Description of Research Expertise

Research Interests
cell proliferation, differentiation and injury in the mammalian liver

Key words: liver, cell cycle, apoptosis, cancer.

Description of Research
The main goal of the Greenbaum lab is to understand the molecular mechanisms that govern cell proliferation, differentiation and mechanisms of injury and repair in the mammalian liver. These processes are of vital importance to human health given the recent rise in hepatocellular carcinoma.

We are investigating the molecular pathways responsible for hepatocyte growth and proliferation using the partial hepatectomy model in mouse models. Understanding the mechanisms that regulate the cell cycle in normal hepatocytes in response to growth, injury and metabolic signals will be applicable to understanding abnormal regulation of these pathways in hepatocellular carcinoma and in patients with inadequate liver recovery associated with fulminant liver failure. In addition to standard molecular analyses, we also utilize high throughput genomics and computational approaches.

1. Regulation of DNA replication licensing proteins by C/EBPβ

Proliferation in the liver, which is normally characterized by very low cell turnover, can be dramatically stimulated in the partial hepatectomy model. After surgical removal of 2/3 of the liver, the remaining hepatocytes exit G0 and synchronously enter the cell cycle in order to restore liver mass. We have investigated the role of the E2F transcription factors during this process, as these factors have been shown to control the expression of a large number of genes involved in DNA replication and cell cycle progression. Liver cell proliferation after partial hepatectomy is one of the best mammalian in vivo models of synchronized exit from G0 and entry into the cell cycle. One of the fundamental questions in the field of cell cycle reguation is “How is biological specificity within the E2F family achieved?” The bZIP transcription factor CCAAT enhancer binding protein beta (C/EBPβ) is phosphorylated in response to activation of metabolic, cytokine and growth factor pathways linked to cell growth, making it a likely candidate for participating in the regulation of cell cycle associated genes in the liver. We have identified a group of E2F-regulated genes including several that are involved in licensing origins of replication that are markedly reduced in C/EBPβnull mice. These findings identify C/EBPβ as a direct activator of E2F promoters and cell cycle progression in the mammalian cell cycle. We have recently established that C/EBPβ and E2Fs synergistically activate the promoter of one of these licensing proteins, CDC6. Current studies are focused on defining the mechanism through which this synergistic activation occurs and are investigating the contribution of posttranslational modifications to the C/EBPβ protein downstream of various signaling pathways that modulate C/EBPβ activation of cell cycle gene expression and hepatocyte proliferation.


2. Coactivator protein regulation of liver regeneration
Transcriptional coactivator proteins are recruited to target gene promoters in response to cellular signals and mediate the adaptation to changes in tissue homeostasis. We have recently made the novel observation that the PGC-1alpha coactivator, a critical molecule for gluconeogenesis during fasting, is dramatically induced in the regenerating liver. Current studies are focused on identification of the signaling pathways that regulate its expression and function during liver regeneration.

3. MicroRNA regulation of hepatocyte proliferation
MicroRNAs are small noncoding RNA molecules that inhibit gene expression via degradation of target mRNAs, inhibition of protein translation or chromatin silencing. These molecules have been implicated in a variety of cellular functions including proliferation, apoptosis and metabolism and dysregulation of these molecules has been linked to carcinogenesis. We are currently investigating the contribution of microRNAs to hepatocyte proliferation in the regenerating liver.


Rotation Projects for 2007-2008: (subject to change)

Analysis of the mechanism of E2F target gene regulation by C/EBPβ.
1. Identification of C/EBPβ protein domains that mediate binding to E2F proteins and effect synergistic activation of cell cycle genes using mutagenesis and co-immunoprecipitation.
2. Analysis of signaling pathways that contribute to C/EBPβ regulation of hepatocyte proliferation. Construction of adenoviral vectors expressing mutated C/EBPβ molecules will be assessed for their ability to rescue proliferation and E2F target gene expression in primary mouse hepatocytes.


Investigation of microRNAs for hepatocyte proliferation
Northern blot analysis will be used to identify differentially expressed microRNAs in the regenerating liver. Antisense constructs targeted to candidate microRNAs will be injected via tail vein to determine the function of these microRNAs during liver regeneration.


Lab personnel:
Yan Gao, Postdoctoral researcher
Jenny Yan, Research Specialist
Akivaga Tsingalia, Research Specialist
Sarah Muse, Graduate student in the CAMB program