Overexpression of G-Protein-Coupled Receptor 40 Enhances the Mitogenic Response to Epoxyeicosatrienoic Acids

Abstract

The cytochrome P450 epoxygenase-dependent arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), are potent survival factors and mitogens for renal epithelial cells, but the molecular identity in the cells that initiates the mitogenic signaling of EETs has remained elusive. We screened kidney cell lines for the expression of G-protein-coupled receptor 40 (GPR40) and found that the porcine renal tubular epithelial cell line LLCPKcl4, which has been previously demonstrated to be sensitive to the mitogenic effect of EETs, expresses higher levels of GPR40 mRNA and protein than the human embryonic kidney cell line HEK293. EETs induced only a weak mitogenic EGFR signaling and mild cell proliferation in HEK293 cells. To determine whether GPR40 expression level is what mediates the mitogenic sensitivity of cells to EETs, we created a human GPR40 (hGPR40) cDNA construct and transfected it into HEK293 cells and picked up a number of stable transfectants. We found that GPR40 overexpression in HEK293 cells indeed significantly enhanced EET-induced cell proliferation and markedly augmented EGFR phosphorylation ERK activation, which were inhibited by the EGFR tyrosine kinase inhibitor, AG1478, or the HB-EGF inhibitor, CRM197. EETs significantly enhanced release of soluble HB-EGF, a natural ligand of EGFR, into the culture medium of hGPR40-transfected HEK293 cells, compared to empty vector-transfected cells. In mouse kidneys, markedly higher level of GPR40 protein was found in the cortex and outer stripe of outer medulla compared to the inner stripe of outer medulla and inner medulla. In situ hybridization confirmed that GPR40 mRNA was localized to a subset of renal tubules in the kidney, including the cortical collecting duct. Thus, this study provides the first demonstration that upregulation of GPR40 expression enhances the mitogenic response to EETs and a relatively high expression level of GPR40 is detected in a subset of tubules including cortical collecting ducts in the mammalian kidney.

GPR40 is a low-affinity epoxyeicosatrienoic acid receptor in vascular cells

“…Additionally, EET regulation of Cx43 and COX-2 requires ERK phosphorylation (11,12). Activation of GPR40 also stimulates ERK phosphorylation in several cell types (38,51,52). We observed that the GPR40 antagonist GW1100 and GPR40 targeted siRNA and the MEK inhibitor U0126 blocked 11,12-EET-induced ERK phosphorylation in HUVECs.…”

“…In endothelial and epithelial cells, EETs stimulate ERK phosphorylation and cell proliferation (14,38,70).…”

“…Specifically, GPR40 is activated by µM concentrations of fatty acids of 12-22 carbons in chain length and is coupled to the G proteins Gs, Gq/G 11 and Gi (31)(32)(33). In cells overexpressing GPR40, 11,12-and 8,9-EET also stimulate an increase in intracellular calcium in µM concentrations as do long chain free fatty acids (32,38). GPR40 is highly expressed in the pancreas and central nervous system.…”

Epoxygenated Fatty Acids Inhibit Retinal Vascular Inflammation

“…The mechanistic details of EET and EDP bioactivities remain undetermined. A specific receptor for epoxygenated fatty acids is yet to be identified, although they do signal through a number of pathways, including PPARs and GPCRs30787980. The majority of evidence supporting EET anti-inflammatory activity points to signaling pathways that converge on the transcription factor NFκB.…”

Cytochrome P450 and Lipoxygenase Metabolites on Renal Function

“…EETs stimulated thymidine incorporation in LLCPKc14 cells with 14,15-EET being the most potent (27). In addition, upregulation of G-protein-coupled receptor 40 (GPR40) enhances the mitogenic response to EETs in HEK293 cells (84). Sulfonamide (SI) EET analogs, 11,12-EET-SI and 14,15-EET-SI were also potent proximal tubule cell mitogens (27).…”