Bile acids activated receptorsregulate the integrity of gastrointestinal mucosa

Stefano Fiorucci, MD

Department of Surgical and Biomedical SciencesUniversity of Perugia

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

VDR

LXR

PXR

Neutral pathway Acidic pathway

AndrogenEstrogenMineralcorticoidsGlucocorticoids

Fiorucci S., et al. Trends Mol Med 2007

FXR

PR

GP-BAR1

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

Fiorucci S., et al TIPS 2012

Me

Me

HOH

COOH

R

24

5

Me

H

OH

OHA B

C24 5b-bile acids inmammalian

A/B ring junction cisBent scape

R=OH R1=H CDCAR=OH R1=OH CAR=H R1=OH DCAR=H R1=H LCA

R1

Bile acids and their receptors

The Superfamily of Human Nuclear Receptors

Endocrine Hormone Receptors

1. Androstan Receptor (CAR)

2. Estrogen Related Receptor- (ERR)

3. Farnesoid X Receptor (FXR)

4. Hepatocyte Nuclear Factor-4 (HNF-4)

5. Liver X Receptor (LXR)

6. Peroxisome Proliferator-Activated Receptor (PPAR)

7. Pregnane X Receptor (PXR)

8. Retinoid X Receptor (RXR)

Adopted Orphan ReceptorsMetabolic Nuclear Receptors

1. Chicken Ovalbumin Upsteram (COUP)

2. Dosage-sensitive Sex Reversal (DAX)

3. Germ Cell Nuclear Factor (GCNF)

4. Liver Related Homologue-1 (LRH-1)

5. NGF-induced clone B (NGFI-B)

6. Photoreceptor Nuclear Receptor (PNR)

7. Reverse ErbA (RevErbA)

8. Small Heterodimer Partner

9. Steroidogenic Factor-1 (SF-1)

10.Testis Receptor-2 (TR-2)

Orphan Nuclear Receptors

1. Estrogen Receptor-b (ER-b)

2. Estrogen Receptor- (ER-)

3. Glucocorticoid Receptor (GR)

4. Mineralcorticoid Receptor (MR)

5. Androgen Receptor (AR)

6. Progesterone Receptor (PR)

7. Retinoic Acid Receptor (RAR)

8. Tyroid Hormone Receptor (TR)

9. Vitamin-D Receptor (VDR)

Metabolic NRs expressed in the liver and gastrointestinal tract

Estrogen Related Receptor- (ERR) Androstan Receptor (CAR) Farnesoid X Receptor (FXR) Hepatocyte Nuclear Factor-4 (HNF-4) Liver X Receptor (LXR) Peroxisome Proliferator-Activated Receptor (PPAR) Pregnane X Receptor (PXR) Retinoid X Receptor (RXR) Vitamin D Receptor (VDR)

Bile Acids

Nuclear Receptors Membrane receptors

Receptor Rank of potency

FXR/RXR CDCA>CA>LCA

PXR/XRX LCA>CDCA

CAR/RXR CDCA>CA>LCA

VDR/RXR LCA

Receptors Rank of potency

GP-BAR1 (TGR5) LCA>DCA>CDCA

Muscarinic LCA, DCA

EGF-R Transactivation

FMLP-R Antagonism

Distrutti et al. J Gastroenterology 2015

FXR works as FXR/RXR heterodimer

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

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; Rizzo et al. Mol Pharmacol 2006

An FXR – RXR heterodimer is essential for

FXR activation

RXR

FXR

FXR

NTCP

CYP7A1

SHP

Cholesterol CA

CDCA

CYP3A4/Cyp3a11, UGT2Bs, Sult2a1

MDR1

ABCBs

MRP2

Glycineconjugation

MRP3 MRP4

DCA

LCA

SMAL INTESTINE

Intestinalmicrobiota

L cells

FXR

FGF-15

GPBAR1

GLP-1

LIVER

FGF-15

CA, DCA, CDCA, LCA

GLP1-R

InsulinGLP-1

FGF-R4

PORTAL VEIN

Enterocytes

Β-cells

BILE DUCTS

BSEP

OSTα/β

Conjugated and Unconjugated BA

OATPs

Uptake

Synthesis

Excretion

PANCREAS

Future Med Chem 2015, in press

Cholesterol biliary excretion

SR-B1

↑ABCG5

↑ ABCG8

Bile acid biosynthesis

↑ BSEP (IR1)

↑ I-BABP (IR1) (intestine)

hIBAT/ASBT (intestine)

↑ hMDR3, rMDR2 (IR1)

↑ MRP2 (ER8)

NTCP

OATP B1

↑ OATP B3 (IR1)

↑ OATP8 (IR1)

CYP7A1

CYP8B1

↑ hFGF-19

↑ mFGF-15 (small intestine)

↑ SHP

Bile acid conjugation

↑ BACS (IR1)

↑ BAT (IR1)

Bile acid & xenobiotics transport

Bile acid detoxification

↑ Cyp3A4 (IR1,DR3,IR8)

↑ SULT2A1 (IR0)

↑ UGT 2B4 (1/2 FXRE)

Triglyceride metabolism

↓ApoAI (1/2 FXRE)

↓ ApoB

↑ ApoCII (IR1)

↓ ApoCIII (HNF4 RE)

↑ ApoA V (IR8)

↑ ApoE

↓ h hepatic lipase

↑ PLTP (IR1)

↑ hPPARα (imp. DR5)

SREBP-1c

↑ VLDL-R (BA, not FXR)

↑ Syndecan (DR1)

↑ Complement C3 (IR1) intestine

MTP

Glucose metabolism

↑PDK4

Cellular protection

↑ A-Crystallin (IR1)

TRB3,Akt?

LXR

RXR/RAR

LRH-1

RXR/RAR(h)

FTF/LRH1(rb)

FGF15 or 19,

JNKFoxo1

HNF4 PEPCK

F 1,6-biPase

G6Pase

Inflammation, coagulation

↑Fibrinogen

↑ h kininogen (IR-1)

↑ ICAM (FXRE in RARE)

FXRHepatocyte

FXRα

CYP7A1

CYP8B1

NTCP

BSEPBile

Canaliculus

BAs

synthesis

Bile acids

MDR2

MRP3

SHP

sinusoidal

capillaries

OSTα

OSTβ

Genes in red are inhibited by BAs

MDR2

MRP2

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

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

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/b

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/b

Gal

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

-Festa, C. et al. J. Med. Chem. 2014,

COOH

OHHOH

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 and cholestasis: 6-ECDCA /INT-747/obeticholic acid

0 15 30 45 60 75 900

25

50

75

100

E27

6-ECDCA 10 mg/kg

6-ECDCA 5 mg/kg

Control

*

**

6-ECDCA 1 mg/kg

Time (min)

Bile f

low

L

/Kg

/min

0 15 30 45 60 75 900

25

50

75

100

Control

6-ECDCA 10 mg/kg

CDCA 10 mg/kg

E27 **

**

Bil

e f

low

L

/Kg

/min

Time (min)

0 15 30 45 60 750

25

50

75

100

E217 alone

E217 + GW4064

Control

*

**

Time (min)

Bil

e f

low

L

/Kg

-min

B

C

A

Fiorucci et al. JPET 2005

0

1

2

3

4

5

6

7Medium

6-ECDCA

SHP Cyp71 Cyp8b1 Bsep Ntcp Oatp1

*

**

*

*

CDCA

qR

T-P

CR

mR

NA

*

* *

*

*

FXR orchestrates the innate Immunity in the GI and liver

FXR is expressed by and exerts counterregulatory effects in 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

eig

ht (%

)

*

* * *

b

Control

FXR+/+

TNBS

FXR-/-

TNBS

4X20X 20X

20X

20X

20X

20X

4X

4X

c

0 20 40 60 100 1000

SMA

TIMP-1

MMP-2

MMP-1

Fibronectin

1(I) collagen

TGFb1

IFN

IL-1b

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

ºº^

a

0

1000

2000

3000

IL-1b

(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

gic

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

TGFb1

IFN

IL-1b

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-1b

(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-b

LX

R

LX

Rb

PX

R

CA

R

TR

2

TR

3

TR

4

FX

R

SH

P

GR

VD

R

PP

AR

PP

AR b

PP

AR

RA

R

RA

R

RO

R

RX

R

RX

Rb

0

1

2

3

4

5

6

7

*

*

Fo

ld c

ha

ng

e

(C

pG

ve

rs

us

no

t t

re

ate

d C

D1

4 d

eriv

ed

PB

MC

)

- + - +0

1

2

3

4

CpG

TLR9+/+ TLR9-/-

*

FX

R

rel. e

xpr.

(2

-

Ct )

*

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

IFNb

FXR

Nucleus

CpG

C.

FXR, microbiota and probioticsa bidirectional liason

LA David et al. Nature 1-5 (2013) doi:10.1038/nature12820

Changes in the faecal concentration of bile acids and biomarkers

for Bilophila on the animal-based diet.

Sama I. Sayin , Annika Wahlström , Jenny Felin , Sirkku Jäntti , Hanns-Ulrich Marschall , Krister Bamberg , Bo An...

Gut Microbiota Regulates Bile Acid Metabolism by Reducing the Levels of Tauro-beta-muricholic Acid, a Naturally

Occurring FXR Antagonist

Cell Metabolism, Volume 17, Issue 2, 2013, 225 - 235

The intestinal microbiota modulates the bile acid metabolism

Hepatic metabolic and signaling pathways modulated by the gut microbiota.

Swann J R et al. PNAS 2011;108:4523-4530

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

iglitaz

one

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/ b

gal

NT

calcife

diolo

50nM

100% 50

%10

%

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/b

gal

*

*

*

NT

CDCA 1

0M

100% 50

%10

%

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/b

gal

*

*

PPARγ VDR FXRA. B. C.

0

1

2

3

WTNaive

ApoE-/-

Naive VSL#3 DSS

Control VSL#3

#

PP

AR

(mR

NA

rela

tive e

xpre

ssio

n)

* *

**

0

1

2

3

WTNaive

ApoE-/-

Naive VSL#3 DSS

Control VSL#3

#

*

VD

R(m

RN

A r

ela

tive e

xpre

ssio

n) #

0

1

2

WTNaive

ApoE-/-

Naive VSL#3 DSS

Control VSL#3

#

FX

R(m

RN

A r

ela

tive e

xpre

ssio

n)

D. F. G.

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

U/b

gal

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

0

10000

20000

30000

40000

50000

60000

70000

80000

Wild type ApoE-/-

Untreated INT-747 INT-747 Rosiglitazone

3 mg/kg 10 mg/kg 20 mg/kg

*

**

****

Lesio

n a

rea (

pix

els

)

Oil Red O H&E CD11b

A B

D

C

E

G H

I

J K

L

M N O

P

Wild

type

APOE-/-

unt

reat

ed

INT-7

47 1

0 m

g/kg

Ros

iglitaz

one

20

mg/

kg0

20

40

60

80

Lesio

n a

rea (

pix

els

)

Q

R*

** **

Mencarelli A., et al, Am J Physiol 2009

FXR/GP-BAR1 activation reduces atherosclerotic plaque formation

GI lab Perugia University

• Barbara Renga

• Sabrina Cipriani

• Claudio D’Amore

• Andrea Mencarelli

• Adriana Carino

• Eleonora Distrutti

• Luca Santucci

• Franco Baldelli

• Annibale Donini

IRD-Tahiti

Dr. Cécile Debitus

Prof. ssa Maria Valeria D’Auria

Dr. Simona De Marino

Dr. Carmen Festa

Dr. Valentina Sepe

Dr. Raffaella Ummarino

Department of Pharmacy, Salerno

Prof. Giuseppe Bifulco

Dr. Maria Giovanna Chini

Dr. Gianluigi Lauro

Department of Pharmacy, Napoli

Prof. Ettore Novellino

Dr. Vittorio Limongelli

Dr. Francesco Saverio Di Leva

Dr. Valentina Sepe

Dr. Carmen Festa

Dr. Dario Masullo

Dr. Claudia Finamore

Also thanks to