FALK WORKSHOPMechanisms of intestinal inflammation

Dresden October 9-10, 2007

TGF-β1 and Smad7 in the regulation of IBD

Giovanni MonteleoneDept of Internal Medicine

UNIVERSITY TOR VERGATA

OF ROME

TGF-β1 is a negative regulator of mucosal inflammation

• TGF-β1 null mice die of inflammation, including the gut

• Transgenic mice expressing a dominant-negative TGF-βRII chain develop severe colitis and pulmonary inflammation

• In mouse models of IBD, synthesis of TGF-β1 associates with either the protection from the development of colitis or greatly diminished severity of colitis

Activated TGF-β1 is produced in the human normal gut

Ileal and colonic biopsies taken from 5 normal subjects

pg/1

00 μ

g to

tal p

rote

ins

50

40

30

20

10

0Ileum colon

TGF-β1 transcripts in laser captured epithelium (E) and lamina propria (LP) mononuclear cells from normal ileum and colon

5 samples in each group

TGF-

βtr

ansc

ripts

(rel

ativ

e un

its)

0

20

40

60

80

100

10

30

50

70

90

E LP E LP

Ileum

Colon

Adapted from Smythies LE et al. JCI 2005; 115: 66

Mast cells, not macrophages, are an intestinal source of TGF-β

TGFβ-producing LPMC C-kit-positive cells

HAM56-positive cells TGFβ-producing epithelial cells

•

T-be

t/β-a

ctin

(a.u

.)

IgG aTGFβ02

64

810

1412 p<0.0001

02

64

IgG aTGFβ

810

1412 p<0.0001

Mucosa

IgGPatient 1 Patient 2

aTGFβT-bet

β-actin

LPMC

Blockade of TGF-β1 in cultures of normal colon LPMC or mucosal explants increases the Th1 transcription factor T-bet and IFN-γ

IFN

-γ(p

g/m

l)

p=0.005

0

40

80

120

160

••••

••

•

•

•

•

•

•

•

•

• •••

•

p=0.020

0

2040

60

100

80

• •• •• •• ••

••

•

• ••

•

••

•

T-be

t/β-a

ctin

(a.u

.)

IgG aTGFβ

IgGPatient 1 Patient 2

aTGFβ IgG aTGFβT-bet

β-actin

IFN

-γ(p

g/m

l)

IgG aTGFβ

IgG aTGFβ

0

300

600

900

1200

1500

1800

2100

IgG aTGFβ IgG aTGFβ IgG aTGFβ

IL-17

p=0.024p=0.047

IL-8

p=0.002

Cytokine production by LPMCs(pg/ml)

0

5

10

15

20

25

IL-2

0

5

10

15

20

25

••

30

35

40

••• •

• •••

•

•

•

•

• ••

•••

•

•

••

• •

•• •

30

35

40

•

•

••

•

• •••

• ••

•

Blockade of TGF-β1 in cultures of normal intestinal LPMCincreases the secretion of inflammatory cytokines

Blockade of endogenous TGF-β1 in cultures of normal LPMC does not affect the percentage of Foxp3-positive cells

CD4

Foxp

3

CD4

Foxp

3

IgG anti-TGF-β0,15% 0,35%

60,8% 38,7%

0,32% 0,34%

69 % 30,3%

3

2

1

0% F

oxp3

+ C

D4+

LP

L

IgG anti-TGF-β(3 days culture)

TNF - + + + + + +TGF-β1 - - + - - - -NF-kB/p65 ab - - - + - - -NF-kB/p50 ab - - - - + - -excess of specific probe - - - - - + -excess of non specific probe - - - - - - +

P50 homodimerP65/p50 heterodimer

TGF-β1 inhibits TNF-induced NF-kB DNA binding activityin normal lamina propria mononuclear cells

4

3

2

1

0 ActiveCD

InactiveCD

ActiveUC

InactiveUC

Normal

Adapted from Babyatsky et al, Gastro 1996; 110:975

Enhanced TGF-β RNA expression in the inflamed colonic mucosa of patients with Crohn’s disease and patients with ulcerative colitis

TGF-

β/G

AP

DH

Real-time PCR analysis

Blockade of TGF-β1 in cultures of CD mucosal explants does not affect the expression of T-bet

T-be

t RN

A re

lativ

e ex

pres

sion 2,0

1,5

1,0

0,5

0IgG anti-TGF-β

P50 homodimer

P65/p50 heterodimer

NF-kB/p65

Medium + - -TNF - + +TGF-β1 - + +

Histone-1

NF-kB activation is barely decreased by TGF-β1 in IBD LPMC

IIIP

P

TGFβ1

Smad2/3

Smad4

DNA

Target gene

TGFβ signalling through Smads

P

P

P-SMAD3

SMAD3

SMAD3-boundSMAD4

Controls CD UC

Active SMAD3 is down-regulated ininflammatory bowel disease mucosa-biopsies

P

P

III P

TGFß1

Smad2/3

Smad4

Monteleone G, et al. J Clin Invest 2001; 108:601-9

SMAD7

controls1 2 3 1 2 3

CD1 2 3

UC

CD UC C CD UC C

non CD3+

SMAD7

CD3+

Increased SMAD7 in inflammatory bowel disease mucosa

TGFß

III PSmad2/3

Smad7

Monteleone G, et al. J Clin Invest 2001; 108:601-9

-

+ + +

U S AS SMAD7

P-SMAD3

SMAD3

Crohn’s disease

Smad7 anti-sense (AS) inhibits Smad7 and restores TGF-β signalling

IIIP

TGFβ

Smad2/3

P

Smad7

Smad3

U S AS

TGF-β

Crohn’s disease

Monteleone G, et al. J Clin Invest 2001; 108:601-9

TNF

SEB +

0

10000

20000

30000

40000

50000

0 0,1 1 10

trans

crip

ts/μ

g to

tal R

NA

mediumsenseantisense

TGF-β1 (ng/ml)

Anti-sense to Smad7 allows TGF-β1 to inhibitTNF transcripts in Crohn’s

Monteleone G, et al. J Clin Invest 2001; 108:601-9

SMAD7

p-SMAD3

SMAD3

TNF-α

IFN-γ

medium sense antisense

IFN

-γ(p

g/μg

tiss

ue) 750

500

250

medium sense antisense

SMAD7 antisense restores TGF-β1 signaling and inhibitscytokine production in Crohn’s disease (CD) tissue

CD tissue culture supernatants

CD tissue

Question?

What induces Smad7 in IBD?

TGF-β1

P

P

STAT1Smad7

nucle

us

Smad7 gene promoter

IFN-γ

p-STAT1

NF-kB

TNF-α

IFN-γ/STAT1 and TNF/NF-kB induce SMAD7 RNA expression in cell lines

Smad7 RNA

p-Tyr-STAT1

Inhibition of either STAT1 or NF-kB activationdoes not change SMAD7 protein expression in CD

LPMC UNST TPCK Vehicle

NuclearNF-kB/p65

Histone-1

Smad7

β-actin

N-p-Tosyl-L-phenylalaninechlorometyl ketone (TPCK) =

NF-kB inhibitor

Smad7

β-actin

STAT1βSTAT1α

UNST TB42 Vehicle

Tyrphostin B42 (TB42) =JAK2/STAT1 inhibitor

Paired biopsies from IBD and normals

Smad7: real time RT-PCR and Western blotting

To examine the molecular mechanisms underlying the induction of Smad7 in human intestine

transcriptionally or post-transcriptionally regulated?

Sm

ad7/

β2-m

icro

glob

ulin

RN

A

(f.u.

x 10

-7)

0

2,5

5

7,5controls

1 2 3 4 Crohn’s disease

61 KD50 KD

38 KD

50 KD

38 KD

Smad7 protein but not RNA is increased in IBD mucosa

Smad7

β-actin

1 2 3 4

Smad7

β-actin

controls1 2 3 4

Crohn’s disease1 2 3 4

controls CD UC

Real-time PCR data

In IBD, Smad7 is regulated at the post-transcriptional level

Is the high Smad7 due to:

• enhanced de novo protein synthesis

• increased stability of the protein

Question?

1,5 1,5 3 3 6 6- + - + - +

Smad7hours

IkBα

β-actin

Per

cent

age

of re

mai

ning

prot

ein

afte

r CH

X

0 1,5 3 6 Time (h)

100

50

0

Smad7IkBα

CD LPMCbasal CHX

Normal LPMC

In IBD, high Smad7 does not relyon a sustained protein synthesis

1,5 1,5 3 3 6 6- + - + - +

hours

basal CHX

Smad7β-actin

Is the high Smad7 due to:

• enhanced de novo protein synthesis

• increased stability of the protein

Question?

Smad7 degradation

Smad7

Arkadia

JAB1

ubiquitin

proteasome

Checkpoints in the control of Smad7 protein stability

- - +

1 2 3

0 3 5 7 0 3 5 7

IP: Smad7Blot: ubiquitin ab

Crohn’s disease controls ve -1 2 3

IP: Smad7Blot: Smad7

Smad7β-actin

control CD Hrs of incubation

0 5 5 Hrs of incubationProteasome

inhibitors

Ubiquitination and degradation of Smad7occur in controls but not Crohn’s disease (CD)

control

Smad7β-actinMonteleone G, et al. Gastroenterology 2005; 129: 1420

Normal LPMCMG132+

UNS MG115 dmso

CD LPMC

Inhibition of the proteasome pathway enhances Smad7 protein expression in normal intestinal LPMC

Smad7

β-actin

MG132+MG115= proteasome inhibitors

MG132+UNS MG115 dmso

Smad

7/β-

actin

prot

ein

(a.u

.)

MG132+UNS MG115 DMSONormal LPMC

MG132+UNS MG115 DMSO

CD LPMC

0

0,3

0,6

0,9

1,2

Smad7 degradation

Smad7

Arkadia

JAB1ubiquitin

proteasome

Smad7

acetylationAcetylated

Smad7ubiquitin

Control of SMAD7 stability by competitionbetween acetylation and ubiquitination

Acetylated- Smad7ns

Total Smad7

Smad7 is acetylated in IBD

1 2 CDcontrols ve -

1 2 1 2 UC

0

0,25

0,5

0,75

1

controls CD

Ace

tyla

ted/

tota

l Sm

ad7

(a.u

.)

UC

P=0.001

P=0.02

P= NS

Monteleone G, et al. Gastroenterology 2005; 129: 1420

Smad7 degradation

Smad7

Arkadia

JAB1ubiquitin

proteasome

Smad7

acetylationAcetylated

Smad7ubiquitin

Control of SMAD7 stability by competitionbetween acetylation and ubiquitination

ROLE OF p300

p300

p300IP: Smad7blot: p300 ab

Smad7IP: Smad7blot: Smad7 ab

p300 interacts with Smad7 in CD

1 2 3Crohn’s disease controls ve -

1 2 3

p300

p300

IgG

IgG

CD

Con

trol

Monteleone G, et al. Gastroenterology 2005; 129: 1420

Inhibition of p300 by siRNAreduces Smad7 expression in CD

control p300Uns siRNA siRNA

p300

CD LPMC

β-actin

control p300Uns siRNA siRNA

ve -

acetylated Smad7

Ubiquitinated Smad7

CD LPMC

Smad7β-actin

control p300Uns siRNA siRNA

CD LPMC

Monteleone G, et al. Gastroenterology 2005; 129: 1420

What is the functional role of Smad7 inmurine models of IBD?

Question?

TNBS-colitis (Crohn’s disease-like colitis)

oxazolone-colitis (which resembles Ulcerative colitis)

naive EtOH TNBS

Smad7

β-actin

p-Smad3

Smad3

High TGF-β1 associates with a diminished p-Smad3 and enhanced Smad7 expression in TNBS-induced colitis

0

200

400

600

800

naive EtOH TNBS

TGF-β1pg/100 μg total proteins

naive EtOH TNBSCD3+

CD3-

LPMC

naive EtOH TNBS

Sm

ad7

1 2naive Oxa

1 2Smad7

β-actinp-Smad3

Smad3

1

10

100

1.000

10.000

naive Oxa

TGF-β1pg/100 μg total proteins

High TGF-β1 associates with a diminished p-Smad3 and enhanced Smad7 expression in oxazolone-induced colitis

1 2naive Oxa

1 2

Administration of Smad7 antisense oligonucleotide in mice with experimental colitis

days

SJL mice

Intrarectal administration of 150 μl of Ethanol 50%

or ethanol 50% + TNBS or oxazolone

Histology andProtein analysis

0 1 2 3 4

Intra-gastric administration ofSmad7 antisense or sense50, 125,250, 500 μg/mouse

ileum colonproximal small

intestine

Inhibition of Smad7 by antisense DNA restores p-Smad3 and down-regulates TNBS-induced colitis

naive TNBS TNBS TNBSsense AS

Smad7p-Smad2/3

Smad3

Intestinal uptake of labelled Smad7 AS

TNBS ->ASTNBS

p<0.01

Time 0

8 hours

0.70.750.80.850.90.951

0 1 2 3 4

wei

ght c

hang

e

days

TNBS->S

*

*

Naive TNBS

TNBS + sense TNBS + AS

Smad7 antisense (AS) DNA inhibitsTh1 type cytokines in TNBS-induced colitis

0

1000

2000

3000

naive EtOH TNBS TNBS TNBS+ S +AS

IFN

-γpg

/100

μg

pro

tein

s

01020304050

IL-1

2p70

pg/2

50 μ

g p

rote

ins

0

1000

2000

3000

4000

IL-1

2p40

pg/1

00 μ

g p

rote

ins TNBS

Sense AS

T-betp-Stat1

β-actinnaive EtOH TNBS TNBS TNBS+ S +AS

naive EtOH TNBS TNBS TNBS+ S +AS

1 2 1 2

Inhibition of Smad7 by antisense DNA restores p-Smad3 and down-regulates oxazolone (oxa)-induced colitis

oxa oxa oxa ++S AS

Smad7p-Smad2/3β-actin

0,8

0,85

0,9

0,95

1

0 1 2 3days

Wei

ght c

hang

e (%

of d

ay 0

)

oxa

OXA+AS

oxa

oxa + sense

oxa + AS

0255075

100IL-4 (pg/250 μg total protein)

Naive oxa oxa+S oxa+AS

High Smad7 in established TNBS-induced colitis

TNB

S

TISSUE ANALYSIS

BALB/c mice

0 1 2 3 4

weeks

Intrarectaladministrationof TNBS (mg) 0,5 0,5 0,7 1

naiv

e

p-Smad3

naive TNBSSmad7

β-actin

Smad7 antisense oligonucleotidedown-regulates established TNBS-induced colitis

days

body

wei

ght c

hang

e (%

ofd

ay 0

)

TNBS+AS

TNBS+Sense90

92

94

96

98

100

102

104

0 1 2 3 6

4th TNBS administration

Oligoadministration

sense

antisense

Francesco PalloneGiovanna Del Vecchio BlancoIvan MonteleoneDaniele FinaRoberta CarusoLivia Biancone Massimo FantiniAngelamaria RizzzoMassimiliano SarraCarmine StolfiFlavio CaprioliUniversity “Tor Vergata”, Rome

Giampiero PalmieriAlfredo ColantoniSerena Battista Luigi Giusto Spagnoli University “Tor Vergata”, Rome

Markus NeurathUniversity of Mainz, Germany

Jelena MannDerek MannSylvia PenderUniversity of Southampton (UK)

Sergio BernardiniSabrina BalleriniGiorgio FedericiUniversity “Tor Vergata”, Rome

Monica BoirivantClaudia Di Giacinto, Mariarosaria MarinaroIstituto Superiore di Sanita’, Rome

Giuliani SpA, MilanBMRP Foundation, Los Angeles

Thomas T MacDonaldBarts and the London School of Medicine and Dentistry, London, UK.

Warren StroberNIH, Bethesda, MD, USA

MMP-3

MMP-3

β-actinMuc

osa

Supe

rnat

ant

IgGPatient 1 Patient 2

aTGFβ

0

4

IgG aTGFβ

8

12 p<0.001

0

4

8

12 p<0.001

MM

P-3

(a.u

.)M

MP-

3/β-

actin

(a.u

.)

TIMP-1

TIMP-1

Blockade of TGF-β1 reduces MMP-3 production in organ cultures of normal colonic mucosal explants

IgG aTGFβ

IgG aTGFβ IgG aTGFβPatient 1 Patient 2

TIM

P-1/β-

actin

(a.u

.)

IgG aTGFβ

IgG aTGFβ IgG aTGFβ

TIM

P-1

(a.u

.)

2

4

6

8

0

4

8

12