Spontaneous Skin Erosions and Reduced Skin and Corneal Wound Healing Characterize CLIC4NULL Mice
2012; Elsevier BV; Volume: 181; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2012.03.025
ISSN1525-2191
AutoresV. C. Padmakumar, Kelsey F. Speer, Sonali Pal‐Ghosh, Katelyn E. Masiuk, Andrew Ryscavage, Samuel L. Dengler, Shelly Hwang, John C. Edwards, Vincenzo Coppola, Lino Tessarollo, Mary Ann Stepp, Stuart H. Yuspa,
Tópico(s)Dermatologic Treatments and Research
ResumoCutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4NULL mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4NULL mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4NULL skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4NULL keratinocytes relative to in wild-type cells, and CLIC4NULL keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4NULL keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway. Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4NULL mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4NULL mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4NULL skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4NULL keratinocytes relative to in wild-type cells, and CLIC4NULL keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4NULL keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway. CLIC4 belongs to the family of chloride intracellular channel proteins, which is composed of seven family members: p64, CLIC1 to CLIC5, and parchorin.1Shukla A. Yuspa S.H. CLIC4 and Schnurri-2: a dynamic duo in TGF-β signaling with broader implications in cellular homeostasis and disease.Nucleus. 2010; 1: 144-149Crossref Google Scholar Although the nature of participation of the membrane-associated CLIC4 in chloride transport is unsettled,2Littler D.R. Harrop S.J. Goodchild S.C. Phang J.M. Mynott A.V. Jiang L. Valenzuela S.M. Mazzanti M. Brown L.J. Breit S.N. Curmi P.M. The enigma of the CLIC proteins: ion channels, redox proteins, enzymes, scaffolding proteins?.FEBS Lett. 2010; 584: 2093-2101Crossref PubMed Scopus (137) Google Scholar the soluble protein is multifunctional and resides in multiple cellular compartments, including the cytoplasm. In the CLIC4 protein sequence is a C-terminal nuclear localization signal that promotes its translocation to the nucleus on several types of cellular stress.3Suh K.S. Mutoh M. Nagashima K. Fernandez-Salas E. Edwards L.E. Hayes D.D. Crutchley J.M. Marin K.G. Dumont R.A. Levy J.M. Cheng C. Garfield S. Yuspa S.H. The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleus in response to cellular stress and accelerates apoptosis.J Biol Chem. 2004; 279: 4632-4641Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 4Suh K.S. Malik M. Shukla A. Yuspa S.H. CLIC4, skin homeostasis and cutaneous cancer: surprising connections.Mol Carcinog. 2007; 46: 599-604Crossref PubMed Scopus (29) Google Scholar CLIC4 is a direct downstream response gene for p53- and c-Myc–mediated transcription and is essential for p53- and c-Myc–mediated apoptosis.5Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. p53 and tumor necrosis factor α regulate the expression of a mitochondrial chloride channel protein.J Biol Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 6Shiio Y. Suh K.S. Lee H. Yuspa S.H. Eisenman R.N. Aebersold R. Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4.J Biol Chem. 2006; 281: 2750-2756Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar CLIC proteins are highly conserved throughout evolution, and a CLIC homologue, EXC-4, is essential for excretory canal tube formation in Caenorhabditis elegans.7Berry K.L. Bulow H.E. Hall D.H. Hobert O. A C. elegans CLIC-like protein required for intracellular tube formation and maintenance.Science. 2003; 302: 2134-2137Crossref PubMed Scopus (127) Google Scholar CLIC4 has a similar function in vascular tubulogenesis in vitro, where its expression is induced in endothelial cells exposed to vascular endothelial growth factor-A, and its silencing decreases capillary network formation and sprouting and lumen formation in vitro.8Bohman S. Matsumoto T. Suh K. Dimberg A. Jakobsson L. Yuspa S. Claesson-Welsh L. Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis.J Biol Chem. 2005; 280: 42397-42404Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Tung J.J. Hobert O. Berryman M. Kitajewski J. Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro.Angiogenesis. 2009; 12: 209-220Crossref PubMed Scopus (70) Google Scholar Recently, defective angiogenesis was the primary phenotype reported in a CLIC4NULL mouse model.10Ulmasov B. Bruno J. Gordon N. Hartnett M.E. Edwards J.C. Chloride intracellular channel protein-4 functions in angiogenesis by supporting acidification of vacuoles along the intracellular tubulogenic pathway.Am J Pathol. 2009; 174: 1084-1096Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar A function for CLIC4 in transforming growth factor-β (TGF-β) signaling was first suggested subsequent to an mRNA analysis of genes modified during the TGF-β–mediated conversion of fibroblasts to myofibroblasts.11Ronnov-Jessen L. Villadsen R. Edwards J.C. Petersen O.W. Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-β1-mediated conversion of fibroblasts to myofibroblasts.Am J Pathol. 2002; 161: 471-480Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar This connection and the mechanism involved was solidified by the discovery that nuclear CLIC4 binds to phospho-Smads (p-Smads) 2 and 3 and inhibits dephosphorylation by its selective phosphatase PPM1a, thus prolonging the intracellular TGF-β signal and enhancing downstream responses.12Shukla A. Malik M. Cataisson C. Ho Y. Friesen T. Suh K.S. Yuspa S.H. TGF-β signalling is regulated by Schnurri-2-dependent nuclear translocation of CLIC4 and consequent stabilization of phospho-Smad2 and 3.Nat Cell Biol. 2009; 11: 777-784Crossref PubMed Scopus (74) Google Scholar To study the broader consequences of CLIC4 depletion in vivo, we generated mice lacking its expression. These results confirm the participation of CLIC4 in TGF-β signaling and reveal an important role for CLIC4 in skin and corneal wound healing. The experimental results suggest that these two findings are related. Recombineering technology was used for generation of the targeting vector.13Tessarollo L. Palko M.E. Akagi K. Coppola V. Gene Targeting in Mouse Embryonic Stem Cells.in: Wolfgang Wurst R.K. Humana Press, a part of Springer Science+Business Media LLC, New York2009Crossref Scopus (12) Google Scholar Briefly, bacterial artificial chromosome RP23-57P18 (BSP001) containing a 184-kb DNA sequence spanning 134,699,828 and 134,884,152 of mouse chromosome 4 containing the CLIC4 locus was purchased from Invitrogen (Carlsbad, CA). pBluescript light vector (Agilent Technologies Inc., Santa Clara, CA) containing the thymidine kinase gene (TK1) was used as a backbone for the targeting vector. The region of interest, including 4.2 kb upstream and 3.3 kb downstream of the second exon of the CLIC4 locus, was recombined into the targeting vector (see Supplemental Figure S1A at http://ajp.amjpathol.org). One loxP site was inserted 1.1 kb upstream, and the second loxP site was introduced 643 bp downstream of CLIC4 exon 2. The final targeting vector also contained a neomycin cassette flanked by FLP recognition target sites for positive selection and a TK gene for negative selection of embryonic stem cell clones. Generation of chimeric CLIC4 conditional knockout mice was obtained by standard technology. Briefly, the targeting vector (BSP031) was linearized by NotI digestion and was electroporated into mouse embryonic stem cells v6.4 (hybrid 129Sv/C57BL/6). Embryonic stem cell clones were subsequently selected with G418 and ganciclovir. Correctly targeted clones were identified by Southern blot analysis using probes specific to regions external to the 5′ and 3′ homology arms (see Supplemental Figure S1B at http://ajp.amjpathol.org). The 5′ probe was amplified using oligonucleotides 5′-GGGATTGGTCAGTTTGAAGACA-3′ (OSP048) and 5′-AGTGAGAGACTAGGAGCTAGAC-3′ (OSP049), and the 3′ probe was amplified using oligonucleotides 5′-CATGAAGAATCTTACTTGCACT-3′ (OSP050) and 5′-TGAGACATAAAACTGAAAAC-3′ (OSP051). Correctly targeted clones were injected into blastocysts, and germline transmission was achieved from mating an 85% chimera to a C57BL/6 wild-type (WT) female. PCR was routinely used for genotyping. WT and CLIC4FLOX mice were identified by genotyping with OSP147 and OSP190 oligonucleotides, which gives rise to a 604-bp WT band and a 446-bp flox band. Two sets of oligonucleotides were used for genotyping WT and CLIC4NULL mice. Oligonucleotides OSP147 and OSP190 produce a 496-bp WT band, and oligonucleotides OSP146 and OSP148 give rise to a 372-bp CLIC4NULL band. The sequences of the oligonucleotides used for genotyping are as follows: 5′-TCCCCATCTCCCTTTGAATCTTG-3′ (OSP146), 5′-CATGTTATTTCATGGAGCAAGAA-3′ (OSP147), 5′-CACGGTTTAGCCAGGCTGACTGG-3′ (OSP148), and 5′-AAGTAAACAAGCAGGGGACTTC-3′ (OSP190). Full-thickness and epidermal abrasion wounds were performed on mice that were approximately 50 days old during the resting phase of the hair cycle. An 8-mm-diameter, circular, full-thickness wound was made on the back, and the mice were observed for 12 days. Pictures were taken intermittently, and samples were collected for immunohistochemical (IHC) analysis at required time points. Epidermal abrasion was performed using a small felt wheel attached to a handheld electric tool (Dremel tool). A 2 cm2 region on the lower back was abraded. Wound depth was controlled by applying the same rotation speed and pressure. Abraded skin was clearly identified by the pink color of the deepidermalized area, and mice that bled during the procedure were discarded from the study and immediately euthanized. Skin samples were collected for IHC analysis 8 days after wounding. All the animal experiments were performed in accordance with the guidelines and approval of the National Cancer Institute Animal Care and Use Committee. Corneal epithelium debridement was performed on 24 WT and 27 CLIC4NULL mice to study the effect of loss of CLIC4 on corneal wound healing.14Pal-Ghosh S. Pajoohesh-Ganji A. Brown M. Stepp M.A. A mouse model for the study of recurrent corneal epithelial erosions: α9β1 integrin implicated in progression of the disease.Invest Ophthalmol Vis Sci. 2004; 45: 1775-1788Crossref PubMed Scopus (50) Google Scholar After anesthesia with ketamine and xylazine, proparacaine ophthalamic ointment eye drops were used to eliminate blink eye reflex. After demarcating a 1.5-mm wound area at the center of the cornea using a trephine, the epithelium was removed using a dulled blade as described previously15Pal-Ghosh S. Tadvalkar G. Jurjus R.A. Zieske J.D. Stepp M.A. BALB/c and C57BL6 mouse strains vary in their ability to heal corneal epithelial debridement wounds.Exp Eye Res. 2008; 87: 478-486Crossref PubMed Scopus (51) Google Scholar; wound closure was evaluated 18 hours after wounding. The area of the remaining wound was determined using ImageJ software version 1.44p (NIH, Bethesda, MD). Skin keratinocytes were isolated from newborn WT and CLIC4NULL mice as described previously.16Lichti U. Anders J. Yuspa S.H. Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice.Nat Protoc. 2008; 3: 799-810Crossref PubMed Scopus (354) Google Scholar Keratinocytes were cultured in minimum essential medium (Gibco S-MEM; Invitrogen) containing 8% serum and a low calcium level (0.05 mmol/L). The methods for the migration assay have been reported previously.17Stepp M.A. Liu Y. Pal-Ghosh S. Jurjus R.A. Tadvalkar G. Sekaran A. Losicco K. Jiang L. Larsen M. Li L. Yuspa S.H. Reduced migration, altered matrix and enhanced TGFβ1 signaling are signatures of mouse keratinocytes lacking Sdc1.J Cell Sci. 2007; 120: 2851-2863Crossref PubMed Scopus (50) Google Scholar In brief, keratinocytes were seeded on 24-well plates and were allowed to grow for 2 days in low-calcium 8% serum containing media before imaging using an Olympus IX81 research microscope (Olympus America Inc., Center Valley, PA) equipped with a ProScan motorized stage (Prior Scientific Inc., Rockland, MA) and a temperature- and CO2-controlled chamber (LiveCell incubation system; Neue Biosciences, Camp Hill, PA). Using relief contrast optics, 10× images were taken per well every 10 minutes for 16 hours 40 minutes (100 images). For each variable, triplicate wells were tracked and images managed using Metamorph image analysis software (Molecular Devices Corp., Chicago, IL), where velocities of 15 cells per well were calculated using the track cell module. From each cell tracked, average velocity, net displacement, and total displacement were determined. For TGF-β neutralizing antibody studies, 1 μg/mL of neutralizing antibody (AB-101-NA; R&D Systems, Minneapolis, MN) or control IgY (AB-101-Cl; R&D Systems) was added to serum containing media at day 2, and cells were tracked between days 2 and 3. Results of two independent experiments are reported. For experiments involving TGF-β treatment, TGF-β (No. 101-B1-001; R&D Systems) was dissolved in 4 mmol/L HCl containing 0.1% serum albumin at concentrations indicated on the figures. Cell attachment assays were performed on plastic dishes coated with matrix previously secreted by WT keratinocytes and extracted with ammonium hydroxide.18Dlugosz A.A. Glick A.B. Tennenbaum T. Weinberg W.C. Yuspa S.H. Isolation and Utilization of epidermal keratinocytes for oncogene research.Method Enzymol. 1995; 254: 3-20Crossref PubMed Scopus (140) Google Scholar WT and CLIC4NULL keratinocytes cultured for 5 days were trypsinized, and 300,000 cells were plated onto the matrix. After 30 minutes of attachment, the medium was removed and the dishes were rinsed twice with low-calcium medium, and attached cell number was assessed by MTT assay as described (No. G4102; Promega Corp., Madison, WI). Protein lysate was prepared from whole skin or cultured cells in lysis buffer (No. 9803; Cell Signaling Technology Inc., Beverly, MA). EDTA-free Halt protease inhibitor (No. 78437; Thermo Scientific, Waltham, MA) and phosphatase inhibitor (No. 78420; Pierce Biotechnology, Rockford, IL) were used during lysis buffer preparation. Protein concentration was measured using Bradford reagent (No. 500–0006; Bio-Rad Laboratories, Hercules, CA), and 10 to 50 μg of protein lysate was loaded in premade 12.5% Tris-HCl protein gels (Nos. 345–0014 and 345–0016; Bio-Rad Laboratories). The protein was transferred to either a polyvinylidene difluoride (No. 162–0177; Bio-Rad Laboratories) or a nitrocellulose (No. RPN303E; GE Healthcare, Piscataway, NJ) membrane and was probed with appropriate antibodies. CLIC4 and CLIC1 monoclonal antibodies were obtained from BD Biosciences, San Jose, CA. The polyclonal antibodies against β4 integrin, α3 integrin, and β1 integrin that were used for immunoblots were described previously.19Sta Iglesia D.D. Gala P.H. Qiu T. Stepp M.A. Integrin expression during epithelial migration and restratification in the tenascin-C-deficient mouse cornea.J Histochem Cytochem. 2000; 48: 363-376Crossref PubMed Scopus (38) Google Scholar P-Smad2 (ser465/467) (No. 3101; Cell Signaling Technology Inc.), p-Smad3 (a gift from Ed Leof, Mayo Clinic), E-cadherin (No. sc-7870; Santa Cruz Biotechnologies, Santa Cruz, CA), and p21 (No. sc-6246; Santa Cruz Biotechnologies) antibodies were used. Blots were developed using SuperSignal West Dura (No. 34076) and SuperSignal West Pico (No. 34078) chemiluminescent substrates from Thermo Scientific. Skin samples were fixed in 10% neutral buffered formalin overnight and then were embedded in paraffin. The Pathology/Histotechnology Laboratory of the National Cancer Institute, Frederick, MD, performed some of the IHC staining. The sections were stained with the following antibodies: BrdU (No. A21301MP; Invitrogen), Ki-67 (No. ab16667; Abcam Inc., Cambridge, MA), CD45 (No. 550539; BD Biosciences), CD31 (No. sc-1506; Santa Cruz Biotechnologies), myeloperoxidase (No. A0398; Dako, Carpinteria, CA), and F4/80 (No. 14–4801; eBioscience, San Diego, CA). CLIC4, p-Smad2, and keratin 6 staining used previously published methods.20Grondahl-Hansen J. Christensen I.J. Briand P. Pappot H. Mouridsen H.T. Blichert-Toft M. Dano K. Brunner N. Plasminogen activator inhibitor type 1 in cytosolic tumor extracts predicts prognosis in low-risk breast cancer patients.Clin Cancer Res. 1997; 3: 233-239PubMed Google Scholar Slides were scanned and analyzed using a ScanScope XT scanner and ImageScope viewing software version 11.0.2.725 (Aperio Technologies Inc., Vista, CA) or were photographed using an Eclipse E800 microscope (Nikon Instruments, Melville, NY) or an Axioplan microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany). Corneal tissues were processed for confocal imaging as described elsewhere.21Pajoohesh-Ganji A. Pal-Ghosh S. Simmens S.J. Stepp M.A. Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse.Stem Cells. 2006; 24: 1075-1086Crossref PubMed Scopus (75) Google Scholar After sacrifice, the eyes were removed; the corneas were fixed; the retina, lens, and iris were discarded; and four incisions were made in the cornea to permit flattening. After staining with the indicated antibodies, DAPI or propidium iodide was used to visualize the nuclei, and the corneas were placed with the epithelial side up in Fluoromount G mounting media (No. 17984–25; EMS, Hatfield, PA) and coverslipped. Images were acquired using the Zeiss LSM 710 Axio Examiner upright microscope and LSM 710 confocal system (Carl Zeiss MicroImaging GmbH) with 34 spectral detection channels and two transmitted light photomultipliers, five laser lines (458, 488, 514, 561, and 633 nm), control electronics, ZEN 2009 software, and x/y/z motorized scanning stage. Eighteen to 20 optical sections were acquired sequentially (z = 1 μm) using a 63× objective lens (numerical aperture = 1.4). Three-dimensional images were obtained using Volocity image analysis software version 5.0 (PerkinElmer, Waltham, MA). Images captured using confocal microscopy were merged and presented en face and/or rotated at 90° for cross-sectional views. For assessment of corneal thickness, the number of 1-μm z sections required to image the entire corneal thickness and the epithelium alone were determined at three locations for five corneas for each genotype. The attachment assays were repeated at least three times. Statistical analysis was performed using unpaired two-tailed t-tests, and data with P < 0.05 were considered significant. The statistical analysis for the migration assay was performed by one-way analysis of variance, and a minimum of 45 cells were evaluated for each genotype and condition in each well. The experiment was repeated twice. To study the in vivo function of CLIC4, we generated conditional CLIC4NULL mice. Mouse chromosomal locus 4D harbors the CLIC4 gene; it consists of six exons that code for a 253–amino acid protein. Sequence analysis of the CLIC4 gene revealed that removal of the second exon would be the most appropriate strategy, resulting in the loss of functional CLIC4 protein. Therefore, we designed the target vector to insert loxP sites surrounding the second exon of the CLIC4 gene (see Supplemental Figure S1A at http://ajp.amjpathol.org). Loss of the second exon would lead to a truncated form of CLIC4 consisting of the first 24 amino acids lacking any recognizable domain. Coincidently, a CLIC4 conventional knockout mouse being generated simultaneously in another laboratory targeted the identical location.10Ulmasov B. Bruno J. Gordon N. Hartnett M.E. Edwards J.C. Chloride intracellular channel protein-4 functions in angiogenesis by supporting acidification of vacuoles along the intracellular tubulogenic pathway.Am J Pathol. 2009; 174: 1084-1096Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Conditional CLIC4NULL animals were mated with C57BL/6 transgenic mice expressing Cre recombinase under the control of EIIa promoter22Lakso M. Pichel J.G. Gorman J.R. Sauer B. Okamoto Y. Lee E. Alt F.W. Westphal H. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.Proc Natl Acad Sci U S A. 1996; 93: 5860-5865Crossref PubMed Scopus (916) Google Scholar to abrogate the expression of CLIC4 in the entire mouse. Immunoblot analysis of CLIC4 in spleen, heart, testis, lungs, liver, kidney, skeletal muscle, and skin confirmed the ubiquitous loss of CLIC4. Expression of CLIC1 was modestly increased in spleen, lungs, and skin in CLIC4NULL mice, suggesting a potential compensation (Figure 1A). Even the loss of one allele of CLIC4 led to considerable reduction in its expression (see Supplemental Figure S1C at http://ajp.amjpathol.org). Statistical analysis of littermates from heterozygous parents showed 22% of the live births to be of the genotype CLIC4NULL, not significantly different from the expected 25% ratio (data not shown). Analysis of the litter size from 32 WT parents and 28 CLIC4NULL parents revealed a statistically significant difference in average litter size (8.3 and 5.3, respectively) (Figure 1B). Ten percent of CLIC4NULL mice weighed visibly less than their WT littermates (Figure 1C). Because previous studies have indicated important functions for CLIC4 in cultured skin keratinocytes,1Shukla A. Yuspa S.H. CLIC4 and Schnurri-2: a dynamic duo in TGF-β signaling with broader implications in cellular homeostasis and disease.Nucleus. 2010; 1: 144-149Crossref Google Scholar we first focused our attention to determine whether a skin phenotype would emerge from deletion of CLIC4 in vivo. CLIC4NULL mice are viable and not readily distinguishable from their WT littermates when full size. On standard H&E staining, WT and CLIC4NULL skin were identical (see Supplemental Figure S2, A and B, at http://ajp.amjpathol.org), and Ki-67 and BrdU staining revealed no statistically significant difference in proliferation of epidermal keratinocytes between WT and CLIC4NULL skin (see Supplemental Figure S2, C–F, at http://ajp.amjpathol.org). The absence of cutaneous CLIC4 did not evoke an inflammatory infiltrate as measured by CD45 staining (see Supplemental Figure S2, G and H, at http://ajp.amjpathol.org). However, some CLIC4NULL mice developed skin erosions around their face and neck and across the entire body. These erosions occur in approximately 25% of mice in CD1 background developed in a coauthor's laboratory (J.E.) (Figure 1D) and in 40% of mice in C57Bl/6 background that are older than 6 months monitored for 4 months at the National Cancer Institute mouse facility (Figure 1, E and F). During this time, 1 of 22 WT animals also developed a milder dermatitis-like skin abnormality. Histologic analysis of the skin erosions revealed hyperplasia as evidenced by keratin 6 and Ki-67 staining and neutrophilic infiltration as detected by myeloperoxidase staining (see Supplemental Figure S3 at http://ajp.amjpathol.org). We investigated whether the spontaneous skin erosions in CLIC4NULL mice occur because of defective healing of wounds that develop in caged housing. We first studied the expression of CLIC4 in wounded skin. In normal resting WT skin, CLIC4 is localized to a distinct population of nuclei along the basal layer of the epidermis and in hair follicles (Figure 2A). Specific expression of CLIC4 is highly up-regulated in the epidermis and in the dermal compartment in experimentally induced skin wounds (Figure 2B). In contrast, CLIC4 is absent from resting and wounded areas of CLIC4NULL skin (Figure 2, C and D). CLIC4 is an integral component of TGF-β signaling, and TGF-β is involved in wound healing. However, neither constitutive nor wound-induced nuclear p-Smad2 was impaired by the absence of CLIC4 in mouse skin (Figure 2, E–H). In both genotypes, p-Smad2 was detected in nuclei of basal and suprabasal epidermis at the wound margins and in infiltrating cells in the dermis 4 days after wounding. To study the wound-healing process, we made 8-mm full-thickness wounds on a cohort of eight WT and eight CLIC4NULL mice and observed them for 12 days. Wound closure was delayed in CLIC4NULL mice after 8 days compared with in WT mice (Figure 3, A–H). WT wound closure was complete after 12 days, but the CLIC4NULL wounds remained open at this time point (Figure 3, I–P). We confirmed the complete reepithelialization of WT wounds histologically after 12 days, but areas remained without closure in all wounds on CLIC4NULL mice 12 days after wounding (Figure 3, Q–X).Figure 3The loss of CLIC4 delays wound healing. An 8-mm, circular, full-thickness wound was made on the backs of eight WT and eight CLIC4NULL mice, and the mice were subsequently monitored for 12 days. After 8 days, four independent WT mice (A–D) and four independent CLIC4NULL mice (E–H) were photographed. I–P: The same mice were photographed after 12 days to monitor the extent of wound closure. Skin from the back wound site of four independent WT (Q–T) and CLIC4NULL (U–X) mice was collected after 12 days, fixed in 10% neutral buffered formalin, and H&E stained. Sections show the epidermis and dermis at the wound site. Scale bar = 200 μm (Q–X).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Epidermal abrasion wounding involving a group of six mice for each genotype also revealed moderately delayed wound resolution in CLIC4NULL mice compared with their WT counterparts (see Supplemental Figure S4 at http://ajp.amjpathol.org). One aspect of wound healing is resolution of the dermal inflammatory infiltrate invariably associated with induced wounds. IHC staining of full-thickness wounds from early healing (2 and 4 days) and near resolution (12 days) indicated similar early recruitment of CD45+ and F4/80+ macrophages into the wound site but sustained a statistically significant presence of these inflammatory cells in 12-day-old CLIC4NULL wounds (Figure 4). Skin wound healing involves reepithelialization and angiogenesis, and CLIC4 is known to influence angiogenesis in vitro and in vivo.8Bohman S. Matsumoto T. Suh K. Dimberg A. Jakobsson L. Yuspa S. Claesson-Welsh L. Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel 4 (CLIC4) in tubular morphogenesis.J Biol Chem. 2005; 280: 42397-42404Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Tung J.J. Hobert O. Berryman M. Kitajewski J. Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro.Angiogenesis. 2009; 12: 209-220Crossref PubMed Scopus (70) Google Scholar, 10Ulmasov B. Bruno J. Gordon N. Hartnett M.E. Edwards J.C. Chloride intracellular channel protein-4 functions in angiogenesis by supporting acidification of vacuoles along the intracellular tubulogenic pathway.Am J Pathol. 2009; 174: 1084-1096Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Corneal debridement wounds remove the corneal epithelium only and reepithelialize without angiogenesis. To sort out the underlying cause of the healing delay in the CLIC4NULL skin, we used corneal wound healing as a model of rapid and highly quantitative reepithelialization in response to superficial debridement.23Buck R.C. Cell migration in
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