MAP3K19 Is Overexpressed in COPD and Is a Central Mediator of Cigarette Smoke-Induced Pulmonary Inflammation and Lower Airway Destruction.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis

<div><p>Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.</p></div

Inhibition of MAP3K19 kinase activity by Compound A inhibits Smad2/3-mediated transcription.

<p>A549 cells were stably transfected with a construct containing the Smad binding element (SBE) linked to a luciferase reporter gene. The cells were plated out in 96-well plates, incubated with the designated concentrations of Compound A, and treated with 0.1 ng/ml of TGF-β1 for 18 hours. At that point, the cells were assayed for luciferase activity, indicative of Smad 2/3 transcriptional activation. Wells that received no Compound A, only TGF-β1, were normalized to 100% luciferase activity. The mean percentage of luciferase activity is shown ± SEM. A representative experiment is shown (n = 3).</p

Inhibition of MAP3K19 blocks TGF-β1-mediated gene transcription.

<p>A549 lung epithelial cells were treated with 1 μM of Compound A, pirfenidone or nintedanib, individually or in combination, and TGF-β1 (1 ng/ml) for 12 hrs. RNA was harvested and relative mRNA transcript levels were measured by RT-qPCR. Results are shown for (A) collagen 1A1, (B) N-cadherin, (C) PAI-1 and (D) αB-crystallin. All samples were normalized to GAPDH, and results are expressed as fold change compared to untreated (No Tx) samples. The RT-qPCR was run in duplicate samples and the mean ± SEMs shown. This experiment was repeated three independent times with similar results and a representative experiment is shown. Similar results were obtained for HeLa cells and Beas-2B cells.</p

Immunohistochemical analysis showed that MAP3K19 expression in normal lung is predominantly limited to bronchial epithelial cells and interstitial and alveolar macrophage.

<p>Normal human lung was stained with either the 1B9C2 anti-MAP3K19 mouse monoclonal antibody (brown staining) or RabK19 rabbit polyclonal antibody (brown staining) and counter-stained with hematoxylin.</p

Histological analysis of the lungs from bleomycin-treated mice in the presence or absence of Compound A or pirfenidone treatment.

<p>Representative histology from Masson’s Trichrome stained sections of saline-instilled mice, vehicle treated, Compound A treated and pirfenidone treated mice are shown in panel (G). MAP3K19 staining of bleomycin treated mouse lungs, from a separate experiment, is shown in the Supplementary Data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154874#pone.0154874.s004" target="_blank">S4 Fig</a>).</p

MAP3K19 protein is predominantly localized to the nucleus.

<p>(A) HeLa cells were grown on glass cover slips and stained with either MAP3K19 (RabK19 polyclonal antibody) or an isotype control antibody. (B) Western analysis of HeLa cell cytoplasmic or nuclear lysates immunoblotted with anti-MAP3K19 antibody (RabK19) showing nuclear localization of the protein.</p

Therapeutic dosing of Compound A protected mice from bleomycin-induced pulmonary fibrosis.

<p>For the therapeutic arm of this experiment, the cohorts were (1) i.t.-delivered saline control group (n = 5 mice), (2) bleomycin-instilled no treatment group (n = 5), (3) bleomycin-treated vehicle control cohort (n = 12), (4) bleomycin-instilled Compound A treated (10 mg/kg) group (n = 12), (5) bleomycin-instilled pirfenidone treated (10 mg/kg) cohort (n = 12), and (6) bleomycin-instilled group with dexamethasone (3 mg/kg delivered i.p.) administered every other day (n = 12). Treatment for mice that received bleomycin started on day 6 and continued to day 13. As with the prophylactic study, mice were scored for fibrosis (A), collagen deposition (B) and the soluble lung collagen content (C). The mean scores ± SEM are shown.</p

Transfection of MAP3K19 renders HEK cells susceptible to Compound A-mediated inhibition of P-Smad2 nuclear translocation.

<p>HEK cells, which express very low levels of MAP3K19 as determined by RT-qPCR analysis, were transfected with either empty vector (pcDNA) or MAP3K19. Following an overnight culture, the cells were pretreated with Compound A (1 μM) for 30 minutes and treated with TGF-β1 (1 ng/ml) for one hour. The cells were then harvested, and the nuclear lysates were assayed by Western analysis for nuclear phospho-Smad2 localization. The blot was then re-hybridized with HDAC-1 to show equal protein loading. Densitometric analysis of P-Smad2/HDAC-1 levels from the Western blot is shown. This experiment is representative of two independent experiments.</p

MAP3K19 antagonist inhibited TGF-β1-induced protein and acted additively with therapeutic doses of pirfenidone.

<p>A549 cells were treated with 30 μM, 3 μM, 300 nM or 30 nM of Compound A or 5000 μM, 1700 μM, 560 μM or 190 μM of pirfenidone alone or in combination, and TGF-β1 (1 ng/ml) for 24 hrs. Cell lysates were isolated and PAI-1 (Serpin-1) protein levels were determined by ELISA. Results shown were the percentage of PAI-1 protein compared to levels from cells treated only with TGF-β1. Results are representative of two independent experiments.</p