INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Integrins form one family of cell adhesion receptors that play a prominent role in the adhesive interactions between cells and their surrounding extracellular matrix (ECM) (Hynes, 1992). All integrins are heterodimers composed of noncovalently linked  and  subunit transmembrane glycoproteins containing large extracellular domains, short transmembrane domains, and carboxyl-terminal cytoplasmic domains of variable length (Hynes, 1992). These adhesive receptors are endowed with both structural and regulatory functions, linking extracellular matrix to the actin cytoskeleton at focal adhesion sites and providing bidirectional transmission of signals across the plasma membrane (Schoenwaelder and Burridge, 1999; Critchley, 2000). The cytoplasmic domain of the  subunit has been shown to play a critical role in focal adhesion and actin stress fiber organization and both outside-in and inside-out integrin signaling (Liu et al., 2000).

Through their molecular interactions integrins regulate a number of critical cellular processes, including proliferation, differentiation, survival, migration, and gene expression (Giancotti, 1997; Giancotti and Ruoslahti, 1999). It is now clear that altered, modulated, or regulated adhesive interactions can change the way cells interact with their environment with dramatic consequences for both normal and pathological conditions. Cells can vary their adhesive properties by selectively expressing different integrins and by modulating their integrin specificity and affinity for ligands (Hynes, 1996). However, cells often display multiple integrins capable of interacting with a particular ECM protein and, conversely, individual integrins can recognize several extracellular matrix molecules (Hynes, 1992). Thus, integrin expression and ligand specificity are often apparently redundant, at least in terms of simple adhesion. The biological significance of this phenomenon is not clear yet; nevertheless, there is increasing evidence that individual integrin receptors mediate distinct functions and can convey unique information (Giancotti, 2000).

Most integrins belong to one of two major subfamilies defined by the ₁ and _V subunits. The ₁ subunit pairs with at least 12 different  subunits (₁-₁₁, _V) to comprise receptors for a variety of ECM proteins, including collagen, laminin, fibronectin, and vitronectin (Hynes, 1992). A large body of literature (Brakebusch et al., 1997; Giancotti, 1997; reviewed in Schoenwaelder and Burridge, 1999) has addressed the role of ₁ integrins in mediating important cell adhesion and signal transduction events. Four different ₁ isoforms have been identified (₁A, ₁B, ₁C, and ₁D), which differ in their cytoplasmic domains and differentially affect many integrin functions (Belkin et al., 1997; Fornaro and Languino, 1997; Belkin and Retta, 1998; Pfaff et al., 1998; Retta et al., 1998).

The _V subunit is known to associate with at least five different  subunits (₁, ₃, ₅, ₆, and ₈). Among these _V integrins, _V3 and _V5 have been extensively studied. The _V3 integrin, in particular, has a relatively limited cellular and tissue distribution (Yamada et al., 1995), but its expression and activity are tightly regulated during a variety of biological processes, including cell proliferation and survival (Montgomery et al., 1994), wound healing (Clark et al., 1996a), angiogenesis (Brooks et al., 1994), bone remodeling (McHugh et al., 2000), tumor progression (Albelda et al., 1990) and metastasis (Yun et al., 1996). This integrin can bind to a variety of ECM proteins, including vitronectin, fibronectin, fibrinogen, thrombospondin, Von Willebrand factor, and denatured collagen (Kühn and Eble, 1994), and it is able to recruit cytoskeletal and signaling proteins to focal adhesion sites (Lewis et al., 1996). In addition, _V3 is one of the integrins that promotes the assembly of fibronectin matrix (Wennerberg et al., 1996; Wu et al., 1996; Retta et al., 1998).

In contrast to _V3, _V5 is among the most widely expressed integrins. This receptor can specifically and efficiently bind its ligand vitronectin but remains randomly distributed over the surface of the cells and does not trigger the assembly of focal adhesion structures (Wayner et al., 1991; Leavesley et al., 1992). Moreover, _V5 integrin has different requirements than _V3 for mediating adhesive events, such as cell spreading and migration, to the common ligand vitronectin (Klemke et al., 1994; Lewis et al., 1996), and it can induce differential biological responses (Friedlander et al., 1995).

Perturbation experiments with antibodies, blocking peptides, and antisense oligonucleotides demonstrated that both ₁ and _V integrins play a primary role in important physiological and pathological processes (reviewed in Varner and Cheresh, 1996; Brakebusch et al., 1997; Bader et al., 1998). However, recent genetic analyses have clearly increased questions as to the primacy of these integrins, and instead have pointed to a cross talk model where spatiotemporal regulation, combinatorial expression, and activation of several integrin receptors generate a high degree of specificity in cell adhesion (Fassler et al., 1996; Hynes, 1996, 1999; Brakebusch et al., 1997; Bader et al., 1998; Hodivala-Dilke et al., 1999; McHugh et al., 2000). Several observations indicate the existence of a cross talk between ₁ and _V integrins, which usually takes the form of one integrin influencing the functional behavior of another integrin expressed on the same cell (Yang and Hynes, 1996; Belkin et al., 1997; Retta et al., 1998; Blystone et al., 1999; Corbett and Schwarzbauer, 1999). However, in most cases, the mechanistic basis of this receptor cross talk is not completely understood, and it is unknown whether and how the integrin cross talk can regulate the ratio of integrin cell-surface expression levels.

GD25 cells, derived from ₁-null mouse embryonic stem cells (Wennerberg et al., 1996), are a valuable model for examination of integrin cross talk. In fact, these cells express _V3, _V5, and ₆₄ as major integrin complexes but do not express integrins of the ₁ subfamily, thus permitting a variety of genetic experiments exploring the basis of integrin cross talk. We have previously transfected GD25 cells with cDNAs encoding for the isoform A, B, or D of the human ₁ integrin subunit or two ₁ mutants lacking either the entire cytoplasmic domain (₁TR) or only the cytoplasmic domain "variable" region that characterizes each isoform (₁COM) (Retta et al., 1998). With the use of these cells, we investigated the specific functional properties of the isoform B and D of the human ₁ integrin subunit, showing the existence of a functional cross talk between these two ₁ isoforms and the endogenous _V integrins. In particular, both ₁B and ₁D expression prevented different fibronectin (FN)-dependent _V integrin functions, including its ability to mediate cell adhesion, to localize to focal adhesions, and to assemble an FN matrix (Belkin et al., 1997; Retta et al., 1998).

In the present study, we show that the cross talk between ₁ and _V integrins is mainly based on the regulation of ₃ and ₅ integrin subunit expression exerted by ₁ integrins. In fact, the ectopic expression of either ₁A, ₁B, or ₁D in GD25 cells induces a drastic down-regulation of ₃ and an up-regulation of ₅ integrin cell surface levels. Moreover, analysis of GD25 cells expressing ₁ integrins lacking either the entire ₁ cytoplasmic domain (₁TR) or only its variable region (₁COM) demonstrate that the "common" region of the ₁ cytoplasmic domain is required for these effects. We further demonstrate that ₁ exerts its control over _v3 expression level by modulating the ₃ mRNA stability, whereas the up-regulation of _V5 is mainly due to translational or posttranslational events leading to an increased recruitment of the ₅ subunit at the cell surface.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Antibodies and Reagents

The mouse anti-human ₁ monoclonal antibody (mAb) TS2/16 was obtained from American Type Culture Collection (Manassas, VA). The rat anti-mouse ₆ mAb GoH3 was a gift from A. Sonnenberg (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The rabbit polyclonal antisera to _V, ₃, and ₅ integrin cytoplasmic domains, produced in our laboratory, were previously described (Retta et al., 1998). The polyclonal antisera to ₃ and ₅ were produced with the use of a previously described protocol (Defilippi et al., 1995). Briefly, rabbits were immunized against a GST-₃ fusion protein containing the cytoplasmic domain of the mouse ₃ integrin subunit and against a synthetic peptide reproducing an amino acid sequence from the carboxy terminus of mouse ₅ integrin subunit, respectively. The ₅ peptide EKAQLKPPATSDA was synthesized by solid phase methods with the use of an LKB Biolynx synthesizer (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and coupled to keyhole limpet hemocyanin with the use of glutaraldehyde. The mouse anti-paxillin mAb was purchased from Transduction Laboratories (Nottingham, United Kingdom). The affinity-purified rhodamine-labeled goat anti-mouse and goat anti-rabbit IgG were from Sigma (St. Louis, MO). Poly-L-lysine and monensin were from Sigma. Vitronectin and fibronectin were purified from human plasma as previously described (Balzac et al., 1994; Retta et al., 1999). Protein A-Sepharose and protein G-Sepharose were from Amersham Pharmacia Biotech AB.

Cells and Culture Conditions

The mouse GD25 fibroblast line, which lacks expression of ₁ integrin heterodimers because of disruption of the ₁ gene by homologous recombination, was established after differentiation of ₁-null embryonic stem cells and immortalization with simian virus 40 large T antigen (Wennerberg et al., 1996). GD25 cells expressing the human ₁A, ₁B, or ₁D integrin isoforms or the ₁TR and ₁COM human ₁ mutants, lacking the entire cytoplasmic domain and the cytoplasmic domain variable region, respectively, were obtained as previously described (Belkin et al., 1997; Retta et al., 1998). To avoid selection for anomalous functional traits, no efforts were made to establish clonal cell lines; instead, bulk cell populations expressing ₁ integrins were selected. Cells were cultured in DMEM (Invitrogen Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The ₁-expressing GD25 cells were cultured in the same medium plus 300 µg/ml hygromycin B (Roche Molecular Biochemicals, Mannheim, Germany). Cell populations expressing high levels of the ₁ forms used were selected by the panning method and monitored by flow cytometry as described previously (Retta et al., 1998).

Biotinylation of Cell Surface Proteins

Adherent cells, grown to 80-90% confluence in 90-mm tissue culture dishes, were washed twice with ice-cold buffer A (1.3 mM CaCl₂, 0.4 mM MgSO₄, 5 mM KCl, 138 mM NaCl, 5,6 mM D-glucose, 25 mM HEPES, pH 7.4) and incubated with 0.5 mg/ml membrane-impermeable biotinylation reagent Sulfo-NHS-Biotin (Sigma) in buffer A at 4°C for 30 min. The reaction was quenched with DMEM containing 0.6% bovine serum albumin (BSA) and 25 mM HEPES, pH 7.4. The cells were then washed four times with ice-cold buffer A and lysed on ice in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.4) containing 0.5% Triton X-100 and the protease inhibitors aprotinin (10 µg/ml), leupeptin (10 µg/ml), phenylmethylsulfonyl fluoride (1 mM), and benzamidine (1 mM) (all from Sigma). Cell lysates were centrifuged at 12,000 × g for 30 min at 4°C, and total protein concentration in the supernatants was determined with the use of a bicinchoninic acid protein assay (Pierce, Rockford, IL). Supernatants containing equal amounts of proteins were precleared with a mixture of protein A-Sepharose and protein G-Sepharose and used in immunoprecipitation experiments.

Immunoprecipitation and Analysis of Integrins

Integrins were immunoprecipitated from precleared cell lysate supernatants by incubation with appropriate dilutions of specific antibodies and a mixture of protein A-Sepharose and protein G-Sepharose beads for 1 h at 4°C. Complexes were washed four times with the lysis buffer then the proteins were eluted with Laemmli's sample buffer (62.5 mM Tris-HCl, pH 6.8, 20% glycerol, 2% SDS) and subjected to SDS-polyacrilamide (7.5%) gel electrophoresis under nonreducing conditions. To visualize the biotinylated proteins, the gel was electroblotted onto Hybond-C transfer membrane (Amersham Pharmacia Biotech AB). The blot was then blocked with 5% BSA in phosphate-buffered saline (PBS) for 1 h at 42°C, incubated with streptavidin-peroxidase (Sigma) (1:10.000 in PBS/1% BSA) for 1 h at room temperature, and further processed by the Western blotting enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech AB).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Northern Blot Analysis

Total RNA was isolated from 1 × 10⁷ cultured cells with the use of the RNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Where indicated, before RNA isolation confluent cells were detached by treatment with 5 mM EDTA in PBS, washed twice with serum-free DMEM, resuspended in the same medium containing 1 µM monensin, and plated for 2 h on tissue culture dishes that had been coated with 10 µg/ml polylysine, fibronectin, or vitronectin as previously described (Retta et al., 1998). A multiplex semiquantitative RT-PCR was used to detect the relative levels of ₃ and ₅ or ₁ integrin mRNAs. cDNA was synthesized from 5 µg of cytoplasmic RNA with the use of the 1st Strand cDNA Synthesis kit (Roche Molecular Biochemicals), and subjected to 28 (₃/₅) or 32 (₁ and ₁/₃/₅) PCR cycles. The reaction conditions and oligonucleotide PCR primers used were optimized so that the amplification products fell within the range of PCR amplification linearity. PCR was performed with each reaction mixture containing 5 µl of cDNA, 1× reaction buffer (Amersham Pharmacia Biotech AB), 1.5 mM MgCl₂, 200 µM dNTP, 0.5 µM of each primer, and 2 U of Taq DNA polymerase (Amersham Pharmacia Biotech AB) in a total volume of 50 µl. The following stages were used for each PCR cycle: 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, with a prolonged extension stage of 72°C for 5 min after the final cycle. The primers were derived from nonhomologous regions of the mouse ₃ and ₅ and the human ₁ cDNA sequences, and led to 705-, 570-, and 857-bp PCR products, respectively. PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. Gels were photographed under UV light and intensities of the amplified cDNA fragments were quantitated with the use of a densitometric software (Molecular Analyst; Bio-Rad, Hemel Hempstead, United Kingdom). Molecular size standards (123-bp DNA ladder) were from Sigma.

For Northern blot hybridization, equal amounts of the purified total RNA (25 µg/lane) were separated by electrophoresis on a 1.2% agarose gel containing 1.8% formaldehyde and 1× FA Gel buffer [20 mM 3-(N-morpholino)propanesulfonic acid, 5 mM NaAc, 1 mM EDTA, pH 7.0], transferred to a Nytran SuPerCharge transfer membrane (Schleicher & Schuell, Dassel, Germany) with the use of the TurboBlotter blotting device accordingly to manufacturer's instructions (Schleicher & Schuell), and UV cross-linked to the membrane. The membrane was prehybridized by incubation in Church's buffer (0.5 M Na-phosphate buffer, 10 mg/ml BSA, 7% SDS, 1 mM EDTA, 0.1 mg/ml salmon sperm DNA, pH 7.4) for 8 h at 65°C and hybridized with ³²P-labeled probes overnight at 65°C in Church's buffer. After hybridization, the membrane was washed once in 2× SSC + 0.1% SDS, once in 1× SSC + 0.1% SDS, once in 0.2× SSC + 0.1% SDS, and once in 0.1× SSC + 0.1% SDS for 15 min each at 65°C. The membrane was then exposed to x-ray film for 24-72 h at 80°C with an intensifying screen. Probes were synthesized by random priming with cDNA fragments of mouse ₃ and ₅ integrin subunits amplified by PCR and cloned in our laboratory. The same blots were rehybridized with a probe of the housekeeping gene -actin to ensure equal loading.

Measurement of mRNA Stability

The measurement of mRNA stability was performed as described by Xu and Clark (1996). Briefly, cells were divided into three plates and cultured in 10% FBS/DMEM for 24 h before the addition of 60 µM 5,6-dichloro-1--D-ribofuranosyl-benzimidazole (DRB; Sigma), an inhibitor of transcription initiation. After addition of DRB, the cells were collected at 0, 4, 8, 12, and 24 h for RNA analysis. Total RNA isolation and Northern analysis were performed as described above.

Immunofluorescence Microscopy

Immunofluorescence studies were performed as described previously (Retta et al., 1996). Briefly, cells were seeded onto fibronectin-coated glass coverslips and allowed to spread for 3 h in complete culture medium. Cells were then washed with cold PBS, fixed for 10 min with 3.7% paraformaldehyde in PBS, permeabilized with ice-cold 0.5% Triton X-100, 3.7% paraformaldehyde in PBS for 5 min, and incubated with 1% BSA in PBS for 30 min. To localize _V and ₁ integrins, the cells were stained with the rabbit antiserum to _V (1:200 in PBS/1% BSA) or the mAb TS2/16 to ₁ (10 µg/ml in PBS/1% BSA). Bound primary antibodies were visualized by appropriate rhodamine-labeled secondary antibodies (1:100). Photographs were taken on an Olympus BX-60 epifluorescence microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of ₁ Integrins Affects Subcellular Localization of _V Integrins

To determine the distribution of _V and ₁ integrin heterodimers on GD25 and GD25-₁A cells attached to fibronectin, indirect immunofluorescence experiments with specific antibodies were performed. GD25 cells, which do not express ₁ integrin heterodimers (Figure 1b), formed _V-containing prominent focal adhesions when allowed to attach and spread on coverslips coated with fibronectin (Figure 1a), consistent with the reported ability of _V3 to localize to focal adhesions in these cells (Wennerberg et al., 1996; Retta et al., 1998). In contrast, the amount of _V-containing focal adhesions was consistently reduced on GD25-₁A cells attached to fibronectin (Figure 1c), whereas ₁A-containing focal adhesions were abundant (Figure 1d).

View larger version (88K): [in this window] [in a new window]   Figure 1.   Subcellular localization of V and 1 integrins on GD25 and GD25-1A cells plated on fibronectin. GD25 (a and b) and GD25-1A (c and d) cells were allowed to attach and spread on coverslips coated with fibronectin (30 µg/ml in PBS) for 3 h at 37°C. Cells were then fixed, permeabilized, and incubated with primary antibodies against V (rabbit anti-V) and 1 (mAb TS2/16) integrin subunits. The V and 1 antibody-antigen complexes were then detected with rhodamine-conjugated anti-rabbit and anti-mouse secondary antibodies, respectively. Representative fields were photographed with the use of an Olympus BM11 microscope fitted with epifluorescence. Notice that 1 integrins displace V integrins from focal adhesions. Bar, 15 µm.

Thus, ₁A, by localizing to focal adhesions, displaces the _V-containing heterodimers from these structures. Interestingly, we have previously shown that the expression of two other human ₁ isoforms, namely, ₁B, that does not localize to focal adhesions, and ₁D, that is efficiently targeted to focal adhesions, also causes the delocalization of _V heterodimers on the cell surface (Belkin et al., 1997; Retta et al., 1998). Taken together, these data indicate that in GD25 cells cultured on fibronectin _V3 takes over the function of ₁ integrins in mediating focal adhesion assembly; however, when expressed, ₁ integrins behave as trans-dominant molecules with respect to _V integrins.

Expression of the ₁ Integrin Subunit in GD25 Cells Induces Drastic Reduction of Surface Level of _V3 and an Up-Regulation of _V5

Previous results showed that transfection of GD25 cells with cDNA constructs of human ₁ integrin led to surface expression of the ₁ integrin subunit associated with the endogenous ₃, ₅, and ₆ subunits but not with the _V subunit (Retta et al., 1998). In addition, no obvious differences in _V integrin expression were seen by immunoprecipitation from ¹²⁵I-surface-labeled GD25 and GD25-₁A cells with an anti-_V antiserum (Retta et al., 1998). To understand the cellular mechanism(s) controlling the effect of ₁ over _V integrins, we analyzed more in detail GD25 and GD25-₁A cells for the expression levels of their _V integrin heterodimers, namely, _V3 and _V5. Untransfected or ₁A-transfected GD25 cells were surface-labeled with Sulfo-NHS-Biotin then _V3 and _V5 integrins were immunoprecipitated from nonionic detergent cell extracts and analyzed by Western blot. As expected, a polyclonal serum to the _V integrin subunit coimmunoprecipitated _V together with its associated ₃ and ₅ subunits (Figure 2A). The biotinylated _V, ₃, and ₅ proteins resulted as distinct bands in Western blots and, surprisingly, we noticed that, whereas expression of the _V subunit did not change significantly, the relative amounts of ₃ and ₅ proteins in ₁A-expressing GD25 cells were clearly different from those of untransfected GD25 cells (Figure 2A). With the use of antibodies specific for ₃ and ₅ subunits, we confirmed this evidence: ₃ protein levels were much lower in GD25-₁A than in GD25 cells, whereas the opposite was true for ₅ protein levels (Figure 2, B and C). Thus, although the expression of the human ₁A integrin isoform in GD25 cells did not modify the surface expression level of the _V integrin subunit, it led to a down-regulation and an up-regulation of the levels of its associated ₃ and ₅ subunits, respectively. These data suggest that the trans-dominant effect of ₁ integrin isoforms over the subcellular localization of _V integrins in GD25-₁ cells is due to a switching of the relative amounts of the cell-surface expression levels of _V3 and _V5 integrins.

View larger version (50K): [in this window] [in a new window]   Figure 2.   Surface expression of v, 3 and 5 integrins in GD25 and GD25-1 cells. Integrin heterodimers were immunoprecipitated from surface biotinylated untransfected (/) or 1A- and 1B-transfected GD25 cells with polyclonal antibodies specific for v (A), 3 (B), and 5 (C) integrin subunits, respectively. After separation by nonreducing SDS-PAGE and Western blot, the immunoprecipitated proteins were detected with the use of peroxidase-conjugated streptavidin and ECL as described in MATERIALS AND METHODS. Notice that, although the expression of either integrin 1 A or B isoforms does not alter significantly the expression level of the V integrin subunit, it induces a net change of the V3/V5 ratio.

Expression of ₁ Integrins Differentially Regulates mRNA Steady-State Levels of ₃ and ₅ Integrin Subunits

To address at what level the control of ₃ and ₅ protein expression in GD25-₁ cells was exercised, we first compared mRNA steady-state levels of these two integrin subunits in GD25-₁A with those of untransfected GD25 cells, with the use of both RT-PCR and Northern blot procedures.

A duplex RT-PCR assay with two sets of primers was developed for the simultaneous detection of the relative levels of ₃ and ₅ integrin subunit mRNAs. As shown in Figure 3A, a great difference in ₃ mRNA steady-state levels was observed between GD25 and GD25-₁A cells. Interestingly, the lower mRNA levels of ₃ in GD25-₁A compared with those in GD25 cells reflected what we observed at the level of protein cell-surface expression (compare with Figure 2, A and B). On the contrary, there was little difference in ₅ mRNA levels among untransfected and ₁-transfected GD25 cells, with a small elevation observed in GD25-₁A cells (Figure 3A). RT-PCR for ₁ mRNA was performed as control (Figure 3B). Thus, the presence of ₁ integrins in GD25 cells differently modulates ₃ and ₅ mRNA expression.

View larger version (75K): [in this window] [in a new window]   Figure 3.   1 integrins differentially regulate mRNA steady-state levels of 3 and 5 integrin subunits. Total RNA was isolated from 1 × 107 cultured GD25 (/) and GD25-1A cells, and 3 and 5 mRNAs were evaluated by RT-PCR and Northern blot analyses as described in MATERIALS AND METHODS. (A) Duplex RT-PCR assay for the simultaneous detection of 3 and 5 mRNAs. (B) RT-PCR for 1 mRNA performed as control. Molecular size standards (123-bp DNA ladder) are shown on the left. (C) Northern blot: equal amounts of total RNA (25 µg/lane) were probed sequentially by 32P-labeled mouse integrin 3 and 5 cDNA fragments, and by a 32P-labeled -actin probe as a control for RNA loading. Notice that the presence of 1 integrins causes a marked down-regulation of 3 and a little up-regulation of 5 mRNA expression levels.

Northern blot analysis demonstrated that our cDNA probes to ₃ and ₅ specifically recognized mRNAs of ~6.6 and 3.5 kb, respectively, consistent with what has been previously described (Yamada et al., 1995). As resulted from this analysis, the mRNA steady-state level of ₃ was much lower in GD25-₁A than in GD25 cells (Figure 3C, ₃), thus reflecting the difference observed by RT-PCR and protein analysis (see above). In addition, the Northern blot analysis confirmed that the difference in ₅ mRNA steady-state levels between GD25 and GD25-₁A cells (Figure 3C, ₅) did not fully correlate with the difference in ₅ cell-surface expression level (compare with Figure 2, A and C). Thus, in GD25-1A cells the down-regulation of _V3 cell-surface expression strictly correlates with the down-regulation of ₃ mRNA steady-state level, whereas the up-regulation of _V5 is mainly due to translational or posttranslational events leading to an increase of ₅ subunit cell-surface recruitment.

₁ Effect over _V Integrins Occurs Irrespective of the Type of ₁ Isoform and Is Dependent on the Presence of the ₁ Cytoplasmic Domain Common Region

We have previously characterized some of the functional properties of ₁B and ₁D integrin isoforms, comparing these properties with those of the common ₁A isoform. In particular, we have shown that the unique cytoplasmic sequences of ₁B and ₁D endow these molecules with distinctive functional properties with respect to a number of cellular functions (Balzac et al., 1994; Belkin et al., 1997; Belkin and Retta, 1998; Calì et al., 1998; Retta et al., 1998).

To analyze more in detail the effects of ₁ over ₃ and ₅ integrins, we tested GD25 cells expressing ₁B and ₁D isoforms as well as two ₁ deletion mutants lacking almost the entire cytoplasmic domain (₁TR) or the cytoplasmic domain variable region (₁COM) (Retta et al., 1998). Untransfected or ₁-transfected GD25 cells were surface-labeled with Sulfo-NHS-Biotin, and the expression of _V3 and _V5 integrin heterodimers was examined by immunoprecipitation and Western blot analysis as described (see MATERIALS AND METHODS). The _V subunit was expressed at a constant level in all the examined cells (Figures 2A and 4A), whereas a down-regulation of ₃ and an up-regulation of ₅ cell-surface levels were seen in either ₁B- (Figures 2 and 4, B and C) or ₁D-expressing GD25 cells (Figure 4, A-C). Moreover, the expression of the ₁COM cytoplasmic domain mutant induced similar effects, although to a lower extent (Figure 4, A-C). However, GD25-₁TR cells, expressing a ₁ mutant carrying the deletion of the cytoplasmic domain (₁TR), behaved equivalently to the ₁-deficient GD25 cells (Figure 4, A-C).

View larger version (45K): [in this window] [in a new window]   Figure 4.   Comparative analysis of surface expression of v integrins in GD25 cells expressing different integrin 1 forms. (A) v integrin heterodimers were immunoprecipitated from surface biotinylated GD25 cells expressing either the 1A or 1D isoforms or two 1 deletion mutants, lacking the entire cytoplasmic domain (1TR) or the cytoplasmic domain variable region (1COM), with the use of a polyclonal antibody against the v subunit. After separation by nonreducing SDS-PAGE and Western blot, the immunoprecipitated proteins were detected with the use of peroxidase-conjugated streptavidin and ECL as described in MATERIALS AND METHODS. (B and C) Scanning densitometry analysis of 3 (B) and 5 (C) integrins as detected by Western blot. Data are displayed as percentage of the control (GD25) and are representative of three independent experiments. Notice that the 1 effect over the V3/V5 ratio occurs irrespective of the type of the 1 isoform and is dependent on the presence of the 1 cytoplasmic domain common region.

The spectrum of relative ₃ integrin levels in untransfected or ₁-transfected GD25 cells compared well with our subsequent mRNA analysis. In fact, when we analyzed by RT-PCR and Northern blot the mRNA steady-state level of the ₃ subunit in GD25 cells expressing either ₁B, ₁D, ₁COM or ₁TR, we found that in GD25-₁B and GD25-₁D cells it was as low as in GD25-₁A cells, whereas in GD25-₁COM cells it was also reduced but to a lower extent (Figure 5, A-C). On the contrary, the ₃ mRNA level in GD25-₁TR was higher and similar to that of ₁-deficient GD25 cells (Figure 5, A-C). On the other hand, although a little increase of ₅ mRNA steady-state level was observed in GD25 cells expressing ₁B, ₁D, or ₁COM (Figure 5, B and D), it did not fully reflect the high increase observed at the ₅ protein level in the same cells.

View larger version (69K): [in this window] [in a new window]   Figure 5.   Comparative analysis of 3 and 5 mRNA steady-state levels in GD25 cells expressing different integrin 1 forms. Total RNA was isolated from 1 × 107 cultured cells as described in MATERIALS AND METHODS. (A) Multiplex RT-PCR assay for the simultaneous detection of 1, 3, and 5 mRNAs in GD25 cells expressing either the 1A, 1B, or 1D isoforms or two 1 deletion mutants lacking the entire cytoplasmic domain (1TR) or the cytoplasmic domain variable region (1COM). (B) Northern blot: equal amounts of total RNA (25 µg/lane) were probed sequentially by 32P-labeled mouse integrin 3 and 5 cDNA fragments and by a 32P-labeled -actin probe as a control for RNA loading. The positions of 28s and 18s rRNAs are indicated as markers for RNA sizes. (C and D) Scanning densitometry analysis of 3 and 5 mRNA levels as detected by Northern blot. Northern signals were normalized to -actin and displayed as percentage of the control (GD25). Data are representative of three independent experiments. Notice that the down-regulation of 3 mRNA steady-state level occurs irrespective of the type of the 1 isoform and is dependent on the presence of the 1 cytoplasmic domain common region.

These results indicate that the ₁-dependent modulation of ₃ and ₅ integrin subunit expression was not confined to GD25-₁A cells, but that it was also present in GD25 cells expressing two other ₁ isoforms. In addition, the fact that a down-regulation of ₃ and an up-regulation of ₅ were also observed in GD25-₁COM, but not in GD25-₁TR cells, strongly suggests that the control of the expression level of ₃ and ₅ integrin subunits was dependent on the presence of the ₁ cytoplasmic domain common region.

Cell Adhesion to ECM Proteins Is not Required for ₁ Effect on ₃ mRNA Steady-State Level

To determine whether cell adhesion to ECM proteins was required for ₁ effect over ₃ expression levels, we performed Northern blot analysis of ₃ mRNA steady-state level in cells plated on tissue culture dishes coated with either polylysine or two ECM proteins, namely, fibronectin and vitronectin. GD25-₁TR and GD25-₁COM cells were cultured to confluence in complete culture medium and then resuspended in serum-free medium, containing 1 µM monensin, and allowed to attach and spread on polylysine-, fibronectin-, and vitronectin-coated dishes for 2 h at 37°C before RNA isolation for Northern blot analysis.

The results, shown in Figure 6, indicate that the ₃ mRNA steady-state level was constitutively low in GD25-₁COM cells compared with that of GD25-₁TR cells. Similar results were obtained by comparing ₃ mRNA steady-state levels in cells kept in suspension in serum-free medium for up to 2 h with those of long-term adherent cells. Thus, these data suggest that ligation of ECM proteins is not required for ₁ effect over ₃ expression.

View larger version (59K): [in this window] [in a new window]   Figure 6.   Cell adhesion to ECM proteins is not required for 1 effect on 3 mRNA steady-state level. GD25-1TR and GD25-1COM cells were cultured to confluence in complete culture medium. Cells were then resuspended in serum-free medium containing 1 µM monensin and allowed to attach and spread on polylysine- (PL), FN-, or vitronectin (VN)-coated tissue culture dishes for 2 h at 37°C before lysis. Total RNA was isolated as described in MATERIALS AND METHODS, and equal amounts (25 µg/lane) were analyzed for 3 mRNA steady-state level by Northern blot hybridization with the use of 32P-labeled mouse integrin 3 cDNA fragments as probe. Equal loading was confirmed by hybridization of the same blot with a 32P-labeled probe for -actin. The positions of 28s and 18s rRNAs are indicated as markers for RNA sizes. Notice that the 3 mRNA steady-state level is constitutively low in GD25-1COM compared with that of GD25-1TR cells.

De Novo Surface Expression of ₁-associated  Subunits Does Not Affect the Level of ₃ Integrin Subunit

Because human ₁ expression in GD25 cells leads to the assembly and cell-surface recruitment of integrin complex with endogenous ₃, ₅, and ₆ subunits (Retta et al., 1996), it was possible that these ₁-associated  subunits could play a direct role in the down-regulation of _V3 integrin.

To exclude this possibility we took advantage of GD25-₁TR cells by extending our observations with this cell line. The expression of the ₁TR mutant at the surface of GD25-₁TR cells was comparable with that of the ₁A isoform in GD25-₁A cells, as previously determined by flow cytometry analysis (Retta et al., 1998). In addition, by immunoprecipitation experiments we did not see any detectable change in the pattern of  subunits associated with ₁TR in GD25 cells compared with GD25-₁A cells (Figure 7). Nevertheless, the presence of ₁TR integrin heterodimers did not lead to any detectable effect over _V3 or _V5 integrins (Figures 4, A-C, and 5, A-D). Taken together, these data strongly suggest that the down-regulation of ₃ is not directly due to de novo surface expression of the ₁-associated  subunits and confirm that for the effect of ₁ integrins over _V3/_V5 integrin ratio a ₁ subunit carrying, at least, the common region of the cytoplasmic domain is required.

View larger version (85K): [in this window] [in a new window]   Figure 7.   Pattern of  subunits associated with the 1TR cytoplasmic domain deletion mutant. (A) 1 Integrins immunoprecipitated from surface biotinylated 1A- and 1TR-transfected GD25 cells with the mAb TS2/16. (B) Integrin heterodimers immunoprecipitated from surface biotinylated GD25-1TR cells with the mAb GoH3 against 6 and polyclonal antibodies against 3 and 5 integrin subunits. After separation by nonreducing SDS-PAGE and Western blot, the immunoprecipitated proteins were detected with the use of peroxidase-conjugated streptavidin and ECL as described in MATERIALS AND METHODS. Notice that the 1TR mutant correctly associates with three major  subunits at the surface of GD25-1TR cells.

Expression of ₁ Integrins Induces a Marked Decrease in ₃ mRNA Stability

Because modulation of mRNA stability is a potential regulatory mechanism for integrin expression (Sachs, 1993; Feng et al., 1999), we next asked whether the changes in mRNA steady-state levels were due to changes in integrin mRNA stability. The rate of turnover of ₃ and ₅ mRNAs was determined by inhibition of RNA synthesis with 60 µM DRB followed by quantitative blot hybridization analysis of ₃ and ₅ mRNA as a function of time. In GD25-₁A cells grown on tissue culture dishes a clear decrease of ₃ mRNA stability was detected compared with GD25 cells (Figure 8A). The ₃ mRNA decayed with an apparent half-life of >8 h in GD25 cells but <4 h in GD25-₁A cells (Figure 8, A and B). In contrast, the stability of ₅ mRNA was much higher than that of ₃, and no significant difference was observed when GD25 and GD25-₁A cells were compared. Therefore, the effects of ₁ expression over ₃ and ₅ mRNA levels clearly involve a regulation of ₃, but not ₅, mRNA stability

View larger version (57K): [in this window] [in a new window]   Figure 8.   The presence of the 1 cytoplasmic domain doubles the decay rate of the 3 mRNA in GD25 cells. GD25-1A and GD25-1TR cells grown to confluence were divided into three 10-cm Petri dishes and cultured in DMEM containing 10% FBS for 24 h before the addition of 60 µM DRB, an inhibitor of transcription initiation. After addition of DRB, the cells were collected at 0, 4, and 8 h for RNA analysis. Total RNA isolation and Northern analysis were performed as described in MATERIALS AND METHODS. (A) Northern blot: equal amounts of total RNA (25 µg/lane) were probed sequentially by 32P-labeled mouse integrin 3 and -actin cDNA fragments. (B) Scanning densitometry analysis of 3 mRNA levels as detected by Northern blot. 3 Northern signals were normalized to -actin and displayed as percentage of the baseline (time 0). Data presented are the mean values ± SE of three independent experiments. Notice the lower stability of 3 integrin subunit mRNA in GD25-1A than in GD25-1TR cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

There is increasing evidence that a coordinated cross talk between integrin receptors is crucial for an integrated and functional response of a single cell to the extracellular environment (Porter and Hogg, 1997; Blystone et al., 1999; Hynes, 1999). However, the molecular mechanisms of integrin cross talk remain mostly undetermined.

Previously, we showed that the expression of either ₁B or ₁D integrin isoforms in ₁-null GD25 cells prevented different FN-dependent functions of endogenous _V integrins, including their ability to mediate cell adhesion, to localize to focal adhesions, and to assembly an FN matrix, thus indicating the existence of a functional cross talk between these two ₁ isoforms and _V integrins (Belkin et al., 1997; Retta et al., 1998). The present study was undertaken to examine this integrin cross talk and establish the regulatory mechanism(s) whereby ₁ integrins exert their trans-acting functions. The main findings are that 1) de novo expression of the ₁ integrin subunit in ₁-null GD25 cells induces a drastic down-regulation of _V3 and an up-regulation of _V5 integrin cell surface levels; 2) this ₁ effect occurs irrespective of the type of ₁ isoform but is dependent on the presence of the common region of the ₁ cytoplasmic domain; and 3) the down-regulation of _V3 is due to a decreased mRNA stability of the ₃ subunit, whereas the up-regulation of _V5 is mainly due to translational or posttranslational events. These findings provide the first evidence of a cross talk between ₁ and _V integrins based on mechanisms of control of mRNA and protein levels.

Expression of the ₁ Integrin Subunit in GD25 Cells Induces a Drastic Reduction of Surface Level of _v3 and an Up-Regulation of _v5

Despite the apparent high degree of integrin-ligand binding redundancy (Hynes, 1992), the localization of distinct integrins to focal adhesions is usually very restricted (Fath et al., 1989). In GD25 cells two integrins are believed to be able to localize to focal adhesions