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

Abstract

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