[Show abstract][Hide abstract] ABSTRACT: To study the (patho)physiological role of transforming growth factor-beta (TGF-beta), potent and selective inhibitors are necessary. Since TGF-beta signaling is initiated by the high affinity binding to the type II receptor (RII), the extracellular part of RII (solRII) can function as a TGF-beta antagonist. SolRII was cloned and large-scale protein synthesis was performed in the yeast Pichia pastoris expression system. Our results indicate that via this system, high levels of pure concentrated solRII can be obtained. Moreover, purified solRII is an active protein as shown by ELISA and bioassay. In conclusion, our large-scale protein expression procedure results in high quantities of purified solRII, which is a powerful tool to study the natural role of TGF-beta.
Journal of Chromatography B 04/2003; 786(1-2):271-7. DOI:10.1016/S1570-0232(02)00717-1 · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-beta1 and -beta3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-beta on osteophyte formation and articular cartilage, TGF-beta activity was blocked via a scavenging soluble TGF-beta-RII. Our results clearly show that inhibition of endogenous TGF-beta nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-beta-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-beta is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.
The Journal of Immunology 08/2002; 169(1):507-14. DOI:10.4049/jimmunol.169.1.507 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To examine the impact of prolonged TGF-beta exposure on cartilage and ligamentous joint structures in vivo, to investigate involvement of TGF-beta in osteoarthritis pathology.
TGF-beta was injected into murine knee joints once or repeatedly, whereafter articular cartilage proteoglycan (PG) synthesis and content, and histological changes in knee joints were studied over a 2-month period.
A single injection of TGF-beta stimulated patellar cartilage PG synthesis for 3 weeks and PG content for 2 weeks. Triple TGF-beta injections prolonged the increase in PG content to 3 weeks. Patellar cartilage showed no histological abnormalities at 1 and 2 months after the last injection. In contrast, 2 months after triple TGF-beta injections the superficial layer of tibial cartilage still had an increased proteoglycan content, while severe PG depletion was found in deeper layers of the posterior part of the lateral tibia in particular. Eventually, lesions occurred at the level of the tide-mark, exactly the site where cartilage is torn off in experimental and spontaneous osteoarthritis in mice. Additionally, multiple TGF-beta injections induced formation of chondroid structures along the margins of articular cartilage. These chondroid structures were transformed into osteophytes via endochondral ossification. Formation of chondroid tissue was also observed in collateral ligaments.
Multiple intra-articular injections of TGF-beta induce changes in articular cartilage and surrounding tissues that have strong resemblance to features of experimental and spontaneous osteoarthritis in mice, suggesting a role for TGF-beta in the OA process.
Osteoarthritis and Cartilage 01/2000; 8(1):25-33. DOI:10.1053/joca.1999.0267 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The related molecules bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-beta 1) have both been shown to stimulate chondrocyte proteoglycan (PG) synthesis in vitro. We investigated the in-vivo effects of these factors on articular cartilage PG metabolism.
Several doses of BMP-2 or TGF-beta 1 were injected into the murine knee joint, once or repeatedly. Patellar cartilage PG synthesis was measured by [35S]-sulfate incorporation and reverse transcriptase polymerase chain reaction (RT-PCR). PG content was analyzed by measuring safranin O staining intensity on histologic sections.
A single injection of 200 ng BMP-2 induced a much earlier and more impressive stimulation of articular cartilage PG synthesis, than 200 ng TGF-beta 1. RT-PCR revealed that both factors upregulated mRNA of aggrecan more than that of biglycan and decorin. However, 21 days after a single injection of 200 ng TGF-beta 1 PG synthesis still was significantly increased, while stimulation by BMP-2 only lasted for 3 to 4 days. Stimulation by BMP-2 could be prolonged to at least 2 weeks by triple injections of 200 ng each, at alternate days. Remarkably, even after this intense exposure to BMP-2, stimulation of PG synthesis was not reflected in long-lasting enhancement of PG content of articular cartilage. In contrast, even a single injection with 200 ng of TGF-beta 1 induced prolonged enhancement of PG content. After repeated injections, both BMP-2 and TGF-beta 1 induced chondrogenesis at specific sites. 'Chondrophytes' induced by BMP-2 were found predominantly in the region where the growth plates meet the joint space, while those triggered by TGF-beta 1 originated from the periosteum also at sites remote from the growth plates.
BMP-2 and TGF-beta stimulate PG synthesis and PG content with different kinetics, and these factors have different chondro-inductive properties.
Osteoarthritis and Cartilage 10/1998; 6(5):306-17. DOI:10.1053/joca.1998.0129 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor-beta (TGF-beta) is a potent regulator of cell metabolism, proliferation, and differentiation. To study the role of endogenous TGF-beta in processes such as tissue repair and inflammation, potent and specific inhibitors are required. Because the type II TGF-beta receptor (TGF beta RII) has a high affinity for TGF-beta, the extracellular domain of TGF beta RII (TGF-beta sRII) was expressed in Pichia pastoris and Escherichia coli. Expression of the soluble TGF beta sRII using P. pastoris resulted in a soluble, heterogeneously glycosylated protein which was secreted into the medium. Although expression of TGF beta sRII in E. coli resulted in the formation of insoluble inclusion bodies, solubilization and refolding resulted in a biologically active protein. Because in both systems a C-terminal 6x His coding sequence was inserted behind the coding sequence for the extracellular domain of TGF beta RII the recombinant proteins could be purified by a powerful, single-step procedure using a Ni-NTA agarose. The purified proteins appeared to be potent inhibitors of TGF-beta 1 and TGF-beta 3. In contrast, TGF beta sRII was less effective in neutralization of TGF-beta 2. In conclusion, biologically active TGF beta sRII can be produced using P. pastoris and E. coli expression systems. The ease of these expression systems, the powerful single step purification and low costs makes it possible to produce TGF beta s RII in large amounts to further elucidate the role of TGF-beta 1 and TGF-beta 3 in physiological processes like tissue repair and inflammation.
Protein Expression and Purification 04/1998; 12(2):201-7. DOI:10.1006/prep.1997.0819 · 1.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A severe consequence of rheumatoid arthritis is depletion of proteoglycans (PGs) from articular cartilage leading to functional impairment of this tissue. We investigated whether local administration of anabolic factors (transforming growth factors-beta1 and -beta2 [TGF-beta1 and -beta2, respectively] and bone morphogenetic protein-2 (BMP-2) into joints could stimulate cartilage repair during arthritis. A unilateral arthritis was induced in mice by intra-articular injection of zymosan. Starting on Day 4 after the induction of arthritis, three injections of TGF-beta1 (200 ng) were given (Days 4, 6, and 8). On Day 11, articular cartilage PG synthesis was measured by 35S-sulfate incorporation, and histologic knee joint sections were prepared, which were used to analyze cartilage PG content by quantification of safranin O staining. Additionally, histologic sections were used to analyze inflammation and chondrophyte-formation. Local administration of TGF-beta1 did not modify inflammation but clearly stimulated PG synthesis and restored PG content of depleted cartilage. TGF-beta2 appeared to be as potent as TGF-beta1 in the stimulation of cartilage repair, and both TGF-beta isoforms also stimulated the formation of chondrophytes in this rodent model. In contrast to TGF-beta, three intra-articular injections with 200 ng BMP-2 did not stimulate the repair process. In summary, this study demonstrates for the first time that local administration of TGF-beta into arthritic joints stimulates the replenishment of PGs in depleted cartilage.
[Show abstract][Hide abstract] ABSTRACT: To study the effect of bone morphogenetic protein 2 (BMP-2) on articular cartilage proteoglycan (PG) synthesis in vivo and to investigate whether BMP-2 is able to counteract the effects of interleukin-1 (IL-1) on articular cartilage PG synthesis and content.
BMP-2 alone or in combination with IL-1alpha was injected into murine knee joints. PG synthesis was measured by 35S-sulfate incorporation using an ex vivo method or autoradiography. Cartilage PG content was analyzed by measuring Safranin O staining intensity on histologic sections.
BMP-2 appeared to be a potent stimulator of articular cartilage PG synthesis in vivo. However, BMP-2 was not able to counteract the deleterious effects of IL-1alpha on articular cartilage PG synthesis and content. In addition, intraarticular injections of BMP-2 induced chondrophytes.
Although BMP-2 is a very potent stimulator of cartilage PG synthesis in vivo, the therapeutic applications of BMP-2 are limited due to the inability of BMP-2 to counteract the effects of IL-1 and the induction of chondrophytes.
[Show abstract][Hide abstract] ABSTRACT: Recently, a new isoform of the type II transforming growth factor beta receptor (TGF-beta RII) was identified. This isoform (TGF-beta RII2) contains an insertion of 25 amino acids in the extracellular domain of the receptor. Using RT-PCR the authors demonstrated that both TGF-beta RII1 and TGF-beta RII2 are expressed by chondrocytes in murine and human articular cartilage. Bovine articular chondrocytes expressed TGF-beta RII1 mRNA but did not express detectable levels of TGF-beta RII2 mRNA, suggesting that the new isoform does not play an important role in normal bovine cartilage physiology. Because TGF-beta responses seem to be age related and differential TGF-beta responses have been described between normal cartilage and cartilage undergoing repair the authors studied if the relative mRNA expression between these isoforms is altered during cartilage repair and aging. No differences in the relative mRNA expression of the two isoforms of the type II TGF-beta receptor could be demonstrated in murine cartilage during aging or during the repair phase after mild PG depletion indicating that it is unlikely that age-related TGF-beta responses and differential TGF-beta responses between normal cartilage and cartilage undergoing repair are the result of differences in the relative expression of the two TGF-beta RII isoforms.
[Show abstract][Hide abstract] ABSTRACT: To elucidate the role of transforming growth factor-beta (TGF-beta) and the small proteoglycans biglycan and decorin in the repair of articular cartilage after proteoglycan depletion.
Limited and reversible proteoglycan depletion was induced by injection of murine knee joints with 0.5% papain. Proteoglycan content of patellar cartilage was examined by safranin O staining on histological sections and overall proteoglycan synthesis was measured by incorporation of 35S sulfate. Changes in mRNA expression of TGF-beta, aggrecan, decorin, and biglycan were determined by semiquantitative reverse transcription polymerase chain reaction.
Papain injection led to rapid depletion of proteoglycans, which was partly overcome 7 days after injection, while total replenishment of the cartilage matrix with proteoglycans was observed on Day 24. The incorporation of radiolabeled sulfate in patellar proteoglycans was initially decreased (up to Day 3), but significantly enhanced on Days 4 and 7 after papain injection. Upregulation of TGF-beta, decorin, and biglycan mRNA in patellar cartilage was observed on Day 2, markedly before elevation of overall proteoglycan synthesis. mRNA levels were less augmented on Day 7, and on Day 24 all messenger RNA levels had returned to control values. As well, in the soft tissue adjoining the patella swift upregulation of TGF-beta mRNA was observed.
mRNA of both TGF-beta and the small proteoglycans decorin and biglycan are elevated at an early phase during cartilage repair after moderate proteoglycan depletion, implying a functional role for these molecules in this repair process.
The Journal of Rheumatology 04/1997; 24(3):543-9. · 3.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor beta (TGF-beta) is a multipotent regulator of cell proliferation and extracellular matrix production. The effect of TGF-beta on chondrocyte matrix production was studied in relation to the expression of TGF-beta binding proteins. The effect of TGF-beta on proteoglycan synthesis of isolated articular chondrocytes depended on the culture period. Proteoglycan synthesis of chondrocytes which were cultured for one day was inhibited by TGF-beta whereas proteoglycan synthesis of chondrocytes cultured in monolayer for seven days or longer was stimulated by TGF-beta. To investigate if this differential response is related to a distinct expression of TGF-beta receptors, this parameter was studied by affinity labelling.
Chondrocytes were incubated with 100 pM TGF-beta labelled with iodine-125. Crosslinking was performed using 0.25 mM disuccinimidyl suberate. Membrane proteins were extracted and analysed by denaturating sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.
Freshly isolated and cultured chondrocytes expressed types I, II, and III TGF-beta receptors. The type II TGF-beta receptor of cultured chondrocytes appeared to be about 15 kilodaltons smaller than the type II TGF-beta receptor expressed on freshly isolated chondrocytes, however.
As the type II TGF-beta receptors appears to be involved in signal transduction, this change in size of the type II TGF-beta receptor might be related to the differential effect of TGF-beta on proteoglycan synthesis of freshly isolated and cultured bovine articular chondrocytes.
Annals of the Rheumatic Diseases 12/1993; 52(11):812-6. DOI:10.1136/ard.52.11.812 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: TGF-beta inhibits the proteoglycan synthesis of freshly isolated articular chondrocytes while TGF-beta stimulates the proteoglycan synthesis of articular chondrocytes cultured for 14 days. To investigate if this differential effect was the result of differences in TGF-beta receptor expression this parameter was studied by affinity labeling in combination with SDS-PAGE. Using this method we demonstrated a difference in size of the TGF-beta type II receptor between freshly isolated and cultured chondrocytes. This difference might explain the differential effect of TGF-beta on the proteoglycan synthesis of chondrocytes cultured for 1 or 14 days.
Agents and actions. Supplements 02/1993; 39:139-45.