Entactin gene expression in normal and fibrotic rat liver and in rat liver cells.

BACKGROUND

Entactin, a constituent of basement membranes, is a sulfated glycoprotein with binding sites for laminin, collagen type IV, fibronectin, and cell surfaces. As it is known that excess matrix deposition and sinusoidal basement membrane formation is a central characteristic of liver fibrogenesis, we investigated whether the entactin gene is expressed in normal and in damaged rat liver and which cell types are able to express this gene. In addition, we were interested in the cellular origin and time course of laminin synthesis, a matrix protein closely associated with entactin.

EXPERIMENTAL DESIGN

Entactin gene expression was analyzed in normal, acutely and chronically damaged rat livers (CCl4-model) by immunohistochemistry and in situ detection of specific transcripts. Rat fat-storing cells (FSC) (Ito), hepatocytes, Kupffer cells, liver endothelial cells, arterial smooth muscle cells (SMC), and skin fibroblasts (SF) were isolated according to standard techniques. Entactin gene expression in cultured cells was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitates, Northern blot analysis, and immunocytochemistry.

RESULTS

In normal liver, entactin was detected in the vessel walls as continuous deposits and in a spot-like fashion along the sinusoids. Entactin was detectable among the cells of the inflammatory infiltrates of acutely damaged liver and in connective tissue septa, in the walls of newly formed vessels and bile ducts of fibrotic liver. Laminin distribution in the vessels was similar, but it was additionally present in the space of Dissé of damaged liver. By in situ hybridization, few entactin-positive cells were found in normal liver sections. Strongly positive cells were scattered over the injured parenchyma of acutely and chronically damaged livers. Northern blot analysis of total RNA extracted from normal and damaged liver tissue showed a distinct increase of entactin specific transcripts during development of fibrosis. Hybridization of total RNA from cultured FSC, hepatocytes, Kupffer cells, endothelial cells, SMC, and SF revealed entactin specific mRNA in FSC, SMC, SF, and endothelial cells; laminin mRNA was found in FSC and SF. Synthesis and secretion of both proteins were found in FSC, SMC and SF. Entactin and laminin gene expression increased in parallel to FSC during time in culture.

CONCLUSIONS

Among the main liver cells, FSC show the highest entactin gene expression and might therefore play the dominant role in the synthesis of this protein in normal and fibrotic liver. However, endothelial cells and liver myofibroblasts could also contribute to entactin production.