Collagen VI is a basement membrane component that regulates epithelial cell–fibronectin interactions

Matrix Biology 30 (2011) 195–206

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Matrix Biology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t b i o

Collagen VI is a basement membrane component that regulates epithelial cell–fibronectin interactions Jean-François Groulx, David Gagné, Yannick D. Benoit, Denis Martel, Nuria Basora, Jean-François Beaulieu ⁎ CIHR Team on the Digestive Epithelium, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

a r t i c l e

i n f o

Article history: Received 11 September 2010 Received in revised form 14 February 2011 Accepted 4 March 2011 Keywords: Basement membrane Cell–matrix interaction Collagen Fibronectin Cytoskeleton Epithelial cell

a b s t r a c t Collagen VI is a heterotrimer composed of three α chains (α1, α2, α3) widely expressed throughout various interstitial matrices. Collagen VI is also found near the basement membranes of many tissues where it serves as an anchoring meshwork. The aim of this study was to investigate the distribution and role of collagen VI at the epithelial–stromal interface in the intestine. Results showed that collagen VI is a bona fide epithelial basal lamina component and constitutes the major collagen type of epithelial origin in this organ. In vitro, collagen VI co-distributes with fibronectin. Targeted knockdown of collagen VI expression in intestinal epithelial cells was used to investigate its function. Depletion of collagen VI from the matrix led to a significant increase in cell spreading and fibrillar adhesion formation coinciding with an upregulation of fibronectin expression, deposition and organization as well as activation of myosin light chain phosphorylation by the myosin light chain kinase and Rho kinase dependent mechanisms. Plating cells deficient for collagen VI on collagen VI rescued the phenotype. Taken together, these data demonstrate that collagen VI is an important basal lamina component involved in the regulation of epithelial cell behavior most notably as a regulator of epithelial cell– fibronectin interactions. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Type VI collagen is a unique member of the collagen family that is ubiquitously expressed throughout all ECMs (Bruns et al., 1986). The collagen VI monomer is composed of three distinct polypeptide chains (α1, α2, α3) each containing a short triple helical domain and two large globular N and C terminal regions assembled in a stoichiometric ratio. (Bruns et al., 1986; Engvall et al., 1986). Collagen VI is expressed in human connective tissues such as those of the joint capsule ligament, tendons, skin, large blood vessels, placenta (Garrone et al., 1997; Hessle and Engvall, 1984; Michelacci, 2003; von der Mark et al., 1984), adipose tissues (Khan et al., 2009) and in some specialized regions like, the pancreas islet–exocrine interface (Hughes et al., 2006) and dermal and skeletal muscle fibers closely associated with the BM (Keene et al., 1988; Sipila et al., 2007). In fact, the major function proposed for collagen VI is as an anchoring meshwork that connects collagen fibers and other structures such as nerves and blood vessels to the surrounding matrix (Bonaldo et al., 1990; Keene et al., 1988).

Abbreviations: BM, basement membrane; BL, basal lamina; colVI, collagen type VI; FA, focal adhesion; FB, fibrillar adhesion; MLC, myosin II light chain; MLCK, MLC kinase; ROCK, Rho kinase. ⁎ Corresponding author at: Département d'anatomie et de biologie cellulaire, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4. Tel.: +1 819 564 5269; fax: +1 819 564 5320. E-mail address: [email protected] (J.-F. Beaulieu). 0945-053X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matbio.2011.03.002

Interestingly, collagen VI has been found to interact directly with an exclusive basement membrane protein, collagen IV (Kuo et al., 1997) supporting the proposed anchoring function for collagen VI. In fact, mutations in the genes encoding collagen VI result in Bethlem myopathy and Ullrich congenital muscular dystrophy caused by faulty cell attachment of muscle fibers to the underlying ECM (Bonaldo et al., 1998; Lampe and Bushby, 2005). While collagen VI has been clearly established as an ECM component and as BM-anchoring molecule, its presence in the BM is less recognized. In fact, up to now only the glomerular BM of the kidney has been found to contain collagen VI although its precise role in this structure, well characterized for its complex pattern of type IV collagen expression, is not known (Zhu et al., 1994). Interestingly, the kidney does not appear to be affected in diseases involving mutations in the collagen VI genes (Bonaldo et al., 1998). Like in the kidney, the intestine is another organ in which a single polarized epithelium lies on a complex BM containing distinct forms of type IV collagen (Beaulieu et al., 1994; Simoneau et al., 1998). Cell attachment to the underlying BM is critical for the integrity and functioning of this highly dynamic epithelium (Benoit et al., 2009; Bouchard et al., 2008; Chaturvedi et al., 2009; Dydensborg et al., 2009; Gagne et al., 2010; Gayer et al., 2009). While collagen VI has been identified in the stroma and at the epithelial–stromal interface in the intestine (Groos et al., 2003), its precise location relative to the epithelial BM remains unknown. The aim of the present study was to complement and extend previous studies on collagen VI expression in relation to simple epithelia, namely the intestinal epithelium. Herein, we established the

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ultrastructural distribution of collagen VI in the basal lamina (BL) showing for the first time that it is a bona fide BM molecule in this tissue and demonstrated its epithelial origin. We also investigated the possible effects of its knockdown on epithelial cells.

2. Results 2.1. Immunolocalization of collagen VI in the epithelial BL of the small intestine The expression of collagen VI was investigated by indirect immunofluorescence in the adult human intestine (Fig. 1A). As in the fetal intestine (see below), positive staining for collagen VI was found throughout the lamina propria, surrounding blood vessels and appeared to be concentrated in regions at the epithelial/stromal interface along the crypt–villus axis. At mid-gestation, the human fetal small intestine is fully functional and considered to be similar to the adult. To more precisely define collagen VI localization, indirect immunofluorescence was carried out on 1 μm thick Lowicryl sections of 17–19 week fetal tissue (Fig. 1B, C). These experiments revealed that collagen VI was indeed localized in close association with the basement membrane underlying the epithelial cell monolayer both in the crypt and villus regions.

Immuno-electron microscopy was used to further define the localization of collagen VI at the base of the intestinal epithelium. Immunogold particles associated with collagen VI were found to be concentrated at a high density in the epithelial BM with scattered detection in the interstitial matrix in association with fibrillar material (Fig. 1D). Higher magnification of the epithelial BM region showed that the gold particles were predominantly found in the lamina densa of the basal lamina (BL) (Fig. 1E). Negative controls showed no specific staining of these structures (Fig. 1G). Positive control for BM detection with laminin EHS (Fig.1F) showed localization under the basal cell membrane of epithelial cell.

2.2. Epithelial origin of collagen VI Expression of collagen VI mRNA was analyzed by RT-PCR in fetal and adult tissue fractions. As shown in Fig. 2A, mRNA for the α1 chain of collagen VI was detected in purified epithelial fractions of both the fetal and adult intestine as well as in the mesenchymal fraction of the fetal intestine indicating that collagen VI was synthesized by epithelial cells in addition to stromal cells. Epithelial fraction purity was confirmed by screening for the epithelial specific marker E-cadherin and the lack of the mesenchymal markers tenascin and vimentin.

Fig. 1. Localization of collagen VI in the human small intestine. (A) Localization of collagen VI in the adult human small intestine showing localization at the base of epithelial cells (e) uniformly distributed along the crypt/villus axis (arrows). (B and C) Immunolocalization of collagen VI on Lowicryl 1 μm thick sections of 17-week ileon. Collagen VI distributed in the BM (arrows) underlying the epithelium (e) in the villus (B) and in the crypt (C). Elements in the interstitial matrix (im), such as myofibroblasts and blood vessels, were also positive for collagen VI. Non-specific staining was found in the epithelial cells. (D-G) Electron microscopy immunolocalization of collagen VI on Lowicryl 80 nm thick sections of fetal small intestine. (D) Low magnification of the BM (bm) region under an intestinal epithelial cell (e) showed positive staining of collagen VI along the BM (arrow). (E) High magnification of an intestinal epithelial BM was positive for collagen VI in the basal lamina (bl). The epithelial basal membrane (ebm) is identified by arrowheads. (F) Positive control for detection of laminin EHS in BM. (G) Negative control, without primary or with another rabbit primary antibody (HNF-1) showed no staining in the BL. Epithelium (e); interstitial matrix (im); basement membrane (bm); basal lamina (bl); epithelial basal membrane (ebm). Bar, A = 50 μm; B–C = 25 μm; D = 500 nm; E = 200 nm; F = 200 nm; G = 200 nm.

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2.4. Collagen VI regulates recruitment of tensin to epithelial adhesion structures

Fig. 2. Collagen VI is of epithelial origin. (A) Expression of the collagen VI α1 chain transcript in the human fetal intestinal epithelium (e) and corresponding mesenchymal (m) fractions; and in the adult epithelium (e). Vimentin and tenascin were used as mesenchyme markers and E-cadherin as an epithelial marker. (B) Expression of the collagen VI α1 chain transcript in the human epithelial cell lines HIEC and Caco-2/15 at confluence. RPLPO was used for quantity control. (C) Total production of collagen VI (α1/α2 chains at 130–140 kDA) by HIEC and Caco-2/15 cells analyzed by western blot. Higher exposition time was necessary to detect collagen VI in Caco-2/15. Representative results of three independent experiments.

Collagen VI expression was also investigated in the crypt-like HIEC and villus-like post-confluent Caco-2/15 cells, which represent the proliferative and differentiated compartments, respectively. Our results showed that collagen VI was detectable at both transcript (Fig. 2B) and protein (Fig. 2C) levels. Interestingly, Caco-2/15 cells produced relatively lower amounts of collagen VI compared to HIEC.

2.3. Loss of collagen VI alters epithelial cell morphology To investigate the potential role of collagen VI on intestinal epithelial cells, its expression was knocked down with an shRNA approach using the epithelial HIEC cell line. These cells were infected with a lentivirus containing either an shRNA directed against the α1 chain of collagen VI (shcolVI) or a control shRNA (shctl). Knockdown was evaluated by western blot and these experiments confirmed that the production of the collagen VI protein was strongly reduced in shcolVI cells versus control cells (Fig. 3A). This reduction translated into an apparent decreased incorporation of collagen VI in the insoluble matrix as visualized by indirect immunofluorescence. Indeed, as compared to shctl cells where collagen VI was deposited around the cell body (Fig. 3B), shcolVI cells showed only very weak staining (Fig. 3C). These results confirmed that knockdown of collagen VI was efficient enough to translate into a loss at its acknowledged location in the ECM. During the selection of stable epithelial shcolVI and shctl cell populations grown on tissue culture plastic, a striking difference in cell morphology became perceptible, where collagen VI knockdown cells appeared to be larger and flatter, suggesting that the absence of collagen VI altered cell spreading ability. These differences in cell phenotype were analyzed by indirect immunofluorescence using anti-vinculin to identify focal adhesion (FA) points (Zamir and Geiger, 2001) and phalloïdin to visualize the actin cytoskeleton. In shctl cells FAs were located at the cell periphery in a discrete punctuate pattern and at the base of the cells in a more elongated pattern, while the actin cytoskeleton was organized into parallel stress fibers (Fig. 3D). In shcolVI cells anti-vinculin staining revealed a noticeable increase in the number of FAs under the cell body and appeared to be accompanied by a reorganization of stress fibers (Fig. 3E). We found that in addition to the perceived increase in FA number, shcolVI cells were indeed 3-fold significantly more spread than control cells when cultured on tissue culture plastic, as determined by measuring the surface area (Fig. 3F).

We then investigated the ability of exogenous collagen VI to rescue the observed phenotype and compared this to adhesion on other RGD ligands–fibronectin, collagen IV, collagen I and serum (providing minimal fibronectin and vitronectin) for 24 h (Fig. 4A, Supplementary 4). Adhesive capacity was correlated by counting positive anti-vinculin FA structures. Shctl cells plated on serum, collagen IV and collagen I coatings displayed a similar number of FAs per cell. When plated on exogenous fibronectin, however, the number of FAs significantly increased in contrast to exogenous collagen VI which caused a significant decrease in adhesive ability. ShcolVI cells displayed a general increased ability to bind the different RGD containing molecules with a significant increase in the number of FAs when plated on serum, collagen IV, collagen I or fibronectin (Fig. 4A). Plating collagen VI knockdown cells on exogenous fibronectin had a striking effect on the phenotype which led to a 2-fold increase in the number of FAs formed when compared to other substrates and a 4-fold increase when compared to shctl cells plated on exogenous fibronectin. Most striking was the ability of exogenous collagen VI to significantly reverse the observed phenotype of shcolVI cells, reducing the number of FAs to control levels. These results suggested that the presence of collagen VI interfered with cell adhesion even under conditions where fibronectin was present in large amounts. In addition, this role for collagen VI appeared to be specific since the presence of other RGD ligands, serum, fibronectin, collagen IV and collagen I did not have the same effect. The extensive staining of anti-vinculin in elongated points in the cell periphery and cell body of shcolVI cells strongly suggested an increase in the formation of fibrillar adhesions (FB). Adhesion structures formed in collagen VI knockdown cells were characterized by comparing the immunocolocalization patterns of vinculin and tensin, a marker of FBs (Zamir et al., 1999), when plated on either collagen VI or fibronectin substrates. As shown in Fig. 4B, analyses of vinculin and tensin immunofluorescence in shctl cells plated on exogenous collagen VI revealed that tensin was present in small punctuates in the cell body and colocalized with vinculin at the cell periphery. On exogenous fibronectin these cells showed heterogeneous tensin/vinculin colocalization at the cell periphery and tensin was also organized in tensin-rich FBs in the cell body. ShcolVI cells plated on collagen VI showed the same heterogenic pattern of tensin/vinculin staining as control cells and tensin was found more frequently in tensin-rich FBs. In shcolVI cells plated on a fibronectin matrix, tensin was found to be more extensively organized in long and parallel tensin-rich FBs throughout the cell body. Tensin and vinculin staining were colocalized only at the tips of these structures in the cell body, tensin being much more weakly detected than vinculin at the cell periphery as compared to other tested conditions. No variation in cellular vinculin and tensin protein levels was observed (Supplementary Fig. 1).These results suggested that collagen VI is important for the regulation of FB formation.

2.5. Collagen VI affects the production, deposition and organization of fibronectin To investigate the increase in FB numbers formed by shcolVI cells, we analyzed the behavior of endogenous fibronectin, which is the requisite ligand for FB formation. The expression of fibronectin transcript levels were analyzed by qPCR in both cell populations under tissue culture plastic and on collagen I matrix culture conditions. In shcolVI cells qPCR confirmed that fibronectin mRNA levels were increased 2.3 fold (p ≤ 0.01) over control levels under conditions where collagen VI mRNA levels were inhibited by 94% (Fig. 5A). Identical results were obtained when shcolVI cells were cultured on collagen I (Fig. 5A). No significant variation was observed in the unrelated laminin β1 mRNA transcript expression.

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Fig. 3. Validation of shRNA directed against the α1 chain of collagen VI in human epithelial HIEC cells cultured on tissue culture plastic. (A) Western blot of whole cell lysates confirmed the abolition of collagen VI production. β-actin served as a loading control. Representative immunofluorescence images of collagen VI in shctl (B) and shcolVI cells (C). Shctl cells showed collagen VI deposition contrary to shcolVI cells. Bar, 100 μm. Immunofluorescence for detection of the actin cytoskeleton (red), vinculin (green) and DAPI (blue) on shctl (D) and shcolVI cells (E) showed an increase in spreading and FA number when cell are plated on serum (n = 3 separate experiments). Bar, 25 μm. (F) Quantification of cell surface area of shcolVI compared to shctl cells plated on serum for 24 h (n = 3 separate experiments); at least 100 cells were counted in each experiment. Results are expressed as the mean ± S.D. ***p ≤ 0.0001, t-test.

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Fig. 4. Collagen VI affects adhesion structures. (A) Graph of FA number per cell determined by counting vinculin positive structures. Shctl and shcolVI cells were seeded on serum, collagen I, fibronectin and collagen VI (n = 3 separate experiments), at least 45 cells were counted per experiment and per condition tested. Results are expressed as the mean ± S.D; ***p ≤ 0.0001, t-test. (B) Colocalization of tensin and vinculin. Representative immunofluorescence images of vinculin (red), tensin (green), and merged with DAPI (blue) in HIEC/ shctl and HIEC/shcolVI cells on fibronectin or collagen VI coatings. Bar, 50 μm. Higher magnification of the merged (merge’) images highlights co-localization of these two proteins. Arrows denote adhesion points positive for both vinculin and tensin and arrowheads identify adhesion points positive for tensin alone. Bar, 10 μm.

The increase in fibronectin mRNA observed by qPCR in shcolVI cells plated on plastic or exogenous collagen I translated directly into proportional significant increases of both cellular amounts of fibronectin protein (3.25 fold increase, p ≤ 0.01) and fibronectin incorporated into the ECM (3.62 fold increase, p ≤ 0.005) from cells grown on plastic (Fig. 5B and C). Protein levels of laminin β1γ1 chains showed no significant changes (Fig. 5B and Supplementary Fig. 2) and served as a loading control for matrix extracts. Indirect immunofluorescence on cell monolayers cultured on tissue culture plastic confirmed the more extensive endogenous fibronectin extracellular

deposition in shcolVI cell populations compared to shctl cells (Fig. 5D, E). In addition, shctl cells exhibited endogenous fibronectin deposition underlying the cell body in short fibrils while in the ECM of shcolVI cells endogenous fibronectin was organized in long parallel fibers underlying the cell body. The most common receptors for fibronectin are integrins α5β1 and αv-containing integrins. Expression levels of integrin subunits α5, αv and β1 were analyzed by western blot to determine if the observed modulation of fibronectin in shcolVI cells cultured on tissue culture plastic was accompanied by an increase in receptor

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Fig. 5. Increased fibronectin deposition in the absence of collagen VI from the ECM. (A) qPCR analyses of collagen VI α1 chain, fibronectin and laminin β1 chain mRNA levels on tissue culture plastic and on collagen substrates. ShcolVI were compared with expression levels in control cultures (n = 3 separate experiments). Results are expressed as the mean ± S.D; **p ≤ 0.005, ***p ≤ 0.0001, t-test. (B-C) Representative western blots and signal quantification by densitometry for fibronectin and laminin β1γ1 expression in whole cell lysates and in matrix extracts. β-actin served as a loading control for total extracts and laminin β1γ1 for matrix extracts (n = 3). Results are expressed as the mean ± S.D; *p ≤ 0.01, **p ≤ 0.005, t-test. (D-E) Immunofluorescence images of fibronectin deposition in the ECM of shctl (D) and shcolVI cells (E). n = 3 separate experiments. Bar,100 μm.

expression. No significant variation was observed for α5, αv and β1 subunit production (Fig. 6A, B). These results indicated that collagen VI knockdown did not affect expression of fibronectin receptors. To investigate a possible interaction between collagen VI and fibronectin, indirect immunofluorescence was carried out in order to determine the deposition patterns of these glycoproteins in the insoluble matrix of wildtype intestinal epithelial cells. Double-labeling immunofluorescence of endogenous collagen VI and fibronectin on epithelial cell monolayers showed that collagen VI was homogenously and abundantly deposited into the ECM around the cell in short fibers (Supplementary Fig. 3A). A highly similar pattern was observed for

fibronectin (Supplementary Fig. 3B) and the two molecules co-localized extensively as seen in the overlay image (Supplementary Fig. 3C). 2.6. Integrin β1 recognition of collagen VI Adhesion assays were performed on wildtype epithelial cells plated on exogenous fibronectin and collagen VI coatings (Fig. 6C) in order to confirm the involvement of integrin receptors. Cell adhesion on fibronectin was normalized to 100%, and cell adhesion on collagen VI was found to be at 70.14%. Preincubation of cell cultures in the presence of a β1 integrin blocking antibody caused adhesion on

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Fig. 7. Collagen VI regulates MLCK activity. (A) Representative western blot and (B) densitometric quantification of MLC phosphorylation (pMLC) levels and total MLC levels in shctl and shcolVI cells treated with no inhibitor, ML7 or Y27632 (n= 3 separate experiments) on tissue culture plastic. Results are expressed as the mean ± S.D; n.s., not significant; **p ≤ 0.001, t-test. (C) Representative western blot and (D) densitometric quantification of MLC phosphorylation levels and total MLC levels in shctl and shcolVI cells plated on fibronectin (FN) and collagen VI (colVI) (n= 3 separate experiments). Results are expressed as the mean± S.D; n.s., not significant; *p≤ 0.05, t-test.

Fig. 6. Collagen VI does not influence the expression of fibronectin-binding integrins but uses β1 integrins for cell adhesion. (A) Western blot for detection of integrin α5, αV and β1 subunits in total extracts of shctl and shcolVI cells when cell were cultured on tissue culture plastic. (B) Densitometric quantification of the relative amounts of these integrin subunits normalized to β-actin levels (n = 3 separate experiments). Results are expressed as the mean ± S.D, t-test. (C) Graph of epithelial cell adhesion on fibronectin and collagen VI exogenous matrix in the presence of control antibody, IgG, or a neutralizing anti-β1 antibody. Results are expressed as the mean ± S.D; **p ≤ 0.005, *p ≤ 0.05, n = 3 separate experiments.

fibronectin to drop by half and effectively abolished adhesion on a collagen VI coating. This result showed that β1 integrins were involved in fibronectin and collagen VI substrate recognition and were the main receptor subtype for collagen VI binding by intestinal epithelial cells. 2.7. Collagen VI regulates MLC phosphorylation Phosphorylation of MLC is a necessary event in the regulation of stress fiber and FB assembly. When cells were seeded on tissue culture plastic, MLC phosphorylation levels were found to be significantly higher in cells deficient for collagen VI expression (Fig. 7A, B). Net changes in the proportion of phosphorylated MLC occur following a shift in the activities of two principal kinases, MLCK and ROCK. We analyzed

the extent to which these two kinases were involved in the stress fiber reorganization observed following the loss of collagen VI plated on tissue culture plastic. MLC phosphorylation was assessed in cells treated with or without MLCK inhibitor (ML7) or ROCK inhibitor (Y27632). As shown in Fig. 7A, B, MLC phosphorylation levels in shctl cells were not affected by ML7 but were significantly reduced by Y27632. Conversely, in shcolVI cells, the high levels of MLC phosphorylation were inhibited by both ML7 and Y27632. Taken together, these results suggest that ROCK can regulate phosphorylation of MLC in both shctl and shcolVI cells while MLCK is only involved in cells lacking collagen VI expression. Therefore, the capacity of the exogenous matrix to regulate MLC phosphorylation was further analyzed. Shctl and shcolVI cells were plated on fibronectin and collagen VI matrices and analyzed for MLC phosphorylation (Fig. 7C, D). Exogenous fibronectin or collagen VI had no visible effect on the relative amounts of MLC phosphorylation in shctl cells. Plating of shcolVI cells on fibronectin resulted in significantly higher levels of phosphorylated MLC, a phenomenon also observed when shcolVI cells were plated on plastic (Fig. 7B). In contrast, plating of shcolVI cells on purified collagen VI restored basal levels of phosphorylated MLC confirming a rescue phenomenon. Taken together, these results indicate that stress fiber reorganization following loss of collagen VI is MLCK dependent and can be rescued by exogenous collagen VI.

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Fig. 8. Collagen VI regulates migration of HIEC. (A) Representative graph of migration rate of shctl and shcolVI cells on tissue culture plastic, n = 3. Results are expressed as mean ± S.D. ** = p ≤ 0.005, t-test. (B) Representative images of DAPI staining of shctl and shcolVI cells which have migrated over the wound edge. Bar, 100 μm.

2.8. Loss of collagen VI increases epithelial cell migration Given the observed increases in spreading and fibronectin adhesion, the migratory capacity of these two cell populations was investigated. Scratch assays revealed that depletion of collagen VI resulted in a significant increase in the migratory capacity of shcolVI cells compared to shctl cells (Fig. 8A). As determined by DAPI staining, a significantly higher number of cells migrated through the wound edge and appeared to migrate further (Fig.8B). These data indicate that collagen VI plays a crucial role in modulating epithelial cell migration. 3. Discussion This study has identified collagen VI as a bona fide BL component found along the length of the intestinal crypt–villus axis, suggesting a new role for this protein in the homeostasis of this rapidly renewing epithelium. Collagen VI is a well established component of most interstitial matrices and its importance in tissue structure is emphasized in the muscle, where mutations in the collagen VI genes lead to muscular dystrophies (Lampe and Bushby, 2005). To our knowledge, the only other BL-like structure in which collagen VI has been observed is the renal glomerular BM (Magro et al., 1996; von der Mark et al., 1984). However, the role collagen VI plays at this site remains unknown. To investigate the function of collagen VI on epithelial cells, we exploited the finding that collagen VI is synthesized by intestinal epithelial cells, in contrast to the other main intestinal BL collagen ([α1(IV)]2α2(IV)) that is of stromal origin (Perreault et al., 1998), to knockdown its expression in normal intestinal epithelial cells. Reduction of collagen VI led to a striking increase in cell spreading and was accompanied by significant increases in the number of stress fibers and of adhesion structures in the central body of the cell. These adhesion structures were found to be tensin-rich, suggesting that collagen VI modulated the extent of fibronectin assembly in epithelial cells. The increased expression and deposition of fibronectin most likely contributed to the enhanced levels of spreading and FB formation, in the absence of collagen VI as suggested by the observation that a similar phenotype was obtained when control cells were seeded on exogenous fibronectin. These observations are not without precedent since an increase in cell size was also observed in adipocytes isolated from colVI α1 (−/−) mice (Khan et al., 2009)

while a relationship between the expression of collagen VI and the pattern of organization of fibronectin was reported in cultured fibroblasts (Sabatelli et al., 2001). The relatively close co-distribution of collagen VI and fibronectin observed in the ECM of normal epithelial cells has not been reported before although it appears consistent with the fact that the collagen VI globular domain can bind to immobilized fibronectin (Kuo et al., 1997; Tillet et al., 1994). In this context, our observations that i) depletion of collagen VI production led to an increase in both fibronectin expression and extracellular deposition which became rearranged into long and parallel fibronectin fibrils while ii) exogenous collagen VI is able to restore the knockdown phenotype, even in the presence of large amounts of fibronectin, are of interest. Indeed, they suggest that collagen VI is an important regulator of fibronectin fibrillogenesis. The effect of collagen VI on intestinal epithelial cell morphology and adhesions (FA, FB) seem to be specific to this collagen type since collagen I and basement membrane collagen IV could not rescue the phenotype. The increased expression of fibronectin was observed in tissue culture plastic and collagen matrix conditions, suggesting that this feature was not just a result of the loss of a major ECM component. The exposure of binding sites on the fibronectin molecule is critical for cell binding and fibronectin matrix assembly (Mao and Schwarzbauer, 2005; Pankov and Yamada, 2002). One possible explanation is that interaction of collagen VI with fibronectin in the ECM modulates cell binding to fibronectin by limiting accessibility. Consistent with this possibility, studies using blocking antibodies have demonstrated that collagen VI could be recognized by α5 and αv integrins (fibronectin receptors) and that cyclic-RGD peptides blocked adhesion to collagen VI (Aumailley et al., 1989; Du et al., 2007; Pfaff et al., 1993). However, cell binding to collagen VI by integrin receptors is complex and appears to be cell type dependent (Tulla et al., 2001). Integrin receptors can bind this molecule either in an RGD or a non-RGD dependent manner (Andresen et al., 2000; Aumailley et al., 1989; Doane et al., 1998; Du et al., 2007; Leitinger and Hohenester, 2007; Pfaff et al., 1993; Tulla et al., 2001). Herein, we showed that the anti-β1 integrin antibody blocked approximately half of the intestinal cell adhesion to fibronectin, consistent with the presence of both β1 and αv integrins on these cells (Benoit et al., 2009), but almost completely blocked cell adhesion to collagen VI indicating that αv integrins are not involved. It is therefore possible that collagen VI and fibronectin compete for binding to common β1 integrin receptor(s) offering a possible mechanism explaining how the presence of collagen VI interferes with cell– fibronectin interaction. This possibility is supported by the significant increase in tensin-enriched FB complexes and fibrillar deposition of fibronectin observed in collagen VI-depleted cells exposed to fibronectin. Indeed, in contrast to FA which contain various integrins, paxillin and vinculin, FB are enriched in tensin and integrin α5β1 (Berrier and Yamada, 2007; Mao and Schwarzbauer, 2005; Zaidel-Bar et al., 2004; Zamir et al., 2000), a fibronectin receptor considered to be of major importance for the fibronectin fibril formation of FB (Leiss et al., 2008; Mao and Schwarzbauer, 2005). Interestingly, generation of FB from FA depends on functional actomyosin forces and can be blocked by ROCK or MLCK inhibitors (Zamir et al., 2000). Indeed, actin functional contractility depends on the phosphorylation of MLC which is principally mediated by the two kinases, MLCK and ROCK, mainly acting on MLC directly and myosin phosphatase, respectively (Fukata et al., 2001; Iizuka et al., 1999; Kimura et al., 1996; Totsukawa et al., 2004). In control cells exposed to an endogenous ECM containing both collagen VI and fibronectin, the relative amounts of phosphorylated MLC were relatively low and only affected by the ROCK inhibitor. In contrast, in epithelial cells grown in a low collagen VI/rich fibronectin ECM environment, MLC phosphorylation was found to be significantly higher than in control cells while being repressed by both MLCK and ROCK consistent with their higher levels of tensin-enriched FB complexes and fibronectin fibrillar deposition. The MLCK-dependent activation of MLC phosphorylation

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in cells exposed to a low collagen VI/rich fibronectin environment suggests that Ca2+/calmodulin activity (Murthy, 2006; Xie et al., 2009) is altered under these conditions. Interestingly, collagen VI deficiency in the muscle has been reported to cause disruption in calcium homeostasis (Bernardi and Bonaldo, 2008; Irwin et al., 2003). Taken together, these observations further emphasize the possibility that collagen VI influences epithelial cell behavior by restraining cell– fibronectin interactions and their downstream events. In summary, this study has identified collagen VI to be a true integral BL component, and not simply associated with the BM it as in other tissues, such as the muscle. Collagen VI is the major collagen type synthesized by intestinal epithelial cells and is subsequently secreted and deposited at the base of epithelial cells. Using 2D epithelial cell cultures, we identified a role for collagen VI as a regulator of fibronectin synthesis and fibrillogenesis. We thus propose that collagen VI is a basement membrane component that regulates epithelial cell–fibronectin interactions. While this study focused on 2D cultures, it would be of interest to further investigate basement membrane collagen VI in 3D culture systems in the future.

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4.4. Cell culture The crypt-like human intestinal epithelial cells, HIEC, were grown as described previously (Perreault and Beaulieu, 1996). The human intestinal epithelial cell line Caco-2/15, a stable clone of the parental Caco-2 cell line (ATCC, Manassas, VA) has been fully characterized in previous studies (Beaulieu and Quaroni, 1991; Pageot et al., 2000) and was grown as described previously (Dydensborg et al., 2009). 4.5. Generation of shRNA stable cell lines HIEC cells were plated at 60% confluence 24 h prior to infection with virus prepared from MISSION® shRNA (Sigma-Aldrich) plasmids for human collagen, type VI, alpha 1, containing the shRNA sequence: 5′-CCG GCC TTT GGA CTG AAA GGA GAA ACT CGA GTT TCT CCT TTC AGT CCA AAG GTT TTT G. The negative control shRNA (Sigma-Aldrich) sequence (shctl): 5′: CCG GGT GGG CAT CAA AGA CGT GTT TCT CGA GAA ACA CGT CTT TGA TGC CCA CTT TTT G had no effect on collagen VI α1chain protein levels. At 3 days post infection, stable cell lines were selected by the addition of 1 μg/ml puromycin to the culture medium (Qiagen, Mississauga, ON) and used after 14 days.

4. Experimental procedures 4.1. Human intestinal tissue samples All tissues were obtained in accordance with protocols approved by the local Institutional Human Research Review Committee. The preparation and embedding of tissues for cryosectioning and RNA extraction were performed as described previously (Ni et al., 2005; Teller et al., 2007).

4.2. Epithelial and mesenchymal fractions Preparation of epithelial and mesenchymal fractions by nonenzymatic dissociation was carried out as described previously (Perreault and Beaulieu, 1998; Perreault et al., 1998).

4.3. Antibodies Mouse primary antibodies used in this study were: anti-β-actin (WB:1/75000) (Santa Cruz Biotechnology, Santa Cruz, CA), antifibronectin (WB:1/500, IF:1/100) (HFN 7.1, Developmental Studies Hybridoma Bank, Iowa City, IA) and anti-vinculin (clone 7 F9; IF:1/ 500) (Chemicon, Temecula, CA). Rabbit primary antibodies used in this study were: anti-collagen VI (WB:1/5000, IF:1/2000) (Fitzgerald, Concord, MA), anti-laminin EHS (WB:1/1000) (Sigma-Aldrich, Oakville, ON), anti-integrin α5 (WB:1/2000)(AB1928, Millipore, Billerica, MA), anti-integrin αv (WB:1/1000)(AB1930, Millipore), anti-p-MLC (WB:1/1000) (Cell Signaling, Danvers, MA), anti-MLC (WB:1/1000) (Cell Signaling), anti-tensin (IF:1/100) (a kind gift from Dr. Su Hao Lo, Center for Tissue Regeneration and Repair, Department of Biochemistry and Molecular Medicine, University of California-Davis, Sacramento, CA). Rat primary anti-integrin β1 antibody (MAB13, WB:1/ 500, blocking antibody: 10 μg/ml) (a kind gift from Dr. Stephen K. Akiyama, National Institutes of Health/NIEHS, Research Triangle Park, NC (Akiyama et al., 1989)) and anti-HNF-1 (Santa Cruz Biotechnology). Mouse IgG (Sigma-Aldrich) was used at 10 μg/ml as a negative control for adhesion assay. Secondary antibodies used were Alexa Fluor 488 goat anti-mouse, Alexa Fluor 594 goat anti-rabbit, Alexa Fluor 488 goat anti-rabbit (Invitrogen, Burlington, ON). The actin cytoskeleton was stained using TRITC-conjugated phalloidin (IF:1/ 1000; Chemicon). The myosin kinase inhibitor, ML7 (Sigma-Aldrich) and the specific inhibitor of Rho-kinase, (ROCK) Y-27632 (SigmaAldrich) were used at 20 μM.

4.6. Coating preparations For FBS coating, glass cover slips were incubated with serum for 1 h at 37 °C. For fibronectin coating, 3 μg/cm2 of human plasma fibronectin (Chemicon) in 1X PBS was incubated for 2 h at 37 °C. Collagen I (BD Bioscience, San Jose, CA), collagen IV (Invitrogen) and collagen VI (Chemicon) coatings were performed at 3 μg/cm2 in a solution of 0.02 μM acetic acid for 2 h at 37 °C. Any remaining potential adhesion sites were blocked with 2% BSA-PBS (pH 7.4) for 1 h at 37 °C prior to plating cells. 4.7. RNA extraction and RT-PCR RNA extraction and reverse transcription were performed as previously described (Teller et al., 2007). Primers used to amplify the collagen VI α1 chain were colVI-F: 5′-GAC TTC ATC CCA GGC TCA GA, and colVI-R: 5′-CAG CAG GAT GGT GAT GTC AG. Amplification was for 35 cycles with an annealing temperature of 60 °C to generate a product of 222 bp. Other primers used were vimentin, RPLPO (ribosomal protein, large, PO), E-cadherin, and tenascin-C as previously described (Francoeur et al., 2009; Gagne et al., 2010). 4.8. Quantitative RT-PCR For quantitative PCR experiments, primers for the α1 chain of collagen VI and RPLPO were the same as above. Other primers targeted fibronectin, FN-F: 5′-GTT GTT ACC GTG GGC AAC TC, and FNR 5′-CTG ACG GTC CCA CTT CTC TC; the laminin β1 chain, LAMB1-up: 5′-GGA ACA GCT CTC CAA GAT GG, and LAMB1-down: 5′-CTG CTT CAA TGC TGT CCA AA. Assessment of differences in gene expression between controls and experimental conditions were established according to the Pfaffl mathematical model (Pfaffl, 2001) using RPLPO for normalization. Real-time experiments were performed using an Mx3000P (Stratagene, La Jolla, CA) as previously described (Dydensborg et al., 2006). 4.9. ECM extracts Newly confluent HIEC and Caco-2/15 cells cultured on plastic were washed with PBS (pH 7.4) twice and incubated with 27 μM ammonium hydroxide (NH4OH) at RT until the cells were removed followed by incubation with double distilled H2O for 15 min to remove cellular debris. ECM proteins were lysed in 100 μl 1× Laemmli

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buffer (2.3% SDS, 10% glycerol, 0.001% bromophenol blue in 62.5 mM Tris–HCl, pH 6.8).

4.10. Western blot Western blots were performed on SDS-PAGE gels under denaturing conditions as previously described (Beaulieu et al., 1989; Vachon and Beaulieu, 1995). Total protein (50 μg/ml) was separated on 10% or 15% gels and electrotransferred onto a nitrocellulose membrane (BioRad, Hercules, CA). Non-specific protein binding was blocked using 10% Blotto-0.1% Tween followed by incubation with the primary antibody diluted in the blocking solution, overnight at 4 °C. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (anti-mouse, anti-rabbit, Amersham) and developed using the Immobilon Western® kit (Millipore).

4.11. Indirect immunofluorescence For standard immunofluorescence, human intestinal tissues were embedded in OCT (Optimum Cutting Temperature; CanemcoMarivac, Lakefield, QC) as previously described and 3 μm thin sections were cut with a Leica CM3050S cryostat and placed on silane-coated glass slides (Basora et al., 1999; Beaulieu et al., 1994; Simoneau et al., 1998). For immunofluorescence of collagen VI on the human small intestine, fetal human intestinal tissues of 17–19 weeks were embedded in Lowicryl K4M at − 20 °C as previously described (Bendayan and Benhamou, 1987) and 1 μm thin sections were cut with an Ultramicrotome LKB8800 and placed on silane-coated glass slides. Sections were fixed in 2% PFA and non-specific sites were blocked for 1 h at RT with 10% Blotto-PBS (pH 7.4). Both primary and secondary antibodies were diluted in 10% Blotto-PBS (pH 7.4) and incubated for 1 h at RT. Slides were mounted as previously described (Teller et al., 2007) and viewed with a Reichart Polyvar 2 microscope (Leica, St-Laurent, QC) equipped for epifluorescence with 10×, 25× and 40× objectives and digital images were captured using a DFC300FX camera (Leica). For immunofluorescence on cells, 5 × 104 cells were seeded on glass cover slips and treated as described above. After 24 h, cells were fixed in 2% PFA. Both primary and secondary antibodies were diluted in 2% BSA-PBS (pH 7.4) or 10% Blotto-PBS (pH 7.4). Cells were treated with 0.2% Triton X-100 for 5 minutes except for immunodetection of ECM molecules (collagen VI and fibronectin). Nuclei were counterstained 4 minutes at room temperature with 10 ng/ml 4′,6-diamidino-2-phenylindole (DAPI)-PBS (pH 7.4), and samples were mounted as previously described (Teller et al., 2007) and viewed with a DMRXA microscope (Leica) equipped for epifluorescence and digital imaging (RTE/CCD Y/Hz-1300 cooled camera) with a Reichart Polyvar 2 microscope (Leica) for ECM proteins. 4.12. Electron microscopy immunolocalization Electron microscopy immunolocalization was carried out on fetal human intestinal tissues at 17–19 weeks of gestation. The preparation, fixation of samples and immunolocalization was carried out as previously described (Calvert et al., 1993). Briefly, tissues were embedded in Lowicryl K4M at − 20 °C as previously described (Bendayan and Benhamou, 1987) and 80 nm sections were cut with an Ultramicrotome LKB8800. The primary rabbit polyclonal antibodies (collagen VI, laminin EHS and HNF-1) diluted 1:50 in 1% BSAPBS (pH 7.4) and incubated overnight. The secondary anti-rabbit antibody conjugated to 10 nm gold particles was used for detection as previously described (Calvert et al., 1993). Images were taken with a HITACHI H7500 electron microscope. Controls omitted incubation with the primary antibody.

4.13. Adhesion assay HIEC cells were plated 24 h prior to experimentation. Cells were harvested with 0.5 mM EDTA/PBS (pH 7.4) and centrifuged at 100 ×g for 5 min at 4 °C. Cell pellets were resuspended in OptiMEM culture medium supplemented with 0.2% FBS and 0.3 mM MnCl2. Cell were incubated 30 min with mouse IgG or β1 integrin blocking antibody (Mab13). 2 × 106 cells were seeded on glass cover slips treated with fibronectin or collagen VI as described above for 1 h at 37 °C. Nonadherent cells were eliminated by washing in PBS (pH 7.4) and the remaining adherent cells were fixed in 2% PFA for 45 min at 37 °C, permeabilized with 0.2% Triton X-100 and stained with 10 ng/ml DAPI-PBS (pH 7.4) for counting. 4.14. Inhibitor assays 5 × 104 cells were seeded on glass cover slips treated with serum as described above. After 24 h, ML7 or Y27632 were added to the cell culture medium for 1 h and cells were lysed in 1× Laemmli buffer and processed for western blot as described above. 4.15. Migration assays Cells were plated at high density in a 100 mm dish and before they reached conflurence 2 mM hydroxyurea (Sigma) was added to stop cell proliferation. After 48 h, a scratch was made with razor blade. After 48 h, cells were fixed in 2% PFA, permeabilized with 0.2% Triton X-100 and stained with 10 ng/ml DAPI-PBS (pH 7.4). The relative migratory capacity was established by comparing the percentage of cells having migrated accros the scratch in a define area over the number of cells behind the wound border in the same area. Supplementary materials related to this article can be found online at doi: 10.1016/j.matbio.2011.03.002. Acknowledgements The authors would like to thank Dr. Su Hao Lo (Center for Tissue Regeneration and Repair, Department of Biochemistry and Molecular Medicine, University of California, Sacramento, CA) for the kind gift of tensin1 antibody and Dr. Stephen K. Akiyama, (National Institutes of Environmental Health Sciences-NIH, Research Triangle Park, NC) for the kind gift of integrin β1 antibody. We also thank Dominique Jean for virus production and Elizabeth Herring for reviewing the manuscript and technical support. Electron microscopy was performed at the EM Facility of the Faculty of Medicine and Health Sciences and the Centre de caractérisation des matériaux of the Université de Sherbrooke. This work was supported by a Canadian Institutes of Health Research Grant MOP 57727. JFB is the recipient of the Canada Research Chair in Intestinal Physiopathology. JFB is a member of the FRSQ-funded Centre de Recherche Clinique Étienne Le Bel of the Centre Hospitalier Universitaire de Sherbrooke. References Akiyama, S.K., Yamada, S.S., Chen, W.T., Yamada, K.M., 1989. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J. Cell Biol. 109, 863–875. Andresen, J.L., Ledet, T., Hager, H., Josephsen, K., Ehlers, N., 2000. The influence of corneal stromal matrix proteins on the migration of human corneal fibroblasts. Exp. Eye Res. 71, 33–43. Aumailley, M., Mann, K., von der Mark, H., Timpl, R., 1989. Cell attachment properties of collagen type VI and Arg–Gly–Asp dependent binding to its alpha 2(VI) and alpha 3 (VI) chains. Exp. Cell Res. 181, 463–474. Basora, N., Herring-Gillam, F.E., Boudreau, F., Perreault, N., Pageot, L.P., Simoneau, M., Bouatrouss, Y., Beaulieu, J.F., 1999. Expression of functionally distinct variants of the beta(4)A integrin subunit in relation to the differentiation state in human intestinal cells. J. Biol. Chem. 274, 29819–29825.

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