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Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology

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Abstract

Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.

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Acknowledgments

We would like to thank Drs. Mayumi Kisaka, Aya Yokota and Aki Shiomi, and Messrs Ki-ichi Takeuchi and Masaaki Hoshino for technical assistance. The authors declare no conflicts of interest with regard to the authorship and/or publication of this paper. This work was supported by a Grant-in-Aid for the Promotion of Science (No. 21659455) to T.M., in part by a Grant-in-Aid for Scientific Research (B) (No. 22390371) to K.I. from the Japan Science and Technology Agency and research for Promoting Technological Seeds from Independent Administrative Corporation Japan Science and Technology Agency (No. 05-033) to K.I.

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  1. Hiroko Kato

    Present address: Department of Oral and Maxillofacial Surgery, University of Michigan, MSRB II, Rm A560, Ann Arbor, MI, 48109-0654, USA

Authors and Affiliations

  1. Division of Reconstructive Surgery for Oral and Maxillofacial Region, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Hiroko Kato & Chikara Saito

  2. Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata, 951-8514, Japan

    Hiroko Kato, Kenji Izumi, Taro Saito, Hisashi Ohnuki, Michiko Terada, Yoshiro Kawano, Kayoko Nozawa-Inoue & Takeyasu Maeda

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  1. Hiroko Kato
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Correspondence to Kenji Izumi.

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Kato, H., Izumi, K., Saito, T. et al. Distinct expression patterns and roles of aldehyde dehydrogenases in normal oral mucosa keratinocytes: differential inhibitory effects of a pharmacological inhibitor and RNAi-mediated knockdown on cellular phenotype and epithelial morphology. Histochem Cell Biol 139, 847–862 (2013). https://doi.org/10.1007/s00418-012-1064-7

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