J Meikai Dent Med 35 1/2, 21 34, 2006 21 RA SMG RA SMG SMG RA RA RA RAR X RXR EGF IGF- FGF TGF -β 1 Sonic hedgehog Shh SMG Regulation Mechanism of Retinoic Acid-induced Branching Morphogenesis in Fetal Mouse Submandibular Glands Takahiro NAKA Meikai University Graduate School of Dentistry Director : Prof Shuji OHKAWA Abstract : Retinoic acid RA, one of the metabolites of vitamin A, is known to be akey signaling molecule in regulating morphogenesis during the development and differentiation of bone, lung, and kidney tissues. The branching morphogenesis of the fetal mouse submandibular gland mouse SMG is abasic developmental and cytodifferentiation process involved in the formation of ducts and acini. Moreover, branching morphogenesis is known to be dependent on reciprocal epithelio-mesenchymal interactions, and is stimulated by many growth factors, such as EGF, IGF- and FGF. However, the effect of RA on the development of fetal mouse submandibular gland is poorly understood. In this study, I investigated the mechanism of the induction of branching morphogenesis by RA in organ cultures of fetal mouse SMG. The addition of RA to the culture of the mouse SMG rudiments at 13 days after the start of pregnancy E 13 resulted in the induction of epithelial branching morphogenesis in a dose-dependent manner. Western blot analysis demonstrated the expression of retinoic acid receptor RAR -α β γ and retinoid X receptor RXR at the stage of E 12 ande13, followed by the decline of their expression at the stage of E 14. Immunohistochemical staining revealed that the RAR-α, β and RXR were localized in the epithelial tissues, whereas RAR-γ was weakly expressed in the mesenchymal tissues. The branching morphogenesis induced by RA was inhibited by anti- FGF-10 antibody, anti-tgf-β 1antibody, SU 5402 inhibitor of FGF receptor phosphorylation, SB431542 inhibitor of TGF-β receptor phosphorylation or cyclopamine inhibitor of sonic hedgehog shh signal transduction, but not by anti-egf and anti-igf- antibodies, genistein inhibitor of EGF receptor phosphorylation or AG 538 inhibitor of IGF- receptor phosphorylation. RAfailed toinduce the branching morphogenesis in the isolated epithelial tissues depleted of mesenchymal tissues. These results suggest that the RA-induced branching morphogenesis is mediated by its receptors, and positively regulated by FGF, TGF and Shh, rather than by EGF or IGF- through the epithelio-mesenchymal interactions. Key words : fetal mouse submandibular gland, retinoic acid, branching morphogenesis, induced cytodifferentiation
22 SMG 10 11 E 10-11 1 E 12-13 branching morphogenesis SMG 2 3 RA A 4 4 7 Strickland 5 F9 RA parietal endodermal cell Tickle 7 RA RA RA polarizing region RA RA 8 11 12 14 15 17 RA 963-8611 31-1 17 E 35, 2006 RA SMG RA RA 1 ICR K K 0 E 0 12 14 E 12-14 SMG Ca 2 Mg 2 PBS 2 ml BGJb Gibco-BRL, Gland Island, NY, USA Anocell 6 Transwell clear filters, Corning inc, Corning, NY, USA 37 5 CO2 100 0 24 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate 50 µg /ml transferrin 12 24 RA 18 E13 SMG PBS 1.5 U/ml Dispase 1 Rosche, Indianapolis, IN, USA 37 20 27 G Anocell matrigel BD Biosciences, Two Oak Park, MA, USA 2 1 RA all-trans RA Sigma-Aldrich, St Louis, MO, USA BGJb 0.001 1 µg/ml
23 0.1 2 Epidermal growth factor EGF, Upstate Biotechnology, Lake Placid, NY, USA fibroblast growth factor-10 FGF-10, Pepro Tech EC, London, England insulin-like growth factor- IGF- TECHNE, Minneapolis, MN, USA transforming growth factor-β 1 TGF-β 1, TECHNE BGJb 3 EGF Chemicon International, Temecula, CA, USA IGF- Upstate Biotechnology FGF-10 Santa Cruz Biotechnology, Santa Cruz, CA, USA TGF-β 1 TECHNE BGJb RA RAR X RXR RAR-α RAR-β RAR-γ RXR Santa Cruz Biotechnology Santa Cruz, CA, USA Western blot RAR-α, β,rxr RAR-γ Bio-Rad Lab Inc, Hercules, CA, USA RAR-α, β,rxr RAR-γ DAKO LSAB kit, Dako, Glostrap, Denmark 4 Genistein EGF AG 538 IGF- SU 5402 FGF Merck Biosciences Darmstadt, Germany SB 431542 TGF-β 1 Tocris Ellisville, MI, USA cyclopmamine Shh Biomo Resecrch Laboratories Plymouth Meeting, PA, USA 3 SMG RARs RXR E 12-14 SMG buffer 20 mm Tris-HCl ph 7.5 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β -glycerophosphate, 1 mm Na3VO4, 1 µg/ml leupetin, 100 nm calyculin A 4 16,000 x g 15 SMG 20 µl SDS-sample buffer 50 mm Tris-HCl ph 6.8 2 SDS, 4 2- mercaptoethanol, 0.1 bromphenol blue, 10 glycerol 100 5 SDS-10 PVDF 30 V PVDF 5 skim milk RAR-α RAR-β RAR-γ RXR 1 1000 60 PBS RAR-α, β RXR 1 200 RAR-γ 1 200 20 NBT 4 SMG E13 4 0.2 M 30 0.2 M 2 µm 0.3 TritonX-100/ PBS 60 PBS 0.3 H2O2 10 PBS 10 30 PBS RAR-α RAR-β RAR-γ RXR 1 1,000 PBS RAR-α, β,rxr
24 明海歯学 35, 2006 中 貴弘 抗体を 抗ヒト RAR-γ 抗体に対してはビオチン標識抗 結 マウスモノクロナール抗体と 室温で 60 分間反応させ た PBS で洗浄後 ペルオキシダーゼ標識ストレピト 果 1 マウス SMG に対する RA 単独投与の影響 アビジン DAKO LSAB kit と室温で 60 分間反応さ E 13 のマウス SMG に 0.001 1 µ g/ml の濃度の RA を せ 発 色 は 0.01 H2O2 加 DAB 溶 液 で 行 っ た そ の 添加し 直後および 24 時間器官培養した後の形態 Fig 後 水洗 脱水 透徹 封入を行い 各受容体の局在性 1A と 分枝数 Fig 1B の変化を示す 分枝形態形 を光学顕微鏡下で観察した なお 陰性対照標本は 一 成は ほぼ RA の濃度依存的に促進された RA の分枝 次抗体の代わりに PBS を反応させ 同 様 に染色 形態形成促進作用は 0.001 µ g/ml から認められ 0.1 µ g し 非特異的な免疫反応が無いことを確認した /ml で最大になった 従って 以後の実験は 0.1 µ g/ml 5 分枝数の測定 の濃度の RA を用いて行った RA の分枝形態形成促進 種々の時間培養したマウス SMG における分枝数を顕 作用 Fig 1A は 分枝 bud の数を増加させる作用 微鏡下で実測し 培養開始前の分枝数との比率で表した が強いものと考えられた Spooner Ratio19 により評価を行った 測定は 各組織に 2 RA 受容体の発現 対して 3 回ずつ行い それらの平均値をもって各器官の 値とした 6 統計学的処理 次に E 12-14 における RA の受容体の発現をウエス タンブロット法を用いて検討した Fig 2 E 12 および E 13 においては RAR-α, β, γ ならびに RXR 分子 すべての実験結果については 統計学的処理を行い 量 約 55 60 kda の発現が観察された しかし E 14 2 群間の比較は Student s t-test を用いて 5 以下の危 になると それらの発現は各受容体とも著明に減弱し 険率で有意差を検定した た 特に RXR はほとんど検出できない程であった このことは RA の各受容体とも その発現は胎生期に Fig 1 Effect of retinoic acid RA on branching morphogenesis in the fetal E 13 mouse SMG. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. * : p 0.05, ** : p 0.01 vs. control. Each value represents the mean±s.d. from 7 determinations A Morphological observations B Quantification of bud formation.
25 RA 3 RA E13 SMG RAR Fig 3 RAR-α Fig 2 Western blotting analysis of retinoic acid receptors RAR-α, β, γ and retinoid X receptor RXR in fetal mouse SMG. E 12, E 13 and E 14 represents the stage of 12, 13, 14 days after the start of pregnancy. RAR-β SMG RAR-γ RXR 4 SMG EGF 1, 20 22 FGF-10 23 25 IGF- 1 TGF-β 1 26 RA 1, 20 26 data not shown EGF IGF- RA Figs 4, 5 RA EGF IGF- Fig 3 Immunohistochemical localization of RAR-α A RAR-β B RAR-γ C and RXR D in fetal E 13 mouse SMG. Arrows indicate SMG epithelial tissue. Magnification bars are equal to 20 µm.
26 中 貴弘 明海歯学 35, 2006 Fig 4 Effect of anti-egf antibody on induction of branching morphogenesis in the RA-treated fetal E 13 mouse SMG. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 2 µ g/ml anti-egf antibody and/or RA 0.1 µ g/ml B Quantification of bud formation. Fig 5 Effect of anti-igf-蠢 antibody on induction of branching morphogenesis in the RA-treated fetal E 13 mouse SMG. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 20 µ g/ml anti-igf-蠢antibody and/or RA 0.1 µ g/ml B Quantification of bud formation.
レチノイン酸による分枝形態形成の誘導機構 Fig 6 Effect of anti-fgf-10 antibody on induction of branching morphogenesis in the fetal E 13 mouse SMG treated with RA. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morpho B Quantification of bud formalogical observations with 2 µ g/ml anti-fgf-10 antibody and/or RA 0.1 µ g/ml tion. Fig 7 Effect of anti-tgf-β 1 antibody on induction of branching morphogenesis in the RA-treated fetal E 13 mouse SMG. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents mean±s.d. from 7 determinations A Morphological observations with 2 µ g/ml anti-tgf-β 1 antibody and/or RA 0.1 µ g/ml B Quantification of bud formation. 27
28 中 貴弘 明海歯学 35, 2006 Fig 8 Effect of Genistein on RA-induced branching morphogenesis in the fetal E 13 mouse SMG The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 7.4 µ M genistein and/or RA 0.1 µ g/ml B Quantification of bud formation. Fig 9 Effect of AG 538 on induction of branching morphogenesis in the fetal E 13 mouse SMG treated with RA. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 841 µ M AG 538 and/or RA 0.1 µ g/ml B Quantification of bud formation.
レチノイン酸による分枝形態形成の誘導機構 Fig 10 Effect of SU 5402 on number of epithelial buds formed by treatment of the fetal E 13 mouse SMG with RA. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 8 µ M SU 5402 and/or RA 0.1 µ g/ml B Quantification of bud formation. Fig 11 Effect of SB 431542 on RA-induced branching morphogenesis in the fetal E 13 mouse SMG. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 1 µ M SB 431542 and/or RA 0.1 µ g/ml B Quantification of bud formation. 29
30 中 貴弘 明海歯学 35, 2006 Fig 12 Effect of cyclopamine on induction of branching morphogenesis in the fetal E 13 mouse SMG by RA. The number of buds T 24/T 0 represents the ratio of the number of buds at 24 hr/the number of buds at 0 hr. ** : p 0.01 vs. culture with RA alone. Each value represents the mean±s.d. from 7 determinations A Morphological observations with 5 µ M cyclopamine and/or RA 0.1 µ g/ml B Quantification of bud formation. Fig 13 Effect of RA on cultures of epithelial tissues taken from the fetal E 13 mouse SMG and denuded of their mesenchyme. The epithelium was covered with Matrigel and cultured for 0, 12 or 24 hours in medium containing RA 0.1 µ g/ ml No branching morphogenesis occurred, and the outline of the epithelium was even and round.
31 FGF-10 RA FGF-10 Fig 6 TGF-β 1 RA Fig 7 RA FGF-10 TGF-β 1 5 SMG 23 RA EGF genistein Fig 8 IGF- AG 538 Fig 9 RA EGF IGF- Figs 4, 5 RA EGF IGF- FGF SU 5402 Hoffman 23 RA Fig 10 TGF-β 1 SB 431542 RA Fig 11 SB 431542 SB 431542 RA FGF-10 TGF-β 1 Shh cyclopamine RA Fig 12 RA Shh 6 Dispase 1 RA RA SMG Fig 13 12 24 Fig 13 RA RA 1 SMG RA RA E13 SMG RA 7 RA SMG cystic dysplasia 16 SMG RA Jaskoll 1 1 pre bud stage 2 initial bud stage 3 pseudoglandular stage 4 canalization stage 5 terminal bud stage 5 E12 initial bud stage, E 13 late initial bud stage, E 14 pseudoglandular stage RA 5 7 RA SMG RA 2 RA Fig 2 4 E 12, 13 E 14 1 initial bud stage late initial bud stage RA E 13 RA RA RA E14 SMG RA RA 4 RA Fig 3 RAR-γ RA EGF
32 20, 21 RA RA RA RA RA RA RA E14 SMG RA In situ hybridization RA mrna E 14.5 SMG RAR-α RAR-β γ 16 RA 3 EGF IGF- RA RA FGF-10 TGF-β 1 EGF IGF- RA SMG EGF 28 EGF 20 RA FGF-10 E 12.5 SMG FGF-10 FGF-10 cleft cleft 27 TGF-β SMG TGF-β 1 26 SMG 35, 2006 EGF genistein 20, 21 SMG IGF- IGF- AG 538 28 FGF-10 SU 5402 23 Shh cyclopamine 29 RA genistein AG 538 SU 5402 SB 431542 cyclopamine RA FGF TGF-β Shh 4 RA RA RA RA 30 33 Fig 14 Possible scheme for RA-mediated regulation of branching morphogenesis in the fetal mouse SMG.
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