oration of class switching and proliferative responses in cells with CD154 mutations. If C4BP binds the mutant CD154 protein it could promote more effective crosslinking of CD154 and CD40 and thereby restore binding and functionality. Our data confirm that C4BP does not prevent the binding of sCD154, or in our hands, the C4BP/sCD154 complex to CD40. Biacore analysis showed dose-dependent saturable binding of C4BP to increasing concentrations of sCD154. Although the Biacore data are convincing in themselves, we thought it important to confirm the formation of 14757152 C4BP/sCD154 complexes using an alternative method, gel filtration, which provided further evidence that CD154 forms stable high molecular weight complexes with C4BP. We then went on to investigate the effect of C4BP/sCD154 complex on the major signalling pathways involved in primary cholangiocyte survival, NFkB, AP-1 and STAT 3. We have previously shown that activation of cholangiocyte CD40 leads to transient RelA expression and sustained cJun/cFos and STAT 3 expression resulting in apoptosis. Surprisingly, despite the ability to bind CD40 when the C4BP/sCD154 complex was used to activate CD40 on cholangiocytes, we were unable to detect transient activation of NFkB or sustained STAT3 phosphorylation and saw only transient activation of the cFos subunit of the AP-1 C4BP/CD154 Prevents Apoptosis heterodimer. This provides a possible reason why the presence of C4BP can prevent sCD154 mediated cholangiocyte apoptosis and suggests further complexities to CD40 mediated signalling in relation to cholangiocyte survival. Our previous comparisons of CD40 mediated NFkB and AP-1 signalling in cholangiocytes and endothelial cells provide evidence for cell-specific differences in transcription factor activation following CD40 ligation. In contrast to cholangiocytes, activation of CD40 on endothelial cells resulted in sustained activation of NFkB and no effect on the AP-1 pathway, the net result being cell proliferation. In the present study, our initial predictions were that the C4BP/sCD154 complex would function to crosslink CD40 more efficiently than sCD154 alone leading to more effective receptor crosslinking and as a consequence, an increase in NFkB and AP-1 activation in cholangiocytes. However, this was not the case and the C4BP/sCD154 complex suppressed the activation of these signalling pathways resulting in cell survival in the absence of proliferation. The ability of C4BP to modulate the outcome of sCD154 interactions with CD40 suggests 
the complement system may be involved in regulate epithelial cell survival in the liver. Prolongation of epithelial cell survival is important to facilitate normal protective immune responses, but if sustained could theoretically facilitate 15863272 malignant transformation. Furthermore, C4BP could potentially modulate CD40 signalling in other cells including endothelial cells, infiltrating leukocytes and stromal cells, which could lead to perpetuation of inflammation. A link between inflammation and malignancy is supported by our finding that C4BP is expressed strongly in the reactive stroma at tumour margins in association with CD40 expression on tumour cells. Interestingly, C4BP protein was also strongly expressed in NVP-BGJ398 custom synthesis inflammatory cells and proliferating bile ductules in PSC, a disease associated with a high risk of for malignant transformation of biliary epithelium and the development of cholangiocarcinoma. Thus the presence of C4BP in close proximity to tumor
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