RESEARCH ARTICLE 1847 HEMCAM/CD146 downregulates cell surface expression of β1

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. , 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 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 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. . 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- 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-; and MCAM/CD146, a kDa extracellular matrix of the laminin family marker for human 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-+ hemopoietic progenitor cells in the 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 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. HEMCAM downregulates β1 integrin expression 1849

Fig. 2. Expression pattern of HEMCAM in avian embryos at 2.5 days of embryonic development. (A) Frozen transversal embryon sections were stained with a pAb specific for HEMCAM. Bound anti-HEMCAM antibody was detected by biotine-conjugated anti-rabbit antibody and streptavidine-FITC. The same expression pattern was obtained using different monoclonal anti-HEMCAM antibodies. Control performed with pre-immune serum gave no staining. (B) In situ hybridizations were performed using digoxygenin-labeled probes on whole embryos (a); these embryos were paraffin-embedded and 10 µm sections were obtained (b). Photographs were taken under bright field. (a) a 2.5 days old chick embryo hybridized with HEMCAM probe showing expression in migrating neural crest cells, and dermomyotome. (b) Transversal section of the embryo in (a) showing expression of HEMCAM in the , migrating neural crest cells, and dermomyotome. The position of the section in the A-P axis is indicated (arrow). d, dermomyotome; e, epidermis; en, endoderm; n, notochord; ncc, neural crest cells; nt, neural tube.

Cell adhesion assays 5% CO2 for 12 hours before analysis. Cell migration and cell motility Microtiter plates (96 flat-bottomed wells) were coated with different were also analyzed under videomicroscopy. purified extracellular matrix proteins, in PBS, for 16 hours at 4°C. Alternatively, cell migration was analyzed using lateral migration Coated plates were washed three times with PBS and the remaining assays (adapted from Koivisto et al., 1999). Briefly, 24-well plates binding sites on the plastic surface were blocked with 1% BSA for 1 were coated with extracellular matrix molecules. Each well was hour at 37°C. Cells were trypsinized and resuspended in serum-free seeded with 30,000 L cells into glass cloning cylinders with an DMEM and counted. 50 µl of cell suspension containing about 3000 opening of 4 mm. Cells were left to attach for 3 hours before cells were added to individual substrate-coated wells and incubated at cylinders were removed, and afterwards cells were allowed to 37°C. Plates were washed twice with PBS to remove the non-adherent migrate for 24, 48 or 72 hours. After fixation in 3.7% formaldehyde cells, and fixed in a 3.7% formaldehyde solution for 15 minutes at in PBS, cells were stained with 0.25% Coomassie blue in 50% room temperature. Attached and spread cells were counted under a methanol-10% acetic acid for 15 minutes at room temperature. Cells Nikon inverted phase-contrast microscope. In all tests, the same were washed twice with 50% methanol-10% acetic acid. To measure number of microscopy fields was analyzed. the average diameter of the cell colony a 20-fold-magnified image Integrin activation assays were performed similar to adhesion was used. Cell migration was calculated by subtracting the area of assays but Puck’s A saline buffer (Life technologies Inc., the original colony to the area of the colony obtained after cell Gaithersburgh, MD) was used to dilute laminin 1, Mn2+ solution and migration. to wash cells before adhesion (Chen et al., 1999). Divalent cations were mixed with cells just before being added to laminin-coated wells. Cell labelling, immunoprecipitation and immunoblotting To determine which integrins were responsible for adhesion to laminin For surface protein labelling, cells were incubated with 1 mg/ml EZ- 1, antibodies against different integrins were added to the cell Link sulfo-NHS-LC-biotin (Pierce Chemical Co.) for 45 minutes suspension just before adhesion assays. at 4°C. Cells were washed three times with TBS, and lysed in lysis buffer (2% NP-40, 150 mM NaCl, 2 mM PMSF, 2 mM CaCl2, 2 mM Proliferation assays MgCl2, 50 mM Tris pH 8.0). For total protein labelling, cells were Kinetics of proliferation were performed in 24-well plates. On each metabolically labelled, incubating them overnight with 30 µCi/ml well, 20,000 cells were seeded in 1 ml of complete medium. After [35S]/cysteine in 1% FCS-supplemented DMEM medium different times of culture, cells were trypsinized and counted. Each without methionine and cysteine. Proteins were extracted for 1 hour point of kinetics corresponds to the average of three wells (n=3). at 4°C in 1 ml of lysis buffer. Pulse-chase experiments were carried out to study the maturation rate of β1 chain. Cells were incubated in Cell migration assays methionine and cysteine-free DMEM for 1 hour before labelling with Migratory ability of L cells was analyzed using wound assays. Cells [35S]methionine/cysteine for 5 hours. Cells were then washed twice were grown at confluence and the cell layer was wounded with a with PBS and incubated in their complete medium. Proteins were yellow tip. Adherent cells were washed twice and incubated at 37°C, extracted after 0, 12, 24 and 36 hours of chase. 1850 JOURNAL OF CELL SCIENCE 114 (10)

For immunoprecipitation, cell extracts were centrifuged for 30 minutes at 13,000 rpm at 4°C. The supernatant was cleared with mouse pre-immune serum coupled to protein G-agarose beads (Boehringer Mannheim) and incubated overnight with the corresponding antibody previously adsorbed to protein G-agarose. For β1 immunoprecipitation, rabbit pre-immune serum and protein A- agarose beads were used to pre-clear extracts. Beads were washed three times with lysis buffer and twice with TBS, boiled in Laemmli buffer, and run on 6% acrylamide SDS-PAGE gels. After the transfer of proteins onto nitrocellulose sheet, analysis of biotinylated proteins was performed with HRP-coupled streptavidine and ECL system (Amersham). For metabolically labelled proteins, gels were autoradiographed following a treatment with an autoradiography enhancer (Dupont). For western blot experiments, proteins from 75,000 cells were extracted and boiled in Laemmli buffer before separating on a 6% acrylamide SDS-PAGE gel under nonreducing conditions. Proteins were electroblotted onto nitrocellulose. Nitrocellulose membranes were blocked with nonfat milk (5% w/v in TBS, 0.5% Tween-20; TBSTw) and incubated for 1 hour at room temperature with 363E antibody in TBSTw. After washing, membranes were incubated with HRP-coupled anti-rabbit antibody for 1 hour at room temperature. After washing in TBSTw and TBS, anti-rabbit antibodies were detected using the ECL system, following the manufacturer’s instructions (Amersham). In situ hybridization Chick embryos at 2.5 days of embryogenic development were fixed in 4% paraformaldehyde/PBS overnight at 4°C. HEMCAM digoxygenin (DIG)-labelled antisense riboprobe was synthesized by in vitro transcription using T7 RNA polymerase. The coding sequence fragment transcribed was localized between positions 425 and 935 of HEMCAM cDNA. Whole-mount in situ hybridization was performed as described previously (Prince and Lumsden, 1994). Immunohistochemistry Japanese quail (Corturnix corturnix japonica) embryos were used for immunohistochemistry. Embryos were routinely fixed at room temperature in 3.7% formaldehyde in PBS for 12 hours. After washing in PBS for one hour, embryos were embedded in increasing concentrations of sucrose solution in PBS (5-15% w/v) and subsequently frozen with OCT compound (Tissue-Tek, Life Sciences Inc.) in liquid nitrogen. Using a cryostat, 10 µm sections were obtained (Bright Instrument Co. Ltd, Huntington, UK) and mounted on superfrost slides (CML, France). Nonspecific staining was blocked by incubating the sections on 1% BSA in PBS for 1 hour at room temperature. The sections were incubated with polyclonal anti- HEMCAM serum (1/1000) for 1 hour at room temperature, washed three times in PBS and incubated for 1 hour with biotin-conjugated donkey anti-rabbit antibody (Amersham). PBS-washed sections were incubated for 1 hour with strepavidin-conjugated fluorescein (Amersham), mounted in Immunomount (CML) and observed under a Leitz Orthoplan epifluorescent microscope. Photographs were taken Fig. 3. Characterization of HEMCAM-transfected cells. (A) Surface using TMX-400 Kodak film. expression of HEMCAM in transfected cells. Immunostaining was performed with pAb specific for HEMCAM and a secondary anti- mRNA amplification, cloning and sequencing rabbit Ig antibody coupled to FITC. HEMCAM was expressed at the Cytoplasmic regions of HEMCAM, murine and human MCAM were cell surface and notably in the cellular filopodia. No staining was amplified by RT-PCR. Total RNA from chicken muscle, mouse obtained with parental or mock-transfected cells under those staining muscle and HUVEC was isolated using the guanidium isothiocyanate conditions. (B) Kinetics of proliferation shows that HEMCAM method (Chomczynski and Sacchi, 1987). 5 µg of total RNA were expression delays proliferation. In spite of this delay, the rate of used as a template for the synthesis of randomly primed single-strand proliferation is the same for all cell lines. cDNA using mouse mammary leukemia virus reverse transcriptase (Life Technologies Inc.) in a reaction volume of 20 µl, according to the supplier’s instructions. cDNA reaction volume was subsequently MCAM were amplified by PCR using specific primers. The made up to 50 µl with water and heated to 94°C for 2 minutes to oligonucleotides used for HEMCAM and MCAM were: 15667 inactivate the enzyme. (HEMCAM, from nucleotide 1876) 5′TTCCTGCACAAGAAGGGC; Cytoplasmic regions of HEMCAM, murine MCAM and human 15668 (HEMCAM-l, from nucleotide 2068, antisense) HEMCAM downregulates β1 integrin expression 1851

Fig. 4. HEMCAM transfection modifies L-cell properties. (A) HEMCAM transfection reduced L-cell migration at the wound edge. Cells were grown at confluence and cell layers were wounded by a yellow tip. Adherent cells were washed twice and incubated in complete medium for 12 hours before analysis. Arrows indicate the size of the wound. Cells were finally fixed and stained with Coomassie blue. Differences observed for the wound healing between mock-transfected and HEMCAM-transfected L cells were related to different migration properties and did not depend on differences of proliferation rates because the duration of cell cycle is superior to the time of incubation for both cell lines. Transfection by HEMCAM-s or HEMCAM-l gave similar results. (B) HEMCAM transfection inhibits lateral migration of L cells on culture plastic dishes. Cells were left to attach for 3 hours in cloning cylinders in 24-well plates. After removing the cloning cylinders, cells were allowed to migrate for 24, 48 or 72 hours. The average diameter of the cell colony was measured. Cell migration was calculated by subtracting the area of the original colony from the area of the colony obtained after migration. Data corresponds to the mean of three independent experiments. Videomicroscopy analysis indicated that HEMCAM blocks cellular mobility but no cellular motility (data not shown). c, mock-transfected cells; l, HEMCAM-l-transfected cells; s, HEMCAM-s-transfected cells (C) Effect of HEMCAM-l transfection on L-cell spreading on laminin 1 (15 µg/ml coating concentration) and fibronectin (5 µg/ml coating concentration). Mock-transfected cells (control) adhered well to laminin 1 and 50% of them exhibited flat and spread cell morphology. By contrast, HEMCAM-l- transfected cells adhered poorly to laminin 1, producing a rounded morphology. No difference in cell morphology was observed between HEMCAM- and mock-transfected cells on fibronectin.

Northern blot analysis Electrophoresis of RNA samples was carried out in a 1.2% agarose gel in the presence of formaldehyde. The RNA was then transferred to a nylon membrane by capillary blotting and fixed to the filter by exposure to UV (Stratalinker, Stratagene, La Jolla, CA). RNA was hybridized with an α6 cDNA probe obtained by PCR labelling with [α-32P]dATP. Hybridization was carried out at 42°C in 50% formamide, 5× SSC, 5× Denhardt’s solution, 0.1% SDS and 300 µg/ml denatured salmon sperm DNA. Filters were washed twice in 1× SSC, 0.1% SDS at room temperature and once at 65°C in 0.1× SSC, 0.1% SDS. Filters were exposed to X-ray film (BIOMAX MR, Kodak) at −80°C with intensifying screens.

RESULTS 5′TTAGTTTCTCAGATCGATC; 11674 (human MCAM, from nucleotide 1336) 5′GTGTTGAATCTGTCTTGTGAA; MEMCAM-1 HEMCAM is the avian homologue of mouse and (murine MCAM, from exon 12) 5′AGTGTACAGCCTCCAAC; human MCAM/CD146 MEMCAM-2 (murine MCAM from exon 16 and human MCAM from nucleotide 1919, antisense) 5′ATGCCTCAGATCGATG (Lehman et Given the homology between HEMCAM and MCAM, we al., 1989; Sers et al., 1993; Vainio et al., 1996). Primers for murine address whether they might in fact be homologous molecules. α6 integrin subunit were 5′ATCTCTCGCTCTTCTTTCCG and Because three HEMCAM mRNA splice variants exist in birds, 5′GACTCTTAACTGTAGCGTGA (Tamura et al., 1991). The 550 bp one way of investigating this possibility was to determine fragment corresponded to the α6A cytoplasmic domain isoform and whether a similar splicing exists for MCAM in mammalian the 420 bp fragment to the α6B isoform. Primers for murine β1 cells. Indeed, two transcripts were detected by RT-PCR in integrin subunit were 5′TTGTGGAGACTCCAGACTGTTCTACT mouse and human tissues using MCAM-specific and 5′TCATTTTCCCTCATACTTCGGATT (Belkin et al., 1996). The β oligonucleotides (Fig. 1A). Cloning and sequencing of PCR 324 bp fragment corresponded to the 1D cytoplasmic domain products demonstrated that two transmembrane isoforms of isoform and the 243 bp fragment to the β1A isoform. Amplified DNA fragments were gel-purified and cloned into pCRTM2.1TOPO vector MCAM were generated by a splicing event and that these two (Invitrogen). Sequences were determined from denatured double- forms differed in the cytoplasmic portion (Fig. 1B). Nucleotide stranded recombinant plasmid DNA using SequenaseTM (Amersham) sequence analysis and comparison with the human in the chain termination reaction. Sequences were analyzed and organization (Sers et al., 1993) indicated that exon 15 was assembled with the software package of Infobiogen (Paris, France). excised in the short MCAM form and that this alternative 1852 JOURNAL OF CELL SCIENCE 114 (10)

Fig. 5. Comparative attachment and spreading of HEMCAM-s and A HEMCAM-l-transfected L cells on laminin 1 and fibronectin. 100 (A) Comparative attachment and spreading of mock, HEMCAM-s-, 160 FN FN HEMCAM-l- and HEMCAM-t-transfected cell lines at various 80 120 concentrations of coated laminin 1 and fibronectin. Tests were 60 performed for 2 hours at 37°C. Note that whereas adhesion and s 80 ll 40 spreading on laminin 1 were similar for HEMCAM-s and e

c ᭿ HEMCAM-l, they differed when fibronectin was used. Mock ( )-,

t 40 20 ) HEMCAM-l (᭜)-, HEMCAM-s (᭝)- and HEMCAM-t (᭺)- %

0 ( 0

eren transfected cell lines. (B) On vitronectin (5 µg/ml coating g h 0 5 10 15 0 5 10 15 concentration), HEMCAM expression decreased cell adhesion and ad spreading but no difference was observed between cells expressing of 250 100 different isoforms of HEMCAM. c, mock-transfected cells; l,

er LN LN Spreadin b 200 80 HEMCAM-l-transfected cells; s, HEMCAM-s-transfected cells. m 60 150 (C) Kinetics of adhesion to laminin 1 and fibronectin. Coating Nu 40 concentrations were 15 and 5 µg/ml for laminin 1 and fibronectin, 100 20 respectively. Note that adhesion to laminin 1 reached a plateau by 40 50 0 minutes of incubation of mock-transfected cells and adhesion of ᭿ 0 -20 HEMCAM-transfected cells is inhibited to 85%. Mock ( )-, 0 5 10 15 20 25 0 5 10 15 20 25 HEMCAM-l (᭜)-, HEMCAM-s (᭝)-transfected cell lines. Coating Concentration (µg/ml) HEMCAM (Vainio et al., 1996). Exhaustive analysis of transcripts was performed around this region in mouse, human B and chicken tissues but no other splicing event was detected. 200 100 To analyze the expression profile of HEMCAM, we VN VN ) s 80 ll performed immunohistochemistry and whole mount in situ %

of 150 e (

c

hybridization experiments in chick. At day 2.5 of development, g er t 60 b 100 HEMCAM was expressed in migrating neural crest cells, an m 40 eren embryonic cell population known to give arise to . h Nu

50

ad 20 HEMCAM immunoreactivity was also detected in epidermis Spreadin 0 0 and endoderm, as well as in the notochord. c l s c l s Whole mount in situ hybridization results clearly show that neural crest cells emigrating from the dorsal part of the neural tube expressed HEMCAM mRNA, which was also observed C in dermomyotome cells (Fig. 2B). This expression pattern is 200 200 confirmed by transverse sections (Fig. 2Bb), which also

s LN FN underline labelling of epidermis. This expression profile was ll 160 160 of e

c observed along the whole A-P axis.

er t 120 120 b

m 80 80

eren HEMCAM transfection delayed proliferation and h Nu 40 40 decreased integrin-dependent adhesion of L cells ad 0 0 Because it was shown that human MCAM could alter cell 10 20 40 60 80 100 120 10 20 40 60 80 100 120 adhesion to laminin (Xie et al., 1997), we analyzed whether its Adhesion time (min) chicken orthologue exerted this function and if its alternative forms exhibited different activities. Stable transfections of each isoform of HEMCAM into L cells were performed. HEMCAM splicing event was identical in birds and mammals. expression was localized at the cell surface and preferentially Interestingly, excision of exon 15 led to a frame shift for exon in the cellular protrusions. This expression pattern was 16 that changed the last six amino acids at the C terminus. confirmed in cell clones and sorted transfected cells (Fig. 3A). Amino acid sequence comparison showed that the short Several properties of L cells were modified by HEMCAM cytoplasmic form of HEMCAM and MCAM contained a transfection. A first consequence was a delay in proliferation putative PDZ domain interaction site (T/S-X-I/V) (Songyang of transfected cells after trypsinization (Fig. 3B). To determine et al., 1997). In the two isoforms of HEMCAM and MCAM, the involvement of HEMCAM in cell migration, wounding the constant sequence of the cytoplasmic part codes for a and lateral migration assays were performed. HEMCAM putative PKC phosphorylation site (RSGK). An additional expression inhibited cell migration on tissue culture dishes PKC phosphorylation site (KSDK) and a YXXL sequence (Fig. 4A,B) and on fibronectin substrate (data not shown). were conserved in the long cytoplasmic form of chicken Motile activity was studied by videomicroscopy 24 hours after HEMCAM, and mouse and human MCAM. The YXXL plating the cells in culture dish plates. Mock- and HEMCAM- sequence was first described as a half immunoreceptor transfected cells were motile but the structure of protrusion was tyrosine-based activation motif (ITAM) and, more recently, as different between cell lines. Mock-transfected cells exhibit an endocytosis motif (Deschambeault et al., 1999). The region long and thin filopodia, whereas transfected cells gave rise to corresponding to exon 13 of human MCAM was absent in short, puffy protrusions. These experiments suggest that these HEMCAM downregulates β1 integrin expression 1853 alterations of motility were involved in the inhibition of clones of transfected cells whatever the HEMCAM spliced migration. variant was expressed (HEMCAM-l or HEMCAM-s). Using To further investigate the role of HEMCAM in this process, mAb 9EG7, which recognizes activated β1 integrins (Bazzoni adhesion to laminin 1, fibronectin and vitronectin was analyzed et al., 1998; Lenter et al., 1993), we found residual cell at graded concentrations of each extracellular matrix protein surface expression in HEMCAM-transfected cells, suggesting but also in a kinetics test (Fig. 4C; Fig. 5). On laminin-1-coated that some β1 integrins are still present (Fig. 7). plates, expression of HEMCAM-l, HEMCAM-s, or Immunoprecipitation studies confirmed the FACS analysis HEMCAM-t inhibited adhesion of L cells by 85%, compared (Fig. 8B; Fig. 9A). In L cells, α6 is expressed exclusively with that in mock-transfected cells. Cell spreading was associated with the β1 chain, because no band of appropriate completely abolished by transfection of all HEMCAM forms. molecular size of the β4 chain was coprecipitated with anti-α6 On fibronectin, HEMCAM-s transfection reduced cell antibodies from surface-biotinylated cells. In addition to the adhesion and spreading, whereas HEMCAM-l and HEMCAM- α6β1 integrin, other laminin binding integrins, such as α3β1 t had a weak effect on cell spreading (Fig. 5A). These results and α7β1, were downregulated by HEMCAM (Fig. 8). The suggested that the cytoplasmic domain of HEMCAM could be low residual β1 integrin detected at the cell surface might involved in the regulation of cell spreading on fibronectin. In addition, HEMCAM reduced cell adhesion and cell spreading on vitronectin, whichever A construct was expressed at the cell surface (Fig. 5B). 200 These results were confirmed using several independent LN 200 FN L-cell clones transfected with the different HEMCAM constructs, and sorted populations of recently 150 150 transfected L cells. 100 100 To address whether HEMCAM could affect integrin expression or function in L cells, we first characterized 50 50 the repertoire of LN and FN integrin receptors in these 0 0 cells using blocking antibodies (Fig. 5; Fig. 6; Fig. 8). number of adherent cells v v v v α α6 α α6 α α6 α α6 On laminin 1, anti-α6 integrin antibodies EA-1 and i- i- i- i- i- i- i- i- nt nt nt nt nt nt nt nt GoH3 decreased the adhesion of mock-transfected L controla a controla a controla a controla a cells by 75%. As expected, anti-αv integrin antibody control HEMCAM-l control HEMCAM-l RMV-7 had no effect (Fig. 6A). These results demonstrate that α6 integrins were the main laminin receptors at the L-cell surface. Thus, the inhibition of B L-cell adhesion to laminin 1 induced by HEMCAM might be due to inactivation of α6 integrin. Integrin receptors can be activated by Mn2+ addition (Shaw 140 control HEMCAM-l and Mercurio, 1993; Shaw et al., 1993). To test 120 whether HEMCAM expression can induce the switch HEMCAM- s from an active to an inactive conformation of the 100 integrin, we replaced Ca2+ by Mn2+ in the adhesion assay. Whereas graded concentrations of Mn2+ 80 increased adhesion of mock-transfected cells to 60 laminin 1, no effect was observed with HEMCAM- transfected cells (Fig. 6B). This suggests that integrin 40 receptors are not sensitive to Mn2+ modulation after overexpression of HEMCAM, either because they number of adherent cells 20 cannot be activated or because they are not present at 0 the cell surface. 0 0.01 0.1 1 medium

HEMCAM inhibits cell surface expression of Mn2+ (mM) β1 integrins Flow cytometry results revealed that HEMCAM Fig. 6. L cells adhere to laminin 1 via α6 integrins. (A) Effect of antibody transfection does indeed induce downregulation of α5, against integrins on L-cell adhesion to laminin 1 and fibronectin. Cell α6 and α7 integrins at the cell surface (Fig. 7; Fig. 8A). adhesion assays were performed at a coating concentration of 15 and 5 µg/ml By contrast, HEMCAM transfection had no effect on for laminin 1 and fibronectin, respectively. Mock- and HEMCAM-l- α transfected cells were incubated for 2 hours at 37°C in the presence or absence the expression levels of v integrins (Fig. 7, Fig. 8B). α α α of functional antibodies. anti- v integrin antibody, RMV-7; anti- 6 integrin The downregulation of 6 was observed with two antibody, EA-1. (B) Mn2+ had no effect on adhesion of HEMCAM-transfected different mAbs each recognizing all conformational L cells to laminin 1. Control and HEMCAM-transfected cells were washed states of this integrin. This inhibition was reversible, and resuspended in a cation-free medium (Puck’A Buffer) before undergoing because loss of HEMCAM expression restored α6β1 an adhesion assay at graded Mn2+ concentrations. Laminin 1 coating expression by L cells (data not shown). The concentration was 15 µg/ml and cells were incubated for 2 hours at 37°C. A disappearance of β1 integrins was observed in different control experiment was carried out with cells in complete medium (medium). 1854 JOURNAL OF CELL SCIENCE 114 (10)

control

HEMCAM-l

HEMCAM-s

HEMCAM-t

2-264 RMV7 EA-1 GoH3 9EG7 HEMCAM αv integrin α6 integrin α6 integrin β1 integrin Fig. 7. HEMCAM transfection of L cells induces disappearance of α6β1 from the cell surface. L cells transfected with pcDNA3 (control), HEMCAM-l, HEMCAM-s and HEMCAM-t were analyzed by flow cytometry for surface staining with antibodies against HEMCAM (c264), αv integrins (RMV-7), α6 integrins (EA-1 and GoH3) and activated β1 integrins (9EG7). All secondary antibodies were FITC-conjugated and flow cytometry was performed on ELITE ESP Coulter. Negative controls (grey profiles) were obtained with secondary antibody alone. correspond to a low accumulation of various α chains HEMCAM induced a redistribution of α6β1 integrin in associated with the β1 subunit. This residual β1 integrin cannot intracellular compartments. Immunoprecipitation of α6β1 correspond to αvβ1 because immunoprecipitation experiments integrin was performed using metabolically labelled L cells. indicate that the αv chain is associated with the β3 subunit in In HEMCAM-transfected cells, a complete disappearance of all cell lines (Fig. 8B). Together, these results demonstrate that α6β1 was observed, indicating that α6β1 integrin was not HEMCAM overexpression dramatically alters the β1 integrin synthesized during labelling (Fig. 9B). Thus this absence at repertoire expressed on the surface of L cells. the cell surface was probably not due to α6β1 integrin redistribution. Western blot experiments using anti- HEMCAM regulates neither the cellular distribution cytoplasmic β1-integrin antibody were performed to detect nor the transcription of the α6β1 integrin the β1 integrin precursor. Although the precursor form of β1 As α6β1 integrin was the major laminin receptor of L cells, integrin subunit was clearly present in all cell lines we have analyzed how HEMCAM might regulate its (transfected and mock-transfected), the mature β1 protein expression. RT-PCR experiments established that HEMCAM was barely detectable in HEMCAM-transfected cells expression did not modify the transcription level of α6 and (Fig. 9B), supporting the data obtained by FACS and β1 integrin (Fig. 10A). These results were confirmed immunoprecipitation experiments (Fig. 7; Fig. 9A). Pulse- by northern blot experiments (Fig. 10B). These experiments chase experiments confirmed that β1 integrin precursor is showed that L cells expressed the β1A and α6A integrin synthesized in all cells (transfected and not transfected) and splice variants and that no modification of splicing was stabilized in this state in HEMCAM-transfected cells (Fig. induced by HEMCAM transfection. Because HEMCAM 9C). The presence of β1 integrin precursor in transfected cells transfection did not interfere with transcription of β1 and α6 showed that HEMCAM transfection interfere on β1 integrin integrin genes (Fig. 10), we tested whether overexpression of expression at the post-translational level. HEMCAM downregulates β1 integrin expression 1855 DISCUSSION of HEMCAM such as cellular localization or regulation of protein expression. HEMCAM/gicerin was described as an adhesion molecule Our studies suggest that an important role of HEMCAM in that exhibits homophilic cell-cell adhesion and heterophilic cell adhesion could be the regulation of β1 integrin expression. interaction with NOF, a laminin-like molecule. In this report, Regulation of α6β1 integrin expression was already seen for we have established that HEMCAM is the avian orthologue of extracellular signals such as 1α,25 dihydroxybutyrate vitamin MCAM/CD146. In addition, our studies demonstrate that an D3, which reduced α6 integrin expression on melanoma cells, additional function of HEMCAM is to regulate cell adhesion by downregulating the expression of mature β1 integrins at the cell surface of fibroblasts. Based on amino acid comparison, we previously showed that chicken HEMCAM and mammalian MCAM/CD146 were related molecules (Vainio et al., 1996). Here, we further demonstrate that the gene products of these molecules are similarly spliced, coding for proteins with a long and a short cytoplasmic tail. Moreover, human and avian molecules have similar functions: MCAM expression in MCAM-negative melanoma cells and HEMCAM expression in L cells inhibited cell adhesion and spreading to laminin 1. In addition, MCAM, absent from melanocytes, is re- expressed by melanoma cells, and HEMCAM was detected in avian neural crest cells, the precursors of melanocytes. Taken together, our results show that HEMCAM is the avian homologue of MCAM. The main difference between the mammalian and the chicken molecule is the absence of 34 amino acids in chicken HEMCAM located between the last Ig domain and the transmembrane domain. This region is encoded by exon 13 in the mammalian MCAM gene (Sers et al., 1993) and corresponds to a repeat (Gum et al., 1989). Interestingly, such structural differences have previously been encountered in orthologous molecules of different species. For example, in mouse MAdCAM-1, the large mucin-like domain is replaced by an Ig domain in human MAdCAM-1 (Shyjan et al., 1996). Comparison of amino acid sequences of the long cytoplasmic tail of mammalian MCAM and avian HEMCAM shows two conserved putative PKC phosphorylation sites and the YXXL motif. The latter can be involved in endocytosis and sorting of integral membrane proteins to specific cellular compartments (Deschambeault et al., 1999; Eng et al., 1999; Kamiguchi and Lemmon, 1998). The short cytoplasmic tail of Fig. 8. HEMCAM downregulates α3β1, α5β1 and α7β1 integrin expression α β α β MCAM is generated by excision of exon 15 and in L cells. (A) HEMCAM transfection downregulates 5 1 and 7 1 conserves the first PCK phosphorylation site. In addition, integrin expression at the surface of L cells. Cells were analyzed by flow cytometry for surface staining with antibodies against α7β1 integrin (CY8) the short forms of mammalian MCAM and avian or α5β1 integrin (MFR5). The secondary antibody was FITC conjugated. HEMCAM both contain a PDZ interaction site generated Negative controls (grey profiles) were obtained with secondary antibody by a shift in the reading frame due to splicing. Proteins alone. Like α6β1 integrin, α5β1 and α7β1 integrin were not expressed at that bind to such sites contain PDZ domains, which are HEMCAM-transfected cell surface. α5β1 integrin downregulation was found in diverse membrane-associated proteins including confirmed by immunoprecipitation experiments (data not shown). members of the MAGUK family of guanylate kinase (B) Detection of αv and α3 integrin subunits expressed at the cell surface. homologues, several protein phosphatases and kinases, Cell surface proteins were biotinylated and cell lysates were neuronal nitric oxide synthase, and several dystrophin- immunoprecipitated with antibodies against αv integrins (RMV7 mAb) and α associated proteins (Ponting et al., 1997). Most PDZ- 3A integrin cytoplasmic region (7A3 mAb). Electrophoresis of domain proteins localize to specific compartments of the immunoprecipitates (IP) was carried out on a 6% polyacrylamide gel under nonreducing conditions. Immunoprecipitated proteins were detected using plasma membrane and participate in formation of cellular streptavidine-HRP and ECL system. Note that the αv subunit is associated junctions, receptor clustering, channel formation, or not with the β1 chain, but probably with the β3 chain (molecular mass 95 intracellular signalling. The functions of different kDa). c, mock-transfected cells; l, HEMCAM-l-transfected cells; s, cytoplasmic sites of HEMCAM-s and HEMCAM-l are HEMCAM-s-transfected cells. Molecular mass standards (kDa) are indicated not yet clear; they might be involved in other functions on the right. 1856 JOURNAL OF CELL SCIENCE 114 (10)

Fig. 9. Immunoprecipitation of α6 and β1 integrins from mock-, HEMCAM-s- and HEMCAM-l- transfected L cells. c, mock- transfected cells; l, HEMCAM-l- transfected cells; s, HEMCAM-s- transfected cells. (A) Detection of α6 and β1 integrins expressed at the cell surface. Cell surface proteins were biotinylated and cell lysates were immunoprecipitated with antibodies against α6 integrins (EA- 1 mAb), β1 integrin cytoplasmic region (363E pAb) and HEMCAM (c264 mAb). Electrophoresis of immunoprecipitates (IP) was carried out on a 6% polyacrylamide gel under nonreducing conditions. Immunoprecipitated proteins were detected using streptavidine-HRP and ECL system. (B) Detection of total α6 and β1 integrins in L cells. Mock- and HEMCAM-transfected cells were metabolically labelled with [35S]methionine and cysteine. Cell lysates were immunoprecipitated with anti-α6 integrin (EA-1 mAb) or anti-β1 integrin antibodies (363E pAb). Electrophoresis of immunoprecipitates was carried out on a 6% polyacrylamide gel under nonreducing conditions and analyzed by fluorography. Western blot of unlabeled cell extracts was assayed with the anti-β1 antibody to determine precisely the bands corresponding to the β1 integrin subunit. Molecular mass is indicated on the right (kDa). (C) Pulse chase experiment. Mock- and HEMCAM-s-transfected cells were incubated with [35S]methionine/cysteine. After different times of chase, proteins were immunoprecipited with anti-β1 antibodies. Radiolabelled proteins were analyzed by fluorography. In HEMCAM-s-transfected cells, the mature form of β1 integrin was barely detected after 36 hours of chase, whereas it was visible after 12 hours of chase in mock-transfected cells. and IL-12, which increased its expression on human type 1 because immunoprecipitation of metabolically labelled α6 helper T cells (Colantonio et al., 1999; Hansen et al., 1998). integrin confirmed its absence from any other cellular Transfection of L cells by HEMCAM-s, HEMCAM-l or compartments. HEMCAM-t reduces expression of mature β1 integrins at the More likely, transfection of HEMCAM either induced the surface of fibroblasts. This phenomenon is thus independent of rapid degradation of β1 integrins or downregulated its the cytoplasmic part of HEMCAM, like other adhesion translation and/or maturation. Regulation of integrin mRNA characteristics of this molecule (Taira et al., 1999). translation resulting in cell adhesion has been previously Consequently, HEMCAM may regulate integrin expression by reported: integrin occupancy induced formation of focal controlling the translation and/or maturation (cell surface adhesion complexes and translation of pre-existing messengers expression) of β1 and/or α integrin subunits. The (Benecke et al., 1998). Moreover, HUVEC interaction with disappearance of α6 integrin from HEMCAM-transfected cells ECM leads to a relocation of mRNAs and ribosomes to focal was not due to downregulation of transcription because the adhesion complexes (i.e. at sites of signal reception) (Chicurel mRNA transcription level remained constant. In addition, no et al., 1998). As MCAM can promote phosphorylation of the modification of splicing was observed for the α6 or β1 integrin focal adhesion kinase (FAK) leading to interaction with transcripts. Another mechanism for reducing the amount of paxillin (Anfosso et al., 1998; Anfosso et al., 2000), it might integrins from the cell surface has been observed in sensory interfere with the translation of specific mRNAs possibly at neurones that redistribute α6β1 in different intracellular focal adhesion plaques. Because overexpression of HEMCAM compartments (Condic and Letourneau, 1997). However, this led to the disappearance of different β1 integrins, HEMCAM mechanism is unlikely to play a major role in our system, might perturb β1 integrin maturation. Indeed, in HEMCAM- HEMCAM downregulates β1 integrin expression 1857

Fig. 10. HEMCAM overexpression does not regulate α6 integrin gene transcription. (A) HPRT expression was used to standardize cDNA amounts and to allow semiquantitative PCR analysis. Templates were cDNA from mock- and HEMCAM-transfected cell lines and from mouse muscle. Controls correspond to PCR experiments performed in the absence of template. After 30 cycles of amplification, PCR products were electrophoresed, stained with ethidium bromide and photographed. Letters on the left indicate the different spliced isoforms: β1A and β1D integrin subunits, α6A and α6B integrin subunits (Bardin et al., 1996; Tamura et al., 1991). (B) Northern blot analysis of α6 integrin mRNA in cell lines. Total RNA was analyzed by northern blot using a [α-32P]dATP- labelled murine α6 cDNA probe. The relative amounts of RNA loaded for each cell line can be evaluated on the ethidium bromide staining gel (right). The hybridization signal is detected as a single band and the intensity is the same for each cell line RNA, confirming results obtained by RT-PCR. transfected cells, β1 integrin subunit is stabilized in its Fournier-Thibault and Dominique Alfandari for critical reading and precursor form. Thus, HEMCAM might interfere with a improvement of the manuscript. We also thank Randall Kramer, chaperone molecule, such as calnexin, known to participate in Douglas De Simone, Arnoud Sonnenberg and Annemieke de Melker integrin maturation (Lenter and Vestweber, 1994). Finally, for the kind gifts of the CY8, 363E, GoH3 and 7A3 antibodies, HEMCAM might be involved in the regulation of the respectively. This work was supported by the CNRS contract Biologie α cellulaire no. 96108, by the Association pour la Recherche contre le degradation pathway of integrin subunits. Cancer (ARC) no. 6982 and 9738, by the Fondation pour la Recherche The role of HEMCAM/CD146 in cell adhesion is complex. Médicale (FRM), the Ligue Nationale Contre le Cancer (LNCC), the This molecule interacts, on the one hand, with NOF, a laminin- Human Frontier Science Programme Organization (HFSP) no. like molecule of the extracellular matrix and, on the other hand, RG366/96, the Academy of Finland and by the Swiss National probably with an unknown cell adhesion molecule at the Science Foundation. S. A. was supported by a LNCC and a ARC surface of other cells. Moreover, several experiments suggest Fellowship. that HEMCAM interacts homophilically (Taira et al., 1994; Taira et al., 1995; Taira et al., 1999; Vainio et al., 1996). In addition, our data show that, in L cells, HEMCAM regulates REFERENCES cell adhesion by controlling β1 integrin expression at the cell surface. Thus the level of HEMCAM expression might Anfosso, F., Bardin, N., Francès, V., Vivier, E., Camoin-Jau, J., Sampol, J. determine the specific laminin that is recognized by fibroblasts: and Dignat-Georges, F. (1998). 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