QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 17, Issue 8, 3664-3677, August 2006

This Article Abstract Full Text (PDF) All Versions of this Article: E05-11-1070v1 17/8/3664    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mak, G. Z. Articles by Matlin, K. S. Search for Related Content PubMed PubMed Citation Articles by Mak, G. Z. Articles by Matlin, K. S.

Regulated Synthesis and Functions of Laminin 5 in Polarized Madin-Darby Canine Kidney Epithelial Cells

Grace Z. Mak^*, Gina M. Kavanaugh^*, Mary M. Buschmann^*, Shaun M. Stickley^*, Manuel Koch, Kathleen Heppner Goss^*, Holly Waechter^*, Anna Zuk, and Karl S. Matlin^*

*Laboratory of Epithelial Pathobiology, Department of Surgery, University of Cincinnati, Cincinnati, OH 45267-0581; Center for Biochemistry, Center for Molecular Medicine, and Department of Dermatology, University of Cologne, Cologne 50923, Germany; and Genzyme Corporation, Framingham, MA 01701

Submitted November 22, 2005; Revised May 31, 2005; Accepted June 1, 2006
Monitoring Editor: Asma Nusrat

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical–basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical–basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin 5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Epithelia line the surfaces of multicellular organisms, acting as semipermeable barriers between the internal milieu and the outside world. To carry out this function, epithelial cells are structurally and functionally polarized (Nelson, 2003). Epithelial cell polarity arises during embryonic development and regenerates after injury (Fish and Molitoris, 1994; Zuk et al., 1998; Nelson, 2003). Although the details are still not clear, it seems many of the factors responsible for signaling polarization of epithelial cells are shared with other types of cells that respond asymmetrically to particular stimuli (Nelson, 2003). What sets epithelial cells apart, however, is that they are capable of forming a continuous sheet of polarized cells, each with exactly the same orientation relative to each other and the extracellular environment.

There is abundant evidence that the environmental signals or "spatial cues" for coordinated polarization of epithelial cells are adhesive interactions, both between cells and with the extracellular matrix (Nelson, 2003). When suspended individually, epithelial cells are depolarized; cell–cell or cell–matrix interactions alone induce at least a degree of polarity, whereas both classes of adhesion are required for full polarization (Vega-Salas et al., 1987). Cell–cell adhesion is facilitated primarily by E-cadherin and its associated proteins, and signals from these molecules are required for polarization (Vega-Salas et al., 1987; Nelson, 2003). In contrast, the exact identities and particular functions of both extracellular matrix receptors and ligands responsible for signaling polarization from the substratum are unknown.

In tissues, epithelial cells reside on a basal lamina composed primarily of isoforms of laminin (LN), collagen type IV, and heparan sulfate proteoglycans (Yurchenco, 1994; Li et al., 2005). Of these molecules, there is evidence that laminins are critically involved in polarization (Klein et al., 1988; Sorokin et al., 1990; Zinkl et al., 1996; Ekblom et al., 1999; Li et al., 2003; Miner and Yurchenco, 2004). In particular, laminin has been implicated in the polarization of both the developing kidney tubular epithelium and the Madin-Darby canine kidney (MDCK) cell line through the use of function-blocking antibodies (Klein et al., 1988; Sorokin et al., 1990; Zinkl et al., 1996). Furthermore, deletion of the 1 subunit of laminin in mouse embryonic stem cells blocks epiblast polarization in vitro, whereas mutations in genes for both laminin subunits and laminin receptors lead to disrupted epithelial polarity in Caenorhabditis elegans and Drosophila (Li et al., 2003; Miner and Yurchenco, 2004).

Laminins are a family of large heterotrimeric glycoproteins consisting of at least 15 members composed of combinations of five , three , and three subunits (Colognato and Yurchenco, 2000; Miner and Yurchenco, 2004). The prototypical laminin structure is cruciform, with three arms contributed by fibrous and globular domains, and a rigid stalk made up of a coiled-coil of all three subunits. The terminal regions of each arm are capable of associating with similar domains on other molecules to form a polymer network, whereas the end of the stalk interacts with a variety of cellular receptors (Miner and Yurchenco, 2004). Assembly of laminin into a basal lamina is believed to be initiated by monomer association with the cell surface, followed by polymerization dependent on laminin concentration and divalent cations (Yurchenco, 1994; Cheng et al., 1997; Colognato et al., 1999; Odenthal et al., 2004; Li et al., 2005). Some laminin isoforms diverge from this model by possessing one or more truncated subunits that render them less able to polymerize (Cheng et al., 1997; Miner and Yurchenco, 2004; Odenthal et al., 2004).

Our laboratory has been studying the involvement of laminin in the polar morphogenesis of epithelia both in vitro using MDCK cells and during regeneration of the renal epithelium after ischemic injury in vivo. In the rat kidney, we found that LN5, a truncated isoform moderately expressed only in the renal papilla under normal conditions, is expressed at high levels in many regions of the kidney during regeneration (Carter et al., 1991; Rousselle et al., 1991; Miyazaki et al., 1993; Miner, 1999; Zuk and Matlin, 2002). Based upon these results, we hypothesized that LN5 might be important in facilitating the reconstitution of an intact, polarized epithelium after injury.

In this article, we report that LN5 is transiently synthesized by MDCK cells during regeneration of a confluent epithelium and is required to both stimulate proliferation and regulate cell spreading. It is not, however, necessary for apical–basal polarization per se, at least in two-dimensional, monolayer culture. These findings indicate that LN5 may have a widespread role in facilitating epithelial regeneration in more instances than previously recognized.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture
Stock cultures of MDCK cells (strain II, Heidelberg isolate, passages 7–35) were grown in DMEM (high glucose) supplemented with 5% fetal bovine serum (FBS) and 10 mM HEPES-KOH, pH 7.3, at 37°C in an atmosphere of 5% CO₂ as described previously (Matlin et al., 1981).

In polarization experiments, cells were grown on uncoated Transwell permeable supports (2.4 cm in diameter, pore size, 0.4 µm; Corning-Costar, Acton, MA). After prewetting the supports with medium, 1.5 ml of complete growth medium containing 1.5 x 10⁶ cells was added to the upper chamber, and 2.5 ml of medium was added to the bottom. Under these conditions, a confluent and polarized monolayer formed after 18–24 h incubation, as determined by immunofluorescence of apical and basolateral markers and measurements of transmonolayer resistance (Zinkl et al., 1996).

Antibodies
Laminin Isoforms. Antibody 8LN5 (affinity-purified rabbit polyclonal anti-kalinin; 0.43 mg/ml) was obtained from Dr. Robert Burgeson (Massachusetts General Hospital, Boston, MA), and was used for immunofluorescence, immunoprecipitation, and immunoblotting. This antibody reacts with all three subunits of human LN5 by immunoblotting and specifically stains the basement membrane underlying human epidermis (Zuk and Matlin, 2002). Mouse monoclonal anti-kalinin B1 (0.25 mg/ml), directed against the 3 laminin subunit, was purchased from BD Biosciences Transduction Laboratories (Lexington, KY) (catalog no. 610424) and used for immunoblotting; it was not suitable for immunofluorescence or immunoprecipitation. Affinity-purified rabbit anti-LN1 (0.6 mg/ml) was purchased from Sigma-Aldrich (St. Louis, MO) (catalog no. L9393) and used for immunofluorescence and immunoprecipitation.

Integrins. Antibody AIIB2 (monoclonal rat anti-mouse 1 integrin) was used for adhesion assays as a culture supernatant prepared from hybridomas obtained from the Developmental Studies Hybridoma Bank (Department of Biological Sciences, The University of Iowa, Iowa City, IA) (Hall et al., 1990). Antibody GoH3 (monoclonal rat anti-human 6 integrin; 1 mg/ml) was purchased from BD Biosciences PharMingen (San Diego, CA) (catalog no. 555734); and was used for adhesion assays and immunoprecipitation (Sonnenberg et al., 1987). Rabbit polyclonal anti-1 integrin was prepared against a synthetic peptide corresponding to the carboxy-terminal 37 amino acids of human 1 integrin and was used for immunoblotting.

Other Primary Antibodies. Culture supernatants containing mouse monoclonal antibodies (mAbs) against the apical MDCK protein gp135 (Ojakian and Schwimmer, 1988; Meder et al., 2005) and the basolateral marker E-cadherin were prepared from the 3F21D8 hybridoma (a kind gift of George Ojakian, SUNY Downstate, Brooklyn, NY) and the rr1 hybridoma (a kind gift of Barry Gumbiner, University of Virginia School of Medicine, Charlottesville, VA) (Gumbiner and Simons, 1986), respectively. The supernatants were used without dilution to stain MDCK cells grown on Transwell supports. Staining of cells labeled with bromodeoxyuridine was accomplished with rat monoclonal anti-bromodeoxyuridine (BrdU) (catalog no. ab6326-250, 1 mg/ml; Abcam, Cambridge, MA).

Secondary Antibodies. For immunofluorescence, affinity-purified goat anti-mouse, -rabbit, and -rat secondary antibodies conjugated to Alexa Fluor-488 or -555 were purchased from Molecular Probes-Invitrogen (Carlsbad, CA) (2 mg/ml). For immunoblotting, affinity-purified polyclonal donkey anti-rabbit or anti-mouse IgG (heavy and light chains) conjugated to peroxidase (0.4 mg/ml) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). When available, all secondary antibodies were highly cross-adsorbed against other species.

Other Reagents
Collagen (type I from rat tail tendon, 4 mg/ml) for coating culture surfaces was purchased from Upstate Biotechnology (Lake Placid, NY) (catalog no. 08-115). LN5 was purified from human keratinocyte conditioned culture medium by affinity chromatography using the 6F12 mAb against the laminin 3 chain as described previously (Eble et al., 1998). The preparation was pure and intact as judged by SDS-gel electrophoresis (our unpublished data) and supported the adhesion of epidermal carcinoma cells. Phalloidin conjugated to Alexa Fluor-488 was purchased from Molecular Probes-Invitrogen and used for fluorescence microscopy by diluting a methanol stock (200 U/ml) 1:80. All other chemicals were reagent grade.

SDS-Gel Electrophoresis and Immunoblotting
SDS-PAGE was conducted in a minigel apparatus (Bio-Rad, Hercules, CA) using Laemmli buffers (Laemmli, 1970). For immunoblotting, cell cultures were extracted directly with SDS extraction buffer (50 mM Tris-HCl, pH 8.8, 2% SDS, and 5 mM EDTA) supplemented with Complete protease inhibitors (Roche Diagnostics, Indianapolis, IN). The extract was then sheared by multiple passes through a 22-gauge syringe needle, immediately heated for 3 min at 95°C, mixed in a 1:3 ratio with 40% sucrose, 5 mM dithiothreitol (DTT), and 0.08% bromphenol blue, and reheated for 3 min at 95°C. Samples were then alkylated at 37°C for 15 min with 4 mM iodoacetamide before loading on the SDS gel. MDCK cell endogenous extracellular matrix was prepared by removing the cell layer with Triton X-100 and NH₄OH as described previously (Le Beyec et al., 1997) and then extracting the matrix proteins directly with SDS extraction buffer.

Immunoblotting was carried out with a Bio-Rad semidry apparatus exactly as described previously (Zuk and Matlin, 2002). Primary antibodies were incubated with membranes sealed in plastic bags overnight at 4°C followed, after washing, by a 1-h incubation with a horseradish peroxidase-conjugated secondary antibody diluted at 1:5000. Bands were visualized by reacting membranes with enhanced chemiluminescence reagents (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and exposing them to x-ray film. Primary antibody dilutions were 1:1500 for polyclonal anti-LN5 and 1:1000 for polyclonal anti-1 integrin.

Metabolic Labeling and Immunoprecipitation
Short-term (4-h) metabolic labeling of MDCK cells was accomplished by incubating 35-mm-diameter cultures in serum-free DMEM (high glucose)/10 mM HEPES, pH 7.3, lacking methionine and cysteine (labeling medium) for 15 min at 37°C, 5% CO₂ to deplete amino acid pools, and labeling with 100 µCi of [³⁵S]Met)/Cys for 4 h. Long-term (18–24 h) labeling was conducted (without washing or preincubation) in DMEM/10 mM HEPES, pH 7.3/5% FBS containing 1/10 the normal amount of unlabeled methionine and cysteine and 100 µCi [³⁵S]Met/Cys at 37°C, 5% CO₂. After completion of labeling, the medium was removed and the cultures were washed twice with ice-cold phosphate-buffered saline (PBS) before extraction.

For immunoprecipitation, cells were extracted on ice with radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.5, 0.5% SDS, 1.0% Igepal-CA630, 0.15 M NaCl, and 1.0% sodium deoxycholate) supplemented with Complete protease inhibitors. The extracts were filtered through Ultrafree-CL centrifugal filters (Millipore, Billerica, MA) and then incubated with antibodies overnight at 4°C. LN5 and presumptive LN10 were immunoprecipitated with 1:200 dilutions of antibody 8LN5 and anti-LN1, respectively. Integrin 6 was immunoprecipitated with 1:250 dilution of GoH3 antibody. Immune complexes were captured with 30 µl of washed protein A-Trisacryl or protein G-agarose beads (Pierce Chemical, Rockford, IL). Immunoprecipitated proteins were solubilized and reduced by heating for 3 min at 95°C in SDS-gel sample buffer (0.16 M Tris-HCl, pH 8.8, 4 mM EDTA, 16% sucrose, 0.16% bromphenol blue, 20 mM DTT, and 2% SDS) and then alkylated before loading on an SDS-gel. To visualize radioactive bands, gels were infiltrated with Enhance (PerkinElmer Life and Analytical Sciences, Boston, MA), dried, and exposed to x-ray film.

In some cases, LN5 was immunoprecipitated not only from extracts of long-term labeled cells but also from the culture medium. To accomplish this, culture medium was collected, centrifuged to remove any particulate matter, and antibodies were added directly to the supernatant.

Immunofluorescence and Confocal Microscopy
Indirect immunofluorescence staining of MDCK cells grown on glass coverslips was conducted on formaldehyde-fixed cells as described previously (Prahalad et al., 2004). LN5 was detected by staining with 10 µg/ml 8LN5; presumptive LN10 was detected with a 1:100 dilution of anti-LN1. Alexa Fluor-conjugated secondary antibodies were diluted 1:500. Stained coverslips were viewed on a Zeiss Axioskop fluorescence microscope with a 63x, 1.4 numerical aperture (NA) PlanApo or 40x, 1.3 NA PlanNeofluor oil immersion objective and Nomarski optics, and photographed with an Axiocam MRm digital camera using Axiovision software (Carl Zeiss, Thornwood, NY).

MDCK cells grown in Transwell chambers were fixed for 30 min at room temperature with 3% formaldehyde in PBS at room temperature and quenched for 15 min with freshly prepared 0.1% NaBH₄/PBS at room temperature with gentle agitation. Cells were permeabilized by incubation with 0.1% Triton X-100 in PBS for 4 min. At this point, the filters were cut from their plastic support with a scalpel blade, transferred cell side up to individual 35-mm plastic Petri dishes, blocked for 30 min at room temperature with 10% goat serum in PBS, and cut into quarters for staining.

Antibody staining was conducted in a moist chamber at room temperature by placing filter quarters cell side up over 100-µl droplets of antibody solutions on sheets of Parafilm and then gently covering the cell side with an additional 100 µl of antibody. The filters were then incubated with the antibody for 30 min at room temperature and washed four times with PBS for 5 min each with agitation in individual 35-mm dishes. Staining with secondary antibodies was conducted identically. When filamentous actin was labeled, dilute fluorescent phalloidin was included with the secondary antibody.

After antibody staining, filter quarters were washed with PBS and 0.1% Triton X-100/PBS and postfixed with 3% formaldehyde/PBS. They were then mounted on glass slides in hard-set Vectashield Antifade mounting solution (Vector Laboratories, Burlingame, CA) under a 22-mm square coverslip supported by four hardened nail polish "feet," and edges sealed with additional nail polish.

Stained filter quarters were imaged with a Zeiss LSM-510 confocal laser scanning microscope in either the XY plane or as a Z-line orthogonal image using a 63x, 1.4 NA PlanApo oil immersion objective. For XY images, the optical slice was 1.0 µm with a frame size of 1024 x 1024 pixels. For Z-lines, the frame size was 2048 x 2048 pixels with a step size of 0.48 µm. For each experiment, images were collected at identical dynamic range settings. Conventional and confocal digital images were optimized with Adobe Photoshop (Adobe Systems, Mountain View, CA) using linear adjustments of levels, contrast, and brightness.

Adhesion Assay
Adhesion assays in 96-well microtiter plates with and without function-blocking anti-integrin antibodies were conducted essentially as described previously (Schoenenberger et al., 1994).

Polymerase Chain Reaction
Laminin 3 and 5 transcripts were detected by combined reverse transcriptase (RT)-PCR. Total MDCK cell RNA was prepared with an RNAeasy kit (QIAGEN, Valencia, CA). RT-PCR was conducted with 1.0 µg of RNA/50-µl reaction with a 10-min annealing step at 25°C, a 12-min reverse transcriptase reaction at 42°C, and 30–40 PCR cycles. Laminin primers were based on the sequences of human transcripts. Laminin 3 primers were 5'-ACAGATGGAGAGGGAAACAAC-3' (forward) and 5'-ATTTGCCTGCTTGGC TTGG-3' (reverse), corresponding to nucleotides 1000–1299 of the human sequence. Laminin 5 primers were 5'-GCGACAACTGCCTCCTCTAC-3' (forward) and 5'-CCAGGTGGTCCTGGGTATC-3', corresponding to nucleotides 3189–3154 of the human sequence. Canine -actin primers were 5'-CAAAGCCAACCGTGAGAAG-3' (forward) and 5'-CAGAGTCCATGACAATACCAG-3' (reverse), corresponding to residues nucleotides 772-1028 in the canine sequence.

Suppression of Laminin 3 Synthesis with Small-Interfering RNA (siRNA)
A partial canine laminin 3 sequence was determined by automated DNA sequencing at the University of Cincinnati College of Medicine core facility(Cincinnati, OH) using a PCR product obtained with human specific primers (see PCR protocol). Possible siRNA targets were identified in the PCR product sequence using a computerized algorithm at the Ambion (Austin, TX) Web site (Elbashir et al., 2001, 2002). One such sequence that mapped specifically to the laminin 3 transcript was chosen. The sequence (5'-AAATGACTACGAAGCCAAACT-3') is located at nucleotide 1257 of the canine laminin 3 transcript and nucleotide 1224 of the human laminin 3 transcript (Ensembl Genome Browser; www.ensembl.org).

The efficacy of the target sequence was first tested by transient transfection of RNA duplexes into MDCK cells. Synthetic RNA molecules corresponding to the target sequence and its complement, and control RNAs not corresponding to any known gene, were purchased from Ambion. These were annealed according to recommendations from Ambion and transfected into MDCK cells as siPORT (Ambion) complexes in serum-free DMEM. After 48-h incubation, RNA was prepared from the transfected and mock-transfected cells, and the RNA analyzed by RT-PCR. Based on this, the selected target was determined to knock down laminin 3 mRNA (Figure 5A).

Adenoviral vectors capable of expressing the siRNA targeted to laminin 3 were engineered using the pSIREN-Shuttle vector and Adeno-X-expression vector (Clontech, Mountain View, CA). cDNA oligonucleotides containing the sense and antisense sequences of the siRNA on the same strand separated by a hairpin sequence and flanked by terminator, 3'-EcoRI, and 5'-BamHI restriction sites were purchased from Integrated DNA Technology (Coralville, IA). These were annealed and ligated into the BamHI and EcoRI sites in the pSIREN vector. The hairpin construct and RNA polymerase promoter sequences were then cut from the vector with I-CeuI and Pl-SceI and ligated into equivalent sites in Adeno-X DNA. The completed construct was verified by DNA sequencing.

Adenovirus was produced by transfection of the Adeno-X-hairpin construct into human embryonic kidney (HEK)-293 cells according to the manufacturer’s directions (Clontech). The primary viral stock was titered with an Adeno-X Rapid Titer kit (Clontech) and was used to grow subsequent viral stocks. Adenoviral vector stocks capable of laminin 3 knockdown were called "Ad-D5." A stock of adenovirus expressing Lac-Z (Ad-LacZ) was prepared in HEK-293 cells from original virus provided by Andrew Lowy (University of Cincinnati, Department of Surgery); this was used in experiments as a control for adenovirus infection.

In some cases, adenovirus was purified by centrifugation in OptiPrep (iodixanol; Axis-Shield USA, Norton, MA) step gradients of 15–25–40–54% centrifuged overnight at 100,000 x g and 4°C. The viral band was harvested between the 25 and 40% layers and used directly without dialysis. Typical stock virus titers were 10⁹–10¹⁰ infectious units/ml.

For laminin 3 knockdown experiments, MDCK cells were plated for 1.5–2 h on uncoated surfaces until well-attached, gently washed twice with PBS, and infected with virus suspended in a small volume of serum-free Opti-MEM (Invitrogen) for 1 h at 37°C, 5% CO₂. Complete MDCK cell growth medium was then added, and incubation continued. In typical experiments, the multiplicity of infection was 60 infectious units/cell. Under these conditions, knockdown of laminin 3 synthesis, as measured by metabolic labeling, was almost completely effective after 18 h infection. To eliminate any morphogenetic effects of LN5 synthesized before knockdown, infected cultures were most often trypsinized and replated 18 h postinfection.

Cell Proliferation Assay
Cell proliferation was estimated by labeling with BrdU. Control cultures of MDCK cells or cultures infected with either Ad-D5 or Ad-Lac Z for 18 h were trypsinized and replated at low density on glass coverslips and incubated overnight. The next day, cultures were incubated with 50 µM BrdU in complete medium for 6 h. To stain for BrdU, cells were washed, fixed with 3% formaldehyde in PBS, blocked and permeabilized with 5% goat serum and 0.1% Triton X-100 in PBS, and stained with a 1:500 dilution of anti-BrdU in the same blocking buffer supplemented with 20 U of DNAse I (Roche Diagnostics) and 20 mM MgCl₂. After washing and staining with a fluorescent secondary antibody, the coverslips were mounted in hard-set Vectashield containing 4,6-diamidino-2-phenylindole (DAPI). Digital micrographs of total (DAPI-stained) nuclei and anti-BrdU-stained nuclei were counted with MetaMorph (Molecular Devices, Sunnyvale, CA), and the fraction of BrdU-positive cells calculated. Labeling experiments were conducted three times with an average of seven fields/treatment (control, Ad-LacZ, and Ad-D5) and 119 cells/field counted.

Wound Healing Assay
Control (uninfected) and Ad-D5– and Ad-LacZ–infected cells were trypsinized and replated on gridded glass coverslips (Bellco Biotechnology, Vineland, NJ). After incubating overnight, approximately one-half of each coverslip was scraped off using a rubber policeman. After changing medium, four regions per coverslip at the wound edge were digitally photographed on an inverted phase microscope at low power, and the cultures incubated for 24 h at 37°C, 5% CO₂. After 24 h, the same regions were rephotographed. To quantitate movement of the wound edge, digital photographs were printed, and the distances measured between fixed points of the grid and the wound edge at the two different time points. Results of three independent experiments with two to four measurements/experiment/treatment condition were averaged.

Analysis of Cell Spreading
Control cultures of MDCK cells or cultures infected with either Ad-D5 or Ad-Lac Z for 18 h were trypsinized and replated at low density on glass coverslips and incubated overnight. The next day, the cells were fixed and permeabilized, and the actin cytoskeleton visualized with fluorescent phalloidin and the nuclei with DAPI. Total spread area was measured from digital micrographs using phalloidin staining and the threshold setting in MetaMorph. Area/cell was then calculated by counting DAPI-stained nuclei.

Statistical Analysis
Statistical analysis was conducted using Sigma-Stat (SPSS, Chicago, IL) by either the Kruskal–Wallis one-way analysis of variance (ANOVA) on rank with multiple comparisons done by Dunn’s test, or by a one-way ANOVA with multiple comparisons carried out by the Holm–Sidak method. Graphs of means + SEs were generated with Sigma-Plot (SPSS).

Sequence Identification
The canine laminin 5 sequence provided here has the EMBL accession no. AY 552590.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
MDCK Cells Synthesize LN5 Only When Subconfluent
In the injured kidney, synthesis of LN5 is turned on during regeneration of the epithelium (Zuk and Matlin, 2002). With epithelial cell lines such as MDCK, the equivalent of this regenerative phase is a subconfluent culture, where cells must spread and proliferate to reestablish a continuous epithelium. To determine whether MDCK cells synthesize LN5 under these conditions, cells were plated at low density in uncoated plastic culture dishes. At various times up to 24 h, cells were detached by treatment with Triton X-100 and NH₄OH, and endogenous matrix proteins deposited on the culture dish extracted with SDS. These were then separated by SDS gel electrophoresis and Western blotted with an antibody against the 3 subunit of LN5.

As illustrated in Figure 1A, small amounts of deposited 3 were detectable as little as 6 h after plating, with increasing amounts evident throughout the remaining time points. In addition to the 3 band, another smaller, faint band was also detected with the antibody (Figure 1A, *). This presumably corresponds to a 3 proteolytic fragment.

View larger version (67K): [in this window] [in a new window]   Figure 1. LN5 is only expressed in MDCK cells at early times after plating. (A) Endogenous matrix proteins deposited by MDCK cells (8 x 106 cells/10-cm2 culture dish) for the indicated times were immunoblotted with an antibody against the laminin 3 subunit. Immunoreactive material corresponding to deposited LN5 was detected beginning at 6 h plating, with increasing amounts afterward. An apparent 3 degradation product was also observed (*). (B) Immunodetection of endogenous matrix proteins deposited on the culture dish by MDCK cells 18 h, 4 d, or 7 d after plating. Under these conditions, LN5 deposition declines significantly after 4 d and is nearly absent after 7 d. As in A, a 3 degradation product is visible (*). (C) MDCK cells (7.5 x 105 cells/35-mm dish) were plated on coverslips and fixed and stained with a polyclonal antibody against LN5 at either 18 h, 4 d, or 7 d after plating. Coverslips were observed by confocal fluorescence microscopy; only sections through the center of the cell are shown. After 18 h, LN5 staining (red) is apparent throughout the cytoplasm in a pattern resembling the endoplasmic reticulum. By 4 d, only a few cytoplasmic vesicles per cell are visible, with only isolated staining (arrows) by 7 d. Green, actin. Bar, 10 µm. (D) Pulse labeling and immunoprecipitation of LN5 in MDCK cells. MDCK cells (1 x 106 cells/35-mm dish) were pulse labeled for 30 min at the indicated times after plating with 50 µCi of [35S]Met/Cys. After labeling, the cells were extracted with RIPA buffer, the extracts were immunoprecipitated with polyclonal anti-LN5, and the immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. After 18 h plating, all three LN5 subunits are detected, whereas little 3 or 2 is detected at later times.

To confirm this, similar cultures on glass coverslips were fixed and stained with a polyclonal antibody against LN5. As shown in Figure 1C, most cells plated for 18 h stained extensively with anti-LN5 antibody (Figure 1C, red) in a dispersed pattern, suggesting that of the endoplasmic reticulum, consistent with continued biosynthesis of LN5. By 4 d of culture, staining was largely absent, being confined to only a few reactive vesicles per cell. By 7 d, staining was almost completely absent (Figure 1C).

To look more directly at the biosynthesis of LN5, cells plated for various times were metabolically labeled for 30 min, and immunoprecipitated LN5 was visualized by fluorography. As shown in Figure 1D, 18 h after plating, all three LN5 subunits, 3, 3, and 2, were detected. At all other times corresponding to fully confluent cultures, only small amounts of 3 and 2 were observed, whereas significant quantities of 3 continued to be made. Thus, the decline in LN5 production and deposition observed by Western blotting and immunofluorescence (Figure 1, A–C) was due to differential inhibition of LN5 chain synthesis. Presumably, the 3 chain detected by metabolic labeling in confluent cells (4–10 d after plating; Figure 1D) was degraded in the endoplasmic reticulum and was not secreted in the absence of the other chains because it was not observed at these times by either immunofluorescence (Figure 1C) or Western blotting of deposited matrix proteins (Figure 1B). To determine whether the differential regulation of LN5 chain synthesis was regulated at the transcriptional level, RT-PCR was performed on RNA isolated from subconfluent and confluent cultures. In subconfluent cultures, mRNA for all three chains was detected, whereas only 3 mRNA was found in confluent cultures (our unpublished data).

MDCK Cells Also Synthesize at Least One Other Laminin Isoform
Previous studies from other laboratories demonstrated that MDCK cells synthesize laminin, although the particular isoform was not identified (Caplan et al., 1987; Boll et al., 1991; O’Brien et al., 2001). In most, if not all cases, the antibodies used in these studies were made against mouse LN1 (111), and some were specifically reactive with the 1 subunit. Because no subunits of LN1 are shared with LN5, it is unlikely that the laminin observed in these previous studies was LN5. However, as at least 10 of the 15 known laminin isoforms contain the 1 subunit, the identity of this other MDCK cell laminin could not be determined based upon existing data (Miner and Yurchenco, 2004).

Studies of the kidney suggest that LN10 (511) is a prominent isoform in the tubular basement membrane (Miner, 1999). To determine whether MDCK cells, which are of renal origin, express LN10 in addition to LN5, RT-PCR analysis of MDCK RNA was conducted using primers derived from the human laminin 5 sequence. As shown in Figure 2A, RT-PCR yielded a product of the appropriate size predicted by the human sequence. When sequenced, this product was highly homologous to both the human and mouse laminin 5 sequences, strongly suggesting that this corresponded to the canine homologue (Figure 2B).

View larger version (78K): [in this window] [in a new window]   Figure 2. MDCK cells synthesize the laminin 5 subunit. (A) PCR products corresponding to laminin 5 and -actin were detected in RNA from MDCK cells by RT-PCR using primers specific to human laminin 5 and canine -actin. Lanes 1 and 2, laminin 5 primers; lane 3, actin primers. (B) The PCR product shown in A was sequenced and the sequence compared with that of human and mouse laminin 5. The canine sequence is 85% identical to the human sequence and 81% identical to the mouse sequence. (C) MDCK cells (7.5 x 106/35-mm dish) were plated for 42 h and then fixed and stained for either LN5 or putative LN10, the latter using a polyclonal antibody against LN1. Confocal sections from either the extreme base of the cells (top) or through the center of cells (bottom) are illustrated. In the case of LN5, significant amounts of deposited LN5 are visible at the base, but the cytoplasm has only limited amounts of perinuclear staining. For LN10, both the base and cytoplasmic compartments are strongly stained, indicating not only deposition of the protein but also continued synthesis. Bar, 20 µm.

To determine whether the synthesis of putative LN10 is regulated the same or differently from LN5, newly confluent cultures of MDCK cells were stained for immunofluorescence with either a polyclonal antibody against LN5 or an antibody prepared against LN1 that reacts with the laminin 1 and 1 subunits (Yu et al., 2005). The stained samples were examined in the confocal microscope to visualize either the cytoplasmic focal plane or the basal focal plane where deposited matrix molecules can be detected. As illustrated in Figure 2C, under these conditions LN5 staining is present in the basal plane, corresponding to deposited matrix, but nearly absent from the intracellular plane (Figure 2C). In contrast, staining with the anti-LN1 antibody, which presumably detects LN10, is evident both on the basal surface and intracellularly (Figure 2C). Thus, synthesis of LN5 and putative LN10 are clearly induced at different times during MDCK cell morphogenesis.

MDCK Cells Use Two Different Integrins to Attach to and Spread on LN5
Keratinocytes and other epithelial cell types have been shown to attach to LN5 via the integrins 31 and 64 (Niessen et al., 1994; Kreidberg, 2000; Kreidberg and Symons, 2000; Nguyen et al., 2001). MDCK cells express both of these integrins as well as 21 and V3 (Schoenenberger et al., 1994). To determine which integrins MDCK cells use for adhesion to LN5, purified LN5 was coated on microtiter plates and adhesion of MDCK cells measured after 90 min in the presence or absence of function-blocking anti-integrin antibodies. As shown in Figure 3, MDCK cells attached readily to wells coated with collagen I to an extent corresponding to nearly 100% adhesion of the applied cells (our unpublished data). Adhesion to collagen was completely inhibited by treatment of cells before assay with a function-blocking anti-1 integrin antibody, AIIB2, indicating that it is mediated by 1 integrins (Figure 3). Cells added to wells coated with purified LN5 adhered to the same extent as to collagen (Figure 3). However, in contrast to collagen, anti-1 had no effect on adhesion to LN5 (Figure 3). A function-blocking antibody against the integrin 6 subunit, GoH3, inhibited adhesion of MDCK cells to LN5 by 33% (Figure 3). However, when GoH3 and AIIB2 were combined, inhibition increased to about two-thirds (Figure 3), suggesting that both 1- and 6-containing integrin receptors are involved in MDCK cell adhesion to LN5.

View larger version (28K): [in this window] [in a new window]   Figure 3. MDCK cells use 1 integrins and 64 integrin to adhere to LN5. A single-cell suspension of MDCK cells was plated in wells of a 96-well plate coated with either bovine serum albumin (BSA) (–control), collagen I, or LN5 in the presence or absence of function-blocking antibodies against 1 integrin (AIIB2) or 6 integrin (GoH3). After 90-min incubation at 37°C, unattached cells were washed away, and numbers of attached cells were estimated after fixation and staining by measuring absorbance of solubilized stain. Almost no cells attach to BSA-coated wells, whereas nearly all cells attach to wells coated with either collagen I or LN5. Anti-1 integrin abolishes all adhesion to collagen I, but by itself has no effect on adhesion to LN5. Anti-6 integrin reduces cell adhesion to LN5 somewhat, but is much more effective in combination with anti-1 integrin. *p < 0.05 and **p < 0.001 relative to no antibody LN5 control.

View larger version (62K): [in this window] [in a new window]   Figure 4. MDCK cells spread extensively on LN5 using a 1 integrin. (A) MDCK cells were plated on coverslips coated with collagen I (i) or LN5 (ii–iv) for 2 h at 37°C in the presence or absence of function-blocking anti-1 integrin (AIIB2; iii) or 6 integrin (GoH3; iv). At the end of the incubation, the cells were fixed and stained with fluorescent phalloidin and viewed by Nomarski DIC microscopy (i–iv) or fluorescence microscopy (ii; inset). On collagen, cells spread asymmetrically and extend narrow lamellipodia (i); on LN5, cells spread more extensively and symmetrically than on collagen (ii; arrowheads mark cell edges). By fluorescence, the spread cells on LN5 display thick bundles of filamentous actin at the cell periphery (ii; inset). In the presence of anti-1 integrin, cells attach to LN5 but do not spread, and blebs are present at the cell peripheries (arrowheads; iii). In the presence of anti-6 integrin, the cells on LN5 spread but not as extensively or uniformly as in the absence of antibody (iv). Bar, 10 µm. (B) MDCK cells were plated at subconfluent density for 24 h at 37°C on uncoated glass coverslips or coverslips coated with either collagen I or LN5. At the end of the incubation, the cells were fixed and stained with fluorescent phalloidin and DAPI. The area occupied by spread cells was then measured using MetaMorph. Cells plated on LN5 spread significantly more than those plated on either glass or collagen I. *p < 0.001 relative to uncoated control and collagen coating.

Inhibition of Laminin 3 Synthesis with siRNA
To investigate further the function of LN5 in MDCK cell morphogenesis, LN5 synthesis was disrupted by expression of a siRNA targeted to the laminin 3 subunit (Elbashir et al., 2001, 2002). In preliminary experiments, a synthetic RNA duplex based on the canine 3 sequence was transiently transfected into MDCK cells. A control RNA duplex was also transfected. As shown in Figure 5A, transfection of RNA targeted to laminin 3 eliminates mRNA detectable by RT-PCR, whereas transfection of the control RNA or the transfection reagent alone had no effect.

View larger version (57K): [in this window] [in a new window]   Figure 5. Knockdown of laminin 3 with siRNA. (A) RNA was harvested from MDCK cells transfected for 2 d with either a synthetic RNA duplex specific for a target sequence in canine laminin 3 (siRNA), a control duplex not representing any known gene (coRNA), or transfection reagent alone (TRS). RNA was also harvested from confluent (Cnfl) or subconfluent (SCnfl) MDCK cell cultures. Laminin 3 and actin transcripts were detected by RT-PCR. RT, reverse transcriptase added to the reaction. Note the absence of a laminin 3 band with transfected siRNA. (B) LN5 was immunoprecipitated from detergent extracts of metabolically labeled control MDCK cells (Co) or cells infected with siRNA-expressing adenovirus (D5) or a control virus (LZ). In both Co and LZ samples, all three LN5 bands are visible; in the D5 siRNA sample, little laminin 3 can be seen, whereas the levels of laminin 3 and 2 chains are increased (arrowheads). (C) Extracts from control (Co), Ad-LacZ–infected, and Ad-D5–infected MDCK cells were immunoblotted with anti-LN5. Laminin 3, which is weakly detected by the antibody, is absent in cells expressing siRNA (arrowhead). The laminin 2 chain is not detected by the antibody. (D and F) The labeled cell extracts used to immunoprecipitate LN5 in B were sequentially immunoprecipitated with anti-6 integrin (D) and anti-LN1 (F). No significant differences between the samples are evident, suggesting that siRNA-mediated knockdown of laminin 3 is specific. The two bands in D running more slowly than 6 (* and arrows) are unidentified but may correspond to forms of integrin 4. In F, the higher molecular weight band is likely laminin 5 and the lower doublet 1 and 1. (E) The immunoblot shown in C was stripped and reprobed with anti-1 integrin. No significant differences between samples can be seen. 1, mature 1 integrin; 1p, precursor 1.

Similarly, when extracts from D5-infected, LZ-infected, and noninfected cells were immunoblotted with the same antibody, no 3 band was detected (Figure 5C, note that the antibody does not react with 2). Under these conditions, the intensity of the 3 band did not change significantly upon knockdown, indicating that the steady-state levels of 3 as detected by Western blotting were not detectably changed by reduction in the 3 chain (Figure 5C).

Knockdown of laminin 3 seemed to be specific. In addition to the continued expression of the other LN5 chains, there were little or no differences between control, LZ, or D5 samples in the amounts of integrin 6 or presumptive LN10 immunoprecipitated from metabolically labeled cells (Figure 5, D and F), or in the amount of integrin 1 detected by Western blotting of cell extracts (Figure 5E).

Knockdown of Laminin 3 Affects Cell Spreading
Based upon the observation that plating of normal MDCK cells on surfaces coated with LN5 led to extensive cell spreading, our expectation was that knockdown of laminin 3 and the resulting disruption of LN5 production would yield cells unable to spread effectively. Instead, when cells were infected overnight with the D5 virus, and then replated on uncoated surfaces, they spread much more extensively than either uninfected control cells or cells infected with the LacZ-expressing virus, yielding round, flat cells resembling fried eggs (Figure 6A). Spreading was so extreme that nuclei were flattened, looking enlarged, and was apparently facilitated by extensive formation of actin stress fibers (Figure 6A, arrows). The amount of spreading also did not seem to be affected by the formation of cell–cell contacts (Figure 6A, Ad-D5, inset). Quantitation of spreading indicated that cells with 3 synthesis knocked down occupied, on average, three times more area than either control group (Figure 6B).

View larger version (28K): [in this window] [in a new window]   Figure 6. Knockdown of laminin 3 leads to increased spreading of MDCK cells. (A) Uninfected MDCK cells and cells infected with control Ad-LacZ virus or siRNA-expressing Ad-D5 virus were replated on uncoated glass coverslips overnight and then fixed and stained with fluorescent phalloidin (green) and DAPI (blue) to visualize the actin cytoskeleton and nuclei, respectively. Micrographs show that cells infected with Ad-D5 spread much more than either control or Ad-LacZ–infected cells and are so flat that the nucleus is distorted and seems larger. Bundles of actin filaments and extensive actin stress fibers are also apparent (arrows). Bar, 20 µm. Inset in Ad-D5 panel, formation of cell–cell contacts does not suppress extensive cell spreading. Bar, 20 µm. (B) Control MDCK cells or cells infected with Ad-LacZ or Ad-D5 were replated on uncoated glass coverslips (–) or coverslips coated with purified human LN5 (LN5). Fixed cells were stained with fluorescence phalloidin and DAPI, and spread area/cell was measured from digital micrographs. Cells expressing siRNA targeted against laminin 3 spread much more than either control or Ad-LacZ–infected cells. Spreading of all cells is stimulated further by plating on LN5-coated coverslips. *p < 0.05 relative to uncoated control and Ad-LacZ. **p < 0.05 relative to LN5-coated control and Ad-LacZ replated on LN5. Ad-D5 on uncoated glass (*) compared with Ad-D5 on LN5-coated glass (**p < 0.001 by t test).

Based upon these observations, we conclude that LN5 does not simply induce spreading of cells but also may regulate spreading in a manner that cannot be duplicated by exogenous LN5. Indeed, provision of exogenous LN5 seems to further exacerbate the dysregulation of cell spreading.

Inhibition of Laminin 3 Synthesis Leads to Secretion of Laminin 3 and 2
When uninfected control cells and Ad-LacZ–infected cells were stained with polyclonal anti-LN5 and examined in the confocal microscope, deposition of LN5 was observed under the cells in a typical uneven pattern with the largest amounts tending to occur near the edges of multicell islands (Figure 7A). Cells infected with Ad-D5, in which laminin 3 synthesis is knocked down, also deposit antibody-reactive material on the substratum. However, the pattern of this deposition, although still uneven, often is whorled to resemble a rose blossom. This pattern may result from circular movement of cells as they deposit the proteins on the substratum (Aumailley, personal communication). Because the anti-LN5 antibody used for immunofluorescence strongly reacts with laminin 3 and is apparently less reactive with canine laminin 3 (Figure 5C), it was impossible to determine whether this pattern was caused by residual heterotrimeric LN5 secreted before knockdown was complete, or secreted laminin 3, possibly in a dimeric complex with 2 (Hirosaki et al., 2000).

View larger version (61K): [in this window] [in a new window]   Figure 7. Laminin 3 and 2 are secreted after siRNA-mediated knockdown of laminin 3. (A) Control MDCK cells or cells infected for 18 h with Ad-LacZ or Ad-D5 were replated on uncoated glass coverslips for 24 h and stained with antibodies against LN5 (red), fluorescent phalloidin (green), and DAPI (blue), and viewed by conventional immunofluorescence microscopy. Anti-LN5 antibody staining is visible at the base in all three cases, although the staining pattern under cells expressing siRNA (Ad-D5) exhibits a characteristic "rose petal" pattern. Bar, 20 µm. (B) Control MDCK cells or cells infected with Ad-LacZ or Ad-D5 were metabolically labeled overnight. Detergent cell extracts and culture medium were immunoprecipitated with anti-LN5, and precipitated polypeptides were visualized on SDS-gels by fluorography. In extracts from Ad-D5–infected cells, the laminin 3 band is absent and laminin 3 and 2 are increased in intensity (D5 lane). In the medium, laminin 3 is detectable but diminished, and the ratio of the 3 and 2 bands to 3 is higher than controls, suggesting secretion of 3 and 2 uncomplexed to 3 (compare D5 with Co and LZ). (C) Control MDCK cells or cells infected for 42 h with Ad-D5 were replated on uncoated glass coverslips for 24 h, stained with antibodies against LN5 (red) and fluorescent phalloidin (green), and viewed by confocal fluorescence microscopy. Individual optical sections taken at either the base of the cells or through the cell center are shown. Cells infected with Ad-D5 are exceedingly spread and still exhibit some staining with anti-LN5 at the base of the cell. Inside the same cells, anti-LN5 staining is intense in what seems to be the endoplasmic reticulum (arrow). Control (uninfected) cells seem normal with some anti-LN5 staining both within the cell and at the cell base. Ad-LacZ–infected cells seemed identical to uninfected controls (our unpublished data). Bar, 10 µm.

Because these experiments indicated that the Ad-D5–induced knockdown was incomplete in the time period tested, infected cells were incubated an additional 24 h, and LN5 was visualized by immunofluorescence and confocal microscopy. As shown in Figure 7C, Ad-D5–infected cells continued to spread even further relative to control cells as incubation was continued, providing additional evidence that knockdown of laminin 3 leads to dysregulation of spreading (Figure 7C, basal image). Under these conditions, deposited immunoreactive material was still apparent, but it was somewhat diminished and spotty relative to earlier times (Figure 7A). In confocal optical sections traversing the cytoplasm, extensive bright staining with the LN5 antibody was observed, apparently corresponding to the endoplasmic reticulum engorged with the 3 and 2 chains.

On the basis of these experiments, we believe that knockdown of laminin 3 likely leads to secretion and deposition of the other LN5 chains. This secretion, however, seemed somewhat inefficient, leading to their accumulation in the endoplasmic reticulum. Although it is likely that secreted 3 and 2 are present in a heterodimeric complex (Kariya et al., 2002), this cannot be ascertained definitively with the current data.

Knockdown of Laminin 3 Inhibits Proliferation and Wound-Edge Migration
A previous study used function-blocking antibodies against LN5 to demonstrate that LN5 stimulated cell proliferation (Gonzales et al., 1999). To determine whether this was also the case in MDCK cells in which synthesis of laminin 3 was suppressed, MDCK cells infected with the D5 adenovirus or the LacZ adenovirus, or noninfected control cells, were replated at equal subconfluent densities onto uncoated coverslips and labeled with BrdU for 6 h. Afterward, cultures were fixed and stained with a specific antibody against BrdU and DAPI to mark all nuclei, and the fraction of positive nuclei scored by fluorescence microscopy. As shown in Figure 8, both control cells and cells infected with the LacZ virus incorporated BrdU into 50% of their nuclei, whereas D5-infected cell nuclei were <10% BrdU positive. Clearly, knockdown of laminin 3 synthesis and its resulting effects on LN5 production block cell proliferation.

View larger version (12K): [in this window] [in a new window]   Figure 8. Knockdown of laminin 3 blocks proliferation. Control MDCK cells or cells infected for 18 h with Ad-LacZ or Ad-D5 were replated on uncoated glass coverslips for 24 h, incubated with BrdU for 6 h, stained with antibodies against BrdU and DAPI (blue), and viewed by conventional immunofluorescence microscopy. Proliferation was measured by counting total and BrdU-positive nuclei. *p < 0.05.

View larger version (14K): [in this window] [in a new window]   Figure 9. Knockdown of laminin 3 slows wound-edge migration. Control MDCK cells or cells infected for 18 h with Ad-LacZ or Ad-D5 were replated at confluent density on gridded, uncoated glass coverslips for 24 h, and wounded by scraping. Wound-edge migration relative to the time of wounding was measured on micrographs after 24 h. Note that wound-edge movement occurred under all three conditions but was significantly reduced in cells expressing siRNA (Ad-D5). *p < 0.05.

Knockdown of Laminin 3 Does Not Affect Apical–Basal Polarization of the MDCK Cell Epithelium

As shown in Figure 10, both control and LacZ adenovirus-infected cells formed uniform monolayers that were completely polarized relative to apical (Figure 10A) and basolateral (Figure 10B) proteins. The monolayer formed by the D5 adenovirus-infected cells, although generally intact, was very heterogeneous in cell size and shape. Nevertheless, apical and basolateral proteins were still segregated to the appropriate plasma membrane domains.

View larger version (44K): [in this window] [in a new window]   Figure 10. Knockdown of laminin 3 affects epithelial morphogenesis but not apical–basal polarization. Control MDCK cells or cells infected for 18 h with Ad-LacZ or Ad-D5 were replated at confluent density on uncoated Transwell supports for 24 h and stained with antibodies against either the apical antigen gp135 (A; red) or the basolateral protein E-cadherin (B; red) and fluorescent phalloidin (green). The stained samples were imaged as orthogonal Z-sections by confocal fluorescence microscopy. Note that the epithelium formed by Ad-D5–infected cells is heteromorphic with cells of different sizes and shapes, but it is as polarized relative to both apical and basolateral proteins as control and Ad-LacZ–infected cultures. Cell height in all samples is 10–15 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Through the inhibition of laminin 3 synthesis using siRNA, we have also uncovered additional clues to the functions of LN5 in MDCK cells. On knockdown of laminin 3, MDCK cells spread more rather than less, and this behavior is not suppressed but rather stimulated upon plating on exogenous LN5. Furthermore, in agreement with previous studies (Gonzales et al., 1999), our results indicate that suppression of LN5 production severely inhibits cell proliferation and reduces wound-edge migration, possibly as a partial consequence of the proliferation block. Finally, we show that LN5 is not critically required for apical–basal polarization of MDCK cells, at least in monolayer cultures, despite significant disruption of normal morphogenesis.

Regulated Synthesis of LN5
Our observation that LN5 synthesis only occurs in subconfluent MDCK cells is novel, and, to our knowledge, it has not been reported for other LN5-producing cell lines. These results with MDCK cells closely parallel our previous observation that LN5, which is not expressed to any great extent in the normal adult kidney, is strongly induced in the rat kidney after ischemic injury (Zuk and Matlin, 2002). In that instance, increased LN5 production occurs 24–48 h after injury at the beginning of the period in which the tubular epithelium regenerates. Thus, the implication of both our in vitro results with MDCK cells and our previous in vivo findings is that LN5 is a key participant in the reestablishment of a continuous epithelium. Induction of LN5 has also been reported in human polycystic kidney epithelium, suggesting, perhaps, that LN5 plays a role in the pathogenesis of the disease by causing tubular epithelial cells to be locked in a chronically regenerative state (Joly et al., 2003).

Pulse-labeling experiments indicate that the shutdown of LN5 synthesis may occur by blocking either the transcription or translation of mRNA for the 3 and 2 chains without affecting 3 synthesis. A similar mechanism for regulation of LN5 production may also function in keratinocytes as well as normal and ras-transformed HaCat cells (Aumailley, personal communication; Korang et al., 1995). In addition, this unusual regulatory scheme was previously observed in the normal human mammary epithelial cell line MCF-10A stably overexpressing the p300 transcriptional coactivator (Miller et al., 2000). Under normal conditions, MCF-10A cells constitutively express LN5. When p300 is overexpressed, however, mRNAs for laminin 3 and 2 are significantly decreased, whereas the message for 3 is not, paralleling our results with MDCK cells. Although there is no evidence that p300 is involved in the regulation of transcription after kidney injury or in MDCK cells, its ubiquitous expression certainly makes it a possibility.

LN5 has been previously implicated in the healing of cutaneous wounds (Decline and Rousselle, 2000; Nguyen et al., 2000b<