RESEARCH ARTICLE 1847 HEMCAM/CD146 downregulates cell surface expression of β1 integrins Sandrine Alais1, Nathalie Allioli1, Cristina Pujades1, Jean-Loup Duband1, Olli Vainio2, Beat A. Imhof3 and Dominique Dunon1,* 1UMR-CNRS 7622, Université Pierre et Marie Curie, 75005 Paris, France 2Dept of Medical Microbiology, Turku University, FIN-20520, Finland 3Dept of Pathology, University of Geneva, CH-1211, Geneva, Switzerland *Author for correspondence (e-mail: [email protected]) Accepted 13 February 2001 Journal of Cell Science 114, 1847-1859 © The Company of Biologists Ltd SUMMARY HEMCAM/gicerin, an immunoglobulin superfamily fibronectin depended on the HEMCAM isoform expressed. protein, is involved in homophilic and heterophilic adhesion. Flow cytometry and immunoprecipitation studies revealed It interacts with NOF (neurite outgrowth factor), a molecule that the expression of HEMCAM downregulated expression of the laminin family. Alternative splicing leads to mRNAs of the laminin-binding integrins α3β1, α6β1 and α7β1, and coding for HEMCAM with a short (HEMCAM-s) or a long fibronectin receptor α5β1 from the cell surface. Semi- cytoplasmic tail (HEMCAM-l). To investigate the cellular quantitative PCR and northern blot experiments showed function of these two variants, we stably transfected murine that the expression of α6β1 integrin modified by HEMCAM fibroblasts with either form of HEMCAM. Expression of occurred at a translation or maturation level. Thus, our each isoform of this protein in L cells delayed proliferation data demonstrate that HEMCAM regulates fibroblast and modified their adhesion properties to purified adhesion by controlling β1 integrin expression. extracellular matrix proteins. Expression of either HEMCAM-s or HEMCAM-l inhibited integrin-dependent Movies available on-line: adhesion and spreading of fibroblasts to laminin 1, showing http://www.biologists.com/JCS/movies/jcs1886.html that this phenomenon did not depend on the cytoplasmic region. By contrast, L-cell adhesion and spreading to Key words: Adhesion, Migration, Integrin, HEMCAM, MCAM INTRODUCTION during development (Taira et al., 1994). Gicerin participates in the development of the retina, the cerebellum and the ear, and is Among adhesion receptors a subgroup of the immunoglobulin involved in the regeneration of different organs in the adult superfamily with a characteristic V-V-C2-C2-C2 Ig domain organism (Hayashi and Miki, 1985; Hayashi et al., 1987; structure has recently emerged. Three members of this family Kajikawa et al., 1997; Kato et al., 1992; Takaha et al., 1995; have been identified in humans: BCAM (or as a splice variant, Taniura et al., 1991; Tsukamoto et al., 1998; Tsukamoto et al., the Lutheran blood group antigen); ALCAM, the activated 1999). Gicerin binds to neurite outgrowth factor (NOF), a 700 leukocyte-cell adhesion molecule; and MCAM/CD146, a kDa extracellular matrix glycoprotein of the laminin family marker for human melanoma tumour progression (Campbell et (Taira et al., 1994; Tsukamoto et al., 1996; Tsukamoto et al., al., 1994; Rahuel et al., 1996; Parsons et al., 1995; Bowen et al., 1997), and promotes cell-cell adhesion of transfected cells. 1995; Shih, 1999; Johnson et al., 1996; Lehman et al., 1989). In These data suggest that HEMCAM exhibits both heterophilic chick, motile c-kit+ hemopoietic progenitor cells in the bone and homophilic adhesion activities (Taira et al., 1995; Taira et marrow express another member of this V-V-C2-C2-C2 al., 1994; Vainio et al., 1996). subgroup, HEMCAM (Vainio et al., 1996; Dunon et al., 1998; Among the three known human V-V-C2-C2-C2 Ig molecules, Dunon et al., 1999). Three HEMCAM mRNA splice variants the highest homology is observed between HEMCAM and have been identified (Vainio et al., 1996). One has a short MCAM/CD146 (39%) (Vainio et al., 1996). Although this cytoplasmic tail (HEMCAM-s), another has a long tail homology is relatively low for the extracellular portion of the (HEMCAM-l), whereas the third lacks transmembrane and molecule, the transmembrane and the cytoplasmic domains cytoplasmic regions. Both transmembrane forms are found on show 66% and 69% homology, respectively. MCAM/CD146, the cell surface (Taira et al., 1995) but they appear to be also named MUC18, A32, Mel-CAM and S-Endo-1 (Shih, expressed differentially in the developing nervous system (Taira 1999), was first identified as a human melanoma-associated et al., 1994; Takaha et al., 1995; Tsukamoto et al., 1996) and in antigen with gradually increasing expression as tumours acquire the immune system (Vainio et al., 1996). HEMCAM expression metastatic potential (Johnson et al., 1996; Luca et al., 1993; is observed on embryonic endothelial cells, myocytes and Schlagbauer-Wadl et al., 1999; Wang et al., 1996; Xie et al., epithelial cells of the bursa of Fabricius. HEMCAM is the same 1997). In contrast to ubiquitous expression on melanoma, molecule as gicerin, a molecule involved in neurite outgrowth MCAM expression is restricted to some types of carcinomas, 1848 JOURNAL OF CELL SCIENCE 114 (10) Fig. 1. Two cytoplasmic variants of MCAM/CD146 are expressed in mammals. (A) RT-PCR analysis of chicken muscle using a combination of two primers, depicting mRNA coding for the long or short HEMCAM cytoplasmic forms (15667-15668) (Vainio et al., 1996). The short form appears as the lower band at 90 bp and the long form at 210 bp. Analysis of the same region of MCAM was performed with mouse muscle and HUVEC. The combination of primers for the murine MCAM was MEMCAM-1-MEMCAM-2 (accession number EMBL: X74628). The expected band corresponding to the putative long form appears as the upper band at 567 bp. The combination of primers for the human MCAM was 11674- MEMCAM2 (Lehman et al., 1989; Sers et al., 1993), the expected long form appears as the upper band at B 703 bp. (B) Alignment of amino acid sequences of chicken HEMCAM, and human, murine and bovine Short cytoplasmic form MCAM cytoplasmic forms. Amino acid sequences of Protein PDZ Domain murine and human MCAM were deduced from the Kinase C interaction nucleotide sequences of the PCR bands obtained in transmembrane domain site site (A). Bovine MCAM sequence was obtained in the HEMCAM-s SESKGIIIVAIIVCILVVAVLGSIIYFLHKKGKISCGRSGKQDIARNTSI EMBL database (accession number: U89328) and mMCAM-s P----VV---V---TL-L----AAL--FY----LP-------EME----- HEMCAM from our published sequences (Vainio et al., 1996). The line indicates the putative hMCAM-s P--R-VV---V------L----AVL---Y----LP-R-----EME----- transmembrane domain. b, bovine; h, human; l, long; m, murine; s, short. Long cytoplasmic form Protein Protein Kinase C Kinase C endocytosis transmembrane domain site site signal HEMCAM-l SESKGIIIVAIIVCILVVAVLGSIIYFLHKKGKISCGRSGKQDITKPEARKDKNVVEVKSDKLSEEAGLLQGANGEKRSPADQSEKYIDLRN mMCAM-l P----VV---V---TL-L----AAL--FY----LP-------E--L-PT--SEF---------P--MA----S--D--A-G--G-------H hMCAM-l P--R-VV---V------L----AVL---Y----LP-R-----E--L-PS--NEL---------P--M-----S--D--A-G--G-------H bMCAM-1 --AVL--FY----LP-------E--L-PT--SEF---------P--M-----S--D--A-G--G-------H such as breast carcinoma, where it has been suggested to act as described previously (Yatohgo et al., 1988). c264 monoclonal a tumour suppressor (Shih et al., 1997a). Similar to HEMCAM, antibody (mAb) against HEMCAM has been previously described MCAM/CD146 mediates cell-cell adhesion both in a (Vainio et al., 1996) and was used for cytofluorimetric (ELITE ESP, homophilic manner and through association with another as yet Coulter) and immunoprecipitation assays. Cells were cultured in unidentified partner (Johnson et al., 1997; Shih et al., 1997b). Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal calf serum (FCS), 10 IU/ml penicillin and 100 µg/ml streptomycin (Life- Transfection of MCAM into melanoma cells reduces adhesion Technologies Inc., Gaithersburgh, MD). to laminin 1 and increases invasive migration through Matrigel- Antibodies against integrin subunits were as follows: rat mAb coated filters, although the molecular mechanism for this effect GoH3 and EA-1 anti-mouse α6 (Sonnenberg et al., 1987; Imhof et is not known (Xie et al., 1997). al., 1991; Ruiz et al., 1993), PS/2 anti-α4 (Miyake et al., 1991), RMV- In the present report, we show that HEMCAM is the avian 7 anti-αv (Takahashi et al., 1990), CY8 against α7 (Yao et al., 1996), homologue of MCAM/CD146. We investigated the effect of MFR5 against α5 (Pharmingen), 9EG7 against activated β1 integrins HEMCAM isoforms on cell adhesion, spreading, migration (Lenter et al., 1993), mouse mAb 7A3 anti-human α3 (de Melker et and proliferation on various extracellular matrix molecules al., 1997) and polyclonal antibodies (pAb) 363E against β1 including laminin 1 and fibronectin, using normal mouse cytoplasmic region (DeSimone and Hynes, 1988). FITC-conjugated fibroblasts transfected by HEMCAM isoforms. We goat-anti mouse IgG (Jackson, West Grove, PA), FITC-conjugated rabbit anti-rat IgG (Biosys, Compiègne, France), and biotinylated demonstrate that the adhesion of transfected cells to laminin 1 donkey anti-rabbit IgG (Amersham) were used as secondary is specifically reduced, and by comparing the expression antibodies. Biotin labelling was detected by streptavidine coupled to pattern of the laminin receptor integrins, we show that either FITC (Amersham) or HRP (Pierce Chemical Co.). HEMCAM actively downregulates the cell surface expression Transfection of L cells by pcDNA3 vector coding for each isoform of the β1 integrins. of HEMCAM has been described previously (Vainio et al., 1996). The sequence of the cytoplasmic part of truncated HEMCAM isoform (HEMCAM-t) is KKGKISCGRSSMHLEGPIL (HEMCAM MATERIALS AND METHODS cytoplasmic tail is truncated after the tenth amino acid, underlined sequence). Expression of HEMCAM isoforms was controlled by Adhesion proteins, antibodies and cell cultures western blot experiments (data not shown). All results were obtained Mouse laminin 1 (LN) and bovine fibronectin (FN) were purchased with cloned cell lines and some of them were confirmed with sorted from Sigma. Vitronectin (VN) was purified from bovine plasma as cell populations.