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

Recently, the existence of the large latent transforming growth factor beta (TGF-beta) complex, consisting of TGF-beta, the N-terminal part of its pre cursor (latency-associated peptide [LAP]), and the latent TGF-beta binding protein (LTBP), was demonstrated in rat liver parenchymal cells (PC) and st ellate cells (HSC). However, in contrast to HSC, in freshly isolated PC, no message of these proteins is detectable. This study was performed to inves tigate the subcellular distribution of the proteins forming the latent TGF- beta complex in PC and HSC from rat liver to obtain more information about their origin and potential intracellular functions. PC and HSC were isolate d from rat liver by protease reperfusion and investigated for TGF-beta(1,-2 ,-3), beta 1-LAP and LTBP-1 after cultivation using double-immunofluorescen t staining, followed by high-resolution confocal microscopic analysis. Subc ellular fractions obtained by standard differential centrifugation of rat l iver homogenate were analyzed using a TGF-beta(1) enzyme-linked immunosorbe nt assay (ELISA) and Western blotting for beta 1-LAP and LTBP-1. By confoca l microscopy, a diffuse distribution of TGF-beta and LAP in the cytoplasm o f PC is noticed, whereas the LTBP immunostaining predominates at plasma mem branes. In PC, distinct intracellular granules were superimposed with TGF-b eta, LAP, and LTBP stainings identified as lysosomal compartments and mitoc hondria by ELISA and immunoblotting of subcellular fractions. In HSC, stain ings of colocalized TGF-beta, LAP, and LTBP are strongest in the perinuclea r area, indicating synthesis and secretion via endoplasmic reticulum and Go lgi, respectively. Partially the proteins were also found in HSC nuclei. Du ring the transformation of HSC to myofibroblasts, LAP and LTBP become stron gly colocalized with other components of the cytoskeletal network like smoo th muscle-alpha-actin, desmin, and talin. The results confirm biochemical d ata about the existence and expression of the large latent TGF-beta complex in PC and HSC, respectively. Baseline information is provided from which n ew hypotheses regarding intracellular functions of TGF-beta, LAP, and LTBP in liver parenchymal and stellate cells can be concluded.