Lecture IV. 30 th March 2009

Transcription

1 Lecture IV 30 th March 2009 Mechanisms of regulation of VEGF expression

2 hypoxia reactive oxygen species nitric oxide VEGF cytokines heme oxygenase-1 growth factors

3 Regulation of VEGF expression

4 Structure of VEGF human promoter

5 CYTOKINES Cytokines That Cause Upregulation of VEGF Cytokines IL-1a IL-1b IL-3 IL-15 IL-18 TPO bfgf EGF HGF KGF IGF-1 PDGF TGF TNF MIF Cell Type Blood mononuclear cells, umbilical endothelial cells A549, SRC-3, PC-14, RERF-LC-AI, synovial cells, smooth muscle cells, visceral glomerular epithelial cells Vascular endothelial cells Blood mononuclear cells Blood mononuclear cells in vitro, rheumatoid arthritis, synovial fibroblasts Hematopoietic stem cells Vascular smooth muscle cells upregulation Gastric cancer cells, endometrial stromal cells, prostate cancer cells Breast cancer and leiomyosarcoma, endothelial cells, papillary carcinoma cells, HaCaT cells Keratinocytes Thyroid carcinoma or pancreatic cancer cells, mesangial cells, endothelial cells Vascular smooth muscle and endothelial cells Epidermoid carcinoma cells, vascular smooth muscle cells, endothelial cells, cholangiocarcinoma cells Glioma cells Hepatocellular carcinoma cells

6 CYTOKINES Cytokines That Cause Downregulation of VEGF Cytokines IL-4 Cell Type Visceral glomerular epithelial cells, blood mononuclear cells IL-10 Blood mononuclear cells, visceral glomerular epithelial cells IFN-a Smooth muscle cells, blood mononuclear cells, brain tumor IFN-b Melanoma cells

7 CYTOKINES Cytokines That Cause BOTH Upregulation and Downregulation of VEGF Cytokines IFN-g Cell Type Melanoma cells, ovarian cancer cells, endometrial stromal cells IL-12 Blood mononuclear cells in vitro, breast cancer animal model IL-13 Smooth muscle cells, visceral glomerular epithelial cells

8 GROWTH FACTORS Adenoviral FGF-4 overexpression upregulates endogenous VEGF production and increases vascular permeability, therapeutic angiogenesis, and arteriogenesis. Non-viral liposomal KGF cdna gene transfer improved neovascularization, as well as dermal and epidermal regeneration through stimulation of epithelial (VEGF) and mesenchymal (insulin-like growth factor-i, IGF-I) factors expression in skin cells. Stimulation of the production of VEGF as well as plasminogen activator inhibitor (PAI-I) after TGF-β treatment leads to vascular remodeling during angiogenesis. Numerous data indicate that blocking of TGFβ action inhibits tumor viability, migration, and metastases in mammary cancer, melanoma and prostate cancer model probably due to inhibition of VEGF synthesis. Therefore, reduction of TGFβ production and activity may be a promising target of therapeutic strategies to control tumor growth.

9 Reactive oxygen species Hydrogen peroxide increases VEGF production in different cell lines acting via Sp1 transcription factor Cisowski, Loboda et al. BBRC 2005

10 HYPOXIA HYPOXIA A state where O 2 availability/ delivery is below the level necessary to maintain physiological O 2 tensions for a particular tissue. When tissue demand exceeds its O 2 supply. Different tissues have different oxygenation levels Atmospheric 21% O 2 Lung capillaries 13% O 2 Healthy tissues 2.5-9% O 2 Diseased tissue < 1% O 2

11 HYPOXIA Tumors are hypoxic 21% ~ % ~15-20% ~5 % ~14 % ~ % Brahimi-Horn et al., 2007

12 HYPOXIA Tissue hypoxia 1. A part of physiological event, in particular in embryogenesis 2. In pathological situations in ischemic diseases and cancer

13 HYPOXIA Tumor growth is dependent on angiogensis

14 HYPOXIA Hypoxic area is formed inside growing tumor

15 HYPOXIA Ischemia in cardiovascular diseases Peripheral vascular disease Coronary artery disease JAMA & Archives Journals Currents: Fall 2001 Impaired blood flow, hypoxic simulation of angiogenic factors,

16 HYPOXIA How do cells sense the changes in the oxygen level?

17 HYPOXIA HIF-1: Hypoxia Inducible Factor - 1 The studies of hypoxia responsive element (HRE) of the erythropoietin gene lead to the discovery of HIF-1 by Semenza and Wang in Semenza GL & Wang GL. (1992). Mol. Cell. Biol. 12: HIF-1 is a protein with DNA binding activity. It is composed of two subunits: HIF-1α and HIF-1β.

18 HYPOXIA HIF-1 A heterodimeric member of basic helix-loop-helix (bhlh) family, containing PAS domain Compose HIF-1α and HIF-1β Persistent expression of HIF-1β, identical to aryl hydrocarbon receptor(ahr) nuclear translocator (ARNT) Binding to DNA sequence 5 -(A/G)CGTG HLH PAS ODDD TAD TAD Pro 402 Pro 564 Asn 803 bhlh - basic Helix-loop-helix domain required for dimerization PAS - domain identified to be required for dimerization ODDD O2-dependent degradation domain TAD transactivation domain NLS nuclear localization signal

19 HYPOXIA Activation and degradation of HIF-1α HIF-1β & Mole et al., IUBM, 2001

20 HYPOXIA Stabilisation of HIF-1α protein in hypoxia Hammond et al., Mol Cell Biol 2002

21 HYPOXIA O 2 HYPOXIA NORMOXIA HIF-1α PROLYL HYDROXYLASES O 2 2-OXOGLUTARATE Fe 2+ CO 2 SUCCINATE HIF-1α OH OH pvhl stabilization proteasomal degradation e.g. VEGF

22 HYPOXIA Activation and degradation of HIF-1α Zagórska & Dulak, Acta Biochimica Polonica, 2004

23 HYPOXIA Prolyl hydroxylases are iron-dependent dioxygenases involved in HIF-1α degradation PHD1 hydroxylates P402 & P564 PHD2 - hydroxylates P402 & P564 PHD3 - hydroxylates P564 Co-factors of PHDs: : iron, oxygen,, 2-oxoglutarate, 2 ascorbic acid Asparaginyl hydroxylase (FIH) modifies HIF-1α and prevents binding of p300 co-activator

24 HYPOXIA Activation and degradation of HIF-1α Brahimi-Horn et al., 2007

25 HYPOXIA HIF-1α protein is stabilized in hypoxia Acker & Plate 2007

26 HYPOXIA In hypoxia HIF-1α localizes in the nuclei Brahimi-Horn et al., 2007

27 HYPOXIA More on HIF transcription factors - Both α and β subunits are members of basic helix/loop/helix (Per/Arnt/Sim) (PAS) family - there are three HIFα family members: HIF-1α, HIF-2α, HIF-3α and three HIFβ members: HIF-1β/ARNT1, HIF-2β/ARNT2, HIF-3β/ARNT3, - over 100 HIF-dependent genes - canonical HIF biding site: 5 -CGTG

28 HYPOXIA Various HRE Zagórska & Dulak Acta Biochim Pol 2004

29 HYPOXIA HIF-1 binds to hypoxia responsive element present in regulating regions of many genes Zagórska & Dulak Acta Biochim Pol 2004

30 HYPOXIA Hypoxia inducible factor a a master regulator of oxygen homeostasis Erythropoiesis & iron metabolism Erythropoietin Transferrin Transferrin receptor Ceruloplasmin Heme oxygenase-1 (rodents) O 2 HIF Angiogenesis VEGF VEGFR-1 PlGF PDGF TGFβ Vasomotor control NOS II Cell proliferation & viability IGF-1 IGFBP-1&3 TGFβ3 NOS II Energy metabolism GLUT1,2 & 3 PEPCK LDH A PGK3 Aldolase A & C PFK L & C Pyruvate kinase Enolase

31 HYPOXIA HIF-1 transcription factors HIF-1a knockouts die at E 9.0 HIF-2a knockouts die in utero at days adrenal insufficiency

32 HYPOXIA Protective physiological mechanisms against hypoxia 1. Increased production of tyrosine hydroxylase controls the ventilation through the carotid body 2. Increased expression of glycolytic enzymes 3. Increased synthesis of erythropoietin 4. Increased production of VEGF stimulation of new blood vessels

33 HYPOXIA Hypoxia one of the strongest inducers of VEGF expression Three ways of increasing VEGF expression 1. VEGF transcription 2. VEGF mrna stabilisation 3. VEGF translation Half-life of endogenous VEGF mrna is about 65 min stability increases ~ 3 times in hypoxia HuR a member of Elav-like family of binding proteins; binds to distal AU-rich region in the VEGF 3 UTR. Stabilizes VEGF mrna

34 HYPOXIA HYPOXIA HIF RNA- binding proteins Increased transcription mrna stabilization IRES mediated translation VEGF gene Increased VEGF mrna Increased VEGF proteins Increased angiogenesis

35 HYPOXIA VEGF mrna has two IRES An alternative mechanisms of translation initation, independent of cap using IRES internal ribosomal entry site IRES A is located within the 300 nucleotides upstream from the AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a 49-nucleotide structural domain (D4) essential to IRES A activity. UV cross-linking experiments revealed that IRES A activity was correlated with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR. An element between nucleotides 379 and 483 is required for its activity. Immunoprecipitation experiments demonstrated that a main IRES B-bound protein was the polypyrimidine tract binding protein (PTB), a well-known regulator of picornavirus IRESs Huez I et al., Mol Cell Biol, 1998

36 IRES initiates translation independent of cap

37 Regulation of VEGF expression Banerjee S et al. (2007) Mechanisms of Disease: angiogenesis and the management of breast cancer Nat Clin Pract Oncol 4: doi: /ncponc0905

38 Hypoxia - 1% O 2 Hypoxia was created using a Modular Incubator Chamber (Billups-Rothenberg Inc., Del Mar, CA, USA) by putting the cells into the chambers which were tightly closed and aired for 20 min with the gas mixture containing 1% O2, 5% CO2 and 94% N2.

39 HYPOXIA Activation of VEGF promoter and HRE part by hypoxia VEGF promoter luciferase HRE luciferase HRE normoxia hypoxia normoxia hypoxia Loboda et al., 2006

40 HYPOXIA HIF-1 activation increases VEGF expression in human microvascular endothelial cells RT-PCR ELISA 500 ** QRT-PCR normoxia hypoxia VEGF EF2 VEGF [% of control] normoxia hypoxia hypoxia normoxia VEGF mrna 3 2,5 2 1,5 1 0,5 0 normoxia * hypoxia Loboda et al., 2006 p<0,05 vs normoksja p<0,01 vs normoksja

41 HYPOXIA VEGF % of control Regulation of VEGF expression by hypoxia is cell-type dependent VEGF protein Normoxia HMEC-1 Hypoxia VEGF mrna expression normoxia hypoxia VEGF GAPDH 160 NIH 3T3 fibroblasts % of control Normoxia Hypoxia normoxia hypoxia VEGF EF2 Loboda A et al., 2006

42 HIF inducers Other ways of induction of HIF-1 HYPOXIA NORMOXIA HIF-1α PROLYL HYDROXYLASES O 2 2-OXOGLUTARATE Fe 2+ CO 2 SUCCINATE HIF-1α OH OH pvhl stabilization DMOG Desferrioxamine CoCl 2 proteasomal degradation e.g. VEGF

43 HIF inducers How to modulate HIF-1 1 activity? Hypoxia Iron chelators: desferrioxamine, CoCL 2 Dominant positive/negative forms, sirna, oligonucleotide decoys Chemical compounds: DMOG (dimethyloxallyl glycine)

44 HIF inducers DMOG (Dimethyloxallyl glycine) Competitive inhibitor of the oxygen-sensing enzymes - prolyl hydroxylases (PHDs), which destruct HIF-1α when hydroxylated at a specific proline residues. Stabilizes HIF-1α expression at normal oxygen tensions at concentrations between 0.1 and 1 mm

45 HIF inducers luciferase activity [relative units] DMOG activates HIF-1 in human microvascular endothelial cells HIF1 binding activity ## ## ### EMSA assay h 3h 6h 12h 24h DMOG 250µM HIF-1 unspecific 0 control hypoxia DMOG (µm) HIF1α activity [% of control] ## *** Western blot control DMOG 500 µm HIF-1α tubulin 0 control DMOG DMOG + DMOG + Wild type Mutated competitor competitor Loboda A et al., ARS 2009

46 HIF inducers HIF-1 activation increases VEGF expression in human microvascular endothelial cells control RT-PCR DMOG * VEGF EF2 VEGF [% of control] ELISA ### ### ### VEGF [% of control] control ELISA ## DMOG 0 control 250 µm 500 µm 1000 µm DMOG ### ### * control DMOG control DMOG * * * DMOG 250 µm 6 h 12 h 24 h ## p<0,01 vs control, ### p<0,001 vs control

47 HYPOXIA Activation and degradation of HIF-1α HIF-1β & Mole et al., IUBM, 2001

48 Cofactors are needed for HIF-1 signaling HIF-1β CHETOMIN HIF-1β HIF-1α HIF-1α p300/cbp p300/cbp HIF-1β p300/cbp HIF-1α TRANSCRIPTION VEGF [% of control] # * * * Chetomin an inhibitor of p300 binding 0 control DMOG 500 µm 10 nm 30 nm 90 nm chetomin

49 HIF inducers Other ways of induction of HIF-1 HYPOXIA NORMOXIA HIF-1α PROLYL HYDROXYLASES O 2 2-OXOGLUTARATE Fe 2+ CO 2 SUCCINATE HIF-1α OH OH pvhl stabilization Desferrioxamine CoCl 2 proteasomal degradation e.g. VEGF

50 HIF inducers CoCl 2 is known to potently activate HIF-1 Increase in HIF- protein levels by cobalt stimulation in osteoblast-like cells. Kim at al.,cytokine Jan 7;17(1):14-27.

51 HIF inducers CoCl 2 activates VEGF promoter in its HRE site VEGF promoter luciferase HRE luciferase HRE 400 * * 300 luciferase activity [% of control] control CoCl µm Loboda A et al., 2006

52 HIF inducers Desferrioxamine activates HIF-1 binding luc/protein [% of control] control desferrioxamine 100 um desferrioxamine 500 um

53 HIF-1 signal pathway and cancer angiogenesis

54 HIF-1 1 as a master regulator of a physiological responses to hypoxia Acker & Plate, 2007

55 Mutation in VHL gene causes disruption in HIF-1 degradation process Is it not known which HIF-1α residues are ubiquitinated

56 Diseases caused by disturbances in HIF-1 signaling - von Hippel Lindau disease Diseases dependent on enhanced HIF-1 activity - cancer - atherosclerosis - wound healing

57 VHL disease

58 Von Hippel Lindau disease - rare (1: live births) dominantly inherited cancer syndrome - multiple hemangioblastomas of CNS and retina, renal cell carcinomas pancreatic islet cell tumors and others Desrcibed in 1911 by Eugene von Hippel and further studied in 1926 by Arvid Lindau Defect in VHL tumor suppresor gene in chromosome 3p25-p26 p26 Hemangioblastoma of the retina Brigham and Women s Hospital, Boston Mutations in VHL occur also in the majority of kidney cancers

59 Is hypoxia the only one activator of HIF-1? NO. L-Arg + O 2 NOSI, NOSII, NOSIII cofactors NO. + L-Cit

60 Effect of IL-1b on VEGF mrna expression in rat vascular smooth muscle cells Li e et al., JBC 1995

61 IL-1b induces also the expression of inducible nitric oxide synthase Dulak et al., ATVB 2000: 20:

62 Nitric oxide induces VEGF synthesis L-Arg + O 2 NOS III (enos) NOS II (inos) NO. + L-Cit IL-1β VEGF Dulak et al., ATVB, 2000; Atherosclerosis 2001; JACC 2001; Jozkowicz et al., Cardiovasc Res 2001

63 NO induces VEGF synthesis L-Arg + O 2 NOS II NO. + L-Cit NO NO 2- (µm) IL-1β VEGF Control IL-1β IL-1β +L-NAME VEGF pg/ml Dulak et al., ATVB, 2000 Control IL-1β IL-1β +L-NAME

64 Interleukin-1β induces VEGF through NO VEGF synthesis is enhanced by NO - generated by NOS II after cytokine stimulation - derived from NOS II or NOS III after gene transfer - released from NO-donors VEGF promoter activation 0,15 + luminescence 0,1 0,05 0 VEGF promoter luciferase βgal enos Dulak et al., ATVB, 2000; Atherosclerosis 2001; JACC 2001; Jozkowicz et al., Cardiovasc Res 2001

65 Enhancement of VEGF synthesis by gene transfer of nitric oxide synthases VEGF (pg/ml) nitrite (µm) * * * * * control β-gal transient stable +NAME +NAME cnos transfection +arginine with cnos # # * * $ bovine enos or human inos cdna Dulak et al, ATVB 2000; Józkowicz et al. Cardiovasc Res 2001

66 Role of NO in regulation of VEGF synthesis in pathological conditions 1. Induction of inos expression in tumors and other inflammatory diseases 2. Inhibition of NO production in cardiovascular diseases

67 Role of nitric oxide in tumors

68 Overexpression of enos enhances angiogenesis in hind limb ischemia Namba K et al., Circulation 2003: 108:

69 Role of heme oxygenase-1 in angiogensis

70 HO-1 Heme oxygenase activity HO-1-1 NO cytokines ROS hypoxia 15d-PGJ2 HO-1 a stress-inducible gene HO-2 a constitutive gene

71 HO-1 How HO-1 influences VEGF synthesis? SnPPIX HO-1 sirna iron heme HO-1/HO-2 biliverdin CO

72 HO-1 HO-1 activity is necessary for VEGF synthesis in vascular smooth muscle cells Dulak et al., Antioxid Redox Signal 2002;4:

73 HO-1 Basal VEGF synthesis is lower in aortic endothelial cells from HO-1 knocked-out mice HO-1-/- % of VEGF produced by wild type cells HO-1+/+ mean of 7 experiments HO-1 +/+ HO-1 -/- Cisowski et al., BBRC 2005

74 HO-1 Possible mediators of HO-1 induced VEGF synthesis eg. PGJ 2 iron ferritin Hb ODQ Heme HO-1 CO soluble guanylate cyclase cgmp Biliverdin Bilirubin

75 HO-1 Inhibition of HO-1 activity, scavenging of CO and inhibition of guanylate cyclase attenuates PGJ 2 -induced VEGF synthesis in HMEC-1 VEGF [% of control] CO [µg/l] * control CO VEGF 15d-PGJ 2 15d-PGJ 2 + SnPPIX, # VEGF [% of control] Control * + ODQ + Hb 15d-PGJ 2 Józkowicz et al. Antioxid Redox Signal, 5: , 2003 # #

76 HO-1 CO (1%) enhances VEGF synthesis in vascular smooth muscle cells Dulak et al., Antioxid Redox Signal 2002;4:

77 HO-1 Iron, HIF-1 activation and VEGF synthesis Prolyl hydroxylase O 2 2-oxoglutarate Fe HIF-1α HIF-1β/ΑRNT HRE activity % of control Control Fe µm Dfx 100 µm Iron inhibits HRE activation Deferoxamine (iron chelator) enhances HRE activation Dulak et al. in: Heme Oxygenase in Biology and Medicine, 2002

78 HO-1 HIF-1α stabilisation by carbon monoxide in lung macrophages Proc Natl Acad Sci U S A Mar 20;104(12): Epub 2007 Mar 12. protein EMSA Air CO

79 HO-1 HO-1 products induce VEGF expression in various cell types 1. Endothelial cells CO 2. Vascular smooth muscle cells CO 3. Keratinocytes, some tumor cells biliverdin

80 HO-1 Biliverdin dose- and time-dependently enhances VEGF gene expression in human keratinocytes control 10 µm 30 µm Control 3 h 6 h 12 h 24 h VEGF EF2 30 µm biliverdin dose-dependent time-dependent VEGF (% of controli) Control BV 1 µm * BV 10 µm * BV 30 µm VEGF (% of control) µm biliverdin * * 3h 6h 12h 24h # - p<0,05 vs control Loboda et al., ARS, 2008

81 Effect of HO-1 on angiogenesis in vivo

82 Facilitated angiogenesis induced by heme oxygenase-1 gene transfer in a rat model of hindlimb ischemia Capillary density VEGF Suzuki M et al., Biochem Biophys Res Commun 2003

83 VEGF-induced signaling in endothelial cells Zachary, Cardiovasc Res 2001

84 NO works upstream and downstream of VEGF NO enos/inos induction or over-expression VEGF SMC keratinocytes enos NO VEGFR-2 endothelial cells EC migration/morphogenesis/survival Dulak & Józkowicz, Card Res 2002

85 Does HO-1 work upstream and downstream of VEGF?

86 VEGF induces HO-1 expression in endothelial cells HUVEC HUVEC HO-1 HO-1 EF2 actin Control Hemin VEGF Control VEGF 24 h VEGF 48 h Dulak et al., Antioxid Redox Signal, 2004 Bussolati et al., Blood 2004:103:

87 Inhibition of HO-1 blocks angiogenic activity of endothelial cells control cumulative length of tubes [% of control value] VEGF VEGF + SnPPIX VEGF + NAME VEGF + SnPPIX + NAME control VEGF VEGF SnPPIX * * VEGF NAME * # VEGF SnPPIX NAME VEGF CuPPIX Józkowicz et al. Antioxid Redox Signal, 5: , 2003

88 Overexpression of HO-1 enhances angiogenic activity of endothelial cells VEGF VEGF + VEGF + VEGF + HO-1 transfection β gal transfection CORM, 1 µm cumulative length of sprouts [% of control value] VEGF + HO-1 cdna Józkowicz et al. Antioxid Redox Signal, 5: , 2003 * + β gal cdna * + CORM 1 µm

89 HO-1 deficiency impairs production of VEGF and decreases proliferation of endothelial cells VEGF synthesis VEGF-induced proliferation VEGF [pg/ml] # WT HO-1 -/- * control 1 ** * * ** ** BrdU [OD 450 nm] Wild type cells 0.05 HO-1 0 -/- control d-PGJ 2 [µm] VEGF [ng/ml] Cisowski et al., BBRC 2005: 326: Dulak et al., in: Heme Oxygenase in Biology and Medicine, Nova Press, 2005

90 HO-1 works upstream and downstream of VEGF NO enos overexpression inos induction HO-1 VEGF SMC keratinocytes VEGFR-1/2 EC enos NO CO? HO-1 EC migration/morphogenesis/survival

91 HO-1 plays a role in vasculogenesis and angiogenesis Deshane J, Chen S, Caballero S, Grochot-Przeczek A, Was H, Li Calzi S, Lach R, Hock TD, Chen B, Hill-Kapturczak N, Siegal GP, Dulak J, Jozkowicz A, Grant MB, Agarwal A. Stromal cell-derived factor-1 promotes angiogenesis via a heme oxygenase-1 dependent pathway. J Exp Med, 2007 Mar 19;204(3): Epub 2007 Mar 5. Dulak J, Deshane J, Jozkowicz A. Agarwal A. Heme oxygenase-1 and carbon monoxide in vascular pathobiology; focus on angiogenesis. Circulation, 2008, 117: (review).

92 Take home messages 1. VEGF expression is regulated by many stimulus, including cytokines, growth factors,, ROS, hypoxia,, NO. 2. Hypoxic regulation of VEGF expression occurs on different levels. 3. Enhancement of VEGF mrna expression - activation of VEGF promoter - increased stability of VEGF mrna 4. Enhanced VEGF translation through internal ribosome entry site (IRES) 5. HO-1 works upstream and downstream of VEGF