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INSERM Laboratory U624 Cellular Stress Campus of Luminy, Marseille Acute pancreatitis What is the reaction of the acinar cells at the cellular and at the molecular level ? acinar cell of the pancreas with acute pancreatitis Gene expression in pancreas with acute pancreatitis Experimental acute pancreatitis pancreas RNA extraction mRNA expression mRNA expression in pancreas with acute pancreatitis 150 acute phase reaction % of control Amylase mRNA 100 50 0 0 0.5 1 2 5 10 Time (days) mRNA expression in pancreas with acute pancreatitis 150 Elastase mRNA acute phase reaction % of control Amylase mRNA 100 ChTg B mRNA Tg I mRNA pCpA mRNA 50 0 0 0.5 1 2 5 10 Time (days) mRNA expression in pancreas with acute pancreatitis 300 % of control 250 200 150 acute phase reaction 100 Amylase mRNA Lithostathine mRNA 50 0 0 0.5 1 2 5 10 Time (days) • Changes in mRNA expression in the acinar cell of the pancreas with acute pancreatitis seems to be complex • Some genes became down-regulated whereas others were activated • This is reminder of the response to cellular stress Stress response pancreatitis gene expression phenotypic changes transcription factors second messengers cellular resistance Stress response pancreatitis gene expression transcription factors second messengers novel activated genes (new mechanisms !!!) phenotypic changes cellular resistance Strategy of cloning mRNA cDNA cDNA library acute pancreatitis candidate genes analysis of expression analysis of sequences Systematic sequencing (>2000 clones) over-expressed in pancreas with pancreatitis 1996 Strategy of cloning mRNA cDNA cDNA library acute pancreatitis candidate genes analysis of expression analysis of sequences Systematic sequencing (>2000 clones) over-expressed in pancreas with pancreatitis VMP1 1996 VMP1-EGFP VMP1 EGFP VMP1-EGFP VMP1 EGFP Autophagy ? autophagy = autodigestion Autophagy Stress-induced autophagy Benefit for the cell Cellular damage (for example chemotherapy) Removal of damaged organelles Limitation of infection Intracellular pathogens Nutrient/growth factor deprivation Hypoxia To produce (in extreme cases) : - substrates for protein synthesis - ATP Autophagy during acute pancreatitis (?) Autophagy during acute pancreatitis (?) Electronic microscopy pre-autophagosome autophagosome autophagosome lysosome autophagosome prepre-autophagosome autophagolysosome autophagolysosome VMP1 expression induces LC3 recruitment pre-autophagosome LC3 LC3 Immunohistochemistry control autophagosome lysosome autophagosome autophagolysosome pcDNA4-VMP1 VMP1 expression is induced by autophagic stimuli and is necessary for autophagy starvation rapamycin VMP1 IHC VMP1 GAPDH hours 0 2 4 0 2 4 starvation starvation rapamycin VMP1 β−tubulin hours 0 2 4 0 2 4 rapamycin VMP1 expression is induced by autophagic stimuli and is necessary for autophagy starvation rapamycin VMP1 IHC starvation VMP1-siRNA scble-siRNA VMP1 + - rapamycin + + - VMP1 GAPDH hours + GAPDH 0 2 4 0 2 4 starvation starvation VMP1-siRNA scble-siRNA + + - rapamycin + + - VMP1 starvation rapamycin β−tubulin VMP1 β−tubulin 0 2 4 rapamycin VMP1-siRNA scble-siRNA starvation starvation VMP1-siRNA scble-siRNA rapamycin rapamycin VMP1-siRNA scble-siRNA control control VMP1-siRNA scble-siRNA 100 90 80 70 60 50 40 30 20 10 0 control 4 rapamycin 2 starvation 0 RFP-LC3 positive cell with punctate per transfected cells hours MCF7 MCF7 MCF7 starvation 100 90 80 70 60 50 40 30 20 10 0 pcDNA4 -VMP1 pcDNA4-VMP1 control starvation per transfected cells control %ERFP-LC3 positive puncta VMP1 binds to the beclin1 protein MCF7 MCF7 MCF7 starvation 100 90 80 70 60 50 40 30 20 10 0 pcDNA4 -VMP1 pcDNA4-VMP1 control starvation per transfected cells control %ERFP-LC3 positive puncta VMP1 binds to the beclin1 protein antiprebeclin1 immune pull-down Ni-agarose beclin1 VMP1-V5 beclin1 TCL VMP1-V5 VMP1-V5 VMP1-V5 TCL beclin1 pcDNA4-VMP1 + - pcDNA4-empty - + pcDNA4-VMP1 + + antiprebeclin1 immune anti- preVMP1 immune beclin1 VMP1 IP VMP1 IgG rapamycin IP beclin1 IP beclin1 IgG + + rapamycin + + VMP1 binds to the beclin1 protein MCF7 MCF7 MCF7 starvation 100 90 80 70 60 50 40 30 20 10 0 pcDNA4 -VMP1 pcDNA4-VMP1 control starvation per transfected cells control %ERFP-LC3 positive puncta D B TCL VMP1-V5 beclin1 VMP1-V5 pcDNA4-VMP1 + - pcDNA4-empty - + pcDNA4-VMP1 + + antiprebeclin1 immune anti- preVMP1 immune beclin1 VMP1 IP VMP1 IgG rapamycin TCL beclin1 IP beclin1 IgG + + rapamycin + + GST beclin1 IP GST VMP1-V5 VMP1-V5 GST pull-down Ni-agarose GST antiprebeclin1 immune beclin1 C A A B C D + His6- beclin1 pull-down VMP1 binds to the beclin1 protein MCF7 MCF7 MCF7 starvation 100 90 80 70 60 50 40 30 20 10 0 pcDNA4 -VMP1 pcDNA4-VMP1 control starvation per transfected cells control %ERFP-LC3 positive puncta D B TCL VMP1-V5 IP + - pcDNA4-empty - + B C D pull-down VMP1-V5 TCL beclin1 pcDNA4-VMP1 GST beclin1 A + His6- beclin1 VMP1-V5 beclin1 GST VMP1-V5 GST pull-down Ni-agarose GST antiprebeclin1 immune beclin1 C A pcDNA4-VMP1 + GST-VMP1A His6- beclin1 + - + GST-VMP1B - + GST-VMP1C GST-VMP1D - + IB : anti-beclin1 antiprebeclin1 immune anti- preVMP1 immune beclin1 VMP1 IP IgG rapamycin IP beclin1 IgG + + + IB : anti-GST beclin1 VMP1 - rapamycin beclin1 + + VMP1 binds to the beclin1 protein MCF7 MCF7 MCF7 starvation 100 90 80 70 60 50 40 30 20 10 0 pcDNA4 -VMP1 pcDNA4-VMP1 control starvation per transfected cells control %ERFP-LC3 positive puncta D B TCL VMP1-V5 IP + - pcDNA4-empty - + B C D pull-down VMP1-V5 TCL beclin1 pcDNA4-VMP1 GST beclin1 A + His6- beclin1 VMP1-V5 beclin1 GST VMP1-V5 GST pull-down Ni-agarose GST antiprebeclin1 immune beclin1 C A pcDNA4-VMP1 + GST-VMP1A His6- beclin1 + - + GST-VMP1B - + GST-VMP1C GST-VMP1D - + - IB : anti-GST IB : anti-beclin1 beclin1 antiprebeclin1 immune anti- preVMP1 immune beclin1 VMP1 IP VMP1 IgG rapamycin IP beclin1 IgG + + + rapamycin PI3k beclin1 + + VMP1-beclin1 interaction is necessary for autophagy VMP1ΔD/V5 beclin1 ? beclin1 pull-down Ni-agarose V5 beclin1 + - - pcDNA4-VMP1ΔD/V5 - + - pcDNA4-empty - - + VMP1ΔD/V5 Triton treatment pH11.0 treatment 1.5M NaCl treatment No treatment VMP1ΔD/V5 No treatment pcDNA4-VMP1/V5 empty/V5 TCL V5 VMP1-beclin1 interaction is necessary for autophagy VMP1ΔD/V5 beclin1 ? beclin1 pull-down Ni-agarose V5 beclin1 + - - pcDNA4-VMP1ΔD/V5 - + - pcDNA4-empty - - + Triton treatment pH11.0 treatment 1.5M NaCl treatment No treatment VMP1ΔD/V5 No treatment pcDNA4-VMP1/V5 empty/V5 TCL V5 VMP1ΔD/V5 VMP1/EGFP VMP1ΔD/EGFP VMP1 expression in vivo VMP1 LC3 Merge VMP1 beclin1 Merge 0 3 6 12 control hours experimental pancreatitis β−tubulin control VMP1 experimental pancreatitis experimental pancreatitis VMP1 transgenic mice DNA construct Promotor-Elastase VMP1 EGFP Anti-EGFP ? Anti-EGFP Hypothesis acute pancreatitis PI3k VMP1 mRNA VMP1 gene autophagolysosome beclin1 ER PI3k autophagosome beclin1 ER lysosome LC3 PI3k autophagosome beclin1 ER pre-autophagosome Conclusion I Expression of VMP1 necessary and sufficient induce autophagy is to Conclusion II During the acute pancreatitis, acinar cells respond to the cellular stress induced by this disease, by changing their gene expression profile in a very complex manner but with the aim to survive….. Conclusion III Starting autophagy, during the acute phase of the pancreatitis, could be a protective mechanism for the acinar cell because : 1/ it could provide energy substrates….. 2/ it could remove damaged organelles….. Calanques of Luminy, Marseille at 5 min walking from the lab…