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WO2008128559A1 - Procédé de production d'un composé capable d'interférer avec un phénotype déformé de chondrocytes - Google Patents

Procédé de production d'un composé capable d'interférer avec un phénotype déformé de chondrocytes Download PDF

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Publication number
WO2008128559A1
WO2008128559A1 PCT/EP2007/003729 EP2007003729W WO2008128559A1 WO 2008128559 A1 WO2008128559 A1 WO 2008128559A1 EP 2007003729 W EP2007003729 W EP 2007003729W WO 2008128559 A1 WO2008128559 A1 WO 2008128559A1
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alkl
alk5
compound
expression
cell line
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PCT/EP2007/003729
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English (en)
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Peter Mario Van Der Kraan
Esmeralda Nicole Blaney Davidson
Wilhelmus Berend Van Den Berg
Dennis Florian George Remst
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Stichting Katholieke Universiteit
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/105—Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone

Definitions

  • 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.
  • TGF-beta Transforming Growth Factor beta
  • Smads Phosphorylated Smads
  • Activin receptor-like kinase 1 (ALKl or ACVRLl) and activin receptor-like kinase 5 (ALK5) are both type I TGF- beta receptors .
  • TGF-beta Upon activation of ALKl by TGF-beta, Smadl, Smad5 and/or Smad ⁇ 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 .
  • TGF-beta 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 is the most common joint disease and its incidence strongly correlates with age. Osteoarthritis (OA) is characterized by destruction of articular cartilage.
  • Chondrocytes 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) .
  • MMP-13 matrix metalloproteinase 13
  • This distorted phenotype of articular chondrocytes in osteoarthritis (OA) is analogous to the phenotype of growth plate chondrocytes that have undergone terminal differentiation.
  • 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
  • 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 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) .
  • 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) .
  • 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.
  • 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.
  • the cell line is a chondrocyte cell line, more preferably a chondrocyte cell line (recombinantly) overexpressing the ALKl receptor.
  • the expression of the ALK5 receptor is additionally down- regulated or silenced.
  • 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.
  • 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 Smad ⁇ 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 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.
  • 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.
  • 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
  • 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.
  • 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) .
  • a quantitative PCR was prepared as follows: a primer mix of 1.5 ⁇ l forward primer (5 ⁇ M) , 1.5 ⁇ l reverse primer (5 ⁇ M) and 4.5 ⁇ l dH 2 O, was added to 12.5 ⁇ l SybrGreen. Then, 5 ⁇ l 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 50 0 C and 10 min at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 6O 0 C, with data collection in the last 30 seconds.
  • 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.
  • 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.
  • 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.
  • SDS Sodium Dodecyl Sulfate
  • 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 4 0 C overnight.
  • 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) .
  • ECL Plus Western Blotting Detection System ECL Plus Western Blotting Detection System, GE Healthcare, Eindhoven, the Netherlands
  • 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 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.
  • 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.
  • 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 ⁇ m were prepared.
  • 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.
  • citrate buffer 0.1 M sodium citrate, 0.1 M citric acid
  • biotin labeled secondary antibody was used at a concentration of 2 ⁇ g/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 H 2 O 2 resulting in a brown precipitate. Sections were briefly counterstained with heamatoxylin and mounted with Permount . Image analysis : quantification of positively stained articular chondrocytes
  • 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.
  • 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.
  • 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.
  • 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.
  • Results were analyzed with the Student's t-test and considered significant if the p-value was smaller than 0.05.
  • Chondrocytes express both ALK5 and ALKl and can signal through both receptors.
  • 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.
  • OA-chondrocytes expressed ALK-I or ALK-5 and whether this was different compared to normal chondrocytes.
  • 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 .
  • ALK5 staining had decreased with 88% and 76%, respectively.
  • ALK5 decreased more drastically than ALKl in focal cartilage degeneration 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.
  • ALK5 and ALKl decrease with progression of spontaneous OA with a more drastic decrease of ALK5
  • 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.
  • TGF-beta RI expression 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.
  • ALKl overexpression results in different responses than ALK5 overexpression in chondrocytes
  • ALK5 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) .
  • 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
  • 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.
  • 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.
  • 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.
  • chondrocyte TGF-beta signaling via ALK5 is strongly diminished.
  • 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.

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Abstract

La présente invention porte sur des procédés de production d'un composé capable d'interférer avec un phénotype de chondrocyte tordu et, en particulier, sur un procédé de production d'un composé capable de traiter l'ostéoarthrite. De façon spécifique, la présente invention porte sur un procédé de production d'un composé interférant avec un phénotype déformé de chondrocytes comprenant : (a) la mise en contact d'une lignée cellulaire exprimant ALK1 avec un composé et (b) la mesure de l'effet dudit composé sur la signalisation en aval de ALK1.
PCT/EP2007/003729 2007-04-20 2007-04-20 Procédé de production d'un composé capable d'interférer avec un phénotype déformé de chondrocytes WO2008128559A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046386A1 (fr) * 1998-03-13 1999-09-16 Ludwig Institute For Cancer Research REACTIONS DE ALK-1 AU TGF-β ET DES SIGNAUX TRAVERSANT SMAD-1 ET SMAD-5
WO2001007067A2 (fr) * 1999-07-21 2001-02-01 Omeros Medical Systems, Inc. Solutions et procedes permettant d'inhiber la douleur, les inflammations et la degradation des cartilages
WO2002022871A2 (fr) * 2000-09-14 2002-03-21 Decode Genetics Ehf Gene humain de l'osteoporose
US20070071675A1 (en) * 2005-08-19 2007-03-29 Chengbin Wu Dual variable domain immunoglobulin and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999046386A1 (fr) * 1998-03-13 1999-09-16 Ludwig Institute For Cancer Research REACTIONS DE ALK-1 AU TGF-β ET DES SIGNAUX TRAVERSANT SMAD-1 ET SMAD-5
WO2001007067A2 (fr) * 1999-07-21 2001-02-01 Omeros Medical Systems, Inc. Solutions et procedes permettant d'inhiber la douleur, les inflammations et la degradation des cartilages
WO2002022871A2 (fr) * 2000-09-14 2002-03-21 Decode Genetics Ehf Gene humain de l'osteoporose
US20070071675A1 (en) * 2005-08-19 2007-03-29 Chengbin Wu Dual variable domain immunoglobulin and uses thereof

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ANIE PHILIP: "Regulation of TGF-beta action in chondrocytes: Balance of signaling via two distinct type I receptors", CANADIAN ARTHRITIS NETWORK - INTERNATIONAL RESEARCH & TRAINING PROGRAM, 21 July 2005 (2005-07-21), pages 1 - 3, XP002460875 *
BLANEY DAVIDSON EN ET AL: "Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity", ARTHRITIS RESEARCH AND THERAPY, BIOMED CENTRAL, LONDON, GB, vol. 7, no. 6, 30 September 2005 (2005-09-30), pages R1338 - R1347, XP021011678, ISSN: 1478-6354 *
BLANEY DAVIDSON ET AL: "TGF-beta and osteoarthritis", OSTEOARTHRITIS AND CARTILAGE, BAILLIERE TINDALL, LONDON, GB, vol. 15, no. 6, 31 March 2007 (2007-03-31), pages 597 - 604, XP022075822, ISSN: 1063-4584 *
PUFE ET AL: "Effects of pleiotrophin, a heparin-binding growth factor, on human primary and immortalized chondrocytes", OSTEOARTHRITIS AND CARTILAGE, BAILLIERE TINDALL, LONDON, GB, vol. 15, no. 2, 26 January 2007 (2007-01-26), pages 155 - 162, XP005853748, ISSN: 1063-4584 *
TEN DIJKE P P ET AL: "Signaling inputs converge on nuclear effectors in TGF-beta signaling", TRENDS IN BIOCHEMICAL SCIENCES, ELSEVIER, HAYWARDS, GB, vol. 25, no. 2, February 2000 (2000-02-01), pages 64 - 70, XP004189096, ISSN: 0968-0004 *
VALCOURT U ET AL: "Functions of transforming growth factor-[beta] family type I receptors and Smad proteins in the hypertrophic maturation and osteoblastic differentiation of chondrocytes", JOURNAL OF BIOLOGICAL CHEMISTRY 13 SEP 2002 UNITED STATES, vol. 277, no. 37, 13 September 2002 (2002-09-13), pages 33545 - 33558, XP002460874, ISSN: 0021-9258 *

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