We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via IkappaB kinase (IKK)/nuclear factor-kappaB (NF-kappaB) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of alpha(5)beta(1) integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C-gamma inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca(2+) pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca(2+)](i) rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC-gamma activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC-gamma-PKC-IKK-NF-kappaB signaling cascade. Another crucial factor, [Ca(2+)](i) increase, may at least be required to activate PKC needed for NF-kappaB activation.