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Activation of protease-activated receptor (PAR) 1 by frog trefoil factor (TFF) 2 and PAR4 by human TFF2

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Abstract

Trefoil factors (TFFs) promote epithelial cell migration to reseal superficial wounds after mucosal injury, but their receptors and the molecular mechanisms underlying this process are poorly understood. In this study, we showed that frog TFF2 activates protease-activated receptor (PAR) 1 to induce human platelet aggregation. Based on this result, we further tested the involvement of PARs in human TFF2 (hTFF2)-promoted mucosal healing. hTFF2-stimulated migration of epithelial HT-29 cells was largely inhibited by PAR4 depletion with small interfering RNAs but not by PAR1 or PAR2 depletion. The PAR4-negative epithelial cell lines AGS and LoVo were highly responsive to hTFF2 as assessed by phosphorylation of ERK1/2 and cell migration upon PAR4 expression. Our findings suggest that hTFF2 promotes cell migration via PAR4. These findings will be helpful in further investigations into the functions and molecular mechanisms of TFFs and PARs in physiology and disease.

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Abbreviations

TFF:

Trefoil factor

PAR:

Protease-activated receptor

GPCR:

G protein-coupled receptor

PAR-AP:

PAR-activating peptide

ERK:

Extracellular signal-regulated kinase

FITC:

Fluorescein 5(6)-isothiocyanate

EGF:

Epidermal growth factor

siRNA:

Small interfering RNA

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Acknowledgments

This work was supported by grants from the National Basic Research Program of China (973 Program, 2010CB529800), the Chinese National Natural Science Foundation (30630014, 30570359, and 30870304), and the Chinese Academy of Sciences “Key Research Direction” (KSCX2-YW-R-088).

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Authors and Affiliations

  1. Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 East Jiaochang Road, Kunming, Yunnan, 650223, China

    Yong Zhang, Guoyu Yu, Yanjie Wang, Yang Xiang, Qian Gao, Ping Jiang, Jie Zhang, Wenhui Lee & Yun Zhang

  2. Graduate School of the Chinese Academy of Sciences, Beijing, 100049, China

    Yong Zhang, Guoyu Yu, Yanjie Wang, Yang Xiang, Qian Gao, Ping Jiang & Jie Zhang

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  1. Yong Zhang
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  2. Guoyu Yu
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Correspondence to Yun Zhang.

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18_2011_678_MOESM1_ESM.jpg

Supplementary material 1 Expression of Bm-TFF2 in E. coli. (A) Purification of recombinant Bm-TFF2 in a reverse-phase HPLC Zorbax 300 SB C8 column. Peak IV was able to induce human platelet activation, as indicated by an arrow. Inset: SDS-PAGE analysis of different fractions obtained in the expression of Bm-TFF2. Lane 1, the crude extract of induced E. coli cells; lane 2, TRX-Bm-TFF2 fusion protein isolated in a His-select nickel affinity column; lane 3, fraction after factor Xa cleavage; lane 4, the peak IV of Zorbax 300 SB C8 column corresponding to purified recombinant Bm-TFF2. (B) Western blotting of the fractions of the lanes 2, 3 and 4 in the inset of (A) with rabbit polyclonal antibodies against naturally purified Bm-TFF2. (C) The effective concentrations of recombinant Bm-TFF2 required to induce human platelet aggregation (10-50 nM) was similar to those needed for naturally purified Bm-TFF2 (Zhang J, Zhang Y, Wan SG, Wei SS, Lee WH, Zhang Y (2005) Biochem Biophys Res Commun 330:1027-1033). (JPEG 524 kb)

18_2011_678_MOESM2_ESM.jpg

Supplementary material 2 hTFF2-induced cell migration via PAR4 is CXCR4-independent. CXCR4 antagonist AMD3100 had no effect on the hTFF2-induced migration of HT-29 cells that are CXCR4-positive (inset), but the inhibitor abolished the SDF-1α-induced migration of HT-29 cells. hTFF2, but not SDF-1α, enhanced the migration of AGS-PAR4 cells that are CXCR4-negative (inset). Cell migration was tested by a Boyden chamber assay. The migration of HT-29 cells stimulated by an agonist indicated was examined in the absence (-) and presence (+) of AMD3100 (Sigma). The expression of CXCR4 was analyzed by Western blotting with the anti-CXCR4 antibody (1:1000, 4°C, overnight, 12G5, BD Biosciences, San Jose, CA, USA). Data are means ± S.D. of three independent experiments. **, p<0.01. (JPEG 530 kb)

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Zhang, Y., Yu, G., Wang, Y. et al. Activation of protease-activated receptor (PAR) 1 by frog trefoil factor (TFF) 2 and PAR4 by human TFF2. Cell. Mol. Life Sci. 68, 3771–3780 (2011). https://doi.org/10.1007/s00018-011-0678-6

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