CD98hc (SLC3A2) is a key regulator of keratinocyte adhesion

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Abstract

Background

Adhesion of keratinocytes is crucial for maintaining the integrity of the skin, as demonstrated by the number of dermatological disorders of genetic origin that are associated with a defect of basal keratinocyte adhesion. Integrins are the main component of the molecular networks involved in this phenomenon, but there are many others. In a recent description of proteins associated to caveolae at the plasma membrane of human basal epidermal cells, we demonstrated that CD98hc is localized with β1 integrin.

Objectives

We investigated the CD98hc proteins interactions and the role of CD98hc in keratinocyte adhesion.

Methods

CD98hc protein interaction was identified following co-immunoprecipitation and proteomic analysis using LTQ-FT mass spectrometer. Extinction of CD98hc gene expression using specific short hairpin RNA or over-expression of CD98hc lacking the β1 integrin binding site was used to evaluate the role of this protein in keratinocyte fate.

Results

We show that CD98hc forms molecular complexes with β1 and β4 integrins in primary human keratinocytes and, using immunofluorescence, that these complexes are localized at the plasma membrane, in keeping with a role in adhesion. We confirmed that this protein is a key player of keratinocyte adhesion because in absence of interaction between CD98hc and integrins, β1 integrin failed to translocate from the cytoplasm to the plasma membrane and keratinocytes expressed epidermal differentiation markers.

Conclusions

All these data strongly suggested that CD98hc is involved in integrin trafficking and by consequence, in keratinocyte adhesion and differentiation.

Introduction

Integrity of the human skin requires the formation and strict maintenance of hemidesmosomal and focal adhesion molecular complexes, in which integrins are associated with a limited number of other key players [1]. Hemidesmosomes contain the heterodimeric α6β4 integrin connecting the cells to the major component of the basement membrane, laminin-332, while the cytoplasmic proteins plectin and BP230 connect to the keratin filaments, thereby creating a stable anchoring complex. Default in the formation of these complexes leads to epidermal diseases, as it happens in epidermolysis bullosa [2]. Besides hemidesmosomes, α3β1 integrin is involved in laminin-332 deposition and promotes keratinocyte survival [3], [4]. Basal keratinocytes highly express β1 and α6 integrins which are clustered in caveolae microdomains and associated with caveolin 1 [5].

In a previous study, we have taken the opportunity of this clustering in caveolae to seek potential partners of integrins in molecular complexes associated with keratinocyte adhesion via proteomics, and identified CD98hc [6]. CD98hc has been described in other cell types and involved in biological functions as diverse as amino acid transport, cell proliferation, differentiation, fusion and adhesion [7], [8]. Transport of neutral amino acid has been especially analysed, as it requires association of CD98 heavy chain with its small subunit, named LAT1 [9]. In parallel, and most interestingly within the framework of our studies, CD98hc has been shown to interact with β1 integrin in transfected CHO cells and mouse embryonic fibroblasts (MEF), suggesting a role in cell-cell adhesion [10], [11]. Molecular interaction of the two proteins has been characterized in those models [10], [12], and its failure leads to a defect in focal adhesion, contributing to cell transformation [13]. Following its interaction with β1 integrin, activation of CD98hc increases FAK and AKT phosphorylation in Madin Darby Canine Kidney and mouse inner medullary collecting duct polarized epithelial cells [14]. In association with integrins, CD98hc is also involved in cell spreading [11], [15] and in the formation of a fibronectin matrix, as demonstrated using null MEF and mouse ES cells [16]. Again suggesting a role of the protein in integrin signaling, spreading of CD98hc null mouse ES cells is altered and this defect is rescued by the phosphorylation of AKT and RAC [15]. Nevertheless, such interactions between integrins and CD98hc have never been studied in keratinocytes.

Together with our identification of CD98hc in caveolae enriched in integrins, these data of the literature prompted us to fully characterize the potential role of the protein in human basal keratinocytes adhesion. We first identified partner proteins of CD98hc in those cells by systematic proteomic analysis, pointing in particular to the two heterodimeric integrins that are major components of keratinocyte adhesion, and confirmed the co-localization of β1 and β4 integrins with CD98hc at the cell membrane. We then revealed a functional role of CD98hc in the maintenance of cell adhesion and demonstrated that CD98hc is necessary for addressing β1 integrin at the cell membrane.

Section snippets

Antibodies and reagents

All antibodies used for immunoprecipitation were from Santa Cruz Biotechnology except anti-β4 integrin and anti-Zyxin purchased from Pharmingen (BD Biosciences). DMEM, HAM-F12, phosphate buffer saline X1 (PBS) and trypsin/EDTA were from Invitrogen. KGM2 medium was from Lonza. fetal calf serum (FCS) was from Hyclone. All other molecular grade reagents were from Sigma.

Isolation of CD98hc complexes from primary keratinocytes

Human keratinocytes were isolated from neonatal foreskin from routine circumcisions. Skin biopsies were obtained with the

Protein partners of CD98hc in human keratinocytes

The characterization of CD98hc complex was performed after CD98hc immunoprecipitation and mass spectrometry analysis. Shotgun proteomic strategy was used to characterize immunoprecipitated protein networks. Using a LTQ-FT-Orbitrap mass spectrometer, we identified with high confidence 454 proteins (Supporting information Table 1) in anti-CD98hc immunoprecipitation. As a control, immunoprecipitation using the mouse haematopoietic marker antiboby, anti-CD45R antibody was done (Supporting

Discussion

The main result of this study is the demonstration that CD98hc interacts with integrins to ensure basal keratinocytes adhesion in the human epidermis. Disruption of this interaction alters the addressing of β1 integrin to the plasma membrane and provokes cell detachment and keratinocyte differentiation.

The role of CD98hc in mechanisms associated with basal keratinocyte adhesion has been suggested since its identification as a plasma membrane protein specifically associated to basal, adherent

Acknowledgements

The authors would like to thank Chloé Féral for providing CD98hc and C98T69E98 plasmids, Daniel Stockolm at Genethon (Evry, France) for assistance in confocal imaging, Dr. Serhal at the “Clinique de l’Essonne”, Evry for skin biopsies and Marc Peschanski for constant support and critical reading of the manuscript. B. Monsarrat is supported by the CNRS, the Genopole Toulouse and the Région Midi-Pyrenées.

References (45)

  • X. Meng et al.

    Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa

    J Invest Dermatol

    (2003)
  • C. Miquel et al.

    Establishment and characterization of cell line LSV5 that retains the altered adhesive properties of human junctional epidermolysis bullosa keratinocytes

    Exp Cell Res

    (1996)
  • K. Tasanen et al.

    Keratinocytes from patients lacking collagen XVII display a migratory phenotype

    Am J Pathol

    (2004)
  • H. Tsumura et al.

    The targeted disruption of the CD98 gene results in embryonic lethality

    Biochem Biophys Res Commun

    (2003)
  • J. Blonder et al.

    A proteomic characterization of the plasma membrane of human epidermis by high-throughput mass spectrometry

    J Investig Dermatol

    (2004)
  • E. Calautti et al.

    Phosphoinositide 3-kinase signaling to akt promotes keratinocyte differentiation versus death

    J Biol Chem

    (2005)
  • S.M. Frisch et al.

    Anoikis mechanisms

    Curr Opin Cell Biol

    (2001)
  • X. Liu et al.

    CD98 and intracellular adhesion molecule I regulate the activity of amino acid transporter LAT-2 in polarized intestinal epithelia

    J Biol Chem

    (2003)
  • D. Merlin et al.

    CD98-mediated links between amino acid transport and beta 1 integrin distribution in polarized columnar epithelia

    J Biol Chem

    (2001)
  • E. Nakamura et al.

    4F2 (CD98) Heavy Chain Is Associated Covalently with an Amino Acid Transporter and Controls Intracellular Trafficking and Membrane Topology of 4F2 Heterodimer

    J Biol Chem

    (1999)
  • L. Liu et al.

    Tetraspanin CD151 promotes cell migration by regulating integrin trafficking

    J Biol Chem

    (2007)
  • T. Ozawa et al.

    Dynamic relationship of focal contacts and hemidesmosome protein complexes in live cells

    J Invest Dermatol

    (2010)
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    The authors are sorry to announce that Professor G. Waksman has died on September 22, 2007, during the completion of the study he had initiated.

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