WO2008128559A1

WO2008128559A1 - Method for providing a compound capable of interfering with a distorted chondrocyte phenotype

Info

Publication number: WO2008128559A1
Application number: PCT/EP2007/003729
Authority: WO
Inventors: Peter Mario Van Der Kraan; Esmeralda Nicole Blaney Davidson; Wilhelmus Berend Van Den Berg; Dennis Florian George Remst
Original assignee: Stichting Katholieke Universiteit
Priority date: 2007-04-20
Filing date: 2007-04-20
Publication date: 2008-10-30

Abstract

The present invention relates to methods for providing a compound capable of interfering with a distorted chondrocyte phenotype and particularly a method for providing a compound capable of treating osteoarthritis. Specifically, the present invention relates to a method for providing a compound interfering with a chondrocyte distorted phenotype comprising: (a) contacting a cell line expressing ALKl with a compound and (b) measuring the effect of said compound on the downstream signaling of ALKl.

Description

METHOD FOR PROVIDING A COMPOUND CAPABLE OF INTERFERING WITH A DISTORTED CHONDROCYTE PHENOTYPE

Description

The present invention relates to methods for providing a compound capable of interfering with a distorted chondrocyte phenotype and particularly methods for providing a compound capable of treating osteoarthritis. The present invention further relates to the use of said compound for interfering with a distorted chondrocyte phenotype and particularly for the treatment of osteoarthritis.

Transforming Growth Factor beta (TGF-beta) is a cytokine that elicits its responses via specific receptors. Upon activation of these receptors by TGF-beta, these receptors phosphorylate intracellular effectors such as Smads . Phosphorylated Smads can translocate to the nucleus where they may be involved in transcription activation.

Activin receptor-like kinase 1 (ALKl or ACVRLl) and activin receptor-like kinase 5 (ALK5) are both type I TGF- beta receptors .

Upon activation of ALKl by TGF-beta, Smadl, Smad5 and/or Smadθ are phosphorylated and, as a consequence, transcription in the nucleus is effected. This signal (TGF- beta) transduction pathway is also known as the Smadl/5/8 route .

Upon activation of ALK5 by TGF-beta, Smad2 and Smad3 are phosphorylated and, as a consequence, transcription in the nucleus is effected although different from the Smadl/5/8 route. This signal (TGF-beta) transduction pathway is also known as the SMAD2/3 route.

Osteoarthritis (OA) is the most common joint disease and its incidence strongly correlates with age. Osteoarthritis (OA) is characterized by destruction of articular cartilage.

Chondrocytes, sometimes also designated as chondroblasts, are the only cells found in cartilage. Under normal conditions, chondrocytes are responsible for the forming and homeostasis of the articular cartilage matrix. In osteoarthritis (OA) , chondrocytes show a distorted phenotype and actively start to degrade their own matrix. It is believed that this distorted phenotype is the result of the production by the chondrocytes of cartilage-degrading enzymes such as the matrix metalloproteinase 13 (MMP-13, also designated as collagenase 3) . This distorted phenotype of articular chondrocytes in osteoarthritis (OA) is analogous to the phenotype of growth plate chondrocytes that have undergone terminal differentiation.

It is an objective of the present invention to provide methods for identifying compounds which are capable for interfering with a distorted chondrocyte phenotype leading to the development of osteoarthritis. An "interfering with a distorted phenotype" as is used within the present context is meant to indicate any decrease in the ability of chondrocytes to actively start to degrade their own matrix resulting in the development of osteoarthritis (OA-) . Such "interfering with a distorted phenotype" could also be regarded as reverting the

1 chondrocytes to the "normal" phenotype meaning maintaining the homeostasis of the articular cartilage matrix as is found in individuals not suffering from osteoarthritis (OA) .

This objective of the present invention is met by a method as defined in claim 1.

Specifically, this objective of the present invention is met by a method for providing a compound interfering with a chondrocyte distorted phenotype comprising: a) contacting a cell line expressing ALKl with a compound; b) measuringXthe effect of said compound on the downstream signaling of ALKl . This method is based on the surprising discovery that the TGF-beta type I receptor ALKl plays an important role in the development of osteoarthritis (OA) .

The present inventors have surprisingly discovered that chondrocytes in aged cartilage and experimental osteoarthritis (OA) models show a strongly diminished expression of ALK5 but a less diminished expression of ALKl resulting in a relative dominance of the Smadl/5/8 route (activated by ALKl) over the Smad2/3 route (activated by ALK5) .

As an effect of this surprisingly discovered shift in balance between the two signal transduction pathways towards the SMAD1/5/8 route, chondrocytes start to terminally differentiate and to degrade the cartilage matrix ultimately resulting in the development of osteoarthritis (OA) .

The compounds identified according to the present invention can be any compound capable of acting as an antagonist of the TGF-beta receptor ALKl such as peptides, peptidomimetics, (glyco) proteins, hormones, (co) polymers, TGF-beta analogues and small organic molecules such as acid anhydrides, acyl halides, alcohols, aldehydes, alkanes, alkenes, alkynes, amides, amines, aromatics, azo compounds, carboxylic acids, esters, ethers, haloalkanes, imines, ketones, nitriles, nitro compounds, organqmetallic compounds, pheno'ls and thiols. i The term "small", as used in this context is not intended to define a particular molecular weight range but is a relative term to discriminate the organic compounds from larger compounds such as proteins, DNA, RNA, bacteria, viral particles, polymers etc.

A cell line expressing ALKl according to the present invention can be any cell line as long as this cell line is capable of displaying the ALKl receptor on its surface and, in addition, is capable of transducing or routing the activation of the ALKl receptor into its intracellular space.

In a preferred embodiment of the present invention, the cell line is a chondrocyte cell line, more preferably a chondrocyte cell line (recombinantly) overexpressing the ALKl receptor. In a particularly preferred cell line, the expression of the ALK5 receptor is additionally down- regulated or silenced. Methods for manipulating the expression of receptors in cell lines are well known to the skilled person in the field of Molecular Biology.

For example, a nucleic acid construct comprising the coding sequence of the ALKl receptor, operably linked to transcription and translation signals, can be transformed into a chondrocyte cell line, thereby providing a cell line capable of displaying the ALKl receptor on its surface and, additionally, capable of transducing or routing the activation of the ALKl receptor into its intracellular space.

Down-regulation or silencing of ALK5 expression can be obtained by additionally transforming such cell line with a nucleic acid constructs providing anti-sense RNA or siRNA capable of decreasing or inhibiting ALK5 expression. Measuring the effect of said compound on the downstream signaling of ALKl can be obtained in a number of preferred ways.

For example, because activation of the ALKl receptor results in the phosphorylation of Smadl, Smad 5 and/or Smad8 , measuring the phosphorylation state of one or more of these compounds indicates if a compound is capable of influencing the d'ownst'ream signaling of ALKl thereby providing ■ interference with the distorted phenotype .

Downstream signaling of ALKl can also be measured by determining (increased) expression levels of proteins whose coding genes are controlled by the phosphorylated Smadl, Smad 5 and/or Smadδ molecules. Suitable and preferred examples of such genes are the genes encoding aggrecan, type X collagen, type II collagen, MMP-3 and MMP13.

The method according to the invention is not restricted to (transgenic) cell lines expressing the ALKl receptor for providing a compound capable of interfering with a chondrocyte distorted phenotype. Therefore, according to another preferred aspect, the present invention also relates to a method for providing a compound capable of interfering with a chondrocyte distorted phenotype comprising: a) contacting ALKl with said compound; b) detecting an interaction between said compound and ALKl .

Suitable sources of ALKl receptor protein, complete or truncated, are generally known bacterial, fungal, yeast, insect and mammalian cell based expression systems. After expression, the ALKl receptor produced can be optionally purified using purification techniques such as ion-exchange chromatography, affinity chromatography, size-exclusion chromatography, etc. Binding of the compound to the receptor can be detected by using, for example, ELISA, surface plasmon resonance, staining, fluorescence, etc.

It is contemplated within the context of the present invention to further demonstrate the capability of the compounds identified to interfere with a chondrocyte distorted phenotype using the cell line according to the present invention.

The present invention will be further detailed using the following example. In the example, reference is made to the following figures wherein:

Fig. 1: shows that transfection with Ad-ALKl specifically provides phosphorylation of Smadl/5/8, whereas Ad-ALK5 transfection resulted in Smad2 phosphorylation;

Pig. 2: shows that Smadl/5/8 phosphorylation was up- regulated after 15 minutes at low dosages TGF- beta (0.01 and 0.05 ng/ml) . Smad2 phosphorylation was up-regulated in the Hl and P2 cell lines after 30 minutes at low dosages TGF-beta. The P2 has a high ALPp. expression while the Hl shows a low ALKl expression; Fig. 3: shows that both ALKl and ALK5 expression in stained knee joints sections of mice aged 5 months or 2 years was decreased with age. For ALKl expression, the number of cells staining positive were decreased with 51% in the medial tibial cartilage and 33% in the lateral tibial cartilage, whereas for ALK5 expression these percentages were 88% and 76%, respectively. Fig. 4: shows that, in a DMM model, the ALKl and ALK5 expression in the tibial cartilage, both on the medial and the lateral side of the joint, was not changed on the lateral side of the joint. However, a drastic decrease of ALKl and ALK5 expression was observed in the medial tibial plateau. ALK5 expression was decreased with 91% and ALKl expression with 75%. Fig. 5: shows that both ALKl and ALK5 expression in stained of knee joints sections of mice, aged 3, 6, 9 and 12 months, was decreased with age. However, ALKl expression decreased less pronounced than ALK5 expression with age.

Fig. 6: shows the effects of adenoviral overexpression of constitutive ALK5 and ALKl on mRNA level. Overexpression of constitutive* ALK5 results in ' ' increased expression of aggrecan, a decreased

' expression of collagen type II and collagen type X. Overexpression of ALKl results in an increase in aggrecan. collagen type II, collagen type X, MMP-3 and MMP-13 expression.

Example

Introduction

This example demonstrates that chondrocytes express both ALK5 and ALKl and thaj: ALK5 signals through Smad2/3 and ALKl through Smadl/5/8. It is shown that stimulation of chondrocytes with TGF-beta results in phosphorylation of both signaling pathways, of which the Smadl/5/8 route is stimulated faster.

Transfection with adenovirus constitutively expressing active ALK5 results in aggrecan up-regulation and down-regulation of collagen type II, whereas constitutive expression of active ALKl results in an up-regulation of collagen type II and MMP-13.

It is shown that both ALK5 and ALKl decrease during . ageing and OA, but the ALK5 decrease is more pronounced in comparison with the ALKl decrease. In addition, the decrease in receptor expression is more pronounced in the areas that are known to be firstly affected by OA.

Materials and Methods

In vitro experiments

Several cell lines were obtained by subcloning chondrocytes isolated from murine cartilage of the patella or the hip. RNA was isolated from unstimulated cells and a quantitative PCR was performed. Two cell lines were selected for their divergence in ALK1/ALK5 expression ratios designated P2 (derived from patellar cartilage) and Hl (derived from hip cartilage) .

' These cells were stimulated with TGF-beta (0.01; 0.05 ng/ml) to assess whether TGF-beta signals through ALK5 and/or ALKl, via phosphorylation of Smad2 or Smadl/5/8 respectively. This was evaluated using western blot analysis. Thereafter, cells were transfected with either Ad- ALKl or Ad-ALK5 (adenovirus mediated expression of ALKl and ALK5) to evaluate whether active ALKl resulted in Smadl/5/8 phosphorylation and active ALK5 resulted in Smad2 phosphorylation . To investigate the functional effects of up-regulation of either ALK5 or ALKl, the cells were transfected with the constitutively active receptors and RNA was isolated for quantitative PCR after 3 days.

Quantitative PCR

A quantitative PCR was prepared as follows: a primer mix of 1.5 μl forward primer (5 μM) , 1.5 μl reverse primer (5 μM) and 4.5 μl dH₂O, was added to 12.5 μl SybrGreen. Then, 5 μl of cDNA was added and the quantitative PCR was performed using a "ABI/PRISM 7000 sequence detection system" (Applied Biosystems, Foster City, CA, USA) according to manufacturers protocol .

PCR conditions were as follows: 2 minutes at 50⁰C and 10 min at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 6O⁰C, with data collection in the last 30 seconds. In addition, for each PCR reaction, melting curves were determined to establish the identity of the products formed. The genes that were measured and the corresponding primer sets are presented in Table 1.

Table 1: primer sets used to measure gene expression

Efficiencies (E) for all primer sets were determined (Table 1) using a standard curve of 5 serial cDNA dilutions in water in duplicate. Primers were accepted if the deviation from slope of the standard curve was less than 0.3 compared to the slope of GAPDH standard curve and if the melting curve showed only one product.

For each primer pair, non-template controls were run in duplicate. The cycle threshold values (Ct) of the genes of interest were corrected for the Ct of the reference gene GAPDH. Relative mRNA expression was calculated by 2 to the power of delta Ct.

Western Blot

For Western blot analysis, 20 μg of a protein sample was loaded on a Sodium Dodecyl Sulfate (SDS) 7.5% polyacrylamide gel. Proteins were electrophoretically transferred onto a nitrocellulose membrane (Hybond P; Amersham Pharmacia Biotech, Buckinghamshire, UK) using the iBlot system (Invitrogen, Breda, the Netherlands). After blotting, the membrane was washed with TBS and subsequently incubated with blocking buffer containing 5% non-fat dry milk in TBS/T for 1 hour at room temperature.

After washing with TBS/T, the membrane was incubated with specific primary antibodies against ALKl, ALK5, Smad- 1/5/8P (1:1000) and Smad-2P (1:1000) (ALKl and ALK5 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) , Smad-l/5/8P and Smad-2P were purchased from Cell Signaling Technology (Beverly, MA, USA) . Smad-2P antibodies were diluted in 5% non-fat dry milk and Smadl/5/8P in 5% BSA in TBS/T and incubated at 4⁰C overnight.

After extensive washing with TBS/T, the membrane was incubated with the secondary HRP-conjugated antibody (goat- anti-rabbit 1:1500) (DAKO, Glostrup, Denmark) in 5% non-fat dry milk in TBS/T for an hour. After washing with TBS, the membrane was developed usi,ng an enhanced chemiluminescence detection system (ECL Plus Western Blotting Detection System, GE Healthcare, Eindhoven, the Netherlands) .

Animals

Male C57B1/6 mice aged 5 months (n=7) or 2 years (n=10) were used for comparison of young versus old mice; C57B1/6 mice were used to induce OA by transection of the medial meniscus (n=10); STR/ort mice aged 3 months (n=10) , 6 months (n=20) , 9 months (n=6) and 1 year (n=10) were used as a spontaneous model of OA.

Animals were kept in filtertop cages with woodchip bedding under standard pathogen free conditions . They were fed a standard diet with tap water ad libitum. This study was approved by the local animal committee.

DMM

The DMM-model is a surgical mouse model in which the anterior attachment of the medial meniscus to the tibial plateau is transected. Mice were sacrificed after 8 weeks.

STR/ort

STR/ort mice spontaneously develop OA. Eighty percent of the male STR/ort mice show degenerative cartilage lesions by' 6 months of age, starting with lesions at the interface of the cruciate ligament and the medial tibial plateau.

The lesions range from mild erosion of the cartilage surface to loss of cartilage exposing the subchondral bone. In addition, the mice develop osteophytes at the joint margins. The histological lesions seen in this model resemble those seen in humans. Therefore, the STR/ort mice were used as a model for human primary OA.

Histology

Immunohistochemistry for ALKl, ALK5, Smad-l/5/8P and Smad-2P, as well as Safranin O/Fast Green staining, were performed on paraffin sections from total knee joints.

Knee joints were fixed in phosphate buffered formalin for 7 days. They were the dehydrated using an automated tissue-processing apparatus (Tissue TeJc VIP, Sakura, Ramsey, MN, USA) and embedded in paraffin. Tissue sections of 7 μm were prepared.

Immunohistochemistry

Sections were deparaffinized and washed with PBS. For antigen unmasking, sections were incubated in citrate buffer (0.1 M sodium citrate, 0.1 M citric acid) for 2 hours. Endogenous peroxidase was blocked with 1% hydrogen peroxidase in methanol for 30 minutes.

Thereafter, sections were blocked with 5% normal serum of the species in which the secondary antibody was produced. Specific primary antibodies against ALKl (1:100), ALK5 (1:100), Smadl/5/8P (1:100) and Smad-2P (1:100) were incubated at 4⁰C overnight (ALKl and ALK5 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) , Smad-l/5/8P and Smad-2P were purchased from Cell Signaling Technology (Beverly, MA, USA) ) . As a negative control, the primary antibody was replaced with goat or rabbit Igάs . i After extensive washing in PBS, the appropriate biotin labeled secondary antibody was used at a concentration of 2 μg/ml in 1% bovine serum albumin/PBS for 2 hours (Vector Laboratories Inc., Burlingame, CA, USA), followed by a biotin-streptavidin detection system according to the manufacturers protocol (Vector Laboratories Inc.) Bound complexes were visualized via reaction with 3,3 diaminobenzidine (Sigma Chemicals Co., Zwijndrecht, The Netherlands) and H₂O₂ resulting in a brown precipitate. Sections were briefly counterstained with heamatoxylin and mounted with Permount . Image analysis : quantification of positively stained articular chondrocytes

For the different antigens, the number of positive articular chondrocytes in the tibia was determined by a blinded observer.

The microscopic image was displayed on a computer monitor using the Qwin image analysis system (Leica Imaging Systems, Rijswijk, The Netherlands) and a Leica DC 300F digital camera. The area representing the non-calcified articular cartilage was selected by hand. For each antigen, a threshold was set in such a manner that only chondrocytes that were found to be positive (brown stained cell) as judged by the observer were selected. The computer program determined the number of positive cells in the cartilage for the different antigens. For each knee joint, the expression of the different antigens was measured in at least three tissue sections. The obtained values were averaged and the average per treatment group was determined.

Human cartilage samples

To assess whether human chondrocytes also expressed ALKl,^' samples of human cartilage obtained from patients undergoing hip-replacement were used.

The cartilage was frozen in liquid nitrogen and crushed using a dismembrator . The crushed material was dissolved in RLT-buffer supplied with the RNAeasy Mini Kit (QIAGEN, Venlo, the Netherlands) . Samples were treated with Proteinase K and, subsequently, RNA was isolated using the Rneasy Mini Kit according to manufacturers protocol. Next, a Real-Time PCR was performed.

Statistical analysis

Results were analyzed with the Student's t-test and considered significant if the p-value was smaller than 0.05.

Results

Chondrocytes express both ALK5 and ALKl and can signal through both receptors.

It was investigated whether chondrocytes express both type I TGF-beta receptors: ALKl and ALK5 by quantitative PCR. It was found that both ALKl and ALKb were expressed in murine cartilage, murine chondrocyte cell lines and in human cartilage. The P2 cell line showed a high ALKl mRNA expression while the Hl cell line showed a low ALKl expression (data not shown). Thereafter, chondrocytes were transfected with Ad- ALKl, Ad-ALK5 or Ad-LacZ and a Western blot analysis was performed to detect subsequent phosphorylation of Smad2 or Smadl/5/8. It was found that transfection with Ad-ALKl specifically resulted in up-regulation of Smadl/5/8P, whereas ALK5 resulted in up-regulation of Smad2P. (Figure 1) .

Stimulation of chondrocytes with TGF-beta provided both Smadl/5/8 and Smad2 phosphorylation in the P2 cell line and only Smad2 phosphorylation in the Hl cell line. Smadl/5/8 was phosphorylated within 15 minutes, whereas Smad2 was phosphorylated after 30 minutes.

> Smad2 phosphorylation is maintained at low dosages for 2 hours and for at least 24 hours when stimulated with 1 ng/ml. (Figure 2)

ALKl and ALK5 expression patterns in chondrocytes

It was evaluated whether OA-chondrocytes expressed ALK-I or ALK-5 and whether this was different compared to normal chondrocytes. In addition, the different chondrocyte- cell lines were screened for ALK-I and ALK-5 receptor expression and a correlation with their responses was established. The P2 cell line showed a high ALKl mRNA expression while the Hl cell line showed a low ALKl expression .

It was previously found that ageing, which is a risk factor for OA, was accompanied by a decrease in ALK5. To investigate whether this is also the case for ALKl, sections of knee joints of mice aged 5 months or 2 years were immunohistochemically stained for ALK5 and ALKl for comparison.

It was found that both ALKl and ALK5 expression decreases with age. However, for ALKl, the number of cells staining positive had decreased with 51% in the medial tibial cartilage and 33% in the lateral tibial cartilage, whereas

ALK5 staining had decreased with 88% and 76%, respectively.

Thus, the decrease in ALK5 expression is more pronounced with age. (Figure 3)

ALK5 decreased more drastically than ALKl in focal cartilage degeneration in the DMM model

In the DMM model, the attachment site of the medial meniscus to the tibia is transected resulting in OA starting in the medial plane. ALKl and ALK5 positive cells were scored in the tibial cartilage both on the medial and the lateral side of the joint. No change in expression on the lateral side of the joint was found, but a drastic decrease of ALKl and ALK5 expression in the medial tibial plateau.

However, when comparing the decrease in receptor expression, ALK5 expression was decreased with 91% compared to a 75% decrease of cells positive for ALKl. Again, this underlines that the number of cells expressing ALK5 declines more rapidly than the number of cells expressing ALKl . (Figure 4)

ALK5 and ALKl decrease with progression of spontaneous OA with a more drastic decrease of ALK5

STR/ort mice spontaneously develop OA at an early age. Initial changes can be seen on the medial tibia in mice of two to three months old. To assess the decline of TGF-beta RI expression during spontaneous OA the number of cells staining positive for ALKl and ALK5 were scored in time. A decrease was found in the number of positive cells for both ALKl and ALK5 with age, and thus with progression of OA. When comparing ALK5 expression to ALKl, it was found that the number of cells staining positive for ALK5 was consistently lower than for ALKl. The ALK5/ALK1 ratio declined with progression of OA.

In case the number of cell staining positive for ALK5 was egual to ALKl in the lateral tibia at 3 months old, the number of cells staining positive for ALK5 in the lateral tibia was 82% less than ALKl positive cells. Strikingly, STR/ort mice develop OA first on the medial plateau. This is also the plateau that shows the first signs of decrease in ALK5 expression. These data again confirm that with progression of OA, the ALK5/ALK1 ratio decreases. (Figure 5)

ALKl overexpression results in different responses than ALK5 overexpression in chondrocytes

To assess the result of signaling ,via the different TGF-b'eta receptors, the effect of adenoviral overexpression ^" of' constitutive ALK5 versus ALKl on mRNA level was investigated.

Overexpression of constitutive ALK5 results in an increased expression of aggrecan, a decrease in expression of collagen type II and collagen type X. Overexpression of ALKl results in a different pattern. The ALKl induced increase in aggrecan expression is only 17% compared to the increase induced by ALK5. Moreover, ALKl induced an increase in collagen type II and collagen type X and resulted in a 15- fold increase in MMP-13 (Figure 6) .

Thus, whereas ALK5 is a known repressor of chondrocyte terminal differentiation markers, ALKl induced their expression, thereby revealing an OA-like expression pattern .

ALKl and ALK5 are expressed in human OA cartilage

In order to establish the potential relevance of the findings to humans, cartilage from OA-patients that had received total hip replacement was obtained and RNA was isolated for quantitative PCR. It was found that cartilage of humans also expresses ALKl, besides the already known presence of ALK5. Expression of ALKl was higher in the human OA cartilage samples than in the P2 cell line, indicating that a subpopulation of human OA cells will signal on TGF-beta via the Smadl/5/8 route.

Conclusion

Osteoarthritis is a joint disease characterized by cartilage degradation. One of the major risk factors for OA is age. The present experiments demonstrate that in chondrocytes signaling via ALKl results in the Smadl/5/8 pathway and signaling via ALK5 results in activation of the Smad2/3 pathway. Signaling via the Smadl/5/8 route results in OA-like changes in chondrocytes. In addition, the present experiment's demonstrate that chondrocytes in aged cartilage and experimental OA show a strongly diminished expression of ALK5 but a less diminished expression of ALKl.

In conclusion, during ageing and OA, chondrocyte TGF-beta signaling via ALK5 is strongly diminished. During ageing in OA prone individuals, the balance in TGF-beta signaling shifts from ALK5 to ALKl. Signaling via ALKl results in phosphorylation of Smadl/5/8, terminal differentiation and cartilage degradation.

Claims

1. Method for providing a compound interfering with a chondrocyte distorted phenotype comprising: a) contacting a cell line expressing ALKl with a compound; b) measuring the effect of said compound on the downstream signaling of ALKl.

2. Method according to claim 1, wherein the cell line expressing ALKl is a chondrocyte cell line.

3. Method according to claims 1 or claim 2, wherein the chondrocyte cell line overexpresses ALKl.

4. Method according to any of the claims 1 to 3, wherein the expression of ALK5 in the cell line is down- regulated or silenced.

5. Method according to any of the claims 1 to 4, wherein the measurement comprises determining expression levels of proteins whose coding genes are controlled by the phosphorylated Smadl, Smad 5 and/or Smad8 molecules.

' ^" '6. Method according to any of the claims 1 to 5, wherein proteins are selected from the group consisting of aggrecan, type X collagen, type II collagen, MMP-3 and MMP13.

7. Method according to any of the claims 1 and 5, wherein the measurement comprises determining the phosphorylation of Smadl, Smad5 and/or Smad8.

8. Method for providing a compound capable of interfering with a chondrocyte distorted phenotype comprising: a) contacting ALKI with said compound; b) detecting an interaction between said compound and ALKl .

9. Compound obtainable by a method according to any of the claims 1 to 8.

10. Use of an ALKl antagonist for the preparation of a medicament for the treatment of osteoarthritis.

11. Use of an antagonist of the Smadl/5/8 route for the preparation of a medicament for the treatment of osteoarthritis .

12. Use of an agonist for the Smad2/3 route for the preparation of a medicament for the treatment of osteoarthritis.