明海大学歯学雑誌　４２‐２★／目次（４２‐２）

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1 J Meikai Dent Med 42 2, , HMG-CoA 1 EGF, TGFα, FGF Insig-1, -2 HMG-CoA HMG-CoA 13 HMG-CoA simvastatin μm HMG-CoA simvastatin squalene GTP farnesyl pyrophosphate, geranylgeranyl pyrophosphate simvastatin HMG-CoA HMG-CoA Localization of HMG-CoA Reductase in the Embryonic Mouse Submandibular Gland and its Role on the Branching Morphogenesis Daisuke YAMASAKI, Tomoko EGUCHI and Jun SHIMADA Division 1 of Oral and Maxillofacial Surgery, Department of Diagnotic and Therapeutic Science, Meikai University School of Dentistry Abstract : Growth factors including EGF, TGFα and FGF play an important role in the branching morphogenesis of embryonic mouse submandibular gland. On the other hand, it has been reported that the deficiency of cholesterol synthesis regulatory proteins Insig-1 and -2 causes facial and palatal clefts in mouse fetuses, suggesting the importance of proper levels of sterol metabolites for the normal organogenesis. In order to investigate the possible involvement of sterol metabolites in the developmental process of embryonic mouse submandibular gland, the submandibular gland localization of HMG-CoA reductase, a rate limiting enzyme for cholesterol synthesis, and the role in the branching morphogenesis were analyzed immunohistochemically. HMG-CoA reductase was markedly expressed in the mesenchymal tissue at the embryonic age of 13 days, but was not detectable in the epithelial tissue. Simvastatin, an inhibitor of HMG-CoA reductase, suppressed the branching morphogenesis of cultured embryonic submandibular gland in a dose-dependent manner μm. When mevalonate, which is synthesized by HMG-CoA reductase, was added, the inhibitory effect of simvastatin on branching morphogenesis was rescued. The addition of squalene, a cholesterol precursor, or the increase of isoprenoids farnesyl pyrophosphate, geranylgeranyl pyrophosphate, which are essential for the activity of small GTP-binding proteins, failed to prevent the suppresive effect of simvastatin on the branching morphogene-

2 sis. These results suggest that HMG-CoA reductase is localized in mesenchymal tissue of embryonic mouse submandibular gland, and that the mevalonic acid metabolite s other than cholesterol and isoprenoids may play an important role in branching morphogenesis. Key words : embryonic mouse submandibular gland, blanching morphogenesis, HMG-CoA reductase, mevalonic acid metabolites E E EGF, TGFα, FGF 6 Insig-1, -2 7 Smith-Lemli-Opitz 8 HMG-CoA 3-hydroxy-3-methylglutaryl coenzyme A Fig 1 9 HMG-CoA farnesyl pyrophosphate geranylgeranyl pyrophosphate Fig 1 Pathway of cholesterol synthesis. GTP Ras Rho HMG-CoA Ras Rho HMG-CoA HMG-CoA 1 1 HMG-CoA Simvastatin fluvastatin CALBIO- CHEM, California, USA atrovastatin LKT Laboratories, MN, USA pravastatin sodium salt rosuvastatin Toronto Research Chemicals, North York, Canada DMSO 10 FBS, Sigma- Aldrich, St. Louis, MO, USA DMEM Invitrogen, Grand Island, NY, USA 100 U/ml penicillin G potassium, 100 μg/ml streptomycin sulfate

3 HMG-CoA , 1, 10 μm 2 Cholesterol Mevalonic acid 0.1, 1, 10 mm Squalene DMSO 20, 200 μm Geranylgeraniol, farnesol Sigma-Aldrich DMSO 20, 30 μm 3 GTP Farnesyl transferase inhibitor FTase Inhibitor I, CALBIO- CHEM geranylgeranyltransferase inhibitor GGTI-298, CALBIOCHEM DMSO 10, 30, 100 μm 2 A 0713 ICR SLC 13 E13 Ca 2 /Mg 2 PBS 2ml Anocell 6 Transwell clear filters, Corning, NY, USA 37 5 CO , 48, 72, U/ml dispase 37 5 CO G 13, 14 PBS DMEM 50 matrigel BD Biosciences, Two Oak Park, MA, USA FBS 10 3 E13 24, 48, 72, M PB 1 PB μm hematoxylin-eosin H-E HMG-CoA HMGCR Santa Cruz Biotechnology, CA, USA PBS PBS HMGCR 1:50 PBS Cy2 IgG 1 : 250, Chemicon Internationals, Temecula, CA, USA 60 PBS LSM-510, Carl Zeiss, Berlin, Germany PBS E13 out-growth HMG-CoA 4 24, 48, 72, 96 Spooner Ratio t 5 1 HMG-CoA H-E E13 24 E13 24h

4 E13 48h Fig 2 HMG-CoA E13 96h Fig 3 E13 Fig 2 Localization of HMG CoA reductase in the cultured embryonic mouse submandibular gland. Upper panels represent phase-contrast image of cultured whole submandibular gland. Lower panels represent hematoxylin-eosin stained section of submandibular gland. Numbers indicate the incubation time of submandibular gland. Fig 3 Immunohistochemical staining of HMG CoA reductase in the cultured submandibular gland by fluorescence Cy2 -labelled antibody method. Asterisks indicate the epithelium of embryonic mouse submandibular gland.

5 HMG-CoA 121 HMG-CoA Fig 4 out-growth Fig 5 HMG-CoA 2 HMG-CoA HMG-CoA HMG-CoA Fig 4 Immunohistochemical stainig of HMG CoA reductase in the whole submandibular gland and separated epithelium. Upper panels represent whole submandibular gland, and the lower panels represent the separated epithelium. A : Phase-contrast image, B : Immunoreactivity of HMG CoA reductase. Fig 5 Immunohistochemical staining of HMG CoA reductase in cultured mesenchymal cells from embryonic mouse submandibular gland. A : Phase-contrast image of cultured mesenchymal cells. B : Immunoreactivity of HMG CoA reductase in cultured mesenchymal cells.

6 HMG-CoA simvastatin Fig 1 E13 0.1, 1, 10 μm simvastatin E13 48h simvastatin simvastatin Fig 6 Simvastatin 10 μm HMG- CoA 3 HMG-CoA Simvastatin HMG-CoA simvastatin logp 1.88 fluvastatin logp 1.73 atrovastatin logp 1.53 pravastatin logp 0.47 rosuvastatin logp 0.3 E13 10 μm fluvastatin, atrovastatin simvastatin pravastatin, rosuvastatin Fig 7 4 HMG-CoA simvastatin 10 mm simvastatin Fig 8 10 mm 5 Simvastatin sim- Fig 6 Effect of simvastatin on the branching morphogenesis in the embryonic submandibular gland. A : Phase-contrast image of branching suppression by simvastatin. B : Quantification of branching suppression by simvastatin. The fold induction of buds represents the ratio of buds at 48h/ buds at 0h. *P v.s. the control group. Values are mean SE for 8 10 glands.

7 HMG-CoA 123 Fig 7 Effect of statins on the branching morphogenesis of embryonic submandibular gland. The concentration of each statin was 10 μm. A : Phase-contrast image of branching suppression by statins. B : Quantification of branching suppression by statins. The fold induction of buds represents the ratio of buds at 48h/ buds at 0h. *P v.s. the control group. Values are mean SE for 8 10 glands. Fig 8 Effect of mevalonic acid on the inhibition of submandibular gland morphogenesis induced by simvastatin. A : Phase-contrast image of branching morphogenesis in the presence of simvastatin without or with mevalonic acid. B : Quantification of branching morphogenesis after culturing under the same condition as in A. The fold induction of buds represents the ratio of buds at 48h/ buds at 0h. *P v.s. the control group. Values are mean SE for 8 10 glands.

8 vastatin squalene Squalene 20,200 μm simvastatin Fig 9 simvastatin Fig 9 Effect of squalene on the inhibition of submandibular gland morphogenesis by simvastatin. Quantification of branching morphogenesis in the presence of simvastatin without or with squalene. The fold induction of buds represents the ratio of buds at 48h/ buds at 0h. *P v.s. control group. Values are mean SE for 8 10 glands. 6 GTP farnesyl pyrophosphate geranylgeranyl pyrophosphate farnesol geranylgeraniol simvastatin GTP Ras Rho farnesyl transferase FTase inhibitor I geranylgeranyl transferase GGTI-298 Farnesol geranylgeraniol simvastatin Fig 10 FTase inhibitor I GGTI-298 simvastatin Fig 11 simvastatin 7 Fig 10 Effect of farnesol or geranylgeraniol on the inhibition of submandibular gland morphogenesis induced by simvastatin. Quantification of branching morphogenesis in the presence of simvastatin without or with farnesol A, geranylgeraniol B. The fold induction of buds represents the ratio of buds at 48h/ buds at 0h. *P v.s. the control group. Values are mean SE for 8 10 glands.

9 HMG-CoA 125 matrigel matrigel E13 48h Fig 12 Fig 11 Effect of FTase inhibitor I and GGTI-298 on the branching morphogenesis of embryonic submandibular gland. Fig 12 Effect of mavalonic acid or mesenchymal tissue on the branching morphogenesis of the separated epithelium in matrigel culture. Numbers indicate the incubation time with separated epithelium. Morphological observation of epithelium A, epithelium plus mevalonic acid B and epithelium in the presence of mesenchyme C.

10 Fig 13 Changes in the sensitivity of submandibular gland to the inhibitory action of simvastatin against the branching morphogenesis. Red bars indicate the treatment period with simvastatin 10 μm in cultured medium. 8 HMG-CoA HMG-CoA simvastatin E13 E13 24h simvastatin E13 48h simvastatin Fig HMG-CoA 1, Insig-1, -2 7 Smith-Lemli-Opitz 8 HMG-CoA HMG-CoA 9 EGF, TGFα FGF 6 HMG-CoA HMG-CoA HMG-CoA farnesyl pyrophosphate, geranylgeranyl pyrophosphate GTP Ras Rho

11 HMG-CoA HMG-CoA , PI3K/ Akt 28, 29 HMG-CoA HMG-CoA E13 Figs 3, 4 E13 96 h HMG-CoA E13 E13 24h simvastatin Fig 13 HMG-CoA HMG-CoA simvastatin Fig 8 Simvastatin μm Fig 6 HMG-CoA HMG-CoA 32 Simvastatin fluvastatin, atrovastatin pravastatin, rosuvastatin Fig 7 statin OATP 2 33 pravastatin simvastatin Simvastatin squalene Fig 9 farnesol geranylgeraniol Fig 10 farnesyl transferase geranylgeranyl transferase Fig 11 GTP Ras, Rho matrigel Fig 12 Takahashi Nogawa 13 collagen gel Matrigel Nogawa Takahashi 14 EGF, TGFα, FGF simvastatin E13 E13 24h Fig 13 E13 E 13 24h HMG-CoA

12 HMG-CoA HMG-CoA GTP HMG-CoA 1 HMG-CoA 2 HMG-CoA 3 4 E13 E13 24h HMG-CoA 5 HMG-CoA Jaskoll T and Melnick M : Submandibular gland morphogenesis : stage-specific expression of TGF-alpha/EGF, IGF, TGF-beta, TNF, and IL-6 signal transduction in normal embryonic mice and the phenotypic effects of TGF-beta2, TGF-beta3, and EGF-R null mutations. Anat Rec 256, Borghese E : Explantation experiments on the influence of the connective tissue capsule on the development of the epithelial part of the submandibular gland of Mus musculus. J Anat 84, , 1950 Ball WD : Development of the rat salivary glands. 3. Mesenchymal specificity in the morphogenesis of the embryonic submaxillary and sublingual glands of the rat. J Exp Zool 188, , 1974 Cunha GR : Support of normal salivary gland morphogenesis by mesenchyme derived from accessory sexual glands of embryonic mice. Anat Rec 173, , 1972 Lawson KA : Mesenchyme specificity in rodent salivary gland development : the response of salivary epithelium to lung mesenchyme in vitro. J Embryol Exp Morphol 32, , , , 2002 Engelking LJ, Evers BM, Richardson JA, Goldstein JL, Brown MS and Liang G : Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin. J Clin Invest 116, , 2006 Kelley R and Hennekam R : The Smith-Lemli-Opitz syndrome. J Med Genet 37, , 2000 Ohashi K, Osuga J, Tozawa R, Kitamine T, Yagyu H, Sekiya M, Tomita S, Okazaki H, Tamura Y, Yahagi N, Iizuka Y, Harada K, Gotoda T, Shimano H, Yamada N and Ishibashi S : Early embryonic lethality caused by targeted disruption of the 3-hydroxy- 3-methylglutaryl-CoA reductase gene. J Biol Chem 278, , 2003 Liao JK : Isoprenoids as mediators of the biological effects of statins. J Clin Invest 110, , 2002 Llevadot J, Murasawa S, Kureishi Y, Uchida S, Masuda H, Kawamoto A, Walsh K, Isner JM and Asahara T : HMG-CoA reductase inhibitor mobilizes bone marrow-derived endothelial progenitor cells. J Clin Invest 108, , 2001 Park HJ, Kong D, Iruela-Arispe L, Begley U, Tang D and Galper JB : 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors interfere with angiogenesis by inhibiting the geranylgeranylation of Rho A. Circ Res 91, , 2002 Takahashi Y & Nogawa H : Branching morphogenesis of mouse salivary epithelium in basement menbrane-like substratum separated from mesenchyme by the membrane filter. Development 111, , 1991 Nogawa H and Takahashi Y : Substitution for mesenchyme by basement- membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium. Development 112, , 1991 Spooner BS, Bassett KE and Spooner BS Jr : Embryonic salivary gland epithelial branching activity is experimentally independent of epithelial expansion activity. Dev Biol Jun 133, , 1989 Kashimata M and Gresik EW : Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the alpha 6- integrin subunit. Dev Dyn 208, , 1997 Kashimata M, Sayeed S, Ka A, Onetti-Muda A, Sakagami H, Faraggiana T and Gresik EW : The ERK-1/2 signaling pathway is involved in the stimulation of branching morphogenesis of fetal mouse submandibular glands by EGF. Dev Biol 220, ,

13 HMG-CoA Koyama N, Kashimata M, Sakashita H, Sakagami H and Gresik EW : EGF-stimulated signaling by means of PI3K, PLCgamma1, and PKC isozymes regulates branching morphogenesis of the fetal mouse submandibular gland. Dev Dyn 227, , 2003 Hoffman MP, Kidder BL, Steinberg ZL, Lakhani S, Ho S, Kleinman HK and Larsen M : Gene expression profiles of mouse submandibular gland development : FGFR 1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms. Development Dec 129, , 2002 Jaskoll T, Zhou YM, Chai Y, Makarenkova HP, Collinson JM, West JD, Hajihosseini MK, Lee J and Melnick M : Embryonic submandibular gland morphogenesis : stage-specific protein localization of FGFs, BMPs, Pax6 and Pax9 in normal mice and abnormal SMG phenotypes in FgfR2-IIIc /Delta, BMP7 / and Pax6 / mice. Cells Tissues Organs 170, 83 98, 2002 Jaskoll T, Witcher D, Toreno L, Bringas P, Moon AM and Melnick M : FGF8 dose-dependent regulation of embryonic submandibular salivary gland morphogenesis. Dev Biol 268, , 2004 Jaskoll T, Choy HA and Melnick M : Glucocorticoids, TGFbeta, and embryonic mouse salivary gland morphogenesis. J Craniofac Genet Dev Biol 14, , 1994 Delanty N and Vaughan CJ : Vascular effects of statins in stroke. Stroke 28, , 1997 Rosenson RS and Tangney CC : Antiatherothrombotic properties of statins : implications for cardiovascular event reduction. JAMA 279, , 1998 Vaughan CJ, Gotto AM Jr and Basson CT : The evolving role of statins in the management of atherosclerosis. J A, Coll Cardiol 35, 1 10, 2000 TGF-β BMP-2 47, , 2005 Statin TGF-β1 BMP-2 49, , 2006 DimmelerS,AicherA,VasaM,Mildner-RihmC,AdlerK,Tiemann M, Rütten H, Fichtlscherer S, Martin H and Zeiher AM : HMG-CoA reductase inhibitors statins increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest 108, , 2001 Skaletz-Rorowski A, Lutchman M, Kureishi Y, Lefer DJ, Faust JR and Walsh K : HMG-CoA reductase inhibitors promote cholesterol-dependent Akt/PKB translocation to membrane domains in endothelial cells. Cardiovasc Res 57, , 2003 Kenis I, Tartakover-Matalon S, Cherepnin N, Drucker L, Fishman A, Pomeranz M and Lishner M : Simvastatin has deleterious effects on human first trimester placental explants. Hum Reprod 20, , 2005 Dostal LA, Schardein JL and Anderson JA : Developmental toxicity of the HMG-CoA reductase inhibitor, atorvastatin, in rats and rabbits. Teratology 50, , 1994 Parker RA, Huang Q and Tesfamariam B : Influence of 3- hydroxy-3- methylglutaryl-coa HMG-CoA reductase inhibitors on endothelial nitric oxide synthase and the formation of oxidants in the vasculature. Atherosclerosis 169, 19 29, 2003 Tokui T, Nakai D, Nakagomi R, Yawo H, Abe T and Sugiyama Y : Pravastatin, an HMG-CoA reductase inhibitor, is transported by rat organic anion transporting polypeptide, oatp 2. Pharm Res 16, ,

Effect of Sympathetic and parasympathetic agents on cholesterol metabolism in mice salivary glands. Eriko, NEZU and Haruo, NAKAMURA (Department of Ora

Effect of Sympathetic and parasympathetic agents on cholesterol metabolism in mice salivary glands. Eriko, NEZU and Haruo, NAKAMURA (Department of Oral Physiology, Hokkaido University School of Dentistry,

明海大学歯学雑誌 ４２‐２★／目次（４２‐２）

明海大学歯学雑誌　４２‐２★／目次（４２‐２）