Methods in molecular biology

Ian Adcock

National Heart & Lung Institute,Imperial College, London

Imperial CollegeLondon

Turkish Thoracic Society10th Annual Congress

Asthma: A Chronic Inflammatory Disease

CoordinateGene

Expression

T -Cells (CD4+)–––

Eosinophils–––

Macrophages–––

Epithelial Cells–––

Smooth MuscleCells

Corticosteroids

GeneticFactors

EnvironmentalFactors

(Smoke, allergens)

GeneProducts

Cytokines–––

Chemokines

–––Cell

AdhesionMolecules

–––Enzymes

Pathology

AcuteInflammation

–––Progressive

ChronicInflammation

–––Airway

Remodeling

Airway Inflammation

Infectious agentsInfectious agents

CytokinesCytokines

Cigarette SmokeCigarette Smoke

Epithelial cellEpithelial cell

IL-6IL-8

Proteinases

IL-6IL-8

Proteinases

NeutrophilNeutrophil

AdhesionChemotaxisProteinases

AdhesionChemotaxisProteinases

MacrophageMacrophage

TNFIL-6

Eicosanoids

TNFIL-6

Eicosanoids

LymphocyteLymphocyte

Interferon IL-2IL-4IL-5IgE

Interferon IL-2IL-4IL-5IgE

Inhibitorx x x x

Induction of inflammatory mediators

Allen et al., 2003; Kerr et al., 2005

Detection of abnormal gene expression using genome-wide screens and microarrays

Functional genomics/Proof of concept

Does an asthma/COPD gene have a relevant function?

What is the effect of selective overexpression?

Does loss of function have functional significance?

Localisation of DPP10 in biopsies of asthma and COPD patients. Results were confirmed using Western blot analysis

Normal

Normal

Asthma

Asthma

COPD

COPD

DPP10 expression is increased in bronchial biopsies of asthmatic subjects

β-actin

DPP10

0

50

100

150

200

% o

f con

trol

NormalModerate

asthmaSevere asthma

Effect of CSE on DPP10 mRNA expression in Beas2B cells

0

0.5

1

1.5

2

2.5

3

3.5

* *

GAPDH

DPP10In

cre

ase f

rom

con

trol

Non-stim 1hr 4hr 24hr

CSE- and H2O2-induced DPP10 is attenuated by NAC

0

100

200

300

400

500

% o

f c

on

tro

l

Non-stim NAC(1mM)

CSE CSE+

NAC

H2O2H2O2

+NAC

*

*

#

Overexpression of target genes

Transfection using naked DNA

Transfection aided by Liposomes

Adenoviral infection of cells

Transfection reagent Cell line specific.

Need to optimise individually.Manufacturer Transfection reagent Cell lines tested

Mirus

Qiagen

Invitrogen

TransIT-TKOA549, C6, CHO-K1, COS-7,

NIH3T3, HEK293, HeLa, Hepa1cLc7, HepG2, MCF-7,

Neuro-2a, SK-N-MC(Transfection of siRNA)

COS-7, NIH3T3, HEK293, HeLa, CHO, S2

(Transfection of RNA and siRNA)

Oligofectamine

Transmessenger HeLa, CHO(Transfection of RNA)

Primary cells work best with ds oligos and viral infection

Amaxa

Meja KK et al., JPET 2004

Use of adenoviral vector to inhibit Protein Kinase A

Use of adenoviral vector to inhibit PKA:Effect on 3H-AA release

Meja KK et al., JPET 2004

HDAC2 RNAiRT-PCR

1 2 3 4 5HDAC2

HDAC1

Western Blotting

2 3 4 5

1. Non treatment2. control duplex siRNA3. HDAC2 duplex siRNA-1 (612-)4. HDAC2 duplex siRNA-2 (813-)5. HDAC2 siRNA-1 & siRNA-2

HDAC2 siRNA attenuates HDAC2 expression

72-96 hrs to KD HDAC2 protein/ 24-48h for mRNA

Dex EC50 (10-10M)0.1 1 10 100

50

100

150

0H

DA

C2

exp

ress

ion

(% o

f co

ntr

ol)

100

% o

f co

ntr

ol

-14 -100

50

sc

Dex (log, M)

HDAC2 knockdown attenuates steroid sensitivity

HDAC2 RNAi

-14 -10

**

Em H2 Em H2

non-smoker COPD

NTNT

HDAC2 overexpression restores steroid sensitivity in COPD BAL macrophages

smoker COPDNon-smoker0

2.5

5.0

**

GM

-CS

F (

ng

/ml)

Control

IL-1(1ng/ml)

IL-1 + Dex (10-6M)

HDAC1 overexpression

*

HDAC2 overexpression

Acetylation of K494/495 is important in GR-mediated suppression of NF-B activity

Ito et al., JEM 2006

DPP10 knockdown in BEAS-2B cells

0

20

40

60

80

100

120

% o

f co

ntr

ol

0 10 50 ng siRNA

DPP10

Dex induced apoptosis following DPP10 knock down in the presence of IL-1β/H2O2

Cas

pase

3 a

ctiv

ity a

ssay IL-1β (10ng/ml) H2O2 (50µM)

+0

+ +8 6

--

Scrambled siRNA

+0

+ +8 6

--

DPP10 siRNA

+0

+ +8 6

--

Scrambled siRNA

+0

+ +8 6

--

DPP10 siRNA

Dex (-log [M])

The glucocorticoid receptor

NT (AF1) DBD LBD (AF2)

Classic nuclear receptor structure

Held in cytosol until ligand bindsIncreased transcription

Upstream promoter elements

GREBasal

transcriptional machinery

Co-activator complex

Activated GR

GFP

Mechanisms of GR transrepression

Basal transcriptional

machinery

Upstream promoter elements

Decreased transcription

Activated GR

NF-B p65

Transrepression depends on a physical interaction between GR and NF-B

Similar mechanism has been described for AP-1

We can measure this association in cells using bioluminescent resonance energy transfer technology (BRET)

Dexamethasone nM

0.01 0.1 1 10 100 1000

BR

ET S

igan

l0.06

0.08

0.1

0.12

0.14

0.16

0.18

YFP

CFP

Investigating GR function using reporter genes in vitro

Foetal rat lung epithelial cells cotransfected with an NF-B-dependent reporter to measure transrepression and an MMTV -driven reporter to measure transactivation

[Steroid] nM0.0010.01 0.1 1 10 100 1000

MM

TV

lu

cife

rase

0

500

1000

1500

2000

[steroid] nM0.001 0.01 0.1 1 10 100N

F-kB

act

ivit

y (

% T

NF)

0

20

40

60

80

100

Reporter gene assays can indicate potential regulatory sequences

Newton et al., 1997; Morello et al., 2006

IL-10 gene promoter

IL-10 gene polymorphism and asthma severity

-1082 -819 -592

G/A C/T G/A Normal Mild Severe n=241 n=82 n=113FEV1 (% pred) 96% 90% 64%

GCC IL-10 49.1 28.8 21.8 **

ACC IL-10 43.3 30.5 26.2 **ATA IL-10 38.5 30.5 30.5

Measures of transcription factor activation

Newton et al., 1997, Morello et al.,2006, Lee et al., 2006

Systems analysis approach to determining protein function

mild asthma severe asthma

10

200

Mr (kDa)

pI pI3.5 10 103.5

Identification of affected Pathways

+ +