An MLCK-dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK-p70S6K pathway.

We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant-negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2-dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK-dependent window in late G1 phase of regenerating liver after two-thirds partial hepatectomy. In conclusion, our results underline an MLCK-dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation.

Strain and gender modulate hepatic hepcidin 1 and 2 mRNA expression in mice.

Hepcidin (HEPC) plays a key role in iron homeostasis and an abnormally low level of hepcidin mRNA has been reported in HFE-1 genetic hemochromatosis. Considering the well-known phenotypic variability of this disease, especially between men and women, it is important to define factors susceptible to modulate hepatic hepcidin expression and, consequently, to influence the development of iron overload in HFE-1 hemochromatosis. Therefore, our aim was to analyze the effects of strain and gender on hepatic hepcidin expression in the mouse. C57BL/6 and DBA/2 wild-type mice were included in this study. Liver and splenic iron contents were measured. Specific hepatic Hepc1 and Hepc2 mRNA levels were quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). C57BL/6 mice expressed predominantly Hepc1 mRNA, whereas Hepc2 mRNA was the main form in DBA/2 mice. In both strains, females had higher levels of iron stores and Hepc mRNAs compared to males. Our results demonstrate that the expression of both hepcidin mRNAs varies according to strain and gender. They suggest that sex and genetic background, which are regulators of hepcidin expression, could play a role in the phenotypic expression of genetic hemochromatosis.

Comparative analysis of mouse hepcidin 1 and 2 genes: evidence for different patterns of expression and co-inducibility during iron overload.

In contrast to the human genome, the mouse genome contains two HEPC genes encoding hepcidin, a key regulator of iron homeostasis. Here we report a comparative analysis of sequence, genomic structure, expression and iron regulation of mouse HEPC genes. The predicted processed 25 amino acid hepcidin 2 peptide share 68% identity with hepcidin 1 with perfect conservation of eight cysteine residues. Both HEPC1 and HEPC2 genes have similar genomic organization and have probably arisen from a recent duplication of chromosome 7 region, including the HEPC ancestral gene and a part of the adjacent USF2 gene. Insertion of a retroviral intracisternal A-particle element was found upstream of the HEPC1 gene. Both genes are highly expressed in the liver and to a much lesser extent in the heart. In contrast to HEPC1, a high amount of HEPC2 transcripts was detected in the pancreas. Expression of both genes was increased in the liver during carbonyl-iron and iron-dextran overload. Overall our data suggest that both HEPC1 and HEPC2 genes are involved in iron metabolism regulation but could exhibit different activities and/or play distinct roles.

Regulation and role of p21 and p27 cyclin-dependent kinase inhibitors during hepatocyte differentiation and growth.

Unlike a large number of cell types that undergo terminal differentiation associated with permanent withdrawal from the cell cycle, mature quiescent hepatocytes retain high proliferative potential. We report here a specific behavior of members of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors during development of the rat liver and proliferation of normal hepatocytes. Expression of p21, p27, and p57 transcripts and proteins was downregulated during the differentiation process to low or undetectable levels in adult liver. In contrast to p27, p21 protein increased in a mitogen-dependent manner in isolated hepatocytes and its expression pattern correlated with that of cyclin D1. In proliferating hepatocytes, p21 was predominantly associated with cyclin D1, these proteins were colocalized in the nucleus and p21-associated retinoblastoma protein (pRb) kinase activity increased in parallel with that of cyclin D1. Overexpression of p21 in mitogen-stimulated hepatocytes reduced DNA synthesis. In contrast, inhibition of p21 expression by antisense or small interfering RNAs oligonucleotides accelerated S phase entry. Finally, expression of p21 and cyclin D1, but not p27 proteins was regulated by MAPK kinase/extracellular signal-regulated kinase and phosphatidylinositol 3-kinase-ferric-reducing ability power/mammalian target of rapamycin signal transduction pathways. In conclusion, these results demonstrate a specific and differential regulation of p21 and p27 during hepatocyte differentiation and proliferation that may contribute to the control of quiescent differentiated hepatic cell proliferating activity.

A new subfamily of structurally related human F-box proteins.

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.

C/EBPalpha regulates hepatic transcription of hepcidin, an antimicrobial peptide and regulator of iron metabolism. Cross-talk between C/EBP pathway and iron metabolism.

Originally identified as a gene up-regulated by iron overload in mouse liver, the HEPC gene encodes hepcidin, the first mammalian liver-specific antimicrobial peptide and potential key regulator of iron metabolism. Here we demonstrate that during rat liver development, amounts of HEPC transcripts were very low in fetal liver, strongly and transiently increased shortly after birth, and reappeared in adult liver. To gain insight into mechanisms that regulate hepatic expression of hepcidin, 5'-flanking regions of human and mouse HEPC genes were isolated and analyzed by functional and DNA binding assays. Human and mouse HEPC promoter-luciferase reporter vectors exhibited strong basal activity in hepatoma HuH-7 and mouse hepatocytes, respectively, but not in non-hepatic U-2OS cells. We found that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPbeta were respectively very potent and weak activators of both human and mouse promoters. In contrast, co-expression of hepatocyte nuclear factor 4alpha (HNF4alpha) failed to induce HEPC promoter activity. By electrophoretic mobility shift assay we demonstrated that one putative C/EBP element found in the human HEPC promoter (-250/-230) predominantly bound C/EBPalpha from rat liver nuclear extracts. Hepatic deletion of the C/EBPalpha gene resulted in reduced expression of HEPC transcripts in mouse liver. In contrast, amounts of HEPC transcripts increased in liver-specific HNF4alpha-null mice. Decrease of hepcidin mRNA in mice lacking hepatic C/EBPalpha was accompanied by iron accumulation in periportal hepatocytes. Finally, iron overload led to a significant increase of C/EBPalpha protein and HEPC transcripts in mouse liver. Taken together, these data demonstrate that C/EBPalpha is likely to be a key regulator of HEPC gene transcription and provide a novel mechanism for cross-talk between the C/EBP pathway and iron metabolism.