Tgr5 and intestinal mucosa

TGR5 and gastrointestinalmucosa

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

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

EGF-R Transactivation

FMLP-R Antagonism

Cipriani S. et al., Progress in Lipid Research 2010

FXR works as FXR/RXR heterodimer

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

Published in: Claudio D’Amore; et al.; J. Med. Chem. 2014, 57, 937-954.DOI: 10.1021/jm401873d

Tridimensional model of GP-BAR1

GP-BAR1 (TGR5, M-BAR1)

Energy

expenditure

Carbohydrate

Metabolism

TLCA-DCA

GP-BAR1

Bile Acids

Metabolism

Normal and

pathologic

growth and

differentiation

Immunity

GP-BAR1 is expressed by and regulatesmonocytes

Kawamata Y, et al. J Biol Chem. 2003 Mar 14;278(11):9435-40

Maruyama T. et al. Biochem Biophys Res Commun. 2002 Nov 15;298(5):714-9.

Lithocholic acid (LCA EC50 35 ± 5 nM) >

deoxycholic acid (DCA EC50 575 ± 75 nM) >

chenodeoxycholic acid (CDCA EC50 4004 ± 662

nM) > cholic acid (CA EC50 >10 M).

HEK- 293

LCA, lithocholic acid; DCA, deoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid; UDCA, ursodeoxycholic acid;

DHCA, dehydrocholic acid; P, progesterone; DHEA, dehydroepiandrosterone; DHT, 5-dihydrotestosterone; T, testosterone.

The rank order of potency of bile acids

TLCA, LCA, DCA, CDCA, and CA

(EC50) of 0.33, 0.53, 1.01, 4.43,

and 7.72 M, respectively.

ileum mesenteric blood

bile canaliculus portal blood

Asbt bile

acids

(+) FXR FGF15

Ostα/β

bile

acids cholesterol

Cyp7a1Bsep

FGF15

FGF-R4

Enterocyte

HepatocyteNtcp

Apical Basolateral

Gβ

Gα

Gγ

Phospholipase

Cβ2

IP3 Ca2+

GLP1

tgr5

0

1000

2000

3000

4000

*

GPBAR1-/-

GPBAR1+/+

(ng/m

l)

0.0

0.5

1.0

1.5

0

1

2

3

4

mR

NA

rela

tive e

xpressio

n

0.00

0.25

0.50

0.75

1.00

1.25

*

Zonulin-1 Ocludin E-cadherin Dextrane FITC

A GPBAR1 +/+ GPBAR1 -/-

20x20x

40x

20x

CGPBAR1-/-GPBAR1 +/+

D

10x 10x

20x

B

20x20x

20x20x

20x 20x

Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011). PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637

0 1 2 3 40

1

2

3

4

GPBAR1-/-

GPBAR1+/+

*

Time (days)

Dia

rrhea s

core

0

20

40

60

80

100

GPBAR1 +/+

Naive DSS

GPBAR1 -/-

Naive DSS

MildAbsent Moderate Severe

Ma

cro

sco

pic

Da

ma

ge

(%

)

0

2500

5000

7500

10000

GPBAR1 +/+

Naive DSS

GPBAR1 -/-

Naive DSS

*

**

FIT

C-d

extrane

(ng/m

l)

Naive DSS0.0

0.5

1.0

1.5

2.0

GPBAR1 +/+

*

GP

BA

R1

mR

NA

rela

tive

expre

ssio

n

DSS

GPBAR1 -/-

GPBAR1 -/- DSS

GPBAR1 +/+

GPBAR1 +/+ DSS

GPBAR1 +/+

Naive

A B C

D

F

20x20x

20x

E

20x10x

20x20x

20x

Cipriani S, Mencarelli A, Chini MG, Distrutti E, et al. (2011) The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of

Intestinal Barrier and Immune Response to Experimental Colitis. PLoS ONE 6(10): e25637. doi:10.1371/journal.pone.0025637

Naive TNBS DSS0.0

0.5

1.0

1.5

2.0

2.5

GPBAR1 +/+

* *

GP

BA

R1

mR

NA

rela

tive e

xp

ressio

nNormal Crohn's disease

0.0

0.5

1.0

1.5

2.0*

GP

BA

R1

mR

NA

rela

tive e

xp

ressio

n

20x

40x

Naive TNBS DSS

Crohn’s colitis

A C E

B D F

G

H I

L



0

1

2

3

GPBAR1 +/+

Naive Ciprofloxacin TLCA

GPBAR1 -/-

*

*

Naive Ciprofloxacin TLCA

(10M) (10M)(10M) (10M)

Sple

en m

onocyte

scA

MP

[pm

ol/m

l]

0

25

50

75

100

*

**

*

*GPBAR1 +/+

Naive LPS Alone + Ciprofloxacin

GPBAR1 -/-

Naive LPS Alone + Ciprofloxacin

(10M) (10M)

TN

F

mR

NA

(re

lativ

e e

xp

ressio

n)

NN

HN

F COOH

O

1 2

HO

NH

H

H

O

SO3

A

B

C

D

E

0

5

10

15

20

25

30

0 0.1 1 5 10 100

Ciprofloxacin (M)

[cA

MP

] i

(pg/m

g p

rote

in)

Naive

Ciproflo

xacin

TLC

A

0

10

20

30

40

*

*



0

1

2

3

4TNBS

*

**

-1 0 1 2 3 4 5

Naive TNBS TNBS +ciprofloxacin

Time (days)

Dia

rrhe

a s

core

Naive TNBS TNBS+Ciprofloxacin

10x 10x 10x

20x 20x 20x

0

1

2

3

4

*

**

Naive TNBS

Vehicle Ciprofloxacin

Mic

roscop

ic s

core

0

1

2

3

4

5

6 *

**

Ma

cro

sco

pic

sco

re

Naive TNBS CIPRO0

10

20

30

40*

**

IL1

rel.

exp

ressio

n

Naive TNBS CIPRO0.0

2.5

5.0

7.5

*

**

TN

Fa

rel.

exp

ressio

n

12

14

16

18

20

22

24

26

28

30

32

Naive TNBS TNBS + ciprofloxacin

TNBS

Ciprofloxacin or vehicle

**

*

-1 0 1 2 3 4 5

Time (Days)

We

igh

t (g

)



Naive

LP

S

TLCA 5

0M

LPS+T

LCA

Olene

alolic a

c 1

mM

LPS+

Olean

olic a

c0

1000

2000

3000

4000

5000*

****

RAW macrophages

TN

F

(pg/m

l)

0

10

20

30

0 5 10 100

Oleanolic acid (M)

[cA

MP

](p

g/m

g p

rote

in)

*



0

1

2

3

*

** **

Naive TNBS

10 1 0.1 mg/kg

Oleanolic acid

Macro

scopic

inju

ry s

core

0 1 2 3 422

24

26

28

30

32

34

Naive TNBS alone TNBS + Oleanolic acid 10 mg

TNBS + Oleanolic acid 1 mg TNBS + Oleanolic acid 0.1 mg

*

*

Time (days)

We

igh

t (g

)

0 1 2 3 4 50

1

2

3

Time (days)

*D

iarr

he

a s

core



0

10

20

30

40

Naive Naproxen Naive Naproxen

GPBAR1+/+

*

GPBAR1-/-

**

Inte

sti

nal

dam

ag

e(m

m o

f le

sio

n)

0

10

20

30

40

50

MP

O a

cti

vit

y(m

U/m

g p

rote

in)

*

*

Naive Naproxen Naive Naproxen

A

C

E

G

H

B

D

F

GP-BAR1-/- small intestine

GP-BAR1 gene deletion alters intestinal structure and function.

Cipriani S. et al. Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-

inflammatory drugs and aspirin in mice. Br J Pharmacol. 2013 Jan;168(1):225-37. doi: 10.1111/j.1476-5381.2012.02128.x.

Anti-inflammatory effects of oleanolic acid.



20x

GPBAR1+/+ mouse naive GPBAR1 human

40x

20x

40x

A

B

C

D

GPBAR1

Monocytes Stomach0.0

0.5

1.0

1.5

mR

NA

rela

tive

exp

ressio

nCipriani S. et al. Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-


0

10

20

30

40

50

60

GP-BAR1+/+

GP-BAR1-/-

Naive 10 mg/kg 30 mg/kg 50 mg/kg

ASA

**

**

*

**

**

Gastr

ic i

nu

ry s

co

re(m

m)

GPBAR1+/+ CTRL

GPBAR1-/- CTRL

GPBAR1+/+ ASA

GPBAR1-/- ASA

20x

20x

20x

20x

40x

H I

L M

Cipriani S. et al. Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non-steroidal anti-


TNFα COX-1 COX-2

eNOS iNOS CSE CBS

0

1

2

3

4

*

GPBAR+/+

GPBAR-/-

*m

RN

A r

ela

tive e

xpre

ssio

n

0

1

2

3

mR

NA

rela

tive e

xp

ressio

n

0

1

2

3

4

5

6

*

**

mR

NA

rela

tive e

xp

ressio

n

0

1

2

3

4

5

6

mR

NA

re

lative e

xpre

ssio

n

*

0.00

0.25

0.50

0.75

1.00

mR

NA

re

lati

ve e

xp

ressio

n

*

*

0

1

2

3

Naive ASA Naive ASA

mR

NA

rela

tive e

xpre

ssio

n

*

*

0.0

0.5

1.0

1.5

*

*

Naive ASA Naive ASA

mR

NA

re

lative

exp

re

ssio

n

Cipriani S., et al. British Journal of Pharmacology 2012; 168: 225-237

Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non‐steroidal anti‐inflammatory drugs and aspirin in mice

MPO PGE2 TNFα

eNOS MUC1 MUC6

CSE CBS

0

10

20

30

40

Gastr

ic m

uco

sal

inju

ry s

co

re (

mm

lesio

n)

ASA 50 mg/kg

Betulinic acid - + - +

*

*

0

1000

2000

* * *

*

(pg/

mg

prot

ein)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

mR

NA

rela

tive

exp

ress

ion

0

1

2

3

4

5

6

7*

*

mR

NA

rela

tive e

xpre

ssio

n

0

1

2

3

4*

Naive ASA ASA Naive ASA ASA + + Betulinic Betulinic

mR

NA

rela

tive e

xp

ressio

n

0

1

2

3

**

*

Naive ASA ASA Naive ASA ASA + + Betulinic Betulinic

0.00

0.25

0.50

0.75

1.00

1.25

**

Naive ASA ASA Naive ASA ASA

+ +

Betulinic Betulinic

0

1

2

3

4

5

*

0

10

20

30

40

50

MP

O(m

U/m

g p

ro

t) * *


Betulinic acid a GPBAR1 agonist protects against gastrointestinal injury caused by non‐steroidal anti‐inflammatory drugs and aspirin in mice

http://upload.wikimedia.org/wikipedia/commons/b/b0/Betulinic_acid_structure.png




eNOS MUC1 MUC6

CSE CBS

0

10

20

30

40

GP-BAR1+/+

GP-BAR1-/-

*

ASA 50 mg/kg

Ciprofloxacin - + - +

Gastr

ic m

uco

sal

inju

ry s

co

re(m

m l

esio

n)

*

0

1000

2000

*

*

(pg

/mg

pro

tein

)

0.0

0.5

1.0

1.5

mR

NA

rela

tive e

xpre

ssio

n

0

1

2

3

Naive ASA ASA Naive ASA ASA + + Ciprofloxacin Ciprofloxacin

mR

NA

rela

tive e

xpre

ssio

n

0.0

2.5

5.0

7.5

*


mR

NA

rela

tive e

xpre

ssio

n

MPO PGE2 TNFα

0

1

2

*

mR

NA

rela

tive

exp

ress

ion

0.00

0.25

0.50

0.75

1.00

1.25


mR

NA

rel

ativ

e ex

pres

sion

igure 4

A B C D

E F G

H I

0.0

2.5

5.0

7.5

*

mR

NA

rela

tive e

xpressio

n

0

10

20

30

40

50

MP

O(m

U/m

g p

ro

t) *

*

Activation of the bile acid receptor GPBAR1 protects against gastrointestinal injury caused by non‐steroidal anti‐inflammatory drugs and aspirin in mice


Tgr5 and intestinal mucosa

Health & Medicine

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