Abstract
Non-obese diabetic (NOD) mice have been used as a model for dry mouth. NOD mice lacking the gene encoding E2f1, a transcription factor, develop hyposalivation more rapidly progressively than control NOD mice. However, the model mice are associated with an underlying disease such as diabetes. We have now established E2f1-deficient NOD/severe combined immunodeficiency disease (NOD/SCID.E2f1−/−) mice to avoid the development of diabetes (Matsui-Inohara et al., Exp Biol Med (Maywood) 234(12):1525–1536, 2009). In this study, we investigated the pathophysiological features of dry mouth using NOD/SCID.E2f1−/− mice. In NOD/SCID.E2f1−/− mice, the volume of secreted saliva stimulated with pilocarpine is about one third that of control NOD/SCID mice. In behavioral analysis, NOD/SCID.E2f1−/− mice drank plenty of water when they ate dry food, and the frequency and time of water intake were almost double compared with control NOD/SCID mice. Histological analysis of submandibular glands with hematoxylin–eosin stain revealed that NOD/SCID.E2f1−/− mice have more ducts than NOD/SCID mice. In western blot analysis, the expression of aquaporin 5 (AQP5), a marker of acinar cells, in parotid and in submandibular glands of NOD/SCID.E2f1−/− mice was lower than in NOD/SCID mice. Immunohistochemical analysis of parotid and submandibular acini revealed that the localization of AQP5 in NOD/SCID.E2f1−/− mice differs from that in NOD/SCID mice; AQP5 was leaky and diffusively localized from the apical membrane to the cytosol in NOD/SCID.E2f1−/− mice. The ubiquitination of AQP5 was detected in submandibular glands of NOD/SCID.E2f1−/− mice. These findings suggest that the change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland cause the pathogenesis of hyposalivation in NOD/SCID.E2f1−/− mice.
Similar content being viewed by others
Abbreviations
- AQP5:
-
Aquaporin 5
- NOD:
-
Non-obese diabetic
- SCID:
-
Severe combined immunodeficiency disease
- SS:
-
Sjögren’s syndrome
- BSA:
-
Bovine serum albumin
- RT-PCR:
-
Reverse transcription/polymerase chain reaction
- GAPDH:
-
Glyceraldehyde-3-phosphate dehydrogenase
- B2M:
-
β2 microglobulin
- HPRT:
-
Hypoxanthine phosphoribosyl transferase
- SDS:
-
Sodium dodecyl sulfate
- PAGE:
-
Polyacrylamide gel electrophoresis
- IP:
-
Immunoprecipitation
- WB:
-
Western blot analysis
- Ub-AQP5:
-
Ubiquitinated-AQP5
References
Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S (2002) Aquaporin water channels—from atomic structure to clinical medicine. J Physiol 542(Pt 1):3–16
Akamatsu T, Azlina A, Purwanti N, Karabasil MR, Hasegawa T, Yao C, Hosoi K (2009) Inhibition and transcriptional silencing of a subtilisin-like proprotein convertase, PACE4/SPC4, reduces the branching morphogenesis of and AQP5expression in rat embryonic submandibular gland. Dev Biol 325(2):434–443
Annicotte JS, Blanchet E, Chavey C, Iankova I, Costes S, Assou S, Teyssier J, Dalle S, Sardet C, Fajas L (2009) The CDK4–pRB–E2F1 pathway controls insulin secretion. Nat Cell Biol 11(8):1017–1023
Blazsek J, Varga G (1999) Secretion from minor salivary glands following ablation of the major salivary glands in rats. Arch Oral Biol 44:S45–S48
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chen ZJ, Sun LJ (2009) Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 33(3):275–286
Dibas A, Yang MH, He S, Bobich J, Yorio T (2008) Changes in ocular aquaporin-4 (AQP4) expression following retinal injury. Mol Vis 14:1770–1783
Entesarian M, Matsson H, Klar J, Bergendal B, Olson L, Arakaki R, Hayashi Y, Ohuchi H, Falahat B, Bolstad AI, Jonsson R, Wahren-Herlenius M, Dahl N (2005) Mutations in the gene encoding fibroblast growth factor 10 are associated with aplasia of lacrimal and salivary glands. Nat Genet 37(2):125–127
Epstein AN, Spector D, Samman A, Goldblum C (1964) Exaggerated prandial drinking in the rat without salivary glands. Nature 201:1342–1343
Fox RI, Stern M, Michelson P (2000) Update in Sjögren syndrome. Curr Opin Rheumatol 12(5):391–398
Gresz V, Kwon TH, Gong H, Agre P, Steward MC, King LS, Nielsen S (2004) Immunolocalization of AQP-5 in rat parotid and submandibular salivary glands after stimulation or inhibition of secretion in vivo. Am J Physiol Gastrointest Liver Physiol 287(1):G151–G161
Hamada A, Inenaga K, Nakamura S, Terashita M, Yamashita H (2000) Disorder of salivary secretion in inbred polydipsic mouse. Am J Physiol Regul Integr Comp Physiol 278(4):R817–R823
Helin K, Lees JA, Vidal M, Dyson N, Harlow E, Fattaey A (1992) A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell 70(2):337–350
Henson BS, Inglehart MR, Eisbruch A, Ship JA (2001) Preserved salivary output and xerostomia-related quality of life in head and neck cancer patients receiving parotid-sparing radiotherapy. Oral Oncol 37(1):84–93
Hu Y, Nakagawa Y, Purushotham KR, Humphreys-Beher MG (1992) Functional changes in salivary glands of autoimmune disease-prone NOD mice. Am J Physiol 263(4 Pt 1):E607–E614
Iglesias A, Murge M, Laresgoiti U, Skoudy A, Bernales I, Fullaondo A, Moreno B, Lloreta J, Field SJ, Real FX, Zubiaga AM (2004) Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice. J Clin Invest 113(10):1398–1407
Jaskoll T, Abichaker G, Witcher D, Sala FG, Bellusci S, Hajihosseini MK, Melnick M (2005) FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis. BMC Dev Biol 5:11. doi:10.1186/1471-213X-5-11
Kaelin WG Jr, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, Chittenden T, Li Y, Farnham PJ, Blanar MA, Livingston DM, Flemington EK (1992) Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell 70(2):351–364
Karabasil MR, Hasegawa T, Azlina A, Purwanti N, Yao C, Akamatsu T, Tomioka S, Hosoi K (2011) Effects of naturally occurring G103D point mutation of AQP5 on its water permeability, trafficking and cellular localization in the submandibular gland of rats. Biol Cell 103(2):69–86
King LS, Agre P (1996) Pathophysiology of the aquaporin water channels. Annu Rev Physiol 58:619–648
Konttinen YT, Tensing EK, Laine M, Porola P, Törnwall J, Hukkanen M (2005) Abnormal distribution of aquaporin-5 in salivary glands in the NOD mouse model for Sjögren's syndrome. J Rheumatol 32(6):1071–1075
La Thangue NB (1994) DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci 19(3):108–114
Leitch V, Agre P, King LS (2001) Altered ubiquitination and stability of aquaporin-1 in hypertonic stress. Proc Natl Acad Sci U S A 98(5):2894–2898
Leiter EH, Prochazka M, Coleman DL (1987) The non-obese diabetic (NOD) mouse. Am J Pathol 128(2):380–383
Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS (1999) Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J Biol Chem 274(29):20071–20074
Matsui-Inohara H, Uematsu H, Narita T, Satoh K, Yonezawa H, Kuroda K, Ito T, Yoneda S, Kawarai T, Sugiya H, Watanabe H, Senpuku H (2009) E2F-1-deficient NOD/SCID mice developed showing decreased saliva production. Exp Biol Med (Maywood) 234(12):1525–1536
Matsuki M, Hashimoto S, Shimono M, Murakami M, Fujita-Yoshigaki J, Furuyama S, Sugiya H (2005) Involvement of aquaporin-5 water channel in osmoregulation in parotid secretory granules. J Membr Biol 203(3):119–126
Matsumoto N, Salam MA, Watanabe H, Amagasa T, Senpuku H (2004) Role of gene E2f1 in susceptibility to bacterial adherence of oral streptococci to tooth surfaces in mice. Oral Microbiol Immunol 19(4):270–276
Matsuzaki T, Suzuki T, Koyama H, Tanaka S, Takata K (1999) Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation. Cell Tissue Res 295(3):513–521
Min H, Danilenko DM, Scully SA, Bolon B, Ring BD, Tarpley JE, DeRose M, Simonet WS (1998) Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev 12(20):3156–3161
Murdiastuti K, Purwanti N, Karabasil MR, Li X, Yao C, Akamatsu T, Kanamori N, Hosoi K (2006) A naturally occurring point mutation in the rat aquaporin 5 gene, influencing its protein production by and secretion of water from salivary glands. Am J Physiol Gastrointest Liver Physiol 291(6):G1081–G1088
Nakamura T, Matsui M, Uchida K, Futatsugi A, Kusakawa S, Matsumoto N, Nakamura K, Manabe T, Taketo MM, Mikoshiba K (2004) M(3) muscarinic acetylcholine receptor plays a critical role in parasympathetic control of salivation in mice. J Physiol 558(Pt 2):561–575
Nandigama R, Bonitz M, Papadakis T, Schwantes U, Bschleipfer T, Kummer W (2010) Muscarinic acetylcholine receptor subtypes expressed by mouse bladder afferent neurons. Neuroscience 168(3):842–850
Nevins JR (1992) E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science 258(5081):424–429
Ohashi Y, Ishida R, Kojima T, Goto E, Matsumoto Y, Watanabe K, Ishida N, Nakata K, Takeuchi T, Tsubota K (2003) Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 136(2):291–299
Ohuchi H, Hori Y, Yamasaki M, Harada H, Sekine K, Kato S, Itoh N (2000) FGF10 acts as a major ligand for FGF receptor 2 IIIb in mouse multi-organ development. Biochem Biophys Res Commun 277(3):643–649
Quissell DO, Watson E, Dowd FJ (1992) Signal transduction mechanisms involved in salivary gland regulated exocytosis. Crit Rev Oral Biol Med 3(1–2):83–107
Robinson CP, Yamamoto H, Peck AB, Humphreys-Beher MG (1996) Genetically programmed development of salivary gland abnormalities in the NOD (nonobese diabetic)-scid mouse in the absence of detectable lymphocytic infiltration: a potential trigger for sialoadenitis of NOD mice. Clin Immunol Immunopathol 79(1):50–59
Salam MA, Matin K, Matsumoto N, Tsuha Y, Hanada N, Senpuku H (2004) E2f1 mutation induces early onset of diabetes and Sjögren's syndrome in nonobese diabetic mice. J Immunol 173(8):4908–4918
Satoh K, Seo Y, Matsuo S, Karabasil MR, Matsuki-Fukushima M, Nakahari T, Hosoi K (2012) Roles of AQP5/AQP5-G103D in carbamylcholine-induced volume decrease and in reduction of the activation energy for water transport by rat parotid acinar cells. Pflügers Arch 464(4):375–389. doi:10.1007/s00424-012-1141-8
Schein OD, Hochberg MC, Muñoz B, Tielsch JM, Bandeen-Roche K, Provost T, Anhalt GJ, West S (1999) Dry eye and dry mouth in the elderly: a population-based assessment. Arch Intern Med 159(12):1359–1363
Senpuku H (2010) Model mouse designed for oral biofilm formation studies. Int J Oral-Med Sci 8(3):125–131
Shan B, Lee WH (1994) Deregulated expression of E2F-1 induces S-phase entry and leads to apoptosis. Mol Cell Biol 14(12):8166–8173
Ship JA, Pillemer SR, Baum BJ (2002) Xerostomia and the geriatric patient. J Am Geriatr Soc 50(3):535–543
Soyfoo MS, De Vriese C, Debaix H, Martin-Martinez MD, Mathieu C, Devuyst O, Steinfeld SD, Delporte C (2007) Modified aquaporin 5 expression and distribution in submandibular glands from NOD mice displaying autoimmune exocrinopathy. Arthritis Rheum 56(8):2566–2574
Steinfeld S, Cogan E, King LS, Agre P, Kiss R, Delporte C (2001) Abnormal distribution of aquaporin-5 water channel protein in salivary glands from Sjögren's syndrome patients. Lab Investig 81(2):143–148
Tashiro E, Minato Y, Maruki H, Asagiri M, Imoto M (2003) Regulation of FGF receptor-2 expression by transcription factor E2F-1. Oncogene 22(36):5630–5635
Turner RJ, Sugiya H (2002) Understanding salivary fluid and protein secretion. Oral Dis 8(1):3–11
Yuasa K, Suzue K, Nagahama M, Matsuda Y, Tsuji A (2007) Transcriptional regulation of subtilisin-like proprotein convertase PACE4 by E2F: possible role of E2F-mediated upregulation of PACE4 in tumor progression. Gene 402(1–2):103–110
Acknowledgments
We thank Prof. Yoshiteru Seo (Dokkyo Medical University) for his helpful advice and discussion. This study was supported by a Dokkyo Medical University Research Grant (#2009-01-3), a Grant-in-Aid for Scientific Research from JSPS (#24580433, #21390506), and MEXT-Supported Program for the Strategic Research Foundation at Private University, 2010–2014.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Satoh, K., Narita, T., Matsuki-Fukushima, M. et al. E2f1-deficient NOD/SCID mice have dry mouth due to a change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland. Pflugers Arch - Eur J Physiol 465, 271–281 (2013). https://doi.org/10.1007/s00424-012-1183-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00424-012-1183-y