Osteoarthritis

Assorted effects of TGF-ß and chondroitinsulfate on p38and ERK 1/2 activation levels in human articular chondrocytesstimulated with LPS

J. Holzmann, N. Brandl, A. Zeman, R. Schabus, S. Marlovits, R. Cowburnand M. Huettinger

Center of Physiology and Pathophysiology, A-1090 Vienna, Waehringerstr. 10/13, Austria

structure of cartilage

chondroitinsulfate

keeping the balance...

anabolic statecartilage synthesis

catabolic statecartilage degradation

inflammation

activateMacrophages

NSAID

inflammatory cytokines signalthrough SAPK (p38 / JNK)

the bad guys:

MatrixMetalloProteinases (MMPs) &

MMP-1 (Interstitial Collagenase)

MMP-2 (Gelatinase A)

MMP-3 (Stromelysin 1)

MMP-9 (Gelatinase B)

MMP-13 (Collagenase-3)

cleaves triple-helical fibrillar collagen II

cleaves triple-helical fibrillar collagen II

cleaves gelatin(part. hydrolysed collagen)

cleaves gelatin(part. hydrolysed collagen)

cleaves collagen IVactivates proMMPs

Aggrecanases

Aggrecanase 1 & 2 cleaves aggrecan

ind

uce

d in

res

pon

se to

cyt

oki

nes

a

nd g

row

th fa

cto

rs

working hypothesis

IL-1IL-6TNF-alpha

enhanced MMP& Aggrecanase expression

degradation of Matrixrelease of CS

Feedback signaling of endproducts

moderation of MMP& Aggrecanase expression

synthesisof new Matrix

pharmacological doses of CS: • enhance feedback signaling• providing building blocks for aggrecan

synthesis

CS

?

• is CS able to downregulate the MMP transcription levels in LPS stimulated chondrocytes as a model for osteoarthritis?

• what are the effects of CS on the MAPK members p38 and ERK?

• is there a cross reaction between TGF-ß and CS ?

aim of the study

LPS

given that clinical studies showed a benefit when CS is taken orally…

methods

Cells were grown to confluency in DMEM containing 10 % FCS and incubated with LPS, CS, TGF-ß1

Cell lysis and protein quantitation using BCA Assay Kit

Immunoblot using phosphospecific antibodies against pERK1/2 and p-p38

Cell lysis and cDNA synthesis using random hexamer Primers and reverse Transcriptase

Expression levels of MMPs were determined by RT-qPCR.

pERK1/2 and p-p38 activation levels MMP expression levels

HAC…human articular chondrocytesfrom patients with no history of OAundergoing joint replacement because of femoral neck fracture

> 3 passage => „fibroblast like“

resultseffects on non stimulated chondrocytes

0

0,2

0,4

0,6

0,8

1

1,2

1,4

MMP2 MMP3

fold

ch

ang

e

0

5

10

15

20

25

30

35

40

MMP13

fold

cha

nge C4S

TGF-ß

CS+TGF-ß

30 min incubation

p-p38

p-ERK 2p-ERK 1

*

*** ***

TGF-ß =>induction of MMP13expressionthrough SMAD &MAPKpathway

*

control

72 h 72 h

0

2

4

6

8

10

12

14

MMP1 MMP 2 MMP 3 MMP 13

Rel

ativ

e ac

tivity

resultseffects of LPS

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

p-p38 p-ERK 1 p-ERK 2

Rel

ativ

e ac

tivity

30 min

72 h******

**

**

**

p-p38

p-ERK 2p-ERK 1

30 min 72 h

30 min 72 h

control

control

LPS strongly activates p38LPS transiently activates ERK 1/2

LPS increases transcriptionof MMP1, MMP3 & MMP13

**

72 h

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

MMP1 MMP2 MMP3 MMP13

fold

ch

an

ge

to

LP

S

CS

TGF-ß1

CS + TGF-ß1

resultseffects of TGF-ß and CS in stim. chondrocytes

87,5

*

-200

-100

0

100

200

300

400

% ch

ange

to L

PS tr

eatm

ent

CS

TGF-ß1

TGF-ß1 + CS

p-p38 p-ERK1 p-ERK2 p-p38 p-ERK1 p-ERK2

30 min 72 hCS moderates p-p38 like TGF-ß

CS counteractsthe effect of TGF-ßon ERK 1/2 at 30´

LPS

control

LPS

CS moderatesthe transcription ofMMP13 by 30%

******

***

*****

**

***

*****

***

***

**

**

******

72 h

resultssummary

CS has no effect on p38 / ERK and MMP expression in non stimulated „healthy“

dedifferentiated chondrocytes (but maybey in diff. chondrocytes?)

LPS induces a catabolic state which is characterised by a p38 / ERK activation

and enhanced MMP 1 & MMP 13 expression

CS and TGF-ß are both able to moderate the p38 activation

CS and TGF-ß show contrary effects on ERK activation and MMP13 expression

=> TGF-ß further stimulated MMP13 expression

=> CS suppressed MMP13 expression

may serve an explanation, on the cellular level, for the beneficial effects found in clinical studies with pharmacologic application of chondroitinsulfate

discussion

What are the molecular targets of CS?

membran receptor / cytokine trapping?

How is CS able to modulate TGF-ß effects?

signal crosstalk / cytokine trapping?

Nagarajan Selvamurugan et al. J Biol Chem. 2004 Apr 30;279(18)

TGF-beta enhanced MMP13 expression through crosstalkbetween p38, ERK and SMAD pathway

-200

-100

0

100

200

300

400

% c

hang

e to

LP

S tre

atm

ent

CS

TGF-ß1

TGF-ß1 + CS

0

0,5

1

1,5

2

2,5

MMP 2 MMP 3 MMP 13

fold

ch

an

ge

to

LP

S

CS

TGF-ß1

CS + TGF-ß1

discussion

p-p38 p-ERK1 p-ERK2 p-p38 p-ERK1 p-ERK2

• only transient downregulation of ERK• delayed & alleviated response to TGF-ß in the presence of CS

discussion

glycosaminoglycans (GAG) can bind cytokines like TGF-ßcytokines are then resistent to degradations therefore matrix can act as a reservoir

equlibrium between the TGF-ß bound to matrix and free GAG

supply of free CS could also act as a binding partner „trap“ TGF-ß

heparan / chondroitin sulfate negatively modulates TGF-beta1 responsiveness by decreasing the ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I, facilitating endocytosis and rapid degradation of TGF-beta1

Chen et al. J Biol Chem. 2006 Apr 28;281(17)

summary

soluble chondroitinsulfate modulates signalling events in chondrocytes concurrent with

• p38 and transiently ERK1/2 downregulation• MMP-13 downregulation

may serve an explanation, on the cellular level, for the beneficial effects found in clinical studies with pharmacologic application of chondroitinsulfate.

• the molecular targets of CS has to be elucidated

• CS possibly modulates TGF-beta responsiveness by a combination of „cytokine trapping“ and enhanced TGF-betaRI + II degradation

Groupmembers:

Manfred HuettingerAdolf Zeman

Nina Brandl

thank you for your attention !