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Abstract：
P2X7 receptor (P2RX7) is expressed strongly by most human cancers, including neuroblastoma, where high levels of P2RX7 are correlated with a poor prognosis for patients. Tonic activation of P2X7 receptor favors cell metabolism and angiogenesis, thereby promoting cancer cell proliferation, immunosuppression, and metastasis. Although understanding the mechanisms that control P2X7 receptor levels in neuroblastoma cells could be biologically and clinically relevant, the intracellular signaling pathways involved in this regulation remain poorly understood. Here we show that (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), an allosteric inhibitor of dual specificity phosphatases (DUSP) 1 and 6, enhances the expression of P2X7 receptor in N2a neuroblastoma cells. We found that exposure to BCI induces the phosphorylation of mitogen-activated protein kinases p38 and JNK, while it prevents the phosphorylation of ERK1/2. BCI enhanced dual specificity phosphatase 1 expression, whereas it induced a decrease in the dual specificity phosphatase 6 transcripts, suggesting that BCI-dependent inhibition of dual specificity phosphatase 1 may be responsible for the increase in p38 and JNK phosphorylation. The weaker ERK phosphorylation induced by BCI was reversed by p38 inhibition, indicating that this MAPK is involved in the regulatory loop that dampens ERK activity. The PP2A phosphatase appears to be implicated in the p38-dependent dephosphorylation of ERK1/2. In addition, the PTEN phosphatase inhibition also prevented ERK1/2 dephosphorylation, probably through p38 downregulation. By contrast, inhibition of the p53 nuclear factor decreased ERK phosphorylation, probably enhancing the activity of p38. Finally, the inhibition of either p38 or Sp1-dependent transcription halved the increase in P2X7 receptor expression induced by BCI. Moreover, the combined inhibition of both p38 and Sp1 completely prevented the effect exerted by BCI. Together, our results indicate that dual specificity phosphatase 1 acts as a novel negative regulator of P2X7 receptor expression in neuroblastoma cells due to the downregulation of the p38 pathway.
摘要：
P2X7受体（P2RX7）在大多数人类癌症中强烈表达，包括神经母细胞瘤,其中高水平的P2RX7与患者的不良预后相关。P2X7受体的强效激活有利于细胞代谢和血管生成，从而促进癌细胞增殖，免疫抑制,和转移。虽然了解神经母细胞瘤细胞中控制P2X7受体水平的机制可能是生物学和临床相关的，参与这种调节的细胞内信号通路仍然知之甚少。在这里我们显示（E）-2-亚苄基-3-（环己基氨基）-2,3-二氢-1H-茚-1-酮（BCI），双特异性磷酸酶（DUSP）1和6的变构抑制剂，增强N2a神经母细胞瘤细胞中P2X7受体的表达。我们发现暴露于BCI会诱导丝裂原活化蛋白激酶p38和JNK的磷酸化，同时阻止ERK1/2的磷酸化。BCI增强双特异性磷酸酶1表达，而它诱导了双特异性磷酸酶6转录本的减少，提示双特异性磷酸酶1的BCI依赖性抑制可能是p38和JNK磷酸化增加的原因。BCI诱导的较弱的ERK磷酸化被p38抑制逆转，表明该MAPK参与抑制ERK活性的调节环。PP2A磷酸酶似乎与ERK1/2的p38依赖性去磷酸化有关。此外,PTEN磷酸酶抑制也阻止ERK1/2去磷酸化，可能是通过p38下调。相比之下,抑制p53核因子降低ERK磷酸化，可能增强p38的活性。最后,p38或Sp1依赖性转录的抑制使BCI诱导的P2X7受体表达增加减半。此外，p38和Sp1的联合抑制完全阻止了BCI的作用。一起,我们的结果表明，由于p38通路的下调，双特异性磷酸酶1在神经母细胞瘤细胞中充当P2X7受体表达的新型负调节因子。
