Mice Lacking Smad3 Are Protected Against Cutaneous Injury Induced by Ionizing Radiation
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64926-7
ISSN1525-2191
AutoresKathleen C. Flanders, Catherine D. Sullivan, Makiko Fujii, Anastasia L. Sowers, Mario A. Anzano, Alidad Arabshahi, Christopher Major, Chu‐Xia Deng, Angelo Russo, James B. Mitchell, Anita B. Roberts,
Tópico(s)Cancer Cells and Metastasis
ResumoTransforming growth factor-β (TGF-β) plays a central role in the pathogenesis of inflammatory and fibrotic diseases, including radiation-induced fibrosis. We previously reported that mice null for Smad3, a key downstream mediator of TGF-β, show accelerated healing of cutaneous incisional wounds with reduced inflammation and accumulation of matrix. To determine if loss of Smad3 decreases radiation-induced injury, skin of Smad3+/+ [wild-type (WT)] and −/− [knockout (KO)] mice was exposed to a single dose of 30 to 50 Gy of γ-irradiation. Six weeks later, skin from KO mice showed significantly less epidermal acanthosis and dermal influx of mast cells, macrophages, and neutrophils than skin from WT littermates. Skin from irradiated KO mice exhibited less immunoreactive TGF-β and fewer myofibroblasts, suggesting that these mice will have a significantly reduced fibrotic response. Although irradiation induced no change in the immunohistochemical expression of the TGF-β type I receptor, the epidermal expression of the type II receptor was lost after irradiation whereas its dermal expression remained high. Primary keratinocytes and dermal fibroblasts prepared from WT and KO mice showed similar survival when irradiated, as did mice exposed to whole-body irradiation. These results suggest that inhibition of Smad3 might decrease tissue damage and reduce fibrosis after exposure to ionizing irradiation. Transforming growth factor-β (TGF-β) plays a central role in the pathogenesis of inflammatory and fibrotic diseases, including radiation-induced fibrosis. We previously reported that mice null for Smad3, a key downstream mediator of TGF-β, show accelerated healing of cutaneous incisional wounds with reduced inflammation and accumulation of matrix. To determine if loss of Smad3 decreases radiation-induced injury, skin of Smad3+/+ [wild-type (WT)] and −/− [knockout (KO)] mice was exposed to a single dose of 30 to 50 Gy of γ-irradiation. Six weeks later, skin from KO mice showed significantly less epidermal acanthosis and dermal influx of mast cells, macrophages, and neutrophils than skin from WT littermates. Skin from irradiated KO mice exhibited less immunoreactive TGF-β and fewer myofibroblasts, suggesting that these mice will have a significantly reduced fibrotic response. Although irradiation induced no change in the immunohistochemical expression of the TGF-β type I receptor, the epidermal expression of the type II receptor was lost after irradiation whereas its dermal expression remained high. Primary keratinocytes and dermal fibroblasts prepared from WT and KO mice showed similar survival when irradiated, as did mice exposed to whole-body irradiation. These results suggest that inhibition of Smad3 might decrease tissue damage and reduce fibrosis after exposure to ionizing irradiation. The three mammalian isoforms of transforming growth factor-β (TGF-β1, TGF-β2, and TGF-β3) affect a number of cellular processes including growth, differentiation, apoptosis, chemotaxis, and extracellular matrix (ECM) production.1Roberts AB Sporn MB The transforming growth factors-β. Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors. Springer-Verlag, New York1990: 419-472Google Scholar One of the most extensively characterized biological functions of TGF-β is its role in regulating physiological and pathological inflammation and fibrosis.2Border WA Noble NA Transforming growth factor beta in tissue fibrosis.N Engl J Med. 1994; 331: 1286-1292Crossref PubMed Scopus (2977) Google Scholar, 3Roberts AB Sporn MB Transforming growth factor-β.in: Clark RAF The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 275-308Google Scholar, 4Branton MH Kopp JB TGF-beta and fibrosis.Microbes Infect. 1999; 1: 1349-1365Crossref PubMed Scopus (499) Google Scholar, 5Roberts AB Piek E Bottinger EP Ashcroft G Mitchell JB Flanders KC Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?.Chest. 2001; 120: S43-S47Abstract Full Text Full Text PDF PubMed Google Scholar TGF-β has been implicated in fibrotic diseases, such as cirrhosis, glomerulonephritis, and pulmonary fibrosis, and blocking the actions of TGF-β with antibodies or antagonists abolishes the inflammation and fibrosis in animal models of many of these diseases.4Branton MH Kopp JB TGF-beta and fibrosis.Microbes Infect. 1999; 1: 1349-1365Crossref PubMed Scopus (499) Google Scholar, 5Roberts AB Piek E Bottinger EP Ashcroft G Mitchell JB Flanders KC Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?.Chest. 2001; 120: S43-S47Abstract Full Text Full Text PDF PubMed Google Scholar At the cellular level, TGF-β affects virtually all stages of the chronic inflammatory and fibrotic disease process. It is a potent chemotactic factor for monocytes,6Wahl SM Hunt DA Wakefield LM McCartney-Francis N Wahl LM Roberts AB Sporn MB Transforming growth factor type beta induces monocyte chemotaxis and growth factor production.Proc Natl Acad Sci USA. 1987; 84: 5788-5792Crossref PubMed Scopus (1083) Google Scholar neutrophils,7Wahl SM Allen JB Brandes ME Cytokine modulation of bacterial cell wall-induced arthritis.Agents Actions Suppl. 1991; 35: 29-34PubMed Google Scholar, 8Reibman J Meixler S Lee TC Gold LI Cronstein BN Haines KA Kolasinski SL Weissmann G Transforming growth factor beta 1, a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways.Proc Natl Acad Sci USA. 1991; 88: 6805-6809Crossref PubMed Scopus (179) Google Scholar mast cells,9Gruber BL Marchese MJ Kew RR Transforming growth factor-beta 1 mediates mast cell chemotaxis.J Immunol. 1994; 152: 5860-5867PubMed Google Scholar, 10Olsson N Piek E ten Dijke P Nilsson G Human mast cell migration in response to members of the transforming growth factor-beta family.J Leukoc Biol. 2000; 67: 350-356PubMed Google Scholar and fibroblasts,11Postlethwaite AE Keski-Oja J Moses HL Kang AH Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor beta.J Exp Med. 1987; 165: 251-256Crossref PubMed Scopus (642) Google Scholar active at femtomolar concentrations. After recruitment of inflammatory cells into an area of insult, TGF-β also activates neutrophils and induces macrophages to secrete cytokines, one of which is TGF-β itself. This autoinduction of TGF-β is important for recruitment of additional inflammatory cells and maintaining local elevated levels of TGF-β.5Roberts AB Piek E Bottinger EP Ashcroft G Mitchell JB Flanders KC Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?.Chest. 2001; 120: S43-S47Abstract Full Text Full Text PDF PubMed Google Scholar The TGF-β produced by macrophages can then induce matrix production by fibroblasts. The sustained expression of TGF-β is critical to the maintenance of the inflammatory and fibrotic response. TGF-βs signal through transmembrane receptors with intrinsic serine/threonine kinase activity. Binding of ligand to these heteromeric receptors induces carboxyl-terminal serine phosphorylation of a set of cytoplasmic signal-transducing proteins collectively referred to as “Smad” proteins. After activation/phosphorylation, pathway-specific Smad proteins (Smad2 and Smad3 for TGF-β signaling) heterodimerize with the common mediator Smad4 and this complex translocates to the nucleus to regulate expression of specific target genes.12Massague J How cells read TGF-beta signals.Nat Rev Mol Cell Biol. 2000; 1: 169-178Crossref PubMed Scopus (1627) Google Scholar Smad7 (an inhibitory Smad) can disrupt signal transduction by preventing phosphorylation of Smad2 or Smad3.13Hayashi H Abdollah S Qiu Y Cai J Xu YY Grinnell BW Richardson MA Topper JN Gimbrone Jr, MA Wrana JL Falb D The MAD-related protein Smad7 associates with the TGF-beta receptor and functions as an antagonist of TGF-beta signaling.Cell. 1997; 89: 1165-1173Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar, 14Nakao A Afrakhte M Moren A Nakayama T Christian JL Heuchel R Itoh S Kawabata M Heldin NE Heldin CH ten Dijke P Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.Nature. 1997; 389: 631-635Crossref PubMed Scopus (1534) Google Scholar Mitogen-activated protein kinase pathways, induced by TGF-β or by other inputs, as well as protein kinase C activation also modulate TGF-β signaling by altering phosphorylation of Smads at sites other than the C-terminal serines phosphorylated by ligand-activated receptors.15Kretzschmar M Massague J SMADs: mediators and regulators of TGF-beta signaling.Curr Opin Genet Dev. 1998; 8: 103-111Crossref PubMed Scopus (427) Google Scholar, 16Yakymovych I ten Dijke P Heldin CH Souchelnytskyi S Regulation of Smad signaling by protein kinase C.FASEB J. 2001; 15: 553-555Crossref PubMed Scopus (149) Google Scholar Smad2 and Smad3, although highly homologous, have distinct modes of action. Smad3 regulates target gene activity directly by binding to DNA,17Zawel L Dai JL Buckhaults P Zhou S Kinzler KW Vogelstein B Kern SE Human Smad3 and Smad4 are sequence-specific transcription activators.Mol Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar whereas Smad2 activates transcription instead by binding to other DNA-binding transcription factors to modulate their activity.18Yagi K Goto D Hamamoto T Takenoshita S Kato M Miyazono K Alternatively spliced variant of smad2 lacking exon 3. Comparison with wild-type smad2 and smad3.J Biol Chem. 1999; 274: 703-709Crossref PubMed Scopus (205) Google Scholar The different DNA-binding characteristics of Smad2 and Smad3 result in regulation of distinct sets of target genes. The distinct activities of Smad2 and Smad3 are evidenced by the finding that targeted deletion of the Smad2 gene results in early embryonic lethality19Nomura M Li E Smad2 role in mesoderm formation, left-right patterning and craniofacial development.Nature. 1998; 393: 786-790Crossref PubMed Scopus (502) Google Scholar, 20Waldrip WR Bikoff EK Hoodless PA Wrana JL Robertson EJ Smad2 signaling in extraembryonic tissues determines anterior-posterior polarity of the early mouse embryo.Cell. 1998; 92: 797-808Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 21Weinstein M Yang X Li C Xu X Gotay J Deng CX Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2.Proc Natl Acad Sci USA. 1998; 95: 9378-9383Crossref PubMed Scopus (256) Google Scholar whereas mice null for Smad3 are viable for up to 8 months.22Zhu Y Richardson JA Parada LF Graff JM Smad3 mutant mice develop metastatic colorectal cancer.Cell. 1998; 94: 703-714Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 23Datto MB Frederick JP Pan L Borton AJ Zhuang Y Wang XF Targeted disruption of Smad3 reveals an essential role in transforming growth factor beta-mediated signal transduction.Mol Cell Biol. 1999; 19: 2495-2504Crossref PubMed Google Scholar, 24Yang X Letterio JJ Lechleider RJ Chen L Hayman R Gu H Roberts AB Deng C Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar Studies using fibroblasts derived from embryos null for either Smad2 or Smad3 show that TGF-β1-mediated autoinduction and induction of c-fos are Smad3-dependent.25Piek E Ju WJ Heyer J Escalante-Alcalde D Stewart CL Weinstein M Deng C Kucherlapati R Bottinger EP Roberts AB Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.J Biol Chem. 2001; 276: 19945-19953Crossref PubMed Scopus (353) Google Scholar Many genes, such as the ECM proteins collagen type I and type VII, contain AP-1 binding sites in their regulatory regions, and their induction by TGF-β has been shown to be Smad3-dependent.26Chen SJ Yuan W Mori Y Levenson A Trojanowska M Varga J Stimulation of type I collagen transcription in human skin fibroblasts by TGF-beta: involvement of Smad3.J Invest Dermatol. 1999; 112: 49-57Crossref PubMed Scopus (367) Google Scholar, 27Vindevoghel L Lechleider RJ Kon A de Caestecker MP Uitto J Roberts AB Mauviel A SMAD3/4-dependent transcriptional activation of the human type VII collagen gene (COL7A1) promoter by transforming growth factor beta.Proc Natl Acad Sci USA. 1998; 95: 14769-14774Crossref PubMed Scopus (157) Google Scholar, 28Verrecchia F Vindevoghel L Lechleider RJ Uitto J Roberts AB Mauviel A Smad3/AP-1 interactions control transcriptional responses to TGF-beta in a promoter-specific manner.Oncogene. 2001; 20: 3332-3340Crossref PubMed Scopus (158) Google Scholar, 29Verrecchia F Chu ML Mauviel A Identification of novel TGF-beta/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/promoter transactivation approach.J Biol Chem. 2001; 276: 17058-17062Crossref PubMed Scopus (530) Google Scholar Characterization of the basal phenotype of Smad3-null mice, as well as studies of incisional wound healing in these mice suggest that Smad3 has an important function in both inflammation and fibrosis in vivo. Smad3-null mice die from defects in mucosal immunity suggestive of defects in neutrophil chemotaxis and consistent with the observation that the chemotactic response of Smad3-null neutrophils to TGF-β is impaired both in vivo and in vitro.24Yang X Letterio JJ Lechleider RJ Chen L Hayman R Gu H Roberts AB Deng C Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar Cutaneous incisional wounds in Smad3-null mice show reduced influx of inflammatory cells, decreased accumulation of matrix, and enhanced re-epithelialization.30Ashcroft GS Yang X Glick AB Weinstein M Letterio JL Mizel DE Anzano M Greenwell-Wild T Wahl SM Deng C Roberts AB Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.Nat Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (759) Google Scholar This results, in part, from the impaired ability of Smad3-null macrophages to respond to TGF-β with chemotaxis and autoinduction; reduced levels of TGF-β in the granulation tissue then result in less ECM production. Additionally, Smad3-null keratinocytes lose their ability to be growth inhibited by TGF-β which contributes to the enhanced epithelialization of the wounds. Chronic inflammatory and fibrotic diseases share many common features with wound healing, except that the fibrotic process does not resolve. Because loss of Smad3 interferes with the effects of TGF-β on chemotaxis and autoinduction in inflammatory cells, and because induction of many ECM genes by TGF-β are also dependent on Smad3, we hypothesized that mice null for Smad3 will be resistant to chronic inflammation and fibrosis in which TGF-β has been shown to be involved.5Roberts AB Piek E Bottinger EP Ashcroft G Mitchell JB Flanders KC Is Smad3 a major player in signal transduction pathways leading to fibrogenesis?.Chest. 2001; 120: S43-S47Abstract Full Text Full Text PDF PubMed Google Scholar Radiation fibrosis has features common to other fibrotic diseases, including the involvement of TGF-β in its pathogenesis.31Martin M Lefaix J Delanian S TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target?.Int J Radiat Oncol Biol Phys. 2000; 47: 277-290Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar Ionizing radiation exposure frequently induces damage in the skin and underlying subcutaneous tissue. Early lesions are characterized by erythema, dry and moist desquamation, and ulceration resulting from the infiltration of inflammatory cells and increased vascularization. As a result of ionizing irradiation, the epidermis may be hyperplastic or atrophic. Likewise, activated dermal fibroblasts are characterized by the appearance of cytoskeletal proteins similar to those involved in wound contraction, such as smooth muscle actin, and a deposition of a fibrous matrix that is abnormal in both quantity and quality. These fibrotic sequelae are an unwanted complication of radiotherapy and severely impact on a patient's quality of life. Biopsies removed surgically from fibrotic lesions 6 months to 20 years after radiotherapy show enhanced expression of mRNA for collagen type I and type III and TGF-β1.31Martin M Lefaix J Delanian S TGF-beta1 and radiation fibrosis: a master switch and a specific therapeutic target?.Int J Radiat Oncol Biol Phys. 2000; 47: 277-290Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar Similarly, in an experimental model of cutaneous radiation in the pig, TGF-β1 mRNA was increased at 1 to 12 months after irradiation32Martin M Lefaix JL Pinton P Crechet F Daburon F Temporal modulation of TGF-beta 1 and beta-actin gene expression in pig skin and muscular fibrosis after ionizing radiation.Radiat Res. 1993; 134: 63-70Crossref PubMed Scopus (161) Google Scholar and immunoreactive TGF-β1 was localized to myofibroblasts, endothelial cells, and the collagen matrix. Irradiation of mouse skin also shows increased expression of TGF-β1 mRNA from 6 hours to 9 months after irradiation.33Randall K Coggle JE Long-term expression of transforming growth factor TGF beta 1 in mouse skin after localized beta-irradiation.Int J Radiat Biol. 1996; 70: 351-360Crossref PubMed Scopus (68) Google Scholar Irradiation of other tissues such as lung,34Yi ES Bedoya A Lee H Chin E Saunders W Kim SJ Danielpour D Remick DG Yin S Ulich TR Radiation-induced lung injury in vivo: expression of transforming growth factor-beta precedes fibrosis.Inflammation. 1996; 20: 339-352Crossref PubMed Scopus (60) Google Scholar intestine,35Hauer-Jensen M Richter KK Wang J Abe E Sung CC Hardin JW Changes in transforming growth factor beta1 gene expression and immunoreactivity levels during development of chronic radiation enteropathy.Radiat Res. 1998; 150: 673-680Crossref PubMed Scopus (38) Google Scholar bladder,36Kraft M Oussoren Y Stewart FA Dorr W Schultz-Hector S Radiation-induced changes in transforming growth factor beta and collagen expression in the murine bladder wall and its correlation with bladder function.Radiat Res. 1996; 146: 619-627Crossref PubMed Scopus (40) Google Scholar and liver37Anscher MS Crocker IR Jirtle RL Transforming growth factor-beta 1 expression in irradiated liver.Radiat Res. 1990; 122: 77-85Crossref PubMed Scopus (161) Google Scholar is also associated with increased TGF-β expression. Because elevated levels of TGF-β are associated with radiation-induced inflammation and fibrosis, and because Smad3-null mice show reduced inflammation, TGF-β1, and matrix accumulation after wounding, we hypothesized that loss of Smad3 will decrease inflammation and subsequent fibrosis induced by irradiation. To test this hypothesis, we exposed the skin of Smad3+/+ (WT), +/− (Het), and −/− (KO) mice to high doses of λ-irradiation. Here we report that 6 weeks after irradiation skin from KO mice shows significantly less epidermal acanthosis, dermal inflammation, and immunoreactive TGF-β than does skin from WT mice. Smad3ex8/ex8 mice were generated by targeted disruption of the Smad3 gene by homologous recombination. Targeted embryonic stem-cell clones were microinjected into C57BL/6 blastocysts to obtain germline transmission. Mice heterozygous for the targeted disruption were intercrossed to produce homozygous offspring.24Yang X Letterio JJ Lechleider RJ Chen L Hayman R Gu H Roberts AB Deng C Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed Google Scholar The protocol used in this study was institutionally approved and in accordance with guidelines of the Institute of Laboratory Animal Resources, National Research Council. Animals were exposed to whole-body irradiation using a 137Cesium Gamma Cell 40 (Nordion Int. Inc., Kanata, Ontario) irradiator that had been calibrated with thermoluminescent dosimetry chips (Bicron, Inc., Solon, OH) planted in phantom Plexiglas mice. Animals were irradiated between 7 to 9 weeks after birth. Smad3 WT, Het, and KO mice were placed in circular Lucite containers with holes for ventilation (up to five animals). The container was positioned within the irradiator and exposed for varying times to deliver the desired dose (single radiation doses ranging from 6 to 10 Gy at a dose rate of 0.98 Gy/minute). After irradiation, mice were removed from the container and returned to the cages in which they were housed (five animals/cage) in a climate-controlled environment with free access to food and water. If at any time an animal was unable to acquire food or water because of illness, the animal was euthanized per protocol. Mice were observed daily for survival. Experiments also involved local irradiation to the leg or flank. For these studies animals (without anesthetics) were placed in customized Lucite jigs that allow for immobilization and selective irradiation of the leg or skin of the flank. Special clamps held the skin without decreasing blood flow to the treatment area as determined previously by laser Doppler studies.38Cuscela D Coffin D Lupton GP Cook JA Krishna MC Bonner RF Mitchell JB Protection from radiation-induced alopecia with topical application of nitroxides: fractionated studies.Cancer J Sci Am. 1996; 2: 273-278PubMed Google Scholar Single radiation doses ranging from 30 to 60 Gy were delivered by a Therapax DXT300 X-ray irradiator (Pantak, Inc., East Haven, CT) using 2.0-mm Al filtration (300 kVp) at a dose rate of 1.9 Gy/minute. Special care was taken to avoid irradiation of other body parts by using lead shields specifically designed as a part of the jigs. In addition to WT, Het, and KO mice, four mice each of strains C57BL/6nCr, Tac:N:NIHS-BCFBR, 129S6/SvEv-ATM , and WBB6F1/J KIT/KITW-v (mast cell deficient)39Kitamura Y Go S Hatanaka K Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation.Blood. 1978; 52: 447-452Crossref PubMed Google Scholar also received hind leg irradiation. After irradiation, the animals were placed in cages as indicated above and observed daily for 5 to 6 weeks at which time animals were euthanized and skin was removed from the hind legs or flank and transferred to 10% neutral buffered formalin for 24 hours. After three washes in 70% ethanol, tissues were processed for paraffin embedding. Formalin-fixed, paraffin-embedded tissues were cut at 5 μm and sections were stained with hematoxylin and eosin (H&E), Geimsa (to identify neutrophils), or low pH toluidine blue (to identify mast cells). The numbers of neutrophils and mast cells were counted in 5 400× magnification fields/skin section. Macrophages, blood vessels, and myofibroblasts were identified by staining with rat anti-mouse Mac-3 (BD PharMingen, San Diego, CA), rat anti-mouse CD31(PECAM-1) (BD PharMingen), and mouse anti-smooth muscle actin Ab-1 (NeoMarkers Inc., Fremont, CA), respectively. Staining was performed using the Optimax Plus 2.0 Automated Cell Staining System with research software (BioGenex, San Ramon, CA). For anti-Mac-3 staining, tissue sections were deparaffinized, treated with 1% H2O2 and nonspecific protein binding was blocked for 1 hour with a solution containing 1% bovine serum albumin and 5% rabbit serum. Sections were incubated for 2 hours at 37°C with anti-Mac-3 (2.5 μg/ml) in Tris-buffered saline, pH 7.4/1% bovine serum albumin. Antigen-antibody complexes were detected using the Vectastain Elite rat ABC peroxidase kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. After 30 minutes of incubation with biotinylated secondary antibody followed by a 30-minute incubation with ABC reagent, a 5-minute reaction with diaminobenzidine/H2O2 was used to detect the bound peroxidase. Staining with anti-CD31 was similar except that sections were also pretreated for 30 minutes with 0.25% trypsin (Sigma Chemical Co., St. Louis, MO) and the primary antibody incubation was performed overnight at 4°C at 6.5 μg/ml. Staining for anti-smooth muscle actin was performed with the Vector M.O.M. kit (Vector Laboratories) according to the manufacturer's instructions using a 2-hour incubation with 0.5 μg/ml of smooth muscle actin IgG. For all staining experiments isotype-matched normal IgG at the same concentration as the primary antibody was used as a negative control. The numbers of macrophages, blood vessels, and myofibroblasts were counted in 5 400× magnification fields/skin section. Quantitation of epidermal thickness was done by capturing images of H&E-stained sections (×400 magnification) from a Zeiss Axioplan microscope using an MTI 3 charge-coupled device camera. On each section three areas of interfollicular acanthosis were identified by eye and the thickness of the epidermis from the outer edge to the epidermal-dermal interface was measured using ImagePro 2.0 software. Quantitative results are expressed as mean ± SE mean. Significant intergroup differences were determined by applying the two-sample assuming unequal variance t-test. Immunoreactive TGF-β1, TGF-β2, and TGF-β3, as well as the type I (RI) and type II (RII) receptors were localized to tissue sections using a protocol similar to that described above for staining with the Mac-3 antibody with the following modifications. Sections were pretreated for 30 minutes with 1 mg/ml of bovine testicular hyaluronidase (Sigma) for TGF-β ligand staining, the primary antibody incubation was performed for 2 hours at room temperature, and a Vectastain Elite rabbit ABC peroxidase kit was used. The primary antibodies used to detect TGF-β1 were IgG fractions of LC 1-30-1 (6 μg/ml) and CC 1-30-1 (0.2 μg/ml), which recognize intracellular and extracellular TGF-β1, respectively.40Flanders KC Thompson NL Cissel DS Van Obberghen-Schilling E Baker CC Kass ME Ellingsworth LR Roberts AB Sporn MB Transforming growth factor-beta 1: histochemical localization with antibodies to different epitopes.J Cell Biol. 1989; 108: 653-660Crossref PubMed Scopus (323) Google Scholar Affinity-purified anti-TGF-β2 (catalog no. sc-90; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-TGF-β341Flanders KC Ludecke G Engels S Cissel DS Roberts AB Kondaiah P Lafyatis R Sporn MB Unsicker K Localization and actions of transforming growth factor-betas in the embryonic nervous system.Development. 1991; 113: 183-191PubMed Google Scholar were used at 0.8 μg/ml and 4 μg/ml, respectively. RI and RII were detected with antibodies from Santa Cruz Biotechnology (catalog no. sc-398 and sc-220) that were used at 4 and 0.8 μg/ml, respectively. Epidermal keratinocytes were isolated from polymerase chain reaction-genotyped Smad3 WT and KO newborn mice by standard methods42Dlugosz AA Glick AB Tennenbaum T Weinberg WC Yuspa SH Isolation and utilization of epidermal keratinocytes for oncogene research.Methods Enzymol. 1995; 254: 3-20Crossref PubMed Scopus (139) Google Scholar and were cultured in Eagle's minimal essential medium/8% Chelex-treated fetal bovine serum/0.05 mmol/L CaCl2 with antibiotics. Fibroblasts were isolated by collagenase (Life Technologies, Inc., Grand Island, NY) digestion of the pooled dermal layers from the newborn WT and KO pups according to Lichti and colleagues43Lichti U Weinberg WC Goodman L Ledbetter S Dooley T Morgan D Yuspa SH In vivo regulation of murine hair growth: insights from grafting defined cell populations onto nude mice.J Invest Dermatol. 1993; 101: S124-S129Abstract Full Text PDF PubMed Scopus (119) Google Scholar and were cultured in Dulbecco's modified Eagle's medium/10% fetal bovine serum/1% Pen-Strep. Epidermal keratinocytes were plated and cultured for 2 days before being exposed to 0, 5, or 10Gy of γ-irradiation from a60Co source. Cells were cultured for an additional 48 hours at which time cells were trypsinized and counted using a hemocytometer after trypan blue staining. The number of surviving unirradiated cells was set as 100% and the surviving irradiated cells were referenced to the unirradiated cells. Dermal fibroblasts (passage 2) isolated from WT and KO mice were removed from the tissue culture flask with 0.05% trypsin-ethylenediaminetetraacetic acid (Life Technologies, Inc.) and divided into six equal aliquots. One aliquot was exposed to each of 0.5, 2, 5, and 10 Gy of γ-irradiation, whereas two aliquots were left unirradiated. Equal numbers of cells from each aliquot were plated into 35-mm dishes and allowed to grow for 5 days and then were rinsed, trypsinized, and the number of surviving cells in each dish determined with a Coulter counter. The number of surviving unirradiated cells was set as 100% and the percentage of surviving irradiated cells was compared to this. Based on our previous studies demonstrating that Smad3-null mice show accelerated healing of incisional wounds,30Ashcroft GS Yang X Glick AB Weinstein M Letterio JL Mizel DE Anzano M Greenwell-Wild T Wahl SM Deng C Roberts AB Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.Nat Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (759) Google Scholar we hypothesized that loss of Smad3 might improve healing or protect against tissue damage resulting from other types of insults. To test this hypothesis, the hind legs of a group of WT, Het, and KO mice were exposed to 50 Gy of γ-irradiation. Animals were sacrificed 5 weeks after irradiation to preclude losing KO animals to opportunistic infections resulting from defects in T-cell activation and mucosal immunity.24Yang X Letterio JJ Lechleider RJ Chen L Hayman R Gu H Roberts AB Deng C Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta.EMBO J. 1999; 18: 1280-1291Crossref PubMed
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