Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M606151200 on July 26, 2006

J. Biol. Chem., Vol. 281, Issue 39, 29181-29189, September 29, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/39/29181    most recent
M606151200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Joly, D.
Right arrow Articles by Knebelmann, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Joly, D.
Right arrow Articles by Knebelmann, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Laminin 5 Regulates Polycystic Kidney Cell Proliferation and Cyst Formation*

Dominique Joly, Sophie Berissi1, Amélie Bertrand1, Laetitia Strehl, Natacha Patey, and Bertrand Knebelmann2

From the Université Paris-Descartes, FacultédeMédecine, Inserm U813, AP-HP, Hôpital Necker-Enfants-Malades, Service Néphrologie, 149 Rue de Sévres, 75015 Paris, France

Received for publication, June 27, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal cyst formation is the hallmark of autosomal dominant polycystic kidney disease (ADPKD). ADPKD cyst-lining cells have an increased proliferation rate and are surrounded by an abnormal extracellular matrix (ECM). We have previously shown that Laminin 5 (Ln-5, a {alpha}3beta3{gamma}2 trimer) is aberrantly expressed in the pericystic ECM of ADPKD kidneys. We report that ADPKD cells in primary cultures produce and secrete Ln-5 that is incorporated to the pericystic ECM in an in vitro model of cystogenesis. In monolayers, purified Ln-5 induces ERK activation and proliferation of ADPKD cells, whereas upon epidermal growth factor stimulation blocking endogenously produced Ln-5 with anti-{gamma}2 chain antibody reduces the sustained ERK activation and inhibits proliferation. In three-dimensional gel culture, addition of purified Ln-5 stimulates cell proliferation and cyst formation, whereas blocking endogenous Ln-5 strongly inhibits cyst formation. Ligation of {alpha}6beta4 integrin, a major Ln-5 receptor aberrantly expressed by ADPKD cells, induces beta4 integrin phosphorylation, ERK activation, cell proliferation, and cyst formation. These findings indicate that Ln-5 is an important regulator of ADPKD cell proliferation and cystogenesis and suggest that Ln-5 {gamma}2 chain and Ln-5-{alpha}6beta4 integrin interaction both contribute to these phenotypic changes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autosomal dominant polycystic kidney disease (ADPKD)3 is one of the most common inherited monogenic disorders, occurring in ~1-1000 live births. ADPKD is a systemic disorder characterized by the development of multiple renal cysts and variably associated with liver, cardiovascular, gastrointestinal, and genital abnormalities. In Western countries, ADPKD accounts for 5-10% of end stage renal disease (1). ~85% of families with ADPKD are linked to the PKD1 locus on chromosome 16 (2). In the remaining families, the genetic defect is linked to the PKD2 locus on chromosome 4 (3). The proteins encoded by PKD1 and PKD2, polycystins 1 (Pc-1) and 2 (Pc-2), are transmembrane proteins that are able to interact, function together as a non-selective cation channel (4), and also induce several distinct transduction pathways. The "polycystin complex" may have three different subcellular localizations and associated putative functions (5): at lateral membranes of the cells (with a role in cell-cell interaction) (6); at the basal pole of the cell (with a role in cell-extracellular matrix interaction) (7); and at the apical primary cilia of the cells (with a putative role in the mechanotransduction of the urinary flux) (8, 9). 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, ultimately leading to cyst formation.

ADPKD renal cysts originate from the epithelia of the nephrons and collecting ducts. Cyst-lining cells are distinguished by increased proliferation, poor differentiation, and abnormalities in cell polarity, fluid secretion, and extracellular matrix production (1).

Expanding cysts must remodel their environment as they enlarge. Morphological anomalies of the cyst-surrounding ECM have been described in human and animal models of polycystic kidney disease. Pericystic ECM appears thickened and multilaminated in the earliest detectable cysts (10, 11). Cystic epithelial cells produce an abnormal basement membrane and have an abnormal proliferative response to ECM proteins (12-14). Thus, altered extracellular matrix has been postulated to play a role in the pathogenesis of ADPKD. However, the contribution of specific ECM proteins to the development of renal cysts is ill defined.

We have previously shown that Laminin 5 is strongly expressed by ADPKD cells and by the pericystic ECM of ADPKD kidneys, whereas no Laminin 5 expression could be detected in adult control kidneys (15). Laminins are a family of at least 15 multifunctional {alpha}beta{gamma} heterotrimeric proteins commonly found in basement membranes of epithelia (16). The various isoforms have a cell- and tissue-specific expression and are differentially recognized by integrins. Laminins undergo a sequential series of extracellular proteolytic cleavages that might successively turn on and off one or several of their biological and mechanical functions through interaction with integrins and other cell surface receptors (17). Laminin-5 (Ln-5) is a heterotrimer of {alpha}3, beta3, and {gamma}2 chains. Its cell surface receptors are integrins {alpha}6beta4, {alpha}2beta1, {alpha}3beta1, and {alpha}6beta1. Mature Ln-5 plays a fundamental role in keratinocyte adherence by promoting through its interaction with {alpha}6beta4 integrin the assembly of strong and stable adhesion structures called hemidesmosomes (18). In peritumoral ECM, Ln-5 is frequently overexpressed, and the proteolytic-driven modulation of its biological activity could trigger tumor progression (19) and migration properties with subsequent invasion and metastasis (20).

Based on our recent discovery of Ln-5 aberrant expression in the pericystic ECM in ADPKD, we hypothesized that this molecule could promote cystogenesis. In the present study, we characterize the Ln-5 {gamma}2 chain isoforms produced and secreted by ADPKD cyst-lining cells and provide evidence for a crucial role for Ln-5 in proliferation, signaling, and in vitro cystogenesis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, Matrix Molecules, and Reagents—Monoclonal antibodies were obtained as follows: anti-integrin {alpha}2 (clone P1E6), anti-integrin {alpha}3 (clone P1B5), anti-integrin {alpha}6 (clone GoH3), anti-integrin beta1 (clone 6S6), anti-integrin beta4 (clone 3E1), and anti-Laminin 5 {gamma}2 chain (clone D4B5) were from Chemicon (Temecula, CA); anti-PCNA (clone PC10) was from Novocastra (Newcastle, UK); anti-phosphotyrosine was from Transduction Laboratories (Lexington, KY). Polyclonal antibodies were anti-Laminin 5 L132 raised against purified native Laminin 5, a gift from Dr. Patricia Rousselle (Lyon, France), anti-beta4 (clone H-101) from Santa Cruz Biotechnology (Santa Cruz, CA), anti-phospho ERK1/2 (Thr-202/Tyr-204) from New England BioLabs, and total ERK1/2 from Sigma. Secondary antibodies and nonspecific mouse IgGs were from Jackson ImmunoResearch Laboratories. Fluorescein isothiocyanate-goat anti-mouse antibody, Alexa Fluor, was from Molecular Probes.

Purified human ECM molecules used in this study were Laminin 5 (Ln-5; {alpha}3beta3{gamma}2, purified from SCC25 cells), a kind gift from Dr. Patricia Rousselle (21), and collagen I and collagen IV purchased from Sigma. Culture plates were coated for 3 h at 37 °C with 1-50 µg/ml Laminin 5, collagen I, or collagen IV in PBS; unbound sites were blocked with 1% albumin in PBS at 37° for 2 h.

Dulbecco's modified Eagle's medium, HEPES, and cell dissociation buffer were from Invitrogen. Growth factor-reduced Matrigel® was from BD Biosciences. All other reagents were from Sigma unless specified.

Cell Culture—Primary cell cultures of cystic epithelium ("ADPKD cells") and non-cystic epithelium ("control cells") were established as previously described (15), subcultured, and exclusively used between passages 2 and 4. Cells were grown in Dulbecco's modified Eagle's medium-based medium containing 1% fetal bovine serum, 5 µg/ml insulin, 10 µg/ml transferrin, 5 ng/ml sodium selenite, 6.5 ng/ml triiodothyronine, 10 ng/ml epidermal growth factor (EGF), 500 ng/ml hydrocortisone, and 1% HEPES (Invitrogen), referred to as "defined medium". For functional studies (cell proliferation, see below), cells were cultivated for 24 h in serum-free Dulbecco's modified Eagle's medium, 1% HEPES (referred to as "starvation") to obtain quiescence, harvested with cell dissociation buffer (Invitrogen), washed, and resuspended in basal Dulbecco's modified Eagle's medium (referred to as "basal medium") containing 0.1% fetal bovine serum and 1% HEPES, supplemented with EGF and various antibodies when indicated.

In Vitro Cyst Formation—Gel mix was made of a mixture of ice-cold liquefied growth factors, reduced Matrigel®, and isotonic saline (80/20%). 1.5 104 cells were incorporated to 100 µl of gel mix, dispensed into flat bottomed 96-well culture plate wells, and incubated at 37 °C. After solidifying, gel mix was overlaid with 100 µl of defined medium. Plates were maintained at 37 °C with 5% CO2 and observed daily. Cysts were visualized using a x20 objective and counted under a light microscope at day 7. Six optic fields in triplicate wells were counted for each condition. For functional experiments, purified Ln-5 was added to the gel mix to reach the final concentration of 2-20 µg/ml. Anti-Laminin 5 D4B5-blocking Ab (final concentration 10 µg/ml), anti-{alpha}2,-{alpha}3,-{alpha}6,-beta1 integrin antibodies (final concentration 10 µg/ml) and anti-beta4 integrin 3E1-stimulating Ab (final concentration 2 µg/ml) were added to the defined medium every other day.

Cell Proliferation—In monolayer culture, when ADPKD or control cells reached 70% confluence the defined medium was replaced by basal medium to induce quiescence. After 24 h, cells were harvested with enzyme-free cell dissociation buffer (Invitrogen), washed, and seeded in a 96-well plate at the concentration of 2500 cells/100 µl/individual chamber in basal medium to maintain cell viability during the experimental period. The wells had been previously left uncoated or coated with ECM molecules (as described above). After 12 h of adhesion, the basal medium was replaced by basal medium containing the indicated concentrations of EGF or antibodies. The Promega Cell titer 96 MTT assay method was used to assess number of cells before medium replacement (T0) and the relative rate of cell proliferation after 3 days of culture (T72). This colorimetric assay system measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells, and the relative absorbance at 595 nm measured after incorporation of MTT is linearly correlated to the number of viable cells counted by a classical hema-cytometer technique. In inhibition of proliferation experiments, apoptosis and anoikis were ruled out by daily microscopic analysis of the monolayers and terminal differentiation was ruled out by the ability of cells to proliferate after addition of fresh defined medium (results not shown). In three-dimensional gel culture, defined medium was removed at day 6, and gel mixes were submitted to Promega Cell titer 96 MTT assay to assess the rate of cell proliferation as above. A well containing the gel mix without cells was used as reference for absorbance measurement at 595 nm.

Immunoblot Analysis—In monolayer cultures, cells were harvested with 0.05% trypsin and 0.53 mM EDTA, lysed in buffer (150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA, 5% glycerol, and protease inhibitor tablet (Roche Applied Science)) for 10 min at 4 °C. Supernatants from cells cultured for 48 h were collected and concentrated 40 times using Microcon membranes (Amicon®). Cyst fluids retrieved from ADPKD patients were similarly concentrated 10 times. In three-dimensional gel cultures, the culture medium was discarded and the gel mix was liquefied in ice-cold lysis buffer to retrieve proteins. Equal amounts of protein were denatured in the sample buffer (125 mM Tris, pH 6.8, 5% mercaptoethanol, 6% SDS, 20% glycerol, and 0.2% bromphenol blue) at 95 °C, separated on 10% SDS-PAGE gels, electrotransferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA), and detected with the appropriate antibodies using an enhanced chemiluminescence system (ECL plus; Amersham Biosciences) and Biomax films (Kodak). When monolayer experiments were conducted in 96-well plates, cells were directly harvested, lysed, and denatured using 95 °C heated sample buffer and processed as above.


Figure 1
View larger version (60K):
[in this window]
[in a new window]
  		FIGURE 1.
Ln-5 production by ADPKD cells. A, ADPKD cells formed multicellular cystic structures in three-dimensional Matrigel-based gels visualized under phase contrast microscopy (a). Nuclei are stained with DAPI (b). Immunofluorescent staining with L132 anti-Ln-5 pAb (c), anti-beta1 integrin (d) shows colocalization surrounding cyst-forming cells (e). On a different cyst, immunofluorescent staining with D4B5 anti-Ln-5 {gamma}2 chain mAb shows a similar pattern (f). B, schematic representation of known Ln-5 {gamma}2 chain isoforms. C, 40 µg of proteins were extracted from cell lysates (WCL, lanes 1 and 2), 10 x concentrated supernatants (SN, lanes 3 and 4), and three-dimensional gel mixes (3D, lanes 5 and 6) of ADPKD cells in culture for 48 h. Proteic extracts were resolved by SDS-PAGE, Western blotted, and probed with anti-Ln-5 {gamma}2 chain antibody D4B5. D, proteins were extracted from the cyst fluid of an ADPKD kidney (100 µg after 10 x concentration), resolved by SDS-PAGE, Western blotted, and probed with anti-Ln-5 {gamma}2 chain antibody D4B5.

 
beta4 Integrin Phosphorylation—To obtain selective ligation of {alpha}6beta4 in the absence of any co-stimulus, cells were serum starved, detached with cell dissociation buffer, resuspended at 5·106 cells/200-µl aliquots, and incubated at 37 °C in suspension with the 3E1 monoclonal antibody (50 µg/ml) coupled with protein-Sepharose A/G beads for the indicated times. After cell lysis at 4 °C in lysis buffer, beads were pelleted, washed three times with lysis buffer, and eluted with 1 x sample buffer at 95 °C for 5 min. The eluted proteins were subjected to SDS-PAGE and immunoblot analysis (as described above) with anti-phospho-tyrosine mAb. Membranes were stripped and reprobed with anti-beta4 polyclonal Ab.

Immunofluorescence—Gel mixes were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature followed by 100% methanol at -20 °C for 20 min and saturated for 30 min with 2% bovine serum albumin in PBS, with intermediate washes in PBS. Gel mixes were immunostained with either anti-Laminin 5 D4B5 mAb (1 h at 4 °C, 1:100) or anti-Laminin 5 L132 pAb (1 h at room temperature, 1:100) and anti-beta1 6S6 mAb (1 h at room temperature, 1:100) or anti-PCNA PC10 mAb (2 h at room temperature, 1:50), and a fluorescein isothiocyanate-goat anti-mouse antibody and then probed for 60 min at room temperature with the appropriate secondary antibodies coupled to Alexa Fluor 488 or 594 (Molecular Probes). Nuclei were counter-stained with DAPI (Sigma). For Laminin 5 and beta1 integrin stainings, gel mixes were observed with an inverted fluorescence microscope.

For PCNA staining, semi-liquefied gels were transferred and spread on coverslips, mounted with Aquapolymount antifading solution (Agar) onto glass slides, and observed under a Leica fluorescence microscope. For each culture condition, PCNA-positive nuclei were counted in at least 150 multicellular structures and results were expressed as a percentage of nuclei identified by DAPI counterstaining.

Statistical and Density Scanning Analyses—Experiments were performed at least three times with primary cultures originating from different ADPKD kidneys. Density scanning was performed with NIH image 6.02 software. Data are presented as means ± S.E. or S.D. and analyzed by one-way analysis of variance with the unpaired t test. Values considered as significant were p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Ln-5 Expression and Secretion by ADPKD Cystic Epithelia—In a previous study, we reported that Ln-5 is strongly expressed by the pericystic ECM of ADPKD kidneys, whereas no Ln-5 expression could be detected in non-cystic adult control kidneys. Ln-5 aberrant expression was similarly observed in primary cultures of epithelial cells derived from ADPKD cysts when compared with control renal tubular epithelial cells (15).


Figure 2
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 2.
Ln-5-induced proliferation of ADPKD cells. A, MTT incorporation of control cells (empty bars) and ADPKD cells (gray bars) cultivated for 3 days in basal medium or defined medium. Each point represents the mean of triplicate wells (±S.D.). Representative of three independent experiments. B, MTT incorporation of control cells (empty bars) and ADPKD cells (gray bars) cultivated for 3 days in basal medium supplemented with EGF at the indicated concentrations. Results are expressed as mean ± S.E. (n = 3) of relative cell proliferation, with cell proliferation in basal medium set as 1. C, MTT incorporation of control cells (empty bars) and ADPKD cells (gray bars) cultivated for 3 days in basal medium after plating on plastic or the indicated ECM purified components. p, plastic; C.I, collagen I; C.IV, collagen IV. Results are expressed as mean ± S.E. (n = 3) of relative cell proliferation, with cell proliferation on plastic set as 1. D, MTT incorporation of quiescent ADPKD cells plated in basal medium for 12 h (T0) and after 3 days (T72) of culture in basal medium (0%) ± EGF (20 ng/ml) ± isotypic control IgG (10 µg/ml) or anti-LN-5 D4B5 antibody. Each point represents the mean of triplicate wells (±S.E.). Representative of three independent experiments. E, MTT incorporation of quiescent ADPKD cells cultivated for 3 days in basal medium containing EGF (20 ng/ml) ± anti-Ln-5 D4B5 antibody at the indicated concentrations. Results are expressed as mean ± S.E. (n = 3) of relative cell proliferation, with control IgG set as 100% (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

 
To study Ln-5 production during in vitro cyst formation, ADPKD cells were incorporated in three-dimensional Matrigel-based gels and incubated at 37 °C for 7-10 days. Multicellular cystic structures became apparent at day 3-4 and were allowed to grow until day 6 (Fig. 1A). Immunofluorescent costaining with anti-Ln-5 L132 pAb and integrin beta1 showed exclusive basal staining around cysts, suggesting that Ln-5 is incorporated in the ECM (Fig. 1A, panels a and b). A similar pattern was observed with the anti-Ln-5 {gamma}2 chain D4B5 mAb, suggesting that Ln-5 chains are assembled together in the pericystic ECM (Fig. 1A, panel f).

To further assess Ln-5 production by ADPKD cells, we performed immunoblotting with the anti-Ln-5 {gamma}2 chain D4B5 mAb in various two- and three-dimensional cell culture conditions. Known proteolytic fragments of the {gamma}2 chain are depicted in Fig. 1B. Ln-5 {gamma}2 chain was detected in cell lysates of quiescent and EGF-stimulated ADPKD cells (Fig. 1C, lanes 1 and 2), both in its mature form (155 kDa) and in its processed isoform {gamma}2'(domains I-III, 100 kDa). Ln-5 {gamma}2 chains were also found in the supernatant of ADPKD cells cultured for 48 h, suggesting a cellular secretion (Fig. 1C, lanes 3 and 4). Of note, mature {gamma}2 was not detectable, and in addition to the {gamma}2' isoforms, we identified a 60-kDa fragment that was dramatically overexpressed after EGF stimulation (Fig. 1C, lane 4). These results suggest an overall increased production and secretion of Ln-5 after EGF stimulation. In three-dimensional culture conditions, although our gel mix contained very little Ln-5 (Fig. 1C, lane 5), ADPKD cells undergoing cystogenic growth produced significant amounts of Ln-5 with major bands at 100, 60, and 30 kDa and a faint band at 150 kDa corresponding to unprocessed {gamma}2 chain (Fig. 1C, lane 6). Interestingly, the Ln-5 {gamma}2 chain isoform profiles differed when ADPKD cells were grown in two-dimensional and in cystogenic three-dimensional conditions and the 30-kDa band appearance was restricted to three-dimensional culture conditions, suggesting that a specific proteolytic cleavage of the {gamma}2 chain occurs in this setting. In cyst fluid retrieved from ADPKD patients, 60- and 30-kDa Ln-5 {gamma}2 chain bands were detected (Fig. 1D), indicating that Ln-5 secretion and proteolysis also occurred during in vivo cystogenesis.


Figure 3
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3.
Ln-5-induced ERK activation in ADPKD. A, serum-starved cells were detached and kept in suspension or cultured for 60 min on 6-well plates coated with 10 µg/ml purified Ln-5, collagen I, or collagen IV. Total cellular proteins were subjected to immunoblot analysis using mAb specific for the phosphorylated isoform of ERK 1,2. To evaluate loading efficiency, membranes were stripped and reprobed with actin Ab. B, after EGF stimulation (10 ng/ml), total cellular proteins from quiescent ADPKD and control cells were subjected to immunoblot analysis using mAb specific for the phosphorylated isoform of ERK 1,2 (pp44/42). Membranes were stripped and reprobed with Ab recognizing total ERK1/2 (p44/42). C, bar graph from six experiments as in panel B; after densitometric analysis, the pp42/p42 ratio was plotted to assess the degree of ERK1/2 activation at the indicated times. Results are expressed as mean ± S.E. (n = 6) of relative ERK2 activation, with ERK activation at 5 min of EGF treatment set as 100% (*, p < 0.05). D, quiescent ADPKD cells were stimulated by EGF (10 ng/ml) for the indicated times, with addition of either anti-D4B5 mAb or isotypic IgG control Ab (10 µg/ml). Total cellular proteins were subjected to immunoblot analysis as in panel B. E, bar graph from three experiments as in panel D: after densitometric analysis, the pp42/p42 ratio was plotted to assess the degree of ERK2 activation at the indicated times. Results are expressed as mean ± S.E. (n = 3) of relative ERK2 activation, with ERK2 activation of cells treated with control Ab at 5 min of EGF treatment set as 100% (*, p < 0.05).

 
Ln-5 Induces ADPKD Cell Proliferation—One key feature associated with cyst formation in ADPKD is enhanced cell proliferation. ADPKD cells displayed a higher proliferation rate compared with control cells when grown in defined medium (Fig. 2A) or in basal medium supplemented with 10 or 20 ng/ml EGF (Fig. 2B). Conversely, in basal medium, ADPKD cells did not display any significant proliferation advantage over control cells (Fig. 2A). To selectively assess the effects of Ln-5 on proliferation in the absence of any co-stimulus, quiescent cells were plated on plastic or dishes coated with purified ECM components (purified Ln-5, collagen I, collagen IV) and cultured in basal medium for 3 days. Ln-5, but not collagen I or IV, stimulated cell proliferation in a dose-dependent manner, with a more pronounced effect on ADPKD cells compared with control cells (Fig. 2C). Furthermore, a dose-dependent effect was observed with increasing concentrations of Ln-5-coated dishes from1to50 µg/ml (Fig. 2C).

Upon EGF stimulation, ADPKD cells both produce Ln-5 and proliferate. To investigate whether endogenously produced Ln-5 could participate in ADPKD cell proliferation, quiescent ADPKD cells were stimulated by EGF and incubated with the anti-Ln-5 D4B5-blocking antibody for 3 days. As shown in Fig. 2D, D4B5 did not reduce cell number at T0 (12 h after plating), ruling out a major effect on cell adhesion. By contrast, D4B5 blunted the proproliferative effects of EGF stimulation. No cellular apoptosis or necrosis was detected in D4B5-treated ADPKD cells. Additionally, D4B5 decreased EGF-stimulated proliferation dose dependently, with a maximal effect (-75%) obtained at 10 µg/ml (Fig. 2E). Taken together, these data suggest that aberrant expression of Ln-5 confers a proliferative advantage to ADPKD cells, with a specific role for the {gamma}2 chain of Ln-5.

Ln-5-induced ERK Activation in ADPKD Cells—Upon ligation to their ECM ligands and subsequent activation, integrins induce signaling events and mitogen-activated protein (MAP) kinase ERK1 and ERK2 activity (22). Activation of the mitogen-activated protein kinase pathway in turn leads to transcriptional control of genes important for cell proliferation (23). We examined whether Ln-5 could activate ERK in quiescent ADPKD cells. After 30 min of adhesion, ERK1,2 phosphorylation was barely detectable on a plastic support, significant on collagens I and IV and more robust on Ln-5-coated dishes (Fig. 3A). In addition, a dose-dependent effect of Ln-5-induced ERK activation was observed (5 and 10 µg/ml). Time course experiments revealed that the level of ERK activation peaked at 30 min of adhesion and declined thereafter (not shown).

We next examined the ability of the anti-Ln-5 D4B5 antibody to disrupt EGF-mediated ERK activation. Time course experiments revealed that quiescent ADPKD cells grown on plastic and stimulated by EGF displayed a more sustained ERK1/2 phosphorylation (up to 16 h) when compared with control cells (Fig. 3, B and C). As shown on Fig. 3, Ln-5 blocking by D4B5 antibody reduced EGF-induced prolonged ERK activation, whereas no effect was observed on early ERK1/2 phosphorylation (Fig. 3, D and E). Taken together, these results suggest that endogenously produced Ln-5 may contribute to a sustained ERK activation and therefore increase proliferation of ADPKD cyst-lining cells.

Ln-5 Regulates in Vitro Cyst Formation—To address the role of Ln-5 in cyst formation and cyst enlargement, we used the in vitro system described above in which ADPKD cells incorporated in three-dimensional Matrigel®-based gels progressively form multicellular cystic structures. Incorporation of various amounts of purified Ln-5 to the gel mix enhanced cystic formation, as assessed by the number of cysts per optic field at day 7 (Fig. 4A). Ln-5 incorporation to the gel mix was also associated with an increased number of viable cells, as assessed by MTT test (Fig. 4B).


Figure 4
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 4.
A, ADPKD cells were incorporated in three-dimensional Matrigel®-based gels enriched with the indicated concentrations of purified Ln-5. After 7 days, the number of cystic structures per optic field was numerated. Representative of four independent experiments. B, ADPKD cells were cultivated as in panel A. After 7 days, the number of viable cells was assessed by MTT test. Representative of four independent experiments. C, ADPKD cells were cultivated as in panel A. In control wells, ADPKD cells were incubated with isotypic IgG mAb (10 µg/ml), and the number of cysts per field was set as 100%. Incorporation of anti-Ln-5 D4B5 mAb to the gel supernatant (10 µg/ml every other day) reduced cyst formation by 73 ± 9% (n = four experiments). D, microphotograph (x20 magnification) showing representative multicellular structures from Fig. 4C. E, ADPKD cells were cultivated as in panel A with incorporation of either isotypic IgG mAb (10 µg/ml) or anti-Ln-5 D4B5 mAb to the gel supernatant (10 µg/ml every other day). At day 7, the number of viable cells treated with isotypic IgG (set as 100%) or D4B5 was assessed by MTT test. F, ADPKD cells were cultivated as in panel A in basal medium ± EGF (20 ng/ml) ± isotypic control IgG (10 µg/ml) or anti-LN-5 D4B5 antibody (10 µg/ml). After 5 days, cells were stained with DAPI or anti-PCNA PC10 Ab and observed under fluorescent microscopy. For each culture condition, more than 150 multicellular structures were examined and the PCNA/DAPI-positive nuclei ratio was determined. Top, graph is representative of three independent experiments. Bottom, microphotograph showing PCNA and DAPI stainings of one three-dimensional multicellular structure for each condition. All results for Fig. 4 are expressed as mean ± S.D. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

 


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 5.
A, ADPKD cells were incubated in suspension with anti-beta4 mAb 3E1 coupled with protein G-Sepharose beads for the indicated times. beta4 integrin immunoprecipitates were subjected to Western blot analysis using anti-phospho-tyrosine mAb. B, 12-well plates were precoated with anti-beta4 mAb 3E1 or control isotypic mAb (10 µg/ml). ADPKD cells were allowed to attach for 60 min, and ERK activation was detected by immunoblotting of protein extracts with a phospho-ERK Ab. C, ADPKD cells grown on a plastic support were serum starved for 24 h and incubated in the presence of various concentrations of 3E1 mAb in basal medium. Proliferation was assessed by MTT test performed after 72 h. D, ADPKD cells were cultivated in three-dimensional gels as in Fig. 4 in basal medium + isotypic control IgG (10 µg/ml) or the indicated anti-integrin antibodies (all 10 µg/ml except anti-beta4, 2 µg/ml). After 7 days, the number of cystic structures per optic field was numerated. Results for panels C and D are expressed as mean ± S.D. (number of experiments indicated in parentheses) of relative cyst number per optic field, with control IgG set as 100%.

 
Inversely, addition of anti-Ln-5 D4B5 mAb (10 µg/ml every other day) to the gel mix supernatant drastically reduced the number of cysts formed at day 7 by 73 ± 9% (Fig. 4C). In three-dimensional gel cultures, initially isolated ADPKD cells treated with D4B5 proliferated and ultimately formed predominately non-cystic multicellular structures (Fig. 4D). In this setting, the number of viable cells determined by MTT test was reduced by 19 ± 28% (non-significant) when compared with cells treated with isotypic IgG (Fig. 4E), with no morphologic signs of apoptosis or necrosis. To further delineate the role of Ln-5 in proliferation of three-dimensional cultured cells, we studied PCNA staining of nuclei in multicellular (either cystic or non-cystic) structures. As shown in Fig. 4F, D4B5 significantly reduced the percentage of PCNA-positive nuclei in gel mixes stimulated by EGF. Overall, these results suggest that Ln-5 not only maintains its proproliferative effects in three dimensions but also is a mandatory ECM component to maintain cystic architecture of ADPKD cells.

Among Ln-5 Integrin Receptors, {alpha}6beta4 Is an Important Mediator of ADPKD Cell Proliferation and Cystogenesis—We have previously shown that integrin chains {alpha}2, {alpha}3, {alpha}6, and beta1 are strongly expressed by both ADPKD and control kidneys; conversely, beta4 integrin is not detectable in adult tubular epithelial cells, although strongly expressed by ADPKD cyst-lining cells along with an {alpha}6 chain. beta4 immunoblots also suggested that {alpha}6beta4 integrin aberrant expression was maintained in vitro in both quiescent and growing primary cultures of ADPKD cells (15). We thus elected to focus on the role of Ln-5-{alpha}6beta4 integrin interaction, which is likely to be a distinctive feature of ADPKD cyst-lining cells compared with normal tubular epithelial cells.

To mimic Ln-5-{alpha}6beta4 interaction, we used the stimulating anti-beta4 antibody 3E1 (24). Incubation of 3E1-coupled beads with ADPKD cells in suspension, which ligates and clusters beta4 at the cell surface, induced beta4 integrin tyrosine phosphorylation (Fig. 5A).

To confirm that {alpha}6beta4 integrin receptor was involved in Ln-5-induced ERK activation, quiescent ADPKD cells were plated on dishes precoated with anti-beta4 3E1 mAb. As shown in Fig. 5B, beta4 integrin ligation by 3E1 strongly induced ERK1,2 phosphorylation. Taken together, these results suggest that among Ln-5 receptors integrin {alpha}6beta4 is able to activate ERK signaling pathway.

Incubation of 3E1-soluble antibody in basal medium induced proliferation of quiescent ADPKD cells with a dose response between 0.125 and 2 µg/ml (Fig. 5C). We then assessed the role of {alpha}6beta4 integrin and other Ln-5 integrin receptors in cyst formation. In three-dimensional culture conditions, incorporation of stimulating anti-beta4 mAb 3E1 to the gel supernatant increased the number of cysts formed (+34 ± 31%), whereas cyst formation was reduced by anti-{alpha}6 (-23 ± 20%) and anti-beta1 (-32 ± 6%) and not statistically modified by anti-{alpha}2- and anti-{alpha}3-blocking antibodies (Fig. 5D). Overall, these results indicate that overexpressed Ln-5 and {alpha}6beta4 form a functional axis that contributes to proliferation and cystogenesis of ADPKD cells and that among other LN-5 receptors {alpha}6beta1 integrin also participates in cyst formation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Following our finding that Ln-5 is aberrantly expressed in the pericystic ECM of ADPKD kidneys (15), the immunofluorescence and immunoblot data described in the present study (Fig. 1) indicate that ADPKD cyst-lining cells are able to produce and secrete Ln-5-specific {gamma}2 chains. In our in vitro cyst formation assays, ADPKD cells continue to produce Ln-5 and incorporate it in the ECM at their basal side, as suggested by the colocalization of beta1 integrin with Ln-5 (Fig. 1A).

We assessed the proteolytic profile of the {gamma}2 chain because extracellular proteolysis is critical to Ln-5 activity. MMP2 and MT1-MMP are enzymes known to induce {gamma}2 chain cleavage (25, 26). Prolonged incubation of mature {gamma}2 chain with MMP2 generates 155-, 100-, 80-, 60-, and 30-kDa fragments (see Fig. 1 and Ref. 27). In our study, Ln-5 immunoblots were performed with D4B5 mAb, whose epitope is localized within the DIII domain of {gamma}2 chain. In theory, this Ab will recognize the 155-, 100-, 60-, and 30-kDa fragments; others (in the range of 30 to 100 kDa, see immunoblot in Fig. 1C) are of unknown significance.

ADPKD cells in monolayers stimulated by EGF secrete a large amount of the Ln-5 {gamma}2 chain 60-kDa fragment. The 60-kDa fragment, described by others as an intermediary MMP2-induced cleavage form of the {gamma}2 chain of Ln-5 that would combine domains III, IV, and V, has currently no known biological activity (27). ADPKD cells in three-dimensional cystogenic growth were associated with a specific proteolytic profile of the {gamma}2 chain, revealing predominant 100-, 60-, and 30-kDa fragments. The exclusive appearance of the 30-kDa fragment during cystogenesis is striking. This fragment was described by Quaranta et al. (27) as the DIII fragment and was found to be mainly composed of EGF-like repeats. Quaranta et al. have shown that recombinant DIII was able to interact with EGF receptor and subsequently activate in vitro ERK1/2 phosphorylation, gene transcription, and cell motility; in vivo, DIII secretion by mammary epithelial cells was associated with its morphological changes during involution.

In accordance with our in vitro results, we also detected the 60- and 30-kDa fragments in the cyst fluid of ADPKD patients. The presence of unspecified laminins in the cystic fluid has already been reported (28). One could speculate that DIII fragments secreted at the apical pole of ADPKD cyst-lining cells could interact with mislocalized and overexpressed EGF receptors, thus stimulating autocrine cell signaling.

Ln-5 strong expression at the leading edges of many tumor cell populations suggests that this ECM molecule may play, on top of its well studied effects on cell migration and invasion, a role in cell proliferation. Here we report that plating ADPKD and control renal epithelial tubular cells on dishes coated with purified Ln-5 stimulates their growth, with a more marked effect on ADPKD cells. The promoting role of purified Ln-5 on cell proliferation has been documented in several epithelial cell lines and in vascular smooth muscular cells (29, 30). EGF stimulation induces the selective up-regulation of both ECM proteins and integrin molecule signaling (31). We observed that in ADPKD cells, EGF stimulation promotes secretion of Ln-5 {gamma}2 chain fragments and stimulates proliferation. Our finding that Ln-5-blocking antibody D4B5 decreases EGF-stimulated ADPKD cell proliferation suggests that endogenously produced Ln-5 also promotes proliferation in these cells.

Which mechanisms govern Ln-5-induced proliferation? The {gamma}2 chain of LN-5 interacts with integrin {alpha}2beta1 (32). The anti-LN-5 {gamma}2 D4B5 mAb epitope is localized within the DIII domain, which is distinct from the N-terminal {alpha}2beta1 binding site. Thus, the blocking effect of D4B5 on ADPKD cell proliferation may not result from LN-5-{alpha}2beta1 integrin disruption but rather from the blocking of LN-5 cryptic fragments containing DIII. Similarly, the observation that D4B5 mAb does not block keratinocyte {alpha}2beta1 integrin ligation to Laminin 5 and subsequent adhesion but strongly inhibits haptotactic migration of these cells suggests that the {gamma}2 chain of Ln-5 or DIII-containing fragments of it may exert biological effects independently of integrin {alpha}2beta1 ligation (32). Further studies should address the potential effect of recombinant DIII on ADPKD cell proliferation and cyst formation.

Ln-5 may also regulate cell proliferation upon ligation of its {alpha}3 chain to its receptor integrins {alpha}3beta1 and {alpha}6beta4. In response to Ln-5, both integrins were shown to stimulate epithelial cell proliferation (30, 33). ADPKD cells express {alpha}3beta1 and {alpha}6beta4 integrins, whereas control tubular epithelial cells express {alpha}3beta1 but not {alpha}6beta4 integrin (15). Such a difference in integrin receptor distribution could at least partially explain why ADPKD cells proliferate more than control cells when plated on Ln-5 (Fig. 2C). Indeed, we observed that beta4 integrin ligation to the stimulating anti-beta4 3E1 mAb was sufficient to induce ADPKD cell proliferation (Fig. 5C). These data suggest that Ln-5-{alpha}6beta4 integrin interaction is likely to be a distinctive feature of ADPKD cells that is able to promote proliferation.

Our mitogen-activated protein kinase analyses suggest that this enzyme is a key component of the pathway that transduces signals from Ln-5 in ADPKD cells. Upon adhesion and ligation to purified Ln-5, quiescent ADPKD cells display ERK1/2 activation. beta4 integrin ligation by 3E1-stimulating mAb also induced beta4 integrin tyrosine phosphorylation and ERK activation (Fig. 5, A and B), suggesting the existence of a functional LN-5-{alpha}6beta4-mitogen-activated protein kinase signaling pathway in ADPKD cells, in accordance with results published in several epithelial cell lines (30, 33). Although the role of {alpha}3beta1 integrin was not addressed in this study, others have found that {alpha}3 integrin function-inhibiting antibodies reduce early ERK activation in epithelial cell lines plated on Ln-5, suggesting the existence of a LN-5-{alpha}3beta1-mitogen-activated protein kinase signaling pathway (30).

In EGF-stimulated ADPKD cells, Ln-5 blocking by D4B5 antibody did not impact the intensity of early ERK activation but significantly reduced late ERK1/2 phosphorylation (Fig. 3, D and E). This suggests that the endogenously produced {gamma}2 chain of Ln-5 contributes to the sustained ERK activation of EGF-stimulated ADPKD cells. The duration of ERK signaling is critical for generating specific biological responses to mitogen-activated protein kinase pathway signaling. Prolonged ERK activation promotes its sustained nuclear localization, stabilization of immediate early gene products, and cell cycle progression (34-36). It is well established that cell interaction with growth factors and ECM components ultimately leads to Ras-ERK activation, and several studies suggest that both stimuli may operate in a coordinated and sequential fashion, with growth factors being responsible for transient ERK activation and subsequent integrin signaling being responsible for delayed and sustained ERK activation (37, 38).

In our study however, the mechanism by which D4B5 antibody impacts ERK activity remains uncertain. D4B5 may prevent cell interaction with cryptic fragments of the {gamma}2 chain produced and released upon EGF stimulation, although we cannot exclude that this mAb perturbs {alpha}2beta1 integrin signaling by steric hindrance.

To address the functional role of Ln-5 during cystogenesis, we modulated the ECM environment of ADPKD cells growing in three-dimensional gels. Adding purified exogenous Ln-5 to the gel mix increased the number formed of both viable cells and cysts (Fig. 4, A and B). Inversely, incorporation of blocking anti-Ln-5 mAb D4B5 drastically reduced cystogenesis (-73%) and was able to prevent EGF-stimulated cell proliferation assessed by PCNA staining (Fig. 4, C and F). Although endogenously produced Ln-5 was reported to protect epithelial tumor cell survival from apoptosis in three-dimensional gel culture conditions (39), we found that D4B5-induced inhibition of cyst formation could occur with no morphological evidence of cell apoptosis and no major reduction in the number of viable cells (Fig. 4E). These data mainly suggest that cell proliferation contributes to Ln-5-driven cystogenesis. Other mechanisms, such as maintenance of cell apical polarity by recruitment and assembly of laminins at their basal pole, may also participate in cyst formation (40, 41) but were not assessed in this study.

As discussed above, several non-exclusive mechanisms may account for the promoting role of Ln-5 {gamma}2 chain on cystogenesis. EGF receptor was reported to be overexpressed and mis-localized to the apical pole of the cells in humans and several animal models of polycystic kidney diseases (42). Furthermore, inactivating EGF pathway dramatically improved the cystic phenotype (43). Thus, cryptic Ln-5 {gamma}2 fragments produced and secreted by ADPKD cells, such as DIII, could activate EGF receptor and promote cyst formation.

Ln-5 stimulation of integrin receptor signaling may also promote cystogenesis. Although it has been reported that loss of {alpha}2beta1 integrin, the Ln-5 {gamma}2 receptor, resulted in reduced cyst formation by Madin-Darby canine kidney cells grown in collagen gels (44), blocking {alpha}2 integrin did not reduce cystogenesis in our model (Fig. 5D), suggesting that the {gamma}2 chain of Ln-5 or its fragments stimulates cystic growth independently of {alpha}2beta1 integrin ligation. Conversely, experiments conducted with anti-integrin {alpha}3-, {alpha}6-, beta1-blocking antibodies and anti-beta4-stimulating antibody suggest that {alpha}6beta4, {alpha}6beta1 and to a lesser extent {alpha}3beta1 integrins, all integrin receptors of Ln-5, participate in ADPKD cell cystogenic growth (Fig. 5D). We specifically studied the role of {alpha}6beta4 signaling in cystogenesis, as this integrin is overexpressed in ADPKD (15). We show that stimulating anti-beta4 mAb 3E1 is sufficient to increase the number of cysts formed by ADPKD cells. Autocrine Ln-5-{alpha}6beta4 signaling was shown to be critical for epithelial tumor survival both in three-dimensional gel cultures and in vivo (20, 39, 41). Of note, in primary culture, control renal epithelial cells that do not express {alpha}6beta4 soon underwent apoptosis in three-dimensional gels and never develop cysts in our hands (not shown). Thus, Ln-5-{alpha}6beta4 interaction and subsequent signaling may be one of the mechanisms involved in ADPKD cell cystogenic growth.

Collectively, our results indicate that Ln-5 is overexpressed in the pericystic ECM of ADPKD kidneys, produced, secreted, and processed by ADPKD cells. Ln-5 roles in regulating ADPKD cell growth, prolonged ERK activation, and in vitro cystogenesis suggest that this ECM molecule may contribute to the development of renal cysts. Thus, interfering with cyst-lining cell-Ln-5 interaction may define a new therapeutic target in ADPKD.


   FOOTNOTES
 
* This work was supported by grants from AIRG (Association pour l'Information et la Recherche sur les Maladies Rénales Génétiques), GIS-Maladies Rares, AMGEN Inc., and AURA (Association pour l'Utilisation du Rein Artificiel). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 33-1-44495241; Fax: 33-1-44495450; E-mail: knebelmann{at}necker.fr.

3 The abbreviations used are: ADPKD, autosomal dominant polycystic kidney disease; ERK, extracellular signal-regulated kinase; ECM, extracellular matrix; Ln-5, Laminin 5; PBS, phosphate-buffered saline; EGF, epidermal growth factor; Ab, antibody; mAb, monoclonal Ab; pAb, polyclonal Ab; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4',6-diamidino-2-phenylindole; PCNA, proliferating cell nuclear antigen. Back


   ACKNOWLEDGMENTS
 
We thank Patricia Rousselle for providing purified Laminin 5 and L132 antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wilson, P. D. (2004) N. Engl. J. Med. 350, 151-164[Free Full Text]
  2. Hughes, J., Ward, C. J., Peral, B., Aspinwall, R., Clark, K., San Millan, J. L., Gamble, V., and Harris, P. C. (1995) Nat. Genet. 10, 151-160[Medline] [Order article via Infotrieve]
  3. Mochizuki, T., Wu, G., Hayashi, T., Xenophontos, S. L., Veldhuisen, B., Saris, J. J., Reynolds, D. M., Cai, Y., Gabow, P. A., Pierides, A., Kimberling, W. J., Breuning, M. H., Deltas, C. C., Peters, D. J., and Somlo, S. (1996) Science 272, 1339-1342[Abstract]
  4. Hanaoka, K., Qian, F., Boletta, A., Bhunia, A. K., Piontek, K., Tsiokas, L., Sukhatme, V. P., Guggino, W. B., and Germino, G. G. (2000) Nature 408, 990-994[CrossRef][Medline] [Order article via Infotrieve]
  5. Boletta, A., and Germino, G. G. (2003) Trends Cell Biol. 13, 484-492[CrossRef][Medline] [Order article via Infotrieve]
  6. Streets, A. J., Newby, L. J., O'Hare, M. J., Bukanov, N. O., Ibraghimov-Beskrovnaya, O., and Ong, A. C. (2003) J. Am. Soc. Nephrol. 14, 1804-1815[Abstract/Free Full Text]
  7. Wilson, P. D., Geng, L., Li, X., and Burrow, C. R. (1999) Lab. Investig. 79, 1311-1323[Medline] [Order article via Infotrieve]
  8. Yoder, B. K., Hou, X., and Guay-Woodford, L. M. (2002) J. Am. Soc. Nephrol. 13, 2508-2516[Abstract/Free Full Text]
  9. Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E., and Zhou, J. (2003) Nat. Genet.
  10. Cuppage, F. E., Huseman, R. A., Chapman, A., and Grantham, J. J. (1980) Kidney Int. 17, 372-381[Medline] [Order article via Infotrieve]
  11. Cowley, B. D., Jr., Gudapaty, S., Kraybill, A. L., Barash, B. D., Harding, M. A., Calvet, J. P., and Gattone, V. H., II (1993) Kidney Int. 43, 522-534[Medline] [Order article via Infotrieve]
  12. Taub, M., Laurie, G. W., Martin, G. R., and Kleinman, H. K. (1990) Kidney Int. 37, 1090-1097[Medline] [Order article via Infotrieve]
  13. Beavan, L. A., Carone, F. A., Nakamura, S., Jones, J. K., Reindel, J. F., and Price, R. G. (1991) Arch. Biochem. Biophys. 284, 392-399[CrossRef][Medline] [Order article via Infotrieve]
  14. Wilson, P. D., Hreniuk, D., and Gabow, P. A. (1992) J. Cell. Physiol. 150, 360-369[CrossRef][Medline] [Order article via Infotrieve]
  15. Joly, D., Morel, V., Hummel, A., Ruello, A., Nusbaum, P., Patey, N., Noel, L. H., Rousselle, P., and Knebelmann, B. (2003) Am. J. Pathol. 163, 1791-1800[Abstract/Free Full Text]
  16. Colognato, H., and Yurchenco, P. D. (2000) Dev. Dyn. 218, 213-234[CrossRef][Medline] [Order article via Infotrieve]
  17. Ghosh, S., and Stack, M. S. (2000) Microsc. Res. Tech. 51, 238-246[CrossRef][Medline] [Order article via Infotrieve]
  18. Nievers, M. G., Schaapveld, R. Q., and Sonnenberg, A. (1999) Matrix Biol. 18, 5-17[CrossRef][Medline] [Order article via Infotrieve]
  19. Giannelli, G., Falk-Marzillier, J., Schiraldi, O., Stetler-Stevenson, W. G., and Quaranta, V. (1997) Science 277, 225-228[Abstract/Free Full Text]
  20. Dajee, M., Lazarov, M., Zhang, J. Y., Cai, T., Green, C. L., Russell, A. J., Marinkovich, M. P., Tao, S., Lin, Q., Kubo, Y., and Khavari, P. A. (2003) Nature 421, 639-643[CrossRef][Medline] [Order article via Infotrieve]
  21. Rousselle, P., and Aumailley, M. (1994) J. Cell Biol. 125, 205-214[Abstract/Free Full Text]
  22. Chen, Q., Kinch, M. S., Lin, T. H., Burridge, K., and Juliano, R. L. (1994) J. Biol. Chem. 269, 26602-26605[Abstract/Free Full Text]
  23. Hill, C. S., and Treisman, R. (1995) Cell 80, 199-211[CrossRef][Medline] [Order article via Infotrieve]
  24. Daemi, N., Thomasset, N., Lissitzky, J. C., Dumortier, J., Jacquier, M. F., Pourreyron, C., Rousselle, P., Chayvialle, J. A., and Remy, L. (2000) Int. J. Cancer 85, 850-856[CrossRef][Medline] [Order article via Infotrieve]
  25. Gilles, C., Polette, M., Coraux, C., Tournier, J. M., Meneguzzi, G., Munaut, C., Volders, L., Rousselle, P., Birembaut, P., and Foidart, J. M. (2001) J. Cell Sci. 114, 2967-2976[Medline] [Order article via Infotrieve]
  26. Koshikawa, N., Minegishi, T., Sharabi, A., Quaranta, V., and Seiki, M. (2005) J. Biol. Chem. 280, 88-93[Abstract/Free Full Text]
  27. Schenk, S., Hintermann, E., Bilban, M., Koshikawa, N., Hojilla, C., Khokha, R., and Quaranta, V. (2003) J. Cell Biol. 161, 197-209[Abstract/Free Full Text]
  28. Slade, M. J., Kirby, R. B., Pocsi, I., Jones, J. K., and Price, R. G. (1998) Biochim. Biophys. Acta 1401, 203-210[Medline] [Order article via Infotrieve]
  29. Kingsley, K., Huff, J. L., Rust, W. L., Carroll, K., Martinez, A. M., Fitchmun, M., and Plopper, G. E. (2002) Biochem. Biophys. Res. Commun. 293, 1000-1006[CrossRef][Medline] [Order article via Infotrieve]
  30. Gonzales, M., Haan, K., Baker, S. E., Fitchmun, M., Todorov, I., Weitzman, S., and Jones, J. C. (1999) Mol. Biol. Cell 10, 259-270[Abstract/Free Full Text]
  31. Chiu, S. J., Jiang, S. T., Wang, Y. K., and Tang, M. J. (2002) J. Biomed. Sci. 9, 261-272[Medline] [Order article via Infotrieve]
  32. Decline, F., and Rousselle, P. (2001) J. Cell Sci. 114, 811-823[Abstract]
  33. Mainiero, F., Murgia, C., Wary, K. K., Curatola, A. M., Pepe, A., Blumemberg, M., Westwick, J. K., Der, C. J., and Giancotti, F. G. (1997) EMBO J. 16, 2365-2375[CrossRef][Medline] [Order article via Infotrieve]
  34. Marshall, C. J. (1995) Cell 80, 179-185[CrossRef][Medline] [Order article via Infotrieve]
  35. Murphy, L. O., Smith, S., Chen, R. H., Fingar, D. C., and Blenis, J. (2002) Nat. Cell Biol. 4, 556-564[Medline] [Order article via Infotrieve]
  36. Adachi, T., Kar, S., Wang, M., and Carr, B. I. (2002) J. Cell. Physiol. 192, 151-159[CrossRef][Medline] [Order article via Infotrieve]
  37. Eliceiri, B. P., Klemke, R., Stromblad, S., and Cheresh, D. A. (1998) J. Cell Biol. 140, 1255-1263[Abstract/Free Full Text]
  38. Hood, J. D., Frausto, R., Kiosses, W. B., Schwartz, M. A., and Cheresh, D. A. (2003) J. Cell Biol. 162, 933-943[Abstract/Free Full Text]
  39. Zahir, N., Lakins, J. N., Russell, A., Ming, W., Chatterjee, C., Rozenberg, G. I., Marinkovich, M. P., and Weaver, V. M. (2003) J. Cell Biol. 163, 1397-1407[Abstract/Free Full Text]
  40. O'Brien, L. E., Jou, T. S., Pollack, A. L., Zhang, Q., Hansen, S. H., Yurchenco, P., and Mostov, K. E. (2001) Nat. Cell Biol. 3, 831-838[CrossRef][Medline] [Order article via Infotrieve]
  41. Weaver, V. M., Lelievre, S., Lakins, J. N., Chrenek, M. A., Jones, J. C., Giancotti, F., Werb, Z., and Bissell, M. J. (2002) Cancer Cell 2, 205-216[CrossRef][Medline] [Order article via Infotrieve]
  42. Sweeney, W. E., Jr., and Avner, E. D. (1998) Am. J. Physiol. 275, F387-F394[Medline] [Order article via Infotrieve]
  43. Richards, W. G., Sweeney, W. E., Yoder, B. K., Wilkinson, J. E., Woychik, R. P., and Avner, E. D. (1998) J. Clin. Investig. 101, 935-939[Medline] [Order article via Infotrieve]
  44. Saelman, E. U., Keely, P. J., and Santoro, S. A. (1995) J. Cell Sci. 108, Pt. 11, 3531-3540[Abstract]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 CJASNHome page
Q. Qian, R. P. Hartman, B. F. King, and V. E. Torres
Increased Occurrence of Pericardial Effusion in Patients with Autosomal Dominant Polycystic Kidney Disease
Clin. J. Am. Soc. Nephrol., November 1, 2007; 2(6): 1223 - 1227.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/39/29181    most recent
M606151200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Joly, D.
Right arrow Articles by Knebelmann, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Joly, D.
Right arrow Articles by Knebelmann, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement