A Study On Pulmonary Biological Effects, Gene Expression Modulation And Natural Ligand Screening Of BRS-3

Objectives:A large number of neuropeptides in lung tissues-suggests theinterplays among these molecules might be important in fine-tuning oflung function. The highest BLPs levels were observed in human fetal lung,localized to the pulmonary neuroendocrine cells, which contribute todiverse biological function in lung. To date, three mammalianbombesin-like receptors have been cloned including GRP receptor,neuromedin B receptor, and the orphan bombesin receptor subtype 3.Bombesin receptor subtype 3 (BRS-3) is a 399-amino acid protein and has50% homology to the other two members of the bombesin receptor family.Its expression level in the majority of normal tissues is extremely low,whereas in the developing lung and certain lung carcinomas it is high.BRS-3 cDNA was also first isol-ated from a small-cell lung cancer,suggesting a role of BRS-3 in the regulation of cell growth and woundrepair. Up to now, there is very little known about the pulmonarybiological effects, gene expression modulation and interacted proteins ofBRS-3. This is in great part because the natural ligand of BRS-3 has not-been identified as of today. The present study was designed to observe thepulmonary biological effects, gene expression modulation and naturalligand of BRS-3.Contents:1. Study on pulmonary biological effects of BRS-3New Zealand rabbits were inhaled a mixture of 1.5ppm ozone andfresh air for two, four, and eight days respectively to establish a airwayhyperresponsiveness animal model' and the time and spatial expression ofBRS-3 were examined by ISH. The results showed that, in the normalgroup, the structure of bronchi walls and pulmonary alveoli were intact andregular, which showed no sigri of inflammationary infiltration, mucosalexudation, and cavitary stricture and there was few expression of BRS-3mRNA. However, inflammationary infiltration, mucosal exudation,cavitary stricture and bronchial epithelial denudation were observed in the ozone-stressed group. The expression of BRS-3 mRNA was detectable inthe early two days after ozone stress, increased with the elongation ofozone stress, peaked on the fourth day, and then reduced. BRS-3 mRNAwas predominantly localized in the ciliated columnar-epithelium ofbronchioles, monolayer columnar epithelium cells of terminal bronchioles,scattered mesenchymal cells and Typeâ…¡alveolar cells. No BRS-3mRNA was detectable in Typeâ… alveolar cells. The results also showedthat few expression of BRS-3 mRNA was detectable in the unstressedhuman BECs, but its expression was explicitly increased in ozone-stressedBECs.To investigate the significance of up-regulation of BRS-3 in AHR, theeffects of BRS-3 activation on wound repair and proliferation of BECswere examined. A small wound was made in the confluent monolayer bymechanical scraping and the remaining wound area was measured seriallyper 4 hours in 24h by video microscopy. A linear regression equation ofthe remaining wound area to time was obtained and the slope was used tojudge the repair speed of BECs. The proliferation of BECs was assayed byMTT. The results showed that the wound repair and proliferation of BECswere accelerated in a concentration-dependent manner by P3513, whichcould be inhibited by PKA inhibitor H89. The above effects were sloweddown after treatment with BRS-3 ASO.The effects of protection against injury of BRS-3 were also observed.The ³H-UdR release rate, LDH activity were measured as injury indicatorof BECs and catalase activity was assayed as antioxidant ability. Theresults showed that P3513 decreased the ³H-UdR release rate, LDHactivity induced by ozone and increased catalase activity in ozone-stressedBECs.2. Study gene expression modulation of BRS-3To probe the mechanisms of the induced expression of BRS-3 underozone stress, ten oligonucleotide probes corresponding to various regionsof the BRS-3 promoter were used in EMSA studies. Four were found tohave an enhance mobility shift with extracts from ozone-stressed cells.Based on the assay of mutated probes and antibody supershift, they were verified as MTF-1, PPARα, AP-2αand HSF-1. By ChIP assay, onlyAP-2αand PPARαincreased the ozone-inducible DNA binding on theBRS-3 promoter.Next, site-directed mutagenesis technology and antisenseoligonucleotide technology were used to observe the inhibitory effects ofthe four nuclear factors on BRS-3 promoter activation and expression. Theresults also showed that only AP-2αand PPARαincreased theozone-inducible DNA binding on the BRS-3 promoter and BRS-3expression.The translocation of PPARαwas observed by immunottuorescenceassay, which showed that PPARαnuclear translocation increased afterozone exposure. The time courses of AP-2αand PPARαactivation,followed by BRS-3 expression were also examined. It was shown thatozone-inducible BRS-3 expression and AP-2αand PPARαbinding activitycorrelated during a-48 hour time course.3. Screening of interacted proteins of BRS-3A library screen based on the two-hybrid system was performed toidentify proteins that interact with human BRS-3. In this study, the humanfetal brain BacterioMatch^TM two-hybrid system XR cDNA library in thepTRG plasmid (Stratagene) was screened with the constructed pBT-BRS-3using the protocol provided by the manufacturer with some modification.Briefly, pBT-BRS-3 and pTRG-cDNA library ptasmids were firstintroduced into two-hybrid reporter strain, and transformants were selectedon 3-AT plates. Then, the selected colonies were screened by growth on3oAT and streptomycin. To confirm the detected protein-proteininteractions, we then retransformed the reporter strain with the isolatedtarget plasmid plus bait ptasmid as described by the manufacturer. Theinteracted proteins of BRS-3 were verified by Pull-Down assay. The resultsshowed that BRS-3 can interact with gastrin-releasing peptide, ciliaryneurotrophic factor, protease inhibitor 5, serine protease inhibitor, proteintyrosine kinase, protein kinase C beta 1, protein tyrosine kinase 2 alpha 1.These proteins have very wide function including cell growth,differentiation, apoptosis, cytoskeletal construction, tyrosine kinaseactivities and so on. By employing the bioinformatics method to analysethe four kinds of new genes, it was shown that, except that a kind of gene does not have intact ORF, and can't extend to obtain intact cDNA, other 3kinds are high expressed in fetal brain, fetal lung, embryo stem cell andtumour cells including 2 kinds of membrance proteins and 1 kind of signalprotein.4) Natural ligand search of BRS-3Firstly, a high expression cell line was built by transfection, andtissues, which consist natural ligand of BRS-3, were preliminarily screenedby [Ca²⁺] assay. It was shown that less than 25 kD components of humanfetal lung tissues may consist natural ligand. Then the tansfected andun-transfected COS-7 cells were collected, and incubated with human fetallung tissues at 4℃. Then the cells were lyses, and co-immunoprecipitatedwith anti-flag antibody. The natural ligand was solved by incubation with40μl PBS at 35℃for 30 min, which was assayed by Tricine-SDS-PAGE.The results showed that the elution can induce [Ca²⁺] change, and consistof a kind of 1kD size polypeptide, the concentration of which wasrelatively high in immune precipitate without elution. Cut the band andcarry on N end amino acid sequening. The result reveals that thispolypeptide is 10 peptides small molecules and has no the prominenthomology in various protein databases. By radioligand binding assay andcompetion assay, the result reveals that the receptor quantity is 80fmol/10⁵ cells, K_d is 0.17 nmol/L, and the binding can be competed by 100times unlabeled ligand.Conclusion:The present study demonstrates that BRS-3's natural ligand consistsof 10 amino acids. BRS-3 can interact with many proteins, whichparticipate in cell signaling and modulation. A robust transcriptionalBRS-3 up-regulation occurs during acute ozone-induced airwayhyper-responsiveness and is mediated at least in part by ozone-inducedrecruitment of PPARαand AP-2αto the human BRS-3 promoter therebyameliorating the effects of lung injury.