Localization and cellular sources of activins in normal and fibrotic rat liver

Abstract

Activins are dimeric proteins, members of the transforming growth factor β (TGF-β) gene superfamily, consisting of the β-subunits of inhibin (β(A) and β(B)). Recently, it was shown that activin A (β(A):β(A)) inhibits DNA replication and induces apoptosis in rat parenchymal cells in vitro and in vivo. Cryostat sections of normal livers and livers of rats treated with intraperitoneal injections of CCl4 were stained for the different inhibin subunits and desmin, a marker for stellate cells (SC). Staining for inhibin- α was invariably negative, both in normal and fibrotic rat liver. In normal liver, inhibin-β(A) subunit immunoreactivity was localized in parenchymal cells (PC). Staining for inhibin-β(B) was weaker but similarly distributed. In fibrotic livers, connective tissue septa were strongly immunoreactive for inhibin-β(A). Desmin-positive stellate cells (SC) accumulated in areas strongly immunoreactive for inhibin-β(A) and several calls were positive for both desmin and inhibin-β(A). Staining for inhibin-β(B) was weaker but similarly distributed. As above data pointed to a possible role for PC and SC, we examined by Northern blot analysis, the expression of inhibin-α, - β(A), and -β(B) messenger RNA (mRNA) in total RNA extracted from freshly isolated SC and PC of normal and CCl4-treated liver and in cultured SC. Inhibin-β(A) mRNA was predominantly expressed in PC of normal liver. Expression was lost in PC of CCl4-treated liver. Inhibin-β(B) mRNA expression was induced in SC of CCl4-treated liver. Inhibin-β(A) mRNA, and to a lesser extent, inhibin-β(B) mRNA expression was rapidly induced in cultured SC. The presence of activin A in conditioned media of cultured SC was shown by Western blotting. Apoptotic cells, identified by terminal deoxy- transferase mediated X-dUTP nik end labeling (TUNEL)-staining, were found predominantly in and near the fibrotic septa. In conclusion: 1) while activin A was constitutively expressed in PC of normal liver, its expression was lost in PC of fibrotic liver; 2) expression of activins was induced in activated SC; and 3) apoptotic cells were found predominantly near the septa, in support of the hypothesis that activin A, derived from activated SC in the septa, contributes to the induction of call death in neighboring PC.