Molecular characterization of MC3R and evaluation of its potential role as a modifier of lung function in cystic fibrosis

genetics;cystic fibrosis;modifier;MC3R;characterization;inflammation;NF-kB;not listed

Park, JeenahCutting, Garry R. ;

Johns Hopkins University

关键词: genetics;    cystic fibrosis;    modifier;    MC3R;    characterization;    inflammation;    NF-kB;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/40646/PARK-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Cystic fibrosis (CF), one of the most common lethal autosomal recessive disorder among Caucasians, affects approximately 30,000 individuals in the United States. CF is caused by loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene. Once the disease-causing gene for CF was identified, numerous studies attempted to correlate CFTR mutations with specific CF phenotypes because genotype-phenotype correlations can predict a course of the disease and lead to the design of a genotype-specific therapeutic strategy. Unfortunately, distilling correlation has been challenging for several reasons. First, CF is a multi-system disease that involves different organs and its phenotype is analyzed in context of its various clinical components. Second, patients exhibit a wide range of disease severity although CF is considered a classic Mendelian disorder. In fact, the degree of variability observed in 293 individuals with the identical CFTR mutations suggests that factors other than the CFTR genotype contribute to lung function variation. Given that obstructive lung disease is the cause of death in 90% of CF patients, there has been continued interest in determining these factors that influence the severity of pulmonary disease in CF patients.MC3R has been identified as a compelling candidate for modifying CF lung disease. Before exploring its role as a modifier, we aimed to understand the molecular organization of MC3R. Using 5’ RACE, we discovered a novel upstream exon that extends the length of the 5’ UTR in MC3R without changing the ORF. The full-length 5’ UTR directs utilization of an evolutionarily conserved second in-frame ATG as the primary translation start site. MC3R synthesized from the second ATG is localized to apical membranes of polarized Madin Darby Canine Kidney (MDCK) cells, consistent with its function as a cell surface mediator of melanocortin signaling. Expression of MC3R causes re-localization of a known accessory factor for MC2R, MRAP2, to the apical membrane, which is coincident with the location of MC3R.In contrast, protein synthesized from MC3R cDNAs lacking the 5’ UTR displayed diffuse cytosolic distribution and no effect upon the distribution of MRAP2. Our findings demonstrate that a previously unannotated 5’ exon directs translation of MC3R protein that localizes to apical membranes of polarized cells.To define the mechanism of lung disease in CF that underlies the linkage signal at chr20q13.2, we evaluated MC3R as a potential modifier of lung function. Three rare variants in MC3R were significantly associated with lung function in individuals with CF, suggestingthat variation in MC3R contibutes to CF lung disease severity. In silico prediction tools and in vitro studies were performed to understand the functional effect of the rare variants. We demonstrated that antagonist (siRNA) or agonist ([D-Trp8]-γ-MSH) of MC3R can modulate pro-inflammatory cytokine expression. Evidence for the presence of MC3R in primary airway epithelial cells of the lungs further supported our hypothesis that MC3R is a modifier of lung function in CF. These studies support the role of MC3R as a mediator of the inflammatory response and a potential therapeutic target in the treatment of CF.