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Cellular and Molecular Life Sciences

, Volume 68, Issue 19, pp 3275–3292 | Cite as

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

  • Maria Dolores Gutiérrez-López
  • Alvaro Gilsanz
  • María Yáñez-Mó
  • Susana Ovalle
  • Esther M. Lafuente
  • Carmen Domínguez
  • Peter N. Monk
  • Isidoro González-Alvaro
  • Francisco Sánchez-Madrid
  • Carlos CabañasEmail author
Research Article

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.

Keywords

Tetraspanins CD9 ADAM17 TACE TNF-α ICAM-1 

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

Notes

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.

Supplementary material

18_2011_639_MOESM1_ESM.jpg (406 kb)
Supplementary material 1 (406 kb)
18_2011_639_MOESM2_ESM.tif (2.9 mb)
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)
18_2011_639_MOESM3_ESM.jpg (950 kb)
Supplementary material 3 (950 kb)

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Copyright information

© Springer Basel AG 2011

Authors and Affiliations

  • Maria Dolores Gutiérrez-López
    • 1
    • 7
  • Alvaro Gilsanz
    • 1
  • María Yáñez-Mó
    • 2
  • Susana Ovalle
    • 1
  • Esther M. Lafuente
    • 4
  • Carmen Domínguez
    • 5
  • Peter N. Monk
    • 6
  • Isidoro González-Alvaro
    • 5
  • Francisco Sánchez-Madrid
    • 2
    • 3
  • Carlos Cabañas
    • 1
    • 4
    Email author
  1. 1.Centro de Biología Molecular Severo Ochoa (CSIC-UAM)MadridSpain
  2. 2.Servicio de InmunologíaHospital Universitario de La Princesa, Instituto de Investigacion Sanitaria PrincesaMadridSpain
  3. 3.Departamento de Biología Vascular e InflamaciónCNICMadridSpain
  4. 4.Departamento de Microbiología I (Inmunología)Facultad de Medicina, UCMMadridSpain
  5. 5.Servicio de ReumatologíaHospital Universitario de La PrincesaMadridSpain
  6. 6.University of Sheffield Medical SchoolSheffieldUnited Kingdom
  7. 7.Departamento de Farmacología, Facultad de MedicinaUniversidad ComplutenseMadridSpain

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