Studies On Function Of G-protein Coupled Receptors GPR48 In Mice Bone Development

1. Study on GPR48's function in bone developmentThe integrity of the vertebrate skeletal system needs two distinct regulatory processes: the embryonic developmental and postnatal regulatory processes. During the embryonic developmental, the majority of bone formation is via endochondral ossification, as mesenchymal progenitor cells differentiate into chondrocytes that are eventually replaced by osteoblasts. And the postnatal regulatory processes, referred to bone remodeling, bone is constantly regenerated through continuous formation by osteoblasts and resorption by osteoclasts. Both of processes are well controlled by a complex transcriptional network in which the transcription factors ATF4 play essential roles. The bZip factor activating transcription factor 4(ATF4)was recently shown to regulate osteoblast biology. Yang et al., (2004)showed that ATF4 can regulate osteoblast differentiation, typeâ… collagen synthesis, osteoblast-specific gene BSP and OCN expression, and osteoblast terminal differentiation. ATF4 was shown to regulate osteocalcin gene transcription by binding to its promoter. Mice deficient for ATF4 are runted and harbor low bone mass, and ATF4 can direct physical interact with Runx2, a transcription factor that is essential for osteoblast and hypertrophic chondrocyte differentiation and bone formation during embryogenesis and postnatal life, then modulates Runx2 transcriptional activity.G-protein-coupled receptors(GPCRs, or GPRs)containing seven transmembrane domains and transduction extracellular signals can initiates a cascade of intracellular processes through heterotrimeric G-proteins. And among G-proteins, theα-subunit of the stimulatory G-protein, Gsα, which in turn increases synthesis of cAMP by adenylyl cyclases(AC)and activates protein kinases A(PKA). GPCRs form the largest family of cell surface receptors known and defects in GRCR function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations.2. The other genes' workI also co-work on other project, show as below,(1) A Novel Peptide from Human Apolipoprotein(a) Inhibits Angiogenesis and Tumor Growth via Selectively Blocking c-Src/FAK to ERK Signaling Pathway.Many angiogenesis inhibitor are derived from larger plasma proteins. For example, a truncated fragment of kringle 5(KV)domains from human apolipoprotein(a)was potent antiangiogenic molecules. But whether there are shorter fragments in KV domain that can play a role in angiogenesis together with tumor growth and the mechanism involved in these functions have not been shown. Using bioinformatics software, an 11-amino acid peptide(11peptide) in human aop(a)KV domain was selected for study. Angiogenesis effect of this peptide was measured by human umbilical vein endothelial cell(HUVEC)in vitro, and in vivo chicken embryos chorioallantoic membrane(CAM)assay, together with the mouse corneal angiogenesis model; Further we measured the 11 peptide effect on xenograft tumor growth and its role in signaling pathway involved in angiogenesis. Angiogenesis in vitro was inhibited by 11 peptide in migration(p＜0.01), invasion(p＜0.01), and tube formation assays(p＜0.001). In vivo, 11peptide significantly inhibited angiogenesis in CAM assay(p＜0.001)and mice corneal vascular models(p＜0.001). 11peptie can not affect the growth of tumor cell but inhibit the growth of xenograft tumor(p＜0.01), and the survival of 11peptide-treated group was higher than that in control group(100ï¼…versus 20ï¼…, difference=80ï¼…). Also, 11peptide selectively blocked the expression of some proteins in Cdc42 to extracellular signal-regulated kinase(ERK) signals. 11peptide inhibits angiogenesis in vitro, in vivo and suppress tumor growth by selectively blocking Cdc42 to ERK signaling pathway, and may be in the key active region of KV domain.(2) GCIP's function in myogenic differentiation and tumor suppressorDifferentiation of skeletal muscle is a highly ordered multi-step process called myogenesis, which involves the expression of muscle-specific genes, withdrawal of cell cycle and formation of multinucleated myotube. In this study, we show that GCIP/CCNDBP1, a recently identified HLH leucine-rich protein without a predicted basic DNA binding region, regulate muscle specific gene expression and E47/MyoD heterodimerization. GCIP is highly expressed in muscle tissue. Both the mRNA and protein expression levels of GCIP were up-regulated during myogenic differentiation of C2C12 cells. Over-expression of GCIP in C2C12 cells promotes E47/MyoD complex association, activation of muscle specific transcription, and myotube formation during skeletal muscle cell differentiation while the mutant form of GCIP reduced the E47/MyoD heterodimerization and inefficient muscle differentiation. These findings identify a novel pro-myogenic role for the recently identified GCIP/CCNDBP1 protein in forming a functional protein complex with MyoD/E47 heterodimers that are essential for myogenesis.Deletions and/or loss of heterozygosity(LOH)on chromosome 15(15q15 and 15q21)has been found in several human tumors, including carcinomas of the colorectum, breast, lung, prostate and bladder, suggesting the presence of potential tumor suppressor gene(s)in this particular region of chromosome 15. GCIP also called CCNDBP1, DIP1 or HHM, localized at chromosome 15q15, is a recently identified helix-loop-helix leucine zipper(HLH-ZIP)protein without a basic region like the Id family of proteins. In this study, we reported that the expression of GCIP was significantly down-regulated in several different human tumors, including breast tumor, prostate tumor and colon tumors. In human colon tumors, both mRNA and protein expression levels of GCIP were decreased significantly compared to the normal tissues. Treatment of colon cancer cells SW480 with sodium butyrate(NaB), which induces colon cancer cell differentiation, can induce the up-regulation of GCIP expression, suggesting that the protein functions as a negative regulator in cell proliferation. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation while silencing of GCIP expression by siRNA can promote cell colony formation. Furthermore, overexpression of GCIP inhibited the transcriptional activity of cyclin D1 promoter and the expression of cyclin D1 protein in the cell. Finally, we demonstrate that GCIP specifically interacts with one of the classâ…¢HDAC proteins, SirT6, which is important for maintaining genome stability. Together, our data suggest a possible function of GCIP in tumor suppression.(3)We also prove that Regulation of Ocular Anterior Segment Dysgenesis(ASD)by GPR48 through PITX2 in eye development and Regulation of genitourinary dysplasia by GPR48 through androgen receptor in reproductive system. We found that E₂ dependent transcriptional activation of the KiSS1 gene is mediated by ERαthrough the interaction of Sp1/Sp3 proteins with GC-rich motifs of KiSS1 promoter, providing a molecular mechanism in our understanding how steroid hormones feedback regulate KISS1 expression in the HPG axis. Identification and characterization of human novel genes including ZNF394, ZNF480 and ZNF411, some of which are expressed in theheart tissue. Through gene knockout method, GPR48/LGR4 has been identified as an orphan GPCR involved in bone and eye development.