IPF/UIP is a progressive, fatal disorder that presents a major challenge for clinicians, as there is currently no effective treatment for this disease. The pathogenesis of IPF/UIP is not well understood, but one hallmark of the clinical course is its unresponsiveness to antiinflammatory therapy. In our recent study, we detected significant up-regulation of the BMP antagonist, gremlin, in patients with IPF/UIP (7
), and speculated that this might contribute to fibrosis by preventing antifibrotic BMP signaling. Many mouse models of pulmonary fibrosis have been developed for mechanistic and therapeutic studies, with bleomycin-induced fibrosis being the most commonly used model. We found no evidence in microarray data that would point to gremlin up-regulation or impaired BMP-signaling in the mouse bleomycin model of pulmonary fibrosis (data accessible at http://www.ncbi.nlm.nih.gov/geo/
; National Center for Biotechnology Information, Gene Expression Omnibus database [accession number GSE485
]). The crocidolite asbestos–induced model of fibrosis is progressive, and it can be considered as having similar temporal characteristics to human IPF/UIP lesions. We observed up-regulation of gremlin in asbestos-induced fibrosis in mice, suggesting that the mouse asbestos-induced fibrosis exhibits a similar gremlin response as human IPF/UIP. Notably, we found evidence that impaired BMP signaling was involved in promoting fibrosis, suggesting that gremlin overexpression may directly contribute to the pathogenesis of pulmonary fibrosis.
Immunohistochemical analyses of mouse lungs indicated that gremlin was mainly localized to the epithelial cells adjacent to fibroblast proliferative areas at 14 days after asbestos exposure. In contrast, gremlin was expressed mostly in the interstitium in human IPF lungs. Our previous results have shown very high gremlin levels in human lungs at advanced stages of the disease (patients that had undergone lung transplantation), suggesting that gremlin is a marker of advanced disease, possibly contributing to disease progression (7
). In a biopsy from a patient with advanced asbestos-induced fibrosis, the parenchymal fibroblasts exhibited gremlin positivity similar to that in the IPF lungs. The observed mouse lung histopathology suggests that enhanced gremlin expression may also contribute to early fibrogenesis. Characterization of early changes in the lung fibrogenesis in man is, however, challenging.
Gremlin can inhibit the actions of BMP-2 and -4 and, to some extent, also BMP-7. The levels of chordin and noggin, which can inhibit the very same BMPs, did not change, providing evidence for specificity for the gremlin induction after exposure to asbestos. The levels of noggin and chordin were similarly unchanged in human IPF (unpublished observations). The levels of P-Smad1/5/8, an indicator of BMP signaling, exhibited a dramatic decrease in asbestos-exposed mouse lungs. The expression of the BMP target gene, Id1, was down-regulated in the fibrotic mouse lungs, which is consistent with the known biological actions of gremlin. Accordingly, we detected down-regulation of Id1 expression in biopsies from patients with IPF, further emphasizing the similarities between human IPF and the model of asbestos-induced mouse lung fibrosis.
The role of TGF-β in fibrotic diseases is well known, and it is also an important regulator of fibroblast accumulation and matrix deposition in asbestos-induced pulmonary fibrosis. Recent studies have indicated that EMT is an ongoing process in the fibrotic lung in vivo
and a potential mechanism leading toward the accumulation of fibroblasts (35
). TGF-β–induced EMT can be reversed by BMP-7, and the signaling balance between BMPs and TGF-β seems to be crucial to evoke these phenotypic changes. We have observed that overexpression of gremlin can sensitize cultured epithelial cells to TGF-β–induced EMT (7
). Notably, we found that TGF-β is involved in the regulation of BMP antagonist expression, which will further promote a fibrosis phenotype in response to TGF-β. In cultured primary lung bronchial epithelial cells, we observed that blockade of TGF-β signaling inhibited gremlin mRNA induction by asbestos. TGF-β signaling activity was markedly increased in asbestos-treated cells in vitro
, which probably resulted in increased expression of gremlin, as well as decreased BMP-signaling. Previous studies have indicated that the release of active TGF-β by alveolar epithelial cells in vivo
can induce fibrosis (36
). The current studies provided evidence of increased TGF-β signaling, as measured by Smad2 phosphorylation, in epithelial cells of asbestos-exposed mouse lungs. The similar localization pattern of TGF-β activity and gremlin protein in mouse lungs indicates that TGF-β may play a role in regulating gremlin expression and BMP-signaling in vivo
as well. Interestingly, similar alterations in TGF-β/BMP signaling have recently been found in a hyperoxia mouse model of bronchopulmonary dysplasia (37
), a condition in which fibrosis is also a hallmark of the pathology.
The MEK/ERK signaling pathway is involved in mediating some of the profibrotic activities of TGF-β, including induction of CTGF and collagen expression (27
). We observed that blockade of the MEK/ERK cascade by specific MEK inhibitors could prevent asbestos-induced up-regulation of gremlin mRNA in cultured epithelial cells. Asbestos exposure is known to evoke induction of ERK1/2 phosphorylation through the epidermal growth factor receptor (39
), and thus to contribute to the expression of gremlin, as well as other TGF-β–regulated genes. In our cell culture models, asbestos exposure also induced ERK1/2 phosphorylation (data not shown). Gremlin has BMP-independent functions and, interestingly, it was recently suggested that cell surface binding of gremlin can induce ERK activation in endothelial cells (41
). Recent experimental data suggest that part of the profibrotic effects of TGF-β in mesenchymal cells are Smad independent and mediated by the c-Abl tyrosine kinase (42
). We find here that the involvement of the MEK/ERK pathway in BMP antagonist expression represents another mechanism that should be considered, when the inhibition of TGF-β–triggered profibrotic signals are evaluated for the treatment of IPF. In support of this hypothesis, Liu and colleagues (43
) have proposed that cAMP-induced down-regulation of ERK1/2 phosphorylation can reduce the profibrogenic effects of TGF-β in cardiac fibroblasts.
The in vivo
role of reduced BMP signaling in the development of fibrosis was assessed by treatment with BMP-7. We observed that BMP-7 treatment inhibited asbestos-induced fibrotic changes, when the treatment was started 7 days after asbestos exposure. Hydroxyproline levels, which reflect collagen deposition in the lung, were reduced by about 50%. A tendency toward a diminished neutrophilic cellular response was also observed in the BMP-7–treated mouse lungs, implying that inflammatory responses might also be targets of the BMP therapy. These results are in full agreement with the role of BMP-7 in reversing EMT and fibrosis in the models of kidney and liver injury (32
A common new mechanism related to fibrotic diseases (i.e., down-regulation of BMP signaling) is emerging from our studies, as well as from work by other groups. Up-regulation of gremlin expression has been reported in fibrotic diseases of the lung, kidney, and liver (7
). Endogenous BMP-7 appears to be involved in the regeneration of normal tissue after kidney injury (32
), and perhaps BMPs have a similar function in the lung. If BMP activity is blocked by overproduction of BMP antagonists, the development of fibrosis is enhanced. In experimental kidney injury models, the lack of BMP inhibitor, uterine sensitization–associated gene-1, or administration of recombinant BMP-7 can rescue the normal architecture of the kidneys (32
). Furthermore, mice lacking the BMP signaling enhancer kielin/chordin-like protein (KCP) are hypersensitive to developing renal interstitial fibrosis (47
). The close reciprocal regulation between TGF-β and BMP signaling pathways is further strengthened by the observation that, in addition to enhancing BMP signaling, KCP can suppress TGF-β signaling (48
). CTGF, which also has a chordin-like domain, has been reported to bind directly to BMP-4 and TGF-β and to regulate their activities in a similar fashion as KCP (49
). The current results indicate that the balance between TGF-β and BMP signaling activities is an important regulator for the development of fibrotic diseases. Novel therapeutic treatment strategies may be aimed at inhibiting TGF-β and/or enhancing BMP signaling activities.