Abstract
The early growth response-1 transcription factor (Egr-1) is induced as part of the immediate-early gene expression response during early liver regeneration. In the studies reported here the functional significance of EGR-1 expression during liver regeneration was examined by characterizing the hepatic regenerative response to partial hepatectomy in Egr-1 null mice. The results of these studies showed that liver regeneration in Egr-1 null mice is impaired. Although activation of interleukin-6-STAT3 signaling, regulation of expression of hepatic C/ebpalpha, C/ebpbeta, cyclin D, and cyclin E and progression through the first wave of hepatocellular DNA synthesis occurred appropriately following partial hepatectomy in Egr-1 null mice, subsequent signaling events and cell cycle progression after the first round of DNA synthesis were deranged. This derangement was characterized by increased activation of the p38 mitogen-activated protein kinase and inhibition of hepatocellular metaphase-to-anaphase mitotic progression. Together these observations suggest that EGR-1 is an important regulator of hepatocellular mitotic progression. In support of this, microarray-based gene expression analysis showed that induction of expression of the cell division cycle 20 gene (Cdc20), a key regulator of the mitotic anaphase-promoting complex, is significantly reduced in Egr-1 null mice. Taken together these data define a novel functional role for EGR-1 in regulating hepatocellular mitotic progression through the spindle assembly checkpoint during liver regeneration.
Highlights
The functional role of EGR-1 in regulating liver regeneration was first suggested by studies demonstrating that Egr-1 mRNA expression is increased during hepatocellular G0-G1 progression as part of the immediate-early gene response of the regenerating liver (17). This pattern of hepatic Egr-1 expression following partial hepatectomy is homologous to that seen in a number of other models of cellular proliferation (17–20)
EGR-1 might be predicted to regulate G1-S phase progression during liver regeneration, as has been shown for Crem[1], another immediate-early gene induced in the regenerating liver (16)
The analyses of liver regeneration in Egr-1 null mice reported here show that the early signaling events leading up to re-entry of quiescent hepatocytes into the cell cycle and progression through the first wave of DNA synthesis occur normally, but hepatocellular cell cycle progression after that is markedly impaired in these animals
Summary
Animal Husbandry—Egr-1 null mice were derived from heterozygous (Egr-1 ϩ/Ϫ) mating pairs on a C57Bl/6 background (provided by Dr Jeffrey Milbrandt, Washington University School of Medicine). Real Time RT-PCR-based Gene Expression Analysis—The mRNA expression of C/ebp␣, C/ebp, cyclin D, cyclin E, Egr-1, Prl-1, and Cdc[20] was evaluated by using real time RT-PCR For these studies, total mRNA, prepared as described above from wild type and Egr-1 null mouse liver harvested at serial time points after partial hepatectomy, was reverse-transcribed to cDNA by the SuperScript Choice System (Invitrogen). Quantification of cDNA was based on monitoring increased SYBR fluorescence during exponential phase amplification in a real time PCR Machine (Bio-Rad) and determination of the PCR cycle number at which the amplified product exceeded a defined threshold (the “crossing threshold”) These data were standardized to the expression of 2-microglobulin, a constitutively expressed transcript commonly used as a reference mRNA in analyses of gene expression during hepatic regeneration (15), and the standardized data were used to calculate fold differences in gene expression (User Bulletin 2, ABI Prism 7700 Sequence Detection System, Applied Biosystems, Foster City, CA). Results are reported as means Ϯ S.E. p Ͻ 0.05 was used to determine statistical significance
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