Regulation of proliferation and differentiation of neural progenitor cells by molecules produced by endothelial cells

我们先前已经证明，内皮素(ET)-B受体的刺激部分地通过细胞外基质分子依赖的方式促进神经前体细胞的增殖。在ET存在下培养的神经前体细胞向神经元、神经胶质细胞和平滑肌细胞(SMC)分化。在本研究中，我们首先研究了ET诱导向SMC分化的机制。在转化生长因子-β存在的情况下，ET能有效地促进内皮细胞产生的SMC特异性蛋白的表达。利用平滑肌肌动蛋白启动子-荧光素酶报告基因分析的实验表明，G蛋白和转化生长因子-β的不同信号通路协同促进神经前体细胞中平滑肌肌动蛋白特异性蛋白的表达。G_&lt；q/11&gt；ym-254890抑制剂和百日咳毒素部分抑制ET刺激的Raf-1和ERK的磷酸化。YM-254890可抑制ET对蛋白激酶C的激活作用，而百日咳毒素可减弱ET对RAS的激活作用。另一方面，百日咳毒素和YM-254890可部分抑制ET诱导的FAK和PXLIN的磷酸化，而蛋白激酶C抑制剂和下调蛋白激酶C则可阻止ET诱导的这些蛋白的磷酸化。一种Src家族的蛋白激酶抑制剂PP2可阻断ET刺激的帕西林的磷酸化，但不影响ERK。综上所述，ET主要通过G&lt；q/11&gt；激活PKC，从而协同G_i刺激的RAS通路，刺激ERK级联反应。PKC可能通过Src家族的激酶增加paxlin的酪氨酸磷酸化，增强整合素信号，从而进一步促进DNA的合成和增殖。

We have previously shown that endothelin (ET)-B receptor stimulation increased neural progenitor proliferation in part via G_<i2> in extracellular matrix molecule-dependent manner. Neural progenitor cells cultured in the presence of ET differentiated into neurons, glial cells and smooth muscle cells (SMCs). In the present study, we first investigated the mechanism of ET-induced differentiation into SMCs. ET effectively promoted expression of SMC-specific proteins in the presence of TGF-β, which is also produced by endothelial cells. Experiments using smooth muscle actin (SMA) promoter-luciferase reporter assay indicated that separate signaling pathways of G protein and TGF-β cooperatively promote expression of SMC-specific proteins in neural progenitor cells.Next we investigated whether G_<q/11> is also involved in ET-stimulated proliferation and how G_i and G_<q/11> regulate ERK pathway and enhancement of integrin signaling. An inhibitor of G_<q/11> YM-254890, as well as pertussis toxin partially inhibited ET-stimulated phosphorylation of Raf-1 and ERK. ET stimulated protein kinase C (PKC), which was inhibited by YM-254890, while pertussis toxin attenuated ET-induced Ras activation. On the other hand, pertussis toxin and YM-254890 partially inhibited ET-stimulated phosphorylation of FAK and paxillin, and a PKC inhibitor and down-regulation of PKC prevented ET-induced phosphorylation of these protein. A Src family kinase inhibitor, PP2, blocked ET-stimulated phosphorylation of paxillin without effect on ERK. Taken together, ET activates PKC mainly via G_<q/11> and consequently stimulates ERK cascade in cooperation with Ras pathway stimulated by G_i. PKC seems to increase tyrosine phosphorylation of paxillin via Src family kinase to enhance integrin signals, which further increase DNA synthesis and proliferation.