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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

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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.

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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

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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.

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Correspondence to Keitaro Satoh.

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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) doi:10.1007/s00424-012-1183-y

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Keywords

  • Salivary glands
  • AQP5 protein
  • Down-regulation
  • Dry mouth
  • Ubiquitin
  • Disease model