Prakoura, Niki

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Calreticulin upregulation in renal fibrosisNiki PrakouraSection of Histology, Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, GreeceEuroKUP meeting, Madrid, Spain, 17-19 June 2011FibrosisFibrosis is defined as the accumulation of extracellular matrixleading to structural and functional alterations of severalorgans such as lung, heart, kidney, pancreas, liver.Causes of kidney fibrosisdiabetes, hypertension, infection, inflammation of renal blood vesselsand glomeruli, kidney stones, cysts, genetic mutations.Kidney Fibrosisis a common feature of Chronic Kidney Diseases [CKD] (which affect10-12% of the population) and is characterized by progressive loss ofkidney function and relentless accumulation and deposition of ECMis considered to be an irreversible process that leads to end-stagerenal failure and requires expensive and life-long treatments withdialysis or transplantationis increasing at a rate of approximately 7% per year among the worldpopulationWhy do organs in the body develop fibrosis?Wynn T.A., JCI, 2007A wound-healing response that has gone out of control45% of all the deaths in the developed world can be attributed to some form of fibroproliferative diseaseThe Unilateral Ureteral Obstruction (UUO) model of kidney fibrosis•in vivo model•encompasses many aspects of other models of kidney fibrosis•there are features that occur within 1 week•there is evidence that animal models with UUO are reflective of the molecular changes in human situations•leaves one kidney intact•mimics in a short time a situation that can take years in humansProteomic analysisSham operated 2d UUO 2d Sham operated 8d UUO 8d UUO  2d UUO 8d Kypreou K. et al., Proteomics, 2008Proteomic ApproachClassical 2DE1st dimension: IEF (pI 3-10)2nd dimension: Gel electrophoresisPD Quest Image processing SoftwareSpot excision and tryptic digestionMALDI-TOF/TOF-MSMASCOT Software for protein identificationProteins altered in all repeats of the experiment (3 differentanimals from each group) were selected for further evaluationVenn diagram of identified proteinsSham / Ligated 8Sham / Ligated 2Ligated 2 / Ligated 8 285124918CALRETICULINLigated 2 daysSham operated 2 daysSham operated 8 days Ligated 8 daysCalreticulin is a multifunctional protein• First isolated in 1974 as ahigh-affinity Ca2+-bindingprotein of the ERThere is no correlation with fibrotic processes yet !TRENDS in Cell Biology, Vol.11, 2001Confirmation of Calreticulin upregulationin fibrotic samplesShamoperated2daysLigated 2 daysLigated 2 daysLigated 2 daysLigated 8 daysLigated 8 daysLigated 8 daysSham operated8daysCalreticulinbeta-tubulinShamoperatedLigated 2 daysRatio of calreticulin expression/tubulin expressionLigated 8 days00.10.20.30.40.50.60.70.80.9Quantification of calreticulinprotein expressionFold of calreticulin mRNA expression normalized to 18S rRNA 050150200250300ShamoperatedLigated 2daysLigated 8 days100Quantification of calreticulinmRNA expressionKypreou K. et al., Proteomics, 2008Why is Calreticulin upregulatedin kidney fibrosis? Which is the mechanism of Calreticulin upregulation? What is the role of Calreticulin in fibrosis?1. WHICH IS THE MECHANISM OF CALRETICULIN UPREGULATION?Transforming Growth Factor-β (TGF-β) induces fibroblasts to synthesize ECM and has long been considered as a central mediator of the fibrotic responseTransforming Growth Factor-β (TGF-β)induces HK-2 cells to produce CalreticulinSham operated Ligated 2 days Ligated 8 daysKypreou K. et al., Proteomics, 2008TGF-β-mediated Calreticulin induction is accompanied by Epithelial to Mesenchymal Transition in HK-2 cells-TGF-β+TGF-βa smooth muscle actin x 40--TGF-β+TGF-βEpithelial to Mesenchymal TransitionTGF-β induces Calreticulin mRNA expression in HK-2 cells02468101214161820Control TGF-βRatio of CalreticulinmRNA expression/18S rRNAexpressionQuantification of Calreticulin mRNA expression by Real-time RT-PCRWhat is upstream of this induction?Bioinformatics analysis on Calreticulin promoter www.genomatix.de: Gene2Promoter 4 organisms: Promoter sequence: 2000bp upstream and 1000bp downstream the TSSCalreticulin promoter sequence contains TF binding sites that act downstream of the TGF-β signaling  calreticulinYY1E2F4Sp1SmadTATAMycC/EBP- 1800bp - 300bpCloning of the human Calreticulinpromoter sequenceCALR-1800exon1+1-1000 -500-1500 +86TATA-27Sp1-71Sp1-251Sp1-305Smad-291Smad-691Smad-706Smad-897Smad-1744Myc-223Myc-282Myc-1080C/EBP-926E2F4YY1-175E2F-318pGL3-BasicMCSKpnISacIMIuINheISmaIXhoIBglIILucTGF-β induces Calreticulin promoter activity in HK-2 cellsTGF-β regulates Calreticulin at the level of transcriptional activation0100000200000300000400000500000600000700000800000pGL3-CALR/ -TGF-β pGL3-CALR/+TGF-βNormalized luciferaseactivity(RLU/β-gal)Calreticulin promoter activityupon TGF-β stimulation in HK-2 cellsCalreticulin promoter is regulated by transcription factors E2F4 and p107050000001000000015000000200000002500000030000000Normalized luciferaseactivity(RLU/β-gal)Calreticulin promoter activity upon TF stimulation in HEK293 cellsE2F4 and p107 act downstream the TGF-β signaling pathwayE2F4 has potential binding sites on Calreticulin promoterE2F4 and its cytoplasmic binding partner p107 are cell cycle regulatorsCalreticulin promoter deletion constructsCALR-1800exon1+1-1000 -500-1500 +86TATA-27Sp1-71Sp1-251Sp1-305Smad-291Smad-691Smad-706Smad-897Smad-1744Myc-223Myc-282Myc-1080C/EBP-926E2F4YY1-175E2F-318-66 +86-522 +86-1215 +86-226 +86-165 +86TATASp1YY1c-MycSmadE2F4C/EBPCalreticulin promoter includes activator and repressor elements The region from -1215 to -1800 contains transcriptional activator element(s) The region from -226 to -522 contains transcriptional repressor element(s) The fragment -226/+86 harbors the basal/core promoter sequence whoseactivity is altered by upstream activator and repressor elements02000000400000060000008000000100000001200000014000000pGL3-CALRpGL3-(-1215/+86)pGL3-(-522/+86)pGL3-(-226/+86)pGL3-(-165/+86)pGL3-(-66/+86)Normalized luciferase activity (RLU/β-gal)Deletion constructs activity in HK2 cellsCalreticulin promoter includes activator and repressor elements02000000400000060000008000000100000001200000014000000pGL3-CALRpGL3-(-1215/+86)pGL3-(-522/+86)pGL3-(-226/+86)pGL3-(-165/+86)pGL3-(-66/+86)Normalized luciferase activity (RLU/β-gal)Deletion constructs activity in HK2 cellsCALRTATABasal promoterRepressorActivator-1800 -1215 -522 -226 +1transcription(+)(-)Calreticulin gene regulation is even more complicated…020000004000000600000080000001000000012000000140000001600000018000000Normalized luciferaseactivity(RLU/b-gal) Deletion constructs activity in HK-2 cells…representing the multiplicity of Calreticulin functionsWhich is the mechanism of Calreticulin upregulation in fibrosis?TGF-β is likely to play a role. Other factors should also play a role, taking intoconsideration the complex nature of both fibrosis andCalreticulin regulation.More signaling pathways and transcription factors thatupregulate Calreticulin gene expression need to be studied, andcorresponded to specific regulatory elements on Calreticulinpromoter.AcknowledgementsDr. Aris CharonisDr. Panos PolitisDr. Katerina KypreouValeria KalteziotiFani KaragianniPanos KavvadasThanassis StergiopoulosZozefina FoskolouLila KaltsaIsmini RozaniElena FrangouPrincipal investigatorsLab membersCenter for Experimental SurgeryMichalis KatsiboulasHistochemistry Core FacilityAnna AgapakiFundingBRFAABodossakis FoundationEuropean Social Fund: NSRF-Heracleitus IICollaboratorsProf. D. Vlahakos, AttikonGeneral Hospital, University ofAthens

Calreticulin upregulation in renal fibrosis

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Calreticulin upregulation in renal fibrosis

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Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)

Calreticulin upregulation in renal fibrosis

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Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)