Transforming growth factor beta (TGF-beta) expression in isolated and cultured rat hepatocytes.

It is still a subject of debate whether hepatocytes have the ability to express TGF-beta. Therefore, we investigated in freshly isolated and in monolayer cultures of rat hepatocytes the expression of TGF-beta isoform s at the RNA and protein level applying RT-PCR, immunocytochemistry, immunoblotting, and functional assays of TGF-beta. TFG-beta 1, -beta 2, and -beta 3 transcripts were detected in cultured cells, and the level of m RNA increased up to 48/72 h, but TGF-beta 1 transcripts were absent in freshly isolated cells. Using APAAP stainings the proteins of all three TGF-beta isoforms were observed in hepatocyte cultures from 5-96 h, but in hepatocytes in the liver in situ and in freshly isolated cell suspensions TGF-beta staining was negative. SDS-PAGE under reducing conditions followed by Western blotting detected in cell lysates the subunit of mature TGF-beta at about 13 kd. Analysis of TGF-beta bioactivity with the mink cell (Mv1Lu) proliferation inhibition assay and competitive radioligand assay confirmed in activated (i.e., acidified and subsequently neutralized) hepatocyte-conditioned media the presence of TGF-beta, which, however, is almost entirely in the latent form. It is concluded that TGF-beta can be expressed in cultured hepatocytes and that the level of expression is quickly upregulated under abnormal, not yet known, microenvironmental conditions.

Synergism between hepatocytes and Kupffer cells in the activation of fat storing cells (perisinusoidal lipocytes).

This study concerns the cooperation of hepatocytes (PC) and Kupffer cells (KC) in the activation of rat liver fat storing cells (FSC) in culture. Various dilutions of conditioned media collected from early, serum-free cultures of both cell types were added separately and in combination, either simultaneously or sequentially, to early, non-confluent, primary cultures of FSC maintained under serum-reduced (0.5% fetal calf serum) conditions to determine the effects on proliferation (incorporations of [3H]thymidine and bromodeoxyuridine [BrdUrd], DNA-content, cell number), transformation and morphology (phase contrast microscopy, immunostainings of desmin and smooth muscle-alpha-actin), on the deposition of fibronectin and laminin and on the formation of 35S sulfated medium proteoglycans. Media of both cell types stimulated cell proliferation in a dose-dependent manner but combined PC- and KC-conditioned media was most potent and increased the incorporation of [3H]thymidine to 4-times above control values. The multiplication stimulatory effects visualized by labeling cell nuclei with BrdUrd and the increase of cell number per culture well were additive. The sequential addition of KC-conditioned medium to FSC preexposed to PC-conditioned medium increased the multiplication of FSC further and in an additive manner. The mitogenic activity of the PC-medium and the enhancing effect of KC-induced FSC proliferation was measured also when PC were damaged by incubation under anoxic conditions during generation of the conditioned medium. This observation indicates the release of the mitogen by membrane damage presumably from a cytoplasmic pool. The PC-medium did not induce either significant morphological changes or transformation of FSC towards myofibroblast-like cells. KC, however, generated transformation of FSC as indicated by more elongated cells with spindle-like cellular extensions and a reduction of retinoid droplets. Both these morphological effects were visible when PC and KC media were added simultaneously. Both media act synergistically on the deposition of fibronectin and laminin in FSC cultures and these components were found to be elevated 2.3 and 2.8-fold, respectively, if the cells were exposed to the combined media. Proteoglycan synthesis was also maximally enhanced if FSC were exposed to PC- and KC-media simultaneously. These findings suggest the involvement of (damaged) hepatocytes in the process of FSC activation. A model of sequential, spatial and time-dependent activation of FSC is suggested where cells in the immediate proximity of hepatocytes are primed to proliferate by a mitogenic signal released by membrane damage presumably from a cytoplasmic pool of injured hepatocytes into the pericellular environment. This non-inflammatory stimulation is followed by secretions of activated Kupffer cells and other inflammatory cell types which further enhance the activation of FSC.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and partial characterization of a hepatocyte-derived factor promoting proliferation of cultured fat-storing cells (parasinusoidal lipocytes).

The molecular and cellular mechanisms of activation of fat-storing cells (Ito cells or parasinusoidal lipocytes), a prerequisite of the fibrogenic response of injured liver, were studied by analysis in vitro of some aspects of the intercellular communication between parenchymal liver cells and fat-storing cells. Conditioned medium harvested from early serum-free monolayer cultures of hepatocytes isolated from normal rat liver stimulated strongly, reproducibly and dose-dependently the proliferation of nonconfluent fat-storing cells maintained under serum-reduced conditions. During exposure of fat-storing cells for 48 hr to the conditioned medium, the incorporation of [3H]thymidine into DNA was stimulated four to six times over control values, the DNA content per culture well was elevated by 40% above control values and the immunocytochemical detection of bromodeoxyuridine-labeled cell nuclei was increased from 13% stained nuclei in controls to 70% stained nuclei in treated fat-storing cells. The mitogenic effects of hepatocyte-conditioned medium were similar to or even higher than those of 10% fetal calf serum. No mitoinhibitory activity could be detected in the hepatocyte-conditioned medium when arginase, as a potential inhibitor, was excluded. Rat skin fibroblasts could not be stimulated under conditions where the proliferation activity of fat-storing cells was greatly enhanced. The occurrence of the mitogenic activity in the medium is not dependent on de novo synthesis or secretion because the media of hepatocytes cultured under anoxic conditions in the presence of cycloheximide, brefeldin A or ethylenediaminetetraacetate were highly active in promoting fat-storing cell proliferation, although hepatocyte viability was greatly reduced under some of these conditions. A significant positive correlation (r = 0.95, p < 0.01) was found between lactate dehydrogenase activity and the mitogenic potency of the conditioned medium. The proliferation factor for fat-storing cells could also be demonstrated in the lysate of freshly isolated hepatocytes from normal liver. The stimulatory activity in the medium was partially enriched by a combination of gel permeation and anion exchange fast protein liquid chromatography and characterized as a protein with an apparent molecular weight of about 60 kD that is heat and pH sensitive but insensitive to reducing agents. It does not bind to immobilized heparin; nor does soluble heparin or proteinase inhibitor affect the mitogenic activity of the factor.(ABSTRACT TRUNCATED AT 400 WORDS)