The liver displays a unique ability to grow and regenerate. After resection of two-thirds of the liver, complete hepatic regeneration can occur within days to weeks. However, chronic hepatocellular damage can impair the regulation of regeneration, leading to hepatocellular carcinoma, one of the most common malignancies in the world. Growth factors stimulate liver regeneration by activating receptor tyrosine kinases (RTKs), which in turn increase free Ca2+ within the cytosol and nucleus, although the relative roles of cytosolic and nuclear Ca2+ in the regulation of liver growth are unclear. We found that, in hepatocytes, inositol 1,4,5-trisphosphate (InsP3) mediates changes in both nuclear and cytosolic Ca2+. These findings led us to formulate the hypothesis that RTKs regulate cell growth by inducing InsP3-mediated Ca2+ signals within the nucleus. We are testing this hypothesis by pursuing the following specific aims with regard to the hepatocyte growth factor (HGF) receptor: (1) investigation of the mechanism by which the phosphorylated HGF receptor reaches the nucleus; (2) identification of the mechanism by which the nuclear HGF receptor generates InsP3 locally and hence nuclear Ca2+ signals; and (3) examination of the process by which nuclear Ca2+ regulates cell growth.
Activated RTKs can be found in the nucleus. HGF is a potent mitogen for hepatocytes during liver regeneration. The HGF receptor, also known as c-Met, is a prototypic RTK that can induce cell proliferation, differentiation, and related activities. Upon binding to HGF, the receptor becomes autophosphorylated at multiple intracellular tyrosine residues. In addition, several adapter proteins, including Gab, are recruited to form a signaling complex with the receptor. Gab1 is particularly relevant for Ca2+ signaling because it binds to and then facilitates activation of the phospholipase C (PLC) PLCγ1. The HGF receptor is important in hepatology in that its excessive activation is involved in the pathogenesis of certain liver diseases. Furthermore, receptor stimulation might be of therapeutic benefit in some circumstances. Our preliminary data provide evidence that, like the epidermal growth factor (EGF) and fibroblast growth factor receptors, the HGF receptor can also be found in the nucleus. Aim 1 of the project investigates whether and how the HGF receptor reaches the nucleus.
The nucleus contains the machinery for locally increasing Ca2+. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate InsP3, which then binds to the InsP3 receptor to release Ca2+ from internal stores. This is the only pathway for the formation of Ca2+ signals in liver cells, although PLC can be activated through either G protein-coupled receptors or RTKs. It is well established that components necessary for InsP3-mediated Ca2+ signaling are found in the plasma membrane, but there is evidence that these components are also present in the nuclear envelope. Although, under certain circumstances, Ca2+ can spread passively from the cytosol into the nucleus, intranuclear InsP3 can increase Ca2+ directly within the nucleus, both in isolated nuclei and in nuclei within intact cells. Moreover, RTKs may selectively activate nuclear isoforms of PLC, particularly PLCβ, and may induce PLCγ1 to translocate to the nucleus. Furthermore, we found that InsP3-gated Ca2+ stores are found not only within the nuclear envelope but also along a nucleoplasmic reticulum. No general paradigm has been established to explain whether, how, or when Ca2+ signals are initiated within the nucleus, although our preliminary data suggest that Ca2+ signals induced by HGF begin in the nucleus. Aim 2 of this proposal is to determine whether HGF directly increases InsP3 formation within the nucleus and, if so, to define the pathway by which it occurs.
Increases in Ca2+ within the nucleus have specific cellular effects. Nuclear Ca2+ signaling directly regulates cellular functions such as activation of kinases within the nucleus, protein transport across the nuclear envelope, and transcription of certain genes. For example, nuclear Ca2+ activates calmodulin kinase IV and induces translocation of intranuclear protein kinase C. Transcriptional activation of Elk-1 by EGF also depends on nuclear rather than cytosolic Ca2+. Moreover, Ca2+ can bind to and directly regulate certain nuclear transcription factors and can also affect DNA structure. Nuclear Ca2+ can also negatively regulate the activity of the transcription enhancer factor TEF/TEAD. Exogenous expression of the Ca2+-buffering protein parvalbumin has shown that intracellular Ca2+ regulates cell growth. A novel approach has been developed by which cells are transfected with Ca2+ chelators, such as parvalbumin, that are targeted to be expressed in either the nucleus or the cytosol. Aim 3 of this proposal will take advantage of this new technique to understand whether and how nuclear Ca2+ regulates cell growth.
Clinical relevance of Ca2+ signaling in the nucleus. Given that Ca2+ signaling in the nucleus may regulate the growth of hepatocytes, an increased understanding of this signaling pathway could lead to novel treatment strategies for liver failure. On the other hand, it would be helpful to inhibit rather than stimulate liver growth in clinical situations such as in hepatocellular carcinoma. The actions of growth factors on hepatocytes may play a role in the pathogenesis of this malignancy, and inhibition of growth factors may provide a novel and effective form of therapy. Furthermore, an increased understanding of Ca2+ signaling in the nucleus could also lead to novel treatment strategies to inhibit the development and growth of hepatocellular carcinoma.
Last updated August 2010
