Chang-Yan Li

Nuclear factor p65 interacts with Keap1 to repress the Nrf2-ARE pathway.

Keap1 is an inhibitor of Nrf2 involved in Nrf2-dependent antioxidant response. However, the mechanisms on how Keap1 regulates Nrf2-ARE signaling pathway remains to be determined. Here, by using a yeast two-hybrid technology, p65 subunit of NF-κB transcription factor was identified as a partner of Keap1. We show that Keap1 physically associated with p65 in vivo and in vitro. Overexpression of p65 inhibited Nrf2-dependent transcription induced by diethylmaleate (DEM) or tert-butyl hydroxyquinone (tBHQ). Knock down of Keap1 by RNA interference partially blocked the repression of Nrf2-mediated activation by p65. It was demonstrated that p65 decreased Nrf2 binding to its cognate DNA sequences and enhanced Nrf2 ubiquitination. The N-terminal region of p65 is necessary for both the interaction with Keap1 and its transcriptional suppression activity. Moreover, nuclear translocation of Keap1 was augmented by p65. Taken together, our findings suggest that NF-κB signaling inhibits Nrf2-ARE pathway through the interaction of p65 and Keap1.

PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells.

BackgroundPCBP1 (or alpha CP1 or hnRNP E1), a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive.ResultsWe found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues.ConclusionsWe first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress

To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G(2)-M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.

Specific Expression and Regulation of Hepassocin in the Liver and Down-regulation of the Correlation of HNF1α with Decreased Levels of Hepassocin in Human Hepatocellular Carcinoma

Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1α (hepatocyte nuclear factor-1α) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1α. An electrophoretic mobility shift assay demonstrated that HNF1α interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1α interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1α expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1α in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1α expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1α are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1α causes, at least in part, the transcriptional down-regulation of HPS in HCC.

Recombinant human Hepassocin stimulates proliferation of hepatocytes in vivo and Improves Survival in Rats with Fulminant Hepatic Failure

Background Human hepassocin (HPS) was originally detected by subtractive and differential cDNA cloning as a liver-specific gene that was markedly upregulated during liver regeneration. Previous studies suggested that HPS showed mitogenic activity on isolated hepatocytes in vitro. However, its in vivo functions remained largely unknown. Therefore, the function of recombinant human HPS during liver regeneration and chemically induced liver injury was investigated. Methods The proliferation of primary hepatocytes was examined by [3H]thymidine incorporation and immunohistological staining of proliferating cell nuclear antigen (PCNA). RNA interference was performed to knock down the endogenous expression of HPS. The proliferation of L02 cells was examined by MTS assay. The phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2) was investigated by western blotting analysis. Assessment of liver injury (histology, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels) and of apoptosis, by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay, was performed. Results Purified recombinant human HPS showed specific mitogenic activity on primary hepatocytes and normal liver cell lines in a mitogen-activated protein kinase (MAPK)-dependent manner and stimulated the proliferation of hepatocytes in rats with 70% partial hepatectomy. Administration of HPS to rats after d-galactose and carbon tetrachloride (CCl4) treatment protected against liver injury (minimal liver necrosis, depressed ALT and AST levels, and decreased lethality), reduced apoptosis and enhanced proliferation. Knock-down of endogenous HPS in vivo enhanced the liver injury induced by d-galactose by increasing the apoptosis and elevating ALT and AST levels. Conclusions HPS is a hepatic growth factor which can accelerate hepatocyte proliferation in vivo and protect against liver injury. These data point to the potential interest of HPS in the treatment of fulminant hepatic failure.

Specific expression and regulation of the hepassocin in liver and down-regulation of HNF1α's correlation with decreased levels of hepassocin in human hepatocellular carcinoma

Hepassocin (HPS), is a liver-specific gene with mitogenic activity on isolated hepatocytes. It is up-regulated following partial hepatectomy and down-regulated frequently in heptocellular carcinoma (HCC). However, very little is known about the HPS transcription regulation mechanism. In this study, we identified HNF1α (hepatocyte nuclear factor-1α) as an important liver-specific cis-acting element for HPS using in vivo luciferase assays. Deletion of the HNF1 binding site not only led to a complete loss of HPS promoter activity in vivo but also abolished the induction of the HPS promoter by HNF1α. An electrophoretic mobility shift assay demonstrated that HNF1α interacted with the HPS gene promoter in vitro. Chromatin immunoprecipitation showed that HNF1α interacted with HMGB1 and CREB-binding protein, and all of them were recruited to the HPS promoter in vivo. Moreover, HNF1α expression was lower in HCC cell lines and tissues and correlated significantly with the down-regulation of HPS expression. Re-expression of HNF1α in human hepatoma HepG2 cells reinduced HPS expression. In contrast, knockdown of endogenous HNF1α expression by small interfering RNA resulted in a significant reduction of HPS expression. Furthermore, we found that partial hepatectomy and IL-6 significantly induced promoter activity of HPS, depending on STAT3 and HNF1 binding sites in the HPS promoter. These results demonstrate that the HNF1 binding site and HNF1α are critical to liver-specific expression of HPS, and down-regulation or loss of HNF1α causes, at least in part, the transcriptional down-regulation of HPS in HCC.

THAP11, a novel binding protein of PCBP1, negatively regulates CD44 alternative splicing and cell invasion in a human hepatoma cell line

THAP11 physically interacts with PCBP1 by anti bait coimmunoprecipitation (View interaction) THAP11 physically interacts with PCBP1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) THAP11 and PCBP1 colocalize by fluorescence microscopy (View interaction)

ABRO1 suppresses tumourigenesis and regulates the DNA damage response by stabilizing p53

Abraxas brother 1 (ABRO1) has been reported to be a component of the BRISC complex, a multiprotein complex that specifically cleaves ‘Lys-63’-linked ubiquitin. However, current knowledge of the functions of ABRO1 is limited. Here we report that ABRO1 is frequently downregulated in human liver, kidney, breast and thyroid gland tumour tissues. Depletion of ABRO1 in cancer cells reduces p53 levels and enhances clone formation and cellular transformation. Conversely, overexpression of ABRO1 suppresses cell proliferation and tumour formation in a p53-dependent manner. We further show that ABRO1 stabilizes p53 by facilitating the interaction of p53 with USP7. DNA-damage induced accumulation of endogenous ABRO1 as well as translocation of ABRO1 to the nucleus, and the induction of p53 by DNA damage is almost completely attenuated by ABRO1 depletion. Our study shows that ABRO1 is a novel p53 regulator that plays an important role in tumour suppression and the DNA damage response.

Hepassocin regulates cell proliferation of the human hepatic cells L02 and hepatocarcinoma cells through different mechanisms

Hepassocin (HPS) is a specific mitogenic active factor for hepatocytes, and inhibits growth by overexpression in hepatocellular carcinoma (HCC) cells. However, the mechanism of HPS regulation on growth of liver‐derived cells still remains largely unknown. In this study, we found that HPS was expressed and secreted into the extracellular medium in cultured L02 human hepatic cells; conditional medium of L02 cells promoted proliferation of L02 cells and this activity could be blocked by anti‐HPS antibody. Moreover, we identified the presence of receptor for HPS on L02 cells and HepG2 human hepatoma cells. Overproduction of truncated HPS, which signal peptide was deleted, significantly inhibited the proliferation of HCC cells and induced cell cycle arrest. These findings suggest that HPS promotes hepatic cell line L02 cells proliferation via an autocrine mechanism and inhibits HCC cells proliferation by an intracrine pathway. J. Cell. Biochem. 112: 2882–2890, 2011.

EDAG Positively Regulates Erythroid Differentiation and Modifies GATA1 Acetylation Through Recruiting p300

Erythroid differentiation‐associated gene (EDAG) has been considered to be a transcriptional regulator that controls hematopoietic cell differentiation, proliferation, and apoptosis. The role of EDAG in erythroid differentiation of primary erythroid progenitor cells and in vivo remains unknown. In this study, we found that EDAG is highly expressed in CMPs and MEPs and upregulated during the erythroid differentiation of CD34+ cells following erythropoietin (EPO) treatment. Overexpression of EDAG induced erythroid differentiation of CD34+ cells in vitro and in vivo using immunodeficient mice. Conversely, EDAG knockdown reduced erythroid differentiation in EPO‐treated CD34+ cells. Detailed mechanistic analysis suggested that EDAG forms complex with GATA1 and p300 and increases GATA1 acetylation and transcriptional activity by facilitating the interaction between GATA1 and p300. EDAG deletion mutants lacking the binding domain with GATA1 or p300 failed to enhance erythroid differentiation, suggesting that EDAG regulates erythroid differentiation partly through forming EDAG/GATA1/p300 complex. In the presence of the specific inhibitor of p300 acetyltransferase activity, C646, EDAG was unable to accelerate erythroid differentiation, indicating an involvement of p300 acetyltransferase activity in EDAG‐induced erythroid differentiation. ChIP‐PCR experiments confirmed that GATA1 and EDAG co‐occupy GATA1‐targeted genes in primary erythroid cells and in vivo. ChIP‐seq was further performed to examine the global occupancy of EDAG during erythroid differentiation and a total of 7,133 enrichment peaks corresponding to 3,847 genes were identified. Merging EDAG ChIP‐Seq and GATA1 ChIP‐Seq datasets revealed that 782 genes overlapped. Microarray analysis suggested that EDAG knockdown selectively inhibits GATA1‐activated target genes. These data provide novel insights into EDAG in regulation of erythroid differentiation. Stem Cells 2014;32:2278–2289