Grummer Mary A, Sullivan Jeremy A, Magness Ronald R, Bird Ian M
Department of Obstetrics & Gynecology, Perinatal Research Laboratories, University of Wisconsin, Madison, WI 53715, U.S.A.
Biochem J. 2009 Jan 15;417(2):501-11. doi: 10.1042/BJ20081013.
During pregnancy, VEGF (vascular endothelial growth factor) regulates in part endothelial angiogenesis and vasodilation. In the present study we examine the relative roles of VEGFRs (VEGF receptors) and associated signalling pathways mediating the effects of VEGF(165) on eNOS (endothelial nitric oxide synthase) activation. Despite equal expression levels of VEGFR-1 and VEGFR-2 in UAECs (uterine artery endothelial cells) from NP (non-pregnant) and P (pregnant) sheep, VEGF(165) activates eNOS at a greater level in P- compared with NP-UAEC, independently of Akt activation. The selective VEGFR-1 agonist PlGF (placental growth factor)-1 elicits only a modest activation of eNOS in P-UAECs compared with VEGF(165), whereas the VEGFR-2 kinase inhibitor blocks VEGF(165)-stimulated eNOS activation, suggesting VEGF(165) predominantly activates eNOS via VEGFR-2. Although VEGF(165) also activates ERK (extracellular-signal-regulated kinase)-1/2, this is not necessary for eNOS activation since U0126 blocks ERK-1/2 phosphorylation, but not eNOS activation, and the VEGFR-2 kinase inhibitor inhibits eNOS activation, but not ERK-1/2 phosphorylation. Furthermore, the inability of PlGF to activate ERK-1/2 and the ability of the VEGFR-2 selective agonist VEGF-E to activate ERK-1/2 and eNOS suggests again that both eNOS and ERK-1/2 activation occur predominantly via VEGFR-2. The lack of VEGF(165)-stimulated Akt phosphorylation is consistent with a lack of robust phosphorylation of Ser(1179)-eNOS. Although VEGF(165)-stimulated eNOS phosphorylation is observed at Ser(617) and Ser(635), pregnancy does not significantly alter this response. Our finding that VEGF(165) activation of eNOS is completely inhibited by wortmannin but not LY294002 implies a downstream kinase, possibly a wortmannin-selective PI3K (phosphoinositide 3-kinase), is acting between the VEGFR-2 and eNOS independently of Akt.
在怀孕期间，血管内皮生长因子（VEGF）部分调节内皮细胞血管生成和血管舒张。在本研究中，我们研究了VEGF受体（VEGFRs）和相关信号通路在介导VEGF(165)对内皮型一氧化氮合酶（eNOS）激活作用中的相对作用。尽管来自非妊娠（NP）和妊娠（P）绵羊的子宫动脉内皮细胞（UAECs）中VEGFR-1和VEGFR-2的表达水平相等，但与NP-UAEC相比，VEGF(165)在P-UAEC中能更有效地激活eNOS，且与Akt激活无关。与VEGF(165)相比，选择性VEGFR-1激动剂胎盘生长因子（PlGF）-1仅能适度激活P-UAEC中的eNOS，而VEGFR-2激酶抑制剂可阻断VEGF(165)刺激的eNOS激活，这表明VEGF(165)主要通过VEGFR-2激活eNOS。虽然VEGF(165)也能激活细胞外信号调节激酶（ERK）-1/2，但这并非eNOS激活所必需，因为U0126可阻断ERK-1/2磷酸化，但不影响eNOS激活，而VEGFR-2激酶抑制剂可抑制eNOS激活，但不影响ERK-1/2磷酸化。此外，PlGF无法激活ERK-1/2，而VEGFR-2选择性激动剂VEGF-E能够激活ERK-1/2和eNOS，这再次表明eNOS和ERK-1/2的激活主要通过VEGFR-2发生。VEGF(165)刺激后Akt磷酸化缺乏与Ser(1179)-eNOS缺乏强烈磷酸化一致。尽管在Ser(617)和Ser(635)处观察到VEGF(165)刺激的eNOS磷酸化，但妊娠并未显著改变这种反应。我们的发现表明，渥曼青霉素可完全抑制VEGF(165)对eNOS的激活，而LY294002则无此作用，这意味着在VEGFR-2和eNOS之间存在一种下游激酶，可能是一种对渥曼青霉素敏感的磷脂酰肌醇3激酶（PI3K），其作用独立于Akt。
