An Adhesion Family G Protein-coupled Receptor GPR126Regualtes Angiogenesis And Colorectal Cancer Progression

G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors (7TM receptors) comprise the largest protein family of transmembrane proteins. There are nearly800different human GPCR genes, about4%of the entire protein-coding genome, being predicted from genome sequence analysis. When ligands binding and activating these receptors, they transducer intracellular signal molecules, such as cAMP, cGMP, NO, DAG and IP, which modulating cell proliferation and differentiation, cell migration and death, thus participating in nearly all physiological processes. Due to broad spectrum of physiological functions and structural advantages, GPCRs are the most successful targets for moden medicine.Functional investigation of GPCR, a frontier field for medicinal chemistry, clinical research and drug discovery, has been shown to be important for pharmaceutical industry and human health. However, many members of the GPCRs have not been well characterized, and even more than half of them are still orphans, meaning their endogenous ligands are still unknown. As the second largest family, adhesion GPCRs are commonly considered to be involved in interaction of cell to cell, or cell to ECM, but the precise physiological roles of most of the adhesion GPCRs are to be identified. GPR126, as a member of adhesion GPCRs, is identified to be required for Schwann cell maturation in zebrafish and mouse during peripheral nerve development. GPR126knock-out mice were observed a sharp loss of viability occurs from10.5dpf to12.5dpf, accompanying with intra-embryonic hemorrhage in about50%(24/49) of embryos. And GPR126was initially isolated from HUVEC cultures that had been challenged with lipopolysaccharides or thrombin. All these indicate that GPR126might be implicated in endothelial cell function and in vascular development including processes of vasculogenesis and angiogenesis. In this thesis, we started the investigation of GPR126from its expression in endothelial cells, and subsequently revealed the role of GPR126in vascular angiogenesis and colon cancer progression.I. Role of GPR126in angiogenesis 1. Gpr126is highly enriched in endothelial cells and induced by pro-angiogenic factors in HMEC-1cellsUsing model of embryoid bodies (EB) formation and subsequent differentiation of embryonic stem cells, we identified Gpr126mRNA expression was highly enriched in Flkl-positive cells at day4of EB formation. And immunohistochemistry staining revealed that GPR126positive cells were specifically detected in the endothelial cells of vessels of embryonic limb bud, lung, heart and kidney. Western blot showed that GPR126expresses in HUVEC and HMEC-1cells. Meanwhile, VEGF, IGF-1and FGF-2increased GPR126expression in HMEC-1cells. And also RT-PCR assay revealed GPR126mRNA is highly enriched in Flk+endothelial cells sorted from zebrafish.2. Knockdown of GPR126impaired endothelial sprout formation in three-dimensional collagen, and also inhibited endothelial activity of proliferation, migration and tube formation.3. Inhibition of GPR126expression impairs physiological and pathological angiogenesis. Matrigel Plug assay and oxygen-induced retinopathy assay demonstrated that GPR126regulated VEGF induced angiogenesis in matrigel and ischemia-induced retinal neovascularization.4. Silencing of GPR126causes angiogensis defects during embryogenesis. Knockdown of Gpr126in zebrafish resulted to defects of intersegmental vessel formation, while coinjecting antisense morpholinos targeting Gpr126(Gpr126MO) with human GPR126mRNA in the embryos restored intersegmental vessel formation. Further investigation showed that antisense morpholinos targeting Gpr126dramatically reduced cell numbers of segmental arteries. The phenotypes of zebrafish Gpr126knockdown showed that the formation of dorsal aorta and the subsequent ECs sprouting from dorsal aorta to the horizontal myoseptum were obviously affected, and the subsequent endothelial tip cell migration and proliferation were severely inhibited.5. GPR126regulated VEGFR2expression via STAT5and GATA2.In GPR126deficient HMEC-1cells, VEGF induced ERK and FAK activities were downregulated. Further GPR126was found to regulate VEGFR2transcription. By RT-PCR and western blot methods, we identified both mRNA and protein of STAT5and GATA2were down-regulated. While both GATA2and STAT5were demonstrated to directly bind to the promoter of VEGFR2and regulate expression of VEGFR2in HMEC-1cells. Forskolin, a PKA agonist, increased CREB activity and upregulated GATA2and STAT5expression in dose dependent way in HMEC-1cells. Furthermore, chip assays revealed that there contain direct binding in promoters of GATA2and STAT5respectively.Together, GPR126regulates GATA2and STAT5expression through PKA-CREB pathway, through which further modulates VEGFR2expression in HMEC-1cells.6. STAT5and GATA2restored the angiogenic activity of endothelial cells attenuated by silencing of GPR126in vivo and in vitro. Coinjection of GATA2mRNA or STAT5mRNA with Gpr126MO partially restored ISVs growth which was disrupted by Gpr126MO alone. When STAT5and GATA2mRNA together were introduced with Gpr126MO at the same time, the impaired ISV morphology was greatly rescued than individual injection of STAT5mRNA or GATA2mRNA. And forced expression of STAT5and/or GATA2in GPR126deficit HMEC-1cells restored the accumulated length of tubes significantly compared to that of GPR126knockdown group in tube formation assay, and also regained VEGF induced activation of ERK and FAK which were attenuated by GPR126knockdown.II. Role of GPR126in Colorectal Cancer Proliferation1. GPR126overexpressed in colorectal cancer cellRT-PCR and Western blot experiment showed that GPR126mRNA and protein level was high. Taking advantage of immunohistochemistry staining, we confirmed that GPR126expression in the malignant tissues from colon cancer is significantly higher than that in tissues beside the malignant ones from colon cancer.2. Knockdown of GPR126in vitro inhibited proliferation of colorectal cancer cells.3. Knockdown of GPR126inhibited tumor growth in vivo. Using xenograft nude mice, we demonstrated that silence of GPR126in HT-29, HCT116and Lovo cells inhibited tumor growth.4. GPR126might regulate proliferation of colorectal cancer cells via mTOR pathway. RNA sequencing technology was used to investigate the mechanism of GPR126modulating proliferation of colorectal cancer cells. The result showed that knockdown of GRR126in HT-29cells decreased mTOR protein.