1. Osteoarthritis 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

2. structure of cartilage chondroitinsulfate

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

4. 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 IV activates proMMPs Aggrecanases Aggrecanase 1 & 2 cleaves aggrecan induced in response to cytokines and growth factors

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

6. 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…

7. 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 chondrocytes from patients with no history of OA undergoing joint replacement because of femoral neck fracture > 3 passage => „fibroblast like“

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

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

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

11. 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

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