Bile acids activated receptorsregulate the integrity of gastrointestinal mucosa

focus on FXR

Stefano Fiorucci, MD

Department of Surgical and Biomedical SciencesUniversity of Perugia

The Japanese Society of Gastroenterology (JSGE) COI Disclosure

There are no companies, etc. in a relation of conflict of interest requiring disclosure in relation to the presentation.

There is no state of conflict of interest requiring disclosure

Bile acids are regulatory steroidsgenerated at the interface of cholesterol metabolism

and intestinal microbiota

Cell Metabolism 2012

Composition of bile acids in the gallbladder and feces of healthy individuals.

Ridlon J M et al. J. Lipid Res. 2006;47:241-259

VDR

LXR

PXR

Neutral pathway Acidic pathway

AndrogenEstrogenMineralcorticoidsGlucocorticoids

Fiorucci S., et al. Trends Mol Med 2007

FXR

PR

GP-BAR1

Published in: Claudio D’Amore; Francesco Saverio Di Leva; Valentina Sepe; Barbara Renga; Chiara Del Gaudio; Maria Valeria D’Auria; Angela

Zampella; Stefano Fiorucci; Vittorio Limongelli; J. Med. Chem. 2014, 57, 937-954.

Non genomic effects Genomic effects

FXR is central to bile acids signaling

Lipid

Metabolism

Carbohydrate

Metabolism

Bile acids

FXR

Bile Acids

Metabolism

Normal and

pathologic

growth and

differentiation

Immunity

Fiorucci S., et al. Prog Lipid Res. 2010 Apr;49(2):171-85

hingeLBDNH2 COOHDBDAF1 AF2

DIMERIZATION

A/B C D E

2 3 4 5 6 7 8 9 10 11

FXR3-4FXR1-2

1

(MYTG)

Bile acids

9-cis RA

Histones

De-Acetylation

FXR RXR

AF1

DBD DBD

AF2

AF1

CoRepressor

AF2Histones

Acetylation/Methylation

Ac AcAc

Ac Ac

Ac

Ac Ac

AcAc

Ac Ac

FXR RXR

AF1

DBD

AF2

DBD

AF2

AF1

CoActivator

CARM1

DRIP205PGC1-

PRMT1 SRC-1

AGGTCA n TGACCT

Fiorucci S, et al. Trends in Mol Med 2007

An FXR – RXR heterodimer is essential for

FXR activation

RXR

FXR

FXR the main bile acid sensor

Inagaki T et al. PNAS 2006;103:3920-3925

Cipriani S, et al. 2009, Current Medicinal Chemistry

ER

LXR

LXRb

FXR

PPAR

PPAR

PPAR

VD

GR

Con

trol

0

50

100

150

200

250

Rlu

c/

Gal

0

50

100

150

-10 -9 -8 -7 -6 -5 -4

Agonist concentration (Log M)

GW4064

6-ECDCA

CDCA

Rela

tive

Luci

fera

se/

Gal

Pellicciari R, al. J Med Chem. 2002 Aug 15;45(17):3569-72

1: Sepe VTotal Synthesis and Pharmacological Characterization of Solomonsterol A, a Potent Marine Pregnane-X-Receptor Agonist Endowed with Anti-Inflammatory Activity. J Med Chem. 2011 Jun 3. 2: De Marino S, Towards new ligands of nuclear receptors. Discovery of malaitasterol A, an unique bis-secosterol from marine sponge Theonella swinhoei. Org Biomol Chem. 2011 Jul 7;9(13):4856-62. Epub 2011 May 16. 3: De Marino S, Theonellasterols and conicasterols from Theonella swinhoei. Novel marine natural ligands for human nuclear receptors. JMed Chem. 2011 Apr 28;54(8):3065-75. Epub 2011 Apr 5. PubMed PMID: 21428459.4: Sepe V, Discovery of sulfated sterols from marine invertebrates as a new class of marine natural antagonists of farnesoid-X-receptor. J Med Chem. 2011 Mar 10;54(5):1314-20. 5: Festa C, Solomonsterols A and B from Theonella swinhoei. The First Example of C-24 and C-23 Sulfated Sterols from a Marine Source Endowed with a PXR Agonistic Activity. J Med Chem. 2010 Dec 9.

FXR orchestrates the innate Immunity in the GI and liver

FXR is expressed by and exerts counterregulatory effects on LPS-primed macrophages.

Mencarelli A et al. Am J Physiol Heart Circ Physiol

2009;296:H272-H281©2009 by American Physiological Society

Vavassori P. et al The Journal of Immunology November 15, 2009 vol. 183 no. 10 6251-6261

0

20

40

60

80

100

Control FXR+/+ TNBS FXR-/- TNBS

I F I F I F

His

tolo

gic

cla

ssific

atio

n (

%)

Severe

Moderate

Mild

Absent

0 7 14 21 28 35 42 49

80

90

100

110

120

FXR-/- TNBS

FXR+/+ TNBS

Control

Days

Bo

dy w

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)

*

* * *

b

Control

FXR+/+

TNBS

FXR-/-

TNBS

4X20X 20X

20X

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20X

20X

4X

4X

c

0 20 40 60 100 1000

SMA

TIMP-1

MMP-2

MMP-1

Fibronectin

1(I) collagen

TGF1

IFN

IL-1

TNF

I-BABP

SHP

FXR

°

°

°

°

°

°

°

°

°

*

*

Relative RNA expression

FXR-/- TNBS

FXR+/+ TNBS

Control

d

0

2000

4000

Control FXR+/+

TNBS

FXR-/-

TNBS

LPS - + - + - +

TN

F

(p

g/m

L)

e

*

*º^

*º

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a

0

1000

2000

3000

IL-1

(p

g/m

L)

*

*

*

0

1000

2000

3000

4000

IFN (

pg

/mL

)

*

*

*

I F I F I F0

20

40

60

80

100

Control TNBS TNBS + INT-747

I F I F I F

His

tolo

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cla

ssific

atio

n (

%)

Severe

Moderate

Mild

Absent

a

b

Control TNBS TNBS + INT-747

20X 20X 20X

20X 20X 20X

0 1 2 3 4 5 5 10

SMA

TIMP-1

MMP-2

MMP-1

Fibronectin

1(I) collagen

TGF1

IFN

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TNF

I-BABP

SHP

FXR

15 300

*°

°

°

°

°

°

°

°

*

*

*

*

*

*

°

°

°

°

TNBS + INT-747

TNBS

Control

Relative RNA expression

c

d

0

1000

2000

3000

Control TNBS TNBS + INT-747

LPS - + - + - +

TN

F

(p

g/m

L)

*

*°

*°

0

1000

2000

3000

4000

*

*

*

IL-1

(p

g/m

L)

*

0

1000

2000

*

IFN (

pg

/mL

)

Vavassori P. et al The Journal of Immunology November 15, 2009 vol. 183 no. 10 6251-6261

FXR shapes the intestinal immunity and interacts with Toll like receptors

-

TLR1/ 2

TLR6/ 2

TLR4

TLR5

TLR3

TLR7/ 8

TLR90

1

2

3

4

*

*

* * * *

P la s m a m e m b r a n e T L R s

I n t r a c e l lu la r T L R s

FX

R

re

l.e

xp

r. (

2-

Ct

)

-

TLR1/ 2

TLR6/ 2

TLR4

TLR5

TLR3

TLR7/ 8

TLR90

1

2

3

4

5

6

7

8

9

*

*

**

*

*

TN

F

re

l. e

xp

r. (

2-

Ct )

Re

v-E

rb

A-

Re

v-E

rb

A-

LX

R

LX

R

PX

R

CA

R

TR

2

TR

3

TR

4

FX

R

SH

P

GR

VD

R

PP

AR

PP

AR

PP

AR

RA

R

RA

R

RO

R

RX

R

RX

R

0

1

2

3

4

5

6

7

*

*

Fo

ld c

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ng

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(C

pG

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PB

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1

2

3

4

CpG

TLR9+/+ TLR9-/-

*

FX

R

rel. e

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(2

-

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*

A. B.

C.

D.

Renga et al. PLoS One. 2013;8(1):e54472. doi: 10.1371/journal.pone.0054472. Epub 2013 Jan 25

- - +0

1

2

3

4

6-ECDCA

TNBS

*

#

Mucosa

l dam

age s

core

- - +0

1

2

3

4

6-ECDCA

TNBS

*

#

Mucosa

l dam

age s

core

Wild type TLR9-/-

Renga B. et al. PlosOne 2013

TLR-9

Endosome

MyD88

TRAF3

IKK

IRA

K1

IRF7

PP

IFN

IFN

FXR

Nucleus

CpG

C.

FXR, microbiota and probioticsa bidirectional liason

Composition of cecal microbiota of rats from different diet groups as revealed by sequencing of

16S rRNA gene clone libraries. Population analyses for each diet group at phyla level

K.B.M. Saiful Islam , et al. Bile Acid Is a Host Factor That Regulates the Composition of the Cecal Microbiota in Rats

Gastroenterology, Volume 141, Issue 5, 2011, 1773 - 1781

⁎ Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan

Naive

group

TNBS

group

TNBS +

VSL#3

group

A B C

D E F

G H I

Mencarelli A. et al. Plos One 2012

FXR mediates Probiotics activitities in IBDs

NT

Ros

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100n

M

(100

%)

(50%

)

(10%

)0.0×10 -00

5.0×10 05

1.0×10 06

1.5×10 06

2.0×10 06

4.0×10 06

5.0×10 06

6.0×10 06

7.0×10 06*

*

*

Conditioned Medium

RL

U/

gal

NT

calcife

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50nM

100% 50

%10

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0.0×10 -00

1.0×10 05

2.0×10 05

3.0×10 05

4.0×10 05

2.0×10 06

3.0×10 06

4.0×10 06

Conditioned Medium

RL

U/

gal

*

*

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%10

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0.0×10 -00

1.0×10 06

2.0×10 06

3.0×10 06

4.0×10 06

3.0×10 07

4.0×10 07

5.0×10 07

Conditioned Medium

RL

U/

gal

*

*

PPARγ VDR FXRA. B. C.

0

1

2

3

WTNaive

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Naive VSL#3 DSS

Control VSL#3

#

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(mR

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rela

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Probiotic metabolism generate ligands for nuclear receptors

Mencarelli A. et al. PlosOne 2012

FXR shapes the intestinal immunityby interacting with glucocorticoid receptor

1A11A2

1A31C 1H1I

1B1D

1F

1J

1E exon 2/ATG

-36851 -32300 -5151 0

bp

ATG

hGR proximal promoterhGR distal promoter

ER-8

-34993 -34673

Renga B. et al. FASEB J (2012) Mar 23

FXR enhances the transcription of Glucocorticoid Receptor gene

- + - +0

1000000

2000000

3000000

4000000

pGL3(ER8)3X

pGL3(ER8mutated)3X

*

6E-CDCA

RL

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Renga B. et al. Pharmacological Research, Volume 77, 2013, 1 - 10

The distal promoter of NR3C1 gene (the GR gene) contains an FXRE that facilitates the

formation of a head-to-tail chromatin loop, increases GR gene transcription efficiency and is

essential to mediate the anti-inflammatory effects of FXR in the intestine.

An intact FXR signaling is required to preserve anti-inflammatory activities of GR.

Barbara Renga , Claudio D’Amore , Sabrina Cipriani , Andrea Mencarelli , Adriana Carino , Valentina Sepe , Angela ...

FXR mediates a chromatin looping in the GR promoter thus promoting the resolution of colitis in rodents

Pharmacological Research, Volume 77, 2013, 1 - 10

The steroid inhibitor RU-486 reverses protection exerted by FXR agonism in a mouse model of colitis.

Barbara Renga , Claudio D’Amore , Sabrina Cipriani , Andrea Mencarelli , Adriana Carino , Valentina Sepe , Angela ...

FXR mediates a chromatin looping in the GR promoter thus promoting the resolution of colitis in rodents

Pharmacological Research, Volume 77, 2013, 1 - 10

http://dx.doi.org/10.1016/j.phrs.2013.08.008

FXR, prostanoids, NO and H2S

FXR

Liver Stomach0.0

0.5

1.0

mR

NA

rela

tive

exp

ress

ion

sto

ma

ch

WB -FXR

live

r

WB -tubulin

A

B

C

0

10

20

30FXR

-/-FXR +/+

ASA

10 mg/kg 30 mg/kg 100 mg/kg

*

*

#* **#

Mucosal dam

age s

core

(mm

of

lesi

on)

WT + ASA 10 mg/kg

FXR -/- + ASA 10 mg/kg

E

F

G

FXR IHC

FXR depletion worsens gastric injury induced bu ASA

Fiorucci S, Mencarelli A, Cipriani S, Renga B, Palladino G, Santucci L, Distrutti E. Activation of the farnesoid-X receptor protects againstgastrointestinal injury caused by non-steroidal anti-inflammatory drugs in mice. Br J Pharmacol. 2011 Dec;164(8):1929-38

ICAM-1

0

1

2

3

4

WT FXR-/-

alone ASA alone ASA

mR

NA

levels

COX-1

0

1

2

3

WT FXR-/-

alone ASA alone ASA

mR

NA

levels

COX-2

0.0

2.5

5.0

7.5

10.0

WT FXR-/-

alone ASA alone ASA

mR

NA

levels

0

10

20

30

40 *

WT FXR-/-

alone ASA alone ASA

MPO

(mU

nits

/mg

of p

rote

in)

TNF

0

1

2

3

4

5

WT FXR-/-

alone ASA alone ASA

mR

NA

levels

E

0

500

1000

1500

FXR-/-FXR

+/+

**

* *6-k

eto

PG

F1

(pg /

mg o

f pro

tein

)0

100

200

300

400

ASA

Naive 10 mg/kg 100 mg/kg

PG

E2

(pg /

mg o

f pro

tein

)

* *

**

Gastric protection exerted by FXR is PGE2 independnet

Fiorucci S, Mencarelli A, Cipriani S, Renga B, Palladino G, Santucci L, Distrutti E. Activation of the farnesoid-X receptor protects againstgastrointestinal injury caused by non-steroidal anti-inflammatory drugs in mice. Br J Pharmacol. 2011 Dec;164(8):1929-38

tNSAIDs

COX-1 and COX-2

CSE and H2S

Fiorucci S, Antonelli E, Distrutti E, Rizzo G, Mencarelli A, Orlandi S, Zanardo R, Renga B, Di Sante M, Morelli A, Cirino G, Wallace JL. Inhibitionof hydrogen sulfide generation contributes to gastric injury caused by anti-inflammatory nonsteroidal drugs. Gastroenterology. 2005 Oct;129(4):1210-24

Mechanism of gastric injury caused by NSAIDs

iNOS

0.0

2.5

5.0

7.5

10.0

WT FXR-/-

alone ASA alone ASA

*

mR

NA

levels

CSE

0.0

0.5

1.0

WT FXR-/-

alone ASA alone ASA

*

mR

NA

levels

CSE activity

0

25

50

75

WT FXR-/-

alone ASA alone ASA

*

mA

bs 7

27n

m/3

0

g o

fp

rote

in

eNOS

0.0

0.5

1.0

1.5

WT FXR-/-

alone ASA alone ASA

*

mR

NA

levels

CSE expresion

F G H I L

M

FXR is a positive regulator of CSE, a H2S generating enzyme

Fiorucci S, Mencarelli A, Cipriani S, Renga B, Palladino G, Santucci L, Distrutti E. Activation of the farnesoid-X receptor protects againstgastrointestinal injury caused by non-steroidal anti-inflammatory drugs in mice. Br J Pharmacol. 2011 Dec;164(8):1929-38

Renga B, Mencarelli A, Migliorati M, Distrutti E, Fiorucci S. Bile-acid-activated farnesoid X receptor regulates hydrogen sulfide productionand hepatic microcirculation. World J Gastroenterol. 2009 May 7;15(17):2097-108

FXR binds and activates a FXR-RE in the promoter of CSE a H2S generating enzyme

HEPG2pSG5FXR

pSG5RXR

-

DCA

LCA

CA

CDCA

6E-C

DCA

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

80000000

(25M)

*

**

*

*

*

RL

U/

gal

HEPG2

pSG5FXR

pSG5RXR

- 0,01 0,1 1 100

2500000

5000000

7500000

10000000

12500000

15000000

6E-CDCA (M)

*

*

*

RL

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HEPG2

pSG5FXR

pSG5RXR

- + - + - +0

2500000

5000000

7500000

10000000

12500000

15000000

6E-CDCA (10 M)

pGL3 pGL3(CSE-IR1)4X

pGL3CSE-IR1mutated

*

#

#

RL

U/

gal

HEPG2

pSG5FXR

pSG5RXR

- - + +0

10000

20000

30000

40000

50000

6E-CDCA (10 M)

*

#

- + - + GUGGULSTERONE 50 M

*

RL

U/

gal