Experimental study on mechanism of liver regeneration with special attention to continuous stretch in endothelial cells

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We previously reported that uni-axial continuous stretch in human umbilical vein endothelial cells(HUVECs) induced IL-6 secretion via I κ B kinases(IKKs)/nuclear factor-κB(NF-κB) activation (Kobayashi, et al., 2003). The aim of this study is to clarify the upstream signaling mechanism responsible for this phenomenon.The stretch-induced IKKs activation, IL-6mRNA expression and IL-6 secretion were inhibited by an application of α 5 β 1 integrin inhibitory peptide : GRGDNP, phosphatidylinositol 3-kinase inhibitor : LY294002,phospholipase Cγ inhibitor : U73122,protein kinase C inhibitor : H7 or the depletion of intra-and extra-cellular Ca^<2+> pools by a co-application of EGTA and thapsigargin(TG), but not by the depletion of extracellular Ca^<2+> by EGTA or the depletion of intracellular Ca^<2+> stores by TG. Using fura-2 fluorescence intensity ratiometry, a transient increase in intracellular Ca^<2+> concentration (Ca^<2+>]_i) upon continuous stretch was observed in the presence of Gd^<3+>, EGTA, TG or GRGDNP, but not with a mixture EGTA and TG, suggesting that both a [Ca^<2+>]_i rise and integrin activation are crucial to elicit IKKs activation and IL-6 secretion in HUVECs. It seemed like that either Ca^<2+> influx via SA channels or Ca^<2+> release from intracellular stores is enough to activate the downstream signaling cascade for IL-6 secretion. PKC kinase activity was inhibited by an application of GRGDNP, LY294002 or U73122, and PLC γ kinase activity was by GRGDNP or LY294002.These results indicate that continuous stretch-induced IL-6 secretion in HUVECs is dependent on both outside-in signaling at integrins and a [Ca^<2+>]_i rise followed by a PI3-K/PLCγ/PKC/IKKs/NF-κB signaling pathway.