Adenovirus-mediated overexpression of activin βC subunit accelerates liver regeneration in partially hepatectomized rats
Article Outline
The transforming growth factor (TGF) beta superfamily consists of a large number of structurally similar polypeptide growth factors, which interact with a number of different and related receptors and are important in modulating growth and developmental processes [1], [2]. Key among these factors is the TGF-β/Activin representing one class and the bone morphogenic proteins (BMPs) representing a distinct class of growth and differentiation factors. Multiple levels of regulation of signalling by TGF/Activin ligands are possible. Inhibition of signalling may be mediated by formation of inactive ligand heterodimers, or interactions with inhibitory proteins like follistatin. The ligands, which may be homo or heterodimers, signal via a complicated receptor system consisting of Type I/Type II TGF/Activin serine kinase receptors that signal within cells via both the SMAD and MAPK pathways (Fig. 1). Within cells, the activity of Smads may be further regulated by inhibitory Smads (e.g. Smad 6,7) and other transcriptional inhibitors (e.g. Sno, Ski). The net effect is tight regulation of the activin and TGFβ signalling pathways that is critical for normal control of cellular and organism growth and differentiation.
Fig. 1.
Mechanism of activin and Smad activation. At the cell surface the activin ligand (shown as βA, binds to the heterotetrameric transmembrane Type I/Type II serine kinase receptor. R-Smad (Receptor Smad 1, 5, 8) is then activated by phosphorylation and interacts with Smad 4 and the complex enters the nucleus. The activated transcriptional complex binds DNA and activates or represses transcription of Smad regulated genes. Smad 6 and 7 are inhibitory Smads. Follistatin is able to inhibit activin βA through direct interaction, and βC may also form dysfunctional subunits with βA to inhibit its action.
In this issue of the Journal, Wada et al. [3] focuses on defining a role in liver regeneration and hepatocyte growth for a less-well characterized subunit and member of the activin family, activin βC. Their data suggests that this factor may have a role in blocking or modulating the negative growth effects of activin βA perhaps via the formation of inactive heterodimers of βC/βA when βC levels are high in the liver.
Liver regeneration is a complex process that involves the interactions of multiple cytokine and growth factor pathways [4], [5]. After partial hepatectomy, liver mass is rapidly restored within days through hyperplasia of the remnant lobes of the liver. Proliferation under normal circumstances involves a mitogenic response of nearly all of the normally quiescent hepatocytes and other cells within the liver. A key aspect of understanding this process is identifying the factors that terminate liver regeneration within a few days after hepatectomy or liver injury once the liver has restored its mass.
Perhaps the best-known hepatocyte antiproliferative factors within the liver are TGFβ and related TGFβ family members such as activin A (subunits βA). TGFβ is produced mainly by hepatic stellate cells, and the up-regulated expression of TGFβ, in addition to blocking hepatocyte proliferation, leads to liver fibrosis and apoptosis. However, for unexplained reasons, hepatocytes become resistant to TGFβ and can proliferate despite the presence of TGFβ during liver regeneration [4], [5]. Early studies showed that increased norepinephrine in the regenerating liver might help protect against TGFβ [6]. TGFβ protein is up-regulated during liver regeneration, and its activity can be examined by assessing Smad activation, which occurs downstream of TGFβ. Smad proteins are partially activated in the quiescent liver, but their activation is further enhanced in the regenerating liver. Interestingly, TGFβ–Smad pathway inhibitors SnoN and Ski are also up-regulated during regeneration. SnoN and Ski are transcriptional repressors that might render some cells resistant to TGFβ by binding Smads. Complexes between SnoN, Ski and the activated Smads are detected during the major proliferation phase in regenerating liver. Inhibitory complexes decrease after liver mass restitution, which indicates that persistently activated Smads participate in returning the liver to a quiescent state at the termination of regeneration. SnoN or Ski induction offers a potential explanation for TGFβ resistance during the proliferative phase of liver regeneration [7].
Similarly, activin A made up of βA homodimers is an apoptogen of the TGFβ family that blocks hepatocyte mitogenesis. activin A shows diminished signalling during regeneration when its cellular receptor level is reduced, and the receptor level is restored at the termination of regeneration [8]. TGFβ and activin signalling are controlled at other levels by extracellular factors such as follistatin, as well as by intracellular signalling pathways. Follistatin administration can block activin signalling, accelerate liver regeneration and increase liver size [9]
Recent studies focused on distinguishing between the relative contribution of TGFβ1 and activin A (βA,) in regulating liver size and regeneration after partial hepatectomy [10]. Isolated hepatocytes from liver specific conditional knockouts of the TGFβ II receptor, demonstrate impaired growth inhibition mediated by TGFβ. These data support the importance of the Type II receptor in TGFβ signalling. Although TGFβ receptor II liver-knockout livers show accelerated regeneration after partial hepatectomy, the termination of liver regeneration is normal implying that other factors are important. In these mice introduction of follistatin to block activin A signalling resulted in increased DNA synthesis and reduction of Smad signalling indicating that activin A–βA plays a prominent role in the termination of liver regeneration.
New members of the activin family, activin βC and βE, were recently identified and characterized [11], [12]. Expression of activin subunits βC and βE is interesting in that their expression is limited largely to the liver of adult animals. Studies to try to identify the function of these proteins including gene knockout have shown that they are not required for normal liver development or regeneration [11]. During liver regeneration activin βE is induced and the levels of activin βC also increase in the early phases posthepatectomy implying that these activin subunits may have some regulatory role. Wada et al. showed that βC may play a positive role in the stimulation of DNA synthesis in isolated hepatocytes perhaps by blocking the growth inhibitory effect of activin A [12] and potentially by generating a direct positive mitogenic signal.
In the current manuscript [3] Wada et al. extend these studies to an in vivo model in which subunit activin βC is overexpressed in the liver using an Adenovirus vector. The livers are then subjected to partial hepatectomy, and the timing and rate of liver regeneration is assessed. A key to the approach is that the authors administered the adenovirus four days prior to the partial hepatectomy to allow the hepatocytes to incorporate and begin expressing activin βC. They demonstrate that 20% of the hepatocytes express βC at the time of surgery and 50% express βC 3 days after hepatectomy during which the major growth phase occurs. No significant difference is seen in the degree of injury in the activin βC or control LacZ transfected livers. As assessed by BrdU incorporation into DNA and rate of restitution of liver mass, liver regeneration is significantly accelerated in animals with the activin βC adenovirus. The authors demonstrate that a high level of BrdU incorporation is detected in hepatocytes neighbouring or expressing βC subunits implying that both autocrine and paracrine regulation of hepatocyte mitogenesis may be components of the βC mitogenic signal. Finally the authors demonstrate that livers with adeno βC express high levels of activin βC homodimers and βA -βC heterodimers. Control livers express high levels of subunits of activin βA. These findings allow the authors to speculate that βC may have an important role in limiting the negative regulation of hepatocyte proliferation mediated by activin A- βA (Fig. 1).
Large numbers of TGF superfamily ligands (more than 30) have been identified and only partially characterized. As indicated, multiple levels of regulation within the TGF superfamily have been characterized including both extracellular, intracellular and transcriptional modulators. Activin βC is a liver restricted member of the family that has not yet been thoroughly explored, and its expression occurs within a milieu of TGF family members that makes it difficult to sort out the contribution of any one factor. Thus, it is not surprising that there are some contradictory findings to those of Wada et al. as to the extent and nature of contribution of activin βC to liver regeneration. The authors point out some key differences between their results and those of others- the βC gene knockout paper that did not demonstrate a critical role for activin βC in liver regeneration [11] and another overexpression study where the findings suggest that activin βC could be a negative regulator of hepatocyte growth [3]. Overall the studies of Wada et al. are compelling that βC has a growth stimulatory role during liver regeneration when overexpressed and that this effect is mediated via inhibition of βA subunits of activin A. There is much supporting evidence to suggest that activin A is an important contributor to the termination of liver regeneration once the major proliferative response has occurred. It has not yet been demonstrated that activin βC expressed at the levels normally seen in liver regeneration play a key role in modulating the activity of activin A potentially in concert with other activins. The careful dissection of the complex pathways using tissue specific knockouts and overexpression studies will ultimately allow scientists to determine the contribution of the complex array of activating and inhibitory TGF superfamily signals.
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PII: S0168-8278(05)00499-X
doi:10.1016/j.jhep.2005.07.011
© 2005 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved.
