Abstract: Lack of understanding of the molecular mechanisms and pathogenesis of impaired healing in chronic ulcers is a serious health issue that contributes to excessive limb amputations and mortality. Here we show that β-catenin and its downstream targets in keratinocytes, c-myc, and keratins K6 and K16, play important roles in the development of chronic wounds. In contrast to normal epidermis, we observed a significant nuclear presence of β-catenin and elevated c-myc expression at the nonhealing wound edge of chronic ulcers from 10 patients. In vitro studies indicated that stabilization of nuclear β-catenin inhibited wound healing and keratinocyte migration by blocking epidermal growth factor response, inducing c-myc and repressing the K6/K16 keratins (cytoskeletal components important for migration). The molecular mechanism of K6/K16 repression involved β-catenin and arginine methyltransferase (CARM-1) acting as co-repressors of glucocorticoid receptor monomers. We conclude that activation of the β-catenin/c-myc pathway(s) contributes to impaired healing by inhibiting keratinocyte migration and altering their differentiation. The presence of activated β-cate-nin and c-myc in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention. Lack of understanding of the molecular mechanisms and pathogenesis of impaired healing in chronic ulcers is a serious health issue that contributes to excessive limb amputations and mortality. Here we show that β-catenin and its downstream targets in keratinocytes, c-myc, and keratins K6 and K16, play important roles in the development of chronic wounds. In contrast to normal epidermis, we observed a significant nuclear presence of β-catenin and elevated c-myc expression at the nonhealing wound edge of chronic ulcers from 10 patients. In vitro studies indicated that stabilization of nuclear β-catenin inhibited wound healing and keratinocyte migration by blocking epidermal growth factor response, inducing c-myc and repressing the K6/K16 keratins (cytoskeletal components important for migration). The molecular mechanism of K6/K16 repression involved β-catenin and arginine methyltransferase (CARM-1) acting as co-repressors of glucocorticoid receptor monomers. We conclude that activation of the β-catenin/c-myc pathway(s) contributes to impaired healing by inhibiting keratinocyte migration and altering their differentiation. The presence of activated β-cate-nin and c-myc in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention. The integrity of the skin depends on the specific attachments of its keratinocytes to the extracellular matrix and to each other. Keratinocytes are programmed to maintain this integrity and are the first cells to respond to injury. Wounding promotes activation of keratinocytes that trigger keratinocyte migration and proliferation, which is paralleled by changes in keratinocyte adhesion and the cytoskeletal content.1Jamora C DasGupta R Kocieniewski P Fuchs E Links between signal transduction, transcription and adhesion in epithelial bud development.Nature. 2003; 422: 317-322Crossref PubMed Scopus (494) Google Scholar, 2Kirfel J Magin TM Reichelt J Keratins: a structural scaffold with emerging functions.Cell Mol Life Sci. 2003; 60: 56-71Crossref PubMed Scopus (140) Google Scholar However, sometimes this reliable program fails, resulting in a chronic wound (ulcers).3Tomic-Canic M Magnus SA Oscar MA Epidermal repair and the chronic wound.in: Rovee DT Maibach HI The epidermis in wound healing. CRC Press LLC, Boca Raton2004: pp 25-57Google Scholar, 4Morasso MI Tomic-Canic M Epidermal stem cells: the cradle of epidermal determination, differentiation and wound healing.Biol Cell. 2005; 97: 173-183Crossref PubMed Scopus (167) Google Scholar Consequential to the extended life span of the modern human population and increased prevalence of diabetes, we are faced with epidemic proportions of chronic ulcers.5Jeffcoate WJ Harding KG Diabetic foot ulcers.Lancet. 2003; 361: 1545-1551Abstract Full Text Full Text PDF PubMed Scopus (717) Google Scholar, 6Brem H Tomic-Canic M Taranovskaya A Ehlich HP Baskin-Bay E Gill K Carasa G Weinberger S Entero H Vladeck B Healing of elderly patients with diabetic foot ulcers, venous stasis ulcers, and pressure ulcers.Surg Technol Int. 2003; 11: 161-167PubMed Google Scholar The total prevalence of diabetes in the United States is estimated as high as 18.2 million or 6.3% of the population in the year 2002 (National Diabetes Fact Sheet: General Information and National Estimates on Diabetes in the United States found online at: http://www.cdc.gov/diabetes/pubs/estimates.htm). Diabetic foot ulcers are estimated to occur in 15% of all patients with diabetes, and precede 84% of all lower-leg amputations, which are at epidemic proportions in the elderly, as well as in the diabetic populations.7Reiber GE Boyko EJ Smith DG Lower extremity foot ulcers and amputations in diabetes.in: Harris MI Cowie C Stern MP Diabetes in America. U.S. Government Printing Office, Washington DC1995: pp 409-428Google Scholar, 8Pecoraro RE Reiber GE Burgess EM Pathways to diabetic limb amputation. Basis for prevention.Diabetes Care. 1990; 13: 513-521Crossref PubMed Scopus (1259) Google Scholar, 9Yoshikawa TT Antimicrobial resistance and aging: beginning of the end of the antibiotic era?.J Am Geriatr Soc. 2002; 50: S226-S229Crossref PubMed Scopus (156) Google Scholar The current lack of understanding of pathogenesis of impaired healing in chronic ulcers contributes to excessive amputations and is an extremely serious health issue. Nonetheless, there are only three FDA-approved thera-peutic modalities for such ulcers.6Brem H Tomic-Canic M Taranovskaya A Ehlich HP Baskin-Bay E Gill K Carasa G Weinberger S Entero H Vladeck B Healing of elderly patients with diabetic foot ulcers, venous stasis ulcers, and pressure ulcers.Surg Technol Int. 2003; 11: 161-167PubMed Google Scholar, 10Brem H Young J Tomic-Canic M Isaacs C Ehrlich HP Clinical efficacy and mechanism of bilayered living human skin equivalent (HSE) in treatment of diabetic foot ulcers.Surg Technol Int. 2003; 11: 23-31PubMed Google Scholar, 11Brem H Balledux J Bloom T Kerstein MD Hollier L Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent: a new paradigm in wound healing.Arch Surg. 2000; 135: 627-634Crossref PubMed Scopus (158) Google Scholar, 12Nagai MK Embil JM Becaplermin: recombinant platelet derived growth factor, a new treatment for healing diabetic foot ulcers.Exp Opin Biol Ther. 2002; 2: 211-218Crossref PubMed Scopus (102) Google Scholar, 13Sibbald RG Apligraf living skin equivalent for healing venous and chronic wounds.J Cutan Med Surg. 1998; 3: S24-S28Google Scholar The efforts to develop new therapies are hampered by the lack of knowledge of the mechanisms responsible for the pathologies, and the corresponding molecular targets for intervention. Differentiating keratinocytes are characterized by two major types of cell adhesions: desmosomes and adherens junctions. Desmosomes are multimolecular complexes containing, as major components, two glycoproteins, desmocollin and desmoglein, two armadillo proteins, plakoglobin and plakophilin, and the plakin family protein desmoplakin.14Green KJ Gaudry CA Are desmosomes more than tethers for intermediate filaments?.Nat Rev Mol Cell Biol. 2000; 1: 208-216Crossref PubMed Scopus (323) Google Scholar, 15Green KJ Jones JC Desmosomes and hemidesmosomes: structure and function of molecular components.FASEB J. 1996; 10: 871-881Crossref PubMed Scopus (300) Google Scholar, 16McGrath JA McMillan JR Shemanko CS Runswick SK Leigh IM Lane EB Garrod DR Eady RA Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome.Nat Genet. 1997; 17: 240-244Crossref PubMed Scopus (315) Google Scholar Adherens junctions are characterized by the presence of E-cadherin, α- and β-catenins, and γ-catenin (plakoglobin) at the membrane.17Geiger B Ayalon O Cadherins.Annu Rev Cell Biol. 1992; 8: 307-332Crossref PubMed Scopus (521) Google Scholar, 18Kemler R From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion.Trends Genet. 1993; 9: 317-321Abstract Full Text PDF PubMed Scopus (884) Google Scholar β-Catenin is a multifunctional protein that plays an important role during embryonic development and neoplasia as a mediator of the Wnt signaling pathway.19Millar SE WNTs: multiple genes, multiple functions.J Invest Dermatol. 2003; 120: 7-8Crossref PubMed Scopus (15) Google Scholar, 20Rowlands TM Pechenkina IV Hatsell SJ Pestell RG Cowin P Dissecting the roles of beta-catenin and cyclin D1 during mammary development and neoplasia.Proc Natl Acad Sci USA. 2003; 100: 11400-11405Crossref PubMed Scopus (46) Google Scholar When the Wnt pathway is quiescent, β-catenin participates in adherens junctions.17Geiger B Ayalon O Cadherins.Annu Rev Cell Biol. 1992; 8: 307-332Crossref PubMed Scopus (521) Google Scholar When β-catenin becomes cytoplasmic it gets phosphorylated and targeted for ubiquitination and degradation. Activation of the Wnt pathway inhibits this phosphorylation, leading to cytosolic stabilization of β-catenin, which consequently translocates to the nucleus where it binds Tcf-Lef transcription factors and regulates transcription.21Hecht A Kemler R Curbing the nuclear activities of beta-catenin. Control over Wnt target gene expression.EMBO Rep. 2000; 1: 24-28Crossref PubMed Scopus (151) Google Scholar, 22Watt FM The stem cell compartment in human interfollicular epidermis.J Dermatol Sci. 2002; 28: 173-180Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Among downstream targets of β-catenin transcriptional pathway is the oncogene c-myc.23He TC Sparks AB Rago C Hermeking H Zawel L da Costa LT Morin PJ Vogelstein B Kinzler KW Identification of c-MYC as a target of the APC pathway.Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4102) Google Scholar Activation of c-myc affects epidermal biology directly relevant to wound healing. Although c-myc is required for transition from the G1 to the S phase of the cell cycle and it promotes proliferation of transit-amplifying cells, deregulation of c-myc depletes epidermal stem cells, causing the inability of the tissue to react to injury.24Waikel RL Kawachi Y Waikel PA Wang XJ Roop DR Deregulated expression of c-Myc depletes epidermal stem cells.Nat Genet. 2001; 28: 165-168Crossref PubMed Scopus (289) Google Scholar, 25Arnold I Watt FM c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny.Curr Biol. 2001; 11: 558-568Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 26Biro S Fu YM Yu ZX Epstein SE Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration.Proc Natl Acad Sci USA. 1993; 90: 654-658Crossref PubMed Scopus (146) Google Scholar, 27Gandarillas A Watt FM c-Myc promotes differentiation of human epidermal stem cells.Genes Dev. 1997; 11: 2869-2882Crossref PubMed Scopus (285) Google Scholar Furthermore, targeted overexpression of c-myc in basal keratinocytes leads to impairment of keratinocyte migration and consequently inhibition of wound healing in a transgenic mouse model.24Waikel RL Kawachi Y Waikel PA Wang XJ Roop DR Deregulated expression of c-Myc depletes epidermal stem cells.Nat Genet. 2001; 28: 165-168Crossref PubMed Scopus (289) Google Scholar Experimental results of the roles of β-catenin and c-myc mostly originate from various mouse models whereas, to the best of our knowledge, the roles of β-catenin and c-myc in impairment of wound healing in human skin have never been investigated in patients.24Waikel RL Kawachi Y Waikel PA Wang XJ Roop DR Deregulated expression of c-Myc depletes epidermal stem cells.Nat Genet. 2001; 28: 165-168Crossref PubMed Scopus (289) Google Scholar, 25Arnold I Watt FM c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny.Curr Biol. 2001; 11: 558-568Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 26Biro S Fu YM Yu ZX Epstein SE Inhibitory effects of antisense oligodeoxynucleotides targeting c-myc mRNA on smooth muscle cell proliferation and migration.Proc Natl Acad Sci USA. 1993; 90: 654-658Crossref PubMed Scopus (146) Google Scholar, 27Gandarillas A Watt FM c-Myc promotes differentiation of human epidermal stem cells.Genes Dev. 1997; 11: 2869-2882Crossref PubMed Scopus (285) Google Scholar, 28Cheon SS Nadesan P Poon R Alman BA Growth factors regulate beta-catenin-mediated TCF-dependent transcriptional activation in fibroblasts during the proliferative phase of wound healing.Exp Cell Res. 2004; 293: 267-274Crossref PubMed Scopus (131) Google Scholar, 29Cheon SS Cheah AY Turley S Nadesan P Poon R Clevers H Alman BA Beta-catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds.Proc Natl Acad Sci USA. 2002; 99: 6973-6978Crossref PubMed Scopus (277) Google Scholar Interestingly, we found that c-myc has a particular transcriptional pattern during normal wound healing in human skin and that inhibitors of wound healing, glucocorticoids (GC), induce c-myc expression. In addition, we found that stabilization of β-catenin inhibits keratinocyte migration and wound healing of human skin in organ culture and that β-catenin participates in GC signaling and repression of the keratin genes that participate in cytoskeletal network and keratinocyte migration. Lastly, we found activation of both β-catenin and c-myc in the nonhealing edge of patients with chronic wounds. Therefore, we propose that nuclearization of β-catenin and induction of c-myc participate in inhibition of wound healing and contribute to impairment of healing in chronic wounds. Plasmids pK14CAT, pK6CAT, pK16CAT, β-catenin, and CARM-1 have been described previously.30Koh SS Li H Lee YH Widelitz RB Chuong CM Stallcup MR Synergistic coactivator function by coactivator-associated arginine methyltransferase (CARM) 1 and beta-catenin with two different classes of DNA-binding transcriptional activators.J Biol Chem. 2002; 277: 26031-26035Crossref PubMed Scopus (106) Google Scholar, 31Radoja N Komine M Jho SH Blumenberg M Tomic-Canic M Novel mechanism of steroid action in skin through glucocorticoid receptor monomers.Mol Cell Biol. 2000; 20: 4328-4339Crossref PubMed Scopus (80) Google Scholar The plasmid containing GRE-CAT was a gift from Dr. P. Chambon (France). All plasmids were grown using Promega kit (Promega, Madison, WI) and commercial protocols.32Jho SH Radoja N Im MJ Tomic-Canic M Negative response elements in keratin genes mediate transcriptional repression and the cross-talk among nuclear receptors.J Biol Chem. 2001; 276: 45914-45920Crossref PubMed Scopus (25) Google Scholar RNA isolation and purification was performed using Triazol (Invitrogen, Carlsbad, CA) extraction and subsequently Qiagen RNeasy kit column purification (Qiagen, Valencia, CA) followed by Northern blot as described.31Radoja N Komine M Jho SH Blumenberg M Tomic-Canic M Novel mechanism of steroid action in skin through glucocorticoid receptor monomers.Mol Cell Biol. 2000; 20: 4328-4339Crossref PubMed Scopus (80) Google Scholar c-myc and GAPDH probes were generated as described.33Li D Turi TG Schuck A Freedberg IM Khitrov G Blumenberg M Rays and arrays: the transcriptional program in the response of human epidermal keratinocytes to UVB illumination.FASEB J. 2001; 15: 2533-2535Crossref PubMed Scopus (112) Google Scholar Densitometry tracing of the films was performed using GS-800 calibrating densitometer (Bio-Rad, Hercules, CA) and the image was quantified using Quantity One 4.1.1 program (Bio-Rad). The values were normalized to the loading control (GAPDH) for each condition. Normal human epidermal keratinocytes were grown as described.31Radoja N Komine M Jho SH Blumenberg M Tomic-Canic M Novel mechanism of steroid action in skin through glucocorticoid receptor monomers.Mol Cell Biol. 2000; 20: 4328-4339Crossref PubMed Scopus (80) Google Scholar, 34Randolph RK Simon M Characterization of retinol metabolism in cultured human epidermal keratinocytes.J Biol Chem. 1993; 268: 9198-9205Abstract Full Text PDF PubMed Google Scholar Cells were transfected at 80% confluency using polybrene with the dimethyl sulfoxide shock method as previously described.35Jiang CK Connolly D Blumenberg M Comparison of methods for transfection of human epidermal keratinocytes.J Invest Dermatol. 1991; 97: 969-973Abstract Full Text PDF PubMed Google Scholar Cells were washed and incubated in the basal medium without epidermal growth factor (EGF) and bovine pituitary extract the day before transfection until the time of harvesting. Each transfection contained 5 μg/dish of keratin-CAT construct. The cells were then incubated with or without 0.1 μmol/L glucocorticoid dexamethasone (Sigma Chemical Co., St. Louis, MO) dissolved in ethanol and harvested 48 hours later. CAT assays were performed using FastCat (Molecular Probes, Eugene, OR) following a commercial protocol. Cell extracts used for CAT assay were normalized by total protein determined by protein assay (Bio-Rad). Thirty μg of protein were used for each reaction. CAT assay values were quantified by Fluor Imager 575 (Molecular Dynamics, Piscataway, NJ). All experiments were performed in duplicates, at least three times. Primary human keratinocytes were grown to 80% confluency. Twenty-four hours before the experiment cells were transferred to basal KBM medium (Life Technologies, Inc., Grand Island, NY). Before the scratch, cells were treated with 8 μg/ml mitomycin C (ICN, Irvine, CA) for 1 hour and washed with basal media. Scratches were performed as previously described.36Lee B Vouthounis C Stojadinovic O Brem H Im M Tomic-Canic M From an enhanceosome to a repressosome: molecular antagonism between glucocorticoids and EGF leads to inhibition of wound healing.J Mol Biol. 2005; 345: 1083-1097Crossref PubMed Scopus (52) Google Scholar Cells were incubated with 20 μmol/L LiCl or 25 ng/ml of EGF for 24 and 48 hours, rephotographed, and cell migration was quantified as previously described.36Lee B Vouthounis C Stojadinovic O Brem H Im M Tomic-Canic M From an enhanceosome to a repressosome: molecular antagonism between glucocorticoids and EGF leads to inhibition of wound healing.J Mol Biol. 2005; 345: 1083-1097Crossref PubMed Scopus (52) Google Scholar, 37Zavadil J Cermak L Soto-Nieves N Bottinger EP Integration of TGF-beta/Smad and Jagged1/Notch signalling in epithelial-to-mesenchymal transition.EMBO J. 2004; 23: 1155-1165Crossref PubMed Scopus (643) Google Scholar Thirty measurements were taken for each experimental condition and distance coverage by cells moving into the scratch wound area was quantified. Three images were analyzed per condition, per time point, and averages and standard deviations were calculated. Specimens of normal human skin were obtained as discarded tissue after reduction mammoplasty (approved protocol H #9796-03) and maintained as described.36Lee B Vouthounis C Stojadinovic O Brem H Im M Tomic-Canic M From an enhanceosome to a repressosome: molecular antagonism between glucocorticoids and EGF leads to inhibition of wound healing.J Mol Biol. 2005; 345: 1083-1097Crossref PubMed Scopus (52) Google Scholar Topical GC treatment was performed by daily application of Cormax (Clobetasol Propionate Cream 0.05%; Oclassen Pharmaceuticals, Inc.) using a sterile Q-tip applicator. Wounds were created using 4-mm punch biopsies through the reticular dermis and a rim of cells participating in wound healing was collected by repunching around the initial wounded area. Each time point was collected in parallel with an unwounded skin specimen of the same donor. All specimens were collected and either stored in RNAlater (Ambion, Austin, TX) or frozen in OCT compound (Tissue Tek, Reading, CA) for immunocytochemistry. To activate β-catenin wounded skin was maintained on the air-liquid interface in the presence or absence of 20 mmol/L LiCl.38Shim M Smart RC Lithium stabilizes the CCAAT/enhancer-binding protein alpha (C/EBPalpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity.J Biol Chem. 2003; 278: 19674-19681Crossref PubMed Scopus (47) Google Scholar Wounds were quantified by planimetry as described previously.36Lee B Vouthounis C Stojadinovic O Brem H Im M Tomic-Canic M From an enhanceosome to a repressosome: molecular antagonism between glucocorticoids and EGF leads to inhibition of wound healing.J Mol Biol. 2005; 345: 1083-1097Crossref PubMed Scopus (52) Google Scholar Chronic ulcer skin specimens were obtained as discarded tissue after debridement procedures on consented patients (approved protocol 01-0960(001)03sux). Samples were fixed in formalin and routinely processed for paraffin embedding. Samples from 10 different patients were analyzed independently. Paraffin-embedded tissue was sectioned and 5-μm-thick sections were stained with hematoxylin and eosin. The sections were analyzed using a Nikon microscope and digital images were obtained using a Spot RTcolor camera. For staining human tissues and cultured cells a c-myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:100 dilution at 4°C using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) as previously published.39Wang Z Frederick J Garabedian MJ Deciphering the phosphorylation “code” of the glucocorticoid receptor in vivo.J Biol Chem. 2002; 277: 26573-26580Crossref PubMed Scopus (248) Google Scholar Keratinocytes were grown on chamber slides to 70% confluency (Lab-Tek, Naperville, IL) and treated with 0.1 μmol/L dexamethasone (Sigma) or 20 mmol/L LiCl. Cells were fixed in 70% methanol for 10 minutes and permeabilized with 0.1% Triton X-100 for 10 minutes. Human tissues were stained with β-catenin antibody (BD Transduction Laboratories, Lexington, KY) as previously described40Munne A Fabre M Marinoso ML Gallen M Real FX Nuclear beta-catenin in colorectal tumors: to freeze or not to freeze? Colon Cancer Team at IMAS.J Histochem Cytochem. 1999; 47: 1089-1094Crossref PubMed Scopus (17) Google Scholar or E-cadherin antibody (Santa Cruz Biotechnology) using 1:200 or K6 antibody (a gift from Dr. Pierre Coulombe, Johns Hopkins, Baltimore, MD41McGowan KM Coulombe PA Onset of keratin 17 expression coincides with the definition of major epithelial lineages during skin development.J Cell Biol. 1998; 143: 469-486Crossref PubMed Scopus (259) Google Scholar) at 1:1500 dilution in 5% bovine serum albumin and visualized using a secondary fluorescein isothiocyanate anti-mouse IgG antibody 1:150 (Sigma). All sections were mounted with mounting media containing propidium iodide or 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA) to help visualization of the nuclear staining whereby the nuclear presence of β-catenin was visualized by the change of red color to orange or yellow. All negative controls were prepared by substitution of the primary antibody with an irrelevant antibody. The sections were analyzed using a Carl Zeiss microscope (Carl Zeiss, Thornwood, NY) and digital images were collected using Adobe TWAIN_32 program. Quantification of the nuclei positive for either c-myc or β-catenin was performed by three blinded lab members and medians and SD were calculated. All experiments were performed in triplicates, in which three to five images per condition, per each time point were independently quantified. To characterize the epidermis at the nonhealing edge of chronic ulcers we analyzed the histopathology of chronic wounds from the actual patients (Figure 1; A to C) and compared it with the epidermis of normal skin (Figure 1; D to F). We found epidermis from chronic ulcers to be very thick, hyperproliferative, containing mitotically active cells in suprabasal layers (Figure 1, compare A with D). Lastly, no epithelial migration was detected (absence of the epithelial tongue), suggesting incomplete keratinocyte activation. We also found epidermis to be hyper- and parakeratotic (presence of nuclei in cornified layer), suggesting incomplete keratinocyte differentiation (Figure 1, compare B with E). Interestingly, the abnormalities found are consistent with previous findings of c-myc overexpression in transgenic mouse models24Waikel RL Kawachi Y Waikel PA Wang XJ Roop DR Deregulated expression of c-Myc depletes epidermal stem cells.Nat Genet. 2001; 28: 165-168Crossref PubMed Scopus (289) Google Scholar, 25Arnold I Watt FM c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny.Curr Biol. 2001; 11: 558-568Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 42Frye M Gardner C Li ER Arnold I Watt FM Evidence that Myc activation depletes the epidermal stem cell compartment by modulating adhesive interactions with the local microenvironment.Development. 2003; 130: 2793-2808Crossref PubMed Scopus (165) Google Scholar, 43Waikel RL Wang XJ Roop DR Targeted expression of c-Myc in the epidermis alters normal proliferation, differentiation and UV-B induced apoptosis.Oncogene. 1999; 18: 4870-4878Crossref PubMed Scopus (68) Google Scholar as well as suppression of cytoskeletal components K6 and K16.44Wojcik SM Bundman DS Roop DR Delayed wound healing in keratin 6a knockout mice.Mol Cell Biol. 2000; 20: 5248-5255Crossref PubMed Scopus (127) Google Scholar, 45Wong P Coulombe PA Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair.J Cell Biol. 2003; 163: 327-337Crossref PubMed Scopus (163) Google Scholar Therefore, we hypothesized that induction of c-myc and suppression of K6/K16 may lead to inhibition of keratinocyte migration in human skin, perhaps contributing to the development of chronic wounds. To test the role of c-myc in wound healing we used a human skin organ culture wound model. Normal skin was wounded by 4-mm punch biopsy and maintained at air-liquid interface. Cells participating in the wound-healing response were harvested at 4 hours (immediate-early response) and 96 hours (intermediate response—exponential phase in which keratinocytes are actively proliferating and migrating) after wounding. c-myc expression was measured by Northern blots. Interestingly, we observed a particular expression pattern of c-myc during wound healing: its mRNA was repressed at 4 hours but derepressed by 96 hours after wounding (Figure 2A). Conversely, when we used inhibitors of wound healing, topical GC,46Beer HD Fassler R Werner S Glucocorticoid-regulated gene expression during cutaneous wound repair.Vitam Horm. 2000; 59: 217-239Crossref PubMed Google Scholar we found significant induction of c-myc mRNA (Figure 2B). Thus, we identified a differential expression pattern of c-myc: suppressed in early wound healing and induced by a wound-healing inhibitor. To determine whether GC indeed activate c-myc expression, we incubated primary human keratinocytes with either GC or lithium chloride (LiCl). LiCl is known to, by stabilizing nuclear localization of β-catenin, activate c-myc.47Roeser T Stein S Kessel M Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.Development. 1999; 126: 2955-2965PubMed Google Scholar To determine the nuclear presence of c-myc, cells were stained with c-myc-specific antibody and quantified. As predicted, we found that both GC and LiCl induce expression and nuclear localization of c-myc (Figure 2, C and D). c-myc was found to be nuclear in more than 70% of cells treated with GC and in 65% of LiCl-treated cells (positive control), whereas it was nuclear in only 1% of the untreated cells (negative control). Taken together, our findings that c-myc is repressed in human epidermis during early wound healing and induced by GC, coupled with the results from mouse models,24Waikel RL Kawachi Y Waikel PA Wang XJ Roop DR Deregulated expression of c-Myc depletes epidermal stem cells.Nat Genet. 2001; 28: 165-168Crossref PubMed Scopus (289) Google Scholar, 25Arnold I Watt FM c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny.Curr Biol. 2001; 11: 558-568Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 42Frye M Gardner C Li ER Arnold I Watt FM Evidence that Myc activation depletes the epidermal stem cell compartment by modulating adhesive interactions with the local microenvironment.Development. 2003; 130: 2793-2808Crossref PubMed Scopus (165) Google Scholar, 43Waikel RL Wang XJ Roop DR Targeted expression of c-Myc in the epidermis alters normal proliferation, differentiation and UV-B induced apoptosis.Oncogene. 1999; 18: 4870-4878Crossref PubMed Scopus (68) Google Scholar suggest that overexpression of c-myc in the early stage of wound healing might inhibit keratinocyte migration, causing impairment in healing. To test if the c-myc overexpression participates in chronic wounds we measured its activation in nonhealing wound biopsies from patients with chronic ulcers using a c-myc-specific antibody. We used normal human skin (negative control), skin treated with topical GC (positive control), and an acute wound edge (additional control). As expected, we found that epidermis of normal skin does not contain c-myc (Figure 3A) (in agreement with previous findings24Waikel RL Kawachi Y Waikel PA Wang XJ Roop DR Deregulated expression of c-Myc depletes epidermal stem cells.Nat Genet. 2001; 28: 165-168Crossref PubMed Scopus (289) G