Increased IL-6 receptor expression and signaling in ageing cartilage can be explained by loss of TGF-ß-mediated IL-6 receptor suppression.

OBJECTIVE: Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-ß (TGF-ß) signaling in cartilage. Recently, we showed that TGF-ß suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-ß signaling results in increased IL-6R expression and signaling in ageing cartilage. DESIGN: Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-ß1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR. RESULTS: Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-ß to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-ß-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed. CONCLUSIONS: Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-ß signaling, resulting in loss of TGF-ß-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.

TGF-ß dampens IL-6 signaling in articular chondrocytes by decreasing IL-6 receptor expression.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) is an important homeostatic regulator of cartilage. In contrast, interleukin-6 (IL-6) is a pro-inflammatory cytokine implicated in cartilage degeneration. Cross-talk between TGF-ß and IL-6 is reported in tissues other than articular cartilage. Here, we investigated regulation of IL-6 signaling by TGF-ß in articular chondrocytes. DESIGN: Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with TGF-ß1 or interleukin-1ß (IL-1ß). Expression of IL-6 and IL-6 receptor (IL-6R) was determined on mRNA and protein level. TGF-ß regulation of IL-6 signaling via phosho-STAT3 (p-STAT3) was determined using Western blot, in presence of inhibitors for IL-6R, and Janus kinase(JAK)- and activin receptor-like kinase ALK)5 kinase activity. Furthermore, induction of STAT3-responsive genes was used as a read-out for IL-6 induced gene expression. RESULTS: TGF-ß1 increased IL-6 mRNA and protein expression in both G6 and primary chondrocytes. Moreover, TGF-ß1 stimulation clearly induced p-STAT3), which was abolished by inhibition of either IL-6R, JAK- or ALK5 kinase activity. However, TGF-ß1 did not increase expression of the STAT3-responsive gene SOCS3 and pre-treatment with TGF-ß1 even inhibited induction of p-STAT3 and SOCS3 by rhIL-6. Interestingly, TGF-ß1 potently decreased IL-6R expression. In contrast, IL-1ß did increase IL-6 levels, but did not affect IL-6R expression. Finally, addition of recombinant IL-6R abolished the inhibitory effect of TGF-ß1 on IL-6-induced p-STAT3 and downstream SOCS3, BCL3, SAA1 and MMP1 expression. CONCLUSIONS: In this study we show that TGF-ß decreases IL-6R expression, thereby dampening IL-6 signaling in chondrocytes. This reveals a novel effect of TGF-ß, possibly important to restrict pro-inflammatory IL-6 effects to preserve cartilage homeostasis.

Expression of TGF-ß Signaling Regulator RBPMS (RNA-Binding Protein With Multiple Splicing) Is Regulated by IL-1ß and TGF-ß Superfamily Members, and Decreased in Aged and Osteoarthritic Cartilage.

OBJECTIVE: RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-ß (TGF-ß)-mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1ß (IL)-1ß and TGF-ß superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis. METHODS: Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase-quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry. RESULTS: RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-ß inhibited RBPMS expression while BMP2 and IL-1ß increased its expression. TGF-ß-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis. CONCLUSIONS: Expression of RBPMS in chondrocytes is regulated by TGF-ß superfamily members and IL-1ß, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage.

Resemblance of osteophytes in experimental osteoarthritis to transforming growth factor beta-induced osteophytes: limited role of bone morphogenetic protein in early osteoarthritic osteophyte formation.

OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage damage, synovial fibrosis, and osteophyte formation. Both transforming growth factor beta (TGFbeta) and bone morphogenetic protein 2 (BMP-2) can induce the formation of osteophytes during OA, but their specific role in this process is unclear. The purpose of this study was to investigate the respective contributions of TGFbeta and BMP-2 to OA. METHODS: Mouse knee joints injected with adenovirus (Ad-TGFbeta or Ad-BMP-2) were compared histologically with knee joints from murine models of OA (joints injected with collagenase and joints from STR/Ort mice with spontaneous OA). To further investigate the role of BMP during osteophyte formation, adenovirus Ad-Gremlin was injected into knee joints that had previously been injected with Ad-TGFbeta or collagenase. RESULTS: BMP-2 induced early osteophytes, which bulged from the growth plates on the femur and grew on top of the patella, whereas TGFbeta induced early osteophyte formation on the bone shaft beneath the collateral ligament on the femur as well as on top of the patella. The pattern of osteophyte formation during experimental OA closely resembled that of TGFbeta-induced osteophyte formation, but differed from the pattern induced by BMP-2. Ad-Gremlin proved to be able to totally block BMP-2-induced osteophyte formation. However, blocking BMP activity inhibited neither TGFbeta-induced nor experimental OA-associated osteophyte formation. CONCLUSION: Our findings demonstrate that the role of BMP during the onset of TGFbeta-induced and experimental OA-induced osteophyte formation is limited. The latter finding does not rule out a role of BMP during osteophyte maturation.

TGFbeta inhibits IL-1 -induced iNOS expression and NO production in immortalized chondrocytes.

OBJECTIVE: The balance between anti-inflammatory (e.g. TGFbeta) and proinflammatory cytokines (e.g. IL-1 and TNFalpha), regulates destructive processes in OA cartilage. IL-1 and TNFalpha enhance nitric oxide (NO) production in OA cartilage through the inducible nitric oxide synthase (iNOS) pathway and NO mediates many of the destructive effects of these cytokines. The aim of the present study was to investigate the effects of TGFbeta on NO production in immortalized H4 chondrocytes exposed to IL-1. RESULTS: IL-1 induced NO production in chondrocytes through nuclear factor kappa B (NF-kappaB) sensitive and dexamethasone insensitive expression of iNOS. TGFbeta inhibited IL-1 -induced iNOS expression and NO production in chondrocytes, but it did not have any effect on iNOS mRNA levels. iNOS protein levels were similar in cells treated with IL-1 or IL-1+TGFbeta when measured after 8 h incubation, whereas when measured after 12 h and 24 h incubations, iNOS protein levels were 50% and 80% lower in cells treated with IL-1+TGFbeta than in cells treated with IL-1 alone. CONCLUSION: TGFbeta suppressed IL-1-induced iNOS expression and NO production in chondrocytes, probably by enhancing iNOS protein degradation. This finding suggests an additional mechanism for TGFbeta to counteract the destructive effects of IL-1 in OA.

Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression.

OBJECTIVE: To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases. METHODS: The H4 chondrocyte cell line was validated by comparing metalloproteinase expression profile with intact murine cartilage by reverse transcription polymerase chain reaction. Genome-wide gene expression in the H4 cells in response to IL-1 and TGF-beta, alone and in combination, was analyzed by using oligonucleotide arrays negotiating approximately 12,000 genes. RESULTS: The response of cartilage and the H4 cell line to IL-1 and TGF-beta was comparable. Oligonucleotide array analysis demonstrated a mutual but asymmetrical antagonism as the dominant mode of interaction between IL-1 and TGF-beta. Cluster analysis revealed a remarkable selectivity in the mode of action exerted by TGF-beta on IL-1 regulated genes: antagonistic on pro-inflammatory genes whereas additive on growth regulators such as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). While the former cluster underlined the protective effect of TGF-beta, the latter underscored the adverse effect of TGF-beta. We further identified potentially novel classes of target genes under control of TGF-beta such as ras family, histones, proteasome components, and ubiquitin family, highlighting the importance of such genes in TGF signaling besides the well-characterized SMAD pathway. CONCLUSIONS: We identified a cluster of genes as potential targets mediating the adverse effect of TGF-beta such as fibrosis. Transcriptional regulation of ras GTPase and ubiquitin/proteasome pathways is likely to be a novel mechanism mediating the effect of TGF-beta and its interaction with IL-1. These down-stream genes and pathways can be targets in future therapy.

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint.

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1 beta enhancer region (-3690 to -2720) upstream of the human IL-6 promoter region (-163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (10(6) ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6(-/-) and IL-1(-/-) gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.

Adenoviral overexpression of Smad-7 and Smad-6 differentially regulates TGF-beta-mediated chondrocyte proliferation and proteoglycan synthesis.

OBJECTIVE: To assess if various biological responses to transforming growth factor-beta (TGF-beta) in chondrocytes are differentially regulated by Smad-6 and Smad-7. DESIGN: Adenoviral overexpression of Smad-6 or -7 mRNA in a chondrocyte cell line was determined via semi-quantitative RT-PCR and protein overexpression was studied by immunocytochemistry. Furthermore, the effect of Smad-6 and -7 overexpression on TGF-beta-induced PAI-1 and aggrecan mRNA upregulation was studied via quantitative RT-PCR. The effect of Smad-6 and -7 overexpression on TGF-beta-induced chondrocyte proliferation was studied via DNA quantification, whereas TGF-beta-induced proteoglycan (PG) synthesis was studied by 35S-sulfate incorporation. RESULTS: Adenoviral transfection of chondrocytes with Smad-6 and -7 resulted in strong upregulation of Smad-6 and -7 mRNA levels, respectively. Immunocytochemistry showed overexpression of Smad-6 and -7 proteins in both the nucleus and cytoplasm. Smad-6 overexpression significantly inhibited TGF-beta-stimulated chondrocyte proliferation, although proliferation was not completely abolished. Smad-7 overexpression, however, completely antagonized the TGF-beta effect on proliferation. Smad-6 overexpression had no effect on TGF-beta-induced PAI-1 expression, while overexpression of Smad-7 completely blocked this TGF-beta effect. Additionally, overexpression of Smad-7, but not Smad-6, totally prevented TGF-beta-induced PG synthesis on the mRNA and protein levels. CONCLUSIONS: Adenoviral transfection of chondrocytes with Smad-6 and -7 resulted in strong upregulation of Smad-6 and -7 mRNA and protein levels. Furthermore, overexpression of Smad-7 in chondrocytes totally inhibited important TGF-beta-mediated biological responses such as proliferation and PG synthesis, while overexpressed Smad-6 had no or only a partial inhibitory effect on TGF-beta activity. We conclude that in chondrocytes distinct TGF-beta activities are differentially regulated by Smad-6 and Smad-7.

Phenotypic differences in murine chondrocyte cell lines derived from mature articular cartilage.

OBJECTIVE: To obtain well characterized immortalized murine chondrocyte cell lines. The cell lines were obtained from mature articular chondrocytes, instead of embryonal cells which are used in most other studies. METHODS: Pieces of articular cartilage were cut from murine patellae and femoral heads. Chondrocytes were isolated by digestion with collagenase. These cells were cultured in monolayer and immortalized by transfection of the SV40 large T antigen gene. To preserve the differentiated phenotype, the resulting clones were cultured in three-dimensional carriers, alginate beads. The phenotypes of the cells were characterized using the following parameters: Cell morphology (light microscopy), messenger RNA (RT-PCR) and protein (immunohistochemistry) levels of extracellular matrix molecules. Moreover, responsiveness to interleukin-1(IL-1) was determined by measuring production of proteoglycans ((35)S-sulfate incorporation) and of nitric oxide (Griess reaction). RESULTS: Sixteen clones were obtained, ten (P1 to P10) derived from patellar cartilage, and six (H1 to H6) from femoral head cartilage. In seven cell lines (P2, P5, H1, H3, H4, H5, H6) high production of type II collagen corresponded with high levels of mRNA of type II collagen (and prevalence of the IIB type) and with high IL-1-induced suppression of proteoglycan synthesis. Like intact murine articular cartilage, all cell lines produced type I and type X collagens, but mRNA levels of both types of collagen were never higher in the cell lines as compared with intact cartilage. CONCLUSION: Our results demonstrate that it is possible to immortalize mature murine articular chondrocytes. Each of the obtained chondrocyte cell lines appeared to have a stable phenotype. Both relatively differentiated and relatively dedifferentiated chondrocyte cell lines could be identified.

Loss of transforming growth factor counteraction on interleukin 1 mediated effects in cartilage of old mice.

OBJECTIVE: To investigate if a difference exists between young and old mice in the response of articular cartilage to interleukin 1 (IL1) and transforming growth factor beta (TGFbeta) alone or in combination. METHODS: The interaction of IL1 and TGFbeta was studied in cartilage of young (three months) and old mice (18 months) both in vivo and in vitro. Therefore, IL1, TGFbeta, or IL1 together with TGFbeta was injected into the knee joints of mice on days 1, 3, and 5 before harvest of the patellae on day 6. Alternatively, isolated patellae were stimulated with IL1, TGFbeta, or IL1 together with TGFbeta in culture for 48 hours. Proteoglycan (PG) synthesis and nitric oxide (NO) production were measured. RESULTS: IL1 inhibited PG synthesis and increased NO production in cartilage of both young and old mice. On the other hand, TGFbeta stimulated PG synthesis and reduced NO production in both age groups. Importantly, TGFbeta was able to counteract IL1 mediated effects on PG synthesis and NO production in young but not in old mice. CONCLUSIONS: Contrary to the findings in young mice, the cartilage of old animals does not antagonise IL1 effects via TGFbeta. This loss of responsiveness to the pivotal cytokine TGFbeta on effects of IL1 can be important in the initiation and progression of osteoarthritis (OA).

Growth factors and cartilage repair.

Growth factors are obvious tools to enhance cartilage repair. Understanding of reactivities in normal and arthritic cartilage and potential side effects on other compartments in the joint will help to identify possibilities and limitations. Growth factor responses have been evaluated in normal and diseased murine knees. The main cartilage anabolic factor, insulinlike growth factor-1, shows great safety, but has little contribution in diseased cartilage because of insulinlike growth factor nonresponsiveness of arthritic chondrocytes. Transforming growth factor-beta can overrule interleukin-1 catabolic effects and can enhance cartilage repair in arthritic tissue, unlike bone morphogenetic protein-2 that only is capable of enhancing chondrocyte proteoglycan synthesis in the absence of interleukin-1. Transforming growth factor-beta and bone morphogenetic protein-2 induce chondrophyte formation at the margins of the joint. Studies with scavenging transforming growth factor beta soluble receptor identified endogenous transforming growth factor-beta involvement in spontaneous cartilage repair and chondrophyte and subsequent osteophyte formation in arthritic conditions. Osteophyte induction may hamper intraarticular transforming growth factor-beta application in the joint and warrants targeted growth factor application to cartilage lesion sites only.

Linkage of chondroitin-sulfate to type I collagen scaffolds stimulates the bioactivity of seeded chondrocytes in vitro.

An increasing amount of interest is focused on the potential use of tissue-engineered articular cartilage implants, for repair of defects in the joint surface. In this perspective, various biodegradable scaffolds have been evaluated as a vehicle to deliver chondrocytes into a cartilage defect. This cell-matrix implant should eventually promote regeneration of the traumatized articular joint surface with hyaline cartilage. Successful regeneration can only be achieved with such a tissue-engineered cartilage implant if the seeded cells reveal an appropriate proliferation rate in the biodegradable scaffold together with the production of a new cartilage-specific extracellular matrix. These metabolic parameters can be influenced by the biochemical composition of a cell-delivery scaffold. Further elucidation of specific cell-matrix interactions is important to define the optimal biochemical composition of a cell-delivery vehicle for cartilage repair. In this in vitro study, we investigated the effect of the presence of cartilage-specific glycosaminoglycans in a type I collagen scaffold on the metabolic activity of seeded chondrocytes. Isolated bovine chondrocytes were cultured in porous type I collagen matrices in the presence and absence of covalently attached chondroitin sulfate (CS) up to 14 days. CS did indeed influence the bioactivity of the seeded chondrocytes. Cell proliferation and the total amount of proteoglycans retained in the matrix, were significantly higher (p < 0.001) in type I collagen scaffolds with CS. Light microscopy showed the formation of a more dense cartilaginous layer at the matrix periphery. Scanning electron microscopy revealed an almost complete surfacing of the initially porous surface of both matrices. Histology and reverse transcriptase PCR for various proteoglycan subtypes suggested a good preservation of the chondrocytic phenotype of the seeded cells during culture. The stimulatory potential of CS on both the cell-proliferation and matrix retention, turns this GAG into an interesting biochemical component of a cell-delivery scaffold for use in tissue-engineering articular cartilage.

Overexpression of active TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation.

OBJECTIVE: To investigate the impact of a prolonged and constant active TGF-beta expression by the synovial lining cells on cartilage and ligamentous joint structures in vivo. DESIGN: An adenoviral vector (AdTGF-beta1(223,225)) was used for the overexpression of active TGF-beta1 in knee joints of C57Bl/6 mice. RESULTS: It was found that physiological relevant levels of active TGF-beta1 produced by the synovial lining layer resulted in histopathological changes: hyperplasia of synovium and chondro-osteophyte formation at the so-called chondro-synovial junctions. No histological changes were seen after intra-articular injection of an empty control vector (AdDL70-3) or by overexpression of latent TGF-beta1 (AdTGF-beta1). The predominant site of TGF-beta production in osteoarthritis (OA) and rheumatoid arthritis (RA) is the synovial lining layer. To address the question whether the TGF-beta-induced changes were related to the expression site in the synovial lining, the synovial lining layer was depleted by local treatment with liposomes encapsulating clodronate. Depletion of the lining resulted in a dramatic change of TGF-beta1-induced pathology: markedly reduced chondro-osteophyte formation and increased accumulation of extracellular matrix in the synovium. CONCLUSION: This study shows that overexpression of active TGF-beta1 in the knee joint results in OA-like changes and suggests the synovial lining cells contribute to the chondro-osteophyte formation.

Role of nitric oxide in the inhibition of BMP-2-mediated stimulation of proteoglycan synthesis in articular cartilage.

OBJECTIVE: Bone morphogenetic protein-2 (BMP-2)-mediated stimulation of articular cartilage proteoglycan (PG) synthesis is suppressed in arthritic murine knee joints and by interleukin-1 (IL-1). The goal of this study was to investigate whether the gaseous mediator nitric oxide (NO) plays a crucial role in the inhibition of BMP-2 effects by IL-1. METHODS: Bone morphogenetic protein-2 alone or in combination with IL-1 was injected into the right knee joint of wild-type and NOS2 deficient C57BI/6x129/Sv mice. Proteoglycan synthesis was measured ex vivo by incorporation of 35S-sulfate on day 1, 2 and 3 after injection. To study the role of NO in the inhibition BMP-2-mediated stimulation of PG synthesis in arthritic joints, BMP-2 was injected intra-articularly in the joints of wild-type and NOS2 deficient mice with zymosan-induced arthritis. To check for NOS2 deficiency, NO production was measured in conditioned medium after challenge of patellae with surrounding tissue with IL-1. RESULTS: BMP-2 potently stimulated proteoglycan synthesis in articular cartilage of normal knees (up to 4-fold) but not in arthritic knees. Co-injection of BMP-2 with tumor necrosis factor alpha had no effect on BMP-2-mediated stimulation of PG synthesis but co-injection with IL-1 alpha resulted in a nearly total inhibition of BMP-2-mediated stimulation. In contrast, in NOS2 deficient mice IL-1 had no effect on BMP-2-mediated stimulation of PG synthesis. However, injection of BMP-2 into arthritic knee joints of NOS2 knock out mice did not result in significant stimulation of PG synthesis. CONCLUSIONS: In this study we show that NO plays a role in the inhibition of BMP-2-mediated stimulation of PG synthesis by IL-1. However, NO, or at least NOS2, plays no dominant role in the inhibition of BMP-2 effects in arthritic knee joints.

Responsiveness of bovine chondrocytes to growth factors in medium with different serum concentrations.

Autologous transplantation of chondrocytes is currently under investigation as a potential therapy to stimulate intrinsic repair in articular cartilage defects. The quality of the repair tissue may benefit from the preservation of the characteristic chondrocytic phenotype of the transplanted cells together with the production of a new extracellular matrix composed of collagen type II and larger proteoglycans. A number of growth factors are believed to play an important role in the process of generating new cartilage repair tissue. In this study, the dose-dependent response of bovine chondrocytes to recombinant human insulin-like growth factor-1, recombinant human transforming growth factor-beta2, and recombinant human bone morphogenetic protein-2 was studied in an alginate culture system under different culture conditions. The chondrocytes were cultured in medium with increasing concentrations of fetal calf serum. The cultures were assessed by the total amount of DNA, quantitative and qualitative synthesis of proteoglycan, production of nitric oxide, and histology. Cells cultured in the presence of each growth factor had an equal, nonsignificant stimulation of DNA synthesis compared with those cultured in basal medium alone. Recombinant human insulin-like growth factor-1 and recombinant human transforming growth factor-beta2 stimulated proteoglycan synthesis in a dose-dependent and reversed dose-dependent fashion, respectively. Recombinant human bone morphogenetic protein-2 stimulated proteoglycan synthesis significantly only in the absence of fetal calf serum or in the presence of small amounts of the serum. Overall, proteoglycan synthesis dramatically decreased with the addition of each growth factor as the concentration of fetal calf serum in the medium decreased, and the dose-dependent stimulation pattern, as observed for recombinant human insulin-like growth factor-1 and recombinant human transforming growth factor-beta2, disappeared. Apart from a moderate increase in mRNA for aggrecan and decorin, the growth factors did not greatly affect the type of proteoglycans synthesized. Histological examination confirmed the presence of a dense pericellular matrix deposition, especially when the chondrocytes were cultured in the presence of recombinant human insulin-like growth factor-1 or recombinant human transforming growth factor-beta2. The results indicate that these growth factors can stimulate qualitatively superior matrix production and that the responsiveness of the chondrocytes to the growth factors changes with the culture conditions. Further knowledge about the interaction between chondrocytes, growth factors, and the external environment is important to stimulate chondrocytes to produce adequate repair tissue in cartilage defects in vivo. Insulin-like growth factor-1 especially seems capable of stimulating, in the most consistent and predictable fashion, qualitatively superior proteoglycan synthesis by differentiated chondrocytes. Additional in vivo studies are needed to evaluate the potential of these growth factors as stimulators in cartilage repair.

Osteoarthritis-like changes in the murine knee joint resulting from intra-articular transforming growth factor-beta injections.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Expression of recombinant human soluble type II transforming growth factor-beta receptor in Pichia pastoris and Escherichia coli: two powerful systems to express a potent inhibitor of transforming growth factor-beta.

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.

Stimulation of articular cartilage repair in established arthritis by local administration of transforming growth factor-beta into murine knee joints.

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.

Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation.

OBJECTIVE: 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. DESIGN: 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. RESULTS: 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. CONCLUSIONS: BMP-2 and TGF-beta stimulate PG synthesis and PG content with different kinetics, and these factors have different chondro-inductive properties.

Bone morphogenetic protein 2 stimulates articular cartilage proteoglycan synthesis in vivo but does not counteract interleukin-1alpha effects on proteoglycan synthesis and content.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.