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Insoluble, Speckled Cytosolic Distribution of Retinoic Acid Receptor Alpha Protein as a Marker of Hepatic Stellate Cell Activation In Vitro

Yoshihiro Mezaki, Noriko Yamaguchi, Kiwamu Yoshikawa, Mitsutaka Miura, Katsuyuki Imai, Hideaki Itoh and Haruki Senoo

Department of Cell Biology and Histology, School of Medicine (YM,NY,KY,MM,KI,HS), and Department of Life Science, Faculty of Engineering and Resource Science (HI), Akita University, Akita, Japan

Correspondence to: Haruki Senoo, Department of Cell Biology and Histology, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan. E-mail: senoo{at}ipc.akita-u.ac.jp

Hepatic stellate cells (HSCs) are the major site of retinoid storage, and their activation is a key process in liver fibrogenesis. We have previously shown that expression of the retinoic acid receptor alpha (RAR) is upregulated in activated rat HSCs at a posttranscriptional level and that these RAR proteins showed a speckled distribution in the cytosol, despite their possession of a nuclear localization signal (NLS). In this report, we further characterize these cytosolic RAR proteins by using exogenously expressed RAR protein fragments or mutants tagged with a green fluorescent protein. Substitution of four amino acids, 161–164 from lysine to alanine, abolished the NLS. Exogenously expressed RAR protein fragments containing an NLS were localized exclusively in the nuclei of activated rat HSCs and never colocalized with the endogenous RAR proteins in the cytosol, suggesting that the NLS of endogenous RAR proteins is masked. Biochemical analysis showed that 65% of RAR proteins in activated HSCs were insoluble in a mixture of detergents. The insolubility of RAR proteins makes it difficult to identify RAR proteins in activated HSCs. Therefore, we propose that insoluble, speckled cytosolic distribution of RAR proteins represents a new marker of HSC activation. (J Histochem Cytochem 57:687–699, 2009)

Key Words: hepatic stellate cell • retinoic acid receptor • retinoid • vitamin A • liver fibrosis • nuclear localization signal • nuclear import

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