1] Department of Oncology, School of Medicine, University of Turin, Turin, Italy [2] Unidad de Bioquímica, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia.
Department of Oncology, School of Medicine, University of Turin, Turin, Italy.
J Cereb Blood Flow Metab. 2014 Aug;34(8):1258-69. doi: 10.1038/jcbfm.2014.100. Epub 2014 Jun 4.
In this work, we investigate if and how transducers of the 'canonical' Wnt pathway, i.e., Wnt/glycogen synthase kinase 3 (GSK3)/β-catenin, and transducers of the 'non-canonical' Wnt pathway, i.e., Wnt/RhoA/RhoA kinase (RhoAK), cooperate to control the expression of P-glycoprotein (Pgp) in blood-brain barrier (BBB) cells. By analyzing human primary brain microvascular endothelial cells constitutively activated for RhoA, silenced for RhoA or treated with the RhoAK inhibitor Y27632, we found that RhoAK phosphorylated and activated the protein tyrosine phosphatase 1B (PTP1B), which dephosphorylated tyrosine 216 of GSK3, decreasing the GSK3-mediated inhibition of β-catenin. By contrast, the inhibition of RhoA/RhoAK axis prevented the activation of PTP1B, enhanced the GSK3-induced phosphorylation and ubiquitination of β-catenin, and reduced the β-catenin-driven transcription of Pgp. The RhoAK inhibition increased the delivery of Pgp substrates like doxorubicin across the BBB and improved the doxorubicin efficacy against glioblastoma cells co-cultured under a BBB monolayer. Our data demonstrate that in human BBB cells the expression of Pgp is controlled by a cross-talk between canonical and non-canonical Wnt pathways. The disruption of this cross-talk, e.g., by inhibiting RhoAK, downregulates Pgp and increases the delivery of Pgp substrates across the BBB.
在这项工作中，我们研究了“经典” Wnt 途径的转导子，即 Wnt/糖原合酶激酶 3（GSK3）/β-连环蛋白，和“非经典” Wnt 途径的转导子，即 Wnt/RhoA/RhoA 激酶（RhoAK），是否以及如何合作控制血脑屏障（BBB）细胞中 P-糖蛋白（Pgp）的表达。通过分析 RhoA 持续激活的人原代脑微血管内皮细胞、RhoA 沉默的细胞或用 RhoAK 抑制剂 Y27632 处理的细胞，我们发现 RhoAK 磷酸化并激活了蛋白酪氨酸磷酸酶 1B（PTP1B），从而使 GSK3 的酪氨酸 216 去磷酸化，减少 GSK3 介导的β-连环蛋白抑制。相比之下，抑制 RhoA/RhoAK 轴阻止了 PTP1B 的激活，增强了 GSK3 诱导的β-连环蛋白磷酸化和泛素化，并减少了β-连环蛋白驱动的 Pgp 转录。RhoAK 抑制增加了 Pgp 底物如多柔比星穿过 BBB 的递送，并提高了多柔比星对共培养在 BBB 单层下的神经胶质瘤细胞的疗效。我们的数据表明，在人 BBB 细胞中，Pgp 的表达受经典和非经典 Wnt 途径之间的串扰控制。这种串扰的破坏，例如通过抑制 RhoAK，下调 Pgp 并增加 Pgp 底物穿过 BBB 的递送。
