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Vol. 20, Issue 12, 2954-2962, June 15, 2009
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Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611
Submitted January 16, 2009;
Revised March 24, 2009;
Accepted April 17, 2009
Monitoring Editor: Jean E. Schwarzbauer
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6β4 integrin is a key regulator of epidermal attachment through its function as a surface receptor for laminin-332 (laminin-5) and by nucleating the formation of hemidesmosomes that adhere basal cells in stratified epithelia to the basement membrane (Jones et al., 1991
; Dowling et al., 1996; Borradori and Sonnenberg, 1999; Aumailley et al., 2005; Litjens et al., 2006). In addition to its well established role in adhesion, 6β4 integrin has been reported to regulate cell migration and tumor invasion (Rabinovitz and Mercurio, 1996, 1997; Mercurio et al., 2001; Dajee et al., 2003; Nikolopoulos et al., 2004; Nikolopoulos et al., 2005; Sehgal et al., 2006). Moreover, recent data indicate that β4 integrin modulates motility via Rac1 signaling to the Slingshot family of phosphatases which dephosphorylate and activate the actin-severing protein cofilin (Sehgal et al., 2006; Kligys et al., 2007).
6β4 integrin associates with two members of the plakin family of cytoskeletal linker proteins: plectin and bullous pemphigoid antigen 1e (BPAG1e, BP230; Jones et al., 1998; Borradori and Sonnenberg, 1999). This family consists of high-molecular-weight proteins that link cytoskeleton networks and tether cytoskeleton components to the cell surface (Leung et al., 2001). At the site of the hemidesmosome, plectin and BPAG1e proteins anchor the keratin cytoskeleton to the cell surface via 6β4 integrin (Jones et al., 1998; Borradori and Sonnenberg, 1999). Loss of either BPAG1e or plectin expression in keratinocytes results in the formation of blisters, due to disruption of basal cell/dermal adhesion (Guo et al., 1995; Andra et al., 1997). These data emphasize the importance of plakins in regulating adhesion in the skin. However, BPAG1e-null animals also display impaired wound healing in vivo (Guo et al., 1995). This result indirectly implicates BPAG1e as a potential regulator of keratinocyte migration. This would be consistent with an up-regulation in expression of BPAG1e in invasive squamous cell carcinoma (Herold-Mende et al., 2001). Thus, in our study, we tested the hypothesis that BPAG1e regulates migration by acting as a scaffold for β4 integrin–mediated signaling to Rac1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The cells were maintained in defined keratinocyte serum-free medium supplemented with a 1% penicillin/streptomycin mixture (Invitrogen, Carlsbad, CA) and grown at 37°C. Stable short hairpin RNA (shRNA) cell lines were generated using MISSION shRNA lentiviruses (Sigma Aldrich, St. Louis, MO) according to the manufacturer's protocol. Briefly, 5 x 105 HEKs were seeded overnight in six-well dishes and then infected with lentivirus encoding a BPAG1e-specific shRNA (AGTCTTGATTCAAATGAAA) at a multiplicity of infection of 0.5 in culture medium supplemented with Polybrene (8 µg/ml). The following day, the medium of the infected cells was aspirated and replaced with fresh medium containing puromycin (0.5 µg/ml) for selection of stable transfectants. Multiple individual clones were isolated and tested for expression of BPAG1e by SDS-PAGE/immunoblotting and by immunofluorescence.
Mouse monoclonal antibodies against β4 integrin (3E1), 3 integrin (P1B5), and Rac1 were purchased from Chemicon International (Temecula, CA). Rabbit monoclonal antibodies against plectin, actin, and paxillin were obtained from Epitomics (Burlingame, CA). Antibodies against tubulin, phospho-cofilin (Ser3), total cofilin, mTor, cytokeratin 5, β4 integrin, and the β3 laminin subunit were purchased from Novus Biologicals (San Jose, CA), Cell Signaling Technology (Beverly, MA), Abcam (Cambridge, MA), and BD Biosciences (San Jose, CA). 5E, a human mAb against BPAG1e, was a gift from Dr. Takashi Hashimoto (Keio University, Tokyo, Japan; (Hashimoto et al., 1993). The mouse IgM mAb preparation (1804b) against the N-terminal domain of BP180 was described previously, as were antibodies against 3 laminin (RG13) and 
2 laminin (3D5; Hopkinson et al., 1992; Goldfinger et al., 1998; Gonzales et al., 1999). Secondary antibodies conjugated with various fluorochromes or horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Adenoviruses expressing constitutively active forms of Rac1, RhoA and Cdc42 and Xenopus cofilin were generously provided by Dr. James Bamburg (University of Colorado at Boulder).
Immunoprecipitation and Western Blotting
Cells were extracted in 1% Triton X-100, 25 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2 2 mM NaF, and 100 µM Na3VO4 supplemented with a protease inhibitor mixture (Sigma-Aldrich). The extract was clarified by centrifugation, and either primary antibody or control IgG was added. After shaking overnight at 4°C, TrueBlot anti-mouse IgG-agarose beads (eBioscience, San Diego, CA) were added to the mix, which was incubated for 90 min at 4°C. The beads were washed, collected by centrifugation, and boiled in SDS-PAGE sample buffer.
Cell extracts and matrix preparations were prepared as described previously (Riddelle et al., 1991; Langhofer et al., 1993). Protein samples were processed for SDS-PAGE and immunoblotting as detailed elsewhere (Harlow and Lane, 1988; Riddelle et al., 1991). Western immunoblots were scanned and quantified using a MetaMorph Imaging System (Universal Imaging, Molecular Devices, Downingtown, PA).
Immunofluorescence Microscopy
Cells on glass coverslips were processed as detailed elsewhere (Sehgal et al., 2006). All preparations were viewed with a confocal laser scanning microscope (UV LSM 510, Zeiss, Thornwood, NY). Images were exported as TIFF files, and figures were prepared using Adobe Photoshop and Illustrator software (Adobe Systems, San Jose, CA).
Observations of Live Cells and Motility Assays
Single-cell motility was measured as detailed by us previously (Sehgal et al., 2006). Briefly, cells were plated onto uncoated 35-mm glass-bottomed culture dishes (MatTek, Ashland, MA) 18–24 h before cell motility assays. Cells were viewed on a Nikon TE2000 inverted microscope (Nikon, Melville, NY). Images were taken at 2 min intervals over 2 h, and cell motility behavior was analyzed by a MetaMorph Imaging System (Universal Imaging, Molecular Devices). Lamellipodia number was quantified by scoring the number of sheet-like extensions in phase-contrast images of live cells. Cell surface area was measured from the same phase-contrast images by tracing the outline of individual cells (including lamellipodia but ignoring filopodia/microspikes and retraction fibers) using MetaMorph Imaging software. Statistical analyses were performed using Microsoft Excel (Redmond, WA), and significance was determined by ANOVA and Student's t test as appropriate.
Fluorescent-activated Cell Sorting
For flow cytometry, freshly trypsinized cells were resuspended in phosphate-buffered saline (PBS) containing a 50% dilution of normal goat serum, incubated with monoclonal antibodies against 3 or β4 integrin for 45 min at room temperature, washed with PBS, incubated with FITC-conjugated goat anti-mouse IgG for 45 min, washed, resuspended in PBS, and analyzed using a Beckman Coulter Elite PCS sorter (Beckman Coulter, Fullerton, CA). For negative controls, primary antibody was omitted from the above procedure.
Rac GLISA
Quantitative analysis of Rac activation was performed using a GLISA Rac activation assay (Cytoskeleton, Denver, CO). Briefly, cell lysates were prepared from HEKs or HEKCn2s at 50% confluence, and their protein concentration was normalized. Samples at a protein concentration of 2 mg/ml in 96-well plate format were used for determination of Rac activity according to the manufacturer's instructions using an ELx808 ultramicroplate spectrophotometer (Bio-Tek Instruments, Winooski, VT). Data analyses were performed using Microsoft Excel.
Fluorescence Recovery after Photobleaching
Fluorescence recovery after photobleaching (FRAP) studies were performed as described elsewhere (Tsuruta et al., 2002, 2003). Briefly, HEKs or HEKCn2s were infected with retrovirus encoding green fluorescent protein (GFP)-tagged, full-length β4 integrin (Sehgal et al., 2006). Time-lapse observations were made using a UV LSM 510 confocal microscope (Zeiss) under the following conditions: 100x, 1.4 NA oil immersion objective, maximum power 25 mW, tube current 5.1 A (31% laser power), and pinhole 1.33 airy units (optical slice, 1.0 µm). GFP images were acquired by excitation at 488 nm and emission at 515–545 nm. Cell regions were bleached at the plane of the membrane at 488 nm, 100% laser power using the minimum number of iterations to cause complete bleaching (n = 20–40). Recovery was monitored at 31% laser power at 1-min intervals. For quantitative analyses, the fluorescence intensity of 1) the photobleached region, 2) the extracellular background intensity, and 3) intracellular brightest intensity were determined using Metamorph 4.0 software (Universal Imaging). All data were analyzed using Microsoft Excel. Data were adjusted for sample fading as detailed elsewhere (Tsuruta et al., 2002, 2003).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mercurio et al., 2001; Nikolopoulos et al., 2004, 2005; Pullar et al., 2006; Sehgal et al., 2006). To test for an association between β4 integrin and BPAG1e in actively migrating cells, we created a scratch wound in a confluent monolayer of HEKs. The cells surrounding the scratch were allowed to migrate into the wounded region for 6 h and subsequently were prepared for immunofluorescence microscopy using BPAG1e and β4 integrin probes. There is extensive colocalization of BPAG1e and β4 integrin not only along the basal aspect of the essentially stationary cells distant from the wound margin, but also toward the leading front of the cells migrating into the scratch site (Figure 1). These results suggest that BPAG1e may function in concert with β4 integrin to regulate keratinocyte motility.
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3, β3, and 2; Figure 2C). No change in protein levels is observed in any instance. Furthermore, cell surface proteins were biotin labeled, pulled down with streptavidin-conjugated beads, and probed for β4 integrin. HEKCn2s exhibit no reduction in cell surface levels of β4 integrin (Figure 2B, bottom panel). This result was confirmed by FACS analysis, which also demonstrates that 3 integrin cell surface expression is similar in HEKs and HEKCn2s (Figure 2D). Subconfluent (60–80%) HEKs and HEKCn2s were analyzed by immunofluorescence microscopy for BPAG1e expression. Whereas control cells exhibit BPAG1e staining in arcs and rosette-like patterns that colocalize with β4 integrin along their substratum-attached surface, there is barely detectable BPAG1e antibody staining in HEKCn2s (Figure 2E). In HEKCn2s β4 integrin staining is observed in the same overall patterns seen in HEKs (Figure 2E).
β4 integrin interacts with two plakin family members, namely BPAG1e and plectin (Hopkinson and Jones, 2000). Thus, we evaluated whether a loss in expression of BPAG1e impacts β4 integrin association with plectin. Comparison of staining patterns for plectin in HEKCn2s versus HEKs reveals that plectin localization is not perturbed in near confluent cells as a result of BPAG1e knockdown and is colocalized with β4 integrin (Figure 3A). We also compared staining for plectin and β4 integrin in single, motile HEKs and HEKCn2s. In both, plectin and β4 integrin colocalize, particularly at the edges of cells (Figure 3B).
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Rac1 and Cofilin Activity Levels Are Reduced in BPAG1e Knockdown Cells
The above data indicate that loss of BPAG1e in keratinocytes impacts lamellipodia formation, polarity, and actin cytoskeleton organization. The small Rho GTPase Rac is known to be involved in each of these events. Because β4 integrin regulates Rac activity, we next assayed the levels of active Rac1 in HEKs, HEKCn2s, and HEKCn12s using a Rac G-LISA (Russell et al., 2003; Zahir et al., 2003; Sehgal et al., 2006; Figure 5A). Rac 1 activity is significantly lower in both knockdown clones than in HEKs (Figure 5A). Specifically, Rac1 activity is decreased by 51% in HEKCn2s and by 38% in HEKCn12s. Blotting for total Rac1 in lysates from HEK, HEKCn2s, and HEKCn12s revealed that the observed change in activity levels is not due to a change in Rac1 expression levels but rather is a change in Rac1 activity (Figure 5B).
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6β4 integrin in HEKs (Sehgal et al., 2006). Thus, we investigated if this interaction is perturbed in HEKCn2s. There is a significant decrease in the levels of Rac1 that coimmunoprecipitates with β4 from extracts of HEKCn2s compared with that coprecipitated with β4 integrin from HEKs (Figure 5C). Together with the Rac activation assay detailed above, our data implicate BPAG1e as being necessary for the activation of Rac1 by 6β4 integrin.
In HEKs, Rac activation leads to dephosphorylation of the actin-severing protein cofilin (Sehgal et al., 2006; Kligys et al., 2007). Thus, we analyzed extracts of HEKs, HEKCn2s, and HEKCn12s for the presence of inactivated (serine-3 phosphorylated) cofilin (Soosairajah et al., 2005). Our data indicate that there are significantly higher levels of inactive, phosphorylated cofilin in HEKCn2s and HEKCn12s compared with that of HEKs (Figure 5D).
BPAG1e Knockdown Cells Show Aberrant Motility Patterns Relative to Controls
On the basis of the changes that we have observed in the front/rear polarity of HEKCn2, we hypothesized that this would likely result in a motility defect. To address this possibility, we evaluated the motile behavior of HEKs and HEKCn2s in single-cell assays (Sehgal et al., 2006; Kligys et al., 2007). HEKs generally migrate either back and forth over linear trails or in a persistent manner, whereas HEKCn2s display frequent turning in their migration patterns, often exhibiting circular migration behavior (Figure 6, A and B; Supplemental Videos 1 and 2). The motility defect of HEKCn12s is less severe than HEKCn2s consistent with the less efficient knock down in BPAG1e expression detected in HEKCn12s (Figure 6C).
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β4 Integrin Dynamics Are Reduced in Migratory HEKs Exhibiting BPAG1e Knockdown
Previously, we demonstrated that β4 integrin dynamics are enhanced upon actin depolymerization, suggesting that actin acts as a brake on the movement of 6β4 integrin complexes in the plane of the membrane (Tsuruta et al., 2003). We also have suggested that cofilin activity is involved in this phenomenon (Sehgal et al., 2006). Thus, we reasoned that a decrease in cofilin activity might change β4 integrin dynamics in keratinocytes. Therefore, we retrovirally infected HEKs and HEKCn2s with GFP-tagged β4 integrin and assayed β4 dynamics by FRAP at both the leading edge of cells moving into scratch wounds and in stationary cells (representative images Figure 7, A and B). Consistent with reduced cofilin activity and a consequent reduced actin remodeling, β4 dynamics at the leading edge of migrating HEKCn2s is reduced compared with that observed in HEKs (Figure 7, A and B, graphed in C). In contrast, there is no significant difference in β4 integrin dynamics in confluent, nonmigratory HEKs and HEKCn2s, as determined by FRAP (Figure 7, A and B, graphed in C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6β4 integrin in keratinocyte adhesion is well established and has been extensively studied (Jones et al., 1994, 1998; Borradori and Sonnenberg, 1996; Nievers et al., 1999). Indeed, the skin fragility disorders of the epidermolysis bullosa family exemplify the importance of the linkage from extracellular laminin 332 to intracellular keratin 5/14 provided by 6β4 integrin and its plakin-binding partners BPAG1e and plectin (Christiano and Uitto, 1996; Pulkkinen and Uitto, 1999). In contrast, the role of integrins in regulating keratinocyte motility on laminin matrices is controversial. A number of workers have proposed that 3β1 integrin mediates migration on laminin 332, whereas recent data indicate that 3β1 may actually act to retard keratinocyte motility (Hodivala-Dilke et al., 1998; Hintermann et al., 2001; Margadant et al., 2009). Moreover, although it has been reported that 6β4 integrin negatively regulates migration there is accumulating evidence that, under certain circumstances, 6β4 integrin activates migration promoting signaling pathways (Mercurio et al., 2001; deHart et al., 2003; Raymond et al., 2005; Sehgal et al., 2006). In addition, although keratinocytes genetically null for β4 integrin close epithelial wounds more slowly than their wild-type counterparts, 3 integrin-null epidermal cells display enhanced wound closure rates (deHart et al., 2003; Russell et al., 2003; Sehgal et al., 2006; Reynolds et al., 2008; Margadant et al., 2009). Furthermore, previous studies have demonstrated that keratinocytes deficient in β4 integrin exhibit reduced Rac activity, which leads to their aberrant motility (Russell et al., 2003; Pullar et al., 2006; Sehgal et al., 2006). However, the precise mechanics of the link from β4 integrin to Rac activity have yet to be fully elucidated.
Here, we provide evidence that the plakin molecule BPAG1e mediates β4 integrin regulation of Rac1 activity and is an essential component of the signaling pathway via which β4 integrin determines both cell front/rear polarity and processivity of cell migration. That BPAG1e does so is an unanticipated function for this plakin protein family member that has been thought to be purely a structural linker protein. Indeed, our data now imply that BPAG1e is also a signal scaffold protein, in addition to its ability to interact with and organize the keratin cytoskeleton. In this regard, we do not yet know whether BPAG1e binds Rac1 directly or whether it somehow modulates the association of Rac1 with 6β4 integrin complexes at the cell surface. This will be an interesting avenue of investigation for the future.
Another plakin molecule, namely plectin, also binds β4 integrin and is already known to interact with a number of signaling molecules/complexes (Andra et al., 1998; Osmanagic-Myers and Wiche, 2004; Osmanagic-Myers et al., 2006). However, our results clearly indicate that plectin cannot compensate for the loss of BPAG1e in keratinocytes, at least with regard to Rac1 and cofilin activation, processivity of migration, or the determination of front/rear polarity.
The dynamics of integrin clusters in the plane of the substrate-attached cell membrane are known to play an important role in regulating cellular adhesion and migration (Palecek et al., 1996; Smilenov et al., 1999; Geuijen and Sonnenberg, 2002; Wiseman et al., 2004). In the studies described here we report that in stationary cells β4 integrin is significantly less dynamic than in migrating cells. This is not surprising because, in nonmotile cells, 6β4 integrin is likely incorporated into hemidesmosome-like stable anchoring contacts, which interact with the keratin cytoskeleton (Jones et al., 1991, 1998). The finding that β4 integrin dynamics in stationary cells is unaffected by BPAG1e expression levels is somewhat more surprising because one might assume that the loss of this keratin-binding protein might result in an increase in integrin dynamics in the plane of the membrane. However, we also present evidence that plectin association with 6β4 is retained in confluent, nonmigratory BPAG1e-deficient cells. Thus, plectin likely stabilizes 6β4 integrin dynamics in such cells by providing a link from β4 to the keratin cytoskeleton. Consistent with this, immunofluorescence staining for keratin 5 in BPAG1e-deficient cells reveals very little difference in keratin intermediate filament organization.
We observe a significant increase in β4 integrin dynamics at the leading edge of migrating keratinocytes. We speculate that β4 dynamics are likely to be dependent on localized actin remodeling, leading to a locally permissive environment for β4 integrin movement in the plane of the membrane. Consistent with this premise, we have previously reported that depolymerization of actin, through treatment with cytochalasin D, actually enhances β4 integrin dynamics (Tsuruta et al., 2003). Moreover, we report here that there is a significant reduction in β4 integrin dynamics at the leading edge of BPAG1e-deficient keratinocytes. These data suggest that at the leading edge of migrating cells the recruitment by BPAG1e of Rac1 and cofilin to the cell surface, and their consequent activation leads to localized remodeling of the actin cytoskeleton that releases a brake on the movement of 6β4 integrin complexes in the plane of the membrane.
The reduction in β4 dynamics in BPAG1e-deficient cells may, in part, explain the loss of their processivity of migration. We speculate that there is a feedback loop in which a complex of β4 integrin/BPAG1e activates Rac1 and cofilin, whose activity then drives lamellipodia formation through actin remodeling. The latter, in turn, releases the cytoskeleton brake on 6β4 dynamics, inducing integrin clustering and stabilization of the newly formed lamellipodia (Santoro et al., 2003). Consistent with this model, expression of active Rac1 or cofilin can bypass the requirement for BPAG1e in the presence but not absence of β4 integrin (Pullar et al., 2006; Sehgal et al., 2006).
In summary, our data provide new mechanistic insight into the wound-healing defect observed in BPAG1e-null mouse skin in vivo (Guo et al., 1995). BPAG1e is clearly multifunctional in keratinocytes. In the cells in the basal layer of an intact epidermis, BPAG1e is part of a complex mediating stable adhesion of cells to the extracellular matrix. However, BPAG1e also has an important function during wound healing and possibly tumorigenesis as a regulator of cell polarity and migration.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Jonathan C.R. Jones (j-jones3{at}northwestern.edu)
Abbreviations used: FRAP, fluorescence recovery after photobleaching; FACS, fluorescent-activated cell sorting; HEK, human epidermal keratinocytes.
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) if they migrated in a predominantly linear and/or backtracking manner or circles (
) if they moved over a approximately circular track and were plotted as percentage of total. The percentage of HEKCn2s moving in a trail under each condition was compared with the percentage of HEKs moving in a similar manner (* p < 0.01). (I) Migration rates for cells visualized in A–G were calculated by summing the displacement of cell bodies between each time point in order to calculate total displacement over time. Differences between cell lines/infections were below significance.