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Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK

Nature Cell Biology volume 19, pages 724–731 (2017)Cite this article

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Abstract

The response of cells to mechanical force1,2,3 is a major determinant of cell behaviour and is an energetically costly event4,5. How cells derive energy to resist mechanical force is unknown. Here, we show that application of force to E-cadherin stimulates liver kinase B1 (LKB1) to activate AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis. LKB1 recruits AMPK to the E-cadherin mechanotransduction complex, thereby stimulating actomyosin contractility, glucose uptake and ATP production. The increase in ATP provides energy to reinforce the adhesion complex and actin cytoskeleton so that the cell can resist physiological forces. Together, these findings reveal a paradigm for how mechanotransduction and metabolism are linked and provide a framework for understanding how diseases involving contractile and metabolic disturbances arise.

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Figure 1: AMPK is activated in response to force applied to E-cadherin.
Figure 2: LKB1 is recruited to the cadherin adhesion complex in response to force and activates and recruits AMPK.
Figure 3: LKB1 and AMPK are upstream of Abl-mediated phosphorylation of Tyr822 vinculin and Rho-mediated contractility.
Figure 4: Force-induced AMPK stimulates glucose uptake and increases intracellular ATP levels.
Figure 5: Force-induced increases in ATP reinforce the actin cytoskeleton and the E-cadherin adhesion complex to modulate barrier formation.

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Acknowledgements

We thank T. Moninger and T. Washington at the University of Iowa for their assistance in performing experiments. Research reported in this publication was supported by The National Institutes of General Medicine (Award Number R01GM112805 to K.A.D.) and the National Cancer Institute of the National Institutes of Health (Award Number P30CA086862). Predoctoral fellowships from the American Heart Association (Award Number AHA 16PRE26701111) and National Institutes of Health (Award T32 GM067795) support J.L.B. and C.H., respectively. M.S. was supported by ‘Fondation ARC pour la Recherche sur le Cancer’ (ARC SFI20111203781) and CNRS-AMI Mecanobio (Mecapol_2016-2017).

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Author notes

  1. Hannah K. Campbell and Christy Heidema: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, USA

    Jennifer L. Bays, Hannah K. Campbell & Kris A. DeMali

  2. Interdisciplinary Graduate Program in Molecular and Cellular Biology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, 52242, USA

    Christy Heidema & Kris A. DeMali

  3. Centre de Recherche en Cancérologie de Marseille, Aix Marseille University UM105, Institut Paoli Calmettes, UMR7258 CNRS, U1068 INSERM, Cell Polarity, Cell signalling and Cancer—Equipe labellisée Ligue Contre le Cancer, Marseille, 13273, France

    Michael Sebbagh

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Contributions

J.L.B. designed and performed experiments, analysed all the data, and helped write the manuscript. C.H. and H.K.C. helped with experimental design and procedures. M.S. provided cell lines and advised on their usage. K.A.D. helped with the experimental design, wrote the manuscript, and directed the project. All authors provided detailed comments.

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Correspondence to Kris A. DeMali.

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Integrated supplementary information

Supplementary Figure 1 The force-induced activation of AMPK occurs in multiple epithelial cell lines and requires E-cadherin and force transmission.

MDCK II (a,e) or MCF10A (b,c,f,g) cells were incubated with magnetic beads coated with IgG (as a control), a syndecan-1 antibody, or E-cadherin extracellular domains (E-Cad). The cells were then left resting (−) or tensile forces were applied to the beads (+), the cells were lysed, and total cell lysates were immunoblotted with antibodies that recognize AMPK (a,b,d,e,f), pAMPK (a,b,d,e,f), acetyl CoA carboxylase (c, ACC), acetyl CoA carboxylase phosphorylated at its AMPK specific site (c, pACC), or E-cadherin (e, E-cad). shE-cad indicates cells with E-cadherin silenced. shAMPK indicates cells with E-cadherin silenced. Compound C (Cmpd. C), Blebbistatin (Blebbi) and HECD-1 indicate cells pretreated with these reagents. (d,h) the assembly of cell–cell junctions was stimulated using a calcium switch assay, and the cells were lysed. The levels of pAMPK and AMPK were examined by immunoblotting (d) or E-cadherin was immunoprecipitated and AMPK association was monitored by immunoblotting (h). ′indicates minutes after Ca2+ re-addition. Steady indicates cells maintained in growth media. The graphs beneath the image show the average ± s.e.m. for 3 independent experiments. ∗∗, ∗, and #indicate P values of <0.001, <0.01, and < 0.05, respectively. NS indicates not significant. Unprocessed scans of blots are shown in Supplementary Figure 5.

Supplementary Figure 2 LKB1 and AMPK are upstream of Abl-mediated phosphorylation of Y822 vinculin and RhoA contractility.

MDCK II (a) or MCF10A (b–e) cells were incubated with magnetic beads coated with IgG (as a control) or E-cadherin extracellular domains (E-Cad). The cells were left resting (−) or tensile forces were applied to the beads (+), and the cells were lysed. (a,b) immunoblots of whole cell lysates showing phosphorylation of CrkL at the Abl-specific site (pCrkL). shLKB1 denotes cells with LKB1 silenced. shLuc indicates cells expressing a control vector cDNA, and cl.11 and cl.14 indicate two different clonal cell lines lacking LKB1. (c) immunoblots of whole cell lysates showing phosphorylation of vinculin Y822 (pY822). d, Active Rho (Rho–GTP) was isolated from whole cell lysates with GST–RBD and analyzed by western blotting. Compound C (Cmpd. C) indicates cells pretreated with this AMPK inhibitor. Y822F indicates MCF10A cells expressing a mutant vinculin containing a Y822F vinculin in place of the endogenous protein. (e) immunoblots of whole cell lysates showing myosin light chain (MLC), or MLC phosphorylated in its regulatory chain (pMLC). The graphs beneath each image represent an average of three experiments ± s.e.m.∗∗, ∗, # and ##indicate P values of <0.001, <0.01, <0.05 and <0.005, respectively. NS indicates not significant. Unprocessed scans of blots are shown in Supplementary Figure 5.

Supplementary Figure 3 Average fold activation of the glucose uptake and ATP values.

Bar graphs displaying the average fold activation of force-induced glucose uptake assays (a–d) or of the intracellular ATP levels (e,f) are shown. (a–c) The fold activations for the data presented in Fig. 4a–c are shown in panels S3a, S3b, and S3c, respectively. (d) The amount of glucose taken up into MCF10A cells after a calcium switch assay performed as described in supplemental Fig. 1f. (e,f) The fold activation for ATP levels presented in Fig. 4d, e are shown in panels S3e and S3f, respectively. The values represent the average fold activation from three independent experiments ± the standard error of the mean.

Supplementary Figure 4 Controls for shear stress studies in Fig. 5.

(a–h) MDCK II cells (n = 79) or two clonal MDCK II cells lines (cl.11 and cl.14, n = 51 and 76 respectively) lacking LKB1 were left untreated, treated with inhibitors of AMPK (Compound C = Cmpd. C, n = 55), treated with inhibitors of ATP synthesis (Oligo A, n = 53 or Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone = FCCP, n = 29), treated with inhibitors of myosin II (blebbistatin = Blebbi, n = 30), treated with the AMPK activator (A-769662), or incubated in low glucose containing media (Low Gluc, n = 25). The cells were left resting (no shear) or exposed to shear stress (shear). (a–d) The cells were fixed, stained with antibodies against E-cadherin or with Texas-Red-labelled phalloidin, and visualized using confocal microscopy. Representative images are shown in a and d. Scale bars, 20 μm. The graph in b and c represent the average corrected fluorescence intensity of E-cadherin (b, E-cad) or F-actin (c) in junctions. The data are represented as a box and whisker plot with median, 10th, 25th, 75th, and 90th percentiles shown. (e) the cells were fixed, stained with antibodies against vinculin or β-catenin, and examined by confocal microscopy. The graphs represent the average corrected fluorescence intensity of vinculin (f) or β-catenin (g) in junctions. (h) the cells were lysed and total cell lysates were immunoblotted with antibodies against myosin light chain (MLC) or MLC phosphorylated at Serine 19 (pMLC). The graphs beneath each image represent an average of three experiments ± s.e.m. ∗∗, ∗ and # indicate P values of <0.001, <0.01 and <0.05, respectively. NS indicates not significant. Unprocessed scans of blots are shown in Supplementary Figure 5.

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Bays, J., Campbell, H., Heidema, C. et al. Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat Cell Biol 19, 724–731 (2017). https://doi.org/10.1038/ncb3537

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