 99: 6973–6978.
- 53. Sato M (2006) Upregulation of the Wnt/beta-catenin pathway induced by transforming growth factor-beta in hypertrophic scars and keloids. Acta Derm Venereol 86: 300–307.
- 54. Skubitz KM, Skubitz AP (2004) Gene expression in aggressive fibromatosis. J Lab Clin Med. 2004 143: 89–98.
- 55. Acharya PS, Zukas A, Chandan V, Katzenstein AL, Pure E (2006) Fibroblast activation protein: a serine protease expressed at the remodeling interface in idiopathic pulmonary fibrosis. Hum Pathol 37: 352–360.
- 56. Niedermeyer J, Garin-Chesa P, Kriz M, Hilberg F, Mueller E, et al. (2001) Expression of the fibroblast activation protein during mouse embryo development. Int J Dev Biol 45: 445–447.
- 57. Park JE, Lenter MC, Zimmermann RN, Garin-Chesa P, Old LJ, et al. (1999) Fibroblast activation protein, a dual specificity serine protease expressed in reactive human tumor stromal fibroblasts. J Biol Chem 274: 36505–36512.
- 58. Aertgeerts K, Levin I, Shi L, Snell GP, Jennings A, et al. (2005) Structural and kinetic analysis of the substrate specificity of human fibroblast activation protein alpha. J Biol Chem 280: 19441–19444.
- 59. Armanios MY, Chen JJ, Cogan JD, Alder JK, Ingersoll RG, et al. (2007) Telomerase mutations in families with idiopathic pulmonary fibrosis. N Engl J Med 356: 1317–1326.
- 60. Tsakiri KD, Cronkhite JT, Kuan PJ, Xing C, Raghu G, et al. (2007) Adult-onset pulmonary fibrosis caused by mutations in telomerase. Proc Natl Acad Sci U S A 104: 7552–7557.
- 61. Matsuo T, Hiyama E, Sugita T, Shimose S, Kubo T, et al. (2007) Telomere length and telomerase activity in extra-abdominal desmoid tumors. Anticancer Res 27: 411–415.
- 62. Phatak P, Burger AM (2007) Telomerase and its potential for therapeutic intervention. Br J Pharmacol 152: 1003–1011.
- 63. Holt SE, Shay JW, Wright WE (1996) Refining the telomere-telomerase hypothesis of aging and cancer. Nat Biotechnol 14: 1734–1741.
- 64. Morlá M, Busquets X, Pons J, Sauleda J, MacNee W, et al. (2006) Telomere shortening in smokers with and without COPD. Eur Respir J 27: 525–528.
- 65. Gericke A, Munson M, Ross AH (2006) Regulation of the PTEN phosphatase. Gene 374: 1–9.
- 66. Di Cristofano A, Pesce B, Cordon-Cardo C, Pandolfi PP (1998) Pten is essential for embryonic development and tumour suppression. Nat Genet 19: 348–355.
- 67. Stiles B, Groszer M, Wang S, Jiao J, Wu H (2004) PTEN less means more. Dev Biol 273: 175–184.
- 68. Nho RS, Xia H, Diebold D, Kahm J, Kleidon J, et al. (2006) PTEN regulates fibroblast elimination during collagen matrix contraction. J Biol Chem 281: 33291–33301.
- 69. Thannickal VJ, Horowitz JC (2006) Evolving concepts of apoptosis in idiopathic pulmonary fibrosis. Proc Am Thorac Soc 3: 350–356.
- 70. Lappi-Blanco E, Soini Y, Paakko P (1999) Apoptotic activity is increased in the newly formed fibromyxoid connective tissue in bronchiolitis obliterans organizing pneumonia. Lung 177: 367–376.
- 71. White ES, Atrasz RG, Hu B, Phan SH, Stambolic V, et al. (2006) Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10). Am J Respir Crit Care Med 173: 112–121.
- 72. Hjelmeland AB, Hjelmeland MD, Shi Q, Hart JL, Bigner DD, et al. (2005) Loss of phosphatase and tensin homologue increases transforming growth factor beta-mediated invasion with enhanced SMAD3 transcriptional activity. Cancer Res 65: 11276–11281.
- 73. Mahimainathan L, Das F, Venkatesan B, Choudhury GG (2006) Mesangial cell hypertrophy by high glucose is mediated by downregulation of the tumor suppressor PTEN. Diabetes 55: 2115–2125.
- 74. Tian Q, He XC, Hood L, Li L (2005) Bridging the BMP and Wnt pathways by PI3 kinase/Akt and 14-3-3zeta. Cell Cycle 4: 215–216.
- 75. He XC, Zhang J, Tong WG, Tawfik O, Ross J, et al. (2004) BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-b-catenin signaling. Nat Genet 36: 1117–1121.
- 76. Weaver M, Batts L, Hogan BL (2003) Tissue interactions pattern the mesenchyme of the embryonic mouse lung. Dev Biol 258: 169–184.
- 77. Bellusci S, Furuta Y, Rush MG, Henderson R, Winnier G, et al. (1997) Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development 124: 53–63.
- 78. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, et al. (2003) BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 9: 964–968.
- 79. Gazdhar A, Fachinger P, van Leer C, Pierog J, Gugger M, et al. (2007) Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis. Am J Physiol Lung Cell Mol Physiol 292: L529–L536.