Wei Hua, Wang Fang, Wang Xianyun, Yang Jinjing, Li Zongwei, Cong Xiangfeng, Chen Xi
Chinese Academy of Medical Sciences, Beijing, People's Republic of China.
Biochim Biophys Acta. 2013 Aug;1831(8):1426-34. doi: 10.1016/j.bbalip.2013.05.003. Epub 2013 May 21.
We previously reported that transplantation of lysophosphatidic acid (LPA) treated mesenchymal stem cells (MSCs) enhances capillary density in the myocardium and improves myocardial function in the ischemic heart. This effect may be mediated through the release of paracrine factors by MSC and potentially involves pro-angiogenic molecules such as vascular endothelial growth factor (VEGF). In this study, we examined the pharmacological and molecular regulation of VEGF secretion induced by LPA in rat MSCs. We showed that LPA stimulated VEGF secretion in MSCs but not in cardiomyocytes or cardiac fibroblasts. LPA-induced VEGF secretion occurred at the post-transcriptional levels and was mediated through the classical ER/Golgi-dependent protein secretory route. LPA also increased ORP150 protein expression. Inhibition of ORP150 upregulation by siRNA knockdown attenuated LPA-induced VEGF secretion. On the other hand, diazoxide, an activator of KATP channel, markedly inhibited LPA-induced ORP150 expression and VEGF secretion. Meanwhile, ATP concentration dependently increased VEGF secretion. In addition, l-Glutamate and NH4Cl significantly reduced VEGF secretion. Furthermore, inhibition of two major subtypes of LPA receptors by Ki16425 and specific siRNA for LPA receptors prevented LPA-induced VEGF secretion and ORP150 expression. Lastly, inhibition of Gi protein that couples with LPA receptors by PTX and siRNA knockdown had no effect on LPA-induced VEGF secretion. Taken together, our findings demonstrate that LPA promotes VEGF secretion at the post-translation level by up-regulating ORP150 expression. Both LPA1 and LPA3 are involved in the LPA-induced VEGF secretion that is independent of Gi protein coupling but associated with the inactivation of KATP channels and inhibition of Na(+)/K(+)-ATPase activity.
我们之前报道过，经溶血磷脂酸（LPA）处理的间充质干细胞（MSC）移植可增强心肌中的毛细血管密度，并改善缺血性心脏的心肌功能。这种效应可能是通过MSC释放旁分泌因子介导的，并且可能涉及促血管生成分子，如血管内皮生长因子（VEGF）。在本研究中，我们研究了LPA在大鼠MSC中诱导的VEGF分泌的药理学和分子调控。我们发现LPA刺激MSC分泌VEGF，但不刺激心肌细胞或心脏成纤维细胞分泌VEGF。LPA诱导的VEGF分泌发生在转录后水平，并通过经典的内质网/高尔基体依赖性蛋白分泌途径介导。LPA还增加了ORP150蛋白的表达。通过小干扰RNA（siRNA）敲低抑制ORP150的上调可减弱LPA诱导的VEGF分泌。另一方面，KATP通道激活剂二氮嗪显著抑制LPA诱导的ORP150表达和VEGF分泌。同时，ATP浓度依赖性地增加VEGF分泌。此外，L-谷氨酸和氯化铵显著降低VEGF分泌。此外，Ki16425和LPA受体特异性siRNA对LPA受体两种主要亚型的抑制作用可阻止LPA诱导的VEGF分泌和ORP150表达。最后，百日咳毒素（PTX）和siRNA敲低对与LPA受体偶联的Gi蛋白的抑制作用对LPA诱导的VEGF分泌没有影响。综上所述，我们的研究结果表明，LPA通过上调ORP150表达在翻译后水平促进VEGF分泌。LPA1和LPA3都参与了LPA诱导的VEGF分泌，这一过程独立于Gi蛋白偶联，但与KATP通道的失活和钠钾ATP酶活性的抑制有关。
