Pulmonary fibrosis is a frequent complication and a major cause of death in systemic sclerosis (SSc; scleroderma); however, the pathogenesis is unclear and no safe and effective therapy exists (1–3). The pathology of pulmonary fibrosis in SSc includes features of dysregulated and abnormal repair, fibroproliferation, deposition of collagen, connective tissue growth factor (CTGF), and other extracellular matrix (ECM) proteins (1–4). Extensive deposition of ECM impairs the ability of lung epithelial cells to regenerate, which results in accelerated lung tissue damage and respiratory failure (4).
Hepatocyte growth factor (HGF) was initially identified and cloned as a mitogen for mature hepatocytes (5). Later studies revealed that HGF is widely expressed in many different organs, including the lungs, and has multiple biologic activities (6, 7). In the lungs, HGF acts as a potent regenerative and cytoprotective factor during organogenesis or following acute injury (8–11). Numerous studies have implicated HGF as an endogenous antifibrotic factor, ameliorating fibrotic lesions and preserving organ function in a wide variety of experimental animal models (12–15). However, the mechanisms by which HGF exerts its antifibrotic effects are not yet fully understood.
In vitro studies have shown that HGF specifically counteracts many profibrotic actions of transforming growth factor β (TGFβ), suggesting that a balance between HGF and TGFβ may play a decisive role in the pathogenesis of fibrosis (16, 17). Using electroporation-mediated gene transfer in the bleomycin-injured lung, Gazdhar et al showed that HGF decreased TGFβ1 levels and increased alveolar epithelial cell proliferation and survival (18). Mizuno and colleagues identified HGF as a key ligand to elicit myofibroblast apoptosis and ECM degradation in bleomycin-treated murine lungs (13). Wu et al reported that HGF prevented and ameliorated the symptoms of dermal sclerosis in a mouse model of SSc (19). Several recent studies have characterized antifibrotic effects of HGF on collagen, matrix metalloproteinase 1 (MMP-1), and CTGF in SSc skin fibroblasts (20, 21), but the mechanisms underlying its antifibrotic effects in human lung fibroblasts remain to be clarified.
Increased levels of serum HGF have been observed in scleroderma patients (22). Recently, we showed that HGF protein expression was significantly up-regulated in bronchoalveolar lavage (BAL) fluid and plasma from white but not from African American SSc patients as compared with healthy controls (23). We also showed that the antifibrotic activity of HGF was diminished in lung fibroblasts isolated from African Americans due to limitations in c-Met receptor phosphorylation, whereas in lung fibroblasts from white SSc patients HGF readily down-regulated type I collagen and CTGF accumulation (23). The present study was undertaken to investigate the signaling pathways underlying HGF's effects on type I collagen and CTGF in lung fibroblasts isolated from white SSc patients.
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We recently showed that the antifibrotic activity of HGF is significantly reduced in lung fibroblasts isolated from African Americans (23), a population known to have significantly higher mortality rates and a higher prevalence of the more severe diffuse cutaneous SSc compared with white patients (27, 28). The results were consistent for different cell lines in SSc lung fibroblasts or in normal lung fibroblasts stimulated with TGFβ (23). HGF considerably inhibited type I collagen and CTGF accumulation in lung fibroblasts from white patients. However, HGF had no effect on either collagen or CTGF expression in lung fibroblasts isolated from African Americans; we showed that this was a result of a deficiency of HGF-receptor phosphorylation (23). The present study was undertaken, therefore, to investigate signaling pathways underlying HGF's antifibrotic effects in lung fibroblasts isolated from white SSc patients.
In a previous study (23), we showed that HGF protein expression was significantly up-regulated in BAL fluid and plasma from white but not from African American SSc patients as compared with healthy controls. HGF levels in serum from scleroderma patients ranged from 2.5 to 6.0 ng/ml. In the study of HGF effects in human lung fibroblasts, HGF was administered in a range of doses from 5 to 500 ng/ml. HGF at a dose of 5 ng/ml had clear antifibrotic effects; however, not all of the findings were statistically significant. Therefore, we used 50 ng/ml of HGF to demonstrate the profound antifibrotic effects of HGF in human lung fibroblasts.
Although earlier studies implicated HGF in embryo development and in promoting tissue regeneration after acute injury, evidence is now emerging that HGF is also an intrinsic antifibrotic factor that plays a critical role in preventing tissue fibrosis in various animal models (8–15). Over the past several years, progress has been made in identifying the cellular targets of HGF and in unraveling the molecular mechanisms that underlie its action in tissue fibrosis (13, 14, 16, 17, 21). The biologic effects of HGF are mediated by a membrane-spanning c-Met tyrosine kinase receptor (29). After binding of HGF, the c-Met receptor undergoes autophosphorylation at tyrosine residues in its cytoplasmic domain, recruits a group of downstream molecules or adapter proteins such as Grb2, GAB-1, STAT-3, Shc, SH2-containing inositol phosphatase, and Src tyrosine kinase to its multidocking sites, and initiates a cascade of signal transduction events that eventually leads to specific cellular responses (7). The binding of Grb2 to the guanine nucleotide–releasing factor human son of seven less homolog 1 establishes a connection between receptor tyrosine kinase and Ras signaling (30). The depletion of Grb2 by siRNA disrupts the downstream signaling of c-Met and completely prevents HGF-induced inhibition of CTGF and collagen, while MAPK and MMP inhibitors significantly reduce the antifibrotic effect of HGF.
Previously we showed that the basal levels of phospho–ERK-1/2 are elevated in SSc lung fibroblasts (25). Here we show that HGF further induces ERK-1/2 phosphorylation in a time- and dose-dependent manner and that this persists up to 72 hours. Basal levels of nonphosphorylated ERK-1/2 in SSc lung fibroblasts were not affected by HGF stimulation. Grb2 siRNA prevented HGF-induced ERK-1/2 phosphorylation, indicating that HGF phosphorylates ERK-1/2 via the c-Met/Grb2/Ras pathway. A similar pathway of HGF-induced ERK-1/2 phosphorylation has been reported for several other cell lines (for review, see ref. 31).
It is known that basal expression and activity of MMP-1 are reduced in SSc fibroblasts compared with normal fibroblasts (26, 32). It has been reported that HGF induces expression and activity of MMP-1 in bleomycin-induced pulmonary fibrosis (13) and kidney fibrosis (33). Recently it has been shown that HGF enhanced MMP-1 production in SSc skin fibroblasts (20, 21). We now report that HGF increases MMP-1 expression and activity in SSc lung fibroblasts in a time- and dose-dependent manner. Up-regulation of MMP-1 expression and activity by HGF in SSc lung fibroblasts appears to be regulated by MAPK-dependent and NF-κB–independent pathways.
NF-κB is known to play an essential role in the regulation of a variety of genes involved in immune and inflammatory reactions leading to fibrogenic responses (34, 35). HGF was reported to prevent vascular endothelial growth factor–induced and TNFα-induced NF-κB activation in endothelial cells (36, 37). In an animal model of renal tubular interstitial fibrosis, HGF reduced NF-κB activation by lowering its expression and translocation to the nuclei of tubular epithelial cells (38). It has been postulated that the antiinflammatory action of HGF can be mediated by its inhibition of NF-κB signaling (36–38). We observed that the basal level of active NF-κB in SSc lung fibroblasts was even higher than in TNFα-stimulated HeLa cells. Additionally, the basal level of active NF-κB in SSc lung fibroblasts was significantly increased when compared with that in normal lung fibroblasts (Bogatkevich GS: unpublished observations). HGF considerably reduced NF-κB DNA binding activity, in a time- and dose-dependent manner. Although the depletion of Grb2 by siRNA prevented the inhibitory effect of HGF on NF-κB activity, the MAPK pathway did not appear to be involved in HGF-induced NF-κB activity in SSc lung fibroblasts.
The activation of NF-κB is tightly regulated in living cells (35). In resting cells, NF-κB is retained in the cytoplasm by a family of inhibitory proteins (IκB). The IκBα isoform represents the major proximal regulator of NF-κB. In an activated state, IκBα undergoes degradation in the proteasome, releasing NF-κB for nuclear translocation (35). Here we show that HGF markedly enhances IκBα protein expression in SSc lung fibroblasts in a time- and dose-dependent manner. Similar to NF-κB activity, HGF-induced IκBα accumulation was regulated by a Grb2-dependent pathway and was independent of MAPK signaling.
In summary, we conclude that in lung fibroblasts from white SSc patients, HGF down-regulates the accumulation of CTGF via MAPK/MMP-1 and NF-κB signaling pathways, whereas collagen down-regulation is mediated mainly by a MAPK/MMP-1–dependent pathway. More studies clarifying the mechanisms of HGF signaling pathways in lung fibroblasts are required. Also, any systemic effect of HGF must be taken into consideration because of the oncogenic potential of overexpressed or activated HGF receptor (39, 40). Nevertheless, the antifibrotic, antiinflammatory, and proregenerative properties of HGF suggest that it could be a promising therapeutic agent for some patients with fibrosing disorders. Our recent observation of differences in the responsiveness of SSc lung fibroblasts to HGF according to race (23) indicates the importance of careful characterization of future study patients and their response to this antifibrotic agent.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Bogatkevich had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Bogatkevich, Ludwicka-Bradley, Highland, Nietert, Silver.
Acquisition of data. Bogatkevich, Hant, Singleton.
Analysis and interpretation of data. Bogatkevich, Ludwicka-Bradley, Nietert, Silver.
Manuscript preparation. Bogatkevich, Ludwicka-Bradley, Highland, Hant, Nietert, Silver.
Statistical analysis. Bogatkevich, Nietert.