Search

Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191-270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity.

E2F transcription factors are key regulators of the cell cycle although the relative contribution of each E2F member in regulating cellular proliferation is still poorly defined. Present evidence suggests that E2F2 may act both as a suppressor and promoter of proliferation, depending on the cellular context. We used a loss-of-function mutant mouse model to investigate the function of E2F2 in liver regeneration after partial hepatectomy, a paradigm of cell-cycle progression. Liver mass recovery and histology were examined over 9 days in 70% hepatectomized E2F2(-/-) and wild-type animals. Transcriptome analysis was performed in quiescent and 48-h regenerating liver samples. TIGR MultiExperiment Viewer was used for the statistical analysis of microarray data, significance was determined by Fischer, and P values were adjusted applying Benjamini-Hochberg multiple-testing correction. We show that E2F2 is required for adult hepatocyte proliferation and for timely liver regeneration, as disruption of the E2F2 gene in hepatocytes leads to a reduced rate of S-phase entry and to delayed liver regeneration. Transcriptome analysis followed by ontological classification of differentially expressed genes and gene-interaction network analysis indicated that the majority of genes involved in normal liver regeneration were related to biosynthetic and catabolic processes of all major biomolecules as well as cellular location and intracellular transport, confirming the complex nature of the regeneration process. Remarkably, transcripts of genes included in functional categories that are crucial for cell cycle, apoptosis and wound-healing response, and fibrosis were absent in the transcriptome of posthepatectomized E2F2(-/-) mice. Our results indicate that the transcriptional activity of E2F2 contributes to promote adult hepatocyte proliferation and liver regeneration.

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified “lipid metabolism regulation” as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2+/+) and E2F2 deficient (E2F2−/−) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2+/+ and E2F2−/− mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2−/− mice resembles the phenotype of proliferating E2F2+/+ liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance. [ABSTRACT FROM AUTHOR]

Transcription of microRNAs (miRNAs) is thought to be regulated similarly to that of protein-coding genes. However, how miRNAs are regulated during the cell division cycle is not well understood. We have analyzed the transcription profiles of miRNAs in response to mitogenic stimulation in primary fibroblasts. About 33% of the miRNAs expressed in these cells are induced upon exit from quiescence. Many of these miRNAs are specifically induced by E2F1 or E2F3 during the G1/S transition and are repressed in E2F1/3-knockout cells. At least four miRNA clusters, let-7a-d, let-7i, mir-15b-16-2, and mir-106b-25, are direct targets of E2F1 and E2F3 during G1/S and are repressed in E2F1/3-null cells. Interestingly, these miRNAs do not contribute to E2F-dependent entry into S phase but rather inhibit the G1/S transition by targeting multiple cell cycle regulators and E2F targets. In fact, E2F1 expression results in a significant increase in S-phase entry and DNA damage in the absence of these microRNAs. Thus, E2F-induced miRNAs contribute to limiting the cellular responses to E2F activation, thus preventing replicative stress. Given the known function of E2F of inducing other oncogenic miRNAs, control of miRNAs by E2F is likely to play multiple roles in cell proliferation and in proliferative diseases such as cancer. [ABSTRACT FROM AUTHOR]