| Low density lipoprotein receptor related protein 5 (LRP5), a member of low density lipoprotein receptor family, is a cell surface protein that binds and internalizes ligands in the process of receptor-mediated endocytosis. Full length cDNA of LRP5 gene was first cloned by Merk Lab and Bayer Research Center in 1998. LRP5 is the only gene in the candidate regions responsible for Osteoporosis-Pseudoglioma Syndrome (OPS). Analysis of gene mutation in the patients from different OPS family indicates that LRP5 gene mutation causes OPS. On (he other hand, researches on Lrp5 gene knock-out mice show that homozygote mice present the similar phenotype of human OPS and heterozygote mice show low bone mass.These results suggest that loss function mutation of LRP5 gene decreases the bone density. Other studies on LRP5 gene shows that gain-of-function mutation of LRP5 gene causes high-bone-mass trait. LRP5 is expressed in extensive tissues. The highest level of LRP5 expression is in the liver, with high levels of expression also being observed in pancreas, prostate, placenta and small intestine. The expression regulation elements and mechanism of LRP5 gene is unknown currently. Thus, it is very important to explore the expression regulation mechanism of LRP5 to understand the function of LRP5 gene.PART ONE Analysis of the Structure and Function of Mouse Lrp5 Cis-acling ElementsAccording to the sequence of mouse Lrp5 gene in Genbank, different length upstream fragments in the 1 Kb region from the translation initiation codon of mouse Lrp5 gene were inserted into pGLs-basic vector, which contained Firefly luciferase reporter gene without promoter and enhancer. Seven luciferase expression constructs which might contain mouse Lrp5 gene promoter were inserted into the clone site and sequenced, including PGL3-1O3 (-103 bp~+132 bp) ,pGL3-303 (-303 bp~+132 bp) ,pGL3-499 (-499 bp~+132 bp), pGL3-708 (-708 bp~+132 bp), pGL3-909 (-909bp~+132bp),pGL3-1034(-1034bp~+132bp)andpGL3-1229(-1229bp~ + 132 bp) . COS-7 cell line was cotransfected with seven expression constructs and pRL-TK using Fugene-6 as transfection reagent, then the relative luciferase activity in each cell lysate was measured after 48h. The relative luciferase activity of pGL3-K)3 was the highest, and the activity of pGL3-1229 decreased observably. The results suggest that the region from -103 bp to +132 bp contains an essential promoter for mouse Lrp5 gene transcription, there contains the negative control elements between the -1229 bp and -1034 bp .In order to screen the tissue-specific expression regulatory elements, seven expression constructs were transfected into osteoblast cell line (U2OS) respectively. Seven luciferase expression constructs showed no significant differences between U2OS and COS-7 cells. The results suggest that there is no tissue-specific expression regulatory elements in 1361 bp sequences (-1229 bp~ +132 bp).PART TWO Analysis of the Basal Promoter of Mouse Lrp5 GeneThe results of part 1 suggest that an essential promoter for mouse Lrp5 gene transcription would be located in the region from -103 bp to +132 bp.To identify the basal promoter of mouse Lrp5 gene, the region supposed to contain the basal promoter of mouse Lrp5 gene were amplified by PCR, then PCR products was cloned into the luciferase reporter vector pGL3-basic. and three luciferase expressionconstructs were constructed and sequenced, which were pGL3-16 (-16 bp~+132 bp),pGL3-54(-54 bp~+132 bp)and pGL3-103(-103 bp—+132 bp). Using Fugene-6 as transfeclion reagent, these expression constructs were transfected into COS-7 cell line respectively, then relative Iuciferase activity was measured after 48h. The relative Iuciferase activity of pGL3-103 was the highest, the activity of pGL3-54 was 80% of the highest activity, and the activity of pGL3-16 decreased significantly. The results suggest that the region from -54 bp to -16 bp includes an essential promoter sequence for mouse Lrp5 gene transcription. The region contains three SP1 elements, and these SP1 elements are necessary for the transcription of mouse Lrp5 gene.PART THREE Analysis of the Negative Regulatory Region of Mouse Lrp5 GeneThe results of the first part suggest that the negative control elements might be located in the region between the -1229 bp and -1034 bp .To identify the negative regulatory elements, primers were designed for PCR, PCR products were cloned into the pGL3-103 vector which contained mouse Lrp5 gene basal promoter and Iuciferase reporter gene. Three Iuciferase expression constructs were constructed and sequenced , which were pGL3-1229 (-1229 bp~-914 bp) , pGL3-1130 (-1130 bp~-914 bp) and pGL3-1051 (-1051 bp~-914bp) . Using Lipofectimine? 2000 as transfection reagent ,these constructs were transfected into COS-7 cell line respectively, then relative Iuciferase activity was measured after 48h. The relative Iuciferase activity of pGL3-1229 was 26% of pGL3-113O. The results suggest that the negative control elements are located from -1229 bp to -1130 bp. In the region NF1,GFI1,AP1 and COUP consensus sequences are identified using Mat Inspector V2.2 soft ware. COUP is the good candidate for negative control element in the region, mutation analysis is needed for further confirmation.PART FOUR Analysis of 5' Control Region of Mouse LrpS GeneTo further analyze the structure and function of 5' upstream control region ofmouse Lrp5 gene, the upstream region 3041 bp (-2909 bp~ +132 bp) from the translation initiation codon of mouse Lrp5 gene was amplified by PCR. The PCR product was cloned into the luciferase reporter gene vector pGL3-basic, and the recombinant plasmid pGL3-2909 was constructed. The different upstream regions were amplified using plasmid pGL3-2909 as a template, and then cloned into the pGL3-103 vector which harbored Lrp5 gene basal promoter and kiciferase reporter gene. Twelve luciferase expression constructs containing 5' upstream different regions of mouse Lrp5 gene were constructed, including pGL3-267, pGL3-513, pGL3-535, pGL3-560, pGL3-575, pGL3-623, pGL3-645, pGL3-719, pGL3-770, pGL3-1032, pGL3-1330 and pGL3-1619. All expression constructs were transfected into COS-7 respectively, the relative luciferase activity in each cell lysate was measured after 48h. The relative luciferase activity of pGL3-267 decreased observably compared with pGL3-103, it suggests there contains the negative control elements from -2909 bp to -2642 bp. The relative luciferase activity of pGL3-513 increased observably compared with pGL3-267, it suggests there contains the positive control elements from -2642 bp to -2396 bp.The negative control elements might exist in the region from -2396 bp to -2374 bp, as well as the region from -2349 bp to -2334 bp. The positive control elements might exist in the region from -2334 bp to -2286 bp, as well as the region from -1877 bp to -1290 bp. The consensus sequences in these control regions are identified using Mat Inspector V2.2 soft ware. Among them, deltaEFl might be the the negative control element, VBP and ER might be the the positive control elements of mouse Lrp5 gene. Mutation analysis is needed to further verify which element contributes to the regulation of mouse Lrp5 gene. |
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