Evaluation of intracellular signalling pathways in response to insulin-like growth factor I in apoptotic-resistant activated human hepatic stellate cells

BACKGROUND: Human hepatic stellate cells have been shown to be resistant to apoptotic stimuli. This is likely dependent on the activation of anti-apoptotic pathways upon transition of these cells to myofibroblast-like cells. In particular, previous studies have demonstrated an increased expression of the anti-apoptotic protein Bcl-2 and a decreased expression of the pro-apoptotic protein Bax during the transition of the hepatic stellate cell phenotype from quiescent to myofibroblast-like cells. However, the role and expression of other key anti-apoptotic and survival pathways elicited by polypeptide growth factors involved in the chronic wound healing process remain to be elucidated. In particular, insulin growth factor-I promotes chemotactic and mitogenic effects in activated human hepatic stellate cells and these effects are mediated by the activation of PI 3-K. The role of insulin growth factor-I as a survival factor in human hepatic stellate cells needs to be substantiated. The aim of this study was to evaluate the involvement of other key anti-apoptotic pathways such as PI-3K/Akt/p-Bad in response to insulin growth factor-I. RESULTS: Insulin growth factor-I induced activation of Akt followed by Bad phosphorylation after 15 minutes of incubation. These effects were PI-3k dependent since selective inhibitors of this molecule, wortmannin and LY294002, inhibited both Akt and Bad phosphorylation. The effect of insulin growth factor-I on the activation of two downstream targets of Akt activation, that is, GSK3 and FHKR, both implicated in the promotion of cell survival was also investigated. Both targets became phosphorylated after 15 minutes of incubation, and these effects were also PI-3K-dependent. Despite the activation of this survival pathway insulin growth factor-I did not have a remarkable biological effect, probably because other insulin growth factor-I-independent survival pathways were already maximally activated in the process of hepatic stellate cell activation. However, after incubation of the cells with a strong apoptotic stimuli such as Fas ligand+cycloheximide, a small percentage of hepatic stellate cells underwent programmed cell death that was partially rescued by insulin growth factor-I. CONCLUSION: In addition to Bcl-2, several other anti-apoptotic pathways are responsible for human hepatic stellate cell resistance to apoptosis. These features are relevant for the progression and limited reversibility of liver fibrosis in humans

Extracellular Signal-Regulated Kinase 5 Regulates the Malignant Phenotype of Cholangiocarcinoma Cells

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal‐regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA. APPROACH AND RESULTS: ERK5 expression was detected in two established (HuCCT‐1 and CCLP‐1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT‐1 and CCLP‐1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5‐silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP‐1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA. CONCLUSIONS: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment