FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2070264908> ?p ?o ?g. }

• W2070264908 endingPage "43870" @default.
• W2070264908 startingPage "43860" @default.
• W2070264908 abstract "Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin α2β1 by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins α3β1 and α6β4 and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896–31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a “leading wound cell,” we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin α3β1 and α6β4is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via α3β1 and α6β4in a Rho-independent mechanism. Spreading on endogenous laminin 5 via α3β1 is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via α2β1, all characteristics of leading keratinocytes in the epidermal outgrowth. Wounding of the epidermis signals the transition of keratinocytes from quiescent anchorage on endogenous basement membrane laminin 5 to migration on exposed dermal collagen. In this study, we attempt to characterize activation signals that transform quiescent keratinocytes into migratory leading cells at the wound edge. Previously, we reported that adhesion and spreading on collagen via integrin α2β1 by cultured human foreskin keratinocytes (HFKs) requires RhoGTP, a regulator of actin stress fibers. In contrast, adhesion and spreading on laminin 5 requires integrins α3β1 and α6β4 and is dependent on phosphoinositide 3-hydroxykinase (Nguyen, B. P., Gil, S. G., and Carter, W. G. (2000) J. Biol. Chem. 275, 31896–31907). Here, we report that quiescent HFKs do not adhere to collagen but adhere and spread on laminin 5. By using collagen adhesion as one criterion for conversion to a “leading wound cell,” we found that activation of collagen adhesion requires elevation of RhoGTP. Adhesion of quiescent HFKs to laminin 5 via integrin α3β1 and α6β4is sufficient to increase levels of RhoGTP required for adhesion and spreading on collagen. Consistently, adhesion of quiescent HFKs to laminin 5, but not collagen, also promotes expression of the precursor form of laminin 5, a characteristic of leading keratinocytes in the epidermal outgrowth. We suggest that wounding of quiescent epidermis initiates adhesion and spreading of keratinocytes at the wound edge on endogenous basement membrane laminin 5 via α3β1 and α6β4in a Rho-independent mechanism. Spreading on endogenous laminin 5 via α3β1 is necessary but not sufficient to elevate expression of precursor laminin 5 and RhoGTP, allowing for subsequent collagen adhesion via α2β1, all characteristics of leading keratinocytes in the epidermal outgrowth. basement membrane human foreskin keratinocyte monoclonal antibody phosphoinositide 3-OH-kinase phosphate buffered saline glutathione S-transferase hemidesmosome(s) 4-morpholinepropanesulfonic acid The migratory process of wound repair involves changes in cell adhesion from a laminin 5-based stable anchoring structure via integrin α6β4 to a dynamic transient adhesion system involving β1 integrins. Quiescent epidermal keratinocytes are anchored to laminin 5 in the basement membrane (BM)1 via integrin α6β4 in hemidesmosomes (HDs) (1Borradori L. Sonnenberg A. J. Invest. Dermatol. 1999; 112: 411-418Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 2Nguyen B.P. Gil S.G. Ryan M.C. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar). Wounding the quiescent epidermis (see Transition A below) generates an initial activation (see Transition B) of keratinocytes at the wound edge that still adhered to endogenous laminin 5. In response to initial wound activation, keratinocytes at the wound edge transform into an epidermal outgrowth composed of leading and following subpopulations of keratinocytes (2Nguyen B.P. Gil S.G. Ryan M.C. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar, 3Lampe P.D. Nguyen B.P. Gil S. Usui M. Olerud J. Takada Y. Carter W.G. J. Cell Biol. 1998; 143: 1735-1747Crossref PubMed Scopus (143) Google Scholar).woundingresponseQuiescent epidermis→initial activation→migrationof leading cells in outgrowth(A)(B)(C)TRANSITIONSA–CResearch efforts have characterized both the quiescent epidermis as well as the downstream leading keratinocytes, but little is known about the cell adhesion and cell signal that constitutes the initial wound activation (B). Here, we attempt to characterize the initial adhesion-dependent activation signals (B) that transform quiescent keratinocytes (A) at the wound margin into migratory leading keratinocytes in the epidermal outgrowth (C) (2Nguyen B.P. Gil S.G. Ryan M.C. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar, 4Woodley, D. T., O'Toole, E., Nadelman, C. M., and Li, W. (1999) Progress in Dermatology, Vol. 33, pp. 1–12.Google Scholar, 5Martin P. Science. 1997; 276: 75-81Crossref PubMed Scopus (3762) Google Scholar). Activated leading keratinocytes are distinct from either the quiescent keratinocytes in epidermis or activated following keratinocytes in the outgrowth. Leading keratinocytes express elevated levels of a precursor form of laminin 5, not expressed by quiescent or following keratinocytes. The leading cells migrate over exposed dermal collagen and deposit precursor laminin 5 as a provisional BM (6Ryan M.C. Tizard R. VonDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar, 7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar, 8Goldfinger L.E. Stack M.S. Jones J.C. J. Cell Biol. 1998; 141: 255-265Crossref PubMed Scopus (274) Google Scholar, 9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Surprisingly, expression of precursor laminin 5, but not other BM components including laminin 10/11, heparan sulfate proteoglycan, or type VII collagen, is elevated in leading cells as one of the earliest activation responses even before migration on collagen is detected (6Ryan M.C. Tizard R. VonDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar,9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Ryan M.C. Lee K. Miyashita Y. Carter W.G. J. Cell Biol. 1999; 145: 1309-1323Crossref PubMed Scopus (258) Google Scholar). The precursor form of the laminin 5 heterotrimer (α3β3γ2), expressed by leading cells, contains a 200-kDa α3 chain with a carboxyl-terminal, heparin-binding subdomain. After secretion, the carboxyl-terminal subdomain is proteolytically removed to generate the mature α3 chain present in BM of quiescent epidermis (2Nguyen B.P. Gil S.G. Ryan M.C. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar,7Tsubota Y. Mizushima H. Hirosaki T. Higashi S. Yasumitsu H. Miyazaki K. Biochem. Biophys. Res. Commun. 2000; 278: 614-620Crossref PubMed Scopus (62) Google Scholar, 8Goldfinger L.E. Stack M.S. Jones J.C. J. Cell Biol. 1998; 141: 255-265Crossref PubMed Scopus (274) Google Scholar, 9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Goldfinger et al. (8Goldfinger L.E. Stack M.S. Jones J.C. J. Cell Biol. 1998; 141: 255-265Crossref PubMed Scopus (274) Google Scholar) have suggested that the precursor form of laminin 5 preferentially interacts with α3β1 to mediate migration, whereas the processed form interacts with α6β4. The processed form appears to be the major adhesive ligand in the quiescent epidermis (2Nguyen B.P. Gil S.G. Ryan M.C. Carter W.G. Curr. Opin. Cell Biol. 2000; 12: 554-562Crossref PubMed Scopus (224) Google Scholar). However, cells expressing α3β1 readily adhere, spread, migrate, and scatter on the processed form of laminin 5 in vitro (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 11Xia Y. Gil S.G. Carter W.G. J. Cell Biol. 1996; 132: 727-740Crossref PubMed Scopus (116) Google Scholar,12Kikkawa Y. Akaogi K. Mizushima H. Yamanaka N. Umeda M. Miyazaki K. In Vitro Cell. Dev. Biol. Anim. 1996; 32: 46-52Crossref PubMed Scopus (29) Google Scholar). This suggests that the activation state of keratinocytes, not just the processing of laminin 5, determines whether α3β1 interacts with laminin 5 in the BM. Conceivably, initial activation of keratinocytes at the wound edge would allow them to adhere and spread on the processed form of laminin 5 in the BM before they up-regulate expression of precursor laminin 5 as a downstream response. In vitro adhesion and migration studies with antibodies against integrins as well as targeted disruptions of α3in mice (13DiPersio C.M. Hodivala-Dilke K.M. Jaenisch R. Kreidberg J.A. Hynes R.O. J. Cell Biol. 1997; 137: 729-742Crossref PubMed Scopus (340) Google Scholar, 14Kreidberg J.A. Curr. Opin. Cell Biol. 2000; 12: 548-553Crossref PubMed Scopus (157) Google Scholar) have established that integrins α3β1 and α2β1mediate adhesion and motility on laminin 5 and collagen, respectively. Leading keratinocytes utilize integrin α2β1and RhoGTP, a regulator of actin stress fibers, to adhere and spread on collagen (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Adhesion and spreading of HFKs on laminin 5 via integrins α3β1 and α6β4is dependent on phosphoinositide 3-hydroxykinase (PI3K) and is resistant to toxin B, a bacterial inhibitor of Rho, that blocks adhesion of leading cells on collagen (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). In quiescent tissue, integrins α6β4, α3β1, and α2β1are expressed on the cell surface. In contrast, integrin α5β1 is not expressed in quiescent epidermis and requires synthesis and expression for adhesion to dermal fibronectin (15Toda K. Tuan T.L. Brown P.J. Grinnell F. J. Cell Biol. 1987; 105: 3097-3104Crossref PubMed Scopus (59) Google Scholar). Different factors may contribute to the initial activation at the wound edge. Wounding of confluent quiescent epidermis results in disruption of cell-cell adhesions, influx of soluble growth factors and cytokines, stress responses, and changes in substrate adhesion (4Woodley, D. T., O'Toole, E., Nadelman, C. M., and Li, W. (1999) Progress in Dermatology, Vol. 33, pp. 1–12.Google Scholar, 5Martin P. Science. 1997; 276: 75-81Crossref PubMed Scopus (3762) Google Scholar). Each of these wound-induced changes may contribute to initial activation (B) and transition of the quiescent epithelium at the wound margin (A) to the activated leading keratinocytes in the outgrowth (C). Many of the characteristics of quiescent epidermis in vivo are mimicked by culture of keratinocytes at confluent cell densities (16Wallis S. Lloyd S. Wise I. Ireland G. Fleming T.P. Garrod D. Mol. Biol. Cell. 2000; 11: 1077-1092Crossref PubMed Scopus (144) Google Scholar). Furthermore, wounding of the confluent cultures or suspension and readhesion at sparse cell densities duplicate the wound-induced changes and promote characteristics of the leading keratinocytes in the outgrowth. For example, culture of Madin-Darby canine kidney epithelial cells or keratinocytes at confluence for multiple days promotes assembly of calcium-independent desmosomes that do not disassemble in the presence of calcium chelators (16Wallis S. Lloyd S. Wise I. Ireland G. Fleming T.P. Garrod D. Mol. Biol. Cell. 2000; 11: 1077-1092Crossref PubMed Scopus (144) Google Scholar). Wounding of the confluent epithelial sheet converts the calcium-independent desmosomes to calcium dependence both adjacent to and distant to the wound edge. Similarly, wounding of epidermis disrupts HDs (17Stepp M.A. Spurr-Michaud S. Gipson I.K. Investig. Ophthalmol. Vis. Sci. 1993; 34: 1829-1844PubMed Google Scholar, 18Stepp M.A. Zhu L. Cranfill R. Investig. Ophthalmol. Vis. Sci. 1996; 37: 1593-1601PubMed Google Scholar, 19Gipson I.K. Spurr-Michaud S. Tisdale A. Elwell J. Stepp M.A. Exp. Cell Res. 1993; 207: 86-98Crossref PubMed Scopus (78) Google Scholar) both in vivo and in vitro. Transcription, translation, and deposition of laminin 5 (6Ryan M.C. Tizard R. VonDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar, 9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Ryan M.C. Lee K. Miyashita Y. Carter W.G. J. Cell Biol. 1999; 145: 1309-1323Crossref PubMed Scopus (258) Google Scholar, 20Kainulainen T. Hakkinen L. Hamidi S. Larjava K. Kallioinen M. Peltonen J. Salo T. Larjava H. Oikarinen A. J. Histochem. Cytochem. 1998; 46: 353-360Crossref PubMed Scopus (91) Google Scholar) or expression of keratins 16 and 17 (21, 22) is not detectable in quiescent epidermis or confluent cultures of keratinocytes. However, injury of the confluent epithelium in vivo or in vitro elevates their expression both adjacent to and distant to the wound margin. We have shown previously that deposition of laminin 5 by leading keratinocytes over exposed dermal collagen changes substrate adhesion of the following keratinocytes from collagen and RhoGTP dependent to laminin 5 and PI3K-dependent (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). We characterized changes in adhesion and signaling that transform quiescent epidermis at the wound edge into the leading keratinocytes in the epidermal outgrowth. We observed that confluent cultures of human foreskin keratinocytes (HFKs) fail in Rho-dependent adhesion to collagen but still adhere and spread on laminin 5, independent of Rho. However, HFKs can readily adhere to collagen via α2β1 when cultured at subconfluent cell densities (23Carter W.G. Wayner E.A. Bouchard T.S. Kaur P. J. Cell Biol. 1990; 110: 1387-1404Crossref PubMed Scopus (523) Google Scholar). This suggested that the quiescent keratinocytes (A in Reaction A–C) may be able to adhere and spread on endogenous laminin 5 in the BM prior to adhesion to collagen. Only after initial wound activation (B) and subsequent up-regulation of collagen adhesion and expression of precursor laminin 5 would the keratinocytes at the wound margin become leading keratinocytes and migrate over the exposed dermis (C). We hypothesized that quiescent keratinocytes anchored via α6β4 to endogenous BM laminin 5 at the wound edge would spread via α3β1 as an initial activation event. Rho-independent spreading mediated by integrin α3β1 on endogenous laminin 5 may increase RhoGTP required for subsequent adhesion of leading keratinocytes on collagen via α2β1. To test this hypothesis, we determined whether cell-substrate adhesion and spreading on endogenous processed laminin 5 was necessary to provide initial adhesion-dependent signals required for expression of the characteristics of leading cells. The characteristics used to define leading cells are elevated levels of RhoGTP, collagen adhesion, and expression of precursor laminin 5. Based on these findings, we suggest that adhesion and spreading of keratinocytes at the wound edge via integrins α6β4 and α3β1 on endogenous BM laminin 5, respectively, are necessary to increase levels of RhoGTP, collagen adhesion, and expression of precursor laminin 5 that define the migratory keratinocytes in the epidermal outgrowth. Keratinocytes (unpassaged P0 cells) were isolated fresh from normal human foreskins (HFKs) as described previously (24Boyce S.T. Ham R.G. J. Tissue Culture Methods. 1985; 9: 83-93Crossref Scopus (258) Google Scholar) by digestion with dispase to separate epidermis from dermis, followed by digestion with trypsin/EDTA to separate basal cells from the epidermis. Fresh P0 HFKs were plated onto culture dishes (6 cm) and grown for 3–5 days as subconfluent P1 HFKs. P1 HFKs were passaged with trypsin/EDTA at different seeding densities from 1 × 105 cells per plate for sparse cells to 1 × 106 cells per plate for confluent cells. The cells were grown for 3 days after seeding at the different cell densities. This corresponded to 3 days of culture at 100% confluence at the highest seeding density. P1 and P2 cultures were maintained in serum-free keratinocyte growth media (KGM; Clonetics, Corp., San Diego, CA). Swiss 3T3 cells used as negative base-line controls were grown in RPMI 1640 + 10% fetal bovine serum. Adhesion of HFKs to cryostat sections of epidermis was performed as described previously (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 10Ryan M.C. Lee K. Miyashita Y. Carter W.G. J. Cell Biol. 1999; 145: 1309-1323Crossref PubMed Scopus (258) Google Scholar). Adhesion assays for HFKs on surfaces coated with 10 μg/ml human type I collagen or laminin 5 or immobilized anti-integrin antibodies were performed as described previously (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 25Xia M. Sreedharan S.P. Dazin P. Damsky C.H. Goetzl E.J. J. Cell. Biochem. 1996; 61: 452-458Crossref PubMed Scopus (36) Google Scholar) and briefly as follows. Keratinocytes isolated fresh from neonatal foreskin or from culture plates were washed in PBS and counted. Equal numbers of cells were labeled with 0.5 μm calcein-AM (Molecular Probes, Eugene, OR) and then added to the different adhesion surfaces (in triplicates per experiment). Cells were allowed 1 h to adhere at 37 °C. Non-adherent cells were removed by washing twice with PBS. Fluorescent readings of pre-wash and post-wash cells were done on CytoFluorII, and the percentage of fluorescent cells adhered were then calculated as described previously (9Nguyen B.P. Gil S.G. Carter W.G. J. Biol. Chem. 2000; 275: 31896-31907Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Laminin 5 stimulation of confluent HFKs (see Fig. 8) was performed as follows. Confluent-derived HFKs were adhered and spread on surfaces coated with laminin 5 at subconfluent densities for 30 min in the absence or presence of inhibitory anti-integrin antibodies. These “stimulated” HFKs were resuspended by digestion with trypsin/EDTA, labeled with 0.5 μm calcein-AM, and assayed for adhesion onto surfaces coated with exogenous collagen I or laminin 5 for 1 h. Where indicated (Fig. 4), adherent cells were fixed and stained with rhodamine-conjugated phalloidin (Molecular Probes) to detect actin stress fiber in spreading HFKs on immobilized anti-integrin antibodies.Figure 4Confluent-derived HFKs spread on immobilized anti-α3 integrin antibody but not anti-α2. Adhesion and spreading of sparse-derived HFKs (a and b) were compared with confluent-derived HFKs (c and d) on surfaces coated with immobilized anti-α3 integrin (mAb P1B5; a and c) or anti-α2antibodies (mAb P1H5; b and d). Cells were seeded at equal cell numbers and allowed to adhere and spread for 1 h. Cells were then fixed with formaldehyde and stained with rhodamine-conjugated phalloidin to visualize F-actin and cell spreading. Sparse-derived HFKs adhered and spread on both anti-α3 and anti-α2, whereas confluent-derived HFKs adhered and spread on anti-α3 but did not spread on anti-α2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) HFKs were washed once with PBS and solubilized directly with 1% v/v Triton X-100 in PBS containing 5 mmEDTA, 50 μm VO4, 10 mm NaF, and protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 10 μg/ml aprotinin, 1 μg/ml leupeptin). Detergent-soluble extracts were quantitated and separated on SDS-PAGE gels (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar). Proteins were blotted onto nitrocellulose, blocked in 2% nonfat milk, and incubated with the following antibodies: anti-phosphotyrosine (PY-20, ICN), anti-PI3K (Upstate Biotechnology, Inc.), anti-RhoA (Santa Cruz Biotechnology), or anti-p120 catenin (BD Transduction Laboratories). Blots were then washed four times in PBS + 0.1% Triton X-100 and incubated with secondary antibody (donkey anti-rabbit (Jackson Laboratories)) or rabbit anti-mouse peroxidase-conjugated (Dako) for 1 h. Blots were detected with ECL chemiluminescence (Amersham Pharmacia Biotech) and direct exposure to Hyperfilm MP (Amersham Pharmacia Biotech). Sparse and confluent HFKs were washed once with PBS and incubated for 1 h in KGM-methionine- and cysteine-free media (Clonetics). 100 μCi/ml of [35S]methionine and -cysteine was added to the media, and cells were labeled for 8 h. Alternatively, sparse and confluent HFKs were surface-biotinylated with N-hydroxysuccinimide ester of Biotin (Pierce) for 10 min at 4 °C. Labeled cells were washed twice with cold PBS and solubilized directly with 1% Triton X-100 in PBS. Detergent-soluble extracts were immunoprecipitated with mAbs as follows: P1B5 (anti-α3), P1E6 (anti-α2), P4C10 (anti-β1), or B4–6 (anti-γ2 chain of laminin 5). Precipitates of35S-labeled HFKs were fractionated on 8% SDS-PAGE gels, dried onto blotting paper, and exposed to Hyperfilm (Amersham Pharmacia Biotech). Precipitates of biotinylated proteins were fractionated by SDS-PAGE, transferred to nitrocellulose, blocked in 0.5% BSA + 0.1% Triton/PBS for 1 h, and then incubated with peroxidase-conjugated streptavidin (Zymed Laboratories Inc.) for 1 h. Blots were developed with ECL reagent (Amersham Pharmacia Biotech). The gels were then directly exposed to Hyperfilm. HFKs were grown at sparse and confluent cell densities for 3 days, suspended with trypsin/EDTA, and re-plated at equal cell numbers onto surfaces coated with laminin 5 or mAbs against integrins anti-α3 (P1B5) or anti-α2 (P1H5), or kept in suspension. Total RNA was isolated using TRIzol reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Northern blots were obtained by electrophoresis of total RNA, 20 μg per lane, in formaldehyde-agarose gels (1% agarose, 6.6% formaldehyde, 20 mm MOPS, pH 7.0, 2.5 mm sodium acetate, 1 mm EDTA). Gels were blotted to Zeta-Probe nylon membranes (Bio-Rad) by capillary action using 20× SSC. Blots were hybridized in 0.25 m NaCl, 7% SDS, 1 mm EDTA, 0.25 m sodium phosphate, pH 7.0, 150 mg/ml salmon sperm DNA, 50% formamide at 48 °C. Filters were washed at 55 °C with 0.2× SSC, 0.1% SDS. cDNA clone Epi, specific for the α3 chain of laminin 5 (provided by Dr. Maureen Ryan, Seattle, WA), and cDNA clone specific for c-Myc (provided by Dr. Bob Eisenman, Seattle, WA) were radiolabeled using the Prime-it RmT random primer labeling kit (Stratagene) and used as a hybridization probe for Northern analysis. Determining levels of GTP-bound Rho for activity was done as described previously (27Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1366) Google Scholar) with the following variation. Sparse and confluent grown HFKs were plated onto immobilized mAbs or onto laminin 5-coated dishes for 1 h. Cells were washed with ice-cold Tris-buffered saline and lysed in RIPA buffer (50 mm Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mm NaCl, 10 mmMgCl2, 10 μg/ml each of leupeptin and aprotinin, 1 mm phenylmethylsulfonyl fluoride). Cell lysates were clarified by centrifugation at 13,000 × g at 4 °C for 2 min, and equal volumes of lysates were incubated with GST-Rhotekin binding domain (20 μg) beads at 4 °C for 45 min. The beads were then washed four times in buffer B (Tris buffer containing 1% Triton X-100, 150 mm NaCl, 10 mmMgCl2, 10 μg/ml each leupeptin and aprotinin, and 0.1 mm phenylmethylsulfonyl fluoride) at 4 °C. Bound Rho proteins were detected by Western blotting using a rabbit polyclonal antibody against RhoA (Santa Cruz Biotechnology). Densitometry analysis was performed using AlphaImagerTM system and ImageQuant 4.0 software. The amount of Rhotekin binding domain-bound Rho was normalized to the total amount of Rho in cell lysates for the comparison of Rho activity (level of GTP-bound Rho) in different samples. Triplicate samples were analyzed for each condition with similar results. The collagen receptor, integrin α2β1, is expressed at the cell surface of basal keratinocytes in quiescent epidermis (23Carter W.G. Wayner E.A. Bouchard T.S. Kaur P. J. Cell Biol. 1990; 110: 1387-1404Crossref PubMed Scopus (523) Google Scholar, 28Carter W.G. Kaur P. Gil S.G. Gahr P.J. Wayner E.A. J. Cell Biol. 1990; 111: 3141-3154Crossref PubMed Scopus (378) Google Scholar, 29Wayner E.A. Carter W.G. Piotrowicz R.S. Kunicki T.J. J. Cell Biol. 1988; 107: 1881-1891Crossref PubMed Scopus (338) Google Scholar). We determined whether quiescent HFKs isolated fresh from epidermis were able to adhere and spread on collagen via their endogenous α2β1. Quiescent HFKs were isolated fresh from neonatal foreskin (passage 0 or P0 HFKs) and assayed for cell adhesion and spreading on exogenous laminin 5 or collagen. Equal numbers of P0 HFKs were plated and photographed (Fig.1 A, pre-wash). Cells were allowed 1 h to adhere and spread. Non-adherent cells were then washed away (post wash). Freshly isolated P0 HFKs adhered and spread on laminin 5 (Fig. 1 A, panel d) but not to collagen (Fig. 1 A, panel b). In control experiments, P0 HFKs were also adhered to immobilized anti-α2 or anti-α3 integrin antibodies. P0 HFKs were able to adhere to both antibody-coated surfaces but spread only on anti-α3 antibody (data not shown). This confirmed that P0 HFKs, like HFKs in epidermis (23Carter W.G. Wayner E.A. Bouchard T.S. Kaur P. J. Cell Biol. 1990; 110: 1387-1404Crossref PubMed Scopus (523) Google Scholar, 28Carter W.G. Kaur P. Gil S.G. Gahr P.J. Wayner E.A. J. Cell Biol. 1990; 111: 3141-3154Crossref PubMed Scopus (378) Google Scholar,29Wayner E.A. Carter W.G. Piotrowicz R.S. Kunicki T.J. J. Cell Biol. 1988; 107: 1881-1891Crossref PubMed Scopus (338) Google Scholar), express both α2β1 and α3β1. Adhesion and spreading of passage 1 cultured HFKs (P1) grown at subconfluence were compared with P0 HFKs. P1 HFKs were able to adhere and spread on either laminin 5 or collagen (Fig. 1 B), whereas P0 HFKs were only able to adhere and spread on laminin 5. Thus, α2β1 is expressed on the cell surface of freshly isolated P0 HFKs, but these cells adhere poorly to collagen. Activation of the P0 HFKs in culture to generate P1 HFKs is sufficient to increase adhesion to collagen via integrin α2β1. We wished to understand how suspension and re-adhesion of the P0 HFKs could activate adhesion to collagen. Therefore, we attempted to establish an in vitro culture model for the P0 HFKs. We determined whether sparse cultures of P1 and P2 HFKs that can adhere to collagen would reduce collagen adhesion when maintained at confluent cell densities, mimicking the P0 HFKs (Fig. 1). For comparison, we cultured P1 HFKs at both sparse (Fig.2 A, panel a) and confluent cell densities (Fig. 2 A, panel b). The sparse HFKs display characteristics of activated leading edge keratinocytes in epidermal wounds. Sparse HFKs transcribe elevated levels of mRNA encoding the α3 chain of laminin 5 and c-Myc (Fig.2 B, lane S) (6Ryan M.C. Tizard R. VonDevanter D.R. Carter W.G. J. Biol. Chem. 1994; 269: 22779-22787Abstract Full Text PDF PubMed Google Scholar). In contrast, HFKs cultured at confluence do not transcribe mRNA for α3 laminin 5 or c-Myc (Fig.2 B, lane C) similar to quiescent keratinocytes in epidermis. We compared sparse and confluent HFKs for adhesion to exogenous laminin 5 or collagen-coated assay plates. Sparse HFKs can adhere and spread on either laminin 5 (Fig. 2 C, black bar) or collagen (Fig. 2 C, striped bar). Culturing HFKs at increasing cell densities inhibits adhesion to collagen (Fig. 2 C, striped bars), whereas adhesion and spreading on laminin 5 (solid bars) remain unaffected. Thus, cultures of P1 and P2 HFKs maintained at sparse cell densities mimic the adhesion, spreading, and transcription characteristics of leading keratinocytes of an epidermal outgrowth in wounds. Cultures of P1 and P2 HFKs maintained at confluence mimic the adhesion and transcription characteristics of quiescent P0 HFKs isolated fresh from epidermis. The transition of keratinocytes from sparse to confluence blocks adhesion and spreading on collagen via endogenous α2β1 but not adhesion and spreading on laminin 5 via α3β1 and α6β4. We were con" @default.
• W2070264908 created "2016-06-24" @default.
• W2070264908 creator A5009263125 @default.
• W2070264908 creator A5053509495 @default.
• W2070264908 creator A5068243605 @default.
• W2070264908 creator A5086030901 @default.
• W2070264908 date "2001-11-01" @default.
• W2070264908 modified "2023-10-16" @default.
• W2070264908 title "Ligation of Integrin α3β1 by Laminin 5 at the Wound Edge Activates Rho-dependent Adhesion of Leading Keratinocytes on Collagen" @default.
• W2070264908 cites W1496915816 @default.
• W2070264908 cites W1583843945 @default.
• W2070264908 cites W1895730431 @default.
• W2070264908 cites W1968193801 @default.
• W2070264908 cites W1970420859 @default.
• W2070264908 cites W1973466009 @default.
• W2070264908 cites W1984274933 @default.
• W2070264908 cites W1989021197 @default.
• W2070264908 cites W1989697191 @default.
• W2070264908 cites W1990370414 @default.
• W2070264908 cites W2009793240 @default.
• W2070264908 cites W2012491088 @default.
• W2070264908 cites W2041400374 @default.
• W2070264908 cites W2048845152 @default.
• W2070264908 cites W2055870465 @default.
• W2070264908 cites W2056862856 @default.
• W2070264908 cites W2062926268 @default.
• W2070264908 cites W2065940152 @default.
• W2070264908 cites W2073129389 @default.
• W2070264908 cites W2074029723 @default.
• W2070264908 cites W2091189056 @default.
• W2070264908 cites W2094599028 @default.
• W2070264908 cites W2100837269 @default.
• W2070264908 cites W2102193002 @default.
• W2070264908 cites W2109582895 @default.
• W2070264908 cites W2112680818 @default.
• W2070264908 cites W2118379843 @default.
• W2070264908 cites W2130172628 @default.
• W2070264908 cites W2131179754 @default.
• W2070264908 cites W2131213444 @default.
• W2070264908 cites W2133032561 @default.
• W2070264908 cites W2137949057 @default.
• W2070264908 cites W2146989892 @default.
• W2070264908 cites W2148142890 @default.
• W2070264908 cites W2154000442 @default.
• W2070264908 cites W2154953216 @default.
• W2070264908 cites W2163740123 @default.
• W2070264908 cites W2165843111 @default.
• W2070264908 cites W2338894968 @default.
• W2070264908 cites W4299736390 @default.
• W2070264908 doi "https://doi.org/10.1074/jbc.m103404200" @default.
• W2070264908 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11571278" @default.
• W2070264908 hasPublicationYear "2001" @default.
• W2070264908 type Work @default.
• W2070264908 sameAs 2070264908 @default.
• W2070264908 citedByCount "67" @default.
• W2070264908 countsByYear W20702649082012 @default.
• W2070264908 countsByYear W20702649082014 @default.
• W2070264908 countsByYear W20702649082016 @default.
• W2070264908 countsByYear W20702649082017 @default.
• W2070264908 countsByYear W20702649082019 @default.
• W2070264908 countsByYear W20702649082021 @default.
• W2070264908 countsByYear W20702649082023 @default.
• W2070264908 crossrefType "journal-article" @default.
• W2070264908 hasAuthorship W2070264908A5009263125 @default.
• W2070264908 hasAuthorship W2070264908A5053509495 @default.
• W2070264908 hasAuthorship W2070264908A5068243605 @default.
• W2070264908 hasAuthorship W2070264908A5086030901 @default.
• W2070264908 hasBestOaLocation W20702649081 @default.
• W2070264908 hasConcept C153911025 @default.
• W2070264908 hasConcept C170493617 @default.
• W2070264908 hasConcept C178790620 @default.
• W2070264908 hasConcept C179437574 @default.
• W2070264908 hasConcept C185592680 @default.
• W2070264908 hasConcept C189165786 @default.
• W2070264908 hasConcept C195687474 @default.
• W2070264908 hasConcept C203014093 @default.
• W2070264908 hasConcept C2779814568 @default.
• W2070264908 hasConcept C2780269544 @default.
• W2070264908 hasConcept C55493867 @default.
• W2070264908 hasConcept C84416704 @default.
• W2070264908 hasConcept C85789140 @default.
• W2070264908 hasConcept C86803240 @default.
• W2070264908 hasConcept C95444343 @default.
• W2070264908 hasConceptScore W2070264908C153911025 @default.
• W2070264908 hasConceptScore W2070264908C170493617 @default.
• W2070264908 hasConceptScore W2070264908C178790620 @default.
• W2070264908 hasConceptScore W2070264908C179437574 @default.
• W2070264908 hasConceptScore W2070264908C185592680 @default.
• W2070264908 hasConceptScore W2070264908C189165786 @default.
• W2070264908 hasConceptScore W2070264908C195687474 @default.
• W2070264908 hasConceptScore W2070264908C203014093 @default.
• W2070264908 hasConceptScore W2070264908C2779814568 @default.
• W2070264908 hasConceptScore W2070264908C2780269544 @default.
• W2070264908 hasConceptScore W2070264908C55493867 @default.
• W2070264908 hasConceptScore W2070264908C84416704 @default.
• W2070264908 hasConceptScore W2070264908C85789140 @default.
• W2070264908 hasConceptScore W2070264908C86803240 @default.
• W2070264908 hasConceptScore W2070264908C95444343 @default.

• next