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• W2908728800 abstract "Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression. Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression. Circadian rhythms are dominantly regulated by clock genes. Briefly, Clock/Bmal1 induces Per1/2/3, Cry1/2, and differentiated embryonic chondrocyte gene 1/2 (Dec1/2) transcription through E-box.1Sato F. Kawamoto T. Fujimoto K. Noshiro M. Honda K.K. Honma S. Honma K. Kato Y. Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation. Interaction with BMAL1.Eur J Biochem. 2004; 271: 4409-4419Crossref PubMed Scopus (88) Google Scholar, 2Kawamoto T. Noshiro M. Sato F. Maemura K. Takeda N. Nagai R. Iwata T. Fujimoto K. Furukawa M. Miyazaki K. Honma S. Honma K.i. Kato Y. A novel autofeedback loop of Dec1 transcription involved in circadian rhythm regulation.Biochem Biophys Res Commun. 2004; 313: 117-124Crossref PubMed Scopus (97) Google Scholar, 3Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Dec1 and Dec2 are regulators of the mammalian molecular clock.Nature. 2002; 419: 841-844Crossref PubMed Scopus (516) Google Scholar The complex of Period (Per) and Cryptochrome (Cry) protein then represses their own transcription by inhibiting transcriptional activity of Clock/Bmal1. However, Dec suppresses their own transcription by competing with Clock/Bmal1 for the occupancy of the E-box in their own promoters.1Sato F. Kawamoto T. Fujimoto K. Noshiro M. Honda K.K. Honma S. Honma K. Kato Y. Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation. Interaction with BMAL1.Eur J Biochem. 2004; 271: 4409-4419Crossref PubMed Scopus (88) Google Scholar, 2Kawamoto T. Noshiro M. Sato F. Maemura K. Takeda N. Nagai R. Iwata T. Fujimoto K. Furukawa M. Miyazaki K. Honma S. Honma K.i. Kato Y. A novel autofeedback loop of Dec1 transcription involved in circadian rhythm regulation.Biochem Biophys Res Commun. 2004; 313: 117-124Crossref PubMed Scopus (97) Google Scholar, 3Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Dec1 and Dec2 are regulators of the mammalian molecular clock.Nature. 2002; 419: 841-844Crossref PubMed Scopus (516) Google Scholar This negative feedback regulation by clock genes plays an important role in regulation of circadian rhythm. Dec1 and Dec2 are basic helix-loop-helix transcription factors, are ubiquitously expressed in whole tissues, and show circadian expression in the suprachiasmatic nucleus (SCN), peripheral tissues, mesenchymal stem cells, and tumor cells.3Honma S. Kawamoto T. Takagi Y. Fujimoto K. Sato F. Noshiro M. Kato Y. Honma K. Dec1 and Dec2 are regulators of the mammalian molecular clock.Nature. 2002; 419: 841-844Crossref PubMed Scopus (516) Google Scholar, 4Sato F. Bhawal U.K. Yoshimura T. Muragaki Y. DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression.J Cancer. 2016; 7: 153-159Crossref PubMed Scopus (64) Google Scholar, 5Sato F. Muragaki Y. Kawamoto T. Fujimoto K. Kato Y. Zhang Y. Rhythmic expression of DEC2 protein in vitro and in vivo.Biomed Rep. 2016; 4: 704-710Crossref PubMed Scopus (10) Google Scholar, 6Sato F. Sato H. Jin D. Bhawal U.K. Wu Y. Noshiro M. Kawamoto T. Fujimoto K. Seino H. Morohashi S. Kato Y. Kijima H. Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.Biochem Biophys Res Commun. 2012; 419: 441-446Crossref PubMed Scopus (28) Google Scholar, 7Sato F. Bhawal U.K. Kawamoto T. Fujimoto K. Imaizumi T. Imanaka T. Kondo J. Koyanagi S. Noshiro M. Yoshida H. Kusumi T. Kato Y. Kijima H. Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.Genes Cells. 2008; 13: 131-144Crossref PubMed Scopus (72) Google Scholar, 8Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins.Biochem Biophys Res Commun. 2001; 280: 164-171Crossref PubMed Scopus (85) Google Scholar, 9Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes.Biochem Biophys Res Commun. 1997; 236: 294-298Crossref PubMed Scopus (145) Google Scholar Dec1 and Dec2 are induced by Clock/Bmal1, hypoxia, serum shock, transforming growth factor (TGF)-β, and antitumor drugs, and they regulate circadian rhythm, sleep length, inflammation, lipid metabolism, adipogenic differentiation, the cell cycle, and tumor progression.5Sato F. Muragaki Y. Kawamoto T. Fujimoto K. Kato Y. Zhang Y. Rhythmic expression of DEC2 protein in vitro and in vivo.Biomed Rep. 2016; 4: 704-710Crossref PubMed Scopus (10) Google Scholar, 6Sato F. Sato H. Jin D. Bhawal U.K. Wu Y. Noshiro M. Kawamoto T. Fujimoto K. Seino H. Morohashi S. Kato Y. Kijima H. Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.Biochem Biophys Res Commun. 2012; 419: 441-446Crossref PubMed Scopus (28) Google Scholar, 7Sato F. Bhawal U.K. Kawamoto T. Fujimoto K. Imaizumi T. Imanaka T. Kondo J. Koyanagi S. Noshiro M. Yoshida H. Kusumi T. Kato Y. Kijima H. Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.Genes Cells. 2008; 13: 131-144Crossref PubMed Scopus (72) Google Scholar, 8Fujimoto K. Shen M. Noshiro M. Matsubara K. Shingu S. Honda K. Yoshida E. Suardita K. Matsuda Y. Kato Y. Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins.Biochem Biophys Res Commun. 2001; 280: 164-171Crossref PubMed Scopus (85) Google Scholar, 9Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes.Biochem Biophys Res Commun. 1997; 236: 294-298Crossref PubMed Scopus (145) Google Scholar, 10Fujita Y. Makishima M. Bhawal U.K. Differentiated embryo chondrocyte 1 (DEC1) is a novel negative regulator of hepatic fibroblast growth factor 21 (FGF21) in aging mice.Biochem Biophys Res Commun. 2016; 469: 477-482Crossref PubMed Scopus (18) Google Scholar Per1 knockdown increased tumor proliferation and size in oral cancer cells, decreasing Dec1 and Dec2 expression.11Zhao Q. Zheng G. Yang K. Ao Y.R. Su X.L. Li Y. Lv X.Q. The clock gene PER1 plays an important role in regulating the clock gene network in human oral squamous cell carcinoma cells.Oncotarget. 2016; 7: 70290-70302Crossref PubMed Scopus (21) Google Scholar It has also been reported that Per1 and Dec2 have tissue-specific interactions in circadian rhythm regulation.12Tsang A.H. Sánchez-Moreno C. Bode B. Rossner M.J. Garaulet M. Oster H. Tissue-specific interaction of Per1/2 and Dec2 in the regulation of fibroblast circadian rhythms.J Biol Rhythms. 2012; 27: 478-489Crossref PubMed Scopus (9) Google Scholar Synergistic disruption of circadian rhythm in Per1 and Dec1 or Dec2 double-mutant mice was observed in comparison with that in single-mutant mice.13Bode B. Shahmoradi A. Taneja R. Rossner M.J. Oster H. Genetic interaction of Per1 and Dec1/2 in the regulation of circadian locomotor activity.J Biol Rhythms. 2011; 26: 530-540Crossref PubMed Scopus (12) Google Scholar It seems that Dec1 or Dec2 interacts with Per1 in not only the regulation of circadian rhythm but also in various other phenomena. Smad3 has circadian expression in the gingiva, liver, heart, and mesenchymal stem cells.7Sato F. Bhawal U.K. Kawamoto T. Fujimoto K. Imaizumi T. Imanaka T. Kondo J. Koyanagi S. Noshiro M. Yoshida H. Kusumi T. Kato Y. Kijima H. Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.Genes Cells. 2008; 13: 131-144Crossref PubMed Scopus (72) Google Scholar, 14Sato F. Kohsaka A. Takahashi K. Otao S. Kitada Y. Iwasaki Y. Muragaki Y. Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.Histochem Cell Biol. 2017; 148: 617-624Crossref PubMed Scopus (18) Google Scholar Smad3 is induced by Clock/Bmal1, serum shock, and TGF-β and regulates cell proliferation, tumor progression, cardiac diseases, fibrosis, diabetes, and metabolic syndrome.7Sato F. Bhawal U.K. Kawamoto T. Fujimoto K. Imaizumi T. Imanaka T. Kondo J. Koyanagi S. Noshiro M. Yoshida H. Kusumi T. Kato Y. Kijima H. Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.Genes Cells. 2008; 13: 131-144Crossref PubMed Scopus (72) Google Scholar, 15Soofi A. Wolf K.I. Emont M.P. Qi N. Martinez-Santibanez G. Grimley E. Ostwani W. Dressler G.R. The kielin/chordin-like protein (KCP) attenuates high-fat diet-induced obesity and metabolic syndrome in mice.J Biol Chem. 2017; 292: 9051-9062Crossref PubMed Scopus (17) Google Scholar, 16Biernacka A. Cavalera M. Wang J. Russo I. Shinde A. Kong P. Gonzalez-Quesada C. Rai V. Dobaczewski M. Lee D.W. Wang X.F. Frangogiannis N.G. Smad3 signaling promotes fibrosis while preserving cardiac and aortic geometry in obese diabetic mice.Circ Heart Fail. 2015; 8: 788-798Crossref PubMed Scopus (78) Google Scholar, 17Wu Y. Sato F. Yamada T. Bhawal U.K. Kawamoto T. Fujimoto K. Noshiro M. Seino H. Morohashi S. Hakamada K. Abiko Y. Kato Y. Kijima H. The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer.Int J Oncol. 2012; 41: 1337-1346Crossref PubMed Scopus (58) Google Scholar, 18Yadav H. Quijano C. Kamaraju A.K. Gavrilova O. Malek R. Chen W. Zerfas P. Zhigang D. Wright E.C. Stuelten C. Sun P. Lonning S. Skarulis M. Sumner A.E. Finkel T. Rane S.G. Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling.Cell Metab. 2011; 14: 67-79Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar The loss of Smad3 in mice promotes re-epithelialization and the restoration of connective tissue, resulting in faster wound healing than in wild-type mice.19Lee J.I. Wright J.H. Johnson M.M. Bauer R.L. Sorg K. Yuen S. Hayes B.J. Nguyen L. Riehle K.J. Campbell J.S. Role of Smad3 in platelet-derived growth factor-C-induced liver fibrosis.Am J Physiol Cell Physiol. 2016; 310: C436-C445Crossref PubMed Scopus (26) Google Scholar, 20Duan W.J. Yu X. Huang X.R. Yu J.W. Lan H.Y. Opposing roles for Smad2 and Smad3 in peritoneal fibrosis in vivo and in vitro.Am J Pathol. 2014; 184: 2275-2284Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 21Jinno K. Takahashi T. Tsuchida K. Tanaka E. Moriyama K. Acceleration of palatal wound healing in Smad3-deficient mice.J Dent Res. 2009; 88: 757-761Crossref PubMed Scopus (22) Google Scholar, 22Wang Y. Moges H. Bharucha Y. Symes A. Smad3 null mice display more rapid wound closure and reduced scar formation after a stab wound to the cerebral cortex.Exp Neurol. 2007; 203: 168-184Crossref PubMed Scopus (71) Google Scholar, 23Ashcroft G.S. Yang X. Glick A.B. Weinstein M. Letterio J.L. Mizel D.E. Anzano M. Greenwell-Wild T. Wahl S.M. Deng C. Roberts A.B. Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.Nat Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (768) Google Scholar This result correlates with the alternation of wound healing–associated genes, decreased production of collagen I and fibronectin, and increased expression of p21, S100A4, and PCNA.14Sato F. Kohsaka A. Takahashi K. Otao S. Kitada Y. Iwasaki Y. Muragaki Y. Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.Histochem Cell Biol. 2017; 148: 617-624Crossref PubMed Scopus (18) Google Scholar, 21Jinno K. Takahashi T. Tsuchida K. Tanaka E. Moriyama K. Acceleration of palatal wound healing in Smad3-deficient mice.J Dent Res. 2009; 88: 757-761Crossref PubMed Scopus (22) Google Scholar, 23Ashcroft G.S. Yang X. Glick A.B. Weinstein M. Letterio J.L. Mizel D.E. Anzano M. Greenwell-Wild T. Wahl S.M. Deng C. Roberts A.B. Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.Nat Cell Biol. 1999; 1: 260-266Crossref PubMed Scopus (768) Google Scholar, 24Dobaczewski M. Bujak M. Li N. Gonzalez-Quesada C. Mendoza L.H. Wang X.F. Frangogiannis N.G. Smad3 signaling critically regulates fibroblast phenotype and function in healing myocardial infarction.Circ Res. 2010; 107: 418-428Crossref PubMed Scopus (281) Google Scholar, 25Piek E. Ju W.J. Heyer J. Escalante-Alcalde D. Stewart C.L. Weinstein M. Deng C. Kucherlapati R. Bottinger E.P. Roberts A.B. Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts.J Biol Chem. 2001; 276: 19945-19953Crossref PubMed Scopus (356) Google Scholar Several reports have shown that Smad3 affects Dec1 and Dec2 expression.17Wu Y. Sato F. Yamada T. Bhawal U.K. Kawamoto T. Fujimoto K. Noshiro M. Seino H. Morohashi S. Hakamada K. Abiko Y. Kato Y. Kijima H. The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer.Int J Oncol. 2012; 41: 1337-1346Crossref PubMed Scopus (58) Google Scholar, 26Sato F. Kawamura H. Wu Y. Sato H. Jin D. Bhawal U.K. Kawamoto T. Fujimoto K. Noshiro M. Seino H. Morohashi S. Kato Y. Kijima H. The basic helix-loop-helix transcription factor DEC2 inhibits TGF-β-induced tumor progression in human pancreatic cancer BxPC-3 cells.Int J Mol Med. 2012; 30: 495-501Crossref PubMed Scopus (22) Google Scholar, 27Kon N. Hirota T. Kawamoto T. Kato Y. Tsubota T. Fukada Y. Activation of TGF-beta/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts.Nat Cell Biol. 2008; 10: 1463-1469Crossref PubMed Scopus (103) Google Scholar For example, activation of TGF-β signaling resets the circadian clock via Dec1.27Kon N. Hirota T. Kawamoto T. Kato Y. Tsubota T. Fukada Y. Activation of TGF-beta/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts.Nat Cell Biol. 2008; 10: 1463-1469Crossref PubMed Scopus (103) Google Scholar TGF-β induced Smad3 phosphorylation and then induced Dec2 expression through Smad3-binding elements of the Dec2 promoter in pancreatic cancer cells.17Wu Y. Sato F. Yamada T. Bhawal U.K. Kawamoto T. Fujimoto K. Noshiro M. Seino H. Morohashi S. Hakamada K. Abiko Y. Kato Y. Kijima H. The BHLH transcription factor DEC1 plays an important role in the epithelial-mesenchymal transition of pancreatic cancer.Int J Oncol. 2012; 41: 1337-1346Crossref PubMed Scopus (58) Google Scholar, 26Sato F. Kawamura H. Wu Y. Sato H. Jin D. Bhawal U.K. Kawamoto T. Fujimoto K. Noshiro M. Seino H. Morohashi S. Kato Y. Kijima H. The basic helix-loop-helix transcription factor DEC2 inhibits TGF-β-induced tumor progression in human pancreatic cancer BxPC-3 cells.Int J Mol Med. 2012; 30: 495-501Crossref PubMed Scopus (22) Google Scholar However, it is not yet clear how the loss of Smad3 affects circadian rhythm and Dec1, Dec2, and Per1 expression. Here, we investigated whether Smad3−/− (Smad3 knockout) affects Dec1, Dec2, and Per1 expression in the SCN and peripheral tissues and how these genes interact in epithelial cell migration and proliferation. All animal experiments were performed according to protocols approved by the Animal Care and Use Committee of the Wakayama Medical University (approval number: 660). Six- to 8-week-old male and female Smad3 (+/+) wild-type and (−/−) whole knockout mice with a C57BL/6 × 129SVEV background were generated as previously described.28Yang X. Letterio J.J. Lechleider R.J. Chen L. Hayman R. Gu H. Roberts A.B. 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 Mice were housed, and tissues were prepared as previously described.6Sato F. Sato H. Jin D. Bhawal U.K. Wu Y. Noshiro M. Kawamoto T. Fujimoto K. Seino H. Morohashi S. Kato Y. Kijima H. Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.Biochem Biophys Res Commun. 2012; 419: 441-446Crossref PubMed Scopus (28) Google Scholar, 14Sato F. Kohsaka A. Takahashi K. Otao S. Kitada Y. Iwasaki Y. Muragaki Y. Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.Histochem Cell Biol. 2017; 148: 617-624Crossref PubMed Scopus (18) Google Scholar, 29Kohsaka A. Das P. Hashimoto I. Nakao T. Deguchi Y. Gouraud S.S. Waki H. Muragaki Y. Maeda M. The circadian clock maintains cardiac function by regulating mitochondrial metabolism in mice.PLoS One. 2014; 9: e112811Crossref PubMed Scopus (77) Google Scholar Forty Smad3+/+ and 40 Smad3−/− mice were used in this study. All mice were reared in 12:12 light/dark cycles (lights on at 8 AM, off at 8 PM). With the use of isoflurane anesthesia, mice were sacrificed by cervical dislocation. A dim red light was used to sacrifice at Zeitgeber time (ZT)12 and ZT18. Mouse brain dissection was performed at 0.5 mm thickness, using a mouse brain coronal matrix (BrainScience Idea, Osaka, Japan), and the SCN and prefrontal cortex from the mice were punched out onto a chilled dish with the use of a 0.5-mm diameter needle. The SCN and prefrontal cortex were quickly assessed for mRNA and protein purification. Mouse locomotor activity was monitored by Supermex (Muromachi Kikai, Tokyo, Japan). Starting at 6 weeks of age, activity tests were conducted for 30 days. A sensor counts the movements of the mice by detecting the radiated body heat. The data were recorded continuously in 1-minute bins by a data collection program (CompACT AMS; Muromachi Kikai). The free-running period of locomotor activity was calculated by periodogram analysis with the use of ClockLab software (Actimetrics, Wilmette, IL). Fast Fourier analysis was performed to determine the amplitude of the power spectral density of the circadian peak. Mice were anesthetized by injection of ketamine (100 mg/kg of body weight) and xylazine (5 mg/kg of body weight) in sterile phosphate-buffered saline, and the wound was made by punching out a 3.0-mm hole with a Harris Uni-core puncher (BrainScience Idea) at the apex of the tongue. The mice were sacrificed at ZT6, and the tongue was prepared at 3 and 5 days after wounding. Tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (Wako, Osaka, Japan) and subjected to immunohistochemistry and hematoxylin and eosin staining. Three independent RNA samples were prepared from the mouse SCN, prefrontal cortex, and tongue. In addition, RNA was prepared from Smad3 overexpressing NIH3T3 cells. Total RNA was isolated and first-strand cDNA was synthesized as previously described.6Sato F. Sato H. Jin D. Bhawal U.K. Wu Y. Noshiro M. Kawamoto T. Fujimoto K. Seino H. Morohashi S. Kato Y. Kijima H. Smad3 and Snail show circadian expression in human gingival fibroblasts, human mesenchymal stem cell, and in mouse liver.Biochem Biophys Res Commun. 2012; 419: 441-446Crossref PubMed Scopus (28) Google Scholar, 14Sato F. Kohsaka A. Takahashi K. Otao S. Kitada Y. Iwasaki Y. Muragaki Y. Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.Histochem Cell Biol. 2017; 148: 617-624Crossref PubMed Scopus (18) Google Scholar Real-time PCR was performed with the SYBR Green Master Mix (Bio-Rad Laboratories, Inc., Hercules, CA). The amplification primer sequences were designed as follows: Dec1 forward (F), 5′-ATCAGCCTCCTTTTTGCCTTC-3′, and reverse (R), 5′-AGCATTTCTCCAGCATAGGCAG-3′; Dec2 F, 5′-ATTGCTTTACAGAATGGGGAGCG-3′, and R, 5′-AAAGCGCGCGAGGTATTGCAAGAC-3′; Per1 F, 5′-CCAGGATGTGGGTGTCTTCT-3,′ and R, 5′-GTCCTTGAGACCTGAACCTG-3′; and 18S rRNA F, 5′-GCGCCGCTAGAGGTGAAAT-3′, and R, 5′-GAAAACATTCTTGGCAAATGCTT-3′. The data were normalized by using 18S rRNA. Dec1, Dec2, and Per1 expression in mouse kidney, liver, and tongue tissues was evaluated by using serial deparaffinized sections. Six Smad3+/+ and six Smad3−/− mice were used for immunohistochemistry. Five serial sections of 4 μm per organ were prepared for staining. Immunohistochemistry was performed by using a Discovery Auto-Stainer with automated protocols (Ventana Medical Systems, Inc., Tucson, AZ; Roche, Mannheim, Germany) as previously described.14Sato F. Kohsaka A. Takahashi K. Otao S. Kitada Y. Iwasaki Y. Muragaki Y. Smad3 and Bmal1 regulate p21 and S100A4 expression in myocardial stromal fibroblasts via TNF-α.Histochem Cell Biol. 2017; 148: 617-624Crossref PubMed Scopus (18) Google Scholar NIH3T3 mouse fibroblasts were obtained from ATCC (Manassas, VA). CA9-22 human gingival cancer cells were obtained from the Japanese Cancer Research Resources Bank (Osaka, Japan). These cells were cultured in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum and 1% antibiotics. The construct expression vectors for human Dec1 and Dec2 were used for transfection.1Sato F. Kawamoto T. Fujimoto K. Noshiro M. Honda K.K. Honma S. Honma K. Kato Y. Functional analysis of the basic helix-loop-helix transcription factor DEC1 in circadian regulation. Interaction with BMAL1.Eur J Biochem. 2004; 271: 4409-4419Crossref PubMed Scopus (88) Google Scholar Human Smad3 plasmid (11742) was purchased from Addgene (Cambridge, MA). Transient plasmid transfection and siRNA transfection were performed as previously described.30Sato F. Muragaki Y. Zhang Y. DEC1 negatively regulates AMPK activity via LKB1.Biochem Biophys Res Commun. 2015; 467: 711-716Crossref PubMed Scopus (19) Google Scholar For Dec1 overexpressed-stable cells, empty (CA9-22vector) and Dec1 (CA9-22Dec1) plasmids were transfected in cells. After transfection, medium was changed, including 1000 μg/mL G418 (Roche) solution every 3 days until 3 weeks and selected stable cells. The Dec1 induction was confirmed in the stable cells by Western blot analyses. Smad3 siRNA was synthesized by Qiagen (Hilden, Germany). The sense and antisense siRNA sequences were as follows: Smad3 siRNA1, 5′-r (CAAGGGAUUUCCUAUGGAATT)-3′ and 5′-r (UUCCAUAGGAAAUCCCUUGAT)-3′; Smad3 siRNA2, 5′-r (GAGAUUCGAAUGACGGUAATT)-3′ and 5′-r (UUACCGUCAUUCGAAUCUCTT)-3′, respectively. CA9-22 cells were seeded in a 12-well plate and transfected with control or Smad3 siRNA2. After 8 hours, the cells were transfected with empty, Dec1, or Dec2 plasmid, and then an artificial wound was carefully created at 0 hour by scratching the surface with the tip of a P-200 pipette. Pictures were taken by microscopic digital camera (Nikon DS-L3; Nikon Corporation, Tokyo, Japan) at 0 and 12 hours. CA9-22 cells were seeded in 96-well plates. Transfection was performed as previously described.30Sato F. Muragaki Y. Zhang Y. DEC1 negatively regulates AMPK activity via LKB1.Biochem Biophys Res Commun. 2015; 467: 711-716Crossref PubMed Scopus (19) Google Scholar After 10 hours of transfection, the cells were added to each well by using a cell proliferation kit (XTT based) (Biological Industries, Kibbutz Beit Haemek, Israel) and incubated at 37°C for an additional 2 and 14 hours. The absorbance at OD480 and at OD650 was measured with the use of a 96-well microplate reader (SH-9000; Hitachi, Tokyo, Japan). Individual mouse liver and kidney were lyzed with the use of T-PER lysis buffer (ThermoFisher Scientific, Yokohama, Japan). Three independent SCN samples were pooled and then were lyzed with the use of T-PER lysis buffer. Cells were lyzed with M-PER lysis buffer (ThermoFisher Scientific, Yokohama, Japan). Lysates were run on SDS-polyacrylamide gels followed by Western blot analysis with the use of standard procedures. A WesternBright Sirius Kit (Advansta, CA) was used for antibody detection, and an AE-9300 Ez capture MG (ATTO, Tokyo, Japan) was used for image data capture. The intensity of the bands detected on the Western blot analysis was quantified with ImageJ software version 1.47v (NIH, Bethesda, MD). The following commercial antibodies were purchased: Smad3 (dilution 1:1000; rabbit monoclonal; ab40854; Abcam, Cambridge, UK), Dec1 (dilution 1:5000; rabbit polyclonal; NB100-1800; Novus Biologicals, Centennial, CO), Dec2 (dilution 1:2000; mouse monoclonal; sc-373763; Santa Cruz Biotechnology Inc., Santa Cruz, CA), proliferating cell nuclear antigen (PCNA; dilution 1:10,000; rabbit polyclonal; sc-7907; Santa Cruz Biotechnology Inc.), Per1 (dilution 1:1000; rabbit polyclonal; TransGenic Inc., Hyogo, Japan), E-cadherin (dilution 1:20,000; mouse monoclonal; M106; TaKaRa Bio Inc., Otsu, Japan), and actin (dilution 1:10,000; mouse monoclonal; A5441; Sigma Chemical Co.). It was first examined whether Smad3 deficiency affected circadian rhythm with the use of Smad3−/− mice. Smad3+/+ and Smad3−/− mice were fed under light/dark conditions for 14 days and constant dark conditions for 16 days. Locomotor activities were analyzed by Supermex. The amplitude of locomotor activity significantly decreased in Smad3−/− mice compared with that in Smad3+/+ mice (Figure 1A). However, no significant differences were observed in the circadian period between Smad3+/+ and Smad3−/− mice. Dec1, Dec2, and Per1 mRNA expression was next examined at ZT6 and ZT12 in the SCN of Smad3+/+ and Smad3−/− mice by real-time PCR, because time difference may affect clock gene expression. Dec1 expression was higher, whereas Dec2 and Per1 expression was lower at both time points in Smad3+/+ mice than in Smad3−/− mice (Figure 1, B and C), suggesting that Smad3 up-regulates Dec1 expression and down-regulates Dec2 and Per1 expression. In addition, Dec1, Dec2, and Per1 protein expression was analyzed at ZT6 and ZT12 in the SCN, and the result was similar at mRNA levels (Figure 1D). Dec1, Dec2, and Per1 expression was found to be higher in the SCN of Smad3+/+ mice than in the prefrontal cortex (Supplemental Figure S1). To examine Dec1, Dec2, and Per1 protein expression in Smad3+/+ and Smad3−/− mice, Western blot analysis was performed. Compared with that in Smad3+/+ mice, Dec1 protein expression at ZT6 decreased in the liver and kidneys of Smad3−/− mice, whereas Dec2 and Per1 protein expression increased (Figure 2A). Because the protein levels of Dec2 was virtually undetectable in the liver of Smad3+/+, the circadian expression of Dec2 and other clock genes was examined specifically in the kidneys of Smad3+/+ and Smad3−/− mice. Circadian expression of Smad3 in Smad3+/+ mice was also examined. As expected, Dec1, Dec2, Per1, and Smad3 protein expression showed diurnal variation in Smad3+/+ mice (Figure 2B). Smad3−/− mice also showed diurnal variation in the protein expression of these circadian genes in the kidneys; however, the expression levels were altered. Specifically, Dec1 protein expression decreased from ZT0 to ZT18 in the kidneys of Smad3−/− mice compared with that in Smad3+/+ mice. Interestingly, Dec2 protein expression greatly increased from ZT0 to ZT18 in the kidneys of Smad3−/− mice compared with that in Sm" @default.
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• W2908728800 date "2019-04-01" @default.
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• W2908728800 title "Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1" @default.
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• W2908728800 doi "https://doi.org/10.1016/j.ajpath.2019.01.006" @default.
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