The Constitutively Active Orphan G-protein-coupled Receptor GPR39 Protects from Cell Death by Increasing Secretion of Pigment Epithelium-derived Growth Factor

Abstract

GPR39 is a constitutively active orphan G-protein-coupled receptor capable of increasing serum response element-mediated transcription. We found GPR39 to be up-regulated in a hippocampal cell line resistant against diverse stimulators of cell death and show that its overexpression protects against oxidative and endoplasmic reticulum stress, as well as against direct activation of the caspase cascade by Bax overexpression. In contrast, silencing GPR39 rendered cells more susceptible to cell death. An array analysis of transcripts induced by GPR39 revealed up-regulation of RGS16 (inhibitor of G-protein signaling 16), which suggested coupling to G␣ 13 and induction of serum response element-mediated transcription by the small GTPase RhoA. In line with this, co-expression of GPR39 with RGS16, dominant-negative RhoA, or serum response factor abolished cell protection, whereas overexpression of the serum response factor protected from cell death. Further downstream the signaling cascade, GPR39 overexpression leads to increased secretion of the cytoprotective pigment epithelium-derived growth factor (PEDF). Medium conditioned by cells overexpressing GPR39 contained 4-fold more PEDF, and when stripped off it lost most but not all of its protective properties. We conclude that GPR39 is a novel inhibitor of cell death, which might represent a therapeutic target with implications for processes involving apoptosis and endoplasmic reticulum stress like cancer, ischemia/ reperfusion injury, and neurodegenerative disease. G-protein-coupled receptors (GPCRs)2 constitute the largest family of cell surface transmembrane proteins (1); they are activated by a wide variety of natural ligands, and pharmacological alteration of their signaling constitutes one of the most successful approaches to the treatment of human disease, which makes GPCRs the most targeted protein superfamily in pharmaceutical research (2). We recently presented a screening system able to discriminate protective and detrimental receptors involved in oxidative stress (3) to identify targets for the multitude of human diseases caused or aggravated by oxidative stress. In this proof-of-concept study, we noticed that the relative mRNA content of cytoprotective GPCRs (the most prominent being VPAC 2 , a receptor for the neuroprotective vasoactive intestinal peptide VIP) was increased in glutamate-resistant (HT22R) cells generated by repeated exposure of the parental cell line HT22 to high concentrations of glutamate and further propagation of the few surviving cells. HT22 cells are derived from embryonal mouse hippocampal cells and are considered to be a model system of cell death by oxidative stress. In this model system, increased extracellular glutamate blocks the gradientdriven glutamate/cysteine antiporter X c Ϫ , depleting the cells of cysteine. Cysteine is required for the synthesis of the important antioxidant glutathione, and the sequence of events after depletion of intracellular glutathione involves the activation of 12-lipoxygenase, the accumulation of in...

TOX3 is a neuronal survival factor that induces transcription depending on the presence of CITED1 or phosphorylated CREB in the transcriptionally active complex

“…We became interested in TOX3, when we found it to be induced in concert with CITED1 downstream of a cytoprotective signal 253 TOX3 cell protection transduction cascade in HEK-293 cells stably overexpressing the constitutively active receptor GPR39 (Dittmer et al, 2008). We aimed to reproduce this induction at the protein level in neuronal cells and transiently overexpressed either empty vector or GPR39 in Neuro2a cells (which express TOX3, see supplementary material Fig.…”

“…Overexpression of the pro-apoptotic protein BAX, by contrast, leads to release of cytochrome c from mitochondria and direct activation of the caspase cascade. GPR39, which is upstream of TOX3 and CITED1, protects against both stressors (Dittmer et al, 2008). Cell death was quantified by flow cytometry by gating for early plus late apoptotic cells [cells double-positive for annexin V and 7-aminoactinomycin D (7-AAD)].…”

“…We recently found TOX3 to be expressed in concert with CITED1 (for 'CBP/p300-interacting transactivator with glutamicacid-and aspartic-acid-rich C-terminal domain 1') in human embryonic kidney HEK-293 cells overexpressing the constitutively active orphan G-protein-coupled-receptor GPR39 (Dittmer et al, 2008). GPR39 overexpression protects against a wide variety of cellular stressors by induction of serum-response element (SRE)-mediated transcription (Dittmer et al, 2008).…”

“…GPR39 overexpression protects against a wide variety of cellular stressors by induction of serum-response element (SRE)-mediated transcription (Dittmer et al, 2008). CITED1 is a transcriptional regulator, which, on its own, lacks DNA-binding properties but enhances transcription mediated by diverse transcription factors, such as estrogen receptors (Yahata et al, 2001), SMAD (Shioda et al, 1998), or early growth response (EGR) 2 (also known as Krox20) (Dillon et al, 2007).…”

Genome-wide gene expression profiles of ovarian carcinoma: Identification of molecular targets for the treatment of ovarian carcinoma

“…The high expression level of homeobox member gene HOXB7 mRNA reveals its possible involvement in the invasive characteristics of ovarian cancer cells (37,38). GPR39 was found to be a novel inhibitor of cell death, which might represent a therapeutic target with implications for processes involving apoptosis and endoplasmic reticulum stress in cancer (39). CP is associated with an unfavorable prognosis and poor outcome in patients with ovarian cancer (40).…”