Epidermal growth factor (EGF) increases claudin-4 expression in Madin-Darby canine kidney (MDCK) cells. Here we examined what regulatory mechanisms are involved in the EGF-induced claudin-4 elevation. EGF transiently increased claudin-4 mRNA at 3 h and persistently increased its protein for 24 h Without affecting claudin-1 expression. EGF increased p-ER1/2 levels, which were inhibited by U0126, a MEK inhibitor. The exogenous expression of constitutively activated MEK increased claudin-4 expression. These results indicate that the activation of ERK1/2 is involved in the EGF-induced claudin-4 elevation. EGF increased Sp1 expression within 1 h, which was inhibited by U0126. In immunocytochemistry. Sp1 Was distributed in nucleus in control and the EGF-treated cells, The EGF-induced claudin-4 elevation was inhibited by mithramycin, a Sp1 inhibitor, and Sp1 small interfering RNA. We Suggest that EGF activates a MEK/ERK pathway and increases Sp1 expression, resulting in all elevation of claudin-4 expression. (C) 2009 Elsevier Inc. All rights reserved.