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Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts

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Abstract

Lysophosphatidylcholine (LysoPC) has been shown to induce the expression of inflammatory proteins, including cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6), associated with cardiac fibrosis. Here, we demonstrated that LysoPC-induced COX-2 and IL-6 expression was inhibited by silencing NADPH oxidase 1, 2, 4, 5; p65; and FoxO1 in human cardiac fibroblasts (HCFs). LysoPC-induced IL-6 expression was attenuated by a COX-2 inhibitor. LysoPC-induced responses were mediated via the NADPH oxidase-derived reactive oxygen species-dependent JNK1/2 phosphorylation pathway, leading to NF-κB and FoxO1 activation. In addition, we demonstrated that both FoxO1 and p65 regulated COX-2 promoter activity stimulated by LysoPC. Overexpression of wild-type FoxO1 and S256D FoxO1 enhanced COX-2 promoter activity and protein expression in HCFs. These results were confirmed by ex vivo studies, where LysoPC-induced COX-2 and IL-6 expression was attenuated by the inhibitors of NADPH oxidase, NF-κB, and FoxO1. Our findings demonstrate that LysoPC-induced COX-2 expression is mediated via NADPH oxidase-derived reactive oxygen species generation linked to the JNK1/2-dependent pathway leading to FoxO1 and NF-κB activation in HCFs. LysoPC-induced COX-2-dependent IL-6 expression provided novel insights into the therapeutic targets of the cardiac fibrotic responses.

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Abbreviations

COX-2:

Cyclooxygenase-2

DPI:

Diphenyleneiodonium chloride

DUOX:

Dual oxidase

EP:

Prostaglandin E2 receptor

FoxO:

FoxO Forkhead box protein O

HCFs:

Human cardiac fibroblasts

IL:

Interleukin

LysoPC:

Lysophosphatidylcholine

NOX:

NADPH oxidase

PC:

Phosphatidylcholine

PGE2 :

Prostaglandin E2

PLA2 :

Phospholipase A2

ROS:

Reactive oxygen species

TGF-β:

Transforming growth factor-β

TNF:

Tumor necrosis factor

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Acknowledgements

We thank Ms. Yu-Wen Chen for her technical assistance. This work was supported by the Ministry of Education, Taiwan, Grant numbers: EMRPD1H032 and EMRPD1H055; the Ministry of Science and Technology, Taiwan, Grant numbers: MOST104-2320-B-182A-003-MY3, MOST105-2320-B-182-005-MY3 MOST107-2320-B-182A-011, and MOST107-2320-B-182-020-MY2; Chang Gung Medical Research Foundation, Taiwan, Grant numbers: CMRPD1F0022, CMRPD1F0023, CMRPD1F0551, CMRPD1F0552, CMRPG3E2232, CMRPG3F1532 CMRPG3F1533, CMRPG3H0061, and CMRPG5F0202.

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Authors and Affiliations

  1. Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-San, Tao-Yuan, Taiwan

    Hui-Ching Tseng & Chuen-Mao Yang

  2. Department of Physiology and Pharmacology and Health Ageing Research Center, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan

    Hui-Ching Tseng, Chen-Yu Wang & Chuen-Mao Yang

  3. Department of Anesthetics, Chang Gung Memorial Hospital at Linkuo and Chang Gung University, Kwei-San, Tao-Yuan, Taiwan

    Chih-Chung Lin, Li-Der Hsiao & Chuen-Mao Yang

  4. Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital at Tao-Yuan, Kwei-San, Tao-Yuan, Taiwan

    Chien-Chung Yang

  5. School of Traditional Chinese Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan

    Chien-Chung Yang

  6. Research Center for Chinese Herbal Medicine and Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan

    Chuen-Mao Yang

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  1. Hui-Ching Tseng
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Supplementary Fig.

 1 The expression of NOXs isoforms in HCFs (A) Total RNA was extracted from growth-arrested HCFs. The levels of NOX1-5 mRNA were determined by RT/PCR analysis. β-actin was used as an internal control. The star symbol indicates each replicon. (B-E) HCFs were transfected with Scramble, NOX1, NOX2, NOX4 or NOX5 siRNA for 36 h, and then incubated with LysoPC for 6 h. The levels of NOXs mRNA expression were determined by RT/qPCR analysis (n = 4). Data are expressed as the mean ± SEM, and analyzed by a one-way ANOVA with Tukey’s post hoc tests. *, p < 0.05. #, p < 0.01. (TIFF 799 kb)

Supplementary Fig.

 2 (A) HCFs were pretreated with SP600125 (0.3, 1, 3 μM) for 1 h and then incubated with LysoPC for 6 h. The levels of COX-2 and GAPDH protein were determined by western blot analysis (n = 5). (B-C) Representative data of western blotting are presented in Fig. 2E (DPI: n = 5; SP600125: n = 5). Data are expressed as the mean ± SEM, and analyzed by a one-way ANOVA with Tukey’s post hoc tests. *, p < 0.05. #, p < 0.01. (TIFF 436 kb)

Supplementary Fig.

 3 (A-D) Representative data of western blotting are presented in Fig. 3C (n = 6) and 3D (DPI: n = 7; SP600125: n = 5; Helenalin: n = 7). Data are expressed as the mean ± SEM, and analyzed by a one-way ANOVA with Tukey’s post hoc tests. *, p < 0.05. #, p < 0.01. (TIFF 407 kb)

Supplementary Fig.

 4 (A) Schematic diagram of positions of NF-κB binding elements and ChIP primer pairs on COX-2 promoter. (B-C) HCFs were treated with LysoPC for the indicated time intervals. The DNA binding activity of NF-κB was determined by ChIP assays using an anti-p65 or anti-phosph-p65 antibody. The immunoprecipitated DNA and inputs were analyzed by an SYBR system for qPCR using a pair of primers flanking the NF-κB binding site within the human COX-2 promoter (proximal NF-κB: -320 ~ -171; distal NF-κB: -499 ~ -335). Representative gel (upper panels) and quantification of data are shown as the fold change normalized to input control (n = 5; bottom panels). Data are expressed as the mean ± SEM, and analyzed by a one-way ANOVA with Tukey’s post hoc tests. *, p < 0.05. #, p < 0.01 (TIFF 498 kb)

Supplementary Fig.

 5 (A-D) Representative data of western blotting are presented in Fig. 5C (n = 7) and 5D (DPI: n = 7; SP600125: n = 5; AS1842856: n = 7). Data are expressed as the mean ± SEM, and analyzed by a one-way ANOVA with Tukey’s post hoc tests. *, p < 0.05. #, p < 0.01 (TIFF 410 kb)

Supplementary material 6 (TIFF 588 kb)

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Tseng, HC., Lin, CC., Wang, CY. et al. Lysophosphatidylcholine induces cyclooxygenase-2-dependent IL-6 expression in human cardiac fibroblasts. Cell. Mol. Life Sci. 75, 4599–4617 (2018). https://doi.org/10.1007/s00018-018-2916-7

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