Tight junction proteins and signal transduction pathways in hepatocytes.

Tight junctions of hepatocytes play crucial roles in the barrier to keep bile in bile canaliculi away from the blood circulation, which we call the blood-billiary-barrier (Kojima et al., 2003). Tight junction proteins of hepatocytes are regulated by various cytokines and growth factors via distinct signal transduction pathways. They are also considered to participate in signal transduction pathways that regulate epithelial cell proliferation, gene expression, differentiation and morphogenesis. This review focuses on recent findings about the relationship between tight junction proteins and signal transduction pathways in hepatocytes.

[Expression, purification and polyclonal antibody preparation for a novel gene BC097361].

OBJECTIVE: To express and purify of the BC097361 recombinant protein, and to prepare the BC097361 specific rabbit polyclonal antibody. METHODS: BC097361 cDNA was ligated into the prokaryotic expressive vector pET-32a (+), and the resulting plasmid was transformed into E.coli BL21 (DE3). The protein expression was induced with IPTG and the protein was analyzed with SDS-PAGE and western blotting. The expressed product was purified using Ni+ affinity column chromatography.Then the purified pET-32a (+) -BC097361 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and potency of polyclonal antibody were evaluated by Western blot and ELISA. RESULTS: The BC097361 fusion protein was highly expressed.The protein was mainly in the inclusion body. ELISA indicated the titer of polyclonal antibody more than 1:320000. The high specificity was confirmed with Western blot. CONCLUSIONS: The recombinant BC097361 fusion protein and the BC097361 specific polyclonal antibody will be valuable tools for the investigation on the biological function of BC097361.

[Expression of intrahepatic CD4+ CD25+ regulatory T cells in patients with chronic hepatitis B].

OBJECTIVE: To investigate the expression and distribution of intrahepatic CD4+ CD25+ regulatory T cells in immuno-tolerant and immuno-clearance phase of patients with chronic hepatitis B. METHODS: The expression of FoxP3 was detected in 19 cases of immuno-tolerant phase and 12 cases of immuno-clearance phase by immunohistochemistry. The relation between the intrahepatic expression of FoxP3 and the clinicopathological features were analyzed. RESULTS: The positive signal of FoxP3 is located in nuclear of lymphocyte and mainly aggregated in portal areas as well as occasionally scattered in hepatic sinusoids. The expression of intrahepatic FoxP3 in the group of immuno-tolerant phase was significantly increased than those in normal control (P < 0.01), and greatly decreased than those in immuno-clearance phase (P < 0.01). No correlation was observed among the expression of intrahepatic FoxP3, ALT, levels of HBV DNA, HBeAg positive, in patients of immuno-clearance phase, respectively. There were significant differences between immuno-tolerant phase and immuno-clearance phase age, ALT, TBIL, PTA, HBV-DNA and detection of HBeAg but not in sex and family history of HBV infection. CONCLUSION: CD4+ CD25+ regulatory T cells may play important roles in the clearance of HBV as well as in liver inflammation and injury during chronic HBV infection.

[Detection of CD4+CD25+ regulatory T cells in liver tissues of fibrosing cholestatic hepatitis after liver and kidney transplantation].

OBJECTIVES: To study the expression and distribution of CD4+CD25+ regulatory T cells (Treg) in liver tissues of patients with fibrosing cholestatic hepatitis (FCH) after liver and kidney transplantation and to investigate their roles in the pathogenesis of FCH. METHODS: Liver biopsy specimens from five patients with FCH were studied histopathologically. A specific marker for CD4+CD25+ regulatory T cells in those specimens was detected with anti-FOXP3 monoclonal antibody by immunohistochemistry. Apoptoses of hepatocytes were detected with in situ apoptosis detection TUNEL kit. RESULTS: Fibrosis in portal and around portal areas, cholestasis in some of the hepatocytes and canaliculi, widespread ballooning and ground-glass appearance of liver cells, and positivity of HBsAg and HBcAg and Pre-S1 protein were seen in the livers of all cases. The positive signal of FOXP3 was located in the cytoplasm of lymphocytes and the positive cells were mainly aggregated in the portal areas as well as occasionally appearing in the hepatic sinusoids. There were many more apoptotic hepatocytes near the portal areas. CONCLUSION: Fibrosing cholestatic hepatitis has specific pathological characteristics which might be caused by high expressions of FOXP3 in liver tissues.

Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions.

In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi.

[Pathology of AIDS-related lymphadenopathy and its relationship to expression of CD4 + CD25 + regulatory T cells in lymphoid node tissue].

OBJECTIVE: To observe the pathology of AIDS-related lymphadenopathy and its relationship to the expression and distribution of CD4 + CD25 + regulatory T cells in lymphoid node tissue. METHODS: Totally 22 biopsy and 13 autopsy lymphoid node tissues from HIV-positive patients were examined under microscopy and pathological staging was performed. Specific marker for CD4 + CD25 + regulatory T cells in lymphoid node tissue was detected with anti-Foxp3 monoclonal antibody by immunohistochemistry. RESULTS: Among all the 35 specimens, 5, 4, 14, and 12 specimens were histopathologically staged from 1 to 4, respectively. FoxP3 were detected in all lymphoid node tissues. The distribution of FoxP3-positive lymphocytes were mainly in intermediate zone of follicle and cortical area in stages 1 and 2. The counts of FoxP3-positive lymphocytes remarkably decreased in stages 3 and 4, following depletion of lymphocytes. CONCLUSIONS: CD4 + CD25 + regulatory T cells exist in lymphoid node tissue of patients with HIV infection. Their amounts decrease or deplete along with the progression of AIDS-related lymphadenopathy.

[Effects of traditional Chinese medicine on CD4 + T cell counts and HIV viral loads during structured treatment interruption in highly active antiretroviral therapy].

OBJECTIVE: To explore the impacts of traditional Chinese medicine (TCM) on CD4 + T cell counts and human immunodeficiency virus (HIV) viral loads during the course of structured treatment interruption (STI) in highly active antiretroviral therapy (HAART). METHODS: Nineteen HIV/ADIS patients were treated for 14 months as follows: initiated with zidovudine/lamivudine + efavirdine for 6 months, then discontinued the therapy and treated with TCM instead for 2 months. HAART was then reinitiated for another 3 months, and then discontinued and replaced with TCM for another 3 months. The changes of CD4 + T cell counts and HIV viral loads were measured. RESULTS: During the first STI of HAART, 43.8% of patients had no viral rebounds one month later, and 62.6% had stable or increased immune functions; 18.8% had no viral rebounds two months later, and 43.8% had stable or increased immune functions. Changes of viral loads were not significantly different between these two months (P = 0.097), while CD4 + T cell counts significantly decreased two months later compared with one month later (P = 0.043). During the second STI of HAART, 33.3% of patients had no viral rebounds one month later, and 64.3% had stable or increased immune functions; 13.3% had no viral rebounds 3 months later and 46.6% had stable or increased immune functions. Changes of viral loads had significant difference (P = 0. 017), while CD4 + T cell counts at month 12 elevated significantly compared with the baseline (P = 0.014). CONCLUSIONS: TCM can suppress the viral rebounds during STI-HAART, maintain immune functions. However, this effect may decrease along with the prolongation of STI-HAART.

Phosphorylation of ezrin enhances microvillus length via a p38 MAP-kinase pathway in an immortalized mouse hepatic cell line.

The apical microvilli are closely related with the development and the maintenance of cell polarization, and the length of microvilli varies in a regular way among cell types. Ezrin, a member of the ezrin/radixin/moesin (ERM) family, seems to be involved in the formation and stabilization of the apical microvilli. We found that phosphorylation of ezrin caused elongation of microvilli via a p38 MAP-kinase signaling pathway in an immortalized mouse hepatic cell line. When, in the oncogenic Raf-1-transfected mouse hepatic cell line, epithelial to mesenchymal transition (EMT) indicated as down-regulation of E-cadherin and up-regulation of Snail occurred, loss of microvilli and down-regulation of ezrin but not radixin and moesin were also observed. In the Raf-1 transfectants treated with the MAP-kinase inhibitor PD98059 and the p38 MAP-kinase inhibitor SB203580, the numbers of microvilli and the expression of ezrin, E-cadherin and Snail were recovered. More interestingly, treatment with SB203580 induced elongation of microvilli and increased phosphorylation of ezrin (at Thr-567 and Tyr-353). Phosphorylated ezrin-positive dots were colocalized with actin-positive dots on the surface of some Raf-1 transfectants treated with SB203580. These results suggested that phosphorylation of ezrin via the p38 MAP-kinase signaling pathway might be involved in the formation of microvilli during development of epithelial cell polarization.

Oncogenic Raf-1 regulates epithelial to mesenchymal transition via distinct signal transduction pathways in an immortalized mouse hepatic cell line.

The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RT-PCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [(14)C]mannitol and [(14)C]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.

Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes.

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.

Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver.

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivo and in vitro, including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivo were studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.