Activation of novel signal transduction pathways by human EP1 prostanoid receptors: the G-protein coupled receptors for prostaglandin E2

摘要

The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is PGE₂. It is in the family of G-protein coupled receptors and is known to activate Ca²⁺ signaling, although it is unclear if this is mediated by coupling to G(q/11). Likewise little is known about other aspects of EP1 receptor signaling. In this dissertation, multiple novel signal transduction pathways have been identified and characterized following the activation of EP1 prostanoid receptors. To study EP1 receptor dependent gene regulation, HEK-293 cells stably expressing EP1 receptor were created and cDNA microarray technology was applied using this cell line as a model. One of the genes that was most upregulated is Nurr1. Nurr1 is an orphan nuclear receptor which has been shown to regulate both cell survival and cell death. The upregulation of Nurr1 expression was confirmed by western blot analysis and could be blocked using a dominant negative mutant of CREB and by the NF-κB inhibitor, BAY11-7082. Futhermore, we found a cAMP independent activation of PKA by stimulation of HEK-hEP1 cells with PGE₂, and these EP1 receptor mediated signalings are coupled to G₁₃/Rho signaling pathway. Stimulation of endogenous EP1 receptors in human neuroblastoma cells recapitulated the upregulation of Nurr1 observed in HEK cells. These findings have potential significance for understanding the mechanisms of the EP1 receptor mediated neurotoxicity. The other gene that was studied in this dissertation is HIF-1α, we have found that PGE2 stimulation of the EP1 receptor upregulates the expression of HIF-1α which can be completely blocked by pertussis toxin, indicating coupling to G(i/o). This upregulation of HIF-1α occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor and rapamycin, suggesting the activation of a phosphoinositide-3 kinase/Akt/mTOR signaling pathway. The upregulation of HIF-1α by the EP1 receptor involves increased translation. Stimulation of endogenous EP1 receptors in HepG2 cells recapitulated the normoxic upregulation of HIF-1α observed in HEK cells. HIF-1α is known to promote tumour growth and metastasis and is often upregulated in cancer. Our findings provide a potential mechanism by which increased PGE2 biosynthesis could upregulate the expression of HIF-1α and promote tumorigenesis.