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Vol. 18, Issue 5, 1621-1633, May 2007

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The Short Arm of Laminin 2 Chain of Laminin-5 (Laminin-332) Binds Syndecan-1 and Regulates Cellular Adhesion and Migration by Suppressing Phosphorylation of Integrin 4 Chain

Takashi Ogawa^*,, Yoshiaki Tsubota^*,, Junko Hashimoto^*,, Yoshinobu Kariya^*,, and Kaoru Miyazaki^*,

*Division of Cell Biology, Kihara Institute for Biological Research, and Graduate School of Integrated Sciences, Yokohama City University, Yokohama 244-0813, Japan; and Kihara Memorial Yokohama Biotechnology Foundation, Yokohama 244-0813, Japan

Submitted September 11, 2006; Revised January 10, 2007; Accepted February 9, 2007
Monitoring Editor: Mark Ginsberg

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The proteolytic processing of laminin-5 at the short arm of the 2 chain (2sa) is known to convert this laminin from a cell adhesion type to a motility type. Here, we studied this mechanism by analyzing the functions of 2sa. In some immortalized or tumorigenic human cell lines, a recombinant 2sa, in either soluble or insoluble (coated) form, promoted the adhesion of these cells to the processed laminin-5 (Pr-LN5), and it suppressed their migration stimulated by serum or epidermal growth factor (EGF). 2sa also suppressed EGF-induced tyrosine phosphorylation of integrin 4 and resultant disruption of hemidesmosome-like structures in keratinocytes. 2sa bound to syndecan-1, and this binding, as well as its cell adhesion activity, was blocked by heparin. By analyzing the activities of three different 2sa fragments, the active site of 2sa was localized to the NH₂-terminal EGF-like sequence (domain V or LEa). Suppression of syndecan-1 expression by the RNA interference effectively blocked the activities of domain V capable of promoting cell adhesion and inhibiting the integrin 4 phosphorylation. These results demonstrate that domain V of the 2 chain negatively regulates the integrin 4 phosphorylation, probably through a syndecan-1–mediated signaling, leading to enhanced cell adhesion and suppressed cell motility.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The basement membrane proteins laminins play essential roles not only in the tissue architecture but also in the regulation of cellular functions (Timpl and Brown, 1996). Laminins are large glycoproteins consisting of three different subunits (, , and chains) linked by disulfide bonds, and each subunit has many functional domains (Aumailley et al., 2005). Therefore, they show a variety of biological activities. For example, laminins regulate cellular adhesion, motility, growth, differentiation, and apoptosis, through interaction with specific receptors on the cell surface (Timpl and Brown, 1996; Colognato and Yurchenco, 2000). The COOH-terminal globular (G) domain of the chain, which consists of five laminin globular (LG) modules (LG1-5), is a major site to interact with cell surface receptors such as integrins, syndecans, and dystroglycan. The NH₂-terminal regions, often called short arms, of the three chains are thought to be mainly involved in the matrix assembly of laminins in basement membranes. Several laminin isoforms, which are produced by different combinations of the , , and chains, are expressed in a tissue-specific manner during embryonic development as well as in the adult (Miner et al., 1997; Colognato and Yurchenco, 2000).

Laminin-5 (332; laminin-332), abbreviated here as LN5, is a major laminin isoform in the skin basement membrane (Carter et al., 1991; Rousselle et al., 1991) and widely expressed in other epithelial tissues as a relatively minor component (Miner et al., 1997; Mizushima et al., 1998). In vitro, LN5 strongly promotes cellular scattering, migration, and adhesion through the interaction with integrins 31, 61, and 64 (Kikkawa et al., 1994; Rousselle and Aumailley, 1994; Miyazaki, 2006). The cell adhesion activity of LN5 contributes to the tight adhesion of basal keratinocytes to the underlying connective tissue in the skin, which is mediated by the association of LN5 with integrin 64 in the hemidesmosome structures (Baker et al., 1996). Therefore, structural defects of LN5 subunits, integrin 64, or other hemidesmosome components cause severe skin blistering in humans and experimental animals (Aberdam et al., 1994). In contrast, the cell migration-promoting activity of LN5 is thought to contribute to wound healing (Ryan et al., 1994) and tumor invasion (Pyke et al., 1995). These unique biological activities are likely to depend on its structural feature. All of the three LN5 subunits are truncated in their short arms (NH₂-terminal regions), and the 3 and 2 chains are found only in LN5. However, it is unknown how the apparently opposite functions of LN5, i.e., stable cell adhesion and cell migration, are regulated. In this context, much attention has been focused on the proteolytic processing of LN5 that modulates its biological activities (Miyazaki, 2006).

Human LN5 is synthesized and secreted as a precursor form consisting of a 190-kDa 3 chain, a 135-kDa 3 chain, and a 150-kDa 2 chain. After secretion, the 3 and 2 chains undergo specific extracellular proteolytic processing to convert to the mature form containing a 160-kDa 3 chain and a 105-kDa 2 chain, respectively. For the 3 chain, the 190-kDa 3 chain is almost completely converted to the 160-kDa form immediately after secretion. The proteolytic cleavage of the 190-kDa 3 chain, which occurs between the LG3 and LG4 modules in the G domain (Hirosaki et al., 2000; Tsubota et al., 2000), increases the biological activity of LN5 and laminin-6 (311; laminin-311) (Tsubota et al., 2005; Hirosaki et al., 2002). Structural studies have shown that the major integrin-binding site in LN5 and laminin-6 is located in the LG3 domain of the 3 chain (Hirosaki et al., 2000; Kariya et al., 2003), and the LG4–LG5 domain is important for the matrix assembly of the laminin (Sigle et al., 2004; Tsubota et al., 2005). In contrast, the 150-kDa 2 chain of LN5 is partially processed to the 105-kDa form in many LN5-producing human cell lines. Bone morphogenic protein-1/mammalian Tolloid metalloproteinase family, including mammalian Tolloid-like-1 and mammalian Tolloid-like-2, are thought to be responsible for the 2 chain processing in human LN5 (Veitch et al., 2003). In rat LN5, however, the 2 chain is processed from the 150-kDa form to the 80-kDa mature form by gelatinase A (matrix metalloproteinase [MMP]-2) or membrane type 1-MMP, and this processing enhances the cell motility activity of the LN5 (Giannelli et al., 1997; Koshikawa et al., 2000). In human LN5, the processing of the 150-kDa 2 chain to the 105-kDa mature form decreases the cell adhesion activity but increases the cell migration activity of LN5 (Ogawa et al., 2004). Furthermore, many immunohistochemical studies have shown that the laminin 2 chain is overexpressed at the invasion front of human cancers (Pyke et al., 1995; Koshikawa et al., 1999). These results imply that the short arm of the 2 chain contains an important, functional domain that regulates cellular adhesion and migration. The active site of the 2 chain may bind some cell surface receptors other than integrins, leading to the modulation of the integrin-mediated signaling. To test these possibilities, we examined the biological functions of the 2 chain short arm in this study.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Antibodies and Laminins
Mouse monoclonal antibodies (mAbs) against the human laminin 3 chain (LS3c4) and 2 chain (D4B5) were established and characterized previously (Mizushima et al., 1998; Koshikawa et al., 1999). Mouse mAbs against integrin 4 (1A3) and BP180 (233) were generous gifts from Dr. K. Owaribe (Graduate School of Human Informatics, Nagoya University, Nagoya, Japan) (Okumura et al., 2002; Hirako et al., 2003). Other antibodies used and their sources were as follows: a mouse mAb against the human laminin 3 chain from BD Biosciences Transduction Laboratories (Lexington, KY), a mouse mAb against integrin 4 (3E1) from Chemicon International (Temecula, CA), a mouse mAb against integrin 4 (450-11A) from BD Bioscience (San Jose, CA), a polyclonal antibody against epidermal growth factor (EGF) receptor (Ab4) from Oncogene Research Products (Boston, MA), a mouse mAb against EGF receptor (LA1) from Upstate Biotechnology (Lake Placid, NY), and a mouse mAb against phospho-tyrosine (PY-20) from BD Biosciences Transduction Laboratories. A recombinant human LN5 with a 150-kDa nonprocessed 2 chain (Np-LN5) and a natural LN5 with the 105-kDa processed 2 chain (Pr-LN5) were prepared as reported previously (Ogawa et al., 2004).

Cells and Culture Conditions
The human bladder carcinoma cell line EJ-1, the Buffalo rat liver cell line BRL and the human epideromoid carcinoma cell line A431 have been used in previous studies (Kariya et al., 2003). The human embryonic kidney cell line HEK293 was obtained from American Type Culture Collection (Manassas, VA). These cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS). The spontaneously immortalized human keratinocyte line HaCaT (Boukamp et al., 1988), which was a generous gift from Dr. N. E. Fusenig (Deutsches Krebsforschungszentrum, Heidelberg, Germany), was maintained in DMEM supplemented with 10% FCS. The immortalized human mammary epithelial cell line MCF-10A (ATCC CRL-10317) was obtained from American Type Culture Collection and cultured in DMEM/F12 supplemented with 20 ng/ml EGF, 100 ng/ml cholera toxin, 0.01 mg/ml insulin, 500 ng/ml hydrocortisone, and 5% horse serum.

Construction of Expression Vectors and Cell Transfection
cDNAs for the laminin 2 chain and its NH₂-termianl short arm were cloned from a human gastric carcinoma cell line (STKM-1) in our laboratory (Tsubota, Ogawa, and Miyazaki, unpublished data). Briefly, the total RNA was extracted from STKM-1, reverse-transcribed with random primers, and used as a template for further polymerase chain reaction (PCR). The cDNA encoding the laminin 2 chain (nucleotides 85-3702) was amplified as four overlapping fragments, according to the reported sequence (Kallunki et al., 1992). To construct a cDNA fragment of the 2 short arm (2sa), which consists of the NH₂-terminal domains III (or LEb), IV (or L4), and V (or LEa), a SpeI–NdeI fragment (nucleotides 85-1262) and a NdeI–EcoRI fragment (nucleotides 1020-1942) of the 2 cDNA were ligated into the SpeI–EcoRI site of pBluescript II KS (+). The 2sa cDNA fragment was finally inserted into the mammalian expression vector pBOS-CITE-Neo, which expresses the inserted cDNA and a neomycin-resistant gene within one mRNA molecule (Ogawa et al., 2004). EJ-1 cells, which expressed none of the laminin 3, 3, and 2 chains, were transfected with the expression vector by the calcium-phosphate precipitation method and incubated in medium containing 500 µg/ml Geneticin (G-418; Sigma-Aldrich, St. Louis, MO). A Geneticin-resistant EJ-1 cell clone was used as EJ-1/2sa cells for the purification of the 2sa protein.

The following three additional 2 cDNA fragments were prepared by PCR by using the 2sa cDNA as a template: 2pf cDNA (nucleotides 199-1419) encoding the full length of domains V and IV and a small sequence of domain III, 2dV cDNA (nucleotides 199-705) encoding only domain V, and 2dIII cDNA (nucleotides 1420-1935) encoding most of domain III. The primer sequences used in the PCR were as follows: 2pf sense, 5'-TAGGTACCTGTGATTGCAATGGGAAG-3'; 2pf antisense, 5'-ATCTCGAGCCCCTGAATAACAATCTCCTGT-3'; 2dV sense, 5'-TAGGTACCTGTGATTGCAATGGGAAG-3'; 2dV antisense, 5'-ATCTCGAGCGCAGCTGGCTGAATG-3'; 2dIII sense, 5'-TAGGTACCGATGAGAATCCTGAC-3'; and 2dIII antisense 5'-ATCTCGAGCTGCTCCATGCTCAC-3'. The sense primers contained an additional KpnI site, whereas the antisense primers contained an additional XhoI site to allow insertion to an expression vector. The PCR products were then inserted into the T-easy vector (Promega, Madison, WI). These constructs was subcloned into the KpnI–XhoI sites of the mammalian expression vector pSectag2B (Invitrogen, Carlsbad, CA), which expresses a secreted protein linked with a histidine hexamer at the COOH terminus. The cDNA constructs were transfected into the human embryonic kidney cell line HEK293. Zeocine-resistant cell clones were then selected for high-level secretion of the deletion proteins.

Preparation of LN5 and Recombinant Proteins of Laminin 2 Short Arm
A natural human LN5 consisting of a 160-kDa mature 3 chain, a 135-kDa 3 chain, and a 105-kDa processed 2 chain, tentatively named Pr-LN5, was prepared from the conditioned medium of the human squamous cell carcinoma line HSC-4 as described previously (Ogawa et al., 2004). A recombinant human LN5 consisting of a 160-kDa 3 chain, a 135-kDa 3 chain, and a 150-kDa, nonprocessed 2 chain, tentatively named Np-LN5, was prepared from the conditioned medium of the human gastric carcinoma line HSC-4 expressing an exogenous, mutated 2 chain (Ogawa et al., 2004). Briefly, these LN5 proteins were purified by successively applying the serum-free conditioned media to molecular-sieve chromatography on a Sepharose 4B column and to immunoaffinity chromatography with an anti-2-chain mAb (D4B5), according to the method published previously (Hirosaki et al., 2000). The 2 short arm protein (2sa) was purified from the serum-free conditioned medium of EJ-1/2sa cells by essentially the same method as described above. To purify three 2 short arm fragments, HEK293 cell lines transfected with 2pf, 2dV, or 2dIII cDNA were grown to confluence in serum-containing medium and then incubated in serum-free medium for 2 d (Kariya et al., 2003). The serum-free conditioned media containing secreted His-tagged proteins (2pf, 2dV, or 2dIII) were collected, concentrated 500-fold by ammonium sulfate precipitation at 80% saturation, and dialyzed against 50 mM phosphate, pH 7.8, buffer containing 300 mM NaCl. Proteins from 1 liter of the conditioned medium were applied to a NiSO₄-conjugated chelating Sepharose column (2 ml) (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). The column was washed with 20 ml of the same phosphate buffer, and the remaining proteins on the column were successively eluted with 3 ml of 50, 100, 300 and 500 mM imidazole in 100 mM phosphate buffer, pH 6.0, containing 300 mM NaCl. The majority of 2pf, 2dV, and 2dIII proteins were eluted at 300 mM imidazole and dialyzed against Ca²⁺/Mg²⁺-free phosphate-buffered saline (PBS). The 2pf and 2dV fractions were finally applied to a heparin-Sepharose column (1 ml) (HiTrap Heparin HP column; GE Healthcare) and eluted with a NaCl gradient. 2pf and 2dV were eluted at 0.36–0.40 M NaCl and 0.30–0.4 M NaCl, respectively. 2dIII was finally purified using the immunoaffinity column of the anti-2-chain mAb (D4B5) as described above. Approximately 500 µg of 2pf, 200 µg of 2dV, and 550 µg of 2dIII were purified from 1 liter of the conditioned media, respectively.

Assays of Cell Adhesion Activity
The cell adhesion assay was performed as described previously (Ogawa et al., 2004). Microtiter plates (96-well; Corning Life Sciences, Acton, MA) were coated with appropriate concentrations of substrate proteins in PBS at 4°C overnight and then blocked with 1.2% (wt/vol) bovine serum albumin (BSA) at 37°C for 1.5 h. Cells were suspended in serum-free medium at a density of 24 x 10⁵ cells/ml, and a 100-µl aliquot was inoculated per well of the plates. After incubation at 37°C for 1 h, nonadherent cells were removed after gentle agitation, and adherent cells were fixed with 2.5% (wt/vol) glutaraldehyde and stained with 0.0005% (wt/vol) Hoechst 33342 in 0.001% (wt/vol) Triton X-100 for 1.5 h. The fluorescent intensity of each well of the plates was measured using a CytoFluor 2350 fluorometer (Millipore, Bedford, MA).

Assays of Cell Migration
Cell migration speed was assayed as reported previously (Hirosaki et al., 2000). Each well of 24-well plastic plates was coated with indicated concentrations of test substrates and then blocked with BSA as described above. EJ-1 cells (1.0 x 10⁴ cells in DMEM/F-12 plus 1% FCS) were inoculated per well of the plates. After preincubation for 1 h at 37°C, cell movement was monitored for 10 h by using a time-lapse video, and the cell migration distance was determined by measuring the total length of the random path that each cell covers, using a video micrometer (VM-30; Olympus, Tokyo, Japan).

Identification of Membrane Receptor of 2sa
Membrane proteins of EJ-1 cells were collected according to the method of Hoffman et al. (1998) and then incubated with a 2sa-conjugated beads, which had been prepared by binding the purified 2sa to Affigel-10 beads (Bio-Rad, Hercules, CA), in PBS buffer at 4°C overnight. The incubated beads were washed with the buffer several times, and 2sa-bound proteins were eluted from the beads with 1 M NaCl. The eluted proteins were precipitated by 10% (wt/vol) trichloroacetic acid and dissolved in 20 mM HEPES-NaOH, pH 7.5, buffer supplemented with a protease inhibitor mixture (Wako Pure Chemicals, Osaka, Japan). To identify heparan sulfate proteoglycans (HSPGs), protein samples (2 µg) were treated with 0.5 U/ml heparitinase (Seikagaku Kogyo, Tokyo, Japan) at 37°C for 4 h and analyzed by immunoblotting with the mouse anti-heparan-sulfate mAb 3G10 (Seikagaku Kogyo), which reacts with a heparan sulfate neo-epitope generated by the heparitinase digestion, with the anti-syndecan-1 mAb B-B4 (AbD; Serotec, Oxford, United Kingdom), the goat anti-syndecan-2 polyclonal antibody L-18 (Santa Cruz Biotechnology, Santa Cruz, CA), or the goat anti-syndecan-4 polyclonal antibody N-19 (Santa Cruz Biotechnology).

Analysis of Phosphorylation of Integrin 4 in 2sa-treated Cells
Cells were serum starved for 48 h, harvested, and suspended in serum-free medium at a density of 1 x 10⁶ cells/ml, and a 2-ml aliquot was inoculated per 60-mm culture dish (Sumibe Medical, Tokyo, Japan), which had been precoated with 1 µg/ml LN5 and blocked with BSA as described above. After incubation at 37°C for 2 h, nonadherant cells were removed by washing the cultures with PBS, and the remaining adherent cells were further incubated in serum-free medium supplemented with 50 ng/ml EGF in the presence or absence of 2sa (0.4 or 0.8 µg/ml) for 10 min. The cells were then lysed in a lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride and 1% Triton X-100), harvested, and centrifuged at 15,000 x g for 10 min. The resultant supernatants were incubated with an anti-mouse immunoglobulin G antibody affinity gel (ICN, Aurora, OH) conjugated with the anti-integrin-4 mAb 3E1 at 4°C for 12 h. The immunoprecipitates thus obtained were extensively washed with the lysis buffer, dissolved in the SDS-buffer containing 2-mercaptoethanol, and subjected to immunoblotting with the anti-phospho-tyrosine antibody PY-20. In some experiments, cell lysates were directly subjected to the immunoblotting.

Immunofluorescence Microscopy of Hemidesmosome-like Structures
HaCaT cells were incubated in serum-free DMEM for 48 h. The serum-starved cells were trypsinized, washed with the medium containing 1 mg/ml soybean trypsin inhibitor, and suspended in the serum-free DMEM at a density of 2.5 x 10⁵ cells/ml. A 250-µl portion of the cell suspension was inoculated per well of eight-well Lab-Tek chamber slides (Nalge Nunc, Naperville, IL), which had previously been coated with LN5, and incubated at 37°C for 3 h. Adherent cells were further treated with EGF and/or 2sa as described above. The cultures were then rinsed with cooled PBS, fixed in 10% (wt/vol) Formalin in PBS for 15 min, and washed three times with PBS. The cells were permeabilized with 0.2% (vol/vol) Triton X-100 in PBS for 15 min, blocked with 10% horse serum in PBS for 15 min, and then incubated with a primary antibody diluted in 3% horse serum in PBS at 4°C for 12 h. A fluorescein isothiocyanate-coupled secondary antibody (Vector Laboratories, Burlingame, CA) was used for detection. Fluorecsence images were obtained using a fluorescence microscope (model BZ-8000; Keyence, Osaka, Japan).

SDS-Polyacrylamide Gel Electrophoresis (PAGE) and Immunoblotting Analyses
SDS-PAGE was performed on 5, 6, or 10% polyacrylamide gels under reducing or nonreducing conditions. In analyses of purified proteins, separated proteins were stained with a Wako silver staining kit II (Wako Pure Chemicals). In immunoblotting analysis, proteins resolved by SDS-PAGE were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) and visualized using an enhanced chemiluminescence (ECL) Western blotting kit (GE Healthcare) with specific antibodies.

Suppresion of Syndecan-1 Expression by RNA Interference
A predesigned small interference RNA (siRNA) corresponding to the target sequence for human syndecan-1 and a control short RNA (#4611) were obtained from Applied Biosystems (Foster City, CA). The target sequence was 5'-GGAGGAAUUCUAUGCCUGA-tt-3' (#16704, sense) (Beauvais et al., 2004). To transfect these RNAs, EJ-1 cells were inoculated the day before transfection at a cell density of 20–30% saturation in 60-mm culture dishes and treated with the control RNA or the siRNA by using the HiPerFect reagent (QIAGEN, Tokyo, Japan) according to the manufacturer's protocol. Three days later, the cells were used for the analysis of syndecan-1 expression by immunoblotting and other experiments.

Determination of Protein Concentrations
Protein concentrations were determined using a Bio-Rad protein assay kit with BSA as a standard, unless otherwise noted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Biological Activity of Laminin 2 Chain Short Arm
The 2 chain of human LN5 is cleaved at a specific site of the short arm by extracellular proteinases (Figure 1A). Previously, we found that a recombinant human LN5 with a 150-kDa 2 chain (Np-LN5) has an 5-times higher cell adhesion activity and an 2-times lower cell migration activity than the mature LN5 with the 105-kDa 2 chain (Pr-LN5). This suggests that the short arm of the 2 chain contains a functional domain that regulates cellular adhesion and migration. To examine this possibility, we constructed an expression vector for the short arm of laminin 2 chain, and we introduced it into the human bladder carcinoma cell line EJ-1 (Figure 1B). The 2 short arm, named 2sa, was purified from the conditioned medium of the EJ-1 cells by affinity chromatography on an anti-laminin-2 mAb (D4B5) column. The purified material exhibited a single band of 70 kDa on SDS-PAGE under reducing conditions (Figure 1C).

View larger version (15K): [in this window] [in a new window]   Figure 1. Domain structures of LN5 and recombinant 2 proteins. (A) Schematic structure of LN5 molecule (332) and its proteolytic processing sites. Top closed arrow, cleavage site in the human laminin 2 chain to produce the 105-kDa chain and the 45-kDa 2 fragment (2pf); open arrow, second cleavage site in the rat laminin 2 chain to produce the 80-kDa chain and the 25- and 45-kDa fragments; bottom arrow, cleavage site in the human laminin 3 chain to produce the 160-kDa 3 chain and a LG4–5 fragment; LG, laminin carboxyl-terminal globular domain of the 3 chain. Roman numerals indicate the domains of the 2 chain: domains III (or LEb), IV (or L4), and V (or LEa). (B) Schematic diagram of the structures of the full-length human laminin 2 chain and recombinant 2 proteins. Numerals indicate the positions of amino acid residues from the NH2-terminus. Shadow boxes indicate the signal sequence. (C) SDS-PAGE of the purified 2 chain short arm (2sa) on a 10% gel under reducing condition. Proteins were stained with silver.

View larger version (20K): [in this window] [in a new window]   Figure 2. Cell adhesion activity of 2sa in presence or absence of Pr-LN5. (A) Effect of varied concentrations of 2sa with or without fixed concentrations of Pr-LN5 on adhesion of EJ-1 cells to plastic plates. Plates (96-well) were precoated without () or with 0.1 µg/ml (), 0.2 µg/ml (), or 0.4 µg/ml () of Pr-LN5 and then coated with the indicated concentrations of 2sa, followed by blocking with BSA. EJ-1 cells were plated into each well in serum-free medium and incubated at 37°C for 1 h. After the incubation, relative numbers of adherent cells were determined as described in Materials and Methods. Each value represents the mean ± SD (bar) of triplicate assays. (B) Effect of varied concentrations of Pr-LN5 with or without a fixed concentration of 2sa on adhesion of EJ-1 cells to plastic plates. Pr-LN5 was coated at the indicated concentrations without () or with () 0.8 µg/ml 2sa on 96-well plates. The adhesion of EJ-1 cells onto the plates was measured as described above. (C) Effect of addition of soluble 2sa on adhesion of EJ-1 cells to Pr-LN5. Plastic plates were coated with 0.3 mg/ml Pr-LN5 and blocked with BSA. After EJ-1 cells were plated on the plates, 2sa was directly added to the serum-free culture medium and incubated for 1 h. Adherent cells were quantified as described above.

View larger version (22K): [in this window] [in a new window]   Figure 3. Inhibitory effect of 2sa on cell migration on Pr-LN5 substrate. (A) Effect of insoluble (or coated) 2sa on migration of EJ-1 cells on Pr-LN5 substrate. Plates (24-well) were cocoated with the indicated concentrations of 2sa and 0.3 µg/ml Pr-LN5 and then blocked with BSA. EJ-1 cells (10,000 cells) were inoculated per well in DMEM/F-12 medium supplemented with 1% FCS. After preincubation for 1 h to allow cell spreading, the migration of EJ-1 cells was monitored by video for 10 h. Each point represents the mean ± SD of the cell migration speed of 20 cells. The cell migration speed at 0.8 µg/ml 2sa is significantly lower than the control value in the absence of 2sa (p < 0.001). Other experimental conditions are described in Materials and Methods. (B) Effect of soluble 2sa on migration of four kinds of cell lines. EJ-1 (), BRL (), A431 (), or MCF-10A () cells were inoculated into each well precoated with 0.3 µg/ml Pr-LN5, and the indicated concentration of 2sa was added to the culture medium containing 1% FCS. After preincubation for 1 h, the cell migration speed was measured as described above. In the four cell lines, the cell migration speeds at 0.8 µg/ml 2sa are significantly lower than the respective control value (p < 0.001). (C) Effect of soluble 2sa on EGF-induced migration of EJ-1 cells on Pr-LN5 substrate in serum-free medium. EJ-1 cells were inoculated into each well, which had been coated with 0.3 µg/ml Pr-LN5, in serum-free DMEM/F-12 medium without () or with () 0.05 µg/ml EGF, and the indicated concentration of 2sa was added to the culture medium. The cell migration speed was measured as described above. The differences in the cell migration speeds between 0.0 and 0.4 or 0.8 µg/ml 2sa are statistically significant in the presence of EGF (p < 0.001) but insignificant in its absence (p > 0.05).

Effect of 2sa on EGF-induced Phosphorylation of Integrin 4

View larger version (35K): [in this window] [in a new window]   Figure 4. Inhibitory effect of 2sa on EGF-induced phosphorylation of integrin 4. (A) Serum-starved EJ-1, A431, or HaCaT cells were suspended in serum-free DMEM/F-12 medium and inoculated into culture dishes that had previously been coated with 0.5 µg/ml Pr-LN5 and then blocked with BSA. After incubation for 1.5 h, the cultures were added with 0.05 µg/ml EGF and without (None) or with 0.4 µg/ml soluble 2sa (2sa). After further incubation for 10 min, cell lysate was prepared from each culture. The samples were subjected to SDS-PAGE and subsequent immunoblotting (I.B.) with a mAb against PY-20. The 175-kDa band was identified as EGF receptor (EGF-R), and the 205-kDa band was identified as integrin 4 (Int.4), as shown below. Other experimental conditions are described in Materials and Methods. (B) A431 cells were treated with 0.4 or 0.8 µg/ml 2sa and 0.05 µg/ml EGF on the Pr-LN5 substrate. Integrin 4 present in the cell lysates was subjected to immunoprecipitation (I.P.) with the anti-integrin 4 mAb 3E1. The immunoprecipitates were analyzed by immunoblotting with the antibodies against PY-20, integrin 4 (Int.4) and EGF-R (left). Relative intensity of the 205-kDa immunosignal for the integrin 4 phosphorylation was determined using the NIH Image software (right). Each value represents the mean intensity ± SD of three separate experiments. The relative intensity of the control culture (None) was regarded as 100. (C and D) Serum-starved HaCaT cells were treated with 0.18 or 0.8 µg/ml soluble 2sa in addition to 0.05 µg/ml EGF in culture dishes that had been pre-coated with 1 µg/ml Pr-LN5 (Pr) or Np-LN5 (Np) as described above. The tyrosine phosphorylation of integrin 4 (arrows) and EGF receptor (arrowheads) in the cell lysates were analyzed before (C) and after (D) immunoprecipitation with the anti-integrin 4 mAb 3E1.

To further investigate the role of 2sa in the regulation of integrin functions, we compared the phosphorylation of integrin 4 in HaCaT cells incubated in three different conditions, i.e., Pr-LN5 alone, Pr-LN5 plus 2sa, and Np-LN5 alone (Figure 4, C and D). When Pr-LN5 and Np-LN5 were compared using their total cell lysates, the cells incubated on Np-LN5 showed a slightly lower level of integrin 4 phosphorylation compared with those on Pr-LN5 (Figure 4C). There was no significant difference in the phosphorylation level of EGF receptor between the cells incubated on Pr-LN5 and those on Np-LN5. The differential phosphorylation of integrin 4 between Pr-LN5 and Np-LN5 was more clearly shown by the analysis after immunoprecipitation with the anti-integrin-4 antibody (Figure 4D). When cells were incubated on Pr-LN5 with or without the same molar concentration (0.18 µg/ml) of soluble 2sa, the treatment with 2sa did not significantly affect the EGF-induced phosphorylation of integrin 4 (Figure 4D). However, when the concentration of 2sa was increased to 0.8 µg/ml, it significantly decreased the phosphorylation of integrin 4 compared with the cells incubated on Pr-LN5 alone. These results suggest that the changes of the biological activities of LN5 induced by the proteolytic cleavage of the 2 short arm may be mediated by the increased phosphorylation of integrin 4. The results also imply that the effect of 2sa is greater in its presence within the LN5 molecule than in the released form.

Effect of Laminin 2 Chain Short Arm on Disassembly of Hemidesmosome-like Structures Induced by EGF
Because tyrosine phosphorylation of integrin 4 is known to disrupt hemidesmosomes (Mainiero et al., 1997; Mariotti et al., 2001), we next examined the effect of 2sa on hemidesmosome structures of HaCaT keratinocytes. Under an immunofluorescent microscope, hemidesmosomes look like punctuated structures, which tend to coalesce around circular areas (Mariotti et al., 2001). Immunofluorescent staining with an anti-BP180 antibody revealed many hemidesmosome-like, punctuated structures in the cells treated with Pr-LN5 alone, Pr-LN5 plus soluble 2sa, and NP-LN5 alone (Figure 5A). When these cells were additionally treated with EGF, the punctuated structures of the cells on Pr-LN5 became faint and diffused. However, the immunostaining patterns of the cells on Np-LN5 and those on Pr-LN5 with soluble 2sa were scarcely affected by the EGF treatment. In contrast, the immunofluorescent staining with the anti-integrin-4 antibody of HaCaT cells showed no significant difference among the three different conditions regardless of the EGF treatment (Figure 5B). This suggested that the association of LN5 with integrin 64 was maintained even after the EGF treatment, although the interaction between integrin 64 and BP230/180 in the cells on Pr-LN5 was lost after the EGF treatment. These results demonstrate that the short arm of 2 chain prevents the disassembly of hemidesmosome-like structure induced by EGF.

View larger version (77K): [in this window] [in a new window]   Figure 5. Effect of 2sa on disassembly of hemidesmosome-like structures induced by EGF. Serum-starved HaCaT cells were inoculated in serum-free medium and incubated for 8 h on culture dishes that had been coated with 1.0 µg/ml Pr-LN5 or Np-LN5. The cultures were then treated for 10 min with 0.4 µg/ml soluble 2sa (2sa) in the presence (+EGF) or absence (None) of 0.05 µg/ml EGF. The treated cells were subjected to immnofluorescence staining with an anti-BP180 mAb (A) and the anti-integrin-4 mAb (B). Other experimental conditions are described in Materials and Methods.

Identification of Membrane Receptor for 2sa

To examine this possibility, effect of heparin on the cell adhesion activity of 2sa was examined using EJ-1 cells. As shown above, 2sa promoted the cell adhesion in synergy with Pr-LN5. Heparin almost completely blocked the cell adhesion activity of 2sa (Figure 6, A and B). However, chondroitin sulfate, a different type of sulfated glycosaminoglycans, failed to block the cell adhesion. These results suggest that 2sa specifically interacts with heparan sulfates.

View larger version (65K): [in this window] [in a new window]   Figure 6. Effects of heparin and chondroitin sulfate on adhesion of EJ-1 cells to culture plates coated with both Pr-LN5 and 2sa. (A) Plastic plates were coated with 0.3 µg/ml Pr-LN5 and then with 0.8 or 1.6 µg/ml 2sa, followed by blocking with BSA. EJ-1 cells were suspended in serum-free medium supplemented without (None) or with 20 µg/ml heparin or 20 µg/ml chondroitin sulfate ABC (Chondroitin sulfate), inoculated on the precoated plates, and incubated at 37°C for 1 h. The resultant cultures were examined under a phase-contrast microscope. Original magnification, 300x. (B) EJ-1 cells were incubated in serum-free medium without () or with heparin () or chondroitin sulfate () on plates that had been coated with 0.3 µg/ml Pr-LN5 and the indicated concentrations of 2sa. Adherent cells were quantified as shown in Figure 2. Other experimental conditions are described in Materials and Methods.

View larger version (28K): [in this window] [in a new window]   Figure 7. Identification of receptor for 2 short arm. (A) Surface membrane proteins of EJ-1 cells were biotinylated with Biotin-OSu (Dojindo, Kumamoto, Japan), and the labeled proteins were passed through a 2sa-conjugated column (2sa) or a control column without 2sa (Cont.). Bound materials were eluted with 1 M NaCl, separated by nonreducing SDS-PAGE on a 5–12% gradient gel, and electrotransferred to a nitrocellulose membrane. The transferred membrane proteins were detected with the horseradish peroxidese-avidin D/ECL method. The arrow indicates molecules specifically bound to the 2sa column. (B) The labeled membrane proteins were passed through columns conjugated with 0, 0.25, or 1.0 µg/ml 2sa in the absence (–) or presence (+) of 1.0 µg/ml heparin. Bound materials were eluted from the column, treated with heparitinase, and immunoblotted with an anti-heparan sulfate antibody (top) and an anti-syndecan-1 antibody (bottom). Arrowheads, syndecan-1 core proteins and their apparent molecular sizes in kilodaltons.

Identification of Active Site of 2 Chain

View larger version (21K): [in this window] [in a new window]   Figure 8. Biological activities of three NH2-terminal 2 fragments: 2pf, 2dV, and 2dIII. (A) SDS-PAGE of purified 2dIII (dIII), 2pf (pf), and 2dV (dV) on a 12.5% gel under reducing conditions. Proteins were stained with Coomassie brilliant blue R-250. (B) Effects of varied concentrations of 2dIII, 2pf, and 2dV on adhesion of EJ-1 cells to Pr-LN5. Plates (96-well) were coated without () or with 0.125 µg/ml (), 0.25 µg/ml µg/ml (), or 0.5 µg/ml () of Pr-LN5 and then with the indicated concentrations of 2dIII (top left), 2pf (top right), or 2dV (bottom). After blocking with BSA, EJ-1 cells were plated on each substrate and incubated at 37°C for 1 h. Relative numbers of adherent cells were determined as described in Figure 2 and Materials and Methods. Each value represents the mean ± SD of triplicate assays. One micorgram per milliliter is equivalent to 32.3 nM for 2dIII, 16.7 nM for 2pf, and 35.7 nM for 2dV. (C) Inhibitory effects of 2pf and 2dV on migration of EJ-1 cells on Pr-LN5 substrate. Plates (24-well) were coated with 0.3 µg/ml Pr-LN5 and then with 0.8 µg/ml (13.3 nM) 2pf or 0.4 µg/ml (14.3 nM) 2dV, followed by blocking with BSA. The cell migration speed on the plates was determined as described in Figure 3. (D) Inhibitory effects of 2pf and 2dV on EGF-induced phosphorylation of integrin 4. Serum-starved A431 cells were inoculated into culture dishes precoated with 1 µg/ml Pr-LN5 and incubated without or with 0.8 µg/ml (13.3 nM) 2pf, 0.4 µg/ml (14.3 nM) 2dV, or 0.8 µg/ml (11.4 nM) 2sa in serum-free medium supplemented with 0.05 µg/ml EGF for 10 min. The tyrosine phosphorylation of integrin 4 was analyzed after immunoprecipitation. Other experimental conditions are described in Figure 4 and Materials and Methods.

Next, inhibitory effects of 2pf and 2dV on cell migration activity of Pr-LN5 were assayed. When 2pf and 2dV were cocoated at nearly the same molar concentration with Pr-LN5, both of them significantly suppressed the migration of EJ-1 cells (Figure 8C). The inhibitory activity of 2dV seemed to be slightly higher than that of 2pf.

Furthermore, we examined effects of 2pf and 2dV on the tyrosine phosphorylation of integrin 4 by using A431 cells (Figure 8D). As expected, both 2pf and 2dV suppressed EGF-induced tyrosine phosphorylation of integrin 4 on the plates precoated with Pr-LN5.

Cationic Nature of the 2 Short Arm Proteins
As described above, the HSPG syndecan-1 was identified as a possible receptor of 2sa, and both 2pf and 2dV had similar biological activities to 2sa. Because HSPGs and heparin bind cationic proteins, the biological activities of the 2 short arm proteins are likely to depend on their cationic nature. Therefore, we compared heparin-binding activity between 2pf and 2dV. When each protein was applied to a heparin-agarose column and the bound protein was eluted by a NaCl gradient, 2pf and 2dV were eluted at 0.360.4 M NaCl and 0.30.33 M NaCl, respectively. This indicates that 2dV has slightly lower heparin-binding affinity than 2pf.

To further test whether the activities of the 2 short arm proteins simply depend on their cationic nature, we compared effects of 2pf and a highly cationic peptide, poly-L-lysine. When only poly-L-lysine or 2pf was coated on plastic plates, poly-L-lysine, but not 2pf, supported adhesion of EJ-1 to the plates (data not shown). When cocoated with Pr-LN5, both poly-L-lysine and 2pf promoted the adhesion of EJ-1 cells dose-dependently (Figure 9A). The cell adhesion activity was stronger for poly-L-lysine than 2pf. However, their adhesion activities were comparable when A431 cells were used instead of EJ-1 cells (Figure 9B).

View larger version (21K): [in this window] [in a new window]   Figure 9. Effects of 2pf and poly-L-lysine on cell adhesion, cell migration, and phosphorylation of integrin 4. (A and B) Effect on adhesion of EJ-1 (A) and A431 (B) cells to Pr-LN5. Plates (96-well) were precoated with 0.25 µg/ml (A) or 0.125 µg/ml (B) Pr-LN5 and then coated with the indicated concentrations of 2pf () and poly-L-lysine (), followed by blocking with BSA. The adhesion of EJ-1 (A) and A431 (B) cells onto the plates was measured as described in Figure 2A. (C) Effect on migration of A431 cells. A431 cells were inoculated into each well precoated with 0.3 µg/ml Pr-LN5, and 0.4 µg of 2sa or poly-L-lysine (PL) was added to the culture medium containing 1% FCS. After preincubation for 1 h, the cell migration speed was measured as described in Figure 3A. (D) Effect on integrin 4 phosphorylation of A431 cells. A431 cells were treated with 0.8 µg/ml 2pf or PL and with 0.01 µg/ml EGF on the Pr-LN5 substrate. Integrin 4 present in the cell lysates was immunoprecipitated with the anti-integrin 4 mAb 3E1. The immunoprecipitates were analyzed by immunoblotting with the mAb against PY-20. Left, immunoblotting patterns. Right, quantitative analysis of the integrin 4 phodphorylation. Other experimental conditions are described in Figure 4 and Materials and Methods.

All these results indicate that although the cationic nature, or heparin-binding activity, is prerequisite but not sufficient for the biological activities of the 2 short arm proteins, especially for the suppression of the integrin 4 phosphorylation and cell migration, it is clear that the 2 short arm has a specific activity.

Suppression of Syndecan-1 Expression by RNA Interference
To further confirm the specific interaction between domain V of the 2 chain and syndecan-1, we carried out an RNA interference experiment to block the expression of syndecan-1 gene. An siRNA and a control RNA were transfected into EJ-1 cells. Some HSPGs, including syndecans-1 and -2, were detected in control cells by immunoblotting (Figure 10A). Syndecan-4 was undetectable by immunoblotting (data not shown). Transfection with siRNA efficiently blocked the expression of syndecan-1 in EJ-1 cells, which was a major HSPG band in the control cells. The siRNA treatment did not significantly affect the amounts of syndecan-2 and other HSPG species. The cell adhesion activity of 2dV in the presence of Pr-LN5 was assayed with the control and siRNA-treated cells 3 d after the RNA transfection. 2dV negligibly promoted the adhesion of the EJ-1 cells treated with the siRNA (Figure 10B, left), whereas it clearly promoted the adhesion of the control cells (Figure 10B, right).

View larger version (24K): [in this window] [in a new window]   Figure 10. Effect of suppression of syndecan-1 expression by RNA interference on cell adhesion and integrin 4 phosphorylation. (A) Changes of HSPGs in EJ-1 cells after transfection with a control RNA (Cont. RNA) and a syndecan-1 siRNA (siRNA). EJ-1 cells were grown in 60-mm dishes, transfected with either 50 nM Cont. RNA or with 10 or 50 nM siRNA and further incubated for 72 h. The cultured cells (1 x 106) were used for the determination of syndecan-1 expression. Total cell lysates (200 µg of protein) were digested by heparitinase, resolved by SDS-PAGE, and transferred to a PVDF membrane. Total HSPG species, syndecan-1 (Synd-1) and syndecan-2 (Synd-2) present in the cell lysates were detected by immunoblotting with the anti-heparansulfate mAb, the anti-syndecan-1 mAb, or the anti-syndecan-2 antibody, as described in Figure 7B. The protein concentrations of the cell lysates were determined by the Lowry–Folin method. (B) Effect of syndecan-1 knockdown on 2dV-dependent adhesion of EJ-1 cells to Pr-LN5. The cell adhesion activity of 2dV in the presence of Pr-LN5 was assayed as described in Figure 2, by using EJ-1 cells that had been transfected with 50 nM control RNA (Control RNA; right) or 50 nM syndecan-1 siRNA (siRNA; left) as shown above. (C) Effect of syndecan-1 knockdown of EJ-1 cells on 2dV-mediated suppression of integrin 4 phosphorylation. Serum-starved EJ-1 cells, which had been transfected with 50 nM control RNA (Cont. RNA) or 50 nM syndecan-1 siRNA (siRNA), were inoculated into culture dishes precoated with 1 µg/ml Pr-LN5 and incubated without or with 0.8 µg/ml 2dV in serum-free medium supplemented with 0.1 µg/ml EGF for 15 min. The tyrosine phosphorylation of integrin 4 was analyzed after immunoprecipitation as described in Figure 4. Left, immunoblots. Right, quantitative analysis of phosphorylated integrin 4. Other experimental conditions are described in Materials and Methods.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study demonstrates that the short arm of laminin 2 chain (2sa) in both soluble and insoluble (coated) forms promotes cell adhesion but suppresses cell migration on the processed LN5 substrate. These activities of 2sa are consistent with our previous finding that the nonprocessed LN5 (Np-LN5) is more adhesive but less active with respect to the cell motility activity than Pr-LN5 (Ogawa et al., 2004). Interestingly, 2sa suppressed the EGF-dependent tyrosine phosphorylation of integrin 4, and it seemed to stabilize hemidesmosome structures. All these activities of 2sa seem to be related with one another and mediated by its specific binding to syndecan-1 as a receptor. Furthermore, our results strongly suggest that the NH₂-terminal sequence domain V is responsible for these biological activities of 2sa.

Proteolytic processing of extracellular matrix proteins modulates their biological activities (Schenk and Quaranta, 2003). Giannelli et al. (1997) reported that when a rat LN5 with the unprocessed 150-kDa 2 chain was treated with gelatinase A (MMP-2), the digest containing the LN5 with an 80-kDa processed 2 chain stimulated cell migration more strongly than the untreated LN5 in the Boyden chamber assay. Recently, the same group showed that a 21-kDa recombinant rat 2 fragment containing only the laminin-type EGF-like domain III, which could be produced by cleavage at two sites (Figure 1A), binds and activates the EGF receptor to stimulate cell migration (Schenk and Quaranta, 2003). They suggested that the domain III fragment of 2 chain, rather than the LN5 with the 80-kDa truncated 2 chain, is responsible for the enhanced cell migration activity. Consistent with our results, a recombinant protein containing domains III–V, which corresponds to our 2sa, was unable to activate the EGF receptor. In human LN5, however, the production of the 80-kDa 2 chain and the 21-kDa domain III fragment is thought to be very rare, if any, under ordinary conditions, because the proteolytic processing site of rat 2 chain to produce the 80-kDa 2 chain is not conserved in the human 2 chain (Veitch et al., 2003). The 150-kDa 2 chain of human LN5 is processed mainly to the 105-kDa 2 form, releasing a 45-kDa fragment containing domains IV–V, which corresponds to 2pf in this study (Figure 1A). Our results indicate that the elevated cell migration activity of Pr-LN5 results from the loss of the 45-kDa fragment (domains IV–V) or domain V, which suppresses cell migration but promotes cell adhesion.

Integrin 64 is an essential component of hemidesmosome structures, which support the stable adhesion of basal epithelial cells to the basement membrane. Integrin 64 is associated with EGF receptor, and the activation of EGF receptor induces the phosphorylation of integrin 4 through the Src family kinase Fyn or Yes (Mariotti et al., 2001). The phosphorylation of the integrin 4 tail promotes the hemidesmosome disassembly, which is essential for the epithelial cell migration and invasion (Mainiero et al., 1997; Mariotti et al., 2001). The activity of 2sa to suppress the EGF-stimulated phosphorylation of integrin 4 and subsequent disassembly of