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Studies on Function of G-protein Coupled Receptors GPR48 in Mice Bone Development

Author: LuoJian
Tutor: WuXiuShan;LiuMingYao
School: Hunan Normal University
Course: Genetics
Keywords: Bone development G protein -coupled receptors CAMP-PKA-CREB signaling pathway
CLC: Q954.4
Type: PhD thesis
Year: 2007
Downloads: 143
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Abstract


1. GPR48 function of vertebrate skeletal system development in bone development requires two completely different processes: the process of embryonic development and postnatal development. During embryonic development, the majority of bone formation by endochondral ossification that mesenchymal cells to differentiate into cartilage cells and was eventually replaced by osteoblasts. Postnatal development mainly bone reconstruction and maintenance of Serve bone cells to produce bone and osteoclasts absorb bone. All these processes are controlled by complex signal regulatory networks. In this paper, a G protein-coupled receptor GPR48/LGR4, it is an orphan receptor belongs to the glycoprotein hormone subfamily containing leucine-rich repeat sequences. Has yet to find the receptor ligand. G protein coupled receptors having seven transmembrane domains, it will be outside the cell signal transduction through its mediated three isomers Gα, β, γ to start the cascade of signals within the cell so that the cell signal outside into a cellular response, but about yet known whether the gene-skeletal system development. We use the mouse gene knockout method to study the biological function of G protein-coupled receptor GPR48 in bone development. The study showed that knockout GPR48 mouse embryonic bone formation delay than the wild-type mice, we use the Von kossa staining GPR48 wild-type and mutant mouse embryonic E14.5 period, E16.5 period and E18.5 period. Can be observed GPR48 - / - mutant mice bone mineralization significantly delayed. By in situ hybridization and immunohistochemistry experiments, we found that the three marker genes: the college type II; Indlan hedgehog, and transcription factors sex9 wild-type and mutant mice was no significant difference, suggesting that GPR48 knockout mice chondrocytes proliferation and hypertrophic differentiation of the early normal process unaffected. However, the of chondrocytes hypertrophy marker genes oolα1 (X) in GPR48 - / - mutant mice separation significantly higher than the wild-type night, which shows delayed knockout GPR48 the TERMINALS maturation of the cartilage cells of long bones. Also seriously affected, including bone mineral density, bone volume, bone formation rate and osteoid decreased bone volume / tissue volume, trabecular thickness, trabecular number, mineral adhesion rate of bone formation and bone formation after birth dynamic index reflects the reduction due to the deposition rate of bone mineralization and mineralized surface result in a 2 percent decrease in bone formation rate, a significant increase in trabecular bone gap. Phenotypic defects in bone development and function and gene knock out mice - ATF4 knockout mice phenotype in bone development is very similar. By real-time PCR, Western Blot and in situ hybridization, we found in the GPR48 gene knockout mice ATF4 expression did significantly decrease. Through GPR48 cAiVlP-PKA-CREB signaling pathway to regulate ATF4 promoter, thereby regulating ATF4 gene expression analysis, found by biochemical and signaling pathways. ATF4 in osteoblast differentiation and development process is a transcription factor, it regulates downstream bone matrix proteins OCN, BSP and collagen protein synthesis in GPR48 knockout mice also affected. That GPR48 through ATF4 and its downstream gene signaling pathways involved in bone formation. 2. A lot of other gene research I was also involved in the research work of the other genes, specifically in the following aspects: (1) Apolipoprotein new short peptide c-Src/FAK the selective inhibition of ERK signaling pathway to inhibit angiogenesis and tumor growth angiogenesis inhibitors derived from large plasma proteins. For example, the truncated fragments of human apolipoprotein kringle5 (KV) is a potential anti-angiogenic molecules. However, whether the KV domain of the existence of a short piece to play a role in angiogenesis and is accompanied by the growth of tumors and their mechanism of action is unclear. We choose a human AOP (a) KV domain of 11 amino acids of the peptides (11 peptides) for research. Angiogenesis the effect of this peptide by human umbilical vein endothelial cells (HUVEC) in vitro measurement, measured in vivo by the chick embryo chorionic analysis and mouse corneal angiogenesis model (CAM); We further measured the 11 peptides in tumor growth effect and it is involved in angiogenesis signaling pathways. In vitro angiogenesis in migration (p <0.01), invasion (p <0.01), and analysis (p <0.001) in all 11 peptide inhibit tube formation. In vivo, 11 peptide analysis (p <0.001) and corneal neovascularization model in mice (p <0.001) significantly inhibited angiogenesis in the CAM 11 peptide did not affect the growth of tumor cells, but it inhibits the growth of transplanted tumors (p <0.01), and 11 peptide-treated group, the survival rate higher than that of the control group (100% vs 20%, difference = 80%). In addition, 11 peptides selectively to the extracellular signal-regulated kinase (ERK) signal expression of this process hindered some protein Cdc42. The above experiments show that 11 peptides in vitro and in vivo inhibition of angiogenesis, and by selectively impede Cdc42 to the ERK signaling pathway to inhibit tumor growth, it may be located KV domain key activation zone. (2) GCIP in skeletal muscle differentiation and inhibit the function of skeletal muscle differentiation in cancer is a highly regular multi-step process, studies have shown that GCIP in C2C12 cells during the differentiation of the muscle cells of the mRNA and protein expression levels. a significant increase in the Department of overexpression of GCIP can activate the expression of muscle-specific genes, and to promote the formation of myotubes in C2C12 cells. While The GCIP the overexpression mutants to inhibit the muscle-specific gene expression, thereby inhibiting the occurrence of muscle. And GCIP able to interact directly with the E12, E47 and MyoD. This result suggests that GCIP as a new functional proteins in the muscle during and MyoD/E47 heterologous dimer form a functional complex to regulate myogenesis. Are found in many human cancers, including skin cancer, breast cancer, lung cancer and bladder cancer in chromosome 15 (15q and 15q21) the homozygote deletion and heterozygous Absence. Suggesting that there is a potential tumor suppressor gene in this region of chromosome 15. The GCIP positioning segment on chromosome 15 (15q15). We found that the expression of GCIP can strongly inhibit the occurrence of many human cancers, including breast, prostate, and colon cancer. In human colon cancer, of GCIP the mRNA and protein expression decreased, while colon cancer cells treated with sodium butyrate (sodium butyrate) SW80 can lead GCIP regulated expression. GCIP's function as a negative regulator of cell proliferation factor. Over-expression in colon cancer cell SW80 GCIP can cause significant inhibition of cancer cell colony formation, but with siRNA reduces The GCIP the expression was able to start the formation of cancer cells cloned. Moreover, overexpression of GCIP inhibited cyclin cyclinD1 promoter cyclinD1 thereby inhibiting the transcriptional activity. Our study shows that GCIP as a tumor suppressor gene can inhibit the occurrence of cancer. (3) I was also involved in proved GPR48 in front of the developmental regulation of eye development by regulating Pitx2; GPR48 through the regulation of the expression of the androgen receptor in the reproductive system to regulate the development of the urogenital system; proved E 2 by the ERα activation KiSS1 gene expression, ERα gene promoter on KiSS1 through the combination of the Sp1/Sp3 proteins and the use of the Sp1/Sp3 recognition combined with GC-rich region to stimulate KiSS1 gene transcription. This helps us to understand the role of the steroids on KiSS1 feedback regulation at the molecular level. Besides, I also participate in human ZNF394, ZNF4805NZNP411 and gene cloning, expression and functional studies, the majority of these genes are expressed in the heart, the heart development of candidate genes.

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CLC: > Biological Sciences > Zoology > Animal morphology > Animal Embryology ( of animals,animal viviparous learn )
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