Redox regulated transcription factors

Redox regulated transcription factors Katarina Johansson, PhD Division of Biochemistry Medical Biochemistry and Biophysics Karolinska Institutet Stockholm, Sweden

Outline General introduction How is a redox signal sensed and transmitted? à OxyR E. coli à Yap1 S. cerevisiae Redox regulated transcription factors in mammals How we study redox regulated transcription factors

Redox regulation ² Cellular regulation via reversible reduction/oxidation ² Difficult to study because of fast, transient and complex reactions ² Key factors in redox regulation; Trx- and GSH- systems Prx ASK-1 RNR Ref-1 Etc. (Or TRP14) GPx Grx GST Etc.

Cysteine biochemistry allows redoxdependent signaling Signaling molecules à H 2 O 2 à NO à Lipidhydroperoxides à H 2 S Cysteine biochemistry à Sulfenic form (RSOH) à Disulfides (RSSR) à Glutathionylated cysteines (RSSG) à Nitrosylated cysteines (RSNO) Reducing systems à Trx system à GSH system Finkel T - J. Cell Biol. (2011)

How is the redox signal transferred and how is specificity achieved?

Principles of redox signaling à 1) Thermodynamic model. à 2) Direct targeting. à 3) Facilitated targeting. Winterbourn CC and Hampton MB, 2008, FRBM

OxyR a well studied redox regulated transcription factor in E. coli - Direct targeting Direct targeting OxyR is activated by ROS and induces a number of protective enzymes, including Catalase, Grx1, and Glutathione reductase (GSSG 2GSH) M Zheng et al. Science 1998;279:1718-1722

Yap1 a well studied redox regulated transcription factor in S. cerevisiae - Facilitated targeting The H 2 O 2 signal is sensed by a Cys residue in Orp1, which oxidizes to sulfenic acid (Cys-SOH) Orp1 transduces this signal to Yap1 by formation of an intermolecular disulfide bond Thereafter, intramolecular disulfide bonds are formed in activated Yap1 Activated Yap1 may finally induce several antioxidant genes, including proteins in the thioredoxin and GSH systems (Brigelius-Flohé and Flohé, ARS, 2011)

Localisation of the oxidant may increase the specificity of the redox signaling Growth factors à Induce NOX that produce superoxide Extracellular H 2 O 2 is channelled back through aquaporins Tyrosine kinases (Src) à Inactivate PrxI à Accumulation of H 2 O 2 PTP with low pka thiol à Oxidized and inactivated PTP Protein Tyrosine Phosphatases PrxI Peroxiredoxin I NOX NADPH-dependent oxidases BUT PTPs react poorly with H 2 O 2 likely oxidation mediated by another thiol protein e.g. Prx Finkel, 2011, JCB

Redox regulated transcription factors in mammals Many different transcription factors are redox regulated à Nrf2, NFkB, HIF, p53, Oct-4, AP-1, FOXO, Notch, β-catenin etc Highly important for development à Embryonic lethal - NFkB (p65), AP-1 (c-jun, JunB), HIF (HIF1a, HIF1b, ARNT), Ref-1 Redox regulated transcription factors have been implicated to be involved in the regulation of self-renewal and differentiation of stem cells à Nrf2, NFkB, HIF, p53, Oct-4, AP-1, FOXO, Notch, β-catenin Redox regulated transcription factors are involved in many different diseases such as cancer, chronic inflammation, cardiovascular-, neurological-, and pulmonary diseases Knowledge about the mechanism of redox regulated transcription factors is limited due to complex, transient and fast reactions

Many levels of redox regulation of transcription factors in cells Ufer et al., ARS, 2010

p53 is redox regulated by the GSHand Trx-systems Tumor suppressor protein that activates genes involved in cell cycle arrest, apoptosis, and DNA repair Activates upon stress such as hypoxia, heat shock, DNA damage etc p53 activates many different redox-controlling genes, including Gpx and Grx3 Maillet, A and Pervaiz, S, ARS, 2012

Crosslinks between p53 and ROS/ RNS pathways Maillet, A and Pervaiz, ARS, 2012

Nrf2 nuclear factor erythroid 2 (NF- E2)-related transcription factor Redox regulated transcription factor that induce proteins that protect against oxidative stress and xenobiotics (including the GSH- and Trx systems) An induction of Nrf2 is beneficial for patients with degenerativeand autoimmune diseases like MS, atherosclerosis, Parkinson, arthritis An induction of Nrf2 in cancer make the cancer cells more viable and resistant to anticancer drugs

Nrf2 a redox regulated transcription factor that protect cells from ROS In normal conditions Keap1 targets Nrf2 for proteosomal degradation Upon oxidative stress Keap1 are oxidized releases Nrf2 that can translocate to the nucleus Surh, YJ, Nature Reviews Cancer, 2003

Hypoxia Inducing Factor (HIF) HIF regulates more than 70 genes coding for proteins involved in angiogenesis, erythropoiesis, energy metabolism, cell survival. HIF is a heterodimeric transcription factor consisting of one HIFα (HIF1α, HIF2α or HIF3α) and one HIF1β (ARNT) subunit. The oxygen sensors are, Prolyl-4-hydorxylasese domain 1-3 (PHD1, PHD2, and PHD3) and a second oxygen sensor FIH (Factor inhibiting HIF) HIF are important in stem cells, for development but have also been highly linked to metastatic cancer.

Redox Regulation of HIF Oxidation of Fe(II) to Fe(III) in PHDs induces HIF Direct oxidation of HIF1α Reduction of Cys800 in HIF by APE1/Ref-1 or Trx system facilitates binding to DNA

Different levels of redox regulation of HIF-1 in cells Ufer et al., ARS, 2010

NFκB, a key regulator of the immune response NFkB is a protein complex made up from homo- or heterodimers à p50, p52, p65, c-rel and RelB. à Most common is the p50/p65 complex Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development.

NF-κB (nuclear factor kappa-light-chainenhancer of activated B cells) Stress response genes Viruses Acute phase proteins Antigen presentation Enzymes Early response genes Apoptosis Regulators Cytokines/Chemokines Cell adhesion molecules Immunoreceptors Miscellaneous Growth factors Cell surface receptors Transcription factors/regulators

NFkB can be activated by many different stimuli (Brigelius-Flohé and Flohé, ARS, 2011)

Redox regulation of NFκB via the classical pathway Redox regulation of NFκB occurs at different levels à By Trx and other redoxins (Grx, TRP14, Prx) preventing activation of NFκB à A complex cascade of redox sensitive phosphorylation and de-phosphorylation à On the other hand, Trx system and APE1/Ref-1 have been shown to be crucial for NFκB binding to the DNA. (Brigelius-Flohé and Flohé, ARS, 2011)

Simultaneous assessment of Nrf2, NFκB and HIF activation at Single-Cell Resolution

Redox regulated transcription factors Nrf2, NFκB and HIF SH& SH& KEAP1& Nrf2& ub& ub&ub& NFkB& &&& IkB& Normoxia& pvhl& OH&OH& HIF1α& ub& ub&ub& ub& Inflamma*on$ ub& Nrf2& S& S& KEAP1& NFkB& S& S& &&& IkB& ub& ub&ub& ub& HIF1α& Hypoxia& Cytosol& Nrf2& NFkB& SH& SH& HIF1α& Nucleus& red& ARE& AnHoxidant&genes& NFkB& HRE& Target&genes& Immune&response&gene& AnHoxidant&enzymes,&detoxifying& enzymes,&trx=&and&gsh&systems& Chemokines,&adhesion&molecules& and&enzymes&(il=8&etc)& Glycolysis,&angiogenesis&(VEGF),&& survival&and&invasion&factors&&

Redox regulated transcription factors Nrf2, NFκB and HIF

Luciferase assays to study the activation of Nrf2, NFκB and HIF Nrf2 HIF NFkB Nrf2 Luciferase HIF Luciferase NFκB Luciferase With luciferase assays it is only possible to characterize è one transcription factor response at a time è the whole cell culture response (not individual cells)

New tool: ptraf Plasmid for Transcription factor Reporter Activities based on Fluorescence ² Nrf2, HIF and NFκB response elements coupled to three different fluorescent proteins 8 kbp

Overlay pictures of HEK293 cells transfected with ptraf 24h exposure in normoxia or hypoxia NFκB Nrf2 HIF1α Control - 21% O 2 TNF - 21% O 2 Auranofin - 21% O 2 TNF+Auranofin - 21% O 2 Control - 1% O 2 TNF - 1% O 2 Auranofin - 1% O 2 TNF+Auranofin - 1% O 2

Operetta High Content Imaging System ² Fluorescent Microscope ² High-throughput screens ² 96-384 well plates x 48 ² Incubator ² Live or fixed cells

Operetta - High Content Imaging System 80000 60000 I/cell 40000 20000 0 Ctrl EtOH Aur Sulphor AHQ BHQ tbuooh Cobalt CDNB Dox Cisplatin Etoposide CuOOH GSNO TNF TNF+Aur TNF+Sulphor TNF+AHQ TNF+Cobalt TNF+CDNB TNF+Dox TNF+Cisplain TNF+Etoposide Untransfected

ptraf transfected HEK293 cells analysed with the Operetta Nrf2 HIF NFkB 250000 200000 21% O 2 I/cell 150000 100000 50000 0 Ctrl EtOH Aur Sulphor AHQ BHQ tbuooh Cobalt CDNB Dox Cisplatin Etoposide CuOOH GSNO TNF TNF+Aur TNF+Sulphor TNF+AHQ TNF+Cobalt TNF+CDNB TNF+Dox TNF+Cisplain TNF+Etoposide Untransfected 1% O 2 I/cell I/cell 80000 60000 40000 250000 20000 200000 0 150000 100000 Ctrl EtOH Aur Sulphor AHQ BHQ tbuooh Cobalt CDNB Dox Cisplatin Etoposide Normoxia Total I / cell CuOOH GSNO TNF TNF+Aur TNF+Sulphor TNF+AHQ TNF+Cobalt TNF+CDNB TNF+Dox TNF+Cisplain TNF+Etoposide Untransfected Nrf2 HIF NFkB 50000

ptraf transfected HEK293 cells analysed with Operetta in 21 % O 2 Nrf2 HIF NFκB 100000 80000 I/cell 60000 40000 20000 0 Ctrl TNF Auranofin TNF+Aur ptraf transfected cells in 21% O 2

ptraf transfected HEK293 cells analysed with Operetta at 1% O 2 Nrf2 HIF NFκB 50000 40000 I/cell 30000 20000 10000 0 Ctrl TNF Auranofin TNF+Aur ptraf transfected cells in 1% O 2

Nrf2 activity in HEK293 cells transfected with ptraf exposed for 24h in 21% O 2 100000 80000 I/Cell 60000 40000 20000 0 EtOH Auranofin Sulforophane AHQ Doxorubicin Cisplatin TNF TNF+Aur TNF+Sulf TNF+AHQ TNF+Dox TNF+Cisp

HIF activity in HEK293 cells transfected with ptraf exposed for 24h in 21% O 2 20000 15000 I/Cell 10000 5000 0 EtOH Auranofin Sulforophane AHQ Doxorubicin Cisplatin TNF TNF+Aur TNF+Sulf TNF+AHQ TNF+Dox TNF+Cisp

NFκB activity in HEK293 cells transfected with ptraf exposed for 24h in 21% O 2 200000 150000 I/Cell 100000 50000 0 EtOH Auranofin Sulforophane AHQ Doxorubicin Cisplatin TNF TNF+Aur TNF+Sulf TNF+AHQ TNF+Dox TNF+Cisp

How are Nrf2, HIF and NFκB redox regulated? Prx ASK-1 RNR Ref-1 Etc. Trx-system Trx-S 2 (Or TRP14) Trx-(SH) 2 Se TrxR NADPH + H + NADP + GR GSH-system GSSG 2GSH Se GPx Grx GST Etc. Absorbance 0.25 0.20 0.15 0.10 0.05 TrxR1 activity in HEK293 cells 80000 60000 0.00 Control 5nM Se 25nM Se 500nM Se Nrf2 I/cell 40000 20000 0 100000 Ctrl Aur TNF TNF+Aur -Selenite +Selenite (25nM) 80000 HIF I/cell 60000 40000 20000 -Selenite +Selenite (25nM) 0 15000 Ctrl Aur TNF TNF+Aur NFκB I/cell 10000 5000 0 Ctrl Aur TNF TNF+Aur -Selenite +Selenite (25nM)

How are Nrf2, HIF and NFκB redox regulated? Prx ASK-1 RNR Ref-1 Etc. Trx-system Trx-S 2 (Or TRP14) Trx-(SH) 2 Se TrxR NADPH + H + NADP + GR GSH-system GSSG 2GSH Se GPx Grx GST Etc. Absorbance 0.25 0.20 0.15 0.10 0.05 TrxR1 activity in HEK293 cells 80000 60000 0.00 Control 5nM Se 25nM Se 500nM Se Nrf2 I/cell 40000 20000 0 100000 Ctrl Aur TNF TNF+Aur -Selenite +Selenite (25nM) 80000 HIF I/cell 60000 40000 20000 -Selenite +Selenite (25nM) 0 15000 Ctrl Aur TNF TNF+Aur NFκB I/cell 10000 5000 0 Ctrl Aur TNF TNF+Aur -Selenite +Selenite (25nM) Pader et al, 2014, PNAS

HEK293 cells stably expressing ptraf - Exposed for 24 in 21% O2 and 1% O2 Nrf2 HIF NFκB Control 21%O2 TNF 21%O2 Auranofin 21%O2 Auranofin+TNF 21%O2 Control 1%O2 TNF 1%O2 Auranofin 1%O2 Auranofin+TNF 1%O2

How are the heterogenic induction pattern Nrf2, HIF and NFκB regulated? Sort with FACS Nrf2 HIF NFκB Why? RT-PCR Gene arrays HEK293 cells that stably express ptraf induced with Auranofin and TNFα for 24h in 21%O 2

On going and future ² Mechanistic studies with regards to the Trx- and GSH systems ² Further studies of stochastic responses ² Develop new ptraf variants for studies of other transcription factors (p53, AP-1, Oct-4, c-myc, β-catenin (wnt) ) ² Cancer stem cells ² Mechanism of clinically used drugs as well as new potential drug leads to treat cancer, MS, Parkinson s, atherosclerosis

Conclusions Redox regulation is important for control of many different signaling pathways Knowledge on how redox regulated transcription factors is controlled is limited due to complex, transient and fast reactions Redox regulation involves specific oxidation or reduction of regulatory target proteins, often via uniquely reactive Cys residue(s) as sensors Compartmentalization, direct targeting and facilitated targeting are likely to be highly important in redox regulation of transcription factors in mammals

Reference list: Brigelius-Flohe R., and Flohe L., Basic Principles and Emerging concepts in the Redox Control of Transcription Factors, 2011, Antioxiants & Redox Signaling, 15(8); 2335-2381 Winterbourn CC., and Hampton MB., Thiol chemisty and specificity in redox signaling, 2008, FRBM, 45;549-561 Arner ESJ., and Holmgren A., Physiological functions of thioredoxin and thioredoxin reductase, Eur J. Biochem. 2000, 267;6102-6109 Ufer et al, Redox control in Mammalian Embryo Develoment, 2010, Antioxidants & Redox Signaling, 13;6, 833-875 Finkel, T, Signal transduction by reactive oxygen species, 2011, J. Cell Biol. 194;(1) 7-15 Marinho H.S et al, Hydrogen peroxide sensing, signaling and regulation of transcription factors, 2014, Redox biology 2, 535-562