Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.

Activins are cytokines of the transforming growth factor β family, which plays a central role in the determination of cell fate and the regulation of tissue balance. Family members are composed of two subunits and this dimerization is critical for liganding their cognate receptors and execution of proper functions. In the current study we focused on the localization of activin β A , β B , β C and β E subunits in the adult rat and analyzed the composition of putative activin β dimers. By dissecting tissue distribution of various activins, we found that the liver, in particular the hepatocytes, is the major source for activin β C and β E transcripts, since other tissues almost failed to express these isoforms. In sharp contrast, the emergence of activin β A and β B appeared ubiquitous. Using a highly selective proteome approach, we were able to identify homo-as well as heterodimers of individual activin subunits, indicating a high redundancy of ligand composition. Certainly, this broad potential to homo-and heterodimerize has to be considered in future studies on activin function.

Activins are formed by dimerization of beta-subunits and, as members of the TGF-beta superfamily, have diverse roles as potent growth and differentiation factors. As the biological function of the activin C homodimer (betaC-betaC) is unknown, we sought to compare activin A (betaA-betaA), B (betaB-betaB), and C homodimer bioactivities and to investigate the consequences of activin betaC-subunit overexpression in prostate tumor cells. Exogenous activin A and B homodimers inhibited cell growth and activated activin-responsive promoters. In contrast, the activin C homodimer was unable to elicit these responses. We previously showed that the activin betaC-subunit heterodimerized with activin betaA in vitro to form activin AC. Therefore, we hypothesize that the activin betaC-subunit regulates the levels of bioactive activin A by the formation of activin AC heterodimers. To test this hypothesis, we measured activin AC heterodimer production using a novel specific two-site ELISA that we developed for this purpose. In the PC3 human prostate tumor cell line, activin betaC-subunit overexpression increased activin AC heterodimer levels, concomitantly reduced activin A levels, and decreased activin signaling. Overall, these data are consistent with a role for the activin betaC-subunit as a regulatory mechanism to reduce activin A secretion via intracellular heterodimerization.

Activin ligands are formed by dimerization of activin ss(A)- and/or ss(B)-subunits to produce activins A, AB, or B. These ligands are members of the transforming growth factor-ss superfamily and act as growth and differentiation factors in many cells and tissues. New additions to this family include activin ss(C)-, ss(D)-, and ss(E)-subunits. The aim of this investigation was to examine the localization of and dimerization among activin subunits; the results demonstrate that activin ss(C) can form dimers with activin ss(A) and ss(B) in vitro, but not with the inhibin alpha-subunit. Using a specific antibody, activin ss(C) protein was localized to human liver and prostate and colocalized with ss(A)- and ss(B)-subunits to specific cell types in benign and malignant prostate tissues. Activin C did not alter DNA synthesis of the prostate tumor cell line, LNCaP, or the liver tumor cell line, HepG2, in vitro when added alone or with activin A. Therefore, the capacity to form novel activin heterodimers (but not inhibin C) resides in the human liver and prostate. Activin A, AB, and B have diverse actions in many tissues, including liver and prostate, but there is no known biological activity for activin C. Thus, the evidence of formation of activin AC or BC heterodimers may have significant implications in the regulation of levels and/or biological activity of other activins in these tissues.

The mRNA expression of two activin growth factor subunits ( A-and C-activin), activin receptor subunits (ActRIIA, ActRIIB) and the activin-binding protein follistatin, and peptide expression of A-activin and C-activin subunits, were examined in regenerating rat liver after partial hepatectomy (PHx). Liver samples were collected from adult, male Sprague-Dawley rats, 12-240 h (n=3-5 rats per time point) after PHx or from sham-operated controls at the same time points. Hepatocyte mitosis and apoptosis were assessed histologically and by in situ cell death detection. RT and PCR were used to assess relative gene expression. A-and C-activin peptide immunoreactivity was assessed in liver and serum samples by western blotting, whereas cellular expression was investigated by immunohistochemistry, using specific monoclonal antibodies. A-and C-activin mRNA dropped to,50% of sham control values 12 h after PHx and remained at this level until 168 h post-PHx, when A-activin expression increased to three times sham control values and C-activin mRNA returned to pre-PHx levels. A peak in follistatin expression was observed 24-48 h post-PHx, coincident with an increase in hepatocyte mitosis. No changes were observed in ActRIIA mRNA, whereas ActRIIB expression paralleled that of A-activin mRNA. C-activin immunoreactive homo-and heterodimers were observed in regenerating liver and serum. Mitotic hepatocytes frequently contained C-activin immunoreactivity, whereas apoptotic hepatocytes were often immunoreactive for A-activin. We conclude that A-and C-activin subunit proteins are autocrine growth regulators in regenerating liver and when expressed independently lead to hepatocyte apoptosis or mitosis in a subset of hepatocytes.