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The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9

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Abstract

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.

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Abbreviations

BSA:

Bovine serum albumin

DTSSP:

3,3′-Dithiobis-sulfosuccinimidylpropionate

EGFR:

Epidermal growth factor receptor

FCS:

Fetal calf serum

FITC:

Fluorescein isothiocyanate

HMEC-1:

Human microvasculature endothelial cells-1

HRP:

Horse radish peroxidase

HUVECs:

Human umbilical vein endothelial cells

PBLs:

Peripheral blood lymphocytes

PBS:

Phosphate buffered saline

PMA:

Phorbol myristate acetate

mAb:

Monoclonal antibody

TACE:

Tumor necrosis factor alpha converting enzyme

TBS:

Tris buffered saline

TEMs:

Tetraspanin-enriched microdomains

TNF-α:

Tumor necrosis factor-α

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Acknowledgments

We are very grateful to Dr. M. Humphries for providing ADAM17-Fc cDNA; to Mariano Vitón and Sandra Moreno for their technical assistance, and to Dr. Ricardo Ramos-Ruiz for his assistance with the real-time PCR assays. This work was supported by grants BFU2007-66443/BMC and BFU2010-19144/BMC from Ministerio de Ciencia e Innovación, a grant from Fundación de Investigación Médica Mutua Madrileña and by RETICS Program RD08/0075-RIER (Red de Inflamación y Enfermedades Reumáticas) from Instituto de Salud Carlos III (to C.C.), a grant from Fundación de Investigación Médica Mutua Madrileña (to M.D.G.L.), and grants PI080794 from Instituto de Salud Carlos III (to M.Y-M) and SAF2007-60578 from Ministerio de Ciencia e Innovación (to E.M.L.). M.D.G.L. was supported by a contract associated to grant SAF2004-01715 from Ministerio de Ciencia e Innovación. S.O. was supported by an I3P predoctoral Fellowship from Consejo Superior de Investigaciones Científicas (CSIC) and by a contract associated to grant BFU2007-66443/BMC from Ministerio de Ciencia e Innovación. A.G. has been supported by a predoctoral Fellowship from Instituto de Salud Carlos III and by grant BFU2007-66443/BMC from Ministerio de Ciencia e Innovación.

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  1. Maria Dolores Gutiérrez-López

    Present address: Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, 28040, Madrid, Spain

Authors and Affiliations

  1. Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Nicolás Cabrera 1, Campus de Cantoblanco, 28049, Madrid, Spain

    Maria Dolores Gutiérrez-López, Alvaro Gilsanz, Susana Ovalle & Carlos Cabañas

  2. Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigacion Sanitaria Princesa, 28006, Madrid, Spain

    María Yáñez-Mó & Francisco Sánchez-Madrid

  3. Departamento de Biología Vascular e Inflamación, CNIC, 28029, Madrid, Spain

    Francisco Sánchez-Madrid

  4. Departamento de Microbiología I (Inmunología), Facultad de Medicina, UCM, 28040, Madrid, Spain

    Esther M. Lafuente & Carlos Cabañas

  5. Servicio de Reumatología, Hospital Universitario de La Princesa, 28006, Madrid, Spain

    Carmen Domínguez & Isidoro González-Alvaro

  6. University of Sheffield Medical School, Sheffield S10 2RX, Sheffield, United Kingdom

    Peter N. Monk

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  1. Maria Dolores Gutiérrez-López
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Corresponding author

Correspondence to Carlos Cabañas.

Additional information

M. D. Gutiérrez-López, A. Gilsanz and M. Yáñez-Mó contributed equally to this work.

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Supplementary material 1 (406 kb)

18_2011_639_MOESM2_ESM.tif

Surface expression of CD9, ADAM17, and ADAM10 on PMA-treated THP-1 cells is not affected by incubation with anti-CD9 mAb. THP-1 cells were treated for 24 h with 20 ng/ml PMA in the presence or absence of anti-CD9 mAb VJ1/20 (20 µg/ml). Surface expression of CD9, ADAM17, and ADAM 10 was analyzed by flow cytometry using VJ1/20, H-170, and H300 as primary antibodies, respectively. Thin and thick solid line histograms represent surface expression of each indicated protein in the absence and presence of the anti-CD9 mAb VJ/20, respectively. Negative controls are represented by the gray-filled histograms (2969 kb)

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Gutiérrez-López, M.D., Gilsanz, A., Yáñez-Mó, M. et al. The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9. Cell. Mol. Life Sci. 68, 3275–3292 (2011). https://doi.org/10.1007/s00018-011-0639-0

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  • DOI: https://doi.org/10.1007/s00018-011-0639-0

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