Activation of Novel Signal Transduction Pathways by FP Receptors: The G-Protein Coupled Receptors for Prostaglandin F2alpha

Abstract

Prostaglandin F2 alpha (PGF(2α))is an arachidonic acid metabolite which plays an important role in cardiac hypertrophy and cancer. PGF(2α)is known to activate intracellular signaling pathways by interactions with its cognate G-protein coupled receptor named the FP prostanoid receptor. To date, the signal transduction pathways by which the FP receptor regulates gene expression have yet to be fully characterized. In this dissertation, multiple novel signal transduction pathways by the activation of FP prostanoid receptors involved in the regulation of gene expression have been identified and characterized. To study FP-dependent gene regulation, cDNA microarray technology was applied using HEK293 cells expressing FP receptors as a model. More than 150 genes, which could be classified into diverse functional groups, were identified to be significantly regulated through the stimulation of FP receptor in the cDNA microarray analysis. To confirm the results from microarray analysis, 20 significantly regulated genes from cDNA microarray analysis were subjected to Northern blot analysis. The expression profile of 14 out of these 20 genes was in agreement with that of cDNA microarray data. One of the 14 genes is early growth response gene 1 (EGR-1). EGR-1 is a transcription factor which has been shown to play important roles in the cardiac hypertrophy. Another gene identified is connective tissue growth factor (CTGF). CTGF belongs to CCN family, which play an important role in cancer angiogenesis. Cysteine-rich, angiogenic inducer-61 (Cyr61) was another member of CCN family. Gene regulation of Cyr61 through the FP receptor has previously been reported. FP receptor mediated gene regulation of the above three proteins was confirmed using Western blotting analysis. Following confirmation of FP receptor mediated gene regulation of EGR-1, Cyr61 and CTGF, the pathways responsible were dissected. We found that FP can activate Ras, which in turn activates C-Raf. Activation of C-Raf activates MEK1/2, and leads to the up-regulation of EGR-1. On the other hand, the studies demonstrated that activation of Ras through the FP receptor activate B-Raf, which can lead to the TCF/β-catenin pathway. TCF/β-catenin pathway regulates the expression of Cyr61. We also found that HIF-1α induced by the activation of FP receptor is involved in the regulation of CTGF expression. Moreover, we found that reactive oxygen species (ROS), a well-known activator of HIF-1α, is involved in the activation of TCF/β-catenin pathway, which leads to the gene regulation of HIF-1α. In conclusion, these studies have identified previously unknown signaling pathways and novel downstream effectors that are regulated by the FP prostanoid receptors. The identification of these novel interactions between the pathways activated by the FP receptor may have future applications in the treatment of heart disease and cancer.