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J. Biol. Chem., Vol. 277, Issue 27, 24835-24841, July 5, 2002

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The Selective Regulation of _V1 Integrin Expression Is Based on the Hierarchical Formation of _V-containing Heterodimers^*

Pekka Koistinen^§ and Jyrki Heino^¶

From the  MediCity Research Laboratory and the Department of Medical Biochemistry, ^§ Turku Graduate School of Biomedical Sciences, University of Turku, FIN-20520 Turku and the ^¶ Department of Biology, University of Jyväskylä, FIN-40351 Jyväskylä, Finland

Received for publication, April 2, 2002, and in revised form, May 6, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The integrin ₁ subunit can form a heterodimer with 12 different  subunits. According to the present model, the expression level of any complex is regulated by the availability of the specific  subunit, whereas ₁ subunit is constantly present in a large excess. The expression of several heterodimers containing the _V subunit seems to be regulated by an identical mechanism. The fact that many cells express _V1 heterodimer, and that this fibronectin/vitronectin receptor may be selectively regulated, compromises the present model of the regulation of ₁ and _V integrins. We have tried to solve this problem by assuming that distinct heterodimers are formed with different tendency. To test the hypothesis, we analyzed WM-266-4 melanoma cells transfected with a cDNA construct coding for an intracellular single-chain anti-_V integrin antibody. We could see 70-80% reduction in the cell surface expression of _V subunit. However, the only one of the _V integrins reduced on the cell surface was _V1. This suggests that the cell surface expression level of _V1 is dependent on the number of _V subunits available after the formation of other _V-containing heterodimers. Thus, there seems to be a hierarchy in the complex formation between _V and its different -partners. These observations explain how _V1 can be specifically regulated without concomitant changes in the expression of other _V or ₁ integrins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The members of the integrin family that form receptors for various extracellular matrix proteins can be divided into two major subgroups according to the subunits present in the receptors. Integrin ₁ can form a complex with 12 different  subunits. The ₁-containing heterodimers are receptors for collagens, laminins, tenascins, and fibronectin. The other subset, the _V integrins, are fibronectin and vitronectin receptors, some of which also have the ability to bind various other matrix and plasma proteins.

In many cells the two promiscuous subunits, _V and ₁, can form a heterodimer with each other. _V1 was originally described as a fibronectin or vitronectin receptor (1, 2). It may also have a capacity for binding to osteopontin and to the latent form of transforming growth factor- (3, 4). Some viruses, including parechovirus 1, adenovirus, and foot-and-mouth disease virus, use _V1 as their cellular receptor (5-7). The tissue distribution of _V1 is mostly unknown because of the lack of a specific antibody against _V1 complex. For the same reason the function of _V1 integrin in many human cell types is unknown, or the published information is based on cell transfections or on the use of combinations of function blocking antibodies against different _V and ₁ integrins. In _V-transfected Chinese hamster ovary cells, _V1 integrin has been found to function as a fibronectin receptor while not supporting cell migration on fibronectin (8). On the other hand, in squamous carcinoma cells derived from head and neck tumors, _V1 integrin contributes to migration on fibronectin (9). It has been suggested that _V1 integrin promotes the migration of oligodendrocyte precursors on composite extracellular matrix containing laminin, fibronectin, and vitronectin (10). In avian neural crest cells, _V1 participates in adhesion to vitronectin, whereas it may have a less important role in cell migration (11). The role of _V1 as a vitronectin receptor has been emphasized in studies on smooth muscle cells, suggesting that vitronectin-mediated contractility of smooth muscle is mediated by _V1 integrin (12).

There is some evidence that the cell surface level of _V1 integrin can be specifically regulated, for example, during development (13). Selective regulation of the number of _V1 integrins on the cell surface cannot be explained simply by assuming that the expression of _V or ₁ genes or the synthesis rate of the corresponding proteins is changed. That would lead to concomitant changes in the numbers of all _V or ₁ integrins. The regulation of both _V and ₁ integrins has been studied in detail, and their regulation seems to be based on the same principle; the promiscuous subunit (₁ or _V) is synthesized in an excess, and the formation of any heterodimer is dependent on the availability of the other subunit (14, 15). Therefore, the cell surface copy number of, for example, ₁₁ and _V3 integrin is dependent on the synthesis rate of ₁ and ₃ subunits, respectively. The presence of _V1 heterodimers challenges this model. To address this controversy, we hypothesized that there is a hierarchy in the formation of distinct heterodimers and that _V1 complex is formed only in the presence of sufficient excesses of _V and ₁ subunits in relation to their other partners. To test this hypothesis, we used a previously constructed cDNA coding for an intracellular, single-chain anti-_V antibody and analyzed stably transfected WM-266-4 melanoma cell clones. The antibody could significantly reduce the expression level of _V subunit and selectively diminish the cell surface expression of _V1 integrin. However, it could not affect the cell surface expression of another prominent _V integrin, namely _V3. Thus, the experimental data support our theory, explaining the mechanism of selective regulation of _V1 integrin expression on cell surface.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Lines-- Melanoma cell line WM-266-4 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM)¹ supplemented with heat-inactivated 10% fetal calf serum (FCS, Invitrogen), 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.

Construction and Transfection of cDNA Coding for Intracellular Single-chain Antibody-- Anti-_V integrin intracellular single-chain antibody was constructed as described previously (16-19). Briefly, total RNA was isolated from 5 × 10⁶ cells of hybridoma line L230 expressing anti-_V integrin monoclonal antibody (obtained from the ATCC) by using the Ultraspec RNA isolation system (Biotex Laboratories, Inc.). This RNA was used to prepare cDNA by using primers B (TGM GGA GAC GGT GAC CRW GGT CCC T) and D (ATT TGC GGC CGC TAC AGT TGG TGC AGC ACT). The primer sequences were from Richardson et al. (18). Immunoglobulin heavy and light chain variable domains (VH and VL) were amplified from the cDNA by PCR using primers A (TTT AAG CTT ACC ATG GAA AGG CAC TGG ATC) and B or C (GAG CTC GTG CTC ACM CAR WCT CCA) and D. A DNA segment coding for the interchain linker was amplified from an anti-tat 3 gene (a gift from Wayne A. Marasco, Dana-Farber Cancer Institute, Harvard Medical School, Boston) by PCR using primers E (GGG ACC TGC GTC ACC GTC TCC TCA) and F (TGG AGA CTG GGT GAG CAC GAG CTC AGA TCC). The single-chain antibody gene was assembled from the VH, VL, and interchain linker fragments by overlap extension (20), followed by PCR amplification with primers A and I (TTT TCT AGA TTA TTA CAG CTC GTC CTT TTC GCT TAC AGT TGG TGC AGC ATC). The complete sequence of the assembled intracellular single-chain anti-_V antibody gene was determined by the dideoxy chain termination method (21). The construct was digested with HindIII and XbaI and ligated into the vector pcDNA3 (Invitrogen), which carries the neomycin resistance gene.

Transfections were performed by electroporation. Neomycin analogue G418 (Invitrogen) was added to the culture medium at a concentration of 400 µg/ml. G418-resistant cell clones were selected for 2-3 weeks, isolated, and analyzed for their expression of _V integrin. Control cells were transfected with the pcDNA3 plasmid only. Transfected cells were cultured in 10% FCS/DMEM containing 2 mM glutamine, 100 IU/ml penicillin G, 100 µg/ml streptomycin, and 200 µg/ml G418 (Invitrogen).

Expression of the mRNA coded by the intracellular antibody construct in transfected cells was confirmed by RT-PCR of the total RNA isolated from both anti-_V plasmid and vector control cells. RT-PCR was performed by the GeneAmp^® RNA PCR kit (PerkinElmer Life Sciences) using primers A and D. T_m was 68 °C.

Cell Adhesion and Spreading Assays-- Coating of 96-well immunoplates (MaxiSorp, Nunc, Roskilde, Denmark) was done by exposure to 0.1 ml of phosphate-buffered saline (PBS, pH 7.4) containing 22 µg/ml type XVII collagen (human recombinant COL 15 domain of type XVII collagen from Dr. Kaisa Tasanen, University of Oulu, Oulu, Finland) (22), 22 µg/ml fibrinogen (Sigma), 10.0 µg/ml fibronectin (human plasma fibronectin, Chemicon International Inc., Temecula, CA), or 1 µg/ml vitronectin (purified human vitronectin, Chemicon) for 12 h at 4 °C or 1 h at 37 °C. Before coating, COL 15 was denatured for 20 min at 56 °C. Bovine serum albumin (BSA, 0.1%) was used to measure the nonspecific binding or spreading. Residual protein absorption sites in all wells were blocked with 0.1% bovine serum albumin in phosphate-buffered saline for 1 h at 37 °C. Confluent cell cultures were detached by using 0.01% trypsin and 0.02% EDTA, rinsed in DMEM containing 10% FCS, and then washed twice with DMEM. Cells were suspended in DMEM, transferred into wells (1.5 × 10⁴ cells/well), and incubated for 35-120 min at 37 °C. Saturating concentrations of functional monoclonal antibodies against ₄ (BD PharMingen), ₅ (mAb 16) (23), ₃ (mAb 2023z, Chemicon), and _V5 (PIF6) (24) integrins were incubated with cells for 15-30 min at room temperature before adding them to wells. Nonadherent cells were removed by rinsing the wells with medium; adherent cells were fixed with 8% formalin and 10% sucrose and then washed with distilled water. A spread cell was characterized as one having a clearly visible ring of cytoplasm around the nucleus. The portion of spread cells was expressed as percentage of the number of adherent cells.

Northern Blot Hybridizations-- Cells were cultured in DMEM supplemented with 10% FCS for 24 h. The total cellular RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA), and mRNA levels of specific genes were measured by Northern blot hybridization. RNAs were separated in formaldehyde-containing agarose gels, transferred to nylon membranes (Zeta-Probe, Bio-Rad), and hybridized with ³²P-labeled (Amersham Biosciences) cDNA probes. The following cDNAs were used: human matrix metalloproteinase-1 (MMP-1; Ref. 25), human matrix metalloproteinase-2 (MMP-2; Ref. 26), and rat glyceraldehyde-3-phosphate dehydrogenase (27). [³²P]cDNA-mRNA hybrids were visualized by autoradiography.

Flow Cytometry-- Cells were grown to early confluence, detached with trypsin-EDTA, washed with PBS (pH 7.4), and then incubated with PBS containing 1% FCS for 30 min at 4 °C. Cells were collected by centrifugation, exposed to saturating concentration of antibodies against _V integrin (L230), _IIb integrin (BD PharMingen), ₁ integrin (Endogen, Rockford, IL), ₂ integrin (12F1) (28), ₄ integrin (BD PharMingen), ₅ integrin (mAb 16) (23), ₁ integrin (R-322, rabbit polyclonal) (14), ₃ integrin (Southern Biotech, Birmingham, UK), _V5 integrin (PIF6) (24), or ₆ integrin (E7P6) (24) in 1% FCS/PBS for 30 min at +4 °C. For labeling, cells were incubated with rabbit anti-mouse (1:20 dilution), rabbit anti-rat (1:100 dilution), or swine anti-rabbit (1:20 dilution) IgG coupled to fluorescein (all from DAKO A/S, Glostrup, Denmark) for 30 min at 4 °C, washed twice with PBS, and suspended in the same buffer. Relative amounts of cell surface integrins were determined by comparison of fluorescent emission intensity data as collected using a FACScan apparatus (BD PharMingen). Control samples were prepared by treating cells without primary antibodies.

Immunoprecipitations and Western Blotting-- Cells were metabolically labeled with 50 µCi/ml [³⁵S]methionine (Tran³⁵S-label, ICN Biomedicals Inc., Irvine, CA) for 16 h in methionine-free minimum essential medium. Cell monolayers were rinsed on ice with a solution containing 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and 25 mM Tris-HCl (pH 7.4) and then detached by scraping. Cell pellets were obtained by centrifugation at 500 × g for 5 min. Cells were solubilized in 200 µl of the same buffer containing 100 mM n-octyl--D-glucopyranoside (Sigma) on ice with occasional vortexing. Insoluble material was removed by centrifugation at 1 × 10⁴ × g for 5 min at 4 °C. Radioactivity in cell lysates was counted, and equal amounts of radioactivity was used in each sample. Triton X-100 (0.5% v/v) and bovine serum albumin (0.5 mg/ml) were added to the supernatants, which were then precleaned by incubation with 50 µl of packed protein A-Sepharose (Amersham Biosciences, Uppsala, Sweden). Supernatants were immunoprecipitated with _V integrin or ₁ integrin antibody (L230 or R-322, correspondingly) for 12 h at 4 °C. After incubation with secondary antibody (rabbit anti-mouse, DAKO), immune complexes were recovered by binding to protein A-Sepharose and washing the beads four times with 25 mM Tris-buffered isotonic saline (pH 7.4) containing 0.5% Triton X-100 and 1 mg/ml bovine serum albumin and twice with 0.5 M NaCl and 25 mM Tris-HCl (pH 7.4). The immunoprecipitates were separated by electrophoresis on sodium dodecyl sulfate-containing 6% polyacrylamide gels under reducing (immunoprecipitation with anti-₁) or nonreducing (immunoprecipitation with anti-_V) conditions, followed by autoradiography. In pulse-chase assays, cells were metabolically labeled for 1 h and harvested 0, 2, 4, and 8 h after pulse.

To examine the amount of _V1 heterodimer, immunoprecipitation was performed with anti-₁ (R-322) (14). Immunoprecipitates were separated on a 7.5% polyacrylamide gel under reducing conditions, and the gel was transferred to a nylon membrane (Hybond^TM ECL^TM, Amersham Biosciences). Nonspecific adsorption sites were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween by incubating the membrane at room temperature for 1 h. A saturating concentration (1:100) of polyclonal _V integrin antibody (Chemicon International Inc., Temecula, CA) was added to the blocking solution. The membrane was rinsed twice, washed twice for 15 min, and washed three times for 5 min. Horseradish peroxidase-linked anti-mouse IgG (Amersham Biosciences) was used as a secondary antibody. The antibody was diluted in Tris-buffered saline containing 0.1% Tween containing 5% milk (1:100), and the membrane was incubated for 1 h. Washing was performed as above. ₁ integrin-linked _V integrin was visualized by an enhanced chemiluminescence reaction (ECL kit, Amersham Biosciences).

Migration Assays-- Coating of 24-well immunoplates (MaxiSorp) was done by exposing each well to 0.3 ml of phosphate-buffered saline (PBS, pH 7.4) containing 27.0 µg/ml (5 µg/cm²) of fibrinogen, fibronectin (human plasma fibronectin, Chemicon), or COL 15 domain of type XVII collagen (22) for 12 h at 4 °C. Residual protein absorption sites in all wells were blocked with 0.1% bovine serum albumin in phosphate-buffered saline for 1 h at 37 °C. The trypsinized cells were rinsed with DMEM plus 10% FCS or 0.2% soybean trypsin inhibitor and washed twice and resuspended in DMEM or Opti-MEM^® (Invitrogen, Inc.). Steel cylinders were placed in each well, and cells were added (2 × 10⁴ cells/well). After 3 h the cylinders were removed, 1 ml of either DMEM or Opti-MEM^® was added, and cells were allowed to migrate for 3 days. Cells were fixed with 10% sucrose and 8% formalin in PBS and stained with 0.1% crystal violet and 0.2 M boric acid. Migration was determined by measuring the increase of the cell colony area.

Invasion Assays-- Cell culture inserts (BD PharMingen) contained polyethylene terephthalate membranes of 8 µm pore size. Membranes were coated with human plasma fibronectin (Chemicon), type I collagen gel (Cellon S.A.), or Matrigel^® (Collaborative Research, Bedford, MA). In fibronectin invasion assays, a concentration of 20 µg/ml was used, and the total volume was 80 µl/insert. Inserts were allowed to air-dry in a cell culture hood overnight. In Matrigel^® and type I collagen invasion assays, 30 µg of Matrigel^® and 35 µl of Cellon gel, respectively, were used for each insert. Inserts were used with 24-well cell culture plates (Costar). Cells from cultures in early confluence were trypsinized, and one volume of 0.2% soybean trypsin inhibitor or 10% FCS/DMEM was added to inhibit trypsin activity. Cells were then washed twice in DMEM, resuspended in 0.1% BSA/DMEM, and added to inserts (1 × 10⁵ cells in 200 µl). 700 µl of 10% FCS in DMEM was used as a chemoattractant in the lower chamber. Cells were allowed to invade at 37 °C for 8-9 h through fibronectin and 2 days through Cellon gel and Matrigel^®. Upper chamber was wiped clean with a swab, and the invaded cells on the lower surface of the membrane were fixed with PBS containing 2% paraformaldehyde and stained with 0.1% crystal violet and 0.2 M boric acid. Invaded cells on the lower side of the membrane were counted under a light microscope with a 10× lens. Three representative fields were counted from each insert. The result was reported as the number of invaded cells in three fields.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cell Clones Expressing Intracellular Anti-_V Integrin Antibody Show Reduced Cell Surface Expression of _V Subunit without Changes in the ₃ Subunit-- Sixteen stable cell clones transfected with a cDNA construct coding for an intracellular anti-_V antibody (Fig. 1A) and 6 vector control cell clones were tested for expression levels of _V integrin. In anti-_V cDNA-transfected clones, the cell surface expression level of _V was reduced up to 80% when compared with vector control clones (Fig. 1B). Three anti-_V cDNA-transfected cell clones (a3, a11, and a15) and three vector control clones (v3, v4, and v5) were selected for further experiments. In these clones the presence of mRNA derived from intracellular antibody construct was confirmed by RT-PCR (Fig. 1C). Surprisingly, there were no significant decreases in the expression levels of ₃ or ₅ integrins (Fig. 2A). A third partner of _V subunit, ₆, was not detected on these cells (Fig. 2A). To confirm the unreduced expression level of _V3, metabolically labeled cells were immunoprecipitated with an anti-₃ antibody. The results did not indicate a decrease in the number of ₃-connected, mature _V integrin subunits in anti-_V-expressing cell clones (Fig. 3A). Immunoprecipitations from anti-_V-expressing cell clones had an extra band with an approximated molecular size of 140 kDa, corresponding to the precursor form of _V integrin described in previous papers (15, 19). The higher molecular mass form represents an integrin with a complex-type N-linked oligosaccharide, whereas the lighter precursor form represents an integrin that is still inside the endoplasmic reticulum with high mannose-type oligosaccharides (14). Here, the maturation process was studied by pulse-chase experiments followed by immunoprecipitations with anti-_V antibody. Maturation occurred in 8 h in vector control clones (data not shown) in accordance with our previous studies with Saos-2 osteosarcoma cells (19). The presence of the larger intracellular pool of precursor _V may indicate a decelerated maturation process of the _V integrins because of the intracellular antibodies.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Intracellular single-chain anti-V integrin antibody, its expression, and function in WM-266-4 melanoma cells. A, schematic illustration of intracellular single-chain anti-V integrin antibody. Antigen binding sites are in immunoglobulin heavy and light chain variable domains (Vh and Vl, respectively). Between these domains there is a flexible interchain linker. Amino-terminal pro-sequence leads the antibody to endoplasmic reticulum, and carboxyl-terminal ER retention signal (KDEL) prevents the transport of the antibody outside the ER. B, FACScan analysis of transfected WM-266-4 cell clones. Sixteen anti-V cDNA construct-transfected (a) and six vector control cell clones (v) were tested for the cell surface expression of V integrin. The V integrin expression of six anti-V and three vector control clones is illustrated in this figure. Monoclonal anti-V antibody L230 was used as a primary antibody. Negative control cells (nc.) were stained with fluorescein isothiocyanate-labeled secondary antibody only. Panel C shows the presence of anti-V cDNA construct in anti-V-transfected cell clones. Total RNA of WM-266-4 cell clones was isolated and RT-PCR reaction was performed by using primers A and I (as described under "Experimental Procedures"). The PCR products were separated on 0.8% agarose gel. The 830-bp fragment of anti-V cDNA construct is visible in anti-V-transfected clones (a) but not in vector control clones (v). St means standard marker of 800 bp.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Cell surface expression of V and V-related  subunits of three selected anti-V- () and vector-transfected () cell clones. Analysis was made by FACScan. The clones stained with secondary antibody only () show the unspecific background.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Analysis of two most abundant major V integrin heterodimers and hypothetical model of V integrin heterodimer formation. A, immunoprecipitation (i.p.) of 3 integrin from anti-V-transfected (a) and vector-transfected (v) clones. 3, V, and suggested precursor form of V () are indicated. B, Western blot analysis of 1-associated V integrin. Cell lysates of selected WM-266-4 cell clones were immunoprecipitated with polyclonal anti-1 integrin antibody. The precipitates were separated on SDS-PAGE gel, and the proteins were transferred to Hybond ECL membrane. The membrane was then incubated with monoclonal anti-V antibody and horseradish peroxidase-linked secondary antibody. The 1-linked V integrin was visualized by ECL reaction. C, the suggested model for the hierarchy in V integrin heterodimer formation in WM-266-4 melanoma cells. When the amount of V integrin, which is not bound to  subunit (V(free)), exceeds the combined amount of 3 and 5 integrins in ER, V1 heterodimer can be seen on the cell surface. When the amount of V(free) is reduced to the level of combined amount of 3 and 5 integrins in the ER, V1 integrin cannot be seen on the cell surface because all the V(free) is bound to 3 and 5 integrins.

Intracellular Antibody against _V Integrin Has a Selective Effect on _V1 Expression Suggesting a Hierarchy in the Formation of _V-containing Heterodimers-- The diminished cell surface expression of the _V subunit was not accompanied by a similar reduction of the ₃ subunit, leading us to the hypothesis that the expression of _V1 heterodimer must be affected. In the absence of a specific antibody for _V1, we first immunoprecipitated the total cell lysate with an anti-₁ antibody and then Western-blotted the immunoprecipitates with anti-_V integrin antibody. Nearly all ₁-bound _V integrin was in its precursor form in anti-_V antibody-transfected cell clones, whereas in vector control clones ₁-associated _V was in the mature form only (Fig. 3B). Thus, our data indicate that, when the amount of _V subunit in the endoplasmic reticulum is reduced, very little _V1 ends up on the cell surface. Importantly, we have also tested the mechanism of overexpression of an _V-associated  subunit, ₆ (9). In these experiments, the _V1-related binding to fibronectin was dramatically reduced in a ₆-transfected cell clone derived from cells in which _V1 was a major fibronectin receptor (9). The obvious hierarchy in the formation of different _V-containing heterodimers suggests that the amount of _V1 integrin can be regulated selectively and independently of other integrin heterodimers. In simplified terms, this model proposes that the number of _V3 and _V5 heterodimers is regulated at the level of the ₃ and ₅ genes, respectively, but the activity of the _V gene dictates the number of _V1 heterodimers (Fig. 3C).

Integrin _V1 Mediates Melanoma Cell Migration on Fibronectin-- Selective reduction of _V1 levels in experimental clones allowed direct observations about its functions to be made. To control for integrin-mediated characteristics, expression levels of other integrins in the experimental clones were tested (Fig. 4). In one of the antibody-expressing clones (a3), the expression of ₁ integrin was higher than in any other clone. This can be attributed to simultaneously higher expression of collagen-binding integrins ₁ and ₂. The expression of ₅, another fibronectin-binding integrin, was slightly reduced in one of the clones when compared with vector control clones. One vector control clone (v4) did not have any ₄ integrin on its surface. Platelet integrin _IIb was not detected on the cell surface of any of the clones. The expression levels of ₃ and ₅ subunits were equal in all cell clones.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Cell surface expression of collagen- and fibronectin-binding integrin subunits on three selected anti-V- () and vector-transfected () cell clones (A and B). Analysis was made by FACScan. The clones stained with secondary antibody only () show the unspecific background.

Because of small variations in the levels of ₄ and ₅ integrins, only clones that had the same numbers of these receptors were selected and tested for adhesion. On fibronectin, cell spreading of the selected anti-_V and vector control clones was equal (Fig. 5A). The spreading of both clones could be reduced but not completely prevented when the fibronectin receptors ₃, ₄, and ₅ were blocked by monoclonal antibodies. Treatment with antibodies revealed differences in the spreading behavior of the anti-_V-expressing and vector control cells; in the presence of antibodies, their average spreading was 50 and 30%, respectively. The number of attached cells within each well was counted with similar results (in the presence of antibodies: 75 versus 55%) (Fig. 5B). This indicates that _V1 integrin can contribute to adhesion and spreading on fibronectin but does so preferentially in the absence of other fibronectin receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Adhesion, spreading, and lateral cell migration experiments on fibronectin. A and B, three parallel samples of one vector control (v3) and one anti-V integrin-transfected (a3) clone were incubated with or without anti-4, anti-5, and anti-3 integrin antibodies and plated on fibronectin-coated wells of 96-well plate. C, control. Cells were allowed to attach for 35 min. Cells were washed and fixed, and three representative microscope fields (10×) of adherent cells were counted. The number of spread cells is reported as a percentage from total number of attached cells. C, two independent migration assays were performed. Three anti-V-transfected (a) and three vector control (v) cell clones were allowed to migrate for 3 days in serum-free conditions, after which the cells were fixed and stained, and increase of the cell colony area was measured. There were three parallel samples of each clone, and the increase is reported as an average of individual cell clones ± S.D. The difference between anti-V-transfected and vector control cells was statistically significant when the two experiments were analyzed together (p < 0.0001; two-way analysis of variance).

On vitronectin cell adhesion and spreading was much slower than on fibronectin. No _V1-related differences in spreading behavior were observed for any of the clones when they were plated on vitronectin. Recently, we have suggested that in keratinocytes _V1 contributes to cell spreading on type XVII collagen (COL 15 domain) (29). However, we did not find any _V1-dependent differences in adhesion or spreading of WM-266-4 melanoma cells on COL 15 domain (data not shown). Likewise, similar results were obtained for spreading and adhesion assays on fibrinogen (data not shown). Fibrinogen has previously been reported to function as a ligand for RGD-binding integrins (30).

We also tested whether the lack of _V1 integrin has an influence on WM-266-4 melanoma cell migration. Three antibody-transfected and three vector control clones were allowed to migrate on fibronectin for 3 days in serum-free DMEM. Two independent experiments were performed, each of which comprised two or three parallel wells of each clone (Fig. 5C). Lateral migration was measured as an increase in the surface area covered by the cells. The area covered by the vector control clones increased on average 75-115% more when compared with anti-_V-transfected clones (p < 0.0001; two-way analysis of variance). These results indicate that _V1 integrin participates in the lateral migration of WM-266-4 cells on fibronectin. Importantly, the small variation in the expression levels of ₄ and ₅ integrins did not play a role in these experiments. Similar to adhesion and spreading assays, there were no _V1-related differences in lateral migration on COL 15 domain of type XVII collagen or fibrinogen (data not shown).

Our finding that _V1 integrin is a low affinity fibronectin receptor is in accordance with the previous data (8). Cells may try to compensate for this low affinity with a high expression level. Spreading and adhesion on fibronectin were approximately the same in anti-_V antibody-expressing and control clones. This indicates that ₄₁, ₅₁, and _V3 are mainly responsible for spreading and adhesion on fibronectin. When these receptors were blocked, we could see the influence of _V1 integrin. Moreover, the depletion of _V1 integrin could markedly reduce the lateral migration of WM-266-4 cells. Previously it has been shown that _V1-expressing Chinese hamster ovary-B2 cells that lack ₅ integrin are unable to migrate on fibronectin (8). Thus, it is probable that _V1 integrin is involved in cell migration on fibronectin, but, to migrate, the cells need another fibronectin-binding integrin such as ₄₁ or ₅₁. The low affinity/high expression of _V1 integrin may be an advantage for cancer cells in such a dynamic process as cell migration, where continuous formation and dissolution of adhesion sites plays a major role.

In addition to fibronectin, _V1 integrin has been suggested to show some binding to vitronectin. However, in our experiments with melanoma cells, we found that cell spreading on vitronectin was not affected by _V1 integrin. The main receptor responsible for spreading on vitronectin seemed to be _V3 integrin. However, on vitronectin _V3 integrin did not have an effect on primary adhesion, whereas blocking of _V5 from _V1-depleted cells clearly reduced the number of adherent cells (data not shown). These results stress the diversity of integrin functions on different substrates.

There are a number of studies in which connections between the expression of specific integrin receptors and the production of MMPs have been proposed (31-34). In the published papers, _V1 integrin has not been studied. We made several invasion assays with type I collagen gels, the basement membrane analog Matrigel^®, and fibronectin, but no _V1-dependent differences were observed. Furthermore, the mRNA levels of MMP-1 and MMP-2 were not _V1-dependent. Based on these results, _V1 does not play a major role in the invasion process or in the expression of metalloproteinases.

To conclude, previous studies have shown that _V1 might be selectively regulated during development (35). Our study suggests that it is an important fibronectin receptor that is abundant on some cancer cells. Furthermore, we propose a molecular mechanism that explains the selective regulation of _V1 expression.

	ACKNOWLEDGEMENTS

We thank Drs. T. Hyypiä, W. A. Marasco, and T. Pulli for the cDNAs; J. Hakalax for statistical analysis; and Dr. W. Connors for critical reading of the manuscript. We gratefully acknowledge the expert technical assistance of M. Tuominen.

	FOOTNOTES

^* This work was supported by grants from the Academy of Finland, the Finnish Cancer Association, and the Sigrid Jusélius Foundation (Finland), and by a fellowship (to P. K.) from the Foundation for the Finnish Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biological and Environmental Science, University of Jyväskylä, P. O. Box 35, FIN-40351 Jyväskylä, Finland. Tel.: 358-14-2602240; Fax: 358-14-2602271; E-mail: jyrki.heino@utu.fi.

Published, JBC Papers in Press, May 7, 2002, DOI 10.1074/jbc.M203149200

	ABBREVIATIONS

The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; MMP, matrix metalloproteinase; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FCS, fetal calf serum; ER, endoplasmic reticulum; VH, variable heavy chain; VL, variable light chain.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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