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To whom correspondence should be addressed: Connective Tissue Biology Laboratories, Cardiff School of Biosciences, Cardiff University, Museum Ave., Biomedical Sciences Bldg., Cardiff CF10 3US, Wales, UK.
* This work was funded by the Arthritis Research Campaign, United Kingdom.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF192770 and AF192771. ‡ Arthritis Research Campaign Postdoctoral Research Fellow. § Arthritis Research Campaign Postdoctoral Research Fellow.
This study describes specific molecular mechanisms by which supplementation with n-3 fatty acids (i.e. those present in fish oils) can modulate the expression and activity of degradative and inflammatory factors that cause cartilage destruction during arthritis. Our data show that incorporation of n-3 fatty acids (but not other polyunsaturated or saturated fatty acids) into articular cartilage chondrocyte membranes results in a dose-dependent reduction in: (i) the expression and activity of proteoglycan degrading enzymes (aggrecanases) and (ii) the expression of inflammation-inducible cytokines (interleukin (IL)-1α and tumor necrosis factor (TNF)-α) and cyclooxygenase (COX-2), but not the constitutively expressed cyclooxygenase COX-1. These findings provide evidence thatn-3 fatty acid supplementation can specifically affect regulatory mechanisms involved in chondrocyte gene transcription and thus further advocate a beneficial role for dietary fish oil supplementation in alleviation of several of the physiological parameters that cause and propogate arthritic disease.
tumor necrosis factor
reverse transcription-polymerase chain reaction
Noninvasive, pharmaceutical-based therapies for the treatment of arthritic diseases are primarily limited to oral administration of nonsteroidal antiinflammatory drugs, which inhibit cyclooxygenase (COX)1-mediated production of inflammatory eicosanoids such as prostaglandins (
). Parenthetically, clinical studies on dietary supplementation withn-3 (omega-3) fatty acids (the principle long chain polyunsaturated fatty acids found in fish oils) have also demonstrated modulation of inflammatory symptoms involved in the pathogenesis of arthritis (
). Such epidemiological observations have been largely anecdotal, because they did not investigate the molecular mechanisms whereby dietary n-3 fatty acid supplementation might affect the metabolism of cells within articular joint tissues and thereby provide relief to arthritic symptoms. Significantly, however, dietary supplementation with n-3 fatty acids elicits antiinflammatory effects in neutrophils and monocytes by inhibiting the 5-lipoxygenase pathway responsible for metabolism of arachidonic acid to leukotrienes (
One of the key pathological features common to degenerative joint diseases (arthritis) is the loss of cartilage proteoglycan (aggrecan), which precedes subsequent cartilage erosion. Catabolism of aggrecan is mediated by the proteolytic activity of aggrecanases (
). Aggrecanase activity is up-regulated by cartilage exposure to pro-inflammatory cytokines such as IL-1 and TNF-α, and model cartilage explant and chondrocyte culture systems stimulated with IL-1 or TNF-α mimic the degradative processes involving aggrecan catabolism which occur during arthritis (
). In addition, exposure to these inflammatory mediators propogates the autocrine synthesis of cartilage cytokines, which contribute to the chronic progression of arthritis. Furthermore, cytokine-induced degradative activities in synovial joint tissues can be potentiated via the biosynthesis of inflammatory eicosanoids by the cyclooxygenases COX-1 and COX-2 (
). COX-1, which is constitutively expressed in most tissues, is responsible for key aspects of eicosanoid biosynthesis, which are important in maintaining homeostasis during normal cellular metabolism (
To determine a molecular basis for potential therapeutic properties associated with dietary intake of fish oils, we investigated the effects of different classes of fatty acids on the expression and activity of cartilage aggrecanases, cytokines (IL-1α and TNF-α) and cyclooxygenases (COX-1 and COX-2). The results of these in vitro studies reveal that exposure of articular chondrocytes ton-3 fatty acids can specifically modulate, at the level of gene transcription, key factors involved in articular cartilage degradation.
RESULTS AND DISCUSSION
In this study, articular cartilage chondrocytes were exposed in culture to fatty acids at concentrations which cover the typical range (50–70 μg/ml) for free fatty acid levels in human plasma (
). The results for measurements of the lipid content of chondrocytes supplemented without or with a polyunsaturated n-3 (α18:3 linolenate) or a saturated (16:0 palmitate) fatty acid are shown in Table I. Exposure to the n-3 fatty acid markedly changed the overall lipid composition profile of the chondrocyte with major changes occurring in the supplemented fatty acid (e.g. α18:3 linolenate), with a concomitant reduction in other polyunsaturated fatty acids. Similarly, supplementation with 16:0 palmitate markedly altered the level of this fatty acid in the chondrocyte lipid composition profile, again at the expense of polyunsaturated fatty acid components. Replicate cultures, with or without prior fatty acid supplementation, were then cultured for a further 96 h in the absence or presence of IL-1α. Proteoglycan synthesis, as measured by [35S]sulfate incorporation, was decreased in chondrocyte cultures treated with IL-1α compared with controls as has been reported previously (
). However, prior addition of fatty acids to the cultures had no effect on these changes in proteoglycan synthesis or in cell numbers, DNA content or morphology, nor in levels of lactate secreted into the medium (results not shown), thus demonstrating that fatty acid supplementation was not toxic to the chondrocytes. Furthermore, unaltered expression of mRNAs for aggrecan and collagen type II confirmed maintenance of the chondrocyte phenotype in the experimental cultures (results not shown).
Table IFatty acid analysis of chondrocyte membranes from cultures treated with (+) or without (−) 100 μg/ml n-3 polyunsaturated or saturated fatty acids
The effect of fatty acid supplementation on chondrocyte aggrecanase activity was then investigated (Fig. 1). There was no evidence for aggrecanase activity in conditioned medium from control cultures supplemented with either n-3 (Fig.1A, lanes 1 and 2) or saturated fatty acids (Fig. 1B, lanes 1 and 2). Exposure to IL-1, as expected (
), did induce aggrecanase activity in these conditioned media (Fig. 1, A and B,lane 3, respectively). However, addition of then-3 fatty acid α18:3 linolenate abolished this IL-1-induced aggrecanase activity in a dose-dependent manner (Fig. 1A, lanes 4–6). In contrast, supplementation with 16:0 palmitate had no effect on aggrecanase activity (Fig. 1B, lanes 4–6). In replicate studies, no evidence for matrix metalloproteinase-mediated aggrecan cleavage was observed for cultures maintained in the absence or presence of either n-3 or saturated fatty acids (results not shown). In keeping with the observed loss of aggrecanase-mediated proteolytic activity in IL-1-treated chondrocyte conditioned medium, there was also a decrease in the levels of mRNA transcripts for aggrecanase-1 and aggrecanase-2 in response to n-3 fatty acid supplementation (Fig. 2). Aggrecanase-1 mRNA was detected in IL-1-treated, but not control, cultures, whereas mRNA for aggrecanase-2 was detected both in the presence and absence of IL-1 (Fig. 2, A and B,lanes 1 and 2). However, addition of then-3 fatty acid α18:3 linolenate caused a decrease in both aggrecanase-1 and aggrecanase-2 transcript levels (Fig. 2A,lanes 3–5). In contrast, cultures supplemented with 16:0 palmitate expressed aggrecanase-1 and aggrecanase-2 mRNAs at all concentrations of fatty acid tested (Fig. 2B, lanes 3–5). The addition of the vehicle for fatty acid supplementation (fatty acid-free albumin) had no effect on either aggrecanase-1 or -2 expression in the control or IL-1-treated cultures (results not shown). Expression of GAPDH was used to normalize the amount of mRNA present in all samples.
We next examined the effect of supplementation with n-3 fatty acids versus other fatty acids on COX-1 and COX-2 mRNA expression and on the expression of IL-1α and TNF-α mRNAs in chondrocyte cultures exposed to exogenous IL-1 (Fig.3). Chondrocyte COX-1 mRNA was present in all culture systems, with or without fatty acid supplementation (Fig. 3A, lanes 1–5). However, chondrocyte COX-2 expression was detected in IL-1-treated, but not control, cultures (Fig. 3A, lanes 1 and2). Significantly, COX-2 mRNA expression in IL-1-treated cultures was decreased by n-3 fatty acid (α18:3 linolenate) supplementation (Fig. 3A, lanes 3–5). In contrast, addition of a saturated fatty acid (16:0 palmitate) had no such effect on IL-1-induced COX-2 expression (data not shown). Examination of chondrocyte-derived cytokine expression revealed that the mRNAs for IL-1α and TNF-α were absent in control cultures (Fig. 3B, lane 1), but these were detected following exposure to exogenous IL-1α (Fig.3B, lane 2). However, expression of chondrocyte IL-1α and TNF-α mRNAs was decreased in n-3 fatty acid-treated cultures (Fig. 3B, lanes 3–5), but not in cultures supplemented with saturated fatty acids (data not shown).
Identical results to those shown in Figs. Figure 1, Figure 2, Figure 3 were obtained when two other n-3 fatty acids found in fish oils (eicosapentaenoic acid and docosahexaenoic acid) were added to the culture systems, and no effects were observed when either an n-6 fatty acid (linoleic acid) or a monounsaturated fatty acid (oleic acid) were supplemented (data not shown). These results conclusively indicate that the observed effects on the expression of chondrocyte aggrecanases, COX-2, and autocrine cytokines are specific to supplementation withn-3 (omega-3) fatty acids.
Collectively, these data provide informative novel information on the biological and molecular mechanisms whereby dietary fish oil supplementation can reduce inflammatory and degradative aspects of articular joint disease and thus modulate disease progression. The balance of dietary n-3/n-6 polyunsaturated fatty acids is well known to affect inflammatory responses (
). On the other hand, accumulating evidence suggests thatn-3 fatty acids can regulate gene expression via activation of transcription factors such as peroxisome proliferator-activated receptor-α or by affecting expression levels of sterol regulatory element-binding proteins (
). Whereas such mechanisms remain to be investigated in chondrocytes, our current findings demonstrate thatn-3 fatty acid supplementation can specifically affect molecular mechanisms that regulate the expression of catabolic factors involved in articular cartilage degradation and thus further advocate a beneficial role for dietary fish oils in alleviation of several of the physiological parameters that cause and propogate arthritic disease.