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

我们以前的研究表明，内皮素（ET）-B受体的刺激部分地通过<i2>细胞外基质分子依赖的方式增加神经祖细胞的增殖。神经前体细胞在ET存在下培养可分化为神经元、胶质细胞和平滑肌细胞（SMCs）。在本研究中，我们首先研究了ET诱导平滑肌细胞分化的机制。ET在TGF-β存在下有效地促进SMC特异性蛋白的表达，TGF-β也由内皮细胞产生。利用平滑肌肌动蛋白（SMA）启动子-荧光素酶报告基因实验表明，G蛋白和TGF-β信号通路协同促进神经前体细胞SMC特异性蛋白的表达，我们进一步研究了G_（q/11）是否也参与ET刺激的神经前体细胞增殖，以及G_（q/11）和G_（q/11）如何调节ERK通路和整合素信号通路的增强。G_&lt;q/11&gt;抑制剂YM-254890和百日咳毒素可部分抑制ET刺激的Raf-1和ERK磷酸化。ET刺激蛋白激酶C（PKC），YM-254890抑制，而百日咳毒素减弱ET诱导的Ras激活。另一方面，百日咳毒素和YM-254890部分抑制ET刺激的FAK和桩蛋白的磷酸化，并且PKC抑制剂和PKC的下调阻止ET诱导的这些蛋白的磷酸化。Src家族激酶抑制剂PP 2阻断ET刺激的桩蛋白磷酸化，而对ERK无影响。因此，ET主要通过G_（q/11）激活PKC，并与G_（i）激活的Ras通路协同激活ERK级联反应。PKC似乎通过Src家族激酶增加桩蛋白的酪氨酸磷酸化以增强整合素信号，这进一步增加DNA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.