Epidermal Hyperplasia and Appendage Abnormalities in Mice Lacking CD109
2012; Elsevier BV; Volume: 181; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2012.06.021
ISSN1525-2191
AutoresShinji Mii, Yoshiki Murakumo, Naoya Asai, Mayumi Jijiwa, Sumitaka Hagiwara, Takuya Kato, Masato Asai, Atsushi Enomoto, Kaori Ushida, Sayaka Sobue, Masatoshi Ichihara, Masahide Takahashi,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoCD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109−/−) mice. Although CD109−/− mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109−/− mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109−/− mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109−/− mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109−/− mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3. CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109−/−) mice. Although CD109−/− mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109−/− mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109−/− mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109−/− mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109−/− mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3. CD109, a glycosylphosphatidylinositol-anchored cell surface glycoprotein, is a member of the α2-macroglobulin/C3, C4, C5 family of thioester-containing proteins.1Sutherland D.R. Yeo E. Ryan A. Mills G.B. Bailey D. Baker M.A. Identification of a cell-surface antigen associated with activated T lymphoblasts and activated platelets.Blood. 1991; 77: 84-93Crossref PubMed Google Scholar, 2Haregewoin A. Solomon K. Hom R.C. Soman G. Bergelson J.M. Bhan A.K. Finberg R.W. 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Correlation of pathological grade and tumor stage of urothelial carcinomas with CD109 expression.Pathol Int. 2010; 60: 735-743Crossref PubMed Scopus (38) Google Scholar CD109 expression was significantly higher in well-differentiated SCCs of the oral cavity and in low-grade urothelial carcinomas of the urinary bladder than in moderately or poorly differentiated SCCs and in high-grade urothelial carcinomas, respectively.14Hagiwara S. Murakumo Y. Sato T. Shigetomi T. Mitsudo K. Tohnai I. Ueda M. Takahashi M. Up-regulation of CD109 expression is associated with carcinogenesis of the squamous epithelium of the oral cavity.Cancer Sci. 2008; 99: 1916-1923Crossref PubMed Scopus (14) Google Scholar, 16Hagikura M. Murakumo Y. Hasegawa M. Jijiwa M. Hagiwara S. Mii S. Hagikura S. Matsukawa Y. Yoshino Y. Hattori R. Wakai K. Nakamura S. Gotoh M. Takahashi M. 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Marcoux A. Lepage P. Roy S. Bizet A.A. Philip A. Identification of CD109 as part of the TGF-β receptor system in human keratinocytes.FASEB J. 2006; 20: 1525-1527Crossref PubMed Scopus (111) Google Scholar Our recent study using cultured cells showed CD109 to be cleaved by furin, generating 180- and 25-kDa fragments; the 180-kDa fragment is partially secreted into the medium.26Hagiwara S. Murakumo Y. Mii S. Shigetomi T. Yamamoto N. Furue H. Ueda M. Takahashi M. Processing of CD109 by furin and its role in the regulation of TGF-β signaling.Oncogene. 2010; 29: 2181-2191Crossref PubMed Scopus (58) Google Scholar The negative effect of CD109 on TGF-β signaling requires this furin-mediated cleavage of CD109 because the resulting 180-kDa fragment is responsible for this negative effect.26Hagiwara S. Murakumo Y. Mii S. Shigetomi T. Yamamoto N. Furue H. Ueda M. Takahashi M. Processing of CD109 by furin and its role in the regulation of TGF-β signaling.Oncogene. 2010; 29: 2181-2191Crossref PubMed Scopus (58) Google Scholar CD109 associates with caveolin-1, a major component of caveolae, and promotes localization of TGFBRs into caveolar compartments to facilitate their degradation.27Bizet A.A. Liu K. Tran-Khanh N. Saksena A. Vorstenbosch J. Finnson K.W. Buschmann M.D. Philip A. The TGF-β co-receptor, CD109, promotes internalization and degradation of TGF-β receptors.Biochim Biophys Acta. 2011; 1813: 742-753Crossref PubMed Scopus (97) Google Scholar Although these findings show the importance of CD109 in the TGF-β signaling pathway in vitro, its physiologic functions in vivo have not yet been elucidated. Recombinant CD109 regulates signal transducers and activators of transcription 3 (STAT3) activation in human keratinocytes.28Litvinov I.V. Bizet A.A. Binamer Y. Jones D.A. Sasseville D. Philip A. 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TGF-β down-regulates IL-6 signaling in intestinal epithelial cells: critical role of SMAD-2.FASEB J. 2003; 17: 2130-2132Crossref PubMed Google Scholar, 32Starsíchova A. Lincová E. Pernicová Z. Kozubík A. Soucek K. TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells.Cell Signal. 2010; 22: 1734-1744Crossref PubMed Scopus (21) Google Scholar STAT3 is critical to such biological activities as cell proliferation, differentiation, migration, survival, and oncogenesis.29Yu H. Kortylewski M. Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour microenvironment.Nat Rev Immunol. 2007; 7: 41-51Crossref PubMed Scopus (1456) Google Scholar, 30Sano S. Chan K.S. DiGiovanni J. Impact of Stat3 activation upon skin biology: a dichotomy of its role between homeostasis and diseases.J Dermatol Sci. 2008; 50: 1-14Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar In the present study, we generated CD109-deficient mice (CD109−/− mice) to investigate the physiologic roles of CD109 in vivo. The CD109−/− mice showed transient impairment of hair growth, accompanied by kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands. These findings suggest that CD109 has a role in the normal development of skin. Construction of the targeting vector started by modifying a pBlueScript II KS vector (Agilent Technologies, Inc., Santa Clara, CA) containing a LacZ reporter with a mouse nuclear localization signal upstream of a phosphoglycerine kinase (PGK) promoter driving a neomycin-resistant gene (neo) selection marker. The CD109 5′ homology arm (1666 bp) was generated via PCR with PfuUltra High-Fidelity DNA polymerase (Agilent Technologies) using genomic DNA of the 129S6 mouse strain as a template and inserted into the EcoRV site of the vector. The CD109 3′ homology arm (4893 bp) of intron 2 was inserted into the NotI site. This targeting vector (Figure 1A, middle) for the CD109 locus (Figure 1A, top) was designed to insert a lacZ-PGK-neo cassette 17 amino acids downstream from the methionine start, resulting in disruption of the remainder of the first coding exon and the entire second exon. Structure of the targeted CD109 allele is shown in Figure 1A (bottom). The final targeting vector was confirmed via DNA sequencing and restriction mapping. The targeting vector was linearized by digestion with KpnI, and introduced by electroporation into embryonic stem cells derived from 129S6 mice. After G418/diphtheria toxin A positive/negative selection, 10 embryonic stem cell clones with successful homologous recombination were isolated via Southern blot screening of SpeI-digested genomic DNA with a 5′ probe (Figure 1A). One clone was injected into C57BL/6J blastocysts; chimeric mice were generated by PhoenixBio Co., Ltd. (Higashihiroshima, Japan). The genetic background of the mice used in the present study was C57BL6J/129S6. All mice were housed in polycarbonate cages containing hardwood chip bedding, at 25°C on a 12-hour light-dark cycle. All animal protocols were approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine (approval ID number 23121). Genomic DNA from offspring was extracted from their tails. Genotyping of mice was performed via PCR, based on four primers: primer P1 (forward), 5′-GTCCCGCTTTCTGGTGACAG-3′; primer P2 (reverse), 5′-GTGTGACTGTTAGACAGTGCAG-3′; primer P3 (forward), 5′-CCATCGCCATCTGCTGCACG-3′; and primer P4 (reverse), 5′-ACGATCCTGAGACTTCCACAC-3′ (Figure 1A). The PCR with rTaq polymerase (Takara Bio Inc., Ohtsu, Japan) was performed as follows: 96°C for 2 minutes; 32 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 30 seconds. The expected PCR product sizes from wild-type and targeted alleles were 205 and 603 bp, respectively. After body weight measurement, mice were sacrificed under general anesthesia. Complete necropsies were performed, and resected organs were cut into 5-mm3 specimens and quickly frozen for protein extraction. Primary antibodies used in the present study included anti-CD109 and anti-Smad7 monoclonal or polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti–β-actin and anti-p63 monoclonal antibodies (Sigma-Aldrich Corp., St. Louis, MO); anti-CD3 polyclonal antibody (Dako, Glostrup, Denmark), anti-CD45R and anti-Gr-1 monoclonal antibodies (eBioscience, Inc., San Diego, CA); anti-CK10, anti-CK14, and anti-filaggrin polyclonal antibodies (Covance, Inc., Princeton, NJ); anti-BrdU (5′-bromo-2′-deoxyuridine) monoclonal antibody (BD Biosciences, San Jose, CA); and anti-cleaved caspase-3, anti–phospho-Smad2 (pSmad2), anti-Smad2, anti–phospho-Stat3 (pStat3), and anti-Stat3 monoclonal or polyclonal antibodies (Cell Signaling Technology, Inc., Danvers, MA). Alexa Fluor 488–conjugated anti-rabbit IgG secondary antibody was purchased from Invitrogen Corp. (Carlsbad, CA), and horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody was purchased from Dako (Table 1).Table 1List of Primary AntibodiesAntibodies toCloneSourceApplicationDilutionPretreatmentCD109C-9/mouse monoclonalSanta Cruz BiotechnologyImmunoblotting1:1000NACD109M-250/rabbit polyclonalSanta Cruz BiotechnologyImmunoperoxidase1:500AutoclaveSmad7H-79/rabbit polyclonalSanta Cruz BiotechnologyImmunofluorescence1:20Water bathβ-ActinAC-74/mouse monoclonalSigma-AldrichImmunoblotting1:5000NACD3Rabbit polyclonalDakoImmunoperoxidase1:500Water bathCD45RRA3-6B2/rat monoclonaleBioscienceImmunoperoxidase1:3200Water bathGr-1RB6-8C5/rat monoclonaleBioscienceImmunoperoxidase1:100Proteinase K, room temperature for 10 minp634A4/mouse monoclonalSigma-AldrichImmunoperoxidase1:100Water bathCK10Rabbit polyclonalCovanceImmunoperoxidase1:1000Water bathCK14AF64/rabbit polyclonalCovanceImmunoperoxidase1:1000Water bathFilaggrinRabbit polyclonalCovanceImmunoperoxidase1:500Water bathBrdUB44/mouse monoclonalBD BiosciencesImmunoperoxidase1:1002 nmol/L HCl, room temperature for 30 minC-caspase-3Rabbit polyclonalCell Signaling TechnologyImmunoperoxidase1:100Water bathpSmad2 (Ser465/467)Rabbit polyclonalCell Signaling TechnologyImmunofluorescence1:100Water bathImmunoblotting1:1000NASmad2Mouse monoclonalCell Signaling TechnologyImmunoblotting1:1000NApStat3 (Tyr705)Rabbit monoclonalCell Signaling TechnologyImmunofluorescence1:20Water bathImmunoblotting1:2000NAStat3Rabbit monoclonalCell Signaling TechnologyImmunoblotting1:2000NANA, not applicable. Open table in a new tab NA, not applicable. Frozen mouse tissues were homogenized in SDS sample buffer (62.5 mmol/L Tris HCl [pH 6.8], 2% SDS, 25% glycerol, and 20 μg/mL bromophenol blue) and sonicated until the tissues were no longer viscous. After measuring protein concentration using the DC Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA), the lysates were boiled at 100°C for 2 minutes in the presence of 2% β-mercaptoethanol. The lysates, containing 40 μg proteins, were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA). Membranes were blocked for 1 hour at room temperature in Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) with gentle agitation, and incubated with the primary antibody for 1 hour at room temperature. After the membranes were washed three times with Tris-buffered saline solution and Tween 20 buffer (20 mmol/L Tris HCl [pH 7.6], 137 mmol/L NaCl, and 0.1% Tween 20), they were incubated with secondary antibody conjugated to horseradish peroxidase for 1 hour at room temperature. After the membranes were washed, the reaction was visualized using the ECL Detection Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions. The major organs were resected as described above. All tissues were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin. Sections 4 μm thick were prepared for H&E staining (which was performed using conventional methods) and immunohistochemistry (IHC). Epidermal thickness of the dorsal skin was measured from the basal lamina to the lower border of the stratum corneum using WinROOF software version 5.04 (Mitani Corp., Fukui, Japan). Lipid accumulation was visualized via Oil Red O (Sigma-Aldrich) staining on frozen sections of formalin-fixed skin tissues. After anesthetization, the mice were perfused intravascularly with 4% (w/v) paraformaldehyde solution, and organs were resected and frozen in 2-methylbutane cooled in liquid nitrogen. Frozen sections 10 μm thick were prepared using a cryostat (Leica Microsystems GmbH, Wetzlar, Germany). CD109-specific PCR products of ∼400 bp with SP6 and T7 promoter fragments were generated using primers 5′-CCAAGCTATTTAGGTGACACTATAGAGAAGTGAACCTTCTCAGTGGC-3′ and 5′-TGAATTGTAATACGACTCACTATAGGGGCACAAAGTACAGAAGGACGG-3′. The PCR products were gel purified and transcribed in vitro using SP6 or T7 RNA polymerase (Roche Applied Science, Penzberg, Germany) incorporating 33P-UTP (PerkinElmer, Inc., Waltham, MA). SP6 RNA polymerase generated the sense probe as a control, and T7 RNA polymerase generated the antisense probe. The probes were subsequently DNase treated and purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Slides were hybridized overnight at 60°C using 200 μL per slide hybridization solution consisting of 1 × 106 cpm radiolabeled probe and hybridization buffer (50% formamide, 10% dextran sulfate, 0.5 mol/L NaCl, 1× Denhardt's solution, 10 mmol/L Tris [pH 8.0], 1 mmol/L EDTA, 500 μg/mL yeast transfer RNA, and 10 mmol/L dithiothreitol). After hybridization, slides were immersed in 2× standard saline citrate solution for 15 minutes at room temperature, and RNase buffer (20 μg/mL RNase A, 0.5 mol/L NaCl, 10 mmol/L Tris [pH 8.0], and 1 mmol/L EDTA) for 30 minutes at 37°C. After intensive washing and dehydration, slides were dipped in twice-diluted Kodak Autoradiography Emulsion (type NTB; Eastman Kodak Co., Rochester, NY) and dried at room temperature for 30 minutes in a darkroom. Slides were then stored at 4°C for approximately 2 weeks in the dark, and were developed using Kodak D19 Developer and Fixer (Eastman Kodak). Paraffin sections were prepared as described above. Slides were deparaffinized in xylene, and rehydrated in a graded series of ethanol. For antigen retrieval, they were immersed in Target Retrieval Solution [pH 9.0] (Dako) and heated for 15 minutes at 121°C via autoclaving or incubated for 30 minutes at 100°C in a water bath. In the case of staining with anti–Gr-1 antibody, slides were pretreated with proteinase K (100 μg/mL; Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 10 minutes at room temperature for antigen retrieval. Nonspecific binding was blocked with 10% normal goat serum for 10 minutes at room temperature. Sections were incubated with primary antibodies for 1 hour at room temperature. Endogenous peroxidase was inhibited using 3% hydrogen peroxide in PBS for 15 minutes. The slides were incubated with the secondary antibody conjugated to horseradish peroxidase–labeled polymer (EnVision+; Dako) for 15 minutes at room temperature, except for the slides incubated with anti-CD45R or anti–Gr-1 antibodies, which were incubated with N-Histofine Simple Stain Mouse MAX PO (Rat) (Nichirei Biosciences, Inc., Tokyo, Japan), for 30 minutes at room temperature. Reaction products were visualized using diaminobenzidine (Dako); nuclear counterstaining was performed using hematoxylin. Wild-type (CD109+/+) and homozygous (CD109−/−) mice were injected intraperitoneally with BrdU (Sigma-Aldrich), 5 mg per 100 g body weight, at postnatal day (P) 14 and P28. After 2 hours, the mice were sacrificed under general anesthesia, and dorsal skin was resected. The skin tissues were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin. Sections 4 μm thick were prepared on slides. The slides were deparaffinized, rehydrated, and immersed in 2 mol/L HCl for 30 minutes at room temperature for antigen retrieval. Immunohistochemical analysis using anti-BrdU antibody was performed as described above. Deparaffinization, hydration, antigen retrieval, and blocking steps were performed as described above. The sections were incubated with primary antibody for 2 hours at room temperature. The slides were incubated with Alexa Fluor 488 (1:500)–labeled secondary antibody for 30 minutes at room temperature. The images were visualized under a fluorescence microscope (Olympus Corp., Tokyo, Japan). Both wild-type and CD109-deficient keratinocytes were isolated from neonatal (P0) dorsal skin using the CELLnTEC Advanced Cell Systems (CELLnTEC, Bern, Switzerland). They were maintained in fully defined, low-calcium (0.07 mmol/L) medium, CnT-07 (CELLnTEC) in an incubator at 37°C and 5% CO2. They were then subcultured via trypsinization and plated on 3.5-cm dishes in CnT-07 medium. At 70% to 80% confluency, cells were starved for 6 hours in medium free of growth factor. They were washed once, treated with 0.1 nmol/L TGF-β1 (PeproTech, Inc., Rocky Hill, NJ) for 1 to 4 hours, and then lysed as described above. Male CD109+/+ and CD109−/− sibling mice (approximately 22 to 28 g body weight and 8 to 12 weeks old) were anesthetized; their dorsal skin was shaved and swabbed with 70% ethanol before the procedure. For wounding, 4-mm punch biopsies (Kai Industries Co., Ltd., Seki, Japan) were performed on the shaved area. Wounds were separated by a minimum of 6 mm of uninjured skin. The diameter of the wound areas was measured at 0, 1, 3, 5, and 7 days after wounding, and wound closure was evaluated as a percentage of the initial wound size. The Student's t-test was used to analyze differences in epidermal thickness, wound healing, and IHC-positive ratios between CD109−/− and CD109+/+ mice. P < 0.05 was considered significant. To inactivate the CD109 gene, we engineered a targeting vector in which the 3′ part of exon 1 and the entire exon 2 were replaced with a lacZ-PGK-neo cassette (Figure 1A). The lacZ sequence was placed in-frame with the start codon of CD109. Male chimeras were generated from one of the
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