cinogenesis, inflammation, and tissue fibrosis (3). To date, inves- tigations ... and galectin 3 (Gal3) are involved in the assembly of collagen fibrils and that fibrotic ...
Editorials Let It Be microRNAs Impact Interstitial Lung Disease Pandit and colleagues reported a role for the microRNA (miRNA) let-7d in idiopathic pulmonary fibrosis (IPF) (1). The authors noted that expression of several miRNAs, including let-7d, is markedly attenuated in IPF lungs compared with normal lungs. Using cell culture and mouse model approaches, they demonstrate that let-7d is down-regulated in response to transforming growth factor b (TGF-b), which in turn disinhibits expression of a number of mesenchymal markers in epithelial cells and results in alveolar septal thickening and increased collagen deposition. These findings suggest that miRNAs are directly involved in the pathogenesis of interstitial lung diseases and may represent important new therapeutic targets. miRNAs are short, noncoding RNAs that regulate expression of a group of gene products by complimentary binding to common miRNA-recognition elements in the 39-UTR of target mRNAs to either block translation or reduce stability, leading to degradation. Recently, it has been shown that mammalian miRNAs predominantly act to decrease target mRNA levels (2). Because of their powerful effect on cellular phenotype, miRNAs have been an increasing focus in studies on the regulation of gene expression in multiple fields, including organ development, carcinogenesis, inflammation, and tissue fibrosis (3). To date, investigations of miRNAs in lung diseases have focused primarily on lung cancer, lung development, and inflammatory diseases, including allergic airway inflammation (4). Although miRNAs have been implicated in fibrosis in other organs, prior studies in lung fibrosis are limited to studies defining roles for miR-21 (5) and miR-155 (6) in pulmonary fibroblasts, in contrast to work by Pandit and colleagues that suggests a prominent role for let-7d in alveolar epithelial cells (1). Pandit and colleagues used a miRNA microarray approach to interrogate lung tissue from patients with IPF and normal control subjects, noting that several miRNAs were differentially expressed in IPF compared with controls. Microarray approaches provide a powerful mechanism to gain large-scale information on pathways that may be operational and potentially aberrant in disease states (7). However, with many diseases, including IPF, patients present late in the disease process, and it can be difficult to determine whether such array findings represent primary pathogenic mechanisms or secondary ramifications of long-standing disease processes. Because of this issue, it is important to follow array findings with detailed experiments to uncover relevant pathobiology, as these authors have elegantly done. Choosing let-7d from array data for further analysis was logical since HMGA2 (high-mobility group A protein), one of the targets for the let-7 family, is involved in mediating downstream effects of TGF-b (8, 9), a principal pro-fibrotic cytokine (10). In vitro studies revealed that TGF-b diminished let-7d expression through smad3 binding in the let-7d promoter. Furthermore, HMGA2 and other mesenchymal markers had an
Supported by NIH NHBLI HL85317 (to T.S.B.), HL92870 (to T.S.B.), HL85406 (to W.E.L.), the Department of Veterans Affairs (to T.S.B. and W.E.L.), and CIHR (to J.G.). Am J Respir Crit Care Med Vol 183. pp 1–7, 2011 Internet address: www.atsjournals.org
expression pattern inversely related to levels of let-7d, producing a phenotype consistent with epithelial-to-mesenchymal transition (EMT). When let-7d was inhibited in vivo through use of an antagomir (11), increased collagen deposition in the alveolar septum was noted along with increased expression of fibroblast markers S100A4 and a–smooth muscle actin (a-SMA) as well as in vivo evidence of co-localization of epithelial and mesenchymal markers. These results extend previous studies that demonstrated that TGF-b–induced EMT of NMuMG mouse mammary epithelial cells was dependent on HMGA2 (9) and that let-7 family members have a prominent role in EMT in pancreatic cancer cells (12). Together, these findings provide a strong argument for a role for let-7d in EMT and may help to explain the mechanism by which TGF-b regulates this process (13, 14). While EMT is now recognized in fibrotic conditions in a number of organs, our overall understanding on how EMT impacts fibrosis remains limited by the technologies available to evaluate it. First, EMT is commonly defined by downregulation of epithelial markers and up-regulation of mesenchymal markers, accompanied by a change to morphology consistent with a fibroblast. Especially in vivo, this evaluation is greatly dependent on expression of cell-specific markers. Using lung epithelial cell fate mapping strategies has the potential to improve this detection over simple immunohistochemistry techniques, but only a few manuscripts have used such an approach to define EMT in the lungs in vivo (14–17). What is needed are functional studies in vivo to demonstrate the contributions of EMT-derived fibroblasts to repair and remodeling, but to date these have been elusive. Investigators have targeted EMT-related pathways to determine the effects in lung and other organs, but all of these pathways (including TGF-b) have the potential to alter other processes that impact fibrosis, including epithelial cell apoptosis and proliferation, fibroblast activation, and pro-fibrotic cytokine production. Since most pathways involved in fibrosis are critical to normal wound healing, it is possible that EMT may serve a beneficial process to wound repair, and attenuating EMT may be detrimental. The use of approaches to modulate expression of let-7d or other miRNAs that regulate epithelial cell phenotype could provide better tools to investigate the role and importance of EMT in lung fibrosis. Fibrosis is characterized by the excess deposition of extracellular matrix (ECM) components, which is the end result of an imbalance of metabolism of the ECM molecule. Collagens are the predominant ECM proteins, while other ECM proteins such as fibronectins, elastin, and fibrillins also have an important role in the development of fibrosis (3). We know little about the turnover of ECM components in normal tissue, and virtually nothing in fibrotic tissue, especially IPF tissue. However, we know that molecules including decorin, fibrillin, and galectin 3 (Gal3) are involved in the assembly of collagen fibrils and that fibrotic tissue contains disordered fibril assembly. Interestingly, let-7d has been shown to directly regulate one of these molecules, Gal3; another molecule involved in crosslinking and correctly stabilizing collagen fibrils, tyrosine hydroxylase (18), is affected by Gal3 through its effects on CREB
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protein (19), thus modulating the ECM quality and possible susceptibility to normal turnover. In addition, Gal3 has a direct effect on macrophage function and accumulation in fibrosis (20, 21), a cell with prominent roles in the process of fibrogenesis (22). Let-7d and other miRNAs, including miR-21 and miR-29, could be affecting much more than the process of EMT in fibrosis. It will be of interest to see if let-7d plays a similar role in other organ fibroses, such as liver, or whether there is tissue specificity in the function of specific miRNAs. A significant strength of this manuscript is the use of modalities for targeting miRNAs that could be applied in future therapies for IPF. Antagomirs are chemically modified, cholesterolconjugated oligonucleotides that have single-strand RNA analogs complementary to specific miRNAs. In 2005, Krutzfeldt and coworkers (11) reported silencing of miRNAs with such antagomirs, specifically observing that an antagomir against miR122 affected hepatic cholesterol biosynthesis genes, leading to reduced plasma cholesterol measurements in mice and raising hopes that a similar approach could lead to medical interventions in humans. Given the current findings, one must wonder if manipulation of miRNA levels by overexpression of let-7d or using an antagomir approach to target other up-regulated miRNAs could one day provide a therapeutic benefit in IPF. Author Disclosure: W.E.L. has received sponsored grants from American Thoracic Society ($50,001–$100,000), American Lung Association ($50,001–$100,000), National Institutes of Health (NIH)/National Heart, Lung, and Blood Institute (NHLBI) (over $100,000), Parker B. Francis Fellowship Program ($50,001– $100,000), and Vanderbilt University Grants (over $100,000). T.S.B. has received sponsored grants from NIH/NHLBI (over $100,000). J.G. has received consultancy fees from GlaxoSmithKline (GSK) ($5,001–$10,000). He has received industry-sponsored grants from from GSK ($50,001–$100,000). He has received sponsored grants from NIH and CIHR (each over $100,000).
William E. Lawson, M.D. Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine Vanderbilt University School of Medicine Nashville, Tennessee and Department of Veterans Affairs Medical Center Nashville, Tennessee Timothy S. Blackwell, M.D. Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine Department of Cell and Developmental Biology Department of Cancer Biology Vanderbilt University School of Medicine Nashville, Tennessee and Department of Veterans Affairs Medical Center Nashville, Tennessee Jack Gauldie, Ph.D. Department of Pathology and Molecular Medicine McMaster University Hamilton, Ontario, Canada
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DOI: 10.1164/rccm.201009-1482ED
