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From INSERM 507,* the Departments of Nephrology and Pathology, Necker Hospital, and Université Paris V, Paris; and Institut de Biologie et Chimie des Protéines, CNRS UMR 5086, Université Lyon, Lyon, France
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Abstract |
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 Top Abstract Experimental ProceduresResultsDiscussionReferences |
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6ß4 integrin ligand, was also found to be abnormally expressed in ADPKD. Studies performed with ADPKD cyst-lining epithelial cells (CC) by comparison with normal tubular cells indicate that integrin 6ß4-Ln-5 interactions are involved in cellular events of potential importance for cystogenesis: 1) laminin 5 is a preferential adhesion substrate for CC, mainly through 6ß4 interaction, 2) CC increased haptotactic and chemotactic motility depends on the presence of Ln-5 and requires integrin 3ß1 cooperation, and 3) CC haptotactic or chemotactic migration is specifically increased by mAb-mediated ß4 integrin ligation, through an 3ß1 integrin-dependent and independent pathway, respectively. These results highlight the role of Ln-5 and 6ß4 integrin in adhesive and motility properties of cyst-lining epithelial cells, and further suggest that integrins and extracellular matrix modifications may be of general relevance to kidney epithelial cell cyst formation.
In ADPKD renal cysts, somatic mutations of the wild-type allele of PKD1 and PKD2 and subsequent loss of the functional polycystin complex presumably trigger a cascade of signaling and gene expression events.3 To further understand how the Pc-1/Pc-2 disruption leads to cystogenesis, we performed cDNA array experiments to identify abnormally expressed genes in ADPKD. Among genes differentially expressed between cells derived from control and ADPKD kidneys, we decided to focus on those involved in cell proliferation, cell adhesion, and cell migration, because these events presumably play a key role in cystogenesis.
We were particularly interested by the detection of a strong overexpression of ß4 integrin in ADPKD cyst-derived cells. ß4 subunit associates with 6 to form 6ß4 integrin, which is primarily expressed at the basal surface of most epithelia, and may participate in both cell adhesion and migration. Integrin 6ß4 is a key component of hemidesmosomes4
that link the keratin cytoskeleton with laminins in the basement membrane.4
Integrin ß4 activation can also trigger intracellular signaling through its particularly long cytoplasmic tail. Epidermal growth factor (EGF) stimulation leads to ß4 cytoplasmic tail phosphorylation, disruption of hemidesmosomes, and mobilization of ß4 to actin protrusions.5
Integrin ß4 interaction with laminin 5 (Ln-5) may also trigger intracellular signal transduction.6
Epithelial cells adhere to Ln-5 via two adhesive structures, focal adhesions and hemidesmosomes, through binding to 3ß1 or 6ß1 and 6ß4 integrins, respectively.7
On processing, Ln-5 may also trigger integrin-dependent migration and integrin-independent cell scattering.8,9
Ln-5 ligation to 6ß4 integrin receptor, for instance, may activate PI3K signaling and subsequent stimulation of other integrins implicated in cell motility, such as 3ß1.10
In this study, we identify 6ß4 integrin and Ln-5 aberrant expression in ADPKD, and characterize the functional consequences of 6ß4 integrin-Ln-5 interactions on ADPKD cell adhesion and migration. We report that 1) Ln-5 enhances 6ß4 integrin-mediated adhesion of CC, 2) Ln-5 induces 6ß4- and 3ß1-dependent haptotactic migration of CC, and 3) under EGF stimulation, ß4 integrin ligation promotes migration independently of 3ß1 integrin. These results point to distinct properties of 6ß4 in Ln-5 expressing epithelia, that may contribute in vivo to renal cyst enlargement in ADPKD.
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Experimental Procedures |
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TopAbstractExperimental ProceduresResultsDiscussionReferences |
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We had the unique opportunity to benefit from an ADPKD kidney harvested before the onset of renal failure. This 32-year-old woman was pronounced brain-dead after a ruptured cerebral aneurysm and underwent organ harvesting. However, both kidneys appeared enlarged and multicystic and were refused for cadaveric donation, thus becoming available for research purposes. The serum creatinine at the time of nephrectomy was subnormal (15 mg/dl). The clinical diagnosis of ADPKD was retrospectively confirmed, based on 1) bilateral enlarged polycystic kidneys with typical histological features, 2) intracerebral aneurysm, and 3) a family history of autosomal dominant polycystic kidney disease. We also handled nine other polycystic kidneys, retrieved from ADPKD patients with end-stage renal failure before renal transplantation. As control, we handled normal portions of kidneys containing localized adenocarcinoma obtained from four age-matched patients.
Primary Cell Cultures of Cystic and Non-Cystic Epithelium
ADPKD and control kidneys were used to rise primary cultures of cystic renal tubular epithelial cells (CC) and non-cystic renal tubular epithelial cells (NC). The detailed methods have been published.11 Each primary culture was derived from a pool of all cysts dissected within a kidney. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA) containing 1% fetal bovine serum, 5 µg/ml insulin, 10 µg/ml transferrin, 5 ng/ml sodium selenite, 6.5 ng/ml Triiodothyronin, 10 ng/ml EGF, 500 ng/ml hydrocortisone, and 1% HEPES (Invitrogen). Reagents were purchased from Sigma (St. Louis, MO) unless indicated. For functional studies (adhesion, migration; see below) cells were cultivated 24 hours in DMEM, 1% HEPES (referred as "starvation"), harvested with cell dissociation buffer (Invitrogen), washed and resuspended in DMEM, 1% HEPES, eventually supplemented with various antibodies or EGF. We elected to exclusively use these primary cultures between passages 2 and 4.
cDNA Arrays
RNA was isolated using RNeasy miniKit (QIAGEN, Hilden, Germany) according to manufacturer. Atlas Human cDNA array-1 (BD Biosciences Clontech, Palo Alto, CA) was used for expression analysis per manufacturer’s instructions. Briefly, 5 µg of DNAse-treated total RNA was converted to 32P-labeled first-strand cDNA and purified using column chromatography to remove unincorporated nucleotides. Labeled cDNA was then used to hybridize Atlas cDNA nylon array in ExpressHyb mix (BD Biosciences Clontech) at 68°C overnight. The membrane was then washed twice in 2X SSC at 65°C for 30 minutes each and exposed to autoradiography for 24 hours. The orientation grid supplied by the manufacturer was then used to identify the positions of the hybridization signals.
Antibodies, Extracellular Matrix Proteins, and Reagents
Monoclonal antibodies used in this study were: anti-integrin 2 (clone P1E6), anti-integrin 3 (clone P1B5), anti-integrin 6 (clone NKI-GoH3), anti-integrin ß4 (clone 3E1), and anti-integrin ß1 (clone 6S6) from Chemicon (Temecula, CA); anti-laminin 5: clone BM-165 against the 3 chain (Dr. Patricia Rousselle, Lyon, France) and clone D4B5 against the 
2 chain (Chemicon); anti-cytokeratin (clone AE1-AE3) from DAKO (Glostrup, Denmark). Polyclonal antibody anti-ß4 (H-101) was from Santa Cruz Biotechnology (Santa Cruz, CA). Human laminin 5 (Ln-5; 3ß32) was purified from SCC25 cells;12
collagen I and collagen IV were purchased from Sigma.
Immunohistochemistry
Cryostat sections of tissues were fixed with acetone and stored at -20°C. Before use, specimens were fixed again in chloroform, blocked with 5% goat serum and 1% bovine serum albumin (BSA) in PBS, incubated with the primary antibody for 1 hour, and treated with LSAB2-system horseradish peroxidase (HRP) (DAKO) according to the manufacturer’s recommendations. Peroxidase staining was examined with an Ortho LEICA microscope coupled to a CDD camera (Olympus). Cells grown on coverslips (Costar, Cambridge, MA) were fixed with acetone, incubated with 3% hydrogen peroxide (DAKO), incubated 1 hour with primary antibody or negative control reagent, and subsequently treated as above.
Immunofluorescence
Monolayers were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes at room temperature, treated with 0.25% Triton X-100 for 10 minutes, and saturated for 30 minutes with 2% BSA in PBS. Cell monolayers were successively (after intermediate washes in PBS) incubated for 1 hour with a monoclonal antibody to Ln-5 2 chain (D4B5, 1:100), and a fluorescein isothiocyanate goat anti-mouse antibody (Jackson Immunoresearch Laboratories, West Grove, PA). The coverslips were mounted with Aquapolymount antifading solution (AGAR, Stansted, Essex, UK) onto glass slides and the slides were observed under a Leica fluorescence microscope.
RT-PCR Analysis
Aliquots of total RNA extracted from cells (1 µg, RNeasy mini Kit; QIAGEN) or tissues (20 µg, Trizol reagent; Invitrogen) were used as a template for cDNA synthesis. Reverse transcription was carried with 200 ng/µl random hexamers (C1181; Promega, Madison, WI) and M-MLV reverse transcriptase (Invitrogen). Polymerase chain reaction (PCR) was performed using 50 ng (cells) or 250 ng (tissues) of cDNA samples, 2U TaqDNA polymerase (M1665; Promega) in 10 mmol/L (pH 9) TrisHCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L of each deoxynucleotide, and 500 nmol/L of each primer pair. Primers and PCR conditions are listed in Table 1
. PCR products were analyzed by electrophoresis using 1.5% agarose gels and photographed as ethidium bromide fluorescent bands. GAPDH was used as control, allowing quantitative estimation of gene expression by computed densitometry (NIH Imager 4.1 software).
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Cells were harvested with cell dissociation buffer (Invitrogen-BRL), lysed and centrifuged; proteins (20 µg) were boiled under denaturing condition, separated on 7.5% SDS-PAGE, and blotted to PVDF membranes (Millipore Corp., Bedford, MA). Blots were saturated 1 hour at 37°C with TBST (20 mmol/L/L Tris-HCl, pH 8, 137 mmol/L/L NaCl, 0.1% Tween-20) containing 2% BSA and incubated 1 hour at room temperature with 1:200 anti-ß4 antibody sc-9090 (Santa Cruz); ß4 integrin was visualized using an enhanced chemiluminescence system (ECL plus; Amersham Biosciences Inc., Buckinghamshire, UK) and Biomax films (Kodak). Membranes were rehybridized with anti-ERK1/2 (Sigma) to ensure equal loading.
Cell Adhesion and Motility
Adhesion
Adhesion assays were performed in 96-well plates. Wells were coated 3 hours at 37°C with 10 µg/ml Ln-5, collagen I or collagen IV, washed in PBS, and blocked with 1% BSA (Sigma) at 37° for 2 hours. 1 x 104 cells were then plated and allowed to attach for 1 hour. Non-adherent cells were washed away. Adherent cells were fixed with absolute ethanol, colored (crystal violet 0.2% in 2% ethanol), visualized using a x20 objective, and counted under a light microscope with an eyepiece grid. At least three optic fields in triplicate wells were counted for each condition.
Modified Boyden Chamber Motility Assays
Haptotactic migration (ie, migration toward ECM components) was studied using modified Boyden chambers (Transwell 24-well plates, 8.0-µm pore size; Costar). The lower chambers of the plate were loaded with DMEM and overlaid by a 8.0 µm polycarbonate filter eventually coated with Ln-5, collagen I, or collagen IV (as above). CC or NC starved cells (2 x 104 in 100 µl of DMEM) were then added to the upper chamber of the filters and allowed to migrate through the filters for 12 hours at 37°C in 5% CO2. On completion filters were removed and fixed with absolute ethanol. Cells that had not migrated were removed from the top of the filter with a cotton swab; cells that had migrated (remaining on the bottom side of the filter) were stained (crystal violet 0.2% in 2% ethanol) and numerated as above (cf adhesion assays).
The same modified Boyden chambers were used to quantify EGF-stimulated migration. For these assays, starved CC or NC cells (4 x 104 in 100 µl) were stimulated by EGF at the time of cell plating and allowed to migrate through uncoated filters for 18 hours. Cells that migrated through the filters were identified and counted as outlined above.
Wound Healing
When reaching 90% to 100% confluence, NC or CC cells grown on plastic coverslips (Costar) were starved and the following day, a straight and uniform scratch was made on the monolayer with a 200-µl plastic pipette tip. Monolayers were washed gently and incubated 20 hours at 37°C in DMEM ± EGF. For each experimental condition, the area of the wound was assessed at various time points in 10 fields initially marked for reference, using a 100-unit eyepiece optic grid under a light microscope. The difference in number of optic units filled by cells before and after wound repair is expressed as mean ± SD.
Inhibition Experiments
For adhesion and Transwell migration inhibition assays, cells were first pre-incubated for 30' at 4°C with anti-integrin antibodies. For wound healing inhibition assays, antibodies (and EGF) were added to the medium later, at the time of stimulation. The concentrations of the antibodies used for inhibition experiments (10 µg/ml) were equal or up to two times the concentration giving the maximal inhibition in preliminary dose-response experiments.
Statistical Analyses
Experiments were performed in triplicate at least three times each, with primary cultures of different origin. Data are expressed as means ± SD. Statistical significance was determined using Student’s t-test. P < 0.05 was considered to be significant.
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Results |
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TopAbstractExperimental ProceduresResultsDiscussionReferences |
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mRNA isolated from a set of ADPKD cyst-derived cells (CC, n = 3) and control kidney tubular cells (NC, n = 3) were used to perform cDNA arrays. Comparison of hybridization signals by optic density scanning suggested that the gene encoding ß4 integrin was overexpressed in ADPKD cyst-derived cells (not shown). These results were confirmed by semiquantitative RT-PCR: the ß4/GAPDH ratio was consistently higher (two to four-fold) when tested in 10 CC when compared to four NC primary cultures (Figure 1A)
. To confirm that this difference holds true at the protein level, we performed Western blot analysis of cell lysates using H-101 polyclonal antibody. Indeed, a much stronger ß4 protein expression was detected in CC than in NC (Figure 1B)
. To test whether ß4 integrin overexpression was specific, or a more general feature of integrin expression in ADPKD, we performed immunocytochemical experiments. ß4 integrin was undetectable in NC and positive in CC with two different anti-ß4 antibodies, 3E1 (Figure 1C)
and ASC-9 (not shown); by contrast, 3, 6, and ß1 integrins were strongly expressed in both types of cells (Figure 1C)
.
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. Immunohistochemistry with the 3E1 antibody, directed against the extracellular domain of ß4 integrin, showed no detectable ß4 integrin expression in normal adult kidney [Figure 2B(a)
]. By contrast, in five different adult ADPKD patients, 70% to 80% of the kidney cyst-lining epithelia exhibited a strong ß4 basolateral expression [Figure 2B(b)
]. Of note, aberrant ß4 expression was also observed on undilated tubules in a patient with APDKD and normal renal function [Figure 2B(c)
], suggesting that it is an early event. Since ADPKD epithelial cells have been reported to express fetal markers, we also studied ß4 immunostaining in two fetal kidneys (33 weeks of gestation), which was found positive in the medullar collecting ducts [Figure 2B(d)
]. Additional stainings performed on serial sections showed a perfect match between ß4 and 6 integrin stainings, as expected [Figure 2B(e to f)
]. The proportion of cysts of proximal origin (stained by lotus tetragonolobus lectin), of distal origin (stained by 3 chain of collagen IV and epithelial membrane antigen antibodies) and originating from the collecting ducts (stained by dolichos biflorus agglutinin lectin) or showing no nephron segment specificity were the same, whether the cysts were positive or negative for ß4 staining (not shown). Positive ß4 staining was also independent of the size of the cysts.
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6ß4 integrin ligand, was expressed in ADPKD kidneys. RT-PCR was performed using primers designed to amplify the LAM2 gene, encoding for the Ln-5-specific 2 chain. LAM2/GAPDH transcript ratio was two- to 3.5-fold higher in ADPKD than in normal renal tissues (Figure 3A)
. Additionally, immunohistochemical staining for Ln-5-specific 2 chain (D4B5 Ab) and Ln-5 3 chain (BM 165 Ab) showed strong positivity in BMs adjacent to cystic epithelia in ADPKD patients [Figure 3B(a and b)
]. By contrast, no significant Ln-5 staining was observed in control adult kidneys [Figure 3B(c)
]. Overall, Ln-5 positivity was found in 70% of ADPKD cysts. Importantly, Ln-5 pericystic aberrant expression was already observed in a patient with preserved renal function [Figure 3B(b)
]. Interestingly, Ln-5 and ß4 integrin aberrant expression correlated nicely in individual cysts (not shown). When fetal kidneys were studied, Ln-5 expression was found early in development, at 12 weeks, in the ureteric bud [Figure 3B(d)
]. As for ß4 integrin, this suggested aberrant reexpression of a fetal marker in ADPKD. To test the specificity of this aberrant laminin expression, we studied the expression of laminin 10/11 5 chain; immunohistochemistry showed a strong positivity in ADPKD but similar to control kidneys (not shown).
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6ß4 Integrin Mediates Enhanced Cystic Cells Adhesion to Ln-5
We first assessed the binding of CC and NC on plastic support coated with increasing concentrations of Ln-5 (1 to 50 µg/ml). As shown on Figure 4A
, Ln-5 is a preferential adhesion substrate for CC, in a dose-dependent manner, with an optimal differential adhesion seen at 50 µg/ml. The 10 µg/ml Ln-5 concentration was chosen for subsequent adhesion assays. The Ln-5 specificity of this adhesion preference was demonstrated by comparing adherence of CC and NC to other ECM components, ie, collagen I and IV. No significant differences were seen on plastic, collagen I- or collagen IV- coated dishes, whereas adhesion to Ln-5 was more than twofold higher for CC (Figure 4B)
. A role for 6ß4 integrin in preferential CC adhesion to Ln-5 was studied using monoclonal blocking antibodies (Figure 4C)
. On a Ln-5 support, anti-Ln-5 mAb (BM 165) exerted a 70% inhibition of CC attachment; mAbs against integrins 6 and ß4 similarly decreased to -40% CC adhesion. By contrast, anti-2, -3, and -ß1 mAbs had little effect on cell binding, suggesting that 6ß4 integrin was the major mediator of CC adhesion to Ln-5 (Figure 4C)
. NC adhesion to Ln-5 was not blocked by anti-ß4 mAb, and was partially blocked by anti 6, 3, and ß1 mAb, suggesting that the two other integrin receptors for Ln-5, ie, integrins 3ß1 and 6ß1, were responsible for NC adherence to Ln-5 (not shown). On a collagen I support, a preferential ligand for 2ß1 integrin, CC adhesion was not influenced by anti-Ln-5 and anti-integrins 6 or ß4 mAbs, showing the specificity of the effects observed in Figure 4C
, while anti-2 and anti-ß1 blocking mAbs exerted a strong inhibitory effect, as expected (Figure 4D)
.
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To study the role of Ln-5 in CC motility, we first performed haptotactic migration assays using Ln-5-coated Transwell filters. Quiescent CC displayed a two-fold higher spontaneous migration toward Ln-5 than NC (Figure 5A)
. The Ln-5 specificity of this migration preference was demonstrated by comparing haptotaxis of CC and NC toward other ECM components; both cell types showed weak and comparable spontaneous migration toward collagen I or IV, and virtually no migration through uncoated filters (Figure 5A)
. Ln-5 displayed a dose-dependent haptotactic effect on CC (Figure 5B)
, and a 10 µg/ml coating concentration was chosen for subsequent experiments. To further confirm the specificity of Ln-5-mediated CC haptotaxis, we used an anti-Ln-5 blocking mAb (BM 165), which almost completely blocked CC migration toward Ln-5 (Figure 6A
, column 1).
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. We further tested EGF-stimulated motility in a scratch assay, an in vitro model for wound healing in various epithelial systems. After 20 hours of EGF stimulation (10 ng/ml) CC displayed a 50% increase in wound healing compared to NC (Figure 5D)
. To test the role of Ln-5 in EGF-induced CC migration, we used BM 165 blocking mAb. Surprisingly, BM 165 was able to almost completely block EGF-induced CC migration through Transwell and during wound healing (Figure 6A
, columns 2 and 3). A control isotypic mAb did not modify CC migration in both assays (not shown). To confirm the relevance of these findings, we further tested whether CC were actually synthesizing Ln-5 in our system. Indeed, immunofluorescence staining with anti-2 chain mAb D4B5 revealed a strong Ln-5 network surrounding CC, while staining with control isotypic Ab was negative (Figure 6B)
. Altogether, these data suggest that Ln-5 is a strong haptoattractant for CC, and that EGF-induced CC motility requires Ln-5 production. ß4 Integrin Ligation to 3E1 Antibody Specifically Stimulates CC Migration
The possibility that 6ß4 integrin expression influenced migration of CC was assessed in vitro by using the three migration models described above. Although anti-ß4 integrin antibody 3E1 exerted a blocking effect on adhesion, it consistently stimulated CC migration, either haptotaxis toward Ln-5 (+ 75%) [Figure 7A(a)
], EGF-induced Transwell migration (+ 120%) [Figure 7A(b)
], or EGF-stimulated wound healing (+ 50%) [Figure 7A(c)
]. While increasing 3E1 concentrations had no effect on NC, they exerted a dose-dependent effect on CC haptotaxis toward Ln-5 (Figure 7B)
, consistent with the ß4 integrin overexpression in CC. Another adhesion blocking anti-ß4 mAb (ASC-9) resulted in a similar albeit milder stimulation of CC migration (not shown). The promigratory effect of these anti-integrin ß4 mAbs raised two non-exclusive hypothesis: 1) an effect of ß4 ligation on cell machinery that would directly promote migration, and 2) an indirect effect mediated by increased 3ß1 and/or 6ß1-dependent migration.
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3ß1-Dependent Haptotaxis and 3ß1-Independent Chemotaxis
To further address these hypotheses, we performed migration assays with anti-integrins blocking antibodies. In the Ln-5 haptotaxis model, migration was dramatically reduced by preincubating cells with anti-integrin 3 (-80%), anti-6 (-75%) and anti-ß1 (-70%) mAbs, but not by anti-2 mAb (Figure 8A)
. The stimulating effect of 3E1 mAb was almost completely blocked by antibodies directed against 3ß1, and partially blocked by antibodies directed against 6ß1 (Figure 8A
, last 3 columns), suggesting that the stimulating effects of ß4 ligation operate mainly through an 3ß1-dependent pathway. In the EGF-stimulated wound repair assay, anti-3 mAb strongly inhibited CC migration (-70%), while anti-2, anti-6, and anti-ß1 mAb exerted a moderate inhibition (-27%, -5% and -33%, respectively; Figure 8B
), suggesting that in this assay, CC migration is also mainly 3ß1-dependent. In this assay however, the promigratory effect of 3E1 was not blocked by the combination of anti-ß1 and either anti-3 or anti-6, suggesting that ß4 integrin ligation is able to promote migration independently of these integrins.
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Discussion |
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TopAbstractExperimental ProceduresResultsDiscussionReferences |
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Through cDNA arrays screening of differentially expressed genes, we identified a strong overexpression of ß4 integrin in ADPKD cystic cells. RT-PCR analysis confirmed that ß4 integrin transcripts were overexpressed in ADPKD compared to normal kidneys. Immunohistochemistry further demonstrated a strong ß4 integrin expression in most ADPKD cyst-lining epithelia, whereas no expression was detected in normal adult tubules. Our observation of ß4 integrin overexpression in slightly dilated ADPKD tubules in a patient with preserved renal function [Figure 2B(c)
] suggests it is an early event in the cyst formation process and that this finding is not related to end-stage renal disease. Strong ß4 integrin expression during normal fetal kidney development suggests that ß4 integrin may be either persistently expressed after birth in ADPKD tubules, or reexpressed later during cystogenesis. Costaining with specific markers ruled out ß4 integrin aberrant expression as a nephron segment-specific event. Expression of other integrins (2, 3, 6, and ß1) by immunohistochemistry was similar in ADPKD and control kidneys (not shown), highlighting the specificity of ß4 integrin overexpression in ADPKD. The reason why a few cysts did not express ß4 integrin remains obscure.
Ln-5, the main ligand for 6ß4, was also aberrantly expressed in most of the cyst-surrounding ECM. Other authors have reported the strong expression of unspecified laminin in the Cy rat model of ADPKD,14
and the presence of uncharacterized laminin breakdown products in the cyst fluid of ADPKD patients.15
As expected, we observed a good correlation between Ln-5 and ß4 integrin-positive staining among individual cysts. In accordance with previous data, no expression of Ln-5 was detected in normal adult tubules.16
By contrast, we detected Ln-5 expression in fetal kidney ureteric bud, as reported by others [Figure 3B(d)
].17
To clarify the role of 6ß4 integrin-Ln-5 interactions in ADPKD, we set up in vitro conditions to analyze cell anchorage and cell motility. As shown in Figure 1
, cells derived from ADPKD cysts in primary culture maintain high production of ß4 integrin mRNA and protein. In short-term adhesion studies, CC and NC adhere similarly to collagens I and IV, while Ln-5 induce specifically CC but not NC to adhere more strongly (two-fold) in a dose-dependent manner (Figure 4, A and B)
. This result contradicts the previously reported weaker adhesion of ADPKD cyst-lining cells to laminin than to other ECM components,18
but the laminin composition and experimental design were clearly different in this work. Most of CC adhesion to Ln-5 was blocked by anti-6 or ß4 mAbs, suggesting that the strong and specific adhesion of CC to Ln-5 is mainly 6ß4-dependent, with 3ß1 and 6ß1 playing a minor role (Figure 4C)
.
We then tested in Transwell assays the ability of exogenous matrix substrates to stimulate haptotactic migration, and found that Ln-5 is a much stronger haptoattractant for CC than collagen I or collagen IV (Figure 5A)
. Furthermore, complete inhibition of EGF-stimulated migration by anti-Ln5 mAb [Figure 6(b and c)
] suggests that Ln-5 endogenous production and extracellular deposition as a substrate is necessary for CC to migrate.19
Blocking experiments further indicated that among Ln-5-binding integrins, 3ß1 was the major mediator of CC haptotaxis toward Ln-5 and EGF-stimulated wound repair (Figure 8A)
, in agreement with findings reported in migrating keratinocytes.20-22
These studies failed to demonstrate a role for 6ß4 in migration, but mAbs used to block integrin 6ß4 function were generally raised against the 6 subunit. In this study we show that the anti-ß4 subunit mAb 3E1 stimulates CC migration in the three different models tested (Figure 7)
. Integrin ß4 ligation to 3E1 could indirectly enhance migration by preventing adhesion. However, a similar observation was made on a colon cancer cell line in which 3E1 had no effect on adhesion.23
Indeed, our blocking experiments suggest that that 3E1 ligation to ß4 may transactivate 3ß1 integrin in the Ln-5-driven haptotaxis assays (Figure 8A)
, and even directly stimulate the promigratory cell machinery in the EGF-stimulated CC motility assays (Figure 8B)
. Interestingly, it was reported that both 3E1 mAb ligation to ß4 and EGF receptor binding to 6ß4 could both trigger ß4 cytoplasmic domain phosphorylation, downstream signal transduction, and subsequent epithelial cell migration and tumor invasion.5,24,25
To date, 6ß4 integrin and Ln-5 overexpression have mostly been reported in neoplastic diseases and were described separately. Expression of 6ß4 integrin was positively correlated to the progression of various carcinomas26
and facilitated cell motility, tumor invasion, and metastatic potential.27
Classically, 6ß4 signaling is triggered by its ligation to the tumor-produced laminins,26
but it has also been described in laminin-free ECM environments.28
Overexpression of Ln-5 was described in various tumors29
and was shown to modulate cell adhesion,30
migration, and proliferation.10,31
Furthermore, disruption of 6ß4-Ln-5 interaction was shown to inhibit tumor growth.32
A role for 6ß4 and Ln-5 in kidney tubulogenesis was recently suggested by Zent et al,33
who reported that blocking antibodies to 6 or Ln-5 could inhibit ureteric bud branching morphogenesis in whole embryonic kidney organ culture as well as in isolated ureteric bud culture. Of note, a role for Ln-5 acting not only through 6ß4 but also through 3ß1 was reported in this model, in accordance with the involvement of 3ß1 integrin in CC migration.
In ADPKD, the mechanism of ß4 integrin and Ln-5 cystic aberrant expression remains unknown. However, we found that ß4 integrin mRNA expression by CC was dose-dependently increased by EGF (data not shown), as described in other cell types.34
The positive regulation of ß4 integrin expression by EGF may be biologically relevant in ADPKD because cysts contain high levels of EGF, which is able to interact with apical EGF receptors in vivo.35
Altogether, our in vitro findings suggest a role for 6ß4 and Ln-5 in kidney cystogenesis. Ln-5-6ß4 complex mediates both adhesion and migration of cyst-derived cells. Several factors may account for this apparent duality, including the proteolytic cleavage of Ln-5 2 chain8
and the activation of EGF receptor pathway.5,25
One can speculate that stable anchorage of the epithelium is necessary to maintain the cystic architecture, while cyst growth requires cell migration. Further work is currently under way to clarify the role of Ln-5-6ß4 interaction in dysregulated proliferation, another cardinal feature of ADPKD cystic epithelia.
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Acknowledgements |
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Footnotes |
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Supported by Institute National pour la Santé et la Recherche Médicale, Assistance Publique-Hôpitaux de Paris, Association pour l’Utilisation du Rein Artificiel, Association pour l’Information et la Recherche dans les maladies Génétiques rénales, and Amgen.
Accepted for publication July 15, 2003.
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References |
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6/ß4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 1991, 113:907-9176ß4 integrin are regulated by EGF. J Cell Biol 1996, 134:241-2532 chain in basement membranes of various human tissues. Horm Res 1998, 50:7-14
2ß1-integrin in focal clusters in adherent renal epithelia. Lab Invest 1999, 79:1311-1323[Medline]
3 ß1 in epithelial basement membranes. Cell 1991, 65:599-610[Medline]
6 ß4 integrin: distinct ß4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J 1995, 14:4470-4481[Medline]
6ß4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion. J Cell Biol 2001, 155:447-4586ß4 integrin and epithelial cell migration. Curr Opin Cell Biol 2001, 13:541-545[Medline]
6ß4 integrin promotes carcinoma invasion. Cell 1997, 91:949-960[Medline]
6ß4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells. J Cell Biol 1998, 143:1749-17602ß1. J Cell Sci 1998, 111:1993-2004
3ß1 integrin, and mitogen-activated protein kinase can regulate epithelial cell proliferation. Mol Biol Cell 1999, 10:259-270
B blockade and oncogenic Ras trigger invasive human epidermal neoplasia. Nature 2003, 421:639-643[Medline]
6ß4 integrin subunits in corneal epithelium. J Cell Biochem 2001, 80:397-414[Medline]
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 D. Joly, S. Berissi, A. Bertrand, L. Strehl, N. Patey, and B. Knebelmann Laminin 5 Regulates Polycystic Kidney Cell Proliferation and Cyst Formation J. Biol. Chem., September 29, 2006; 281(39): 29181 - 29189. [Abstract] [Full Text] [PDF]
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 G. Z. Mak, G. M. Kavanaugh, M. M. Buschmann, S. M. Stickley, M. Koch, K. H. Goss, H. Waechter, A. Zuk, and K. S. Matlin Regulated Synthesis and Functions of Laminin 5 in Polarized Madin-Darby Canine Kidney Epithelial Cells Mol. Biol. Cell, August 1, 2006; 17(8): 3664 - 3677. [Abstract] [Full Text] [PDF]
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 M. B. Shannon, B. L. Patton, S. J. Harvey, and J. H. Miner A Hypomorphic Mutation in the Mouse Laminin {alpha}5 Gene Causes Polycystic Kidney Disease J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1913 - 1922. [Abstract] [Full Text] [PDF]
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