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The LIMD1 Protein Represses the Hypoxic Response Through Bridging an Association Between the Phds and VHL

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The LIMD1 Protein Represses the Hypoxic Response Through Bridging an Association Between the Phds and VHL The LIMD1 Protein represses the Hypoxic Response through Bridging an Association Between the PHDs and VHL Dr Daniel Foxler Centre for Molecular Oncology www.bci.qmul.ac.uk LIMD1 is a 3p21.3 encoded tumour suppressor gene Normal epithelia 3p21.3 loss LIMD1 Squamous metaplasia x200 Hyperplasia p53 mutations Dysplasia Resp epithelium & adjacent HG dysplasia x400 Kras mutations Micro-invasion Invasive SCC in subepithelial stroma x200 LIMD1 knockout mice are LIMD1 loss correlates with predisposed to develop poor prognosis in breast lung cancer cancer Sharp et al 2008 Spendlove et al 2008 LIMD1 is a member of the Zyxin family and represses cell cycle progression miRNA silencing (RNA storage/degradation) AGO E2F PHD1 HIF1 is the master transcriptional regulator of the hypoxic response • Hypoxia inducible factor 1 (HIF1) is the master transcriptional regulator that responds to decreases in intracellular O2 tension Enhanced Gene Transcription •VEGF •VEGF receptor 1 •PDGF Angiogenesis •Ang-2 •Epo •Transferrin Red blood cell production •Transferrin receptor HIF1α HIF1β HIF1 •Lactate dehydrogenase A •Pyruvate kinase 1 Protein •Aldolase A/C Energy metabolism •Phosphoglycerate kinase •Hexokinase •Glucose transporter 1 & 3 •BNIP3 •Insulin-like growth factor 2 •Adenylate kinase 3 Cellular proliferation •WT1 •Nip3 The PHDs and VHL bind to discrete regions within LIMD1 WT LIMD1 LIM1 LIM2 LIM3 676 aa LIMD1 Δ472-676 PHD2 LIMD1 Δ1-467 LIM1 LIM2VHL LIM3 LIM domains PHD2 Pre-LIM region VHL region kDa kDa 75 75 INPUT 50 INPUT 50 BLOT: Xpress BLOT: Xpress 37 37 25 25 37 50 INPUT INPUT BLOT: PHD2 BLOT: V5-VHL 37 25 75 75 IP: Xpress 50 IP: Xpress 50 BLOT: Xpress BLOT: Xpress 37 37 25 25 37 50 IP: Xpress IP: Xpress BLOT: PHD2 BLOT: V5-VHL 37 25 LIMD1 endogenously binds to PHD2 and VHL Endogenous Co-immunoprecipitation HEK 293 kDa 37 VHL 25 IgG IP: VHL 20 BLOT: VHL VHL 15 100 LIMD1 75 IP: VHL BLOT: LIMD1 50 IgG 75 IP: VHL 50 BLOT: PHD2 37 20 15 IP: VHL BLOT: elonginB 10 100 IP: VHL 75 BLOT: cullin2 50 LIMD1 enhances the association between PHD2 and VHL LIMD1 PHD2 V5-VHL + + + + V5-VHL + + + + V5-VHL - + - + LIMD1 - + - + LIMD1 kDa PHD2 - - + + kDa - - + + PHD2 37 37 BLOT: V5-VHL BLOT: V5-VHL 25 V5 IP: 25 100 INPUTS BLOT: LIMD1 100 - 75 BLOT: LIMD1 VHL 75 50 BLOT: PHD2 50 BLOT: PHD2 37 37 50 BLOT: β-ACTIN 37 Does loss of LIMD1 inhibit HIF1α protein degradation? LIMD1 PHD2 V5-VHL LIMD1 Loss PHD2 V5-VHL LIMD1 depletion causes an increase in endogenous HIF1α protein siRNA: control LIMD1 PHD2 control LIMD1 PHD2 control LIMD1 PHD2 control LIMD1 PHD2 control LIMD1 PHD2 control LIMD1 PHD2 LIMD1/PHD2 LIMD1/PHD2 LIMD1/PHD2 LIMD1/PHD2 LIMD1/PHD2 kDa LIMD1/PHD2 150 α-HIF1α (1min) α-HIF1α (1min) 100 150 α-HIF1α (10 min) α-HIF1α (10 min) 100 100 α-LIMD1 α-LIMD1 75 50 α-PHD2 α-PHD2 37 50 α-actin α-actin 37 Normoxia 4 16 24 48 72 Hypoxia (hrs) LIMD1 loss causes an increase in endogenous HIF1α protein LIMD1 expression decreases HIF1 driven transcription HRE LUCIFERASE 6 20% O2 5 1% O2 4 3 2 1 Relative Luciferase Activity Luciferase Relative 0 VO HIF1α HIF1α + LIMD1 PHD2 and VHL binding is required for LIMD1 mediated HIF1α degradation Pre-LIM region LIM domains 1 2 3 PHD2 VHL 5’ LTR U6 promoter LIMD1 shRNA Ub promoter LIMD1 cDNA FH SV40 puromycin 3’ LTR 1001. Deplete endogenous LIMD1 75 Anti-LIMD1 50 2. RescueAnti with-β-Actin an RNAi 37 resistant LIMD1 driven by ubiquitin promoter LIMD1 depletion causes an increase in HIF1 driven transcription 5 140 16 si-scrambled LIMD1 si-Scrambled HIF1α si-scrambledHEK 293 120 14 4.5 si-LIMD1 si-LIMD1 * si-LIMD1100 12 mRNA 10 4 of * si-PHD2 80 8 60 3.5 6 40 * * * 4 * * 3 20 Fold Induction Fold 2 % Expression of LIMD1 LIMD1 ofmRNA % Expression 0 1% O2 0 2.5 20% 1O2 * * 20% O2 1% O2 * U2OS * Fold InductionFold * * * * 2 * * * * 16 * * * 16 * *si-scrambled si-scrambled * VEGF BNIP3 1.5 14 14 * si-LIMD1 si-LIMD1 12 12 mRNA mRNA 1 10 * of 10 of 8 8 0.5 6 6 4 4 0 Fold Induction Fold Fold induction Fold 2 2 0 0 20% O2 1% O2 20% O2 1% O2 LIMD1 causes a decrease in nuclear accumulation of HIF1α LIMD1 Protein, mRNA and promoter driven transcription increases in hypoxia 4.5 4 kDa 3.5 100 3 75 BLOT: LIMD1 2.5 2 50 BLOT: PHD2 1.5 37 50 1 BLOT: actin 0.5 Fold Induction of InductionLIMD1mRNA Fold 37 0 Normoxia 8h Hypoxia 16h hypoxia 24h hypoxia Is the hypoxic up-regulation specifically driven by HIF1α? The LIMD1 Promoter ATG...LIMD1... LIMD1 Promoter Sharp et al 2008 The LIMD1 Promoter is Responsive to Hypoxia The LIMD1 Promoter Foxler et al 2011 A The LIMD1 Promoter is Responsive to Hypoxia p>0.1 BA 1200 1000 800 600 p>0.1 400 p>0.1 200 Luciferase Value (Normalised to Renilla)Renilla)toto(Normalised(Normalised ValueValue Luciferase Luciferase 0 N H N H N H N H N H N H N H N H N H N H N H N H VO WT IΔ1 IΔ2 IΔ3 IΔ4 IΔ5 IΔ6 IΔ7 IΔ8 IΔ9 IΔ10 B 4 3.5 3 2.5 2 p>0.1 1.5 1 Luciferase Value (Normalised to Normoxia)to(Normalised Value Luciferase Luciferase Value (Normalised to Normoxia)to(Normalised Value Luciferase 0.5 0 N H N H N H N H N H N H N H N H N H N H N H N H VO WT IΔ1 IΔ2 IΔ3 IΔ4 IΔ5 IΔ6 IΔ7 IΔ8 IΔ9 IΔ10 LIMD1 Transcription is Up-regulated by HIF1α Normoxic Hypoxic Normoxic Hypoxic 150- α-HIF1a 150- 100- 100- α-HIF1a 75- α-LIMD1 75- α-LIMD1 50- α-PHD2 50- α-PHD2 50- α-actin 50- α-actin HeLa U2OS bp Gene 500- 400- LIMD1 300- 400- 300- PHD2 200- 500- 400- DHFR 300- Conclusions PHD/LIMD1/VHL complex PHD PHD VHL VHL Regulation of complex formation represents an LIMD1 PHD VHL VHL additional level of HIF/hypoxic OH OH OH OH OH OH regulation HIF1α ODD HIF1α ODD HIF1α ODD NEW Ub Ub Ub Ub Ub Ub 2+ CO /Succinate 02/Fe /2-OG CO2/Succinate OLD 2 MODEL 2+ MODEL 20% 02/Fe /2-OG 26S Proteosome 26S Proteosome HIF1α ODD HIF1α ODD HIF1α degraded HIF1α degraded Conclusions Decreased O2 High LIMD1/Low HIF Attenuation of hypoxic HIF1α response Low LIMD1/High HIF Nucleus Squamous metaplasia x200 P300/ CBP HIF1α Ub Transcription of HRE Ub containing genes Ub OH OH Ub Ub HIF1α VEGF LIMD1 Epo SAG PHD2 Hypoxia Hypoxic response + adaptive attenuation genes Decreased mitochondrial genes O2 consumption Acknowledgements • Sharp Lab – Katherine Bridge – Sybil Wong – John Foster – Thomas Webb – Maureen Mee – Victoria James – Tyson Sharp • Collaborators – Peter Ratcliffe, University of Oxford, UK – Greg Longmore, Washington University, USA References: Foxler et al, Nat. Cell Biology 2012 Foxler et al FEBS 2011 Sharp et al, PNAS 2008 Spendlove et al Int. J. Cancer Sharp et al, PNAS 2004 .
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