Dual targeting of MEK and PI3K pathways attenuates established and progressive pulmonary fibrosis - PubMed (original) (raw)

Dual targeting of MEK and PI3K pathways attenuates established and progressive pulmonary fibrosis

Satish K Madala et al. PLoS One. 2014.

Abstract

Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Effect of in in vivo inhibition of MEK and PI3K in combination on ERK and S6 phosphorylations.

(A) CCSP/− and CCSP/TGFa mice on Dox for 2 weeks were co-treated with PX-866 (3 mg/kg, every other day) and ARRY (37.5 mg/kg) either once a day (QD) or twice a day (BID) for 7 days. Total lung lysates that represent four separate mice in each group were analyzed by immunobloting. Western blot analysis was used to determine levels of phosphorylated ERK (B) and phosphorylated S6 (C) that normalized to the total ERK and S6 protein in lung homogenates, respectively. Data are means ± SEM, and statistical significance between groups was measured using one-way ANNOVA (n = 4/group).

Figure 2

Figure 2. Inhibition of MEK and PI3K alone or in combination alters lung weights and collagen accumulation during TGFα-induced pulmonary fibrosis.

(A) The treatment protocol is represented schematically. Controls included CCSP/− and CCSP/TGFα mice treated with vehicle for the last 4 weeks while remaining on Dox a total of 8 weeks. (B) Changes in the right lung weights of mice treated with vehicle or inhibitors. (C) Changes in the right-lung hydroxyproline levels of mice treated with vehicle or inhibitors. Data are means ± SEM, and statistical significance between groups was measured using one-way ANNOVA (n = 8–12/group). (D) Percent change in body weights of mice treated with vehicle or inhibitors. Data are means ± SEM, and statistical significance between groups was measured using one-way ANNOVA (n = 8–12/group).

Figure 3

Figure 3. Inhibition of MEK and PI3K alone or in combination alters collagen accumulation and pleural thickening during TGFα-induced pulmonary fibrosis.

(A) Representative photomicrographs of lung tissues stained with Masson Trichrome for each treatment group. Upper panel: area of airways, vessels, and parenchyma. Lower panel: pleural regions. Scale bar, 80 µm. (B) Pleural thickness of lung tissues stained with Masson Trichrome for treatment groups. (D) Inhibition of MEK and PI3K alone or in combination alters lung compliance during TGFα-induced pulmonary fibrosis. Data are means ± SEM, and statistical significance between groups was measured using one-way ANNOVA (n = 8–12/group).

Figure 4

Figure 4. Inhibition of MEK and PI3K in combination alters contraction of collagen gels and SMA deposition in the lungs.

(A) Primary fibroblasts from the lung cultures of CCSP/TGFα transgenic mice on Dox for 4 wks were platted in 3D collagen gels for 4 or 7 days with media, ARRY, PX866 or inhibitors combined. Images were taken to quantify the changes in the surface of 3D collagen gels (B) Representative photomicrographs of lung tissues for each treatment group (n = 4/group) demonstrate changes in SMA staining in the fibrotic lesions of the pleura (upper panel) and adventitia (lower panel).

Figure 5

Figure 5. Inhibition of MEK and PI3K alone or in combination alters proliferating cells in the lung during TGFα-induced pulmonary fibrosis.

Changes in the number Ki67 positive cells of mice in each group on Dox for 8± SEM and statistical significance between groups was measured using one-way ANNOVA (n = 4–5/group).

Figure 6

Figure 6. Inhibition of MEK and PI3K alone or in combination alters the transcriptome of the lung during TGFα-induced pulmonary fibrosis.

Heatmap depicting expression levels of total lung genes with at least a two-fold change from CCSP/− control mice on Dox for 8 wks. Differentially expressed genes (up- or down-regulated) by ≥ two-fold in the fibrosis, ARRY, PX-866, ARRY/PX-866 in combination treatment groups are shown. Expression levels of these genes were either maintained or enriched in the combination group as compared to both single inhibitors. Several known fibrotic genes that are significantly down regulated in combined therapy are highlighted using their gene symbols.

Figure 7

Figure 7. Inhibition of MEK and PI3K alone or in combination alters transcripts involved in the proliferation of cells in the lung during TGFα-induced pulmonary fibrosis.

(A) Heatmap depicting expression levels of several top genes involved in proliferation, based on ingenuity pathway analysis. Up-regulated genes in the lungs of mice treated with vehicle, ARRY, PX-866, or ARRY/PX-866 in combination are shown. (B) MYCN transcript levels in the lungs of each treatment group on Dox for 8 wks, during which mice were treated with either vehicle or inhibitors for the last 4 wks (C) CDK4 transcript levels in the lungs of each treatment group on Dox for 8 wks, during which mice were treated with either vehicle or inhibitors for the last 4 wks. Data are means ± SEM, and statistical significance between groups was measured using one-way ANNOVA (n = 4/group).

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