Subcellular localization of (latent) transforming growth factor β and the latent TGF-β binding protein in rat hepatocytes and hepatic stellate cells

Abstract

Recently, the existence of the large latent transforming growth factor β (TGF-β) complex, consisting of TGF-β, the N-terminal part of its precursor (latency-associated peptide [LAP]), and the latent TGF-β binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and stellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to investigate the subcellular distribution of the proteins forming the latent TGF-β complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolated from rat liver by protease reperfusion and investigated for TGF- β1,-2,2-3, β1-LAP, and LTBP-1 after cultivation using double- immunofluorescent staining, followed by high-resolution confocal microscopic analysis. Subcellular fractions obtained by standard differential centrifugation of rat liver homogenate were analyzed using a TGF-β1 enzyme-linked immunosorbent assay (ELISA) and Western blotting for β1-LAP and LTBP-1. By confocal microscopy, a diffuse distribution of TGF- β and LAP in the cytoplasm of PC is noticed, whereas the LTBP immunostaining predominates at plasma membranes. In PC, distinct intracellular granules were superimposed with TGF-β, LAP, and LTBP stainings identified as lysosomal compartments and mitochondria by ELISA and immunoblotting of subcellular fractions. In HSC, stainings of colocalized TGF-β, LAP, and LTBP are strongest in the perinuclear area, indicating synthesis and secretion via endoplasmic reticulum and Golgi, respectively. Partially, the proteins were also found in HSC nuclei. During the transformation of HSC to myofibroblasts, LAP and LTBP become strongly colocalized with other components of the cytoskeletal network like smooth muscle-α-actin, desmin, and talin. The results confirm biochemical data about the existence and expression of the large latent TGF-β complex in PC and HSC, respectively. Baseline information is provided from which new hypotheses regarding intracellular functions of TGF-β, LAP, and LTBP in liver parenchymal and stellate cells can be concluded.