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Genes to Cells (2009) 14, 835-850. doi:10.1111/j.1365-2443.2009.01315.x
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Intracellular polarity protein PAR-1 regulates extracellular laminin assembly by regulating the dystroglycan complex

 Maki Masuda-Hirata1,b, Atsushi Suzuki1,b,*, Yoshiko Amano1, Kazunari Yamashita1, Mariko Ide1, Tomoyuki Yamanaka1,a, Michihiro Sakai2, Michihiro Imamura3 and Shigeo Ohno1,*

1 Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
2 Department of Anesthesiology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
3 Department of Molecular Therapy, National Institute of Neuroscience, NCNP, 4-1-1 Ogawahigashi-cho, Kodaira, Tokyo 187-8502, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Cell polarity depends on extrinsic spatial cues and intrinsic polarity proteins including PAR-aPKC proteins. In mammalian epithelial cells, cell–cell contacts provide spatial cues that activate the aPKC-PAR-3-PAR-6 complex to establish the landmark of the initial cellular asymmetry. PAR-1, a downstream target of the aPKC-PAR-3-PAR-6 complex, mediates further development of the apical and basolateral membrane domains. However, the relationships between the PAR-aPKC proteins and other extrinsic spatial cues provided by the extracellular matrix (ECM) remain unclear. Here, we show that PAR-1 colocalizes with laminin receptors and is required for the assembly of extracellular laminin on the basal surface of epithelial cells. Furthermore, PAR-1 regulates the basolateral localization of the dystroglycan (DG) complex, one of the laminin receptors essential for basement membrane formation. We also show that PAR-1 interacts with the DG complex and is required for the formation of a functional DG complex. These results reveal the presence of a novel inside-out pathway in which an intracellular polarity protein regulates the ECM organization required for epithelial cell polarity and tissue morphogenesis.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Many biological processes observed in tissue morphogenesis include asymmetric cell division, cell movement and changes in cell shape, all of which rely on the dynamic regulation of the polarity of individual cells. The initial development of cell polarity is elicited by localized polarity cues provided in a cell-autonomous manner or by extrinsic signals, which lead to the asymmetric organization of cytoskeletons, junctional structures and plasma membranes (Yeaman et al. 1999). Accumulating evidence has revealed the presence of several groups of evolutionarily conserved proteins that ubiquitously play indispensable roles in converting the localized cues into the establishment of global cell polarity (Nelson 2003).

One of the pivotal sets of polarity proteins is the PAR-aPKC system comprised of two output devices, namely the aPKC-PAR-3-PAR-6 complex and PAR-1 (Suzuki & Ohno 2006). In response to sperm entry, these devices transiently localize to the cortex of the Caenorhabditis elegans one-cell embryo in a mutually exclusive manner and play essential roles in the segregation of cell fate determinants required for the subsequent asymmetric division. The aPKC-PAR-3-PAR-6 complex and PAR-1 also play roles in the establishment of epithelial apicobasal polarity (Knust & Bossinger 2002; Suzuki et al. 2004). In mammalian epithelial cells, cell–cell contacts of neighboring cells trigger the development of the apical junctional complexes and asymmetric membrane domains required for overall cellular asymmetry. Initially, the aPKC-PAR-3-PAR-6 complex is recruited to cell–cell contact regions and plays a role as a landmark in converting spatial cues into epithelia-specific junction formation and apical domain development (Wang & Margolis 2007). Other sets of polarity complexes, the Crumbs complex and the Lethal giant larvae (Lgl) complex, cooperate with the aPKC-PAR-3-PAR-6 complex to regulate the process positively and negatively, respectively (Yamanaka & Ohno 2008). The serine/threonine kinase PAR-1 is excluded from the apical membrane by the aPKC-PAR-3-PAR-6 complex and plays roles in the further development of the apical and basolateral membrane domains (Suzuki et al. 2004).

Besides cell–cell contacts, cell–extracellular matrix (ECM) interactions provide other spatial cues required for epithelial cell polarity (Ekblom 1989). For example, cultured Madin Darby canine kidney (MDCK) epithelial cells polarize and develop an apical membrane domain, albeit partially, even in the absence of cell–cell interactions (Vega-Salas et al. 1987). Recent studies have further demonstrated that laminin assembly on the basal cell surfaces is essential for the de novo apical domain formation directed by the ECM (O’Brien et al. 2002). Laminins are major components of the basement membrane (BM), a sheet of specialized ECM underlying a variety of tissues such as epithelia, epidermis and muscle, and play critical roles during the morphogenesis, tissue organization and integrity of muscle in vivo (Li et al. 2003). Although the most intensively studied receptors for laminins are integrins, another essential laminin receptor, dystroglycan (DG), forms a large multiple complex containing a spectrin-like actin-binding protein, dystrophin/utrophin (Winder 1997). The DG complex was originally identified in studies on muscular dystrophy (Ervasti & Campbell 1991) and subsequently shown to play important roles in the cell-ECM interactions of not only muscle cells but also epithelial cells (Barresi & Campbell 2006). DG is indispensable for BM formation in several tissues, including epithelia, and is involved in epithelial cell polarity (Durbeej et al. 1995; Michele et al. 2002). DG-null mice die during the early embryonic days owing to failure of extra-embryonic BM formation (Williamson et al. 1997). Conditional ablation of the DG gene in mammary epithelial cells abolishes laminin binding to the cell surface and disrupts polarity development (Weir et al. 2006). Hence, DG plays critical roles in ECM organization and epithelial polarity through the assembly of extracellular laminin. However, the mechanism for how the DG complex is regulated to organize extracellular laminin assembly remains completely unknown.

It has been demonstrated that when single MDCK cells are embedded in type I collagen gels or when two-dimensionally cultured MDCK cells are overlaid with collagen gels, the cells reorganize their polarity to generate a de novo apical free surface at a site as far apart as possible from the cell-ECM contact site (Wang et al. 1990; Schwimmer & Ojakian 1995). Interestingly, a previous study demonstrated that suppression or depletion of PAR-1b inhibits this ECM-directed apical domain reconstruction induced by collagen overlay (Cohen et al. 2004). These observations suggest the involvement of PAR-1b in the ECM-directed regulation of epithelial cell polarity, although the underlying molecular mechanism remains unclear. Here, we extend these observations and show that PAR-1b is involved in extracellular laminin organization, thereby regulating the ECM-directed dynamic remodeling of epithelial polarity. We also provide evidence for the involvement of the DG complex in this PAR-1-mediated laminin assembly. Based on these results, we propose the presence of a novel inside-out pathway, in which the intracellular polarity protein PAR-1b organizes extrinsic polarity cues and thereby coordinates the polarity axis of individual cells to construct the overall tissue architectures in multicellular organisms.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Basolateral colocalization of laminin receptors and PAR-1b in MDCK cells

Previous studies demonstrated that extracellular laminin assembly is crucial for ECM-directed regulation of epithelial cell polarity (O’Brien et al. 2001; Yu et al. 2005). Therefore, we examined a possibility that PAR-1b affects the apical domain reconstruction induced by collagen overlay by regulating extracellular laminin accumulation (Cohen et al. 2004). As a first step to assess this possibility, we analyzed subcellular localizations of PAR-1b, laminin receptors and laminin assembly in MDCK cells.

DG and integrins containing the β1 subunit represent the two major laminin receptors required for BM assembly and epithelial polarity in vitro and in vivo (Ojakian & Schwimmer 1994; Schoenenberger et al. 1994; Ekblom et al. 1998; Li et al. 2003). DG is composed of two non-covalently associated subunits, namely an extracellular {alpha}-DG and a transmembrane β-DG (Fig. 1A), which are post-translationally cleaved from a single precursor protein (Barresi & Campbell 2006). {alpha}-DG is heavily glycosylated with the unique O-mannosyl oligosaccharide and responsible for laminin binding (Barresi & Campbell 2006), while the cytoplasmic tail of β-DG binds to utrophin or dystrophin, the large spectrin-like molecules that interact with a number of proteins including F-actin (Fig. 1A) (Winder 1997). Although basolateral localizations of utrophin and syntrophin have already been reported in MDCK monolayers (Kachinsky et al. 1999), the detailed nature of the DG complex in epithelial cells remains to be clarified.


Figure 1
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  		Figure 1  Localization of laminin receptors and PAR-1b in MDCK cells. (A) Schematic illustration of the DG complex. Utrophin-binding proteins other than syntrophin and actin are omitted for simplification. (B, C) MDCK 3D cysts grown in collagen gels for 8 (B) or 6 (C) days were immunostained for the indicated proteins. Midline confocal sections of each cyst are presented. (D, E) Polarized MDCK cells grown on filter supports for 3 days were stained for the indicated proteins. Z-sectional views are presented. The arrowheads in (E) indicate the apical tip of the lateral membrane where E-cadherin, but not utrophin and PAR-1b, is concentrated. Scale bars: 20 µm.

 
In highly polarized MDCK cysts grown in type I collagen gels, β-DG and utrophin were clearly localized to the basal membrane and invaded into the lateral membrane as observed for β1-integrin in vitro and in vivo (Fig. 1B,C) (Ojakian & Schwimmer 1994). The distribution patterns of these laminin receptors were in sharp contrast to that of E-cadherin, which was excluded from the basal membrane and tended to concentrate to the apical regions of the lateral membrane (Fig. 1C). Interestingly, PAR-1b showed similar localization with the laminin receptors but not E-cadherin in three-dimensional cysts: PAR-1b was dominantly distributed to the basal membrane and only weakly leaked into the lateral membrane, making a sharp contrast to E-cadherin (Fig. 2C, bottom panels).


Figure 2
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  		Figure 2  Laminin assembly on the MDCK cell surface. (A) A midline confocal section of 8-day MDCK cysts doubly stained for E-cadherin and extracellular laminin. (B) Z-sectional views of filter-grown MDCK cells immunostained for the indicated proteins. The arrows with roman numerals I, II and III indicate the focal planes at 0, 0.8 and 3.6 µm from the basal membrane, respectively. (C) Single xy confocal sections of filter-grown MDCK cells taken at three focus planes (I, II and III) corresponding to those indicated in (B). (D) Effects of DG knockdown on laminin assembly on filter-grown MDCK cells. The indicated heterogeneous RNAi stable cells were polarized on filter supports for 3 days. An independent shRNAi sequence for DG knockdown gave similar results (Supporting Fig. S1; data not shown). Scale bars: 20 µm.

 
Colocalization of PAR-1b and the laminin receptors were also confirmed in filter-grown monolayers (Fig. 1D,E; Fig. 5B). In this culture system, the signals of PAR-1b and the laminin receptors at the basal membrane were significantly weaker than those on the lateral membrane probably due to their contact with a non-physiological substrate. However, their reciprocal relationship with E-cadherin distribution was maintained. E-cadherin was free from the basal membrane and the basal side of the lateral membrane where the DG complex, β1-integrin and PAR-1b were mainly localized, while the DG complex, β1-integrin and PAR-1b were excluded from apical junctional regions to which E-cadherin was concentrated (Fig. 1E, white arrows, data not shown). These results suggest a possible functional link between PAR-1b and laminin assembly.

The DG complex is required for laminin assembly on the MDCK cell surface

Next, we examined laminin assembly on the MDCK cell surface. Previous studies have shown that MDCK cysts secrete laminin species containing β1 and {gamma}1 chains (probably laminin-10) and assemble them around the cyst periphery (O’Brien et al. 2001; Yu et al. 2005). We confirmed these observations (Fig. 2A), although the intensity of the laminin varied significantly depending on several uncontrollable factors intrinsic to this assay system staining gel-embedded cysts. In contrast, detection of laminin assembly on filter-grown monolayers gave us reproducible signals. In this case, laminin staining was detected not only on the basal surface (Fig. 2B,C, see focal plane III) but also in the basal regions of intercellular spaces (Fig. 2B,C, see focal plane II). As a result, a honeycomb pattern of laminin signals was observed in xy views slightly above the basal membrane (Fig. 2C, see focal plane II). This lateral invasion of laminin assembly appeared to result from the characteristic distributions of the laminin receptors in filter-grown monolayers being excluded from the basal membrane. In fact, RNAi depletion of DG or utrophin impaired laminin assembly at not only the basal membrane (focal plane III) but also the basal tip of the lateral membrane (focal plane II) (Fig. 2D; Supporting Fig. S1; data not shown). It is also noteworthy that the lateral invasion of laminin assembly was restricted to the regions where laminin receptors but not E-cadherin was localized (Fig. 2B,C). These observations may indicate that E-cadherin directly or indirectly inhibited the activities of the laminin receptors in most areas of the lateral membrane. In any case, these results not only confirm the role of DG in laminin assembly in MDCK cells but also indicate that filter-grown monolayers can be used to quantitatively analyze the effects of PAR-1b depletion on laminin assembly.

PAR-1b is required for extracellular laminin assembly on the MDCK cell surface

Previously, we and others demonstrated that MDCK cells lacking PAR-1b expression exhibit normal development of tight junction and asymmetric localization of aPKC and PAR-3 but show reduced lateral membrane extension and apical domain development (Cohen et al. 2004; Suzuki et al. 2004). Here, we examined whether PAR-1b-depeleted cells show additional defects in extracellular laminin assembly. For this purpose, we transiently transfected MDCK cells with a plasmid vector simultaneously expressing EGFP and a shRNA for PAR-1b. Under these conditions, PAR-1b expression was efficiently and specifically abolished in EGFP-expressing cells (Fig. 3A). Subsequently, we found that laminin signals on the basal surface (Fig. 3B, focal plane III) as well as the basal tip of the lateral membrane (Fig. 3B, > focal plane II) were severely compromised in the EGFP-positive cells. Quantitative analyses utilizing the honeycomb pattern of laminin signals clearly confirmed this effect of PAR-1b knockdown (Fig. 3C, + mock). This defect in laminin assembly was restored by cotransfecting RNAi-resistant wild-type PAR-1b, but not its kinase-negative mutant (kn) (Fig. 3B,C), indicating that the defect was specifically caused by the lack of PAR-1b kinase activity.


Figure 3
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  		Figure 3  PAR-1b is required for laminin assembly on the MDCK cell surface. (A) MDCK cells transfected with a plasmid vector simultaneously expressing EGFP and a PAR-1b shRNA were examined for PAR-1b expression. (B) Laminin assembly on PAR-1b knockdown cells. Cells were transfected with plasmid vectors for the indicated RNAi with or without an expression vector encoding RNAi-resistant T7-PAR-1b (wild-type or kinase knockout mutant (T208A)). Confocal images within 4 µm above focal plane II are projected (> focal plane II). For laminin staining, the signals on the basal membrane (focal plane III) are also presented. Note that the laminin assembly is suppressed in PAR-1b knockdown cells, and significantly restored by T7-PAR-1b expression (cyan) in a kinase activity-dependent manner (white arrows). (C) Quantification of the results in (B). Based on the laminin signals collected within 4 µm above focal plane II, the laminin assemblies on GFP-positive cells were categorized into three types as indicated, and analyzed statistically in each experiment (n > 100; GFP and anti-tag double-positive cells were analyzed when RNAi-resistant T7-PAR-1b was coexpressed). Bars represent the means ± SD of three independent experiments. *P < 0.05, **P < 0.01. (D) MDCK cells were subjected to Lgl-2 RNAi as described in (A) and examined for laminin assembly and PAR-1b localization. (E) Quantification of the results for laminin assembly in (D). **P < 0.01. Scale bars: 20 µm.

 
We previously demonstrated that knockdown of Lgl, another cell polarity protein that localizes to the basolateral membrane, also suppresses the apical domain reorganization induced by collagen overlay (Yamanaka et al. 2006). Hence, we examined whether Lgl knockdown also affected laminin assembly. As reported previously (Yamanaka et al. 2006), depletion of one of the two mammalian Lgl species, Lgl-2, was sufficient to suppress the collagen overlay-induced apical domain reorganization in MDCK cells (Supporting Fig. S2). Nevertheless, Lgl-2 knockdown did not reduce the extracellular laminin signals (Fig. 3D,E; Supporting Fig. S2). We also confirmed that a stable MDCK clone lacking both Lgl-1 and Lgl-2 (Yamanaka et al. 2006) exhibited normal laminin assembly on the basal surface (Supporting Fig. S3). These results clearly indicate that, despite the similar basolateral localizations and involvement in ECM-directed apical domain reorganization, PAR-1b but not Lgl plays an essential role for extracellular laminin assembly. In contrast to the results in the Drosophila oocyte (Tian & Deng 2008), the basolateral localization of PAR-1b was normally observed not only in Lgl-2 knockdown cells but also Lgl-1/Lgl-2 double-knockdown cells (Fig. 3D; Supporting Fig. S3). Taken together, we conclude that PAR-1b and its kinase activity are required for promoting extracellular laminin assembly independently of Lgl.

PAR-1b knockdown does not alter laminin secretion and synthesis

Previous studies have shown that laminin secretion interfered with microtubule (MT) destabilization (Boll et al. 1991). Therefore, we next examine the possibility that PAR-1b affects laminin synthesis and/or secretion through its activity to regulate MT stability (Drewes et al. 1997; Cohen et al. 2004). For this purpose, we established two independent MDCK clones (Q3 and B2) that stably expressed independent shRNAs for PAR-1b knockdown (Fig. 4A). Next, we analyzed the amounts of soluble laminin deposited by confluent monolayers for 3 days. Immunoprecipitation and immunoblotting using an anti-laminin-1 antibody (O’Brien et al. 2001; Yu et al. 2005) revealed that PAR-1b knockdown did not reduce, but rather upregulated, the amounts of the β1/{gamma}1 chains in both apical and basal media (Fig. 4B). Furthermore, analyses of total extracts of the corresponding cells revealed that the amounts of intracellular laminins, which migrate faster than soluble laminins (O’Brien et al. 2001; Yu et al. 2005), were not significantly altered in the PAR-1b knockdown cells (Fig. 4A). These results suggest that the reduced laminin assembly observed in PAR-1b knockdown cells was not due to decreased laminin synthesis and/or secretion.


Figure 4
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  		Figure 4  PAR-1b knockdown does not reduce laminin synthesis and secretion. (A) PAR-1b RNAi stable clones (Q3 and B2) and the corresponding control clones (NG #1 and #2, respectively) were polarized on filter supports. At 3 days after the cells reached confluency, their total extracts were subjected to western blot analysis using antibodies against the indicated proteins. In lane 1, immunoprecipitated laminin from the medium (soluble laminin) was loaded for comparison. (B) Soluble laminins deposited in the culture media were examined for the same cells analyzed in (A). The apical and basal culture media were collected separately, and subjected to immunoprecipitation and immunoblotting with an anti-laminin-1 antibody.

 
PAR-1b is required for the membrane localization of the DG complex

The above results are consistent with the fact that reduced laminin assembly induced by PAR-1b knockdown was restricted to cells lacking PAR-1b expression (Fig. 3B). Together with the finding that PAR-1b colocalizes with laminin receptors, these results raise the possibility that PAR-1b affects the cell surface attachment of secreted laminin by regulating the localization or activity of laminin receptors. To examine this, we investigated the effects of PAR-1b knockdown on the laminin receptor distributions (Fig. 5). In these experiments, we frequently used utrophin as a marker for DG localization, since DG and utrophin were mutually dependent in their membrane localizations (Supporting Fig. S4). Despite of reduced cell height, PAR-1b knockdown cells showed clear localization of E-cadherin and β1-integrin at the middle and basal tip of the lateral membrane, respectively (Fig. 5A,B). In contrast, the basolateral localization of the DG complex was almost completely abolished in these cells (Fig. 5A,B). These effects of PAR-1b knockdown were rescued by overexpressing RNAi-resistant PAR-1b (data not shown), indicating that the observed effects were attributable to reduced expression of PAR-1b. Conversely, DG or utrophin RNAi did not affect the basolateral localization of PAR-1b (Supporting Fig. S5), indicating that the DG complex did not act as an essential membrane anchor for PAR-1b. These results suggest that PAR-1b resides upstream of the DG complex and specifically regulates its localization or expression. We confirmed a similar relationship between the localizations of the DG complex and PAR-1b in 3D cysts (Supporting Fig. S5).


Figure 5
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  		Figure 5  PAR-1b is required for the membrane localization of the DG complex in polarized MDCK cells. (A, B) The indicated heterogeneous RNAi stable cells were examined for the basolateral localizations of PAR-1b, utrophin and E-cadherin or β1-integrin (A or B, respectively). Single confocal images at 2 µm (A) or 0.8 µm (B) upward from the basal membrane (arrowheads) are presented together with z-sectional views. (C) Ectopic expression of PAR-1b enhances the lateral accumulation of the DG complex in MDCK cells. Heterogeneous stable transformants expressing wild-type (wt) T7-PAR-1b or its kinase-negative mutant (kn: T208A) were subjected to immunofluorescence analysis. Single confocal sections at the indicated focal planes defined in Fig. 2B are presented. Scale bars: 20 µm. (D) Quantitative data for the fluorescence intensities at focal plane I along the white lines in (C).

 
The role of PAR-1b in regulating the DG complex was further supported by overexpression experiments. When wild-type PAR-1b was overexpressed, utrophin and β-dystroglycan, but not β1-integrin were enriched on the middle (focal plane I) and the basal tip (focal plane II) of the lateral membrane (Fig. 5C,D, data not shown). Since a kinase-negative mutant of PAR-1b did not exert similar effects, these results indicate that PAR-1b regulates the membrane localization of the DG complex through its kinase activity. Given the finding that the DG complex was indispensable for cell surface laminin assembly in MDCK cells (Fig. 2D), these results imply that PAR-1b regulates extracellular laminin assembly through its effects on the membrane accumulation of the DG complex.

PAR-1b is required for the formation of a functional DG complex

To investigate the mechanism for how PAR-1b regulates the DG complex, we subjected the PAR-1b knockdown MDCK cell lines to biochemical analyses. Western blot analyses of total extracts revealed that PAR-1b knockdown did not consistently alter the expression levels of utrophin and β-DG (Fig. 6A). However, the analysis of β-DG immunoprecipitates revealed that the amounts of utrophin coprecipitated with β-DG were reduced in a clone Q3 and to a lesser extent in a clone B2 that showed less efficiency in PAR-1b knockdown (Fig. 6B). The defect was partially rescued by overexpression of human PAR-1b in B2 clone, suggesting that PAR-1b is required for the stable interaction between utrophin and β-DG. Since utrophin and DG are mutually required for their membrane localizations (Supporting Fig. S4), the reduced utrophin-β-DG interactions may be the primary cause of the loss of the DG complex from the basolateral membrane of PAR-1b knockdown cells.


Figure 6
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  		Figure 6  PAR-1b RNAi impairs the formation of the functional DG complex. (A) Total cell extracts of the stable PAR-1b RNAi MDCK clones and their corresponding control clones were subjected to Western blotting analysis using the indicated antibodies. For {alpha}3-integrin, the cell extracts were prepared under non-reducing conditions to prevent cleavage of the intramolecular disulfide bonds of intact {alpha}3-integrin (150 kDa). In the extra lane indicated by the asterisk, a reduced extract was loaded for comparison. (B) β-DG was immunoprecipitated from 1% digitonin extracts of the indicated stable RNAi cells and the amounts of coprecipitated utrophin were examined. For clone B2, rescue experiments using adenovirally-introduced T7-tagged human PAR-1b were also performed (lacZ indicates the control experiments). (C) Altered processing of {alpha}-DG in PAR-1b knockdown cells. The indicated stable RNAi cells grown on filter supports were subjected to 0.1% NP40 extraction and anti-β-DG immunoprecipitation. {alpha}-DG in the immunoprecipitates was detected by an anti-{alpha}-DG antibody (VIA4-1) or a laminin overlay assay as indicated.

 
As mentioned above, {alpha}-DG, an extracellular component of the DG complex (Fig. 1A), is derived from a precursor protein common to β-DG. Therefore, although commercially available anti-{alpha}-DG antibodies (VIA4-1 and IIH6) failed to detect {alpha}-DG in total extracts of MDCK cells (data not shown), unchanged synthesis of {alpha}-DG was implicated in PAR-1b knockdown cells from the results on the unchanged expression of β-DG (Fig. 6A). However, further analysis of {alpha}-DG revealed an additional, unexpected defect of the DG complex in PAR-1b knockdown cells. Owing to the heavy glycosylation, {alpha}-DG exhibits various molecular masses of approximately 120–180 kDa in a cell type-dependent manner (Ibraghimov-Beskrovnaya et al. 1992). Consistently, we detected {alpha}-DG in β-DG immunoprecipitates from MDCK cells as two discrete bands with molecular masses of ~120 and ~180 kDa (Fig. 6C). To our surprise, the intensities of these {alpha}-DG bands were dramatically decreased in PAR-1b knockdown cells. Because the antibodies used recognize the O-mannosyl oligosaccharide moieties responsible for laminin binding (Ervasti & Campbell 1993), we cannot conclude from these results whether PAR-1b depletion caused disappearance of {alpha}-DG from the DG complex or it only reduced the O-mannosyl glycosylation of {alpha}-DG without affecting the presence in the complex. However, the above results clearly indicate that a substantial proportion of the DG complexes in PAR-1b knockdown cells lack functional {alpha}-DG capable of binding to laminin. Consistently, blot overlay assays using purified laminin-1 revealed that β-DG immunoprecipitates from control but not PAR-1b knockdown cells exhibited laminin binding at the predicted molecular mass of {alpha}-DG (Fig. 6C, middle panels). {alpha}3- and β1-integrins, the other major laminin receptors in MDCK cells (Ojakian & Schwimmer 1994; Yu et al. 2005), did not show any changes in their expressions and mobilities in SDS-PAGE analyses of PAR-1b knockdown cells (Fig. 6A). Furthermore, overexpression of human PAR-1b restored the VIA4-1 immunoreactivity in the B2 clone (Fig. 6C). Taken together, these results indicate that PAR-1b is specifically required for the formation of a functional DG complex containing utrophin and normally glycosylated {alpha}-DG.

PAR-1b specifically interacts with the DG complex

Interestingly, when MDCK cell lysate was subjected to PAR-1b immunoprecipitation, the components of the DG complex, utrophin, β-DG and syntrophin, but not β1-integrins, were coprecipitated with an anti-PAR-1b antibody (Fig. 7A; data not shown). Since the coprecipitation of the DG complex was not observed in PAR-1b knockdown cells, these results suggest the specific interaction between PAR-1b and the DG complex. The interaction was further confirmed by reciprocal experiments: endogenous PAR-1b, but not other polarity proteins such as PAR-3 and Lgl-2, was coprecipitated with utrophin (Fig. 7B). It should be noted that the utrophin immunoprecipitates did not contain actin, indicating that F-actin is not a mediator of the interaction between PAR-1b and the DG complex. The complexity of the DG complex still hampers us to completely reveal the relationship between the interaction between PAR-1b and the DG complex and the defects of the DG complex observed in PAR-1b knockdown cells (see Discussion). However, together with the results that PAR-1b specifically colocalizes with the DG complex, the above results support the notion that PAR-1b regulates the DG complex through direct binding and/or phosphorylation.


Figure 7
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  		Figure 7  PAR-1b specifically interacts with the DG complex in MDCK cells. (A) The indicated RNAi stable MDCK clones were subjected to immunoprecipitation with an anti-PAR-1b antibody. The resultant immunoprecipitates were analyzed for several components of laminin receptors. The asterisk indicates IgG heavy chain in the immunoprecipitate. (B) MDCK cells were subjected to immunoprecipitation with an anti-utrophin antibody. The resultant immunoprecipitates were analyzed for several polarity proteins and actin. Cont. IgG indicates a negative control experiment using normal rabbit IgG for immunoprecipitation (see bottom panel: IgG HC).

 
PAR-1b plays an essential role in ECM-directed apical pole reorganization by regulating extracellular laminin accumulation

Finally, we examine the hypothesis that PAR-1b affects the ECM-induced apical domain remodeling by regulating extracellular laminin accumulation via the DG complex. When two-dimensionally cultured MDCK cells were overlaid with type I collagen gels, cells retracted the original apical domain and reconstruct it at intercellular regions (Fig. 8A) (Schwimmer & Ojakian 1995). In contrast, consistent with the previous results, cells lacking PAR-1b expression showed defects in intercellular lumen formation upon collagen overlay (Fig. 8B, see top panels) (Cohen et al. 2004). As expected, control cells, but not PAR-1b knockdown cells, exhibited extracellular laminin accumulation at new ECM-cell contact sites (Fig. 8C). Furthermore, utrophin, but not E-cadherin, was concentrated beneath new ECM-cell contact sites where laminin was accumulated (Fig. 8C, see arrowheads in cont. RNAi), suggesting that the DG complex is directly involved in the de novo laminin accumulation. We also found that utrophin and DG knockdown cells lost the ability to reconstruct apical domain in response to collagen overlay (Fig. 8B, top panels). Taken together, these results support the notion that loss of extracellular laminin accumulation via the DG complex is the cause of the defective response of PAR-1b knockdown cells to collagen overlay.


Figure 8
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  		Figure 8  PAR-1b plays an essential role in ECM-directed apical pole reorganization by regulating extracellular laminin assembly. (A) MDCK cells were cultured with or without collagen overlay for 48 h and labeled for the indicated proteins (nuclear DNA was stained with TOPRO). Confocal z-sectional views are presented. The arrowheads indicate intercellular lumens labeled for an apical marker protein, gp135. (B) Indicated heterogeneous RNAi stable MDCK cells were overlaid with type I collagen gels supplemented with or without exogenous laminin-1, and stained for the indicated proteins. The formation of intercellular lumen (arrowheads) was identified by conventional microscopy. (C) Indicated heterogeneous RNAi stable MDCK cells were overlaid with type I collagen gels, and labeled for E-cadherin, utrophin and extracellular lmainin. The arrowheads indicate de novo laminin assembly and the corresponding utrophin accumulation observed at new ECM-cell interfaces. (D) The rescue effects of exogenous laminin supplementation shown in (B) were quantified by counting the numbers of intercellular lumens per cell in the indicated RNAi cells (n > 90). Bars represent the means ± SD of three independent experiments. Scale bars: 20 µm.

 
To further confirm the hypothesis, we examined the effect of supplementing the overlaid collagen gel with exogenous laminin. Inclusion of a high concentration of EHS-laminin (laminin-1) in the overlaid collagen gel dramatically rescued the defects in apical domain remodeling observed in PAR-1b, utrophin and DG knockdown cells (Fig. 8B, bottom panels). In all cases, the knockdown cells regained the ability to respond to the collagen overlay by inducing intercellular lumens to a comparable level to control cells (Fig. 8D). In contrast, inclusion of collagen type IV, another component of BMs, in the overlaid type I collagen gel did not show any rescue effects on the RNAi cells. It was shown that DG serves as an essential receptor for the initial binding of laminin and concentrates it on the cell surface, while β1 integrin and perlecan mediate the subsequent polymerization of the concentrated laminin (Henry et al. 2001). Therefore, the above results are consistent with the notion that the presence of excess amount of extracellular laminin enabled cells to skip the DG complex-dependent initial step for laminin accumulation. Taken together, the present results demonstrate a novel PAR-1b function in regulating apical pole orientation by controlling extracellular laminin accumulation through the DG complex.


   Discussion
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 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The BM, a sheet of specialized ECM underlying all epithelial cells, has been implicated in epithelial polarity (Ekblom 1989; Li et al. 2003). However, the functional links between BMs and the general cell polarity machinery, the PAR-aPKC system, have been completely unknown. In the present study, we have demonstrated the specific colocalization of PAR-1b with laminin receptors in cultured epithelial cells. Furthermore, we have provided evidence that PAR-1b plays an indispensable role in the cell surface assembly of laminins by regulating one of the laminin receptors, the DG complex. The following observations support this notion. First, knockdown of PAR1b but not Lgl impaired the cell surface assembly of laminins. Since the introduction of wild-type PAR-1b but not its kinase-deficient mutant rescued this defect, the PAR-1b kinase activity appears to be essential for laminin assembly. Second, PAR-1b depletion did not affect the synthesis and secretion of laminin, suggesting that PAR-1b affects laminin after its secretion. Third, PAR-1b depletion interfered with the membrane localization but not the synthesis of the components of the DG complex. Fourth, PAR-1b depletion affected the integrity of the functional DG complex. Fifth, depletion of either DG subunit caused similar defects in extracellular laminin assembly in MDCK cells, as expected from previous observations (Williamson et al. 1997; Weir et al. 2006). Finally, PAR-1b physically associated with the DG complex. Based on these observations, we can draw a very simple picture, in which PAR-1b regulates the localization and function of the DG complex and thereby regulates extracellular laminin assembly (Fig. 9).


Figure 9
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  		Figure 9  Model of the PAR-1b function in the regulation of the cell surface laminin assembly. PAR-1b plays indispensable roles for extracellular laminin assembly through regulation of the localization and function of the DG complex. PAR-1b, Lgl and Rac1 regulate apical domain remodeling independently, although the precise molecular mechanism for each process remains to be clarified.

 
The PAR-aPKC system has been shown to regulate the intrinsic polarity of individual cells in response to external cues provided by cell–cell contacts (Suzuki & Ohno 2006; Wang & Margolis 2007). The present study provides a novel inside-out pathway by which the essential component of the PAR-aPKC system, PAR-1, actively regulates an extrinsic polarity cue, extracellular laminin. Considering the essential role of the BM in coordinating cell polarity with tissue architecture (O’Brien et al. 2001, 2002), the present results imply that individual cells within epithelial tissues do not passively conform but actively organize external cues to dynamically couple cell polarity with the overriding tissue architectures. In the present study, we have obtained evidence to support this idea by demonstrating that supplementation with ectopic laminin could restore the defect of PAR-1b knockdown cells to dynamically reorganize the apical domain in response to collagen overlay. PAR-1 plays indispensable roles in gastrulation movement in Xenopus and vulva morphogenesis in C. elegans, in which the critical importance of cell-ECM interactions has been suggested (Hurd & Kemphues 2003; Kusakabe & Nishida 2004). Therefore, it is plausible that the novel function of PAR-1b to regulate laminin assembly plays important roles during the tissue morphogenesis of multicellular organisms.

In previous studies, overexpression of a dominant-negative mutant of Rac1 (Rac1DN) was found to result in aberrant accumulation of extracellular laminin and defects in ECM-induced apical domain reorganization (O’Brien et al. 2002; Yu et al. 2005). However, Rac1DN-expressing MDCK cells did not show loss of {alpha}-DG signals in the DG complex or a reduction in the utrophin-β-DG interactions observed in PAR-1b knockdown cells (Supporting Fig. S6). Furthermore, Rac1DN-expressing cells cultured on filter supports did not lose their laminin-binding ability, but instead exhibited abnormal laminin aggregation on their basal surface (Supporting Fig. S6). These findings indicate that the PAR-1b-DG pathway identified in the present study and the Rac1 pathway have independent effects on extracellular laminin accumulation. Together with the results that Rac1DN-expressing cells exhibited reduced F-actin staining (Supporting Fig. S6), the present results may indicate that Rac1 function downstream of PAR-1b by affecting the integrin-dependent rearrangement of cell-associated laminin into a polygonal network through the regulation of the submembranous F-actin structures (Colognato et al. 1999). In the present study, we also found that depletion of Lgl did not significantly affect the cell surface laminin assembly, despite of its suppressive effect on ECM-induced apical domain reorganization (Fig. 3D; Supporting Figs S2, S3). This finding is consistent with our previous study demonstrating that Lgl promotes apical domain reassembly by suppressing the aPKC-PAR-6-PAR-3 complex (Yamanaka et al. 2006). Taken together, the present results suggest that dynamic reorganization of epithelial polarity is achieved by coordination of multiple regulatory pathways in which cell polarity proteins are distinctly involved (Fig. 9).

We have demonstrated that PAR-1b specifically interacts with the DG-utrophin complex and plays essential roles for not only the stable interaction between utrophin-β-DG but also the maintenance of functional {alpha}-DG in the DG complex. Since we observed that utrophin is required for normal glycosylation of {alpha}-DG (A. Suzuki, unpublished results), the reduced interaction between utrophin and β-DG may be the primary cause of the reduced laminin accumulation in PAR-1b knockdown cells. The present results suggest the possibility that PAR-1b directly phosphorylates the components of the DG complex and thus regulates its normal maturation. However, the complicated nature of this multiprotein complex prevented us from demonstrating the direct targets for the novel function of PAR-1b in the present study. Utrophin is a large spectrin-like protein with a molecular mass of ~400 kDa and, compared with the DG-dystrophin complex, the biochemical natures of the utrophin-DG complex in non-muscle cells are still largely unknown (Haenggi & Fritschy 2006). In addition, accumulating evidence has continued to reveal the complexities of DG (Winder 2001), including its characteristic O-mannosyl glycosylation, an unusual type of protein modification in mammals (Grewal & Hewitt 2003). Previous studies have demonstrated that loss of PAR-1 results in altered organization of MT and F-actin in epithelial cells (Cohen et al. 2004; Suzuki et al. 2004). Therefore, at present, we cannot exclude the possibility that PAR-1b indirectly regulates the DG complex by regulating these cytoskeletal components. Further studies are required to completely clarify this issue (Suzuki et al., in preparation).

The components of the DG complex were originally identified in skeletal muscle during studies on muscular dystrophies (Ervasti & Campbell 1991). Notably, the defects in the DG-utrophin complex in PAR-1b knockdown cells are closely related to those observed in the DG-dystrophin complex in several muscular dystrophies. For example, loss of the interaction between dystrophin and DG is the primary cause of Duchenne muscular dystrophy. Hypoglycosylation of {alpha}-DG is a common mechanism for congenital muscular dystrophies, and is caused by defects in known or putative glycosyltransferases responsible for the O-mannosyl glycosylation of {alpha}-DG (Grewal & Hewitt 2003). The present results demonstrate for the first time that the post-translational processing of {alpha}-DG depends on a protein kinase. A recent study further reported that reduced glycosylation of {alpha}-DG is involved in carcinoma progression (Singh et al. 2004). Taken together, these results suggest that further extension of the present study, especially regarding the molecular mechanism underlying the PAR-1-dependent regulation of the DG complex, will open new avenues for studies of not only epithelial polarity but also carcinogenesis and muscular dystrophy.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Expression vectors

The human PAR-1b cDNA used in the present study corresponds to an abundant isoform registered in GenBank as human EMK1/MARK2 (accession number: NM_017490 [GenBank] ) (Suzuki et al. 2004). Various fragments of the PAR-1b cDNA were subcloned with 5' tag sequences (for T7- or HA-tag) into appropriate expression vectors, namely pEB6CAG, pOSTet14.4MCS and pEFHis (Suzuki et al. 2004). PAR-1b K82M and T208A were used as kinase-negative mutants. T7-tagged human PAR-1b, which is resistant to one of the RNAi sequences (Cohen et al. 2004) (see below) was generated by changing the target sequence as follows: gaggtagctgtcaaaatta (the underlined nucleotides were substituted).

Antibodies

The anti-PAR-1b polyclonal antibody (pAb), anti-Lgl2 pAb, anti-utrophin pAb (UT-2) and anti-β-DG pAb were described previously (Imamura et al. 2000; Suzuki et al. 2004). The anti-utrophin monoclonal antibody (mAb) (MANCHO-3) was kindly provided by Dr G.E. Morris (RJAH Orthopaedic Hospital, Centre for Inherited Neuromuscular Diseases, UK) and used for immunofluorescence microscopy. The anti-gp135 mouse mAb (3F2) was generously provided by Dr G. Ojakian (SUNY Health Science Center, NY). The other antibodies used in this study were: anti-{alpha}-DG mAbs (VIA4-1 and IIH6), anti-PAR-3 pAb, anti-Rac1 mAb (23A8) and anti-myc mAb (4A6) (Upstate Biotechnology); anti-syntrophin mAb (ABR-Affinity BioReagents); anti-β1-integrin pAb (AB1952) and anti-{alpha}3-integrin pAb (AB1920) (Chemicon); anti-laminin pAb (L9393), anti-β-actin mAb (AC-15) and anti-uvomorulin/E-cadherin rat mAb (Sigma); anti-β1-integrin rat mAb (A2B2; Hybridoma Bank); anti-T7-tag mAb (omni probe) (Santa Cruz Biotechnology); and anti-GAPDH mAb (6C5) (Abcam). For staining of F-actin and nuclei in cells, rhodamine-phalloidin and TOPRO (Molecular Probes) were used, respectively.

Cell culture, transfection and establishment of stable transformants

MDCK II cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin, 0.1 µg/mL streptomycin and 1 mM glutamine (Suzuki et al. 2001). In general, the cells were seeded on TranswellTM filters (Corning Costar) at 2.2 x 105 cells/cm2 and grown for 3 days to produce polarized monolayers. Plasmid transfection was performed using Lipofectamine 2000 (Invitrogen) or an electroporation system (Amaxa Inc.) according to the corresponding manufacturer's instructions. Heterogeneous stable transformants of MDCK cells were established using the autonomously replicative pEB6 or pOSTet14.4MCS vector as previously described (Suzuki et al. 2004). This method was used to stably express or knockdown appropriate proteins in MDCK cells. Briefly, MDCK cells (2.5 x 105 cells) were transfected with 1.6 µg of EBV-based expression vectors and reseeded the following day at one-fifth of the cell density. After 6 h, the cells were subjected to selection using DMEM containing 800 µg/mL geneticin (Gibco BRL) and the surviving cells were used in the following experiments without cloning. MDCK tet-off cell lines stably expressing myc-Rac1N17 under the control of tetracycline-repressive transactivation were a gift from Dr W. James Nelson (Stanford University, CA). The cells were routinely cultured in the presence of 20 ng/mL doxycycline (DC). Expression of Rac1N17 was induced by removing DC from the culture medium.

3D culture

MDCK cell cyst formation was performed as described previously (Yamanaka et al. 2006). Briefly, MDCK II cells were trypsinized and suspended in ice-cold DMEM containing 1.5 mg/mL calf skin type I collagen (KOKEN), 12 mM HEPES (pH 7.4) and 6% FBS. The resulting suspension was placed on Transwell filters. After formation of a gel at 37 °C, growth medium was added to the wells. The cells were incubated for several days to allow cyst formation.

Collagen gel overlay assays

Collagen gel overlay assays were performed as described previously (Ojakian et al. 1997). Briefly, MDCK II cells (2 x 104 cells/cm2) were seeded on coverslips coated with collagen I. On the following day, the cells were overlaid with a collagen I gel (1.8 mg/mL) containing DMEM/10% FBS and incubated for 48 h, before being subjected to immunofluorescence analysis. For rescue experiments, laminin-1 or collagen type IV purified from EHS tumor cells (Upstate Biotechnology or BD Bioscience, respectively) was added to the collagen gel at a final concentration of 250 or 150 µg/mL, respectively (O’Brien et al. 2001). In these experiments, the coverslips were coated with laminin-1 before coating with collagen I.

RNAi experiments

pEB-Super vectors containing appropriate hairpin oligonucleotide sequences in a tail-to-tail fashion were used to establish heterogeneous RNAi stable transformants (Suzuki et al. 2004). The RNAi sequences were as follows; utrophin, 5'-gaatatagaagaagactag-3'; DG, 5'-gggtattcctgatggcata-3' (#1) and 5'-gggtacagttcaacagcaa-3' (#4). PAR-1b, 5'-gaggtagctgtgaagatca-3' (Cohen et al. 2004); Lgl-2, 5'-GATGAGAGTTTCACACTGC-3' (Yamanaka et al. 2006). In some experiments, pEB-Super-gfp, which expresses EGFP simultaneously with a shRNA, was used to transiently knockdown PAR-1b or Lgl-2 without selection. To establish a stable RNAi MDCK clone for PAR-1b (Q3), the above PAR-1b RNAi sequence was subcloned into pSuper-neo. An independent RNAi sequence (5'-gaggagaggtgtttgacta-3') for PAR-1 RNAi which is ineffective against human PAR-1b was subcloned in pSuper-puro (Yamanaka et al. 2006), and used to establish another RNAi stable clone (B2). The negative control RNAi sequence used in the present study was described previously (Suzuki et al. 2004). Lgl-2 knockdown and Lgl-1/Lgl-2 double-knockdown stable clones (2–10 and 24–15, respectively) were described previously (Yamanaka et al. 2006).

Biochemical analyses of MDCK cells

For immunoprecipitation, cells were lysed in a lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 3 mM Na2VO5, 0.5 mM PMSF, 10 µg/mL leupeptin and 2 µg/mL aprotinin) supplemented with an appropriate detergent. After centrifugation, the supernatants were subjected to immunoprecipitation with antibody-conjugated protein G-Sepharose beads (Amersham). After extensive washing with the lysis buffer, immunocomplexes were solubilized in SDS sample buffer for subsequent analysis. Immunoprecipitation of laminin secreted by MDCK cells was performed as described previously (O’Brien et al. 2001). In this case, MDCK cells were cultured in serum-free medium (OPTIPRO SFM; Gibco) to eliminate bovine laminin contaminating the FBS. Laminin secreted into the medium for 3 days after the cells reached confluency was immunoprecipitated and subjected to SDS-PAGE using 3%–8% gradient gels, followed by immunoblotting. Laminin overlay assays were performed using EHS-laminin (KOKEN) as previously described (Michele et al. 2002). Western blotting analyses were performed using an ECL detection kit (Amersham), and chemiluminescent signals were captured by a Luminoimage analyzer (LAS3000; Fuji Film) for subsequent quantitative analyses.

Immunofluorescence microscopy

Cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS. To specifically detect extracellular laminin, filter-grown cells were incubated with medium containing an anti-laminin antibody for 4 h at 37 °C before fixation (the antibody was added to both apical and basal medium). When staining 3D cysts or collagen-overlaid cells, medium containing the anti-laminin antibody was overlaid on the collagen gels and allowed to penetrate into the gels. Cells overlaid with collagen gels were similarly treated after removing the gel with an aspirator (Ojakian et al. 1997), whereas 3D cysts in collagen gels were processed within the gels after digestion with collagenase VII (Sigma) before fixation. The cells were then subjected to standard immunostaining procedures for intracellular proteins as described previously (Suzuki et al. 2001). Most samples were analyzed using a laser confocal scanning microscope system (LSM 510; Carl Zeiss). To analyze 3D cysts, a microscope system equipped with a disc confocal scanner unit (CSU10; Yokogawa) and an Orca II CCD camera (Hamamatsu Photonics) were used. When quantifying the apical domain remodeling induced by collagen overlay, a conventional immunofluorescence microscope equipped with a cooled CCD camera (AxioImager; Carl Zeiss) was used.


   Acknowledgements
 
The authors are deeply grateful to F. Saitoh, K. Matsumura and M. Yoshida for helpful advice regarding {alpha}-DG detection and laminin overlay assays. This work was supported by Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to S.O. and A.S.), the Japanese Society for the Promotion of Science (to S.O., A.S. and M.M.) and the Ministry of Health, Labor and Welfare (to A.S.; 20B-1 for Nerve and Mental Disorders).


   Footnotes
 
Communicated by: Eisuke Nishida

aPresent address: Laboratory for Structural Neuropathology, RIKEN Brain Sciences Institute, 2-1 Hirosawa, Wako, Saitama 351-198, Japan. Back

bThe first two authors contributed equally to this work. Back

* Correspondence: ohnos{at}med.yokohama-cu.ac.jp or abell{at}med.yokohama-cu.ac.jp


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Received: 29 March 2009
Accepted: 14 April 2009





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