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Targeted Deletion of the Integrin ß4 Signaling Domain Suppresses Laminin-5-Dependent Nuclear Entry of Mitogen-Activated Protein Kinases and NF-B, Causing Defects in Epidermal Growth and Migration

Sotiris N. Nikolopoulos,^1, Pamela Blaikie,^1, Toshiaki Yoshioka,¹ Wenjun Guo,¹ Claudia Puri,² Carlo Tacchetti,² and Filippo G. Giancotti^1*

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York,¹ IFOM Center of Cell Oncology and Ultrastructure, Department of Experimental Medicine, University of Genova, Genoa, Italy²

Received 10 November 2004/ Returned for modification 20 December 2004/ Accepted 28 April 2005

	ABSTRACT

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The 6ß4 integrin—a laminin-5 receptor—mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of ß4. Mice carrying a targeted deletion of the signaling domain of ß4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from ß4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IB in ß4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-B remain in the cytoplasm in ß4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that 6ß4 promotes keratinocyte proliferation and migration through its effect on NF-B and P-JNK. These findings provide evidence that ß4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-B and mitogen-activated protein kinases.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
The integrins mediate cell adhesion to the extracellular matrix and transmit mechanical and chemical signals to cells (13, 23). Integrin signaling imparts a stringent control to the action of receptor tyrosine kinases (RTKs), determining the nature and direction of the cell's response to growth factors and cytokines (14, 34). In spite of considerable amounts of cell biological data, genetic evidence of the significance of integrin signaling remains scarce. In particular, it has been difficult to separate the adhesive and signaling functions of individual integrins in any model system analyzed to date.

The 6ß4 integrin is a laminin-5 receptor expressed in many epithelial cells, in Schwann cells, and in endothelial cells. Integrin 6ß4 signaling proceeds through Src family kinase-mediated phosphorylation of the unique cytoplasmic domain of ß4, recruitment of Shc, and activation of Ras (7, 12, 31) and phosphoinositide 3-kinase (PI-3K) (48, 49). Upon dephosphorylation, the ß4 tail associates with the keratin cytoskeleton, causing assembly of hemidesmosomes and, hence, strengthening adhesion to basement membranes containing laminin-5 (7, 35, 51).

The pattern of expression of 6ß4 in normal and hyperproliferative skin is consistent with a role for 6ß4 signaling in the control of epithelial proliferation (11). We have shown that 6ß4 promotes progression through G₁ and entry in S phase in keratinocytes treated with epidermal growth factor (EGF) (30). In epidermal cells, 6ß4 associates with the EGF receptor (EGF-R) and Ron RTKs (32, 44). Activation of these RTKs enhances phosphorylation of ß4, causing disruption of hemidesmosomes and increased keratinocyte migration and proliferation (7, 32, 44). These results suggest that these RTKs decrease the ability of 6ß4 to mediate stable adhesion but increase its signaling function.

Prior genetic studies have indicated that the ß1 integrins participate in epidermal growth and repair. Whereas mice lacking 3ß1 display defects in epidermal adhesion and assembly of the basement membrane (8, 21), conditional ablation of all ß1 integrins results in profound proliferation defects (4, 40) and aberrant wound healing (15). Despite activating the wound-related vß6 integrin, ß1-null keratinocytes do not migrate efficiently in vitro because of defective FAK-Src-mediated remodeling of their actin cytoskeleton (41). In addition, these cells lose expression of 6ß4 (40). Finally, deletion of 3 impairs keratinocyte migration in vitro (6).

Mice carrying a targeted deletion of the entire cytoplasmic domain of ß4 lack hemidesmosomes and die at birth due to extensive blistering of the skin and upper gastrointestinal tract (35), precluding a definitive investigation of skin homeostasis and repair. To analyze the role of 6ß4 signaling in the absence of loss of adhesion strengthening, we have recently generated mice carrying a deletion of the C-terminal, signaling segment of the ß4 tail (37). We report here that these mice have intact hemidesmosomes but display defective epidermal growth and wound healing. Through studies of primary keratinocytes derived from these mice, we provide evidence that 6ß4 signaling controls epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-B and P-Jun N-terminal protein kinase (P-JNK).

	MATERIALS AND METHODS

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Cells, antibodies, and other reagents. Primary keratinocytes from newborn mice were grown on collagen I in EMEM.06 with 8% Chelex-treated fetal bovine serum, 2 ng/ml EGF, and 0.06 mM CaCl₂ (17). We purchased rat monoclonal antibody (MAb) to ß4 (346-11A) from Pharmingen; rabbit antibodies to P-extracellular signal-regulated kinase (P-ERK), P-JNK, P-Akt (S473), IB, and P-IB (S32) from Cell Signaling; rabbit antibodies to ERK2, NF-B p65 (C-20), green fluorescent protein (GFP) (FL), and histone H3; mouse MAbs to P-ERK (T203/Y204) and P-JNK (T183/Y185); and goat antibodies to Akt from Santa Cruz; MAbs to Rac, paxillin, and Rho GDI from BD Biosciences; MAb to vinculin (hVIN-1) and rhodamine-phalloidin from Sigma; MAb to NF-B p65 (clone 2A12A7) and sheep antibodies to JNK1 from Zymed; and fluorescein isothiocyanate (FITC)- and Cy3-conjugated affinity-purified secondary antibodies from Jackson Laboratories. The rabbit anti-3 cyto antibody was a gift from G. Tarone. Affinity-purified rabbit antibodies to the N terminus of bullous pemphigoid antigen 2 (BPAG-2) and the LE4-6 segment of mouse laminin 2 and MAb 121 to HD-1/plectin were previously described (18, 35, 45). Laminin-5 matrices were prepared as described previously (50). Human fibronectin and rat tail collagen I were from Sigma, and PD98059, U0126, JNK II, BAY11-7082, Y-27632, and LY294002 were from Calbiochem.

Biochemical assays. After growth factor deprivation, keratinocytes were detached, plated on laminin-5- or anti-ß4-coated plates for the indicated times, and then treated with 10 ng/ml EGF for 15 min. Cells were lysed in 50 mM HEPES, pH 7.4, 5 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10% glycerol, and 1% NP-40 with protease and phosphatase inhibitors and subjected to immunoblotting or biphenylylphenyloxadiazole (PBD) pull-down assay as described previously (33). Nuclear and cytoplasmic fractions were prepared with the NE-PER kit (Pierce).

Cell proliferation, survival, and migration. For cell proliferation, keratinocytes were synchronized in G₀ by growth factor deprivation and plated on laminin-5 or fibronectin in the presence of 2 ng/ml EGF and 10 µM bromodeoxyuridine (BrdU) for 18 h. For in vivo labeling, mice were injected intravenously with 5 µM BrdU/100 g of body weight and sacrificed 1 h later. Keratinocytes and paraffin-embedded skin sections were subjected to immunofluorescent or immunohistochemical staining with anti-BrdU antibodies, using the BrdU Labeling and Detection Kit I (Roche). Retroviral infection of keratinocytes with pFB-Neo/IB-2A or PINCO/GFP was as previously described (25). For matrix-dependent survival, keratinocytes were deprived of growth factors for 18 h, detached, and replated on laminin-5 or fibronectin in medium lacking serum and EGF. Apoptotic cell death was monitored at 4 and 18 h by using the In Situ Cell Death Detection kit (Roche). To estimate cell death in vivo, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were performed on paraffin-embedded skin sections. For in vitro wound assay, keratinocytes were grown until confluent on laminin-5-coated plates and deprived of growth factors for 18 h. Monolayers were scratched with a P200 tip and incubated in the presence of serum and 2 ng/ml EGF for 18 h. Wound closure was monitored by digital photography.

Immunohistochemistry, electron microscopy (EM), and immunogold labeling. For nuclear translocation, primary keratinocytes were deprived of growth factors for 18 h, detached, plated on laminin-5-coated glass coverslips, and stimulated with 10 ng/ml EGF for 15 min. After fixation with 3.7% paraformaldehyde, they were permeabilized with 0.2% Triton X-100 and stained with the indicated antibodies. For immunofluorescent detection of hemidesmosome-like adhesions, keratinocytes were extracted with 0.2% Triton X-100 and then fixed with cold methanol as described previously (7). For immunohistochemical analysis, tissues and plugs were embedded in paraffin or snap-frozen in OCT compound (Tissue-Tek). For electron microscopy, skin samples were collected, fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, postfixed with 1% osmium tetroxide, stained en bloc with 1% uranyl-acetate, and embedded in Polybed 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM 902 or a Philips CM10 electron microscope. Immunogold labeling was performed on ultrathin cryosections as previously described (29). Preliminary experiments were conducted to identify the optimal concentration of antibodies for immunolabeling. In order to obtain highly specific labeling, we used the lowest concentration of antibodies able to give a signal (labeling of compartments known to associate with the antigen)-to-noise (labeling of compartments known not to associate with the given antigen) ratio of >5.

Wound repair. Adult mice were anesthetized, shaved, and disinfected with 70% ethanol. Two full-thickness excisional wounds, 2 mm in diameter, were generated on either side of the dorsal midline of each mouse with a disposable biopsy punch tool (Premier, Plymouth Meeting, PA). Wounds were left uncovered and photographed daily. The wound surface remaining open at each time point was measured by using the NIH Image software. For histological analysis, complete wounds including 5 mm of adjacent normal skin were excised at 3, 4, or 5 days after injury. Cryosections across the middle of the wounds were stained with hematoxylin and eosin or the indicated antibodies.

	RESULTS

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The ß4 substrate domain is not necessary for assembly of hemidesmosomes and epidermal adhesion in vivo. We have recently reported that mice carrying a specific deletion of the C-terminal portion of ß4 tail (henceforth termed "substrate domain," Fig. 1A) are viable and fertile but display defective postnatal angiogenesis. Experiments on cells from these mice have provided evidence that deletion of the ß4 substrate domain impairs 6ß4 signaling but not adhesion to laminin-5 (37). Although the N-terminal part of the ß4 tail, upstream of amino acid 1355, is sufficient for interaction with the plakin HD-1/plectin and hence for association with the keratin cytoskeleton, prior studies have suggested that the C-terminal portion of ß4 tail contributes to the assembly of hemidesmosome-like adhesions in vitro by promoting recruitment of BPAG-2 and, hence, BPAG-1 (46). We thus felt it necessary to specifically examine the effect of the ß4 mutation on epidermal adhesion in vivo.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Deletion of the ß4 substrate domain does not affect assembly of hemidesmosomes and epidermal adhesion. (A) Structure of the wild-type ß4 protein (ß4 WT) and its mutant form (ß4 1355T) encoded by the targeted allele. Ovals, fibronectin type III repeats; stars, tyrosinephosphorylation sites. (B) Skin sections from wild-type and mutant (1355T) mice were stained with hematoxylin and eosin (H&E), MAb 346-11A to the extracellular domain of ß4, rabbit antiserum to laminin-5, MAb to HD-1, or rabbit antiserum to BPAG-2. (C) Transmission EM of the dermal-epidermal junction of wild-type and mutant skin. Bars, 0.489 µm (wild type) and 0.436 µm (1355T) (top). Immunogold labeling of BPAG-2 on ultrathin cryosections from newborn skins. Arrowheads indicate gold particles incorporated in hemidesmosomes. Bars, 0.219 µm (wild type) and 0.255 µm (1355T) (bottom). (D) Morphometric analysis of the hemidesmosomes of wild-type and mutant mice. The number of hemidesmosomes per cell profile was derived from the photographic reconstruction of 10 cell profiles per group. Morphometric data represent the means (± standard deviations) of values obtained from the analysis of 80 hemidesmosomes per group. DBL, distance from basal lamina; HBL, thickness of basal lamina. (E) Primary keratinocytes from wild-type and mutant mice were plated on laminin-5 matrix, extracted with 0.2% Triton X-100, and subjected to double staining with anti-ß4 (green) and anti-HD-1 (red) (top) or anti-ß4 (green) and anti-BPAG-2 (red) (bottom). WT, wild type.

To examine the assembly of hemidesmosomes in mutant mice, we used EM. Transmission EM showed that the skin of mutant mice contains well-structured hemidesmosomes (Fig. 1C), and morphometric analysis confirmed that they are similar in number and appearance to those of wild-type skin (Fig. 1D). Furthermore, cryo-immuno-EM demonstrated that BPAG-2 is regularly incorporated in the hemidesmosomes of mutant mice (Fig. 1C). Taken together, these results provide direct evidence that the N-terminal portion of ß4 tail (to amino acid 1355) is sufficient for association with the keratin cytoskeleton and assembly of adhesion-competent hemidesmosomes in vivo.

Immunofluorescence experiments were employed to examine the ability of keratinocytes from mutant mice to form hemidesmosome-like structures in vitro. As shown in Fig. 1E, the mutant keratinocytes assemble in culture hemidesmosome-like structures containing HD-1/plectin but devoid of BPAG-2. These results are in agreement with the observation that the C-terminal portion of ß4 tail contributes to the recruitment of BPAG-2 and BPAG-1 to hemidesmosome-like adhesions in vitro (46).

How do we explain the apparent discrepancy between the EM results in vivo and those of immunofluorescent staining in vitro? It is possible that under physiological conditions BPAG-2 is recruited to hemidesmosomes through its binding to a basement membrane component of dermal origin. Unable to synthesize this component, cultured keratinocytes would be dependent on the C-terminal segment of ß4 tail to recruit BPAG-2, and hence BPAG-1, to hemidesmosome-like adhesions. In addition, or instead, the association of BPAG-2 with the integrin 6 subunit (22) and/or with plectin (27) may be sufficient for assembly of hemidesmosomes in vivo, because this process is intrinsically more robust under these conditions than it is in vitro. Irrespective of the underlying mechanism, these findings indicate that the molecular requirements for hemidesmosome assembly are best studied in their physiological context, and deletion of the ß4 signaling domain does not affect assembly of hemidesmosomes in this context.

Integrin 6ß4 signaling promotes epidermal growth and repair. To examine epidermal proliferation in vivo, newborn mice were "pulse" labeled with BrdU for 30 min and sections of their skins were subjected to anti-BrdU staining. As shown in Fig. 2A, the skin of mutant mice contains significantly fewer BrdU-positive basal keratinocytes per linear millimeter of basement membrane (6.7 ± 2) than does that of wild-type mice (16 ± 2.5) (P < 0.01 by Student's t test). In addition, staining with MAb Ki-67, which binds to a nuclear protein expressed predominantly during the S, G₂, and M phases of the cell cycle, showed that the skin of mutant mice contains fewer proliferating elements per linear millimeter of basement membrane (45.8 ± 5.42) than does that of control mice (66.2 ± 9.39) (P < 0.01). Similar results were obtained by examining the skin of adult mice (not shown). Although we had previously observed that E18.5 embryos carrying a deletion of the entire cytoplasmic domain of ß4 display a defect in epidermal proliferation (35), a subsequent study had failed to detect impaired epidermal proliferation in E16.5 embryos lacking whole ß4 (9). Since epidermal proliferation is not robust at E16.5, it is likely that other integrins compensate for loss of 6ß4 at this embryonic stage. Taken together, our results indicate that 6ß4 signaling is required for optimal proliferation of keratinocytes in newborn and adult epidermis.

View larger version (50K): [in this window] [in a new window]   FIG. 2. The ß4 substrate domain promotes epidermal growth and wound healing. (A) Wild-type and mutant (1355T) mice were injected with BrdU prior to euthanasia. Paraffin-embedded sections of their skins were subjected to immunoperoxidase staining with anti-BrdU MAb and counterstaining with hematoxylin (top). BrdU-positive cells are indicated by arrows. The graph shows the mean number of BrdU-positive cells (± standard deviation) per linear millimeter of basement membrane (BM; bottom), obtained from the analysis of 15 mice per group (n = 15). (B) Full-thickness excisional wounds were monitored by digital photography at the indicated times (top). The percentage of wound closure was estimated from the area of the wound remaining open at each time point and plotted against time. The graph shows the wound closure values (± standard deviations) obtained from the analysis of eight mice per group (n = 8) (middle). Cross sections from a 4-day-old wound from a wild-type mouse and a 5-day-old wound from a mutant mouse were stained with hematoxylin and eosin (bottom). Arrows indicate the position and direction of the advancing epithelia. Bar, 100 µm. WT, wild type.

To study the effect of the ß4 mutation on wound healing, we generated full-thickness excisional wounds in wild-type and mutant mice and monitored wound closure over time. As shown in Fig. 2B, deletion of the ß4 substrate domain decreased the rate of wound repair, while delaying final closure to a lesser degree. Histological analysis indicated that the two opposing epithelial margins had reached the midline and fused at 5 days after wounding in wild-type mice. By contrast, the advancing epithelium was still penetrating the space between the clot and the granulation tissue at the same time in mutant mice (Fig. 2B). Immunohistochemical staining showed that 6ß4 is not expressed in blood vessels in the wound bed or in the fibroblasts and monocytes/macrophages of granulation tissue, suggesting that the delay in wound healing in mutant mice is not due to a defect in vascularization or function of granulation tissue (not shown). In accordance with the observation that leading-edge keratinocytes are not actively proliferating (24), we detected very few Ki-67-positive cells at the leading edge of both wild-type and mutant wounds (not shown). These observations suggest that ß4 signaling promotes extension of the epidermal leading edge during wound healing through an effect on keratinocyte migration.

The ß4 substrate domain opposes spreading but promotes cell migration, proliferation, and resistance to apoptosis in primary keratinocytes. Prior studies have shown that 6ß4 inhibits the ability of the 3ß1 integrin to promote spreading and assembly of focal adhesions (36). Accordingly, 3ß1 relocates from cell-to-cell adhesions to focal adhesions in ß4-null keratinocytes plated on laminin-5 (36). Upon plating on laminin-5 in serum-free conditions, the mutant keratinocytes spread more extensively than wild-type controls (Fig. 3A). This was not a transient effect, as measurement of cell surface areas over a 2-hour time course indicated that the mutant cells spread significantly more—and increasingly better—than wild-type cells at each of the time points examined (Fig. 3B). Furthermore, indirect immunofluorescent staining showed that the mutant cells form focal adhesions and stress fibers more robustly than control cells. Finally, 3ß1 localized within structures resembling focal adhesions in mutant keratinocytes but not in control cells (Fig. 3C). These results suggest that the C-terminal signaling domain of ß4 tail opposes spreading and mediates the transdominant-negative effect that 6ß4 exerts on 3ß1.

View larger version (32K): [in this window] [in a new window]   FIG. 3. The ß4 substrate domain opposes 3ß1-mediated spreading and assembly of actin filaments. (A) Wild-type (WT) and mutant (1355T) keratinocytes were plated on laminin-5 matrix and examined by phase-contrast microscopy. Representative images acquired at 30 min after plating are shown. Bar, 10 µm. (B) Quantitation of cell area during spreading of wild-type (WT) and mutant (1355T) keratinocytes upon plating on laminin-5 matrix for up to 2 h. Total cell area was calculated from the digital images. Data represent the means (± standard deviations) of 50 randomly chosen cells from three different experiments. (C) Wild-type (WT) and mutant (1355T) keratinocytes plated as above were subjected to double staining with FITC-conjugated paxillin and rhodamine-conjugated phalloidin. Bar, 5 µm. (D) Wild-type (WT) and mutant (1355T) keratinocytes plated as above were subjected to double staining with anti-3 cyto rabbit antiserum and FITC-conjugated paxillin followed by Cy3-conjugated anti-rabbit antibody. Bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   FIG. 4. The ß4 substrate domain promotes keratinocyte migration, proliferation, and resistance to apoptosis. (A) Wild-type and mutant (1355T) keratinocytes were plated on laminin-5 and subjected to wound closure assay in the presence of serum and EGF (top). The graph shows the mean percentage of wound closure (± standard deviation) after 18 h from three different experiments (bottom). (B) Wild-type and mutant keratinocytes were synchronized in G0, plated on laminin-5 in the presence of EGF for 18 h, and subjected to double staining with anti-BrdU and 4',6'-diamidino-2-phenylindole (DAPI) (top). The graph shows the mean percentage of BrdU+ cells (± standard deviation) from three experiments (bottom). (C) Wild-type and mutant keratinocytes were deprived of mitogens, plated on laminin-5 in the absence of growth factors for 18 h, and subjected to TUNEL and DAPI staining (top). The graph shows the mean percentage of apoptotic cells (± standard deviation) from three experiments with cells plated for the indicated times on either laminin-5 (Ln-5) or fibronectin (Fn) (bottom). WT, wild type.

To examine the role of 6ß4 signaling in keratinocyte survival in vitro, wild-type and mutant keratinocytes were plated on laminin-5 or fibronectin, deprived of growth factors, and subjected to TUNEL analysis. As shown in Fig. 4C, the mutant keratinocytes survived growth factor deprivation on fibronectin well but underwent massive apoptosis on laminin-5 at 18 h. By contrast, only a minor fraction of wild-type cells underwent apoptosis on both substrates at 18 h of growth factor deprivation. These results indicate that the ß4 substrate domain promotes activation of antiapoptotic pathways. When cultured in the presence of growth factors, the mutant keratinocytes did not display signs of apoptosis (not shown), suggesting that mitogens and possibly other survival factors can compensate for the loss of 6ß4 survival signaling in vivo. Taken together, these observations indicate that the ß4 substrate domain activates signaling pathways necessary for keratinocyte migration, proliferation, and resistance to stress-induced apoptosis.

The ß4 substrate domain does not contribute to EGF-dependent cytoplasmic activation of ERK, phosphorylation of JNK, and degradation of IB. To gain insight into the mechanism by which the ß4 substrate domain promotes cell migration, proliferation, and survival, we monitored the activation of various signaling pathways in wild-type and mutant keratinocytes plated on laminin-5. Adhesion to laminin-5 induced activation of ERK and Akt in wild-type but not in mutant keratinocytes (37). However, EGF stimulation caused a similarly high activation of ERK and Akt in wild-type and mutant keratinocytes adhering to laminin-5, suggesting that the ß4 substrate domain is not required for EGF-R-mediated activation of ERK and PI-3K on this substrate (Fig. 5A). Together, these observations indicate that the ß4 substrate domain is required for laminin-5- but not EGF-R-dependent activation of ERK and PI-3K.

View larger version (21K): [in this window] [in a new window]   FIG. 5. The ß4 substrate domain is not required for EGF-dependent phosphorylation of ERK and Akt, activation of Rac and JNK, and phosphorylation of IB. (A) Wild-type and mutant (1355T) keratinocytes were deprived of growth factors, detached, and either kept in suspension (S) or plated on laminin-5 for 30 min and then stimulated with EGF for 10 min. Equal amounts of total proteins were probed with antibodies to activated ERK and Akt or total ERK2. (B) Growth factor-starved keratinocytes were either kept in suspension (–) or plated on dishes coated with anti-ß4 (ß4) for 2 h and then subjected to GST-PAK-PBD pull-down assay. Levels of active and total Rac were determined by immunoblotting with anti-Rac (top). Cells were either kept in suspension or plated on laminin-5 for 2 h with or without EGF and then subjected either to GST-PAK-PBD pull-down assay or to immunoblotting with antibodies to activated JNK or vinculin as control(bottom). (C) Growth factor-starved keratinocytes were either kept in suspension (–) or plated on dishes coated with anti-ß4 (ß4) for 30 min (top) or laminin-5 for 2 h (bottom) with or without EGF and then probed by immunoblotting with antibodies to phosphorylated IB, total IB, or vinculin. WT, wild type; Ln-5, laminin-5.

We next compared NF-B signaling in wild-type and mutant keratinocytes. As shown in Fig. 5C, ligation of wild-type 6ß4 was sufficient to induce phosphorylation and degradation of IB, which functions to retain NF-B in the cytoplasm. This process was unimpaired in mutant keratinocytes and was not augmented by EGF (Fig. 5C). In addition, IB was degraded efficiently in both wild-type and mutant keratinocytes plated on laminin-5 (Fig. 5C). These observations suggest that 6ß4 is able to induce the first step of NF-B signaling, the degradation of IB, and this process does not require the ß4 substrate domain or EGF-R signaling, as observed for the activation of Rac and JNK.

Together, these results indicate that the ß4 substrate domain does not contribute to activation of Rac, phosphorylation of JNK, and degradation of IB. Therefore, the proliferative and migratory defects of mutant keratinocytes are not caused by defects in these processes.

The ß4 substrate domain promotes nuclear translocation of P-ERK, P-JNK, and NF-B. To further examine the mechanism by which the ß4 substrate domain controls cell proliferation and migration, we monitored EGF-induced nuclear translocation of P-ERK in wild-type and mutant keratinocytes plated on laminin-5. In accordance with the observation that EGF activates ERK to the same extent in wild-type and mutant keratinocytes (Fig. 5A), immunofluorescence documented a similar increase in anti-P-ERK staining in both wild-type and mutant cells plated on laminin-5 in the presence of EGF (Fig. 6A). However, whereas EGF induced entry of P-ERK into the nucleus of the majority of wild-type cells adhering to laminin-5, it did not exert this effect in mutant keratinocytes, implying that the ß4 substrate domain controls EGF-induced nuclear translocation of P-ERK (Fig. 6A). This effect was specific because adhesion to fibronectin, which is mediated by the 5ß1 integrin, rescued EGF-mediated nuclear translocation of P-ERK in mutant keratinocytes (not shown). Furthermore, the ß4 substrate domain did not affect nuclear translocation of Akt in cells treated with EGF (Fig. 6A). To confirm this result, we performed biochemical fractionation experiments. As shown in Fig. 6B, EGF stimulation induced accumulation of ERK in the nuclear fraction of wild-type but not mutant keratinocytes plated on laminin-5. By contrast, it promoted accumulation of Akt in the nuclear fraction of both wild-type and mutant keratinocytes (Fig. 6B). These results indicate that signaling by the ß4 substrate domain promotes nuclear translocation of P-ERK, but not P-Akt.

View larger version (46K): [in this window] [in a new window]   FIG. 6. The ß4 substrate domain controls nuclear translocation of p-ERK, p-JNK, and NF-B. (A) Serum-starved wild-type and mutant (1355T) keratinocytes were plated on laminin-5 for 45 min and stimulated or not with EGF for 15 min. After fixation, the cells were stained with antibodies to P-ERK, p-Akt, P-JNK, and the p65 subunit of NF-B and examined by confocal microscopy. The percentage of cells that had more intense staining in the nucleus than in the cytoplasm was estimated after counting of at least 1,000 cells per coverslip. The graphs show the results (± standard deviations) from three independent experiments. Bar, 10 µm. (B) Growth factor-starved keratinocytes treated as above were subjected to subcellular fractionation. Equal amounts of proteins from the cytoplasmic (C) and nuclear (N) fractions were probed with antibodies to the indicated proteins. (C) Serum-starved wild-type and mutant (1355T) keratinocytes were plated on laminin-5 matrix and subjected to wound closure assay in the presence of serum and EGF (10 ng/ml) for 2 h. They were then fixed and stained with anti-p65 antibodies and examined by confocal microscopy. Cells at the leading edge with nuclear (in wild-type cells) or cytoplasmic (in 1355T cells) NF-B are shown by arrows. The graph shows the percentage of leading-edge cells (± standard deviation) with nuclear NF-B. Bar, 15 µm. (D) Cross sections of wild-type and mutant (1355T) wounds shown in Fig. 2B were stained with anti-p65 antibodies and examined by confocal microscopy. Dashed white lines demarcate the leading edge of the epithelia advancing between the escar and granulation tissue in both cases. Bar, 25 µm. WT, wild type.

To examine the potential relevance of this signaling mechanism during cell migration, we examined nuclear translocation of NF-B during keratinocyte wound closure in vitro. Wild-type and mutant keratinocytes were plated on laminin-5 at confluency and subjected to in vitro wound assay. Two hours after EGF stimulation, the cells were fixed and stained with antibodies to the p65 subunit of NF-B. As shown in Fig. 6C, the wild-type keratinocytes located at the wound edge, but not those behind them, displayed significant nuclear accumulation of NF-B. In contrast, the mutant keratinocytes at the wound edge did not show significant nuclear accumulation of NF-B. These results suggest that the ß4 substrate domain controls nuclear translocation of NF-B at the onset of keratinocyte migration in vitro.

To evaluate the physiological significance of these observations, cross sections of full-thickness excisional wounds from wild-type and mutant mice were stained with anti-p65 antibodies. As shown in Fig. 6D, NF-B accumulated in the nucleus of a significant fraction of keratinocytes at the leading edge of the advancing epithelium in wild-type wounds. By contrast, it did so in only a minor fraction of leading-edge keratinocytes in mutant wounds. These results suggest that the ß4 substrate domain promotes epidermal migration in vivo by inducing nuclear translocation of NF-B in leading-edge keratinocytes.

NF-B and JNK promote keratinocyte proliferation and migration, and PI-3K protects them from apoptosis on laminin-5. To identify the mechanism by which the ß4 substrate domain controls keratinocyte proliferation, we plated wild-type keratinocytes on laminin-5 in the presence of specific inhibitors and used BrdU incorporation to examine their ability to progress through the cell cycle in response to EGF. As shown in Fig. 7A, the NF-B inhibitor BAY11-7082 suppressed keratinocyte cell cycle progression, whereas the PI-3K inhibitor LY294002 and the JNK inhibitor JNK II exerted a partial effect. Notably, the MEK inhibitors PD98059 and U0126 reduced keratinocyte proliferation by a modest extent. Transduction of a retrovirus encoding the dominant-negative mutant IB-2A, which suppresses NF-B signaling, confirmed the role of NF-B in keratinocyte proliferation (Fig. 7B). These results suggest that NF-B, and to a smaller extent PI-3K and JNK, is required for keratinocyte proliferation on laminin-5. Since the EGF-R efficiently activates PI-3K but is unable to promote nuclear translocation of NF-B and P-JNK in mutant keratinocytes plated on laminin-5 (Fig. 6), these results imply that the ß4 substrate domain controls keratinocyte proliferation by promoting nuclear translocation of NF-B and P-JNK.

View larger version (26K): [in this window] [in a new window]   FIG. 7. NF-B or JNK is required for keratinocyte proliferation and migration on laminin-5, whereas PI-3K mediates keratinocyte survival upon EGF withdrawal. (A) Wild-type keratinocytes were plated on laminin-5 matrix in the absence (Ctrl) or presence of inhibitors of NF-B (12.5 µM BAY11-7082), PI-3K (10 µM LY294002), JNK (20 µM JNK II), or MEK (50 µM PD98059 or 50 µM U0126). Entry in S phase and wound closure were in response to EGF, whereas survival was in the absence of growth factors, as in Fig. 3. The graphs show the mean percentages (+ standard deviations) of BrdU-positive cells (top), wound closure (middle), or TUNEL+ cells (bottom). (B) Retroviral transduction of the IB superrepressor IB-2A inhibits keratinocyte proliferation on laminin-5. Primary keratinocytes from wild-type mice were infected with viruses encoding GFP or hemagglutinin (HA)-tagged IB-2A. The cells were synchronized in G0 and plated on laminin-5 in the presence of EGF and BrdU for 18 h. They were then subjected to double labeling with anti-BrdU (green) and anti-GFP or anti-HA (red), respectively. The graph shows the mean percentage (+ standard deviation) of BrdU-positive transduced cells per infection. At least 250 transduced cells per coverslip were counted.

Finally, we investigated the mechanism by which the ß4 substrate domain promotes cell survival. Wild-type keratinocytes were plated on laminin-5, deprived of growth factors, and treated with the inhibitors. TUNEL analysis indicated that LY294002, but none of the other inhibitors, suppresses keratinocyte survival (Fig. 7A), suggesting that 6ß4-mediated activation of PI-3K-to-Akt signaling is necessary for laminin-5-dependent survival.

Taken together, these observations support the hypothesis that the ß4 substrate domain promotes cell proliferation and migration by inducing nuclear translocation of NF-B and P-JNK and cell survival through activation of PI-3K.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeted deletion of the C-terminal segment of the ß4 tail does not affect adhesion to laminin-5 and assembly of hemidesmosomes, allowing a genetic analysis of 6ß4 signaling in the absence of loss of adhesion. The results of this analysis indicate that the ß4 substrate domain controls laminin-5-dependent nuclear translocation of P-ERK, P-JNK, and NF-B and, through this mechanism, plays a role in epidermal growth and wound healing. Indeed, although both ß1 and v integrins can support epidermal proliferation and migration (15, 41), the epidermis of ß4 mutant mice displays a significant proliferative defect and it undergoes reepithelialization at a reduced rate, presumably because of decreased epidermal migration. In addition, the ß4 mutant mice display defective postnatal angiogenesis (37). Thus, whereas the adhesive function of 6ß4 is essential for epidermal stability, and its loss is incompatible with life (10, 53), signaling by the ß4 substrate domain plays a role in epidermal homeostasis and repair and in angiogenesis. The observation that the ß4 mutant mice are viable and fertile and do not display evident abnormalities in the absence of stress suggests that the novel signaling mechanism identified here does not contribute to embryonic development and normal adult life. Alternatively, other integrins may play redundant or compensatory roles.

We find that the ß4 substrate domain performs key functions in epidermal cells. Upon plating on laminin-5, the mutant keratinocytes display poor mitogenic response to EGF, and they rapidly undergo apoptosis upon removal of the growth factor. These defects are not observed in cells plated on fibronectin, indicating that the ß4 substrate domain is necessary for cell proliferation and survival on laminin-5 but not other permissive matrix substrates. In addition, keratinocytes lacking the ß4 substrate domain migrate inefficiently in response to EGF, providing evidence that ß4 signaling controls keratinocyte migration. Although prior studies had suggested that 6ß4 activates promigratory signaling pathways in epidermal cells (43), our results are the first to provide genetic evidence that this is the case. Finally, the mutant keratinocytes show enhanced activation of Rac, spreading, and assembly of 3ß1-containing focal contacts on laminin-5, suggesting that loss of the ß4 substrate domain enhances the adhesive and signaling function of 3ß1, which also binds to laminin-5. These results suggest that the transdominant-negative effect that 6ß4 exerts on 3ß1 (36) is mediated by the ß4 substrate domain. In addition, they imply that the 3ß1 integrin is unable to compensate for the effects of loss of ß4 signaling on cell proliferation, survival, and migration. In fact, it is remarkable that the ß4 mutant keratinocytes are unable to migrate efficiently, despite upregulation of 3ß1, as this latter integrin clearly promotes migration on laminin-5 (6). Taken together, our observations suggest that 6ß4 activates potent promigratory signals able to overcome the partial suppression of 3ß1-dependent spreading.

Our conclusions stand in stark contrast with those reached recently by Sonnenberg and colleagues (42). These authors have used a K14-Cre transgene to delete ß4 from the basal layer of the skin of ß4 floxed mice but have obtained only a mosaic pattern of expression of Cre and, thereby, limited excision of floxed ß4. They report that the ß4-negative areas of the skin of these mice do not contain a decreased number of proliferating keratinocytes (42). We note, however, that it may have been very difficult to identify continuous sections of the epidermis of these mice totally lacking ß4 and, hence, to perform a statistically significant analysis. In addition, it is possible that the ß4-positive keratinocytes rescued the proliferation of their ß4-negative neighboring cells through a paracrine mechanism. Sonnenberg and colleagues have also failed to detect a proliferation defect in immortalized, p53-negative keratinocytes lacking ß4 under standard culture conditions. By contrast, we have documented a significant proliferation defect in early-passage primary keratinocytes lacking the ß4 signaling domain. Notably, this defect was evident when the cells were plated on laminin-5 but not when they were plated on fibronectin. We suspect that serum-derived factors, such as fibronectin or vitronectin, or loss of p53-mediated control of the cell cycle may have allowed the ß4-null immortalized keratinocytes to proliferate despite loss of 6ß4 signaling. Finally, Sonnenberg and colleagues also report that their p53-negative, ß4^–/– keratinocytes migrate more efficiently—not less efficiently—than the ß4^+/+ controls (42). The apparent contrast between the migratory behavior of keratinocytes lacking 6ß4 and those lacking only the ß4 signaling domain is fully consistent with the model that 6ß4-mediated assembly of hemidesmosomes opposes cell migration, whereas 6ß4 signaling promotes it. In line with this conclusion, prior studies have indicated that the EGF-R and other RTKs enhance phosphorylation of ß4, causing disruption of hemidesmosomes and increased keratinocyte migration (7, 32, 44).

What are the molecular mechanisms by which the ß4 substrate domain exerts its biological function? Our biochemical studies indicate that this segment of ß4 mediates activation of PI-3K-to-Akt and Ras-to-ERK signaling independently of the EGF-R, as anticipated from prior studies (30, 31, 48). Since inhibition of PI-3K, but not MEK, induces apoptosis of keratinocytes plated on laminin-5, it is likely that the ß4 substrate domain protects these cells from apoptosis through PI-3K-to-Akt signaling (Fig. 8). Thus, although 6ß4-mediated assembly of hemidesmosomes may impart polarity to epithelial cells and protect them from apoptotic insults (54), 6ß4 promotes cell survival also by a direct signaling mechanism. We note that the ß4 substrate domain is not necessary for efficient activation and nuclear translocation of P-Akt in the presence of EGF, suggesting that the antiapoptotic effect of 6ß4 signaling through PI-3K may be important only when the amounts of trophic factors available to the cell are limiting. In accordance with this model, we have not detected increased apoptosis in the epidermis of mutant mice in vivo.

View larger version (29K): [in this window] [in a new window]   FIG. 8. Model of integrin 6ß4 signaling.

What is then the mechanism by which 6ß4 promotes keratinocyte migration and proliferation? Our studies indicate that the EGF-R is unable to induce nuclear accumulation of P-ERK, P-JNK, and NF-B, and hence presumably transcription of target genes, in mutant keratinocytes adhering to laminin-5. This striking result indicates that the ß4 substrate domain is necessary for nuclear translocation of three distinct transcriptional regulators (Fig. 8). Prior studies have indicated that 5ß1-mediated adhesion to fibronectin promotes nuclear accumulation of P-ERK, but not P-JNK and P-p38, and this occurs through activation of Rac (1, 2, 20). The effect of 6ß4 signaling is more general, as it involves three distinct nuclear effectors, and it does not appear to be mediated by Rac, as the levels of activation of Rac are enhanced, not diminished, in ß4 mutant cells. In addition, whereas the effect of fibronectin-mediated adhesion on nuclear translocation of ERK requires changes in the actin cytoskeleton consequent to cell spreading (2), the ß4 mutant keratinocytes display defective nuclear accumulation of P-ERK, P-JNK, and NF-B in spite of increased spreading on laminin-5. Also, it has been reported that the leukocyte integrin Lß2 promotes nuclear translocation of the c-Jun coactivator JAB1 (3). However, this effect is mediated by cytohesin-1, which binds selectively to the integrin ß2 cytotail (39). These observations suggest that the mechanism by which the ß4 substrate domain promotes nuclear translocation of mitogen-activated protein kinases and NF-B is novel. Future studies will be required to elucidate it.

Our results provide evidence that 6ß4-dependent activation of NF-B and JNK is necessary for keratinocyte proliferation and migration (Fig. 8). In agreement with this hypothesis, genetic studies of dorsal closure in Drosophila melanogaster and wound healing in mice have indicated that JNK signaling and AP-1-dependent transcription are crucial for epidermal migration and proliferation (26, 28, 55). In addition, Ron cooperates with 6ß4 to promote NF-B signaling and keratinocyte migration (44). In apparent contrast to our results, prior transgenic studies have indicated that overexpression of dominant-negative IB promotes proliferation and inhibits differentiation of basal keratinocytes, whereas overexpression of the NF-B subunit p50 exerts the opposite effects (47). We note, however, that epidermis-specific deletion of the IB kinase IKKß, which mediates NF-B signaling in the skin, does not cause cell-autonomous hyperproliferation, or impaired differentiation, but rather inhibits proliferation of keratinocytes (38). Because the EGF-R activates NF-B (52) and NF-B participates in the transcriptional control of cyclin D (16), transient activation of NF-B may be required for progression of keratinocytes through the G₁ phase of the cell cycle. By contrast, persistent activation of NF-B may lead to the upregulation of genes encoding cytokines, such as tumor necrosis factor alpha and interleukin-1, and extracellular factors, such as growth inhibitory factor, which inhibit keratinocyte growth (19). In this model, the apparently contradictory effects of NF-B signaling in the skin are explained by two temporally and kinetically distinct roles of the transcription factor. Similar considerations may apply also to the apparent discrepancy between the reported role of Ras-ERK signaling in the control of epidermal proliferation (5) and our observation that inhibition of MEK with two distinct compounds inhibits keratinocyte proliferation on laminin-5 only modestly.

In conclusion, our results provide genetic evidence that 6ß4 signaling promotes epidermal growth and wound healing by controlling nuclear translocation of NF-B and P-JNK. These findings identify a novel integrin signaling mechanism and highlight its physiological function.

	ACKNOWLEDGMENTS

 
We thank K. Owaribe and R. Timple for reagents, the Transgenic and Knock-out Mouse Facility and the Molecular Cytology Facility of MSKCC for help, and members of the Giancotti lab for discussions.

This work was supported by NIH awards F32 CA97886 (to S.N.N.), R37 CA58976 (to F.G.G.), GTF01018 (to C.T.), and P30 CA08748 (to MSKCC).

	FOOTNOTES

 
* Corresponding author. Mailing address: Memorial Sloan-Kettering Cancer Center, Box 216, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-6998. Fax: (212) 794-6236. E-mail: f-giancotti{at}ski.mskcc.org.

S.N.N. and P.B. contributed equally.

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