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J. Biol. Chem., Vol. 275, Issue 41, 31896-31907, October 13, 2000

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Deposition of Laminin 5 by Keratinocytes Regulates Integrin Adhesion and Signaling^*

Beth P. Nguyen^§, Susana G. Gil^§, and William G. Carter^§¶

From the  Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and the ^§ Department of Pathobiology, University of Washington, Seattle, Washington 98195

Received for publication, July 18, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Deposition of laminin 5 over exposed dermal collagen in epidermal wounds is an early event in repair of the basement membrane. We report that deposition of laminin 5 onto collagen switches adhesion and signaling from collagen-dependent to laminin 5-dependent. Ligation of laminin 5 by integrin ₆₄ activates phosphoinositide 3-OH-kinase (PI3K) signaling. This activation allows for adhesion and spreading via integrin ₃₁ on laminin 5 independent of RhoGTPase, a regulator of actin stress fibers. In contrast, adhesion and spreading on collagen via ₂₁ is Rho-dependent and is inhibited by toxin B, a Rho inhibitor. Deposition of laminin 5 and ligation of ₆₄ increases PI3K-dependent production of phosphoinositide di- and triphosphates, PI3K activity, and phosphorylation of downstream target protein c-Jun NH₂-terminal kinase. Conversely, blocking laminin 5-deposition with brefeldin A, an inhibitor of vesicle transport, or with anti-laminin 5 monoclonal antibodies abolishes the PI3K-dependent spreading mediated by ₃₁ and phosphorylation of c-Jun NH₂-terminal kinase. Studies with keratinocytes lacking ₆₄ or laminin 5 confirm that deposition of laminin 5 and ligation by ₆₄ are required for PI3K-dependent spreading via ₃₁. We suggest that deposition of laminin 5 onto the collagen substratum, as in wound repair, enables human foreskin keratinocytes to interact via ₆₄ and to switch from a RhoGTPase-dependent adhesion on collagen to a PI3K-dependent adhesion and spreading mediated by integrin ₃₁ on laminin 5.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Laminin 5 is a major adhesive component of many epithelial basement membranes (BMs)¹ and is secreted by epithelial cells in tissue and in culture. Injury of epidermis activates transcription and deposition of laminin 5 into the provisional BM by the leading keratinocytes of the epidermal outgrowth that migrates into the wound bed (1-4). The activated expression of laminin 5 occurs within hours after injury and prior to expression of laminin 10/11 or type VII collagen, attesting to the import of laminin 5 for tissue repair. Deposition of laminin 5 over the exposed collagen and fibronectin is required for the repair of the BM and for re-establishing anchorage of the epithelium to the BM via integrin ₆₄ in hemidesmosomes (5-9). Here, we have examined the function of laminin 5 deposited onto exposed dermal collagen in regulating adhesion and signaling in epidermal outgrowths.

Based on in vivo and in vitro studies, keratinocyte interactions with laminin 5 are mediated by integrins ₆₄ and ₃₁; interactions with ₆₄ mediate anchorage, whereas interactions with ₃₁ mediate motility (9-11). Laminin 5, composed of ₃, ₃, and ₂ chains, also interacts with type VII collagen to link the epidermis to the dermis (12-14). Laminin 5 is synthesized and secreted by cultured keratinocytes in a precursor form containing a 200-kDa ₃ chain (₃-200) and a 140-kDa ₂ chain (₂-140) (15). Precursor laminin 5 is deposited into the provisional BM of the wound but is absent from the mature BM (2, 10). Proteolytic processing of ₂-140 by MT1-MMP, a membrane-bound protease, is suggested to increase migration on laminin 5 (16). Similarly, proteolytic processing of ₃-200 occurs extracellularly (15, 17). Cleavage by plasmin has been suggested to convert precursor laminin 5 from a migration ligand to an anchorage ligand in hemidesmosomes (17). However, processed laminin 5 is also reported to promote motility or scatter via integrin ₃₁ (18, 19). Alternatively, proteolytic processing of laminin 5 may regulate deposition of laminin 5 instead of regulating interactions with integrins. Related to this, the carboxyl-terminal end of the ₁ chain of laminin 1 and the ₂ chain of laminin 2 contain heparin-binding subdomains consisting of laminin globular (LG) repeats 4 and 5 (LG4/5). Interactions of LG4/5 with dystroglycan mediates assembly of laminins 1 and 2 in BM (for review, see Ref. 20). It is not clear if the LG4/5 subdomain of laminin 5 ₃-200 interacts with heparin or other cellular or dermal components to facilitate deposition of laminin 5.

Interactions of integrin ₆₄ with laminin 5 generate both an anchorage function and a signaling function (11). Inherited defects in expression of laminin 5 or ₆₄ inhibit assembly of mature hemidesmosomes and generate severe or lethal blistering of quiescent epithelium in individuals with junctional epidermolysis bullosis (5, 21-24) and junctional epidermolysis bullosis combined with pyloric atresia (JEB-PA). Therefore, the anchorage function via ₆₄ to laminin 5 is physiologically significant. The signaling function of ₆₄ regulates the cell cycle and cell invasion (for reviews, see Refs. 25 and 26). Ligation of ₆₄ activates the Ras-ERK and Rac-JNK signaling pathways through Shc (27). In addition, ₆₄ signals through PI3K, a target effector of Ras that activates RacGTPase, to mediate invasion by carcinoma cell lines (28, 29). It has been suggested that ₆₄ mediates invasion of carcinoma cells via both its adhesive and signaling functions through PI3K (28). However, in normal human keratinocytes, ₆₄ plays a primary anchorage role that is independent of actin or FAK phosphorylation (9, 11, 30). Conceivably, ₆₄ may have different adhesion and signaling functions in normal keratinocytes as compared with carcinoma cell lines.

The primary mediator of motility on laminin 5 in normal keratinocytes is integrin ₃₁, not ₆₄ (9, 11, 18, 31, 32). Adhesion dependent signals mediated by ₁ integrins are transmitted via tyrosine phosphorylation, changes in intracellular pH, activation of protein kinases and mitogen-activated protein kinase pathways, and recruitment of regulators involved in focal adhesion formation such as focal adhesion kinase (FAK). Focal adhesion formation and phosphorylation of FAK are downstream of RhoGTP activity (33, 34). Curiously, integrin ₃₁ in keratinocytes is not readily detectable in focal adhesions on laminin 5 in contrast to the robust localization of ₂₁ in focal adhesions on collagen (35, 36). This suggests that interactions of keratinocytes with laminin 5 differ from interactions with collagen and the differences may be attributable to signaling through Rho that affects assembly of focal adhesions. Recently, we showed that laminin 5 interactions with ₃₁ promote assembly of gap junctions and increase intercellular communication in basal keratinocytes (2). Collagen or fibronectin adhesion via ₂₁ and ₅₁, respectively, does not promote gap junction intercellular communication. Ligation of ₂₁ by collagen, however, promotes expression of matrix metalloproteinase 1 (MMP1) that is necessary for migration on collagen in the wound bed (37-40). Therefore, BM laminin 5 provides instructional signals that are distinct from those of dermal collagen or fibronectin. However, the signaling pathways resulting from ₃₁-laminin 5 and ₂₁-collagen ligations have yet to be characterized.

We wished to determine if deposition of laminin 5 onto collagen during wound repair changes signaling from collagen-dependent to laminin 5-dependent and if signaling pathways required for adhesion and spreading on laminin 5 were different than on collagen. We found that laminin 5 is deposited as a precursor form by a subpopulation of keratinocytes migrating at the leading edge of the wound outgrowths. Further, laminin 5 requires and stimulates signaling pathways for adhesion and spreading of keratinocytes that are different then collagen. Deposits of laminin 5 that interact with integrin ₆₄ are required for keratinocyte spreading on laminin 5 in the absence of RhoGTPases. We suggest that deposition of laminin 5 by keratinocytes at the leading edge of the wound outgrowth determines which signaling pathways are used by keratinocytes for motility. The advantage of dual signaling pathways for migration of keratinocytes on laminin 5 are discussed in relation to epithelial cell interactions and wound repair.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Cell Culture-- Primary keratinocytes from normal human foreskins (HFKs) and keratinocytes from individuals with junctional epidermolysis bullosa-pyloric atresia (JEB-PA; inherited defect in the ITGB4 gene) or from individuals with junctional epidermolysis bullosa-gravis (JEB-G; inherited defect in the LAMB3 gene) were prepared as described by Boyce and Ham (41). Primary and secondary keratinocyte cultures were maintained in serum-free keratinocyte growth media (KGM; Clonetics Corp., San Diego, CA). JEB-PA and JEB-G keratinocytes were infected with an LXSN retroviral vector (42) expressing E6 and E7 oncogenes from human papilloma virus to extend life in culture. When JEB-PA and JEB-G keratinocytes were compared with HFKs (Figs. 4 and 5), the HFKs were also infected with the same retroviral vector. Early passages (p1-15) were compared to avoid possible changes in adhesion or signaling due to later passages. JEB-PA individual JF/VS9-3-96 has premature termination mutations in both alleles of the ITGB4 gene encoding the ₄ integrin subunit (JF/VS9-3-96 is proband from family two in Ref. 43). Three other individuals suspect for defects in ITGB4 were also examined. They are classified clinically as JEB-PA based on blistering phenotype, immature hemidesmosome structures, defective anchorage on laminin 5 in assays specific for ₆₄, and decreased expression of ₄ in tissue and culture. Causal mutations are currently under investigation. The individual with JEB-G does not deposit laminin 5 in culture and has homozygous premature termination mutations in both alleles of the LAMB3 gene encoding the ₃ chain of laminin 5.

Explants of foreskin were grown on collagen layers (epibole cultures) as described previously (2) and used as a wound model. The collagen layers were prepared by drying rat tail collagen on Petri dishes (Collaborative Research, acid-soluble, diluted to 100 µg of collagen/ml of water).

Characterization of the Precursor Laminin 5-- mAbs D2-1, C2-5, and B4-6 immunoblot the ₃, ₃, and ₂ chains of laminin 5, respectively, and were prepared as described previously (44). They were used to characterize precursor and mature forms of laminin 5 (see "Results"). Rabbit polyclonal Ab R9883 was prepared against a synthetic peptide (LTTLRIPVWKSFFGCLRNIHVNH) corresponding to residues 1668-1690 near the carboxyl terminus of the LG5 subdomain of the ₃ chain of precursor laminin 5 (45, 46). Peptides ₃-35 and ₃-37 (₃-35/37) derived from the ₃ chain of precursor laminin 5 were found to interact with heparin and were affinity-purified from conditioned culture medium of HFKs by chromatography on immobilized heparin. After further purification by preparative gel electrophoresis, ₃-35/37 was partially sequenced by mass spectroscopy at the Harvard Microsequencing Facility (Harvard, Cambridge MA). This identified the amino acid sequence APVYLGSPPSGKPK in ₃-35/37 as identical to residues 1484-1497 near the amino-terminal end of the LG4 subdomain of the ₃ chain (45, 46).

In Vitro Cell Adhesion Assay-- Human placental collagen type I was prepared in the lab from human placenta as described (47) and coated onto 12-well culture plates overnight at 4 °C at the concentration of 10 µg/ml. Laminin 5-coated plates were prepared as described previously (11) and briefly as follows; HFKs were grown on TC plates, then detached by brief digestion with 0.5% trypsin (w/v)/EDTA (Sigma). Laminin 5 remaining on the TC surface was blocked with 0.5% w/v heat-denatured BSA/PBS for 30 min and used for adhesion studies. Cell adhesion to these laminin 5 plates was completely inhibited with anti-laminin 5 mAb C2-9 (11). To generate laminin 5-conditioned collagen, HFKs were plated onto collagen-coated assay plates for 24 h. This allowed the cells to deposit laminin 5 onto the collagen substratum. The cells were trypsinized off, leaving the substrata for adhesion assay. Coated plates were blocked with 0.5% w/v heat-denatured BSA/PBS for 30 min. Cultured cells were either not treated for controls or pretreated with 50 ng/ml toxin B (gift from Laurie Neville, Tech Labs, Inc.), 50 nM wortmannin (Sigma), or 5 mM LY294002 (Sigma) for 3 h. Cells were then suspended with trypsin (w/v)/EDTA and labeled with 0.5 µM calcein-AM (Molecular Probes, Eugene, OR). Labeled cells were then plated onto ligand-coated plates in the presence or absence of inhibitors (e.g. anti-₆ antibody, G0H3) for 1 h as indicated. Total cell fluorescence (prewash) was read on CytoFluorII fluorescence plate reader (PerSeptive Biosystems, MA). Cells were washed three times with PBS, and remaining cell fluorescence (post-wash) was again read. An empty well containing just buffer medium where no cells were added was read to give base-line fluorescence that was subtracted from raw data. In every adhesion experiment, triplicates of each condition were done. Percent fluorescent cells adhered was calculated as: % fluorescent cell adhered = (post-wash fluorescence reading  base-line fluorescence)/(total (prewash) fluorescence reading  base-line fluorescence). Cells were then fixed with 2% formaldehyde in 0.1 M sodium cacodylate/0.1 M sucrose for 20 min and viewed under phase microscope for documentation of cell spreading.

In Vitro Tissue Adhesion Assay-- The in vitro tissue adhesion assay was performed as a variation of the assay described (5-9). In brief, epidermis of neonatal foreskin was separated from the dermis by incubation with EDTA/NaCl to expose the underlying BM containing laminin 5. The dermis is abundant in interstitial collagen (types I and III) and fibronectin. Tissue sections (6-µm thick) were cut from OCT-embedded frozen split skin and adhered onto lids of 35-mm Petri dishes, washed twice in PBS, and blocked in 0.5% heat-denatured BSA/PBS for 30 min. HFKs were untreated or pretreated with toxin B (50 ng/ml) for 3 h prior to assay. Calcein-labeled HFKs were plated onto the immobilized tissue sections and allowed to adhere for 1 h at 37 °C. Non-adherent cells were washed twice with PBS. Adherent cells were fixed with 2% formaldehyde, mounted, and examined by phase and fluorescence microscopy.

Spreading Assay on Immobilized Antibodies-- Antibody-coated surfaces were prepared as described previously (2). Cells were untreated or pretreated for 3 h with toxin B (50 ng/ml), wortmannin (50 nM), or the combination, then plated onto immobilized anti-₃ (P1B5), anti-₂ (P1H5), or anti-₆ (G0H3) mAbs for 1 h. Cells were fixed, and phase images were taken to document cell spreading.

Immunofluorescence Staining-- Cells were plated onto surfaces coated with immobilized anti-₃ antibodies or laminin 5 for 30 min. Cells were then treated ± toxin B (50 ng/ml) or ± wortmannin (50 nM) for 3 h. Cells were fixed for 20 min with 2% formaldehyde in 0.1 M sucrose, 0.1 M sodium cacodylate (pH 7.2), permeabilized with 0.5% Triton X-100 for 10 min, and then blocked in 0.5% heat-denatured BSA in PBS for 30 min. P1F2 (anti-₃), VIN-11-5 (anti-vinculin; Sigma), and anti-FAK (ICN) were incubated with the cells for 1 h at room temperature. The cells were washed, and bound antibody was reacted with fluorescein isothiocyanate-conjugated, goat anti-mouse antibody (Cappel). Rhodamine-conjugated phalloidin (Molecular Probes) was incubated for 10 min and washed. For laminin 5 staining, attached cells were extracted with Triton, then the matrix was stained with biotinylated anti-laminin 5 mAb (P1E1). Bound biotinylated mAb was detected with rhodamine-avidin. Immunofluorescence was visualized using a Zeiss microscope equipped with epifluorescence. Images were photographed with TMAX 400 ASA film.

Immunoblotting-- HFKs were untreated or pretreated with 50 nM wortmannin for 3 h, trypsin-suspended, and plated onto laminin 5-, collagen-, or immobilized antibody-coated surfaces for 1 h in the absence or presence of inhibitory anti-laminin 5 antibody (C2-9). Cells were solubilized directly with 1% Triton X-100 in PBS containing 5 mM EDTA, 50 µM Na₃VO₄, 10 mM NaF, and protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, 1 µg/ml pepstatin, 10 µg/ml aprotinin, 1 µg/ml leupeptin). Detergent-soluble extracts were quantitated and separated on SDS-polyacrylamide gels (48). Proteins were blotted onto nitrocellulose, blocked in 1% nonfat milk, and incubated with primary antibodies: anti-phosphotyrosine (PY20; ICN), anti-phosphorylated JNK (JNKp(G7); Santa Cruz), anti-JNK all (JNK(c17); Santa Cruz), anti-ERK2 (Transduction Laboratories), anti-keratin (K8.13, Sigma), anti-laminin 5 (D21, C25, R9883). Primary antibodies were reacted with peroxidase-conjugated rabbit anti-mouse IgG (RAMP; Dako) or goat anti-rabbit IgG (ICN) for 1 h. Blots were developed with ECL chemiluminescence (Amersham Pharmacia Biotech) and direct exposure to Hyperfilm MP (Amersham Pharmacia Biotech). Densitometry was performed as described previously (1-4).

Steady-state Cellular Phosphoinositide Levels-- Phospholipid labeling was performed as described previously (44). Briefly, phosphate-free Dulbecco's modified Eagle's medium containing [³²P]P_i (250 µCi/ml) was incubated for 1 h with HFKs that were either adherent (A), trypsin-suspended (S), or plated onto 35-mm dishes coated with laminin 5 (Lam5) or collagen type I (Col, 10 µg/ml). Radioactive culture supernatants were discarded, cells were washed, then extracted with MeOH:CHCl₃ (2:1) +0.63 mg/ml butylated hydroxytoluene, 10 µg/ml phosphatidylinositol carrier. The organic extracts containing labeled lipids were mixed with CHCl₃:HCl (2.4 N) 1:1, and the aqueous phase was discarded. Organic phases dried under nitrogen. Lipids were resuspended in 100 µl of CHCl₃:MeOH (2:1 v/v) for thin layer chromatography using 20 × 20-cm oxalate-coated silica gel plates and developed with CHCl₃:acetone:MeOH:acetic acid:H₂O (80:30:26:24:14 v/v/v/v/v). Phosphoinositide standards were detected with iodine vapor and labeled products with exposure to Hyperfilm.

PI3K Assay-- PI3K enzymatic activity was assayed as described previously by (49). HFKs were suspended, or adherent on immobilized anti-₂ (P1H5), anti-₃ (P1B5), or anti-₆ (G0H3) integrin antibodies for 1 h, then extracted 1% v/v Nonidet P-40 detergent in kinase buffer (100 mM NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 0.1 mM sodium orthovanadate, 20 mM MgCl₂). PI3K proteins were immunoprecipitated from the extract with rabbit polyclonal anti-PI3K (Upstate Biotechnology, Inc.). Immunoprecipitates were incubated with 20 µg of phosphatidylinositol 4,5-biphosphate (Avanti Polar Lipids, Inc.) and [-³²P]ATP (30 µCi) in kinase buffer for 15 min at 37 °C. Immunoprecipitates were removed by centrifugation; supernatant solutions were extracted for lipids, spotted onto TLC silica gel 60 plates precoated with 5% oxalate acid, and developed in propanol plus 2 M acetic acid 65:35 (v/v).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Leading Keratinocytes in Epidermal Outgrowths Express Laminin 5 and Are Sensitive to Toxin B-- We used skin explants placed in organ culture to study keratinocyte migration over collagen and the effects of deposited laminin 5. Biopsies of skin were placed onto collagen-coated surfaces, allowing keratinocytes to migrate out as a continuous epithelial outgrowth. Keratinocytes at the leading edge of the outgrowth are in direct contact with collagen, and express elevated cytoplasmic levels of a precursor form of laminin 5 recognized by mAb D2-1 (45, 46). This distinguishes the leading keratinocytes from the following cells that express little cytoplasmic laminin 5 (Fig. 1a; arrowheads indicate leading cells, arrows identify following cells). Precursor laminin 5 is deposited by the leading cells onto the collagen substratum over which the following cells migrate. Extraction of the epithelial outgrowth with Triton X-100 detergent allows antibody access to the substratum and exposes the deposited laminin 5 under the following cells (Fig. 1b, arrow indicates direction of migration). Note that the extraction also removes the cytoplasmic precursor laminin 5 from the leading cells (dotted line demarks leading edge).

View larger version (151K): [in this window] [in a new window]   Fig. 1.   Leading keratinocytes in epidermal outgrowths express laminin 5 and are toxin B-sensitive. Skin explants were cultured on a collagen surface. a, immunofluorescence staining of precursor laminin 5 with mAb D2-1 shows positive cytoplasmic expression in leading cells (white arrowheads) compared with following confluent cells (white arrows). b, epidermal outgrowth was extracted with 0.5% Triton X-100 and the exposed substrata was stained for laminin 5 with mAb D2-1 to show deposited laminin 5 beneath confluent following cells. White arrow indicates direction of epithelial cell outgrowth. White dashed lines indicate the leading front. c, phase image of an epidermal outgrowth showing leading edge keratinocytes migrating on collagen and the following confluent population migrating on deposits of laminin 5. Black arrow is used as a point of reference. d, the same field as in panel c was treated with 50 ng/ml toxin B for 10 min then re-photographed. e, 2-fold magnification of panel d to show rounding of leading cells (black arrow). Toxin B caused rounding of the leading cells on collagen but not the following cells on the deposited laminin 5.

mAb D2-1 was used to characterize the precursor form of laminin 5 (₃₃₂) synthesized and deposited by leading cells. Results in Fig. 2 establish three points. (i) Precursor laminin 5 immunoprecipitated with anti-laminin 5 mAbs (D2-1, C2-5, B4-6) from the cell layer of culture HFKs is composed of chains ₃-200 + ₃-140 + ₂-140 (Fig. 2A; panel CELLS). mAb D2-l reacts with the 200-kDa form of the ₃ chain of precursor laminin 5 (₃-200), and two peptides ₃-35 and ₃-37 (₃-35/37; Fig. 2A, panel MEDIUM). Rabbit polyclonal Ab R9882 specific for the carboxyl-terminal LG 5 subdomain of ₃-200 reacts with ₃-35/37 establishing the origin of the peptides from the carboxyl terminus of ₃-200 (Fig. 2A, IP:BLOT). (ii) After secretion into the culture medium, precursor laminin 5 is proteolytically processed into mature laminin 5 composed of chains ₃-165 + ₃-140 + ₂-100 (Fig. 2A, MEDIUM). Pulse-chase studies with D2-1 (results not shown) indicated that proteolytic processing of ₃-200 in the medium occurs as a post-secretory event and yields ₃-165 and ₃-35/37. (iii) Peptides ₃-35/37 bound heparin and were affinity purified on immobilized heparin (Fig. 2B). Partial sequencing of purified ₃-35/37 identified the amino acids sequence identical to residues 1484-1497 near the amino-terminal end of the LG4 subdomain of ₃-200 (45, 46). Based on the relative molecular mass of ₃-35/37 and the presence of amino acid sequences from both LG4 and LG5, we suggest that cleavage of ₃-200 occurs extracellularly at two sites between LG3 and LG4 to release the heparin-binding ₃-35/37. Based on the staining with mAb D2-1 (Fig. 1, a and b), we conclude that precursor laminin 5 is synthesized and deposited primarily by the leading keratinocytes in epidermal outgrowths and the precursor and or processed laminin 5 deposited on the collagen substratum interacts with the following keratinocytes.

View larger version (46K): [in this window] [in a new window]   Fig. 2.   Precursor laminin 5 (332) contains a heparin-binding LG4/5 subdomain in 3-200 recognized by mAb D2-1. A, peptide 3-35/37 reacts with mAb D2-1 and derives from the carboxyl terminus of 3-200: HFKs were metabolically labeled with [35S]Met (4 h, 50 µCi/ml) in KGM (Met-free containing 100 µg/ml carrier BSA). Detergent extracts of the labeled cell layer (CELLS; 1% Triton X-100 detergent (v/v) in PBS for 20 min) and the conditioned culture medium (MEDIUM) containing secreted labeled proteins were immunoprecipitated (IP) with mAbs SP2 (lane 1; negative control), D2-1 (lane 2; anti-laminin 3 subunit), C2-5 (lane 3; anti-laminin 3 subunit), B4-6 (lane 4; anti-laminin 2 subunit) and G2'-1 (lane 5; anti-thrombospondin, an unrelated control mAb). Each anti-laminin 5 mAb precipitates similar precursor laminin 5 composed of 3-200 + 3-140 + 2-140 from the cell layer. However, from the MEDIUM panel, C2-5 and B4-6 immunoprecipitate processed laminin 5 composed primarily of 3-165 + 3-140 + 2-100 while D2-1 immunoprecipitates primarily processed 3-35/37 and small amounts of precursor laminin 5. Immunoprecipitation with mAb D2-1 (lane 2) but not C2-5 (lane 3) followed by immunoblotting (IP:BLOT) with rabbit polyclonal Ab R9883 prepared against LG5 subdomain of 3-200 (see "Experimental Procedures") reacts with both 3-35 and 3-37. Ig indicates immunoglobulin heavy chain in the immunoprecipitates. B, 3-35/37 bind immobilized heparin: 3-35/37 were purified from conditioned culture medium of HFKs by affinity chromatography on immobilized heparin (Sigma) followed by washing with PBS and elution with a linear 0-1 M NaCl gradient in PBS. Fractions from the elution profile (FRACTIONS) run on SDS-polyacrylamide gel electrophoresis (12%) were stained with Coomassie Blue for protein. Multiple high molecular mass bands were bound and eluted early in the gradient (fraction 2 indicated by *), and 3-35/37 eluted late in the NaCl gradient (fraction 5). 3-35/37 purified on heparin-agarose (fraction 5) immunoblotted (BLOT) with mAb D2-1.

We examined signaling differences in leading and following keratinocytes that interact with collagen and deposited laminin 5, respectively. We compared the sensitivities of leading and following keratinocytes to toxin B, an inhibitor of RhoGTPase that regulates actin stress fibers. Toxin B glucosylates Thr³⁷ of Rho proteins, thereby rendering them inactive (45, 46). Toxin B (50 ng/ml) was added to cultures of skin explants grown on collagen, and the epidermal outgrowth was photographed at the zero time point (Fig. 1c, black arrow indicates leading edge) and also at 10 min (Fig. 1, d and e; panel e is a 2-fold magnification of d to better show cell rounding at the leading edge). Toxin B selectively rounded the leading cells, whereas following cells remained adherent and spread. Thus, adhesion and spreading of leading keratinocytes on collagen is toxin B-sensitive, whereas adhesion and spreading of following cells on deposits of laminin 5 is toxin B-insensitive.

Integrin ₂₁ Interaction with Collagen Is Toxin B-sensitive Compared with ₃₁-Laminin 5-- We compared toxin B sensitivity of HFKs adherent on exogenous collagen via ₂₁ to cells adherent on laminin 5 via ₆₄ and ₃₁. Cultured HFKs were plated onto exogenous laminin 5 (Fig. 3, a and c) or collagen (b and d) for 30 min. Toxin B (50 ng/ml) was added, and cells were photographed at time 0 (a and b) and at 30 min (c and d). Cells were stained with rhodamine-conjugated phalloidin for actin. Consistent with results in Fig. 1, HFKs in the presence of toxin B on collagen rounded up while cells on laminin 5 retained their spread morphology.

View larger version (151K): [in this window] [in a new window]   Fig. 3.   Toxin B induces rounding of HFKs on collagen but not on laminin 5. Cultured HFKs were plated onto exogenous laminin 5 (Lam5; a and c) or collagen (ColI; b and d) for 30 min. Toxin B (50 ng/ml) was then added and cells were photographed at time 0 (a and b) and at 30 min (c and d) and was stained with rhodamine-conjugated phalloidin for actin. HFKs in the presence of toxin B on collagen, similar to the epibole model, rounded up while cells on laminin 5 retained their spread morphology.

Initial adhesion and spreading of HFKs on collagen was inhibited by toxin B in a dose-dependent manner (Fig. 4A, open circles). In contrast, adhesion and spreading of HFKs on laminin 5 was unaffected by treatment with toxin B (Fig. 4, A (black squares) and B (panel b)). Although both ₆₄ and ₃₁ contribute to adhesion on laminin 5, the spreading on laminin 5 in the presence of toxin B was blocked by anti-₃ antibodies (P1B5; Fig. 4B, panel c) and not by anti-₆ antibodies (data not shown). This confirmed that ₃₁ mediates spreading on laminin 5 in the presence of toxin B. 

View larger version (120K): [in this window] [in a new window]   Fig. 4.   Integrin 31 mediates cell spreading on laminin 5 in the presence of toxin B. A, HFKs were treated with 0, 10, 25, 50, and 100 ng/ml toxin B for 3 h. Treated HFKs were trypsin-suspended, labeled with calcein, and adhered for 1 h at 37 °C to Petri dishes coated with laminin 5 (solid squares) or collagen (open circles). Non-adherent cells were washed away with PBS, and adherent, labeled cells were quantitated on a CytoFluorII reader. B, phase images were taken of HFKs adherent and spread onto laminin 5 following no treatment (Control, panel a) or treatment with 50 ng/ml toxin B for 3 h prior to adhesion (panels b and c) and inhibited with anti-3 (P1B5) antibodies during the adhesion assay (panel c). C, adhesion of untreated HFKs (panels a and c) or toxin B-treated HFKs (panels b and d) to cryostat sections of neonatal foreskin. The foreskin was salt-split to expose the BM-laminin 5 prior to sectioning. HFKs were prelabeled with calcein prior to adhesion to tissue. All assays were done at 37 °C in the presence of anti-6 (G0H3) mAb to block adhesion via 64. Phase images (panels c and d) were taken along with their fluorescent counterparts (panels a and b).

We used a tissue adhesion assay to determine whether native collagen in tissue could support adhesion of HFKs in the presence of toxin B (45, 46). HFKs were treated with toxin B (50 ng/ml for 3 h), fluorescently labeled with calcein, and then assayed for adhesion on cryostat sections of skin (Fig. 4C). For these studies, the epidermis was separated from the skin by incubation with EDTA/NaCl in order to expose the underlying BM containing laminin 5. The dermis is rich in interstitial collagens, including types I and III, and fibronectin. Untreated control cells adhered to both laminin 5 in the BM and to the dermis that contains collagen (Fig. 4C, panels a and c). In contrast, toxin B-treated HFKs only adhered to the BM and not to the dermis (Fig. 4C, panels b and d). Thus, ₂₁ failed to mediate adhesion in the presence of toxin B even on native collagen in dermis. In contrast, toxin B had no observable inhibitory effects on adhesion to laminin 5 in the BM.

Laminin 5 Is Required for Spreading via ₃₁ in the Presence of Toxin B-- As seen in Figs. 1, 3, and 4, HFKs interacting with laminin 5 can spread via ₃₁ in the presence of toxin B. We evaluated the import of laminin 5-deposition on this toxin B-insensitive spreading. HFKs were plated onto immobilized anti-₃ antibody surfaces. The cells adhered, spread, and deposited laminin 5 onto the anti-₃ substratum in the absence (Fig. 5, A (Control, panel a) and B (panel a)) or in the presence of toxin B (Fig. 5, A (ToxB, panel d) and B (panel b)). Blocking laminin 5 deposition with brefeldin A (Fig. 5B, panel d) inhibited HFK spreading in the presence of toxin B (Fig. 5A, panel g). Blocking HFK interactions with laminin 5 using inhibitory antibody C29 also inhibited cell spreading in the presence of toxin B (Fig. 5A, compare panels b and e). Keratinocytes from an individual with JEB-G with inherited defects in laminin 5 expression (50) were unable to spread on anti-₃ immobilized surface in the presence of toxin B (Fig. 5A, panel f compared with untreated control panel c). These JEB-G keratinocytes do not express nor deposit laminin 5 in culture (data not shown). However, addition of exogenous laminin 5 allowed JEB-G keratinocytes to spread in the presence of toxin B (Fig. 5A, panel h). Therefore, either endogenous or exogenous laminin 5 on the substrate is required for the toxin B-insensitive cell spreading via ₃₁.

View larger version (150K): [in this window] [in a new window]   Fig. 5.   Laminin 5 is required for Rho-independent spreading via 31. A, adhesion of HFKs on immobilized anti-3 mAb (P1B5) antibody in the absence (HFK) or presence of inhibitory anti-laminin 5 antibodies (HFK+C29) were compared with adhesion of JEB-G keratinocytes (JEB-G). Cells were untreated (Control, panels a-c) or treated with 50 ng/ml toxin B (ToxB, panels d-h) or treated with toxin B + brefeldin A (ToxB+BrefA, panel g) for 3 h, trypsin-suspended, and plated onto immobilized anti-3 antibodies for 1 h, and then fixed with formaldehyde. Exogenous laminin 5 was added to JEB-G (h) keratinocytes that were treated with 50 ng/ml toxin B. B, laminin 5 deposition onto the immobilized anti-3 mAb surfaces was examined. HFKs were untreated (panel a) or treated with toxin B (panel b), LY294002 (panel c), or brefeldin A (panel d) and then plated onto immobilized anti-3 mAb for 1 h. Cells were extracted and substratum was stained with biotinylated anti-laminin 5 monoclonal antibody (P1E1). Biotinylated anti-laminin 5 mAb was detected with rhodamine-avidin. Deposits of laminin 5 could be detected in untreated cells, and cells treated with toxin B or LY294002. However, little was seen in cells treated with brefeldin A.

Toxin B-insensitive Spreading via ₃₁ on Laminin 5 Requires ₆₄ and PI3K-- Since ₆₄ also contributes to adhesion to laminin 5, we determined if interaction of ₆₄ with laminin 5 participates in the toxin B-insensitive spreading via ₃₁. Individuals with JEB-PA bear mutations in the ITGB4 gene that encodes the integrin ₄ subunit (51, 52) and display severe to lethal defects in anchorage function of ₄ (13). Keratinocytes cultured from a JEB-PA individual (Fig. 6A, striped bars) attached and spread on exogenous laminin 5 via ₃₁ in the absence of toxin B comparable to normal HFK controls (Fig. 6A, solid bars). However, JEB-PA keratinocytes failed to adhere and spread on exogenous laminin 5 in the presence of toxin B (Fig. 6A, + Toxin B, striped bars). Keratinocytes from three additional individuals suspect for JEB-PA were also examined for sensitivity to toxin B. Toxin B inhibited adhesion and spreading on laminin 5 for all three of the suspect JEB-PA keratinocytes (data not shown). Therefore, ligation of ₆₄ by laminin 5 is required for keratinocyte spreading via ₃₁ in the presence of toxin B. 

View larger version (99K): [in this window] [in a new window]   Fig. 6.   Toxin B-insensitive adhesion and spreading mediated by 31 requires 64 and PI3K. A, HFKs (solid bars) and keratinocytes from an individual (JF/VS 9-3-96) with JEB-PA (striped bars) lacking integrin 64 were treated with (+) or without () 50 ng/ml toxin B for 3 h, trypsin-suspended, labeled with calcein, and assayed for adhesion to exogenous laminin 5. B, HFKs were untreated (Control), or treated with toxin B (TxB; 50 ng/ml), wortmannin (Wt; 50 nM), or toxin B + wortmannin combination for 3 h, trypsin-suspended, labeled with calcein, and added to surfaces coated with exogenous laminin 5 (solid bars) or collagen (striped bars). The assay was done in the presence or absence (data not shown) of anti-6 mAb (G0H3). In either case no spreading occurred in the presence of toxin B + wortmannin. In the absence of anti-6, adhesion was unaffected but spreading was inhibited with toxin B + wortmannin. C, phase images of HFKs that were untreated (Control, panels a and b), or treated with toxin B (TxB, d and e), wortmannin (Wort., g and h), or the combination of toxin B and wortmannin (j and k) for 3 h, and plated onto immobilized anti-integrin 3 (P1B5; a, d, g, and j) or anti-integrin 2 (P1H5; b, e, h, and k) for 1 h of adhesion. HFKs were compared with JEB-PA keratinocytes when adherent on immobilized anti-3 antibodies either untreated (panel c), or treated with toxin B (panel f), wortmannin (panel i), or the combination of toxin B and wortmannin (panel l).

We wished to identify the signaling pathway involved in the toxin B-insensitive spreading on laminin 5. A panel of known inhibitors of adhesion was examined for effects on HFK adhesion and spreading on laminin 5 in the presence of toxin B. In preliminary studies, wortmannin or LY294002, inhibitors of phosphoinositide 3-OH-kinase (PI3K; Ref 53), selectively inhibited ₃₁-mediated spreading on laminin 5 when toxin B was also present. Quantitative experiments were performed (Fig. 6B). HFKs were pretreated with toxin B alone, 50 nM wortmannin alone, or the combination of both, then assayed for adhesion to exogenous laminin 5 (solid bars) or collagen (striped bars). Untreated HFKs adhered and spread on both laminin 5 and collagen. Toxin B (TxB) selectively inhibited collagen adhesion whereas on laminin 5 the cells still adhered and spread. Wortmannin (Wt) by itself did not inhibit adhesion or spreading on either laminin 5 or collagen. However, the combination of toxin B and wortmannin (TxB + Wt) inhibited adhesion and spreading on both laminin 5 and collagen. Note that LY294002 generated results similar to wortmannin (results not shown) and had no inhibitory effects on the deposition of laminin 5 (Fig. 5B, panel c). Note that the assay was done in the presence or absence (data not shown) of anti-₆ mAb (GoH3) to block ₆₄ contribution to adhesion. In the absence of anti-₆ mAb spreading was inhibited by toxin B and wortmannin but adhesion occurred. Thus, ₃₁ mediates adhesion and spreading on laminin 5 via two pathways; one is PI3K-dependent, and the other is toxin B-sensitive.

Similar results were obtained when we assayed cell spreading on immobilized antibodies (Fig. 6C). We compared normal HFKs to JEB-PA keratinocytes adherent on immobilized anti-₃ antibodies under all assay conditions. JEB-PA keratinocytes spread in the absence but not the presence of toxin B (Fig. 6C, compare panels c and f) and was unaffected by wortmannin (panel i). HFKs spread on anti-₃ in the absence (panel a) or presence of toxin B (panel d) or wortmannin (panel g). HFK spreading was blocked only in the presence of a combination of inhibitors (panel j). Therefore, integrin ₆₄ plays a critical role in regulating ₃₁ spreading via the PI3K-dependent pathway.

Adhesion of HFKs on immobilized anti-₂ antibody was apparent under all assay conditions but spreading was inhibited in the presence of toxin B or toxin B + wortmannin but not by wortmannin alone (Fig. 6C, panels e, k, and h, respectively). The behavior of HFKs on immobilized anti-₂ is similar to the behavior of JEB-PA keratinocytes on anti-₃.

Inhibition of PI3K Increases Focal Adhesions and FAK Phosphorylation-- Laminin 5 interaction with ₆₄ is required for cell spreading on laminin 5 via ₃₁ in the presence of toxin B (Figs. 5 and 6). Further, PI3K is required for spreading on laminin 5 via ₃₁ in the presence of toxin B. We hypothesized that inhibition of PI3K would increase Rho dependence and increase Rho activity. Rho activity is essential for focal adhesion formation when cells interact with ECM ligands via integrin receptors (33, 34). We examined focal adhesions in HFKs plated on laminin 5 after treatment with toxin B or wortmannin. Untreated control HFKs did not efficiently localize ₃₁, vinculin (VIN), or FAK into focal adhesion structures (Fig. 7, panels a, d, and g). Consistent with inactivating Rho, toxin B treatment decreased localization of all three components in focal adhesions (panels b, e, and h). Note that toxin B caused disappearance of actin stress fibers; however, the cells retained their spread morphology (Fig. 7, compare panels j and k). Inhibiting PI3K with wortmannin caused a major increase in localization of ₃₁, vinculin, FAK, and actin into focal adhesion sites (Fig. 7, panels c, f, i, and l). Note that LY294002 shows the same effects (data not shown).

View larger version (174K): [in this window] [in a new window]   Fig. 7.   Inhibition of PI3K with wortmannin causes 31 to localize into focal adhesions. Sparse HFKs were plated onto laminin 5-coated cover glasses and allowed to adhere and spread for 1 h. Cells were then untreated (Control, panels a, d, g, and j) or treated with toxin B (ToxB; 50 ng/ml, panels b, e, h, and k) or wortmannin (50 nM, panels c, f, i, and l) for 3 h. Cells were then immunostained for integrin 3 (P1F2, panels a-c), vinculin (VIN, panels d-f), focal adhesion kinase (FAK, panels g-i), and with phalloidin (F-actin, panels j-l).

Consistent with immunolocalization studies, FAK phosphorylation was reduced upon toxin B treatment (Fig. 8, A (lane 2) and B (white bar)) compared with untreated control cells (Fig. 8, A (lane 1) and B (black bar)). Wortmannin treatment increased tyrosine phosphorylation of FAK by 2-3-fold over control cells (Fig. 8, A (lane 3) and B (striped bar)). Thus, Rho participation in ₃₁ interactions with laminin 5 increases when PI3K is inhibited.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Inhibition of PI3K increases tyrosine phosphorylation of FAK. A, sparse HFKs were treated with nothing (control, lane 1), toxin B (50 ng/ml, lane 2), or wortmannin (50 nM, lane 3) for 3 h. Lysates were immunoblotted with anti-phosphotyrosine antibody (PY20). Bands corresponding to FAK are shown. B, densitometry reading of relative optical density of phosphorylated signal of one representative blot out of three separate experiments. Sparse HFK: untreated controls (black bar), toxin B-treated (ToxB, white bar), and wortmannin-treated (Wort., striped bar), were analyzed.

Adhesion to Laminin 5 Increases Synthesis of Phosphoinositides and PI3K Activity-- We compared steady-state levels of phosphotidylinositol di-phosphate (PIP₂) synthesized by PI3K in HFKs that are adherent on either laminin 5 or collagen (Fig. 9A). Cells were labeled for 1 h with ³²P_i in original adherent (A) or in suspension (S) or during re-adhesion to laminin 5 (L5) or collagen (Col) with (+) or without () wortmannin. Extracts from the labeled cells were fractionated by thin layer chromatography and levels of PIP₂ were quantitated. HFKs adhering to laminin 5 synthesized over 2 times higher levels of PIP₂ than cells adhering on collagen (L5: 39.35; Col: 19.15) and this increased level could be inhibited by wortmannin (L5+: 18.32; Col+: 13.74). These results suggest that HFKs adhering to laminin 5 had higher levels of PI3K activity compared with cells on collagen.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   Interaction with laminin 5 activates PI3K activity compared with collagen. A, steady-state levels of phosphotidylinositol diphosphate (PIP2) synthesized by PI3K were compared in HFKs that are adherent on either laminin 5 or collagen. Cells were labeled for 1 h with 32Pi in original adherent (A) or labeled in suspension (S) or during adhesion to laminin 5 (L5) or collagen (Col) with (+) or without () wortmannin. B, time course of PIP2 and PIP3 synthesis resulting from PI3K activation upon cell adhesion via 31 and 21. HFKs were labeled for 1 h with 32Pi in suspension (0) and then adhered onto immobilized anti-3 (P1B5) or anti-2 (P1H5) mAbs. Samples were taken at 0, 15, 30, 60, and 120 min of adhesion. C, densitometry reading of PIP2 and PIP3 bands from B, comparing time course between cells on immobilized anti-3 (open circles) or anti-2 (black squares). D, PI3K immunoprecipitated from extracts of HFKs that were suspended, or plated onto immobilized anti-3 (P1B5), anti-2 (P1H5), or anti-6 (G0H3) and PI(3,4)P2 was used as an exogenous substrate (see "Experimental Procedures"). E, densitometry reading of relative ratios of PIP3 on TLC from D.

We examined the time course of PIP₂ and PIP₃ synthesis, both known products of PI3K activation, resulting from HFKs adhesion via ₃₁ and ₂₁ (Fig. 9, B and C). HFKs were labeled for 1 h with ³²P_i in suspension (0) and then adhered onto immobilized anti-₃ (P1B5) or anti-₂ (P1H5) antibodies. Samples were taken at 15, 30, 60, and 120 min of adhesion. Adhesion and spreading of HFKs on anti-₃ (Fig. 9B; quantitated in Fig. 9C, open circles) generated a more rapid and higher levels of PIP₂ and PIP₃ than cells adhering on anti-₂ (Fig. 9, B and C, black squares). This confirms that adhesion to laminin 5 via ₃₁ generates a higher level of PI3K activity than adhesion to collagen via ₂₁.

Enzymatic activity of PI3K was assayed in HFKs adherent via ₂₁, ₃₁, or ₆₄; PI3K enzyme was immunoprecipitated with anti-PI3K rabbit polyclonal antibody from HFKs that were suspended, or adherent to immobilized anti-₂ (P1H5), anti-₃ (P1B5), or anti-₆ (G0H3) integrin mAbs. Immunoprecipitated PI3K samples were incubated with phosphatidylinositol 4,5-biphosphate substrate and [-³²P]ATP. Labeled PIP₃ product was fractionated by thin layer chromatography and quantitated (Fig. 9, D and E; see "Experimental Procedures"). Compared with suspended cells, PI3K activity was higher in all adherent cells. However, cells adhering to anti-₃ and anti-₆ had higher PI3K activity than cells adherent on anti-₂. This relationship was observed in three separate experiments and suggested that ligation of either ₃₁ or ₆₄ promoted PI3K activity as well as production of both PIP₂ and PIP₃.

Deposition of Laminin 5 Regulates PI3K