H.M. van Beuningen

Increase in ALK1/ALK5 Ratio as a Cause for Elevated MMP-13 Expression in Osteoarthritis in Humans and Mice

During osteoarthritis (OA) chondrocytes show deviant behavior resembling terminal differentiation of growth-plate chondrocytes, characterized by elevated MMP-13 expression. The latter is also a hallmark for OA. TGF-β is generally thought to be a protective factor for cartilage, but it has also displayed deleterious effects in some studies. Recently, it was shown that besides signaling via the ALK5 (activin-like kinase 5) receptor, TGF-β can also signal via ALK1, thereby activating Smad1/5/8 instead of Smad2/3. The Smad1/5/8 route can induce chondrocyte terminal differentiation. Murine chondrocytes stimulated with TGF-β activated the ALK5 receptor/Smad2/3 route as well as the ALK1/Smad1/5/8 route. In cartilage of mouse models for aging and OA, ALK5 expression decreased much more than ALK1. Thus, the ALK1/ALK5 ratio increased, which was associated with changes in the respective downstream markers: an increased Id-1 (inhibitor of DNA binding-1)/PAI-1 (plasminogen activator inhibitor-1) ratio. Transfection of chondrocytes with adenovirus overexpressing constitutive active ALK1 increased MMP-13 expression, while small interfering RNA against ALK1 decreased MMP-13 expression to nondetectable levels. Adenovirus overexpressing constitutive active ALK5 transfection increased aggrecan expression, whereas small interfering RNA against ALK5 resulted in increased MMP-13 expression. Moreover, in human OA cartilage ALK1 was highly correlated with MMP-13 expression, whereas ALK5 correlated with aggrecan and collagen type II expression, important for healthy cartilage. Collectively, we show an age-related shift in ALK1/ALK5 ratio in murine cartilage and a strong correlation between ALK1 and MMP-13 expression in human cartilage. A change in balance between ALK5 and ALK1 receptors in chondrocytes caused changes in MMP-13 expression, thereby causing an OA-like phenotype. Our data suggest that dominant ALK1 signaling results in deviant chondrocyte behavior, thereby contributing to age-related cartilage destruction and OA.

Protection from interleukin 1 induced destruction of articular cartilage by transforming growth factor beta: studies in anatomically intact cartilage in vitro and in vivo.

The modulation of interleukin 1 (IL-1) effects on proteoglycan metabolism in intact murine patellar cartilage by transforming growth factor beta (TGF-beta) was investigated in vitro and in vivo. In vitro TGF-beta (400 pmol/l) had no effect on basal proteoglycan degradation. Proteoglycan degradation induced by IL-1, however, was suppressed by TGF-beta in serum free medium alone and in medium supplemented with 0.5 micrograms/ml insulin-like growth factor 1. This suggests a specific regulatory role for TGF-beta under pathological conditions. In contrast with the suppression of breakdown, synthesis of proteoglycans was stimulated by TGF-beta for both basal and IL-1 suppressed proteoglycan synthesis in cultures without insulin-like growth factor. In the presence of insulin-like growth factor no extra effect of TGF-beta on proteoglycan synthesis was observed. With insulin-like growth factor, however, TGF-beta potentiated the ex vivo recovery of IL-1 induced suppression of proteoglycan synthesis. Analogous to the in vitro effects, TGF-beta injected intraarticularly suppressed IL-1 induced proteoglycan degradation. Furthermore, TGF-beta injected into the joint counteracted IL-1 induced suppression of proteoglycan synthesis. This indicates that in vivo also TGF-beta can ameliorate the deleterious effects of IL-1 on the cartilage matrix.

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.

In vivo protection against interleukin-1-induced articular cartilage damage by transforming growth factor-beta 1: age-related differences.

OBJECTIVES--Transforming growth factor-beta (TGF-beta) has been shown to antagonise interleukin-1 (IL-1) effects in different systems. Investigations were carried out to study whether TGF-beta 1 modulates IL-1 induced inflammation and IL-1 effects on articular cartilage in the murine knee joint. METHODS--IL-1, TGF-beta 1 or both factors together were injected into the knee joint. Inflammation was studied in whole knee histological sections. Patellar cartilage proteoglycan synthesis was measured using 35S-sulphate incorporation while patellar cartilage glycosaminoglycan content was determined with automated image analysis on joint sections. RESULTS--Co-injection of TGF-beta 1 and IL-1 resulted in synergistic attraction of inflammatory cells. In contrast, TGF-beta 1 counteracted IL-1 induced suppression of articular cartilage proteoglycan synthesis. Proteoglycan depletion was similar shortly after the last injection of IL-1 or IL-1/TGF-beta 1, but accelerated recovery was found with the combination at later days. This protective effect of TGF-beta 1 could not be demonstrated in older mice. CONCLUSIONS--TGF-beta 1 aggravates IL-1 induced knee joint inflammation, but counteracts the deleterious effects of IL-1 on articular cartilage proteoglycan synthesis and content. The data indicate that TGF-beta 1 could play an important part in articular cartilage restoration after IL-1 induced proteoglycan depletion.

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-1β 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 (106 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.

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 β (TGFβ) alone or in combination. Methods: The interaction of IL1 and TGFβ was studied in cartilage of young (three months) and old mice (18 months) both in vivo and in vitro. Therefore, IL1, TGFβ, or IL1 together with TGFβ 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, TGFβ, or IL1 together with TGFβ 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, TGFβ stimulated PG synthesis and reduced NO production in both age groups. Importantly, TGFβ 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 TGFβ. This loss of responsiveness to the pivotal cytokine TGFβ on effects of IL1 can be important in the initiation and progression of osteoarthritis (OA).

Adenoviral overexpression of Smad-7 and Smad-6 differentially regulates TGF-β-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.

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

Abstract.Objective: The balance between anti-inflammatory (e.g. TGFβ) and proinflammatory cytokines (e.g. IL-1 and TNFα), regulates destructive processes in OA cartilage. IL-1 and TNFα 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 TGFβ 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-κB) sensitive and dexamethasone insensitive expression of iNOS. TGFβ 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 + TGFβ 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 + TGFβ than in cells treated with IL-1 alone.Conclusion: TGFβ suppressed IL-1-induced iNOS expression and NO production in chondrocytes, probably by enhancing iNOS protein degradation. This finding suggests an additional mechanism for TGFβ to counteract the destructive effects of IL-1 in OA.

Direct effect of murine rIL-1 on cartilage metabolismin vivo

The finding of elevated levels of IL-I in synovial fluid from patients with various types of joint diseases, was linked to joint inflammation [1]. In vitro observations on cartilage destruction by IL-1 [2] and its involvement in inflammatory processes [3], suggest that IL-I was the responsible mediator of joint pathology. The main purpose of this study was to elucidate the direct effect of IL-I on patellar cartilage metabolism but also to keep in mind its inflammatory potential in the murine knee joint. Murine recombinant IL-1 was injected intraarticularly into the murine knee joint. A possible relationship between cartilage destruction and inflammation due to IL-1 was investigated.

Role of Growth Factors and Cartilage Repair

Osteoarthritis (OA) is characterized by focal lesions of the articular cartilage, and concomitant hypertrophic reactions in the bone compartment. The latter includes sclerosis in the subchondral bone and new bone formation at the joint margins, the so-called osteophytes. The overall picture resembles a failure in attempt at repair. It is still debated whether the initiating process in human OA originates from changes in the underlying bone, with the articular cartilage trying to cope with this condition, or that the initial failure is in the cartilage itself. Given the heterogeneous character of the ill defined condition of human OA it might well be that both options do apply. It is long recognized that OA may occur as a consequence of multiple causes, ranging from blunt joint trauma, biomechanical overloading and inborn or acquired joint incongruency, to genetic defects in matrix components, inappropriate matrix assembly, or an imbalance of synovial and cartilaginous homeostasis. Probably the joint has only a limited capacity to react to various insults and, in fact, the osteoarthritic lesion does reflect a common endpoint. However, it seems likely that the mediators involved in the process may be different in the initial stages of the various forms.