Elsevier

Journal of Hepatology

Volume 37, Issue 3, September 2002, Pages 322-330
Journal of Hepatology

Expression of transmembrane 4 superfamily (TM4SF) proteins and their role in hepatic stellate cell motility and wound healing migration

https://doi.org/10.1016/S0168-8278(02)00175-7Get rights and content

Abstract

Background/Aims: Migration of activated hepatic stellate cells (HSC) is a key event in the progression of liver fibrosis. Little is known about transmembrane proteins involved in HSC motility. Tetraspanins (TM4SF) have been implicated in cell development, differentiation, motility and tumor cell invasion. We evaluated the expression and function of four TM4SF, namely CD9, CD81, CD63 and CD151, and their involvement in HSC migration, adhesion, and proliferation.

Methods/Results: All TM4SF investigated were highly expressed at the human HSC surface with different patterns of intracellular distribution. Monoclonal antibodies directed against the four TM4SF inhibited HSC migration induced by extracellular matrix proteins in both wound healing and haptotaxis assays. This inhibition was independent of the ECM substrates employed (collagen type I or IV, laminin), and was comparable to that obtained by incubating the cells with an anti-β1 blocking mAb. Importantly, cell adhesion was unaffected by the incubation with the same antibodies. Co-immunoprecipitation studies revealed different patterns of association between the four TM4SF studied and β1 integrin. Finally, anti-TM4SF antibodies did not affect HSC growth.

Conclusions: These findings provide the first characterization of tetraspanins expression and of their role in HSC migration, a key event in liver tissue wound healing and fibrogenesis.

Introduction

Cell adhesion is an essential prerequisite for the action of growth factors, cytokines and other factors. Accordingly, attention has been brought on the interaction between hepatic stellate cells (HSC), key cellular effectors of hepatic wound healing and fibrogenesis [1], [2], and the ECM microenvironment occurring through integrin receptors. Along these lines, activated human HSC express several integrins of the β1 subfamily (particularly α1β1, α2β1, αvβ1) which are involved in different functions, such as cell-matrix adhesion, migration, ECM remodelling [3], and release of chemokines [4], [5].

Recent studies have shown that some integrins can physically interact with other membrane receptors, such as transmembrane 4 superfamily proteins or tetraspanins (TM4SF) [6], [7]. TM4SF are a large family of ubiquitously expressed membrane proteins that have four putative membrane-spanning domains, short N- and C-terminal cytoplasmic domains, a small intracellular loop, and two extracellular loops [8]. Although several TM4SF molecules have been identified and implicated in the regulation of cell development, differentiation, proliferation, motility and tumor cell invasion [9], the biochemical functions of this class of molecules are still largely undefined. Recent experimental evidence suggests that TM4SF may play an important role in cell migration and in the generation of complexes with integrins functionally relevant for cell motility [10], [11], [12], [13].

HSC migration is a crucial step in the recruitment of activated cells in areas of injury during hepatic wound healing/fibrogenesis [14], [15]. In addition, studies addressing the role of TM4SF in the process of wound healing are still scarce and limited to cell types such as keratinocytes and endothelial cells [16], [17], [18]. Accordingly, aim of the present study was to provide information on the expression and function of TM4SF molecules in activated human HSC. In particular, we evaluated the expression and cellular distribution of four TM4SF molecules, namely CD9, CD81, CD63, and CD151, their physical association with integrins of the β1 family, and their role in cell adhesion, cell migration, and cell growth.

Section snippets

Antibodies and reagents

The anti-TM4SF monoclonal antibodies (mAbs) used were: DU-ALL-1, anti-CD9 (Sigma, Chemical Co., St. Louis, MO, USA); JS81, anti-CD81 (Pharmingen, San Diego, CA, USA). The monoclonal anti-CD9 FMC8 was generously provided by Dr H. Zola (Child Health Research Institute, North Adelaide, Australia). The monoclonal anti-CD63 antibody 6H1 and monoclonal anti-CD151 antibody 5C11 were generously provided by Dr F. Berditchevski (CRC Institut for Cancer Studies, University of Birmingham, UK). Monoclonal

Expression and cellular distribution of TM4SF molecules in HSC

The expression of TM4SF molecules in human HSC was first analyzed by flow cytometry. Antibodies against TM4SF proteins showed high level of surface binding for CD9, CD63, CD81 and CD151 (Fig. 1). The cellular distribution of TM4SF proteins in HSC was investigated by indirect immunofluorescence and confocal microscopy. In paraformaldehyde fixed cells, anti-TM4SF mAbs delineated the plasma membrane of individual cells (not shown). Upon permeabilization, CD81 and CD151 showed more diffuse

Discussion

Migration of ECM-producing cells represents a key event in the wound healing and fibrogenic processes, and it is regulated by precise interactions with the ECM microenvironment. Integrin receptors play a fundamental role in these interactions by ensuring optimal cell anchorage to ECM during migration. A continuous turnover of integrin receptors at the leading edge of cell advancement is necessary for cell motility, and it is achieved by endocytosis and subsequent recycling of ‘fresh’ integrin

Acknowledgements

Supported by grants from the Italian MURST (project ‘Cellular and Molecular Biology of Hepatic Fibrogenesis’), the University of Florence, and Banco di Roma, Rome, Italy. Financial support was also provided by the Italian Liver Foundation.

The authors wish to thank Professor Sandra Zecchi Orlandini, Dipartimento di Anatomia, Istologia e Medicina Legale, Università degli Studi di Firenze, for her precious help with confocal microscopy studies.

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