Peirong Lu

Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines.

Most cases of human hepatocellular carcinoma develop after persistent chronic infection with human hepatitis B virus or hepatitis C virus, and host responses are presumed to have major roles in this process. To recapitulate this process, we have developed the mouse model of hepatocellular carcinoma using hepatitis B virus surface antigen transgenic mice. To identify the genes associated with hepatocarcinogenesis in this model, we compared the gene expression patterns between pre‐malignant lesions surrounded by hepatocellular carcinoma tissues and control liver tissues by using a fluorescent differential display analysis. Among the genes that were expressed differentially in the pre‐malignant lesions, we focused on Pim‐3, a member of a proto‐oncogene Pim family, because its contribution to hepatocarcinogenesis remains unknown. Moreover, the unavailability of the nucleotide sequence of full‐length human Pim‐3 cDNA prompted us to clone it from the cDNA library constructed from a human hepatoma cell line, HepG2. The obtained 2,392 bp human Pim‐3 cDNA encodes a predicted open reading frame consisting of 326 amino acids. Pim‐3 mRNA was selectively expressed in human hepatoma cell lines, but not in normal liver tissues. Moreover, Pim‐3 protein was detected in human hepatocellular carcinoma tissues and cell lines but not in normal hepatocytes. Furthermore, cell proliferation was attenuated and apoptosis was enhanced in human hepatoma cell lines by the ablation of Pim‐3 gene with RNA interference. These observations suggest that aberrantly expressed Pim‐3 can cause autonomous cell proliferation or prevent apoptosis in hepatoma cell lines.

The pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen‐induced liver injury in mice

Acetaminophen (APAP) causes a massive production of intrahepatic tumor necrosis factor α (TNF‐α). However, it still remains elusive regarding the roles of TNF‐α in APAP‐induced liver injury. Hence, we examined pathogenic roles of the TNF‐α–TNF receptor with a molecular weight of 55 kDa (TNF‐Rp55) axis in APAP‐induced hepatotoxicity using TNF‐Rp55‐deficient [TNF‐Rp55‐knockout (KO)] mice. When wild‐type (WT) BALB/c and TNF‐Rp55‐KO mice were intraperitoneally injected with a lethal dose of APAP (750 mg/kg), the mortality of TNF‐Rp55‐KO mice was marginally but significantly reduced compared with WT mice. Upon treatment with a nonlethal dose (600 mg/kg), WT mice exhibited an increase in serum transaminase levels. Histopathologically, centrilobular hepatic necrosis with leukocyte infiltration was evident at 10 and 24 h after APAP challenge. Moreover, mRNA expression of adhesion molecules, several chemokines, interferon‐γ (IFN‐γ), and inducible nitric oxide synthase (iNOS) was enhanced in the liver. On the contrary, serum transaminase elevation and histopathological changes were attenuated in TNF‐Rp55‐KO mice injected with APAP (600 mg/kg). The gene expression of all molecules except for IFN‐γ and iNOS was significantly attenuated in TNF‐Rp55‐KO mice. Moreover, anti‐TNF‐α neutralizing antibodies alleviated liver injury when administered at 2 or 8 h after but not at 1 h before APAP challenge to WT mice. Collectively, the TNF‐α–TNF‐Rp55 axis has pathogenic roles in APAP‐induced liver failure.

Essential contribution of a chemokine, CCL3, and its receptor, CCR1, to hepatocellular carcinoma progression

We previously observed that a chemokine, macrophage inflammatory protein‐1 α/CCL3, and its receptor, CCR1, were aberrantly expressed in human hepatocellular carcinoma (HCC) tissues. Here, we show that CCL3 and CCR1 are also expressed in 2 different models of this cancer; N‐nitrosodiethylamine (DEN)‐induced HCC and HCC induced by hepatitis B virus surface (HBs) antigen‐primed splenocyte transfer to myelo‐ablated syngeneic HBs antigen transgenic mice. At 10 months after DEN treatment, foci number and sizes were remarkably reduced in CCR1‐ and CCL3‐deficient mice, compared with those of wild‐type (WT) mice, although tumor incidence were marginally, but significantly, higher in CCR1‐ and CCL3‐deficient mice than in WT mice. Of note is that tumor angiogenesis was also markedly diminished in CCL3‐ and CCR1‐deficient mice, with a concomitant reduction in the number of intratumoral Kupffer cells, a rich source of growth factors and matrix metalloproteinases (MMPs). Among growth factors and MMPs that we examined, only MMP9 and MMP13 gene expression was augmented progressively in liver of WT mice after DEN treatment. Moreover, MMP9, but not MMP13, gene expression was attenuated in CCR1‐ and CCL3‐deficient mice, compared with that of WT mice. Furthermore, MMP9 was expressed mainly by mononuclear cells but not hepatoma cells, and MMP9‐expressing cell numbers were decreased in CCR1‐ or CCL3‐deficient mice, compared with WT mice. These observations suggest the contribution of the CCR1‐CCL3 axis to HCC progression.

Potential interaction between CCR1 and its ligand, CCL3, induced by endogenously produced interleukin-1 in human hepatomas

Hepatoma cell lines can produce a massive amount of chemokines in response to various stimuli including hepatitis viruses and their products. However, it remains elusive on the types of chemokine receptor(s) expressed in the hepatoma tissues and its roles in hepatoma development. To clarify these points, we examined the chemokine receptor expression in six human hepatoma cell lines. All of the hepatoma cell lines constitutively and exclusively expressed CCR1 mRNA and its protein on their cell surface. CCR1 expression was also detected on hepatoma cells and to a lesser degree, on endothelial cells in hepatoma tissues but not in normal liver tissues. Furthermore, CCL3 expression was detected in hepatoma cells, endothelial cells, and to a lesser degree, fibroblast-like cells in hepatoma tissue, whereas only occasional vascular endothelial cells and inflammatory cells in normal liver tissues were weakly positive for CCL3. Moreover, the forskolin-mediated increases in intracellular cAMP concentrations were inhibited by the ligands for CCR1, CCL3, CCL4, and CCL5, suggesting that the expressed CCR1 was functional. Four hepatoma cell lines produced CCL3 only in response to interleukin (IL)-1 alpha and IL-1 beta. Finally, IL-1 alpha and IL-1 beta were detected abundantly in hepatoma tissues but not in normal liver tissues. Thus, IL-1 may enhance the local production of CCL3, which may interact with CCR1 expressed on hepatoma cells, in an autocrine and/or paracrine manner.

Attenuated liver tumor formation in the absence of CCR2 with a concomitant reduction in the accumulation of hepatic stellate cells, macrophages and neovascularization

The liver parenchyma is populated by hepatocytes and several nonparenchymal cell types, including Kupffer cells and hepatic stellate cells. Both Kupffer cells and hepatic stellate cells are responsive to the chemokine CCL2, but the precise roles of CCL2 and these cells in liver tumor formation remain undefined. Hence, we investigated the effects of the lack of the major CCL2 receptor, CCR2, on liver tumor formation induced by intraportal injection of the murine colon adenocarcinoma cell line, colon 26. Wild‐type mice showed macroscopic tumor foci in the liver 10 days after injection of colon 26 cells. After 10 days, CCL2 proteins were detected predominantly in tumor cells, coincident with increased intratumoral macrophage and hepatic stellate cell numbers. Although tumor formation occurred at similar rates in wild‐type and CCR2‐deficient mice up to 10 days after tumor cell injection, the number and size of tumor foci were significantly attenuated in CCR2‐deficient mice relative to wild‐type mice thereafter. Moreover, neovascularization and matrix metalloproteinase 2 expression were diminished in CCR2‐deficient mice with a concomitant reduction in the accumulation of macrophages and hepatic stellate cells. Furthermore, matrix metalloproteinase 2 was detected predominantly in hepatic stellate cells but not in macrophages. We provided the first definitive evidence that the absence of CCR2‐mediated signals can reduce the trafficking of hepatic stellate cells, a main source of matrix metalloproteinase 2, and consequently can diminish neovascularization during liver tumor formation.

Critical Role of TNF-α-Induced Macrophage VEGF and iNOS Production in the Experimental Corneal Neovascularization

PURPOSE We evaluated the roles of tumor necrosis factor (TNF)-α in alkali-induced corneal neovascularization (CNV). METHODS CNV was induced by alkali injury and compared in wild-type (WT) BALB/c mice, and TNF receptor 1-deficient (TNF-Rp55 KO) counterparts, or in mice treated with TNF-α antagonist and recombinant TNF-α. Angiogenic factor expression and leukocyte accumulation in the early phase after injury were quantified by real-time PCR and immunohistochemical analysis, respectively. RESULTS Alkali injury augmented the intraocular mRNA expression of TNF-α and its receptor, together with a transient macrophage and neutrophil infiltration. Compared to WT mice, TNF-Rp55 KO mice exhibited reduced CNV. Intraocular F4/80-positive macrophages and Ly-6G-positive neutrophils infiltration did not change in KO mice compared to WT mice after the injury. Alkali injury induced a massively increased intraocular mRNA expression of angiogenic factors, including vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, E-selectin, and intercellular adhesion molecule (ICAM)-1 in WT mice, whereas these increments were retarded severely in KO mice. Immunofluorescence analysis demonstrated that F4/80-positive cells expressed VEGF and iNOS. Moreover, TNF-α enhanced VEGF and iNOS expression by peritoneal macrophage from WT, but not KO mice. Topical application of TNF-α antagonist reduced CNV, while topical application of recombinant TNF-α enhanced it. CONCLUSIONS TNF-Rp55-KO mice exhibited impaired alkali-induced CNV through reduced intracorneal infiltrating macrophage VEGF and iNOS expression.