Osteoarthritis

1. Assorted effects of TGF-ß and chondroitinsulfate on p38 and ERK 1/2 activation levels in human articular chondrocytes stimulated with LPS J. Holzmann, N. Brandl, A. Zeman, R. Schabus, S. Marlovits, R. Cowburn and M. Huettinger Center of Physiology and Pathophysiology, A-1090 Vienna, Waehringerstr. 10/13, Austria Osteoarthritis

2. structure of cartilage chondroitinsulfate

3. keeping the balance... anabolic state cartilage synthesis catabolic state cartilage degradation inflammation NSAID activate Macrophages inflammatory cytokines signal through SAPK (p38 / JNK)

4. the bad guys: MMP-1 (Interstitial Collagenase) MMP-2 (Gelatinase A) MMP-3 (Stromelysin 1) MMP-9 (Gelatinase B) MMP-13 (Collagenase-3) MatrixMetalloProteinases (MMPs) & Aggrecanases cleaves triple-helical fibrillar collagen II cleaves gelatin (part. hydrolysed collagen) cleaves collagen IV activates proMMPs induced in response to cytokines and growth factors cleaves gelatin (part. hydrolysed collagen) cleaves triple-helical fibrillar collagen II Aggrecanase 1 & 2 cleaves aggrecan

5. working hypothesis Feedback signaling of endproducts enhanced MMP & Aggrecanase expression IL-1 IL-6 TNF-alpha moderation of MMP & Aggrecanase expression synthesis of new Matrix degradation of Matrix release of CS • enhance feedback signaling • providing building blocks for aggrecan • synthesis pharmacological doses of CS:

6. aim of the study given that clinical studies showed a benefit when CS is taken orally… • 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 ? CS ? LPS

7. methods HAC…human articular chondrocytes from patients with no history of OA undergoing joint replacement because of femoral neck fracture Cells were grown to confluency in DMEM containing 10 % FCS and incubated with LPS, CS, TGF-ß1 > 3 passage => „fibroblast like“ Cell lysis and protein quantitation using BCA Assay Kit Cell lysis and cDNA synthesis using random hexamer Primers and reverse Transcriptase Immunoblot using phosphospecific antibodies against pERK1/2 and p-p38 Expression levels of MMPs were determined by RT-qPCR. pERK1/2 and p-p38 activation levels MMP expression levels

8. effects on non stimulated chondrocytes results * 30 min incubation p-p38 control p-ERK 2 p-ERK 1 72 h 72 h *** *** TGF-ß => induction of MMP13 expression through SMAD & MAPK pathway *

9. effects of LPS results 30 min 72 h *** *** p-p38 30 min 72 h * * p-ERK 2 p-ERK 1 control LPS strongly activates p38 LPS transiently activates ERK 1/2 72 h ** ** LPS increases transcription of MMP1, MMP3 & MMP13 ** control

10. effects of TGF-ß and CS in stim. chondrocytes results 30 min 72 h CS moderates p-p38 like TGF-ß *** *** *** *** *** ** LPS CS counteracts the effect of TGF-ß on ERK 1/2 at 30´ ** ** ** control *** *** *** ** p-p38 p-ERK1 p-ERK2 p-p38 p-ERK1 p-ERK2 72 h *** *** 8 7,5 CS moderates the transcription of MMP13 by 30% * LPS

11. summary results 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

12. 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?

13. discussion TGF-beta enhanced MMP13 expression through crosstalk between p38, ERK and SMAD pathway Nagarajan Selvamurugan et al. J Biol Chem. 2004 Apr 30;279(18) 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

14. discussion glycosaminoglycans (GAG) can bind cytokines like TGF-ß cytokines 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

15. 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)

16. 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

17. Groupmembers: Manfred Huettinger Adolf Zeman Nina Brandl thank you for your attention !