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

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 alpha) is upregulated in activated rat HSCs at a post-transcriptional level and that these RAR alpha 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 alpha proteins by using exogenously expressed RAR alpha 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 alpha protein fragments containing an NLS were localized exclusively in the nuclei of activated rat HSCs and never colocalized with the endogenous RAR alpha proteins in the cytosol, suggesting that the NLS of endogenous RAR alpha proteins is masked. Biochemical analysis showed that 65% of RAR alpha proteins in activated HSCs were insoluble in a mixture of detergents. The insolubility of RAR alpha proteins makes it difficult to identify RAR alpha proteins in activated HSCs. Therefore, we propose that insoluble, speckled cytosolic distribution of RAR alpha. proteins represents a new marker of FISC activation. (J Histochem Cytochem 57:687-699, 2009)