The Function Of G Protein-coupled Receptor GPR41in Histone Acetylation

G protein-coupled receptor GPR41is highly conserved and widely expressed in different tissues in human, mouse and rat. GPR41was identified as an orphan GPCR. However, short chain fatty acids were described as the potential endogenous agonists for GPR41in2003. The de-orphaning of GPR41encouraged people to re-assess the role of free fatty acids and to investigate the mechanism of this receptor in physiological and disease processes. Short-chain fatty acids are histone deacetylase inhibitors, which plays important roles in gene regulation via its inhibition of histone deacetylase (HDAC). Studies have shown that the abnormity of histone acetylation may lead to human diseases, which mainly due to remodeling the key protein complex and affecting the normal expression of genes. Histone acetylation in transcriptional regulation has been a hot spot of epigenetics in recent years. It has been reported that butyrate treatment results in the hyperacetylation of histones and multiple biological effects, including inhibition of proliferation, inducement of cell cycle arrest, and/or apoptosis in a variety of cultured mammalian cells. Butyrate, as a histone deacetylase inhibitor, is also able to activate GPR41receptor. This phenomenon may prompt us that GPR41activation may be involved in histone acetylation, and the regulation of gene expression.There are few reports on GPR41receptor agonist, and the specific mechanisms of GPR41are not fully clear. Molecular biology and cell biology methods were performed to establish the GPR41cell line. Furthermore, we established cAMP and calcium assays to screen for the GPR41receptor agonists. Using flow cytometry and Western Blot analysis, we analyzed the role of GPR41in sodium butyrate induced histone acetylation. 1. The establishment of GPR41cell lineThe purpose of this study is to establish a stable hGPR41receptor cell model to screen the agonists of this receptor. First, human GPR41cDNA was cloned from human genomic DNA using PCR method and the sequence of the cDNA clone was confirmed. Using restriction enzyme digestion, ligation and transformation method, the PCR product was subcloned into mammalian expression vector pcDNA3.1-N-myc, and constructed the hGPR41-pcDNA3.1mammalian expression vector. Then, CHO cells were transfected with GPR41expressing vector alone or co-transfected with Ga plasmid. By antibiotic selection, the antibiotic resistant clones were analyzed by real time PCR and Western Blot analysis. Furthermore, forskolin and butyrate were applied to the cell line to evaluate the cell-based functional assay. The cAMP assay results showed that the activation of the receptor could decrease the accumulation of cAMP in GPR41-CHO. The calcium assay results showed that the GPR41activation could induce the calcium flow. These experimental results have shown that we have successfully established GPR41cell line which can be used for screening the agonists of the receptor in vitro.2. Preliminary screening of GPR41receptor agonistsIn the marine living resources, the exploration of marine fungi has received great attention due to its variety, wide distribution, large amount of secondary metabolites and bioactive substances. Using secondary metabolites extracted from aflatoxin c-f-3, which was from gulf seaweed in Putian Fujian, we have tested their receptor binding activity by cAMP assay. Using GPR41receptor cell lines,100nM,1μM and10μM of three concentrations of small molecule compounds were detected by cAMP and compared with control. Based on the primarily screening results, the second screening was performed. The results have shown that the number37compound has GPR41receptor agonist activity with high affinity. By structural analysis, it has been clear that compound37is a new compound, belonging to2-Pyrones. We also found the17th could induce increase of cAMP in a variety of cells. Thus,17th compound was speculated to be a phosphodiesterase inhibitor.3. The effects of GPR41on histone acetylation and related gene expressionHistone acetylation abnormalities may cause human disease, which mainly due to the mutation of key protein. These mutations may affect the normal expression of genes and induce human diseases. Histone acetylation in transcriptional regulation has become hot spot in epigenetics in recent years. It has been reported that butyrate treatment results in the hyperacetylation of histones and multiple biological effects, including inhibition of proliferation, inducement of cell cycle arrest, and/or apoptosis in a variety of cultured mammalian cells. Butyrate, as a histone deacetylase inhibitor, is also able to activate GPR41receptor. This phenomenon prompts us that GPR41activation may be involved in histone acetylation, and the regulation of gene expression. Our results have shown that activation of GPR41can reverse reduction of cell viability and apoptosis induced by sodium butyrate, and can affect cell cycle.4. The activity analysis of mutant GLP-1Glucagon-like peptide-1(GLP-1) and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases. Its short half-life has limited the application of GLP-1in the clinic. We generated a mutated form of human GLP-1(mGLP-1) using site-directed mutagenesis and gene recombination techniques, and found that these modifications significantly prolonged the biological half-life of GLP-1compared with native GLP-1(nGLP-1). This study investigated the role of mGLP-1on inducing PC12cell differentiation. mGLP-1induced PC12cell differentiation with neurite outgrowth and increased the expression of growth-associated protein-43and neuronal class Ⅲ β-tubulin, and significantly increased cyclic adenosine monophosphate level. No significant difference was found between mGLP-1and nGLP-1. The results indicate that mGLP-1activates the GLP-1receptor, induces PC12cell differentiation, and has neurotrophic effects.