The present study provides three novel insights in the TGF-α lung fibrosis model: (1) pharmacologic MEK inhibition prevents the evolution of fibrosis that is already embedded and progressing; (2) inhibition of the MEK pathway prevents fibrosis independent of persistent PI3K activation; and (3) MEK/ERK is primarily activated in mesenchymal cells of fibrotic lesions.
Using biochemical, histologic, and physiologic methods, our data demonstrate that MEK inhibition prevented the progression of established disease when administered as a rescue therapy. To our knowledge, this is the first study to successfully demonstrate that inhibition of the MEK/ERK pathway in vivo
attenuates the progression of fibrosis and physiologic alterations in lung mechanics. The clinical relevance for this finding is the strong evidence demonstrating up-regulation of ERK/MAPK in human fibrotic disease (38
). Yoshida and colleagues (39
) analyzed transcripts for Akt and MAPK signaling pathways in lung tissue of patients with idiopathic pulmonary fibrosis (IPF), and found increased levels of serine/threonine-protein kinase B-Raf (BRAF) compared with normal lung control subjects. BRAF is a major signaling intermediate protein that regulates the MAPK/ERK pathway. Western blot of human lung biopsy samples also demonstrates increased ERK1/2 signaling in IPF samples compared with normal lungs (38
). The recent development of pharmacologic inhibitors of MAPK/ERK that are in clinical oncology trials, coupled with the findings of this study, support additional studies in animal models and humans to verify MAPK/ERK as a therapeutic target in fibrotic disease.
We previously demonstrated that inhibition of the PI3K/Akt pathway with the specific PI3K inhibitor, PX-866, prevented fibrosis in the CCSP/TGF-α model, despite unaltered activation of the MAPK/ERK pathway (18
). In the current study, Akt remained phosphorylated both in vitro
and in vivo
( and ) after TGF-α induction, confirming the specificity of ARRY inhibition. The effectiveness of either PX-866 or ARRY in preventing fibrosis development suggests that inhibition of either the PI3K or MEK/ERK pathway is sufficient to prevent significant fibrosis from becoming established. Together, these findings would suggest that both pathways need to be active in vivo
to induce fibroproliferation, but the mechanism for this observation remains under investigation. One possible explanation for these findings includes cell-selective pathway activation. We previously demonstrated Akt phosphorylation primarily in airway and type II epithelial cells after TGF-α induction (40
). In contrast, we observed elevated ERK activity primarily in α-SMA and vimentin-positive mesenchymal cells of lungs from CCSP/TGF-α mice on Dox for 4 days or 4 weeks (). Interestingly, similar to the CCSP/TGF-α model, immunohistochemistry of human lung sections from patients with IPF demonstrates increased pERK1/2, primarily in smooth muscle cells, with little activation in the epithelium (41
). An alternative explanation includes a common converging pathway downstream of both PI3K and MAPK/ERK, whereby input from both pathways is required for activation. The mTOR is a highly conserved intracellular serine/threonine kinase and a major downstream component of the PI3K pathway (42
). Activation of mTOR, in complex with raptor (mTORC1), leads to phosphorylation of p70S6K and 4E-binding proteins (4E-BPs) (43
). p70S6K and 4E-BPs control the translation of specific mRNAs and protein synthesis involved in cell cycle regulation, with effects on cellular growth, proliferation, and translation (44
). The p70S6K and 4E-BP pathways possess several phosphorylation sites that are points of convergence not only for mTORC1, but also for MAPK (43
). Together, these findings provide a basis to further investigate molecular events and downstream targets in both the PI3K and MEK/ERK pathways that contribute to pulmonary fibrosis initiation.
CCSP/TGF-α mice administered ARRY as a rescue therapy 4 weeks after beginning Dox demonstrated significant improvements with 4 weeks of treatment in lung histology, lung mechanics, body weights, and collagen gene transcripts compared with vehicle-treated mice, demonstrating that Erk inhibition prevented the progression of fibrosis. However, all parameters remained altered compared with control mice. We previously reported that fibrosis in the CCSP/TGF-α model is completely reversible when TGF-α overexpression is extinguished after 4 weeks of induction (18
). Therefore, if ARRY was successful in completely reversing the fibrosis, endpoints in the ARRY-treated rescue group would be expected to return to no fibrosis/control levels. As all endpoints in the ARRY-treated group remained above the control values, these findings demonstrate that prevention of fibrosis does not guarantee reversal once the fibrotic process is initiated. Further resolution of existing fibrosis may have required longer treatment times than 4 weeks. Alternatively, once fibrosis is established and progressing, multiple signaling pathways are likely activated and contribute toward the maintenance of lung fibrosis. We previously demonstrated that TGF-β1 is not activated during the induction of fibrosis in the CCSP/TGF-α model, but is detected in advanced fibrotic lesions (7
). Therefore, inhibition of multiple pathways, such as TGF-β, may be required to accelerate or complete fibrosis resolution. The concept of combined signaling pathway inhibition has proved effective in transgenic and xenograft models of cancer (45
), and our findings support that the resolution of existing and progressing fibroproliferative diseases is likely complex, involving multiple pathways.
In summary, overexpression of TGF-α in the lung epithelium is associated with mesenchymal cell activation of the MEK/ERK signaling pathway. There are strong data supporting activation of ERK in human fibrotic disease, and the MEK/ERK pathway is a logical target for potential fibrosis therapy, as several fibrogenic cytokines signal through MEK/ERK, including noncanonical TGF-β, platelet-derived growth factor, IL-13, and TNF-α (14
). In addition, cell cycle progression in lung mesenchymal cells is regulated through ERK1/2 by altering expression of cyclin D1 and CDK4 (49
). In the TGF-α model, selective inhibition of MEK prevented the development of fibrosis and attenuated the progression of fibrosis when administered as a rescue therapy. These findings support the concept that MEK/ERK activation may play a significant role in human lung fibrotic disease, which could be amenable to targeted therapy.