CELL SIGNALING THROUGH THE THIOL GROUP

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CELL SIGNALING THROUGH THE THIOL GROUP
Yvonne Janssen-Heininger, Ph.D Associate
Professor of Pathology, The University of
Vermont yvonne.janssen_at_uvm.edu
2
INFLAMMATORY SIGNALS ARE GENERATED IN LOCALIZED
COMPARTMENTS WITHIN TISSUES












Homogenization can lead to spurious
results Requires in situ evaluation
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NEED FOR IN SITU ANALYSIS OF REDOX CHANGES OR
INFLAMMATORY SIGNALING

• Strategies in my laboratory
• - Microscopy approaches to image cell signaling
  and redox changes
• - Epithelial cell lines
• - Primary epithelial cell cultures
• - Transgenic mouse technologies

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REDOX CHANGES IN ASTHMA OR MODELS OF ALLERGIC
AIRWAYS DISEASE
Reactive Oxygen Species Increases in H2O2 in
the exhaled breath, or BAL Inactivation of
glutathione peroxidase Inactivation of
superoxide dismutase Inactivation of
catalase Comhair, Lancet 2000, Comhair
Am J Pathol 2005, Ghosh J. Immunol 2006, Gaston,
Lancet 1998, Dweik, PNAS 2001, Saleh, Faseb J
1998, Kaminsky, JACI 1999, Ghosh J. Immunol
2006, Zimmermann J. Clin Invest 2003, Que
Science 2005
Reactive Nitrogen Species NOS2 increases in
airway epithelium Increased levels of NO in
exhaled breath Increased tyrosine nitration
Substantial decrease in S-nitrosothiols Increas
ed activity of GSNO reductase Increased activity
of arginase
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FUNCTIONAL SIGNIFICANCE OF REDOX CHANGES IN
INFLAMMATION?

• Critical questions
• - What are the relevant oxidative events?
• - Does oxidative modification of specific cell
  targets occur?
• - Where do these modifications occur within
  tissues?
• - How do we measure these modifications and
  determine their consequences?

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REVERSIBLE OXIDATIVE MODIFICATIONS POTENTIAL
FOR CELL SIGNALING

• Oxidation of Cysteine residues
• Sulfenic acids SOH
• Disulfides S-S
• S-Nitrosation SNO
• S- Glutathionylation P-SSG
• Targets are many, including proteases,
  signaling molecules,
• transcription factors
• Nuclear Factor kappa B, caspases, phosphatases
• Problems Difficulties in detection
• Reversible nature
• Need for chemical derivatization

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NF-kB

• Transcription factor known to induce expression
  of over 100 genes
• involved in innate and adaptive immune responses
• that allow survival to multiple stresses
• involved in proliferation
• involved in inflammation
• NF-kB activation occurs in pulmonary diseases
  (asthma, COPD)
• Mice that lack c-Rel or p50 subunits of NF-kB do
  not develop allergic airway disease
• NF-kB is a redox sensitive transcription factor

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Canonical
stimulus
TRADD
IKKa
P
P
IKK?
IKKß
P
P
I?Ba
p50
RelA
P
P
p50
RelA
26S
I?Ba
GGGPuNNPyPyCC
Ub
Ub
Ub
Ub
Genes important in inflammation and inhibition of
apoptosis
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MOUSE MODEL OF ALLERGIC AIRWAYS DISEASE
Experimental protocol for inducing inflammation
with Ovalbumin (Ova)
14
21 22 23
25
1
Day
c c c
Treatment
IP
IP
Evaluation
IP intraperitoneal injection of 400 mcg
ovalbumin mixed with alum c challenge with
aerosolized ovalbumin (1 soln.) Alum/Ova
experimental controls Ova/Ova inflamed mice
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RAPID ACTIVATION OF NF-kB IN AIRWAY EPITHELIUM
no 1 Ab
control
15min
30min
6hr
24hr
Nuclei Nuclear RelA RelA
Poynter M et al. Am. J. Pathol, 2002
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HOW DOES ONE DETERMINE THE IMPORTANCE OF NF-kB
ACTIVATION IN A SPECIFIC LOCATION (I.E. THE
AIRWAY EPITHELIUM) IN CAUSING INFLAMMATION?
MOUSE TRANSGENICS
Use a tissue specific promoter that is only
active in the organ of interest Rat CC10 -
Bronchiolar epithelium Rat SPC - Alveolar
epithelium and bronchiolar epithelium TIE2 -
Endothelial cells Distal LCK -
Lymphocytes Villin - Enterocytes FSP-1 -
Fibroblasts Keratin 14 - Epidermis MCK
- Skeletal muscle AlfP -
Hepatocytes LysM - Myeloid cells
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MOUSE TRANSGENICS
Subclone your construct of interest so that it
is expressed under the control of that promoter
(with tag for verification) Include a polyAAA
sequence (from the human growth hormone) to
enhance mRNA stability and expression Submit
to transgene facility for injection into oocytes
and implantation in mice Analyze genotype and
phenotype of offspring
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TRANSGENE-MEDIATED INHIBITION OF NF-kB IN AIRWAY
EPITHELIUM STRONGLY SUPRESSES INFLAMMATION
Transgene negative
Transgene positive
DNA RelA Nuclear RelA
Lumen
Lumen
Histo- pathology
Poynter M. et al. J. Immunol 2003, J. Immunol,
2004
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NF-?B ACTIVATION IN THE AIRWAY EPITHELIUM IS
NECESSARY AND SUFFICIENT TO DRIVE INFLAMMATION
Allergen (Ova)
Airway Lumen
NF-kB
NF-kB
IkB
IkB
NF-kB
Chemokines
NOS 2, MIP 2, Eotaxin, IL-6, MHC-II
inflammatory cell
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DO REDOX CHANGES INFLUENCE NF-kB ACTIVATION IN
LUNG EPITHELIUM?
Focus of presentation Evaluation of activation
and oxidation of IKKb
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PRINCIPLES OF AN IN VITRO KINASE ASSAY
Make cell lysate
Use antibody specific to target of interest
Add Protein A or Protein G beads to bind the
antibody
Centrifuge to precipitate the complex of target,
antibody and Protein A/G beads
Wash
Incubate pellet with substrate of the kinase, in
presence of 32P ATP
Run polyacrylamide gel, dry, exposure to film,
and evaluate incorporation of 32P into substrate
Substrate P
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TRANSIENT INACTIVATION OF IKK ACTIVITY BY H2O2
recovery time
TNFa 0 5 10 30
H2O2
Sham
GST-IkB
IB IKKg
120
100
80
kinase activity
60
40
20
0
TNFa
TNFa
TNFa
TNFa
TNFa
sham
H2O2
0 5 10 30
H2O2 recovery time
Reynaert N PNAS 2006
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H2O2 INHIBITS IKK ACTIVITY THROUGH OXIDATION OF
CYSTEINE 179 OF IKKb
Strategy Transfect wildtype (wt) IKKb, with
Hemeagglutinin (HA) tag Transfect mutant
form of IKKb in which Cys 179 is mutated to Ala
(c179a), with HA tag Co-transfect Nox1 plus
co-activators to enhance H2O2
production Immunoprecipitate wt or c179a mutant
IKKb via the HA tag using a specific antibody
against HA Do in vitro kinase
assay
Reynaert, PNAS 2006
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ASSESSMENT OF S-GLUTATHIONYLATION OF CYSTEINE 179
OF IKKb
Reynaert N et al. PNAS 2006
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MAMMALIAN GLUTAREDOXIN/THIOLTRANSFERASE ENZYMES
Grx 1 12 kD, cytosolic, CSYC active site,
reduced by GSH/GR Grx 2 18 kD, mitochondrial
nuclear, CPYC active site, reduced by GSH/GR and
Trx/TrxR
Fernandes Holmgren, AntioxRedox Sign, 2004
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GRX1 PROTECTS IKK AGAINST INACTIVATION BY H2O2
AND RESTORES NF-kB ACTIVATION
TNFa
Sham TNFa H2O2
GST-IkB
IB IkBa
IB IKKg
pcDNA3
Reynaert, PNAS 2006
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GRX1 KNOCKDOWN INHIBITS TNFa-STIMULATED NF-kB
ACTIVATION IN ASSOCIATION WITH ENHANCED
S-GLUTATHIONYLATION OF IKKb
IKK activity
GRX activity
TNFa
Sham 0 100 200
mM H2O2
6
units
4
GST-IkB
2
IBIKKg
0
1 10.2 7.7 0.3
control siRNA
GRX1 siRNA
Fold induction
control siRNA
IKKb-SSG
TNFa
Sham
Sham
100
100
200
200
Sham 0 100 200
mM H2O2
mM H2O2
GST-IkB
IPbiotin, IBHA
IBIKKg
1 3 0.2 0.1
Fold induction
IBHA
GRX1 siRNA
Control siRNA GRX1 siRNA
Reynaert N et al, PNAS, 2006
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PRIMARY MOUSE TRACHEAL EPITHELIAL CELLS FROM
Glrx1-/- MICE HAVE ENHANCED P-SSG AND FAIL TO
ACTIVATE NF-kB
Sham LPS
WT Glrx1-/-
WT
Glrx1-/-
DNA RelA
LPS 1 mg/mL
WT
Glrx1-/-
WT
Glrx1-/-
1800
30000
NFkB
1600
NS
25000
1400
1200
20000
Free probe
MIP-2 (pg/mg protein)
1000
KC (pg/mg protein)
15000
Fold induction 1 0.5 2.9
0.6
800
600
10000
400
5000
200
0
0
Glrx1-/- sham
WT LPS
Glrx1-/- LPS
WT sham
Reynaert N et al, PNAS, 2006
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INTERIM CONCLUSIONS
Hydrogen peroxide inhibits IKKb via
S-glutathionylation of Cysteine
179 Glutaredoxin-1 reverses S-glutathionylation
of IKKb-Cys 179, and permits activation of the
NF-kB cascade Glutaredoxin-1 knockdown dampens
NF-kB activation and enhances
S-glutathionylation of IKKb-Cys 179 Cells
derived from Glrx-1 -/- mice show enhanced
protein S-glutathionylation, and are refractory
to activation of NF-kB
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GRX1 IS INCREASED IN LUNGS OF MICE WITH
ALLERGIC AIRWAYS DISEASE
Alu/OVA OVA/OVA
IB GRX1
100

80
units
60
40
20
0
Alu/OVA
OVA/OVA
OVAlOVA
Alu/OVA
DNA GRX1
Reynaert, Am J Respir Cell Mol Biol 2006
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HOW CAN WE DETECT REGIONAL DIFFERENCES
IN SULFHYDRYL OXIDATIONS IN THE INTACT LUNG?
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GRX1-CATALYZED DERIVATIZATION TO DETECT
S-GLUTATIONYLATION IN SITU
S - NEM
SH
1.Blocking free SH in the protein with NEM during
lysis
SSG
2. Wash excess NEM
SSG
S-S
S-S
S -NEM
S -NEM
4. Biotinylation of free SH with
maleimidyl propionyl biocytin
3. Reduction of SSG by glutaredoxin-1
SH
S- biotin
S-S
S-S
5. Detection of biotin with streptavidin
fluorophore
In situ detection
Reynaert, BBA-Gen, 2006
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GRX1-CATALYZED REDUCTION TO DETECT
PROTEIN-S GLUTATHIONYLATION IN SITU
preDTT
sham
-GRX
-MPB
sham
BSA
BSA-cys
BSA-GSSG
DNA Protein-SSG
Reynaert, BBA-Gen, 2006
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GRX1-CATALYZED REDUCTION TO DETECT
PROTEIN-S GLUTATHIONYLATION IN SITU
wound 40X
wound 20X
sham
H2O2
DNA Protein-SSG
Reynaert, BBA-Gen, 2006
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GRX1-CATALYZED REDUCTION TO DETECT
PROTEIN-S GLUTATHIONYLATION IN SITU
Alum/Ova
Ova/Ova
Control
DNA Protein-SSG
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CONCLUSIONS
GRX-1 catalyzed cysteine derivatization allows
detection of S-glutathionylated cysteine
residues in situ through visualization by
confocal microscopy. GRX-1 catalyzed cysteine
derivatization readily permits detection of local
oxidative changes in cells, cellular
compartments, or tissues. This approach can be
used to probe oxidative changes in diseased
tissues, and does not require prior
administration of labeling, or trapping
agents. Note that potential limitations include
accessibility of targets, and specificity of
GRX1-dependent catalysis.
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ACKNOWLEDGEMENTS
Department of Pathology Albert van der
Vliet, Ph.D Brooke Mossman, Ph.D
Nicholas H. Heintz, Ph.D Douglas Taatjes,
Ph.D Vermont Lung Center Charles G.
Irvin, Ph.D Matthew E. Poynter, Ph.D
Jason Bates, Ph.D Mercedes Rincon,
Ph.D Maastricht Emiel F.M. Wouters,
MD,Ph.D Wayne State University Ye-Shih
Ho, Ph.D

• Janssen-Heininger Laboratory
• Karina Ckless, Ph.D
• Cristen Pantano, Ph.D
• John Alcorn, Ph.D
• Vikas Anathy, Ph.D
• Jennifer Ather
• Niki Reynaert, Ph.D
• Scott Aesif
• Amy Guala
• Amy Brown
• Stacie Beuschel

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