Activation of p38(MAPK) mediates the angiostatic effect of the chemokine receptor CXCR3-B.

Chemokines binding the CXCR3 receptor have been shown to inhibit angiogenesis via the CXCR3-B isoform, but the underlying molecular mechanisms are unknown. Aim of this study was to elucidate the effects of CXCR3-B on activation of members of the mitogen-activated protein kinase family, and to explore the relevance of defined signaling pathways to the angiostatic effects of CXCR3-B ligands. Human embryonic kidney (HEK) 293 cells were transfected with expression vectors encoding for CXCR3-A or CXCR3-B. In cells expressing CXCR3-A, CXCL10 (IP-10) at nanomolar concentrations induced activation of ERK, Akt, and Src, as previously described in human vascular pericytes. In HEK-293 cells expressing CXCR3-B, exposure to CXCL10 in the micromolar concentration range led to activation of the p38(MAPK) pathway, as indicated by phosphorylation of p38(MAPK) itself, and of MKK3/6 and MAPKAPK-2, that lie upstream and downstream of p38(MAPK), respectively. Similar results were obtained in cells stimulated with CXCL4 (PF4), a specific ligand of CXCR3-B. In contrast, CXCL4 was unable to activate p38(MAPK) in mock-transfected HEK-293 cells. Only a modest induction of ERK or JNK was observed upon CXCR3-B activation. In human microvascular endothelial cells, which selectively express CXCR3-B, in a cell cycle-dependent fashion, CXCL10 and CXCL4 increased the enzymatic activity of p38(MAPK). Pharmacologic inhibition of p38(MAPK) by SB302580 resulted in a significant increase in DNA synthesis and in reversal of the inhibitory action of CXCL10. In conclusion, the p38(MAPK) pathway is a downstream effector of CXCR3-B implicated in the angiostatic action of this chemokine receptor.

Nuclear localization of TRK-A in liver cells.

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.

Prevention of severe toxic liver injury and oxidative stress in MCP-1-deficient mice.

BACKGROUND/AIMS: Administration of carbon tetrachloride determines liver injury, inflammation and oxidative stress, but the molecular mechanisms of damage are only partially understood. In this study, we investigated the development of acute toxic damage in mice lacking monocyte chemoattractant protein-1 (MCP-1), a chemokine which recruits monocytes and activated lymphocytes. METHODS: Mice with targeted deletion of the MCP-1 gene and wild type controls were administered a single intragastric dose of carbon tetrachloride. Serum liver enzymes, histology, expression of different chemokines and cytokines, and intrahepatic levels of oxidative stress-related products were evaluated. RESULTS: Compared to wild type mice, peak aminotransferase levels were significantly lower in MCP-1-deficient animals. This was paralleled by a delayed appearance of necrosis at histology. In addition, MCP-1-deficient mice showed a shift in the pattern of infiltrating inflammatory cells, with a predominance of polymorphonuclear leukocytes. Lack of MCP-1 was also accompanied by reduced intrahepatic expression of cytokines regulating inflammation and tissue repair. The increase in tissue levels of reactive oxygen species and 4-hydroxy-nonenal following administration of the hepatotoxin was also significantly lower in animals lacking MCP-1. CONCLUSIONS: Lack of MCP-1 affords protection from damage and development of oxidative stress in a toxic model of severe acute liver injury.

Thrombopoietin stimulates migration and activates multiple signaling pathways in hepatoblastoma cells.

Thrombopoietin (TPO), a cytokine that participates in the differentiation and maturation of megakaryocytes, is produced in the liver, but only limited information is available on the biological response of liver-derived cells to TPO. In this study, we investigated whether HepG2 cells express c-Mpl, the receptor for TPO, and whether TPO elicits biological responses and intracellular signaling in this cell type. Specific transcripts for c-Mpl were detected in HepG2 cells by RT-PCR, and expression of the protein was demonstrated by Western blot analysis and immunofluorescence. Exposure of HepG2 cells to TPO was associated with a dose-dependent increase in cell migration and chemoinvasion through Matrigel-coated filters. A checkerboard analysis showed that the effects of TPO on cell migration were dependent on both chemotaxis and chemokinesis. Exposure of HepG2 cells to TPO resulted in the activation of different members of the MAPK family, including ERK and JNK, as assessed using phosphorylation-specific antibodies and immune complex kinase assays. TPO also activated phosphatidylinositol 3-kinase (PI3K) and the downstream kinase Akt in a time-dependent manner. Finally, activation of c-Mpl was associated with increased activation of nuclear factor-kappaB. With the use of specific inhibitors, tyrosine phosphorylation and activation of PI3K were found to be required for the induction of migration in response to TPO. We conclude that TPO exerts biological actions on cultured hepatoblastoma cells via activation of c-Mpl and its downstream signaling.

Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.

Leptin upregulates collagen expression in hepatic stellate cells (HSCs), but the possible modulation of other actions has not been elucidated. The aim of this study was to investigate the expression and function of leptin receptors (ObR) in human HSCs and the biological actions regulated by leptin. Exposure of HSCs to leptin resulted in upregulation of monocyte chemoattractant protein 1 (MCP-1) expression. Leptin also increased gene expression of the proangiogenic cytokines vascular endothelial growth factor (VEGF) and angiopoietin-1, and VEGF was also upregulated at the protein level. Activated HSCs express ObRb and possibly other ObR isoforms. Exposure to leptin increased the tyrosine kinase activity of ObR immunoprecipitates and resulted in activation of signal transducer and activator of transcription 3. Several signaling pathways were activated by leptin in HSCs, including extracellular-signal-regulated kinase, Akt, and nuclear factor kappaB, the latter being relevant for chemokine expression. Leptin also increased the abundance of hypoxia-inducible factor 1alpha, which regulates angiogenic gene expression, in an extracellular-signal-regulated kinase- and phoshatidylinositol 3-kinase-dependent fashion. In vivo, leptin administration induced higher MCP-1 expression and more severe inflammation in mice after acute liver injury. Conversely, in leptin-deficient mice, the increase in MCP-1 messenger RNA and mononuclear infiltration was less marked than in wild-type littermates. Finally, ObR expression colocalized with VEGF and alpha-smooth muscle actin after induction of fibrosis in rats. In conclusion, ObR activation in HSCs leads to increased expression of proinflammatory and proangiogenic cytokines, indicating a complex role for leptin in the regulation of the liver wound-healing response.

Differential requirement of members of the MAPK family for CCL2 expression by hepatic stellate cells.

Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including CCL2 (formerly known as monocyte chemoattractant protein-1). In this study, we evaluated the role of different proteins of the MAPK family (ERK, p38(MAPK), and JNK) in the regulation of CCL2 expression by HSC, as an index of their proinflammatory activity. Several mediators activated all three MAPK, including TNF, IL-1, and PDGF. To assess the relative role of the different MAPKs, specific pharmacological inhibitors were used; namely, SB203580 (p38(MAPK)), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system were verified analyzing the enzymatic activity of the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation of downstream substrates. SB203580 and SP600125 dose-dependently inhibited CCL2 secretion and gene expression induced by IL-1 or TNF. In contrast, inhibition of ERK did not affect the upregulation of CCL2 induced by the two cytokines. Finally, activin A was also found to stimulate CCL2 expression and to activate ERK, JNK, p38, and their downstream targets. Unlike in cells exposed to proinflammatory cytokines, all three MAPKs were required to induce CCL2 secretion in response to activin. We conclude that members of the MAPK family differentially regulate cytokine-induced chemokine expression in human HSC.

The chemokine CCL21 modulates lymphocyte recruitment and fibrosis in chronic hepatitis C.

BACKGROUND AND AIMS: The chemokines CCL19 and CCL21 bind CCR7, which is involved in the organization of secondary lymphoid tissue and is expressed during chronic tissue inflammation. We investigated the expression of CCL21 and CCR7 in chronic hepatitis C. The effects of CCL21 on hepatic stellate cells (HSCs) were also studied. METHODS: Expression of CCL21 was assessed by in situ hybridization and immunohistochemistry. CCR7 on T cells was analyzed by flow cytometry. Cultured human HSCs were studied in their activated phenotype. RESULTS: In patients with chronic hepatitis C, expression of CCL21 and CCR7 was up-regulated. CCL21 was detected in the portal tracts and around inflammatory lymphoid follicles, in proximity to T lymphocytes and dendritic cells, which contributed to expression of this chemokine. Expression of CCR7 was also increased in patients with primary biliary cirrhosis. Intrahepatic CD8(+) T lymphocytes isolated from patients with chronic hepatitis C had a significantly higher percentage of positivity for CCR7 than those from healthy controls, and the expression of CCR7 was associated with that of CXCR3. Cultured HSCs expressed functional CCR7, the activation of which stimulated cell migration and accelerated wound healing in an in vitro model. Exposure of HSCs to CCL21 triggered several signaling pathways, including extracellular signal-regulated kinase, Akt, and nuclear factor kappaB, resulting in induction of proinflammatory genes. CONCLUSIONS: Expression of CCL21 during chronic hepatitis C is implicated in the recruitment of T lymphocytes and the organization of inflammatory lymphoid tissue and may promote fibrogenesis in the inflamed areas via activation of CCR7 on HSCs.