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• W2005011918 abstract "E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1−/− keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins α5, α6, β1, and β4 in E2F-1−/− keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1−/− keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-γ1. E2F-1−/− keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G1 and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1−/− mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1−/− mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo. E2F factors are involved in proliferation and apoptosis. To understand the role of E2F-1 in the epidermis, we screened wild type and E2F-1−/− keratinocyte mRNA for genes differentially expressed in the two cell populations. We demonstrate the reduced expression of integrins α5, α6, β1, and β4 in E2F-1−/− keratinocytes associated with reduced activation of Jun terminal kinase and Erk upon integrin stimulation. As a consequence of altered integrin expression and function, E2F-1−/− keratinocytes also show impaired migration, adhesion to extracellular matrix proteins, and a blunted chemotactic response to transforming growth factor-γ1. E2F-1−/− keratinocytes, but not dermal fibroblasts, exhibit altered patterns of proliferation, including significant delays in transit through both G1 and S phases of the cell cycle. Recognizing that proliferation and migration are key for proper wound healing in vivo, we postulated that E2F-1−/− mice may exhibit abnormal epidermal repair upon injury. Consistent with our hypothesis, E2F-1−/− mice exhibited impaired cutaneous wound healing. This defect is associated with substantially reduced local inflammatory responses and rates of re-epithelialization. Thus, we demonstrate that E2F-1 is indispensable for a hitherto unidentified cell type-specific and unique role in keratinocyte proliferation, adhesion, and migration as well as in proper wound repair and epidermal regeneration in vivo. transforming growth factor-β1 extracellular-regulated kinase Jun terminal kinase The E2F family of transcription factors is pivotal in the control of cell proliferation and is involved in patterning during early embryogenesis (1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (737) Google Scholar, 2Myster D.L. Bonnette P.C. Duronio R.J. Development. 2000; 127: 3249-3261PubMed Google Scholar, 3Suzuki A. Hemmati-Brivanlou A. Mol. Cell. 2000; 5: 217-229Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). E2F belongs to a network that also includes cyclins, cyclin-dependent kinases (cdk), cdk inhibitors, and the retinoblastoma family of proteins (for review, see Refs. 1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (737) Google Scholar and4Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1965) Google Scholar). The E2F multigene family consists of two subgroups termed E2F and DP. Functional E2F units are heterodimers composed one E2F and one DP protein. Mammalian E2F-1–5 also have a C-terminal transactivation domain, which mediates binding to retinoblastoma family proteins (for review, see Refs. 1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (737) Google Scholar and 4Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1965) Google Scholar). The precise biological roles played by each E2F protein are poorly understood. To address this issue, mice with targeted inactivation of E2F genes have been generated. These studies have shown that E2F proteins can fulfil tissue-specific functions. For example, E2F-1 is necessary for thymocyte apoptosis and selection (5Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 6Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar), E2F-4 is indispensable for hematopoietic and intestinal epithelial cell maturation (7Humbert P.O. Rogers C. Ganiatsas S. Landsberg R.L. Trimarchi J.M. Dandapani S. Brugnara C. Erdman S. Schrenzel M. Bronson R.T. Lees J.A. Mol. Cell. 2000; 6: 281-291Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8Rempel R.E. Saenz-Robles M.T. Storms R. Morham S. Ishida S. Engel A. Jakoi L. Melhem M.F. Pipas J.M. Smith C. Nevins J.R. Mol. Cell. 2000; 6: 293-306Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), and E2F-5 is critical for choroid plexus function (9Lindeman G.J. Dagnino L. Gaubatz S. Xu Y. Bronson R.T. Warren H.B. Livingston D.M. Genes Dev. 1998; 12: 1092-1098Crossref PubMed Scopus (152) Google Scholar). E2F-1 is one of the most extensively characterized members of the E2F family. E2F-1 is a strong activator of transcription and DNA synthesis (10Johnson D.G. Schwarz J.K. Cress W.D. Nevins J.R. Nature. 1993; 365: 349-352Crossref PubMed Scopus (833) Google Scholar, 11DeGregori J. Leone G. Ohtani K. Miron A. Nevins J.R. Genes Dev. 1995; 9: 2873-2887Crossref PubMed Scopus (201) Google Scholar) and can promote S phase entry and induce apoptosis in a p53-dependent or -independent manner (12Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (534) Google Scholar, 13Lissy N.A. Davis P.K. Irwin M. Kaelin W.G. Dowdy S.F. Nature. 2000; 407: 642-645Crossref PubMed Scopus (288) Google Scholar). E2F-1−/− mice are viable and fertile (5Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 6Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). However, they exhibit testicular atrophy and exocrine gland dysplasia, and one of the two lines reported develops tumors with multiple tissues of origin (6Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). Notably, abnormalities in stratified epithelia have yet to be reported in these animals. To begin to define the biological functions of E2F factors, we analyzed their patterns of expression during development. We have previously shown that E2F-1 is expressed in undifferentiated cells in tissues of neuroectodermal origin, including neuronal progenitors and epidermal keratinocytes (14D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 15Dagnino L. Fry C.J. Bartley S.M. Farnham P. Gallie B.L. Phillips R.A. Mech. Dev. 1997; 66: 13-25Crossref PubMed Scopus (65) Google Scholar). Terminal differentiation of epidermal keratinocytes results in pronounced down-regulation of E2F-1 transcripts and protein, and ectopic expression of E2F-1 is sufficient to promote entry into S phase in keratinocytes at any stage of differentiation (14D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These properties are consistent with a role for E2F-1 in maintenance of the proliferative state in these cells. A fundamental property of the epidermis is its constant state of self-renewal (for review, see Ref. 16Fuchs E. Princess Takamatsu Symp. 1994; 24: 290-302PubMed Google Scholar). This is accomplished through keratinocyte stem cells, which divide to give rise to new stem cells or to differentiating keratinocytes. The epidermis also has the ability to regenerate upon injury through a dynamic process that involves multiple cell types and responses (for review, see Refs. 17Singer A.J. Clark R.A. N. Engl. J. Med. 1999; 341: 738-746Crossref PubMed Scopus (4607) Google Scholar and 18Martin P. Science. 1997; 276: 75-81Crossref PubMed Scopus (3691) Google Scholar). Cutaneous repair requires blood clotting, inflammation, re-epithelialization, and tissue remodeling. Multiple signals control these processes, including cell interactions with other cells or with the extracellular matrix as well as secretion of and responses to a variety of cytokines. To determine the role that E2F-1 plays in epidermal homeostasis, morphogenesis, and repair, we examined the proliferative and regenerative properties of keratinocytes isolated from E2F-1−/− mice. Our studies demonstrate that E2F-1 is indispensable for normal keratinocyte proliferation in culture and cutaneous wound repair in vivo. Our studies also show a novel, previously unidentified biological role of E2F-1 beyond cell proliferation, which includes modulation of cell adhesion, migration, and chemotaxis through integrin expression and function. Poly(A+) RNA was isolated from total RNA obtained from wild type or E2F-1−/−keratinocytes by the guanidinium thiocyanate method (19Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63087) Google Scholar) and reverse-transcribed with Superscript II (Invitrogen) following the manufacturer's recommendations. Representational difference analysis was performed on the cDNAs prepared as described (20Hubank M. Schatz D.G. Nucleic Acids Res. 1994; 22: 5640-5648Crossref PubMed Scopus (780) Google Scholar). E2F-1−/− keratinocyte cDNA was subtracted three times against wild type cDNA (1:100, 1:400, and 1:800) to generate the final difference products. Integrin mRNA levels were measured using a quantitative reverse transcription-PCR method (21Dagnino L. Lavigne J.P. Nemer M. Hypertension. 1992; 20: 690-700Crossref PubMed Scopus (62) Google Scholar) from total RNA prepared from freshly isolated or from cultured keratinocytes. Reverse transcription followed by polymerase chain reaction amplification was conducted on at least two different RNA samples (1 μg of RNA was reverse-transcribed, and cDNA equivalent to 0.1 μg of RNA was used in each PCR reaction). The primers used were: α5integrin, 5′-CATTTCCGAGTCTGGGCCA and 5′-TGGAGGCTTGAGCTGAGCTT (22Ashcroft G.S. Yang X. Glick A.B. Weinstein M. Letterop J.J. Mizel D.E. Anzano M. Greenwell-Wild T. Wahl S.M. Deng C. Roberts A.B. Nat. Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (765) Google Scholar); α6 integrin, 5′-TAGAGCCAGCATCAGAATCCC and 5′-GCTAAGCCTTCGCAGGTGTAT; β1 integrin, 5′-TGTTCAGTGCAGAGCCTTCA and 5′-CCTCATACTTCGGATTGACC (22Ashcroft G.S. Yang X. Glick A.B. Weinstein M. Letterop J.J. Mizel D.E. Anzano M. Greenwell-Wild T. Wahl S.M. Deng C. Roberts A.B. Nat. Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (765) Google Scholar); β4 integrin, 5′-GAGGATCTCCTGCCTAACTAC and 5′-ACTGTTGGTCCATATGAGTGC; glyceraldehyde-3-phosphate dehydrogenase, 5′-CAAAGTTGTCATGGATGACC and 5′-GTTGCCATCAACGACCCCTT or 5′-GCTTCACCACCTTCTTGATGTCATC and 5′-GTTGCCATCAACGACCCCTT. PCR fragments obtained at 18, 20, 22, and 24 amplification cycles were resolved by electrophoresis, transferred to nylon membranes, and hybridized to appropriate 32P-labeled probes. The signals were detected and quantified using a Canberra-Packard phosphorimager, as described (21Dagnino L. Lavigne J.P. Nemer M. Hypertension. 1992; 20: 690-700Crossref PubMed Scopus (62) Google Scholar, 23Dagnino L. Drouin J. Nemer M. Mol. Endocrinol. 1991; 5: 1292-1300Crossref PubMed Scopus (69) Google Scholar), and normalized to glyceraldehyde-3-phosphate dehydrogenase products amplified in the same reactions. For each cDNA, amplification was quantified by phosphorimaging analysis at multiple PCR cycles (18–24 cycles). Amplification of all cDNAs tested was logarithmic within these parameters. In situ hybridization experiments were conducted as described (15Dagnino L. Fry C.J. Bartley S.M. Farnham P. Gallie B.L. Phillips R.A. Mech. Dev. 1997; 66: 13-25Crossref PubMed Scopus (65) Google Scholar) using 8-μm frozen sections from wound tissue samples obtained after debridement. Primary keratinocytes were isolated from 1–3-day-old E2F-1−/−, E2F-1+/+, or E2F-1+/− mice (5Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar) and cultured under non-differentiating conditions as described (14D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Unless otherwise indicated, experiments were conducted on triplicate samples 3–5 days after initial plating in cultures that were 70–80% confluent by the end of the experiment. For proliferation experiments, 2 × 105 cells/well were plated in 12-well culture dishes, and cell numbers were determined daily. To measure DNA synthesis, keratinocyte cultures were incubated with 1.5 μCi/ml [3H]dThd for 3 h, and incorporation into DNA was measured by liquid scintillation counting of trichloroacetic acid-insoluble cell fractions. The 3H activity was normalized to cell numbers. For flow cytometric analyses, 5 × 106 cells/10-cm tissue culture dish were plated and collected at the indicated intervals. Each experiment was conducted on triplicate samples. Pharmacological treatments included culture with human TGF-β1 (Life Science Technologies) at 1, 5, or 10 ng/ml (final), as indicated in individual experiments. Chemicals were purchased from Sigma, unless otherwise indicated Primary keratinocytes cultured for 2 days in low calcium medium (0.05 mm CaCl2) were trypsinized, washed, resuspended at 1.5 × 106 cells/ml in serum-free calcium-free Eagle's minimum essential medium, and 200 μl of this cell suspension were added to tissue culture inserts (8-μm pore size, Becton and Dickinson). Test medium in the lower chamber included EMEM, conditioned medium from E2F-1−/−or E2F-1+/+ keratinocytes, or TGF-β11 (1 ng/ml, final). Cells were cultured at 37 °C for 7 h, and those cells that migrated through the membrane were stained with Hemacolor (EM Science) and counted from microscope images obtained with a Leica DMIRBE photomicroscope. Adhesion experiments were conducted on keratinocytes briefly trypsinized and re-plated (100,000 cells/cm2) for 1 h in non-coated culture dishes or on dishes coated with fibronectin, collagen IV (Becton-Dickinson), or laminin as described (24Hertle M.D. Jones P.H. Groves R.W. Hudson D.L. Watt F.M. J. Invest. Dermatol. 1995; 104: 260-265Abstract Full Text PDF PubMed Scopus (44) Google Scholar). All experiments were conducted on triplicate samples. To obtain ligation of integrins in the absence of other stimuli, keratinocytes were cultured in 0.1% serum and growth factor-free medium for 24 h, detached, and resuspended in serum-free medium containing 1% bovine serum albumin. The cells were incubated in suspension for 45 min and plated onto dishes previously coated with fibronectin (0.5 mg/ml stock) and collagen I (3 mg/ml stock) or with laminin 5-enriched matrix, prepared as described (25Jones P.H. Watt F.M. Cell. 1993; 73: 713-724Abstract Full Text PDF PubMed Scopus (997) Google Scholar). The cells attached and spread equally well on all matrices. Cell lysates from adherent and non-adherent cells were prepared 30 min after plating and subjected to biochemical analysis. Lysis buffer contained 20 mm Tris·HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mmγ-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin and pepstatin, and 1 mmphenylmethylsulfonyl fluoride. Positive controls for JNK and Erk activation were prepared using cell lysates from serum-free adherent cultures exposed to UV irradiation, as described (26Mainiero F. Murgia C. Wary K.K. Curatola A.M. Pepe A. Blumenberg M. Westwick J.K. Der C.J. Giancotti F.G. EMBO J. 1997; 16: 2365-2375Crossref PubMed Scopus (306) Google Scholar). JNK activity was assessed by measuring phosphorylation of glutathioneS-transferase-Jun fusion proteins with a stress-activated protein kinase/JNK assay kit (New England Biolabs) following the manufacturer's instructions or from immunoblots prepared from 50 μg of cell lysates/sample and probed with an anti-phospho-JNK antibody (New England Biolabs). Erk activity was measured from immunoblots using a phospho-Erk antibody (New England Biolabs). Signals from the blots were normalized with anti-tubulin or with anti-Erk (total) antibodies (New England Biolabs). The results shown are representative of results obtained from experiments conducted on duplicate samples and repeated four times. Groups of sixteen 6-week-old mice (8 males and 8 females; E2F-1+/+or E2F-1−/−) were used for each time analyzed after wounding. For wound-healing measurements, tails were clipped with a single stroke of a scalpel blade in halothane-anesthetized animals, following a described procedure (27Guo L. Degenstein L. Fuchs E. Genes Dev. 1996; 10: 165-175Crossref PubMed Scopus (463) Google Scholar, 28Mann G.B. Fowler K.J. Gabriel A. Nice E.C. Williams R.L. Dunn A.R. Cell. 1993; 73: 249-261Abstract Full Text PDF PubMed Scopus (524) Google Scholar). The animals were sacrificed at the indicated times after tail amputation, and tail stumps containing the healing areas were removed, embedded in OCT compound (Miles), and frozen. The tissue was then used to prepare frozen histological sections. Tissue sections (8 μm) were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin following standard procedures. Measurements of cell numbers/unit area or wound area (measured below the clot) and re-epithelialization were done using Openlab software (Improvision). Representative photographs of sections traversing the tissue in its mid-portion were recorded. Following this approach, % re-epithelialization was calculated from the percentage of distance migrated by newly formed epidermis relative to upper wound width. The values shown in Table I for re-epithelialization at any given time represent the minimum and maximum fraction of epithelialized tissue found per animal in each group of 16 mice. A single value of 100% indicates full re-epithelialization in all 16 animals. Inflammation in tissue sections was assessed by determining the number of inflammatory cells (macrophages, neutrophils) per unit area in Giemsa-stained tissue from wild type and mutant animals, as described (29Ashcroft G.S. Lei K. Jin W. Longenecker G. Kulkarni A.B. Greenwell-Wild T. Hale-Donze H. McGrady G. Song X.-Y. Wahl S.M. Nat. Med. 2000; 6: 1147-1153Crossref PubMed Scopus (345) Google Scholar). We scored as (+++) the maximum number of cells observed in wild type wounds (505 ± 79 cells/unit area, mean ± S.E., which were observed at days 1 and 2 post-wounding). Average cell number values in the range 50–166 cells/unit area and 167–333 cells/unit area were scored, respectively, as (+) and (++) in Table I.Table IDelayed wound healing in E2F-1 null miceGenotypeDay 1Day 2Day 3Day 4Day 5Wild typeInflammatory cells (neutrophils, macrophages) (+++)Inflammation (+++)Re-epithelialization 10–15%Partial re-epithelialization 60–70%Re-epithelialized 100%Granulation tissue (++)No re-epithelializationE2F-1−/−Few infiltrating cells (+)Inflammation (+)Inflammation (+++)Granulation tissue (++)Partial re-epithelialization 40–55%1-aFull re-epithelialization at days 7–8.Granulation tissue (+)Granulation tissue (+)Slight re-epithelialization 10–15%No re-epithelializationNo re-epithelializationSixteen age-matched (6-week-old) mice were used per group, 8 males and 8 females. Percent re-epithelialization was calculated from the percentage of distance covered by new epidermis relative to total upper wound width. The maximum number of inflammatory cells observed per unit area (500 ± 79) is represented as (+++). Average cell number values in the range 50–166 cells/unit area and 167–333 cells/unit area were scored, respectively as (+) and (++).1-a Full re-epithelialization at days 7–8. Open table in a new tab Sixteen age-matched (6-week-old) mice were used per group, 8 males and 8 females. Percent re-epithelialization was calculated from the percentage of distance covered by new epidermis relative to total upper wound width. The maximum number of inflammatory cells observed per unit area (500 ± 79) is represented as (+++). Average cell number values in the range 50–166 cells/unit area and 167–333 cells/unit area were scored, respectively as (+) and (++). To gain insight into the role of E2F-1 in epidermal morphogenesis, we employed representational difference analysis (20Hubank M. Schatz D.G. Nucleic Acids Res. 1994; 22: 5640-5648Crossref PubMed Scopus (780) Google Scholar) to identify genes differentially expressed in E2F-1−/− versus wild type undifferentiated primary keratinocytes. We identified 11 distinct products and compared their sequences with those available in GenBankTM (NCBI) using the FASTA program. The isolated products correspond to nine known and two potentially novel cDNAs down-regulated in mutant keratinocytes. 2S. Murkherjee and L. Dagnino, unpublished data. Four of these cDNAs correspond to integrin gene products, specifically integrins α5, α6, β1, and β4. To confirm that the above described integrins are indeed down-regulated in mutant keratinocytes, we isolated RNA from wild type and E2F-1−/− primary undifferentiated keratinocytes and epidermis and estimated integrin mRNA abundance using a quantitative reverse transcription-PCR approach (21Dagnino L. Lavigne J.P. Nemer M. Hypertension. 1992; 20: 690-700Crossref PubMed Scopus (62) Google Scholar, 23Dagnino L. Drouin J. Nemer M. Mol. Endocrinol. 1991; 5: 1292-1300Crossref PubMed Scopus (69) Google Scholar). The results of these experiments confirmed that the transcript levels of integrins α5, α6, β1, and β4 are 2–3-fold lower in mutant keratinocytes (Fig. 1 A). We next examined whether decreased integrin transcript levels resulted in altered signaling via integrin heterodimers. Undifferentiated keratinocytes express several integrin heterodimers, including α6β4 and α5β1integrins, which mediate adhesion to and are stimulated by laminin-5 and fibronectin or collagen, respectively. Stimulation of α6β4 integrin by laminin-5 induces activation by phosphorylation of JNK and is key for normal keratinocyte proliferation (26Mainiero F. Murgia C. Wary K.K. Curatola A.M. Pepe A. Blumenberg M. Westwick J.K. Der C.J. Giancotti F.G. EMBO J. 1997; 16: 2365-2375Crossref PubMed Scopus (306) Google Scholar). To begin to understand the functional consequences of reduced α6 and β4 integrin expression in mutant keratinocytes, we first analyzed the degree of activation of JNK by laminin stimulation of integrin α6β4 in wild type and E2F-1−/− cells. We first compared the abundance of phosphorylated JNK in wild type or E2F-1−/−keratinocyte lysates prepared from suspended keratinocytes (to preclude integrin engagement) or from cells plated on laminin-5. We found that phospho-JNK levels were substantially decreased in laminin-5-stimulated mutant keratinocytes, relative to wild type cells (Fig. 1 B). To confirm these observations, we measured the ability of JNK immunoprecipitates prepared from these lysates to phosphorylatein vitro a glutathione S-transferase-Jun fusion protein. We also found correspondingly similar decreases in phosphorylated glutathione S-transferase-Jun in mutant lysates (data not shown). Thus, the reduced expression of α6 and β4 integrin subunits in mutant keratinocytes is associated with decreased signaling through α6β4 dimers and activation of the JNK pathway. Notably, this phenotype is specific to JNK activation via the integrin pathway, as UV irradiation of quiescent, adherent wild type, and mutant cells resulted in similar degrees of JNK activation (Fig.1 B). Similar to the activation of JNK by laminin, ligation of α5β1 integrin in keratinocytes by fibronectin and/or collagen results in activation of the mitogen-activated protein kinase cascade, with phosphorylation and consequent activation of Erk (p42 and p44). We measured the levels of active phospho-Erk p42/p44 in lysates from keratinocytes in suspension or plated on fibronectin/collagen I-coated dishes (Fig. 1 C). Consistent with the alteration in integrin α5 and β1 levels, phospho-Erk abundance in E2F-1−/− keratinocytes was substantially lower than in wild type cells. To determine whether the reduced Erk activation was specifically associated with integrin signaling, we measured phosphorylated Erk abundance in quiescent, adherent cells exposed to UV radiation. Irradiated mutant cells exhibited similar levels of Erk phosphorylation than those observed in normal keratinocytes, indicating a selective reduction in Erk activation associated with integrin stimulation. Integrins are central for cell adhesion and migration. Having determined alterations in integrin signaling pathways consequent to E2F-1−/− inactivation, we then examined the consequences of reduced integrin levels and function on keratinocyte migration in response to chemotactic agents. Thus we measured the ability of cells to migrate through culture inserts placed over wells in the presence or absence of chemoattractants (Fig.2 A). Wild type keratinocytes showed the same extent of increased migration toward conditioned media from normal or from E2F-1−/− cultures, indicating that disruption of the E2F-1 gene does not appreciably alter the ability of keratinocytes to secrete chemoattractant factors. TGF-β1 was strongly chemotactic toward normal cells. In contrast, E2F-1−/−keratinocytes exhibited greatly reduced migration both toward TGF-β1 and toward growth factors present in keratinocyte-conditioned media (Fig. 2 A). To investigate whether other responses of E2F-1−/− keratinocytes to TGF-β1 were affected, we also measured the ability of this cytokine to inhibit proliferation of cultured mutant and wild type keratinocytes. Treatment with TGF-β1 for 24 or 48 h inhibited DNA synthesis by 80–90% in both cell types (Fig. 2 B). Thus, E2F-1 inactivation and integrin signaling defects in keratinocytes specifically affect chemotaxis but not proliferative responses to TGF-β1. Keratinocyte migration and adhesion to the extracellular matrix are jointly regulated by integrins. Therefore, we determined the integrin-mediated adhesion properties of wild type and E2F-1−/− keratinocytes toward various extracellular matrix proteins using conventional short term adhesion assays. Primary keratinocytes maintained as basal cells in 0.05 mmextracellular Ca2+ for 3 days were re-plated onto untreated plates or plates coated with fibronectin, collagen IV, laminin-1, or laminin-5. Wild type cells adhered more efficiently to non-coated plates than E2F-1−/− keratinocytes (Fig.3). The numbers of wild type cells that adhered extracellular matrix-coated plates were substantially higher than those on uncoated culture dishes (Fig. 3). In contrast, E2F-1−/− keratinocytes exhibited significantly smaller increases in adhesion in the presence of the extracellular matrix proteins (Fig. 3). Thus, decreased integrin expression appears to be a contributing factor to the impaired migration and adhesion exhibited by mutant keratinocytes. One of the most thoroughly characterized functions of E2F-1 is its ability to promote transit to S phase and cell proliferation (for review, see Ref. 1Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (737) Google Scholar). In addition, integrin stimulation is necessary for normal keratinocyte growth (26Mainiero F. Murgia C. Wary K.K. Curatola A.M. Pepe A. Blumenberg M. Westwick J.K. Der C.J. Giancotti F.G. EMBO J. 1997; 16: 2365-2375Crossref PubMed Scopus (306) Google Scholar, 30Haase I. Hobbs R.M. Romero R. Broad S. Watt F.M. J. Clin. Invest. 2001; 108: 527-536Crossref PubMed Scopus (147) Google Scholar). Hence, we next characterized the proliferative capacity of primary epidermal keratinocytes as well as dermal fibroblasts isolated from wild type, E2F-1+/−, and E2F-1−/−mice. We first measured the growth rates of primary cultured keratinocytes and dermal fibroblasts. Wild type and heterozygote E2F-1+/− keratinocytes proliferated at similar rates throughout the experiment (8 days), with a mean doubling time of about 33 h (Fig. 4 A). In contrast, E2F-1−/− keratinocytes did not complete a population doubling within 8 days after plating (Fig. 4 A). Based on the experimental data obtained, the estimated doubling time of these cells in culture is 250 h, about 7-fold greater than that in wild-type cells. This failure of E2F-1−/− keratinocytes to divide was accompanied by a reduced capacity to synthesize DNA (Fig.4 B). DNA synthetic capacity was not altered in E2F-1+/− keratinocytes, indicating the lack of gene dosage effects. The low proliferation rates and DNA synthetic capacity in E2F-1−/− keratinocytes did not arise from increased cell loss, as evidenced by the ability to exclude trypan blue of >95% of cells" @default.
• W2005011918 created "2016-06-24" @default.
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• W2005011918 date "2002-03-01" @default.
• W2005011918 modified "2023-10-01" @default.
• W2005011918 title "E2F-1 Is Essential for Normal Epidermal Wound Repair" @default.
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