Mitogen activated protein kinases a family of selleck chemical Erlotinib ser ine threonine kinases, have a fundamentally important roles as signal transducers. Activation of MAP kinases by various growth factors and cytokines are important mole cules involved in modulating cellular responses. In terms of tight junction regulation the role of MAP kinase signaling has been of interest. MAPK kinase overexpression led to epithelial dedifferentiation in MDCK C7 cells. Tight junction biogenesis was inhibited in MDCK cells expressing constitutively active MAP kinase. pharmacological inhibition of MEK1 signal ing in these cells permitted tight junction formation. Pharmacological inhibition of MEK, a Ras effector known to phosphorylate extracellular signal regulated kinase 1 and 2, attenuated dexamethasone induced tight junction formation in the Con8 mammary tumor cell line.

In these studies, the mitogenic effect of MAP kinase activity is logically opposed to tight junc tion formation. The analysis of the effects of external stim uli on tight junction regulation, specifically the activated signaling pathways, will provide valuable insight into tight junction regulation. The goal of this present study was to characterize the response of MDCK cells to the combination of TNF IFN. We hypothesized that TNF IFN would impair MDCK cell tight junction function. We examined TER, paracellular flux, tight junction protein expression and localization in response to the proinflammatory cytokines.

In a variety of disease states inflammation is thought to negatively GSK-3 impact epithelial barrier function, we report that TNF IFN co administration to MDCK cell monolayers impaired epithelial barrier function as measured by elevated paracellular flux and produced marked elevation in transepithelial electrical resistance. Occludin, claudin 1 and claudin 3 protein expres sion was induced by TNF IFN exposure, whereas clau din 2 levels decreased. tight junction protein localization was modulated contributing to impaired tight junction function. Inhibition of MEK1 and p38 signaling during exposure to TNF IFN, abrogated these cytokine induced effects in MDCK cells. Results Effect of TNF and IFN on cellular cytotoxicity In order to determine whether TNF IFN induced cyto toxic effects in the MDCK cell cultures, we determined the percentage of apoptotic cells in confluent MDCK cultures using the TUNEL assay and measured LDH enzyme activity released from treated conflu ent cultures. No significant differences were found in per cent of TUNEL positive cells following treatment for 24 hours with increasing doses of TNF IFN. As a positive control, cells were serum and glucose starved for 24 hours, and this resulted in a significant increase in TUNEL posi tive cells.