Autocrine lysophosphatidic acid signaling activates β-catenin and promotes lung allograft fibrosis
Pengxiu Cao, … , Eric R. Fearon, Vibha N. Lama
Pengxiu Cao, … , Eric R. Fearon, Vibha N. Lama
Published February 27, 2017
Citation Information: J Clin Invest. 2017;127(4):1517-1530. https://doi.org/10.1172/JCI88896.
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Research Article Cell biology Transplantation

Autocrine lysophosphatidic acid signaling activates β-catenin and promotes lung allograft fibrosis

Abstract

Tissue fibrosis is the primary cause of long-term graft failure after organ transplantation. In lung allografts, progressive terminal airway fibrosis leads to an irreversible decline in lung function termed bronchiolitis obliterans syndrome (BOS). Here, we have identified an autocrine pathway linking nuclear factor of activated T cells 2 (NFAT1), autotaxin (ATX), lysophosphatidic acid (LPA), and β-catenin that contributes to progression of fibrosis in lung allografts. Mesenchymal cells (MCs) derived from fibrotic lung allografts (BOS MCs) demonstrated constitutive nuclear β-catenin expression that was dependent on autocrine ATX secretion and LPA signaling. We found that NFAT1 upstream of ATX regulated expression of ATX as well as β-catenin. Silencing NFAT1 in BOS MCs suppressed ATX expression, and sustained overexpression of NFAT1 increased ATX expression and activity in non-fibrotic MCs. LPA signaling induced NFAT1 nuclear translocation, suggesting that autocrine LPA synthesis promotes NFAT1 transcriptional activation and ATX secretion in a positive feedback loop. In an in vivo mouse orthotopic lung transplant model of BOS, antagonism of the LPA receptor (LPA1) or ATX inhibition decreased allograft fibrosis and was associated with lower active β-catenin and dephosphorylated NFAT1 expression. Lung allografts from β-catenin reporter mice demonstrated reduced β-catenin transcriptional activation in the presence of LPA1 antagonist, confirming an in vivo role for LPA signaling in β-catenin activation.

Authors

Pengxiu Cao, Yoshiro Aoki, Linda Badri, Natalie M. Walker, Casey M. Manning, Amir Lagstein, Eric R. Fearon, Vibha N. Lama

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Figure 6

Autocrine loop of NFAT1 and ATX regulation by LPA.

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Autocrine loop of NFAT1 and ATX regulation by LPA. (A) Representative li...
(A) Representative live cell imaging of non-BOS MCs expressing GFP-NFAT1 in the presence or absence of LPA (10 μM, 1 hour) (n = 5/group). Scale bars: 100 μm. (B) Non-BOS MCs were treated with LPA, and NFAT1 expression in nuclear extract was analyzed by immunoblotting. Lanes were run on the same gel but were noncontiguous, as indicated by the black lines. Mean ± SEM (n = 4/group with paired t test). (C) ATX expression in whole cell lysates of non-BOS MCs treated with LPA (10 μM) for varying time intervals. Mean ± SEM (n = 4/group with 1-way ANOVA). (D) ATX activity as measured in concentrated conditioned medium from non-BOS MCs cultured in the presence or absence (Ctrl) of LPA (n = 3/group with 2-way ANOVA). (E) Non-BOS MCs were transfected with NFAT1 siRNA or scrambled siRNA and treated with LPA for 48 hours (10 μM). The ATX activity in concentrated cell supernatant is shown (n = 9/group with 2-way ANOVA). The results represent mean ± SEM from 2 independent experiments. (F) NFAT1 and ATX protein expression was analyzed by immunoblotting in BOS MCs transfected with siRNAs for LPA1 or scrambled control (n = 5/group). (G) LPA1, NFAT1, and ATX mRNA expression was assayed by real-time PCR in BOS MCs transfected with LPA1 siRNA or scrambled control siRNA. Mean ± SEM (n = 3–6/group with paired t test). Experiments were performed twice. (H) ATX and LPA1 expression in MCs transfected with siATX was assayed by immunoblotting with scrambled siRNA transfected MCs as control. Mean ± SEM (n = 8/group with paired t test). *P < 0.05, **P < 0.01, ****P < 0.0001. (I) Schematic model of the proposed pathway by which autocrine LPA production regulates MC activation. G, G proteins; AM095, LPA1 inhibitor; PF-8380, ATX inhibitor.