Interaction Of Antiflammin-1 With Uteroglobin-binding Protein And Functional Correlates

Uteroglobin(UG),also called Clara cell 10 kDa protein(CC10),is the founding member of the newly formed protein superfamily,secetroglobin 1A1. It is an evolutionarily conserved protein that has multiple physiological functions.In a series of experiments it was demonstrated that UG has potent anti-inflammatory,anti-chemotactic and immunomodulatory properties.UG also imparts a protective effect to the lung during oxidant injury and suppresses allergen induced TH2 differentiation.However,the molecular mechanisms regulating these important cellular events are not well understood.A recent interest in the field has been generated by the observation that UG may bind to a specific membrane protein,UG-binding protein.However,many aspects concerning the UG-binding protein-ligand interaction are very unclear at this moment.The paper is divided into four chapters.The first chapter presents briefly the combined bioinformatic analysis and experimental results to indicate the posibility of mouse UG-binding protein(mUGBP)as a membrane protein.The second chapter outlines evidences for antiflammin-1(AF-1),a nonapeptide fragment of UG,binding with mUGBP on the surface of living cells,while the downstream signaling to extracellular signal-regulated kinase are discussed in the third chapter.The final chapter lists our preliminary findings concerning the functional correlates of mUGBP.The principal findings of this study are threefold,as follows:we provided visual confirmation that UG-binding protein is predominantly present in NIH 3T3 cell membrane.Next,the current data implies that UG-binding protein is essential for AF-1 binding to specific cell types and possibly contributes to the subsequent signaling;and the finding that UG-binding protein is involved in regulating AF-1-mediated ERK phosphorylation gives new insight for a possible receptor-mediated mechanism of action responsible for AF-1.Further, in vivo results point to the likelihood that the induction of mUGBP in bleomycin-injured murine lungs is protective during fibrogenesis,and the latent role of mUGBP likely correlates with collagen expression after fibrotic stimuli, although the precise mechanism awaits further analysis.ChapterⅠBioinformafic Prediction and Experimental Verification of Mouse Uteroglobin-Binding Protein as a Membrane ProteinWe have undertaken a combined bioinformatic and experimental approach to examine the subcellular location of mUGBP protein.Firstly,the bioinformatics analyses revealed that the mUGBP cDNA encodes a membrane protein of 489 amino acids with a predicted molecular mass of 55 kDa.Hydropathy analysis of the deduced amino acid sequence using several computer programs strongly suggests a transmembrane topology with nine putative transmembrane domains.This structural kinship predict that the mUGBP protein is most likely to play a role as a membrane protein(possibisity of 60.9%)for as yet unknown functions,a notion now amenable to molecular investigation.Secondly,we verified cell membrane localization of the protein by expressing enhanced green fluorescent protein(EGFP)-tagged mUGBP in COS-1 cells.We also validated a polyclonal antibody raised against mUGBP;and the cell membrane location of this protein was further confirmed by immunocytochemistry and Western blot analysis of membrane fractions of murine NIH 3T3 cells using the specific mUGBP antibody. ChapterⅡEvidence for Antiflammin-1 Binding with Uteroglobin-Binding Protein on the Surface of Living CellsGrowing evidence supports that UG-binding protein(UGBP)plays critical role in UG mediated physiological activity.However,the specific epitope of UG that interacts with this receptor has not yet been identified.Of interest,out of UG protein more than 400 amino acid long,small peptides drawn for the C-terminal region retain biological activity.Studies have revealed that peptides termed AFs,including AF-1,have been reported to retain most of the effects of the full-length protein in in vivo and in vitro systems.AF-1 has proved to be a potent bioactive nonpeptide derived from UG,and is widely studied for its powerful anti-inflammatory and anti-chemotaxis activity.But how AF-1 activates the intracellular signaling pathway remains elusive.To test the hypothesis that AF-1 might interact with mUGBP,we have used several criteria to determine that in responder cells AF-1 binding is an UG-binding protein-mediated process.Firstly,the interaction between AF-1 and mUGBP was suggested by co-localization of fluorescence dye labeled AF-1 and (EGFP)-tagged UGBP on the living cells under confocal microscope.The observation has provided visual evidence that AF-1 may bind to UG-binding protein.This conclusion was further supported by the results of flow cytometry-based binding assays using labeled AF-1 on NIH 3T3 cells and COS-1 cells.The data showed that,with increasing concentration of unlabeled peptide AF-1,the decrease in fluorescences indicates a progressive occupation of Cy5-AF-1 binding sites on the cell surface.Excess unlabeled peptide AF-1 displaced labeled AF-1 binding with the following values:60%,80%,85%,and 91%for the concentrations of 1μM,5μM,10μM,and 20μM,respectively.The finding that Cy5-and biotin-labeled AF-1 binding were readily displaced by unlabeled peptide is in accord with the essential features of a ligand-receptor interaction.The binding data were then subjected to mass law analysis by the method of Scatchard to yield the K_d and B_maxvalues.The results showed that K_d =37.75+4.003 nmol/L,B_max=93.29±4.267RFI.Taken together,these results indicated that AF-1 specifically bound to UG-binding protein in a ligand-receptor-like manner.Whereas AF-1 was considered to be a PLA2 inhibitor,in light of its ability of interacting with UG-binding protein,it appears that this bioactive peptide derived from UG may act in an even more complex manner,at least in certain cell type,via its specific binding protein or receptor-mediated pathways.The present study opens possibility for further elucidating the intracellular mechanism for AF-1 mediated activity.ChapterⅢInteraction of Antiflammin-1 with Uteroglobin-Binding Protein Induces Phosphorylation of ERK1/2 in NIH 3T3 CellsIt has been previously suggested that uteroglobin(UG)-binding protein functions as a putative receptor of UG,however,the downstream events of UG-binding protein signaling remain unclear.ERK1 and ERK2 are strongly activated by growth factors,serum,phorbol esters and also by ligands of heterotrimeric G protein-coupled receptors and cytokines.It has been demonstrated that in UG knockout mice,lungs manifest altered expression of ERK.Studies have also revealed increased phosphor-ERK (p-ERK)levels after UG gene overexpression in non-small cell lung cancer cells. Although previous observations raised the possibility that exogenous and endogenous UG might signal via ERK,there is no experimental evidence that UG-binding protein is involved in this process.As a consequence of AF-1 specifically binding to UG-binding protein, modulation of ERK pathway has been revealed in this study.The results indicated that incubation of peptide AF-1(1μM and 10μM)with NIH 3T3 cells resulted in an elevated level ofphosphorylated ERK1/2 protein.ERK1 and ERK2 phosphorylation occurred during 30 min of incubation,and higher expression levels of p-ERK were observed at 10 min after AF-1 treatment. Levels of total ERK1/2 protein were not affected.To establish whether AF-1-induced ERK phosphorylation via its specifically binding to UG-binding protein,we took advantage of the RNAi technology.The oligonucleotides containing sense and antisense of the target sequences and loop sequence were synthesized,annealed,and constructed into a pRNAT-U6.1/Neo siRNA expression vector,which had a cGFP gene and a U6 promoter driving to express shRNA.In addition,we constructed an expression plasmid encoding a non-relevant sequence shRNA sequence as a control.The capacity of the shRNA to downregulate expression of the endogenous UG-binding protein was tested by transient transfection of the UG-binding protein shRNA constructs in NIH 3T3 cells,followed by quantitative RT-PCR and Western blots.Finally, one of the most efficient shRNA sequences was used in functional assays in this study.When reduced the level of endogenous UG-binding protein expression in murine fibroblast cell line NIH 3T3 by RNA interference,we found that knockdown of UG-binding protein inhibited AF-1 induced extracellular signal-regulated kinase 1 and 2(ERK1/2)phosphorylation.The present study for the first time provided evidences for AF-1 binding with UG-binding protein with a cellular signaling consequence,and preliminarily characterized UG-binding protein as a point downstream of AF-1 in mediating ERK phosphorylation,further supportting the proposal that UG-binding protein as a functional receptor.However,AF-1-UG-binding protein interactions are very unclear at this moment;the findings in our present study have particularly intriguing implications for further elucidating UG-binding protein-mediated signaling but the story is much more complex.The altered cellular signal transduction may be understood more clearly when the physiological relevance of this property of AF-1 are studied in detail.To better understand changes in downstream gene expression after inhibition of mUGBP,a real-time quantitative PCR-based array was used to compare the gene expression profile of shRNA-transfected NIH3T3 with that of the control. Real-time quantitative PCR array analysis identified 9 downstream genes whose expression seemed specifically regulated by mUGBP.We found that most of the changes were associated with growth,invasion,and translational activity.Atf2 (Activating transcription factor 2),Egr1(Early growth response 1),Fos (FBJ osteosarcoma oncogene),ICAM1(Intercellular adhesion molecule), Mdm2(Transformed mouse 3T3 cell double minute 2),Ptgs2 (Prostaglandin-endoperoxide synthase 2,cox-2),and Tfrc(Transferrin receptor)have significant increased expression(＞3 fold).In addition,Q-PCR array results also revealed changes in some members of insulin pathway(Hk2, Hexokinase 2)and some members of LDL pathway(Selp,Selectin,platelet) not commonly associated with reported activities of UG.The array results need further confirmation. ChapterⅣRegulated Expression of Uteroglobin-Binding Protein in Murine Lungs and in NIH 3T3 Fibroblast Following Fibrotic StimuliAs a small,secreted,multifunctional protein,UG plays important roles in preventing pulmonary fibrogenesis.Recently,others and we have shown that UG-binding protein mediates some cellular functions of UG or its bioactive fragment on several cell types.In this chapter,we demonstrate the expression of mouse UG-binding protein (mUGBP)in bleomycin-induced pulmonary fibrosis in mice as well as its induction by profibrotic TGF-β1 in murine fibroblast NIH 3T3 cells.Real-time PCR showed that mUGBP mRNA in whole lung homogenates began to increase gradually from day 14 to day 28 following bleomycin challenge.In situ hybridization revealed that increased mUGBP positive labeling was preferentially observed in interstitial sites of bleomycin-injured lungs.In vitro studies further showed that treatment of mouse NIH 3T3 cells with TGF-β1 for 48 hours led to increased expression of mUGBP mRNA and protein.TGF-β1 stimulated procollagen 1(I)(pro-collαl(I))expression in NIH 3T3 cells was inhibited by 10μM of AF-1,a known UG-derived bioactive fragment.However, this UG-bioactive fragment did not change the expression levels of pro-collα1(I) under basal condition without TGF-β1.Antisense oligonucleotides(AS-OND) of mUGBP partly blocked the inhibition of TGF-β1-stimulated pro-collα1(I) protein expression by AF-1.These results point to the likelihood that the induction of mUGBP is necessary for AF-1 to inhibit of procollagen I expression,and its induction is protective during fibrogenesis.The current study also promotes a previously undescribed mechanism of UG and its analogues to inhibit fibrogenesis in response to fibrotic stimulation.Further elucidation of the molecular mechanisms involved might yield novel insights into potential roles of mUGBP during pulmonary fibrosis and help develop new therapeutic strategies.