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

我们先前报道了人脐静脉内皮细胞（HUVEC）中的单轴连续拉伸通过I κ B激酶（IKK）/核因子-κB（NF-κB）激活诱导IL-6分泌（小林等人，2003年）。本研究的目的是阐明导致这一现象的上游信号机制。应用α 5 β 1整合素抑制肽GRGDNP、磷脂酰肌醇3-激酶抑制剂LY 294002、磷脂酶Cγ抑制剂U 73122、蛋白激酶C抑制剂LY 294002和蛋白激酶C抑制剂LY 294002可抑制牵张诱导的IKKs激活、IL-6 mRNA表达和IL-6分泌。H7或通过EGTA和毒胡萝卜素（TG）的共同应用来消耗细胞内和细胞外Ca^2+库，但不通过EGTA消耗细胞外Ca^2+或TG消耗细胞内Ca^2+库。使用Fura-2荧光强度比值法，在Gd^<3+>、EGTA、TG或GRGDNP存在下，观察到连续牵张后细胞内Ca^<2+>浓度（Ca^<2+>]_i）的瞬时增加，但在EGTA和TG混合物存在下则没有，这表明[Ca^<2+>]_i升高和整合素激活对诱导HUVEC中IKK激活和IL-6分泌至关重要。似乎无论是通过SA通道的Ca^2+流入还是从细胞内储存的Ca^2+释放都足以激活IL-6分泌的下游信号级联。GRGDNP、LY 294002和U 73122可抑制PKC激酶活性，而GRGDNP和LY 294002可抑制PLC γ激酶活性。提示牵张诱导的HUVECs分泌IL-6依赖于整合素的外向内信号和[Ca^<2+>] i升高，随后通过PI 3-K/PLCγ/PKC/IKKs/NF-κB信号通路。

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.