Leptin promotes methionine adenosyltransferase 2A expression in hepatic stellate cells by the downregulation of E2F-4 via the ß-catenin pathway.

Most obese patients develop hyperleptinaemia. Leptin, mainly produced by adipocytes, demonstrates a promotional role in liver fibrosis. Hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis, requires global reprogramming of gene expression. The remodeling of DNA methylation is a mechanism of the epigenetic regulation of gene expression. The biosynthesis of S-adenosylmethionine, a principle biological methyl donor, is catalyzed by methionine adenosyltransferase (MAT) such as MATⅡ which has been shown to promote HSC activation in vitro. This study was mainly aimed to determine the effect of leptin on MAT2A expression (the catalytic subunit of MATⅡ) in HSCs. Results showed that MAT2A knockdown reduced leptin-induced HSC activation and liver fibrosis in the leptin-deficient mouse model. Leptin promoted MAT2A expression in HSCs and increased MAT2A promoter activity. The axis of the ß-catenin pathway/E2F-4 mediated the effect of leptin on MAT2A expression. Leptin-induced ß-catenin signaling reduced E2F-4 expression and thus abated E2F-4 binding to MAT2A promoter at a site around -2779 bp, leading to an increase in the MAT2A promoter activity. These data might shed more light on the mechanisms responsible for liver fibrogenesis in obese patients with hyperleptinaemia.

Targeting the RB-pathway in cancer therapy.

The RB-pathway, consisting of inhibitors and activators of cyclin-dependent kinases, the retinoblastoma tumor suppressor (RB), and the E2F-family of transcription factors, plays critical roles in the regulation of cell cycle progression and cell death. Components of this pathway, particularly p16Ink4a, cyclin D1, and RB, are frequently altered in sporadic human cancers to promote deregulated cellular proliferation. The consistent disruption of the RB-pathway in human cancers raises the possibility of exploiting tumor-specific RB-pathway defects to improve the efficacy of current therapies and to develop new therapeutic strategies. This article discusses how the RB-pathway status impacts the cellular responses to cytotoxic, cytostatic, and hormone therapies, and how the components of the RB-pathway may be directly targeted to treat cancer.

Cyclin D1/cdk4 can interact with E2F4/DP1 and disrupts its DNA-binding capacity.

The E2F family of transcription factors regulate the expression of many growth-related genes in a cell cycle-dependent manner. These transcription factors can activate or, in conjunction with an Rb-related protein, repress transcription. E2F transcriptional activity is regulated at several different levels that are each linked to cell cycle progression. In many cell types, E2F4 and E2F5 are the predominant E2F species during G(0) and early G(1) and function primarily as repressors of E2F-regulated genes. In this study, co-immunoprecipitation techniques were used to demonstrate that cyclins D1, D2, and D3 are capable of interacting with E2F4, E2F5, and DP1. Overexpression of cyclin D1/cdk4 reduced E2F4-mediated transcription in a simple reporter gene assay and electrophoretic mobility shift analyses using nuclear extracts from transfected cells indicated that cyclin D1/cdk4 disrupts the DNA-binding ability of E2F4. Cell cycle analysis following stimulation of serum-starved 3T3 cells indicated that E2F4 undergoes changes in its phosphorylation pattern coincident with the synthesis of cyclin D1. Examination of a series of E2F4 deletion mutants indicated that a cyclin D1-binding site located close to the carboxyl terminus of E2F4 was critical for the disruption of DNA binding by cyclin D1/cdk4. These data support a model in which E2F4 DNA binding is abolished during mid-G(1) at the same time when E2F interactions with pRb-related proteins are disrupted by cyclin D1/cdk4.

E2F-HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer.

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.