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Fibroblast-specific inhibition of TGF-?1 signaling attenuates lung and tumor fibrosis.

ABSTRACT: TGF-?1 signaling is a critical driver of collagen accumulation and fibrotic disease but also a vital suppressor of inflammation and epithelial cell proliferation. The nature of this multifunctional cytokine has limited the development of global TGF-?1 signaling inhibitors as therapeutic agents. We conducted phenotypic screens for small molecules that inhibit TGF-?1-induced epithelial-mesenchymal transition without immediate TGF-?1 receptor (T?R) kinase inhibition. We identified trihydroxyphenolic compounds as potent blockers of TGF-?1 responses (IC50 ~50 nM), Snail1 expression, and collagen deposition in vivo in models of pulmonary fibrosis and collagen-dependent lung cancer metastasis. Remarkably, the functional effects of trihydroxyphenolics required the presence of active lysyl oxidase-like 2 (LOXL2), thereby limiting effects to fibroblasts or cancer cells, the major LOXL2 producers. Mechanistic studies revealed that trihydroxyphenolics induce auto-oxidation of a LOXL2/3-specific lysine (K731) in a time-dependent reaction that irreversibly inhibits LOXL2 and converts the trihydrophenolic to a previously undescribed metabolite that directly inhibits T?RI kinase. Combined inhibition of LOXL2 and T?RI activities by trihydrophenolics resulted in potent blockade of pathological collagen accumulation in vivo without the toxicities associated with global inhibitors. These findings elucidate a therapeutic approach to attenuate fibrosis and the disease-promoting effects of tissue stiffness by specifically targeting T?RI kinase in LOXL2-expressing cells.

SUBMITTER: Wei Y

PROVIDER: S-EPMC5617667 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Fibroblast-specific inhibition of TGF-β1 signaling attenuates lung and tumor fibrosis.

Wei Ying Y Kim Thomas J TJ Peng David H DH Duan Dana D Gibbons Don L DL Yamauchi Mitsuo M Jackson Julia R JR Le Saux Claude J CJ Calhoun Cheresa C Peters Jay J Derynck Rik R Backes Bradley J BJ Chapman Harold A HA

The Journal of clinical investigation 20170905 10

TGF-β1 signaling is a critical driver of collagen accumulation and fibrotic disease but also a vital suppressor of inflammation and epithelial cell proliferation. The nature of this multifunctional cytokine has limited the development of global TGF-β1 signaling inhibitors as therapeutic agents. We conducted phenotypic screens for small molecules that inhibit TGF-β1-induced epithelial-mesenchymal transition without immediate TGF-β1 receptor (TβR) kinase inhibition. We identified trihydroxyphenoli ...[more]

PMID: 28872461

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Project description:Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Project description:BackgroundPulmonary fibrosis is a progressive and irreversible disease for which therapeutic options are currently limited. A recent in vivo study showed that tenofovir, a nucleotide analogue reverse transcriptase inhibitor, had direct antifibrotic effects on skin and liver fibrosis. Another study in vitro revealed that NS5ATP9 inhibited the activation of human hepatic stellate cells. Because of the similarity of fibrotic diseases, we hypothesized that tenofovir alafenamide fumarate (TAF), the prodrug of tenofovir, and NS5ATP9, is related to and plays a role in the suppression of pulmonary fibrosis.MethodsWe investigated the influence of NS5ATP9 on fibrosis in vitro. Human lung fibroblasts (HFL1) were transfected with short interfering RNAs or overexpression plasmids of NS5ATP9 before stimulation by human recombinant transforming growth factor-β1. The effect of TAF was evaluated in a bleomycin-induced fibrosis murine model. Male C57BL/6 mice were treated with bleomycin on day 0 by intratracheal injection and intragastrically administered TAF or vehicle. Left lung sections were fixed for histological analysis, while homogenates of the right lung sections and HFL1 cells were analyzed by western blotting and quantitative reverse transcription polymerase chain reaction.ResultsNS5ATP9 suppressed the activation of lung fibroblasts. Upregulation of collagen type 3 (α 1 chain) and α-smooth muscle actin was observed in HFL1 cells when NS5ATP9 was silenced, and vice-versa. TAF also showed anti-fibrotic effects in mice, as demonstrated by histological analysis of fibrosis and expression of extracellular matrix components in the lung sections. Additionally, TAF inhibited transforming growth factor-β1 and phosphorylated-Smad3 synthesis in HFL1 cells and the murine model, which was accompanied by upregulation of NS5ATP9.ConclusionsOur results suggest that NS5ATP9 forms a negative feedback pathway in pulmonary fibrosis and TAF has anti-fibrotic properties as it upregulates the expression level of NS5ATP9. As TAF has been shown to be safe and well-tolerated in humans, TAF and NS5ATP9 may be useful for developing novel therapeutics for pulmonary fibrosis.

Dataset Information

Fibroblast-specific inhibition of TGF-?1 signaling attenuates lung and tumor fibrosis.

Publications

Fibroblast-specific inhibition of TGF-β1 signaling attenuates lung and tumor fibrosis.

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