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Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

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Cell Stress and Chaperones Aims and scope

Abstract

Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27.

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Abbreviations

ADT:

5-[p-methoxyphenyl]-3H-1,2-dithiol-3-thione

D3T:

1,2-dithiole 3-thione

Hpf:

hours post-fertilization

DTT:

dithiothreitol

FCS:

fetal calf serum

Hsp27:

heat shock protein 27 (HSPB1)

HUVEC:

human umbilical vein endothelial cells

LC-MS:

liquid chromatography-mass spectrometry

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Acknowledgements

We thank Susan Garfield for guidance with confocal imaging. The authors acknowledge support from the Intramural Research Program of the NIH, NCI, Center for Cancer Research (DDR) and NIH grants GM 1584 (BLK) and CA 128616 (JSI). Contribution Number 934 from the Barnett Institute.

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Author notes

  1. Jeff S. Isenberg

    Present address: Hemostasis and Vascular Biology Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA

Authors and Affiliations

  1. Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA

    Yifeng Jia, Jeff S. Isenberg, John M. Sipes, Lyndsay Field, Bixi Zeng, Russell W. Bandle, Ramani Ramchandran & David D. Roberts

  2. Barnett Institute, Northeastern University, Boston, MA, 02115, USA

    Shiaw-Lin Wu, Shujia Dai & Barry L. Karger

  3. Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA

    Lisa A. Ridnour & David A. Wink

  4. Department of Pediatrics, Children’s Research Institute, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Ramani Ramchandran

  5. NIH, Building 10 Room 2A33, 10 Center Dr MSC1500, Bethesda, MD, 20892-1500, USA

    David D. Roberts

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  1. Yifeng Jia
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Jia, Y., Wu, SL., Isenberg, J.S. et al. Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton. Cell Stress and Chaperones 15, 165–181 (2010). https://doi.org/10.1007/s12192-009-0130-0

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