The Preliminary Study Of Molecular Mechanism Of GPR12in Cell Proliferation,Survival, And Differentiation

G protein-coupled receptors (GPCRs), the largest and wildly expressed seven-transmembrane domain receptor family, were involved in the transduction of various extracellular signals such as photons, odorants, peptides, biogenic amines, proteases, pheromone, hormones, nucleotides, and lipids. The activation of their downstream effectors results in a number of biological and physiological functions through generating intracellular second messengers, such as cyclic AMP (cAMP), inositol1,4,5-trisphosphate(IP3), or Calcium (Ca2+). More and more investigations have revealed the existence of some GPCRs whose natural ligands have not yet been identified. Such GPCRs are thus referred to as "orphan GPCR (oGPCR)"GPR12, GPR3and GPR6belong to the same oGPCR family with constitutive activity, which means they can directly activate the downstream effectors to promote varieties of physiological changes in the absence of ligands. Sphingosylphosphorylcholine (SPC) was proved to be a high-affinity ligand of GPR12and their interaction may promote a number of physiological activities including cell proliferation, outgrowth extension, cell clustering, and maintenance of meiotic arrest. However, signaling pathways and mechanisms involved in the GPR12-mediated physiological and biochemical changes remain unclear. In the present study, heterologous expression of GPR12was demonstrated to promote the proliferation and survival in human embryonic kidney293(HEK293) cells, whereas GPR12overexpression induced neuronal differentiation with enlarged cell size and enhanced neurite outgrowth in pheochromocytoma cells of rat adrenal medulla (PC12). Biochemical analysis including fluorescence real-time quantitative polymerase chain reaction (real-time Q-PCR), immunocytochemistry, western blotting, and intracellular cAMP measurement were used to investigate the possible function and mechanisms of GPR12in cell proliferation, survival, and differentiation. 1. Identification of the functional expression of recombinant human GPR12Molecular biological analysis was used to construct recombinant human GPR12expressing vector which was then transfected into the nonneuronal cell line Chinese hamster ovary (CHO) cells. Real-time Q-PCR, western blotting, and intracellular cAMP assay were performed to detect the functional expression of human GPR12recombinant eukaryotic expressing vector. CHO subclones transfected with GPR12construct showed high expression of GPR12at both mRNA and protein level and an obvious constitutive activity characterized by directly increasing intracellular cAMP.In the previous study, Sphingosine-1-phosphate (S1p) was demonstrated to activate GPR12family and increase the intracellular cAMP level through the activation of the downstream Gs protein. However, SPC binds to GPR12with higher affinity than S1p does. Forskolin was reported as a broad-spectrum agonist of adenylate cyclase. Results from intracellular cAMP assay demonstrated that in the presence of forskolin or not,10μM of SPC can generate more cAMP than S1p, which is consistent with the results of previous study.2. Involvement of GPR12in the proliferation and survival of HEK293cells.Stable heterogenous expression of human GPR12in HEK293cells was firstly performed to confirm its functional expression with constitutive activity. MTT test and Alamar Blue assay were carried out to detect cell viability. The increase in both colourimetry and fluorimetry indicated that GPR12overexpression in HEK293cells resulted in the stimulation of cell growth. Besides, Ki67is an often-used molecular marker for cell proliferation. Immunocytochemistry revealed that Ki67expression ratio evaluated in a time-dependent manner; however, the proportion of cells expressing Ki67in GPR12group was significantly increased compared with the vector group, further confirming that GPR12promotes cell proliferation.Certain stress condition may induce cell apoptosis. MTT test and Alamar blue assay demonstrated that GPR12overexpression could significantly increase cell viability under serum deprivation compared with control cells. Flow cytometry analysis of apoptosis was also performed by using FITC-Anexin V/Propidium Iodide (PI) double-staining to confirm the protection of GPR12from apoptosis induced by serum starvation. Early apoptotic cells can be stained with annexin V/FITC, whereas the late apoptotic or necrotic cells were intensive with PI dye liquor. Present study showed that cell apoptosis could be induced by serum deprivation; HEK293cells highly expressing GPR12increases cell viability under stress and the apoptotic proportion in GPR12group was less than that in control group. The involvement of GPR12in promote cell survival indicated that the role of GPR12may be a critical regulator in cell apoptosis.3. Possible mechanisms of GPR12-mediated signaling pathwayMitogen-activated protein kinase (MAPK) signaling pathway was involved in various physiological phenomena including proliferation, mitosis, migration, apoptosis, survival, and cell interaction. Extracellular relative kinase (Erk) signaling cascade was first discovered as a classical signal-transduction pathway of MAPK family, which plays significant roles in cell proliferation and survival. B-cell lymphoma2(Bcl-2) was reported to be a key protector against cell apoptosis. Previous study showed that Bcl-2can directly activate erk signaling pathway, whereas Erk1/2can increase the expression or phosphorylation of Bcl-2. Bcl-2-associated X protein (Bax), a member of Bcl-2family, was known for its proapoptotic role in cell apoptosis by dimerization. The competitive binding between Bax and Bcl-2can efficiently inhibit cell apoptosis, further promoting cell survival.In the present study, results from western blotting showed that GPR12overexpression may transiently increase the phosphorylation level of erkl/2and the activation of Erk1/2is increased in a dose-dependent manner, revealing that activating Erk signal pathway may be involved in GPR12-mediated cell proliferation and survival.To further investigate the possible involvement of Bcl-2in cell survival promoted by GPR12, transient-transfection and RNA interference (RNAi) were performed to overexpress and knock down the expression of GPR12, respectively. Our data shows that RNAi of GPR12can significantly knock down the expression of GPR12at both mRNA and protein levels; besides, GPR12overexpression was demonstrated to increase Bcl-2and decrease Bax in a dose-dependent manner; whereas opposite changes were incurred by GPR12RNAi, indicating an active role of GPR12in protecting cells from apoptosis.In addition, the expression of tErk1/2was always altered in some physiological or psychological disorder. In the present study, positive alteration of tErk1/2expression level was also found in a dose-dependent manner with GPR12, indicating that GPR12may be a potential target for disease treatment.4. Neuronal differentiation of PC12cells induced by GPR12overexpressionPrevious research demonstrated that members in GPR12family overexpressed in Neuro2a neuroblastoma cells can promote striking morphological changes with neurite extension; SPC, as a high-affinity ligand of GPR12, was reported to induce neurite outgrowth in PC12cells in a dose-dependent manner, revealing that GPR2may be involved in neuronal differentiation of PC12cells.In the present study, PC12cells transfected with human GPR2recombinant construct demonstrated significantly enhanced neurite outgrowth and enlarged cell diameter. TuJ1and MAP2are neuronal markers that detect the early and late stages of neuronal differentiation, respectively. Results from immunocytochemistry demonstrated that Pc12cells could be labeled with both TuJ1and MAP-2in the perikaryon; GPR12overexpression in PC12cells displayed more intensity than those cells expressing vector either in single cell or the pellet, further confirming that GPR12overexpression can induce the transformation of PC12cells into neuron-like cell.5. Possible mechanisms of GPR12-meidated neuronal differentiation in PC12cellsIt was reported that GPR12family was involved in synapse extension and GPR12showed the most potent neurite outgrowth activity by overexpression of GPR12in rat cerebellar neurons; whereas in E15d rat cerebral cortical neurons highly expressing endogenous GPR12, SPC enhances cell clustering and increases the signal intensity of synaptophysin (SYP) immunostaining. Hence in our hypothesis, GPR12may play important roles in neuronal differentiation, neurite outgrowth, and synaptic plasticity. Real-time Q-PCR, western blotting, and immunocytochemistry were carried out to investigate the molecular mechanism of GPR12in PC12cells. GPR12was demonstrated to increase SYP in both mRNA and protein level and co-localize with SYP in the perikaryon and neurite outgrowth.Bcl-2and its family member Bcl-xL were both known for their particular roles in not only regulating cell survival and apoptosis but also promoting synaptic plasticity, neuronal survival, and differentiation. Previous study revealed that Bcl-xL overexpression in cerebellar neurons could promote synapse formation and upregulate SYP. Immunoblotting was performed and showed that GPR12was involved in regulation of Bcl-2, Bcl-xL and SYP by activation of Erk signaling pathway to promote differentiation of PC12cells into neuron-like cells.