There is accumulating evidence that ROS serve as signaling intermediates in a number of intracellular pathways initiated through a variety of receptors including growth factor, cytokine, and integrin receptors [2
]. In the present study, we focused on the chondrocyte α5β1 integrin pathway which is stimulated in response to FN-f and results in increased MMP production. We found that ROS levels in the cell were rapidly increased after exposure to FN-f and that inhibition of ROS with anti-oxidants inhibited FN-f stimulation of MAP kinase phosphorylation as well as phosphorylation of the p65 subunit of NFκB. Importantly, the signaling inhibition was associated with an inhibition of NFκB promoter activity and MMP production, in particular the production of MMP-13.
It is useful to view ROS on a sliding scale – lower levels are required for normal cellular homeostasis while higher levels can participate in pathological conditions [2
]. After exposure to various stimuli, chondrocytes have been shown to produce a variety of ROS including NO, superoxide, H2
, and peroxynitrite (ONOO−
), the latter which can form by the reaction of NO with superoxide [30
]. In the present study, we did not directly identify which specific ROS was produced in response to FN-f stimulation of α5β1 but the results with the inhibitors suggest that the species mediating the observed signaling events and the increase in MMP production was likely H2
. Of the inhibitors we have tested in the present study, MnTBAP was most effective in blocking signaling and blocking the increase in MMPs. MnTBAP is not only an SOD mimetic and peroxynitrite scavenger [24
], it also possess catalase activity and so converts H2
to water [34
]. Because MnTBAP scavenges ONOO−
, we cannot rule out the possible contribution of ONOO−
, formed by NO reacting with O2−
, but against this are the findings of a lack of significant inhibition by NO synthase inhibitors and the finding that overexpression of catalase or glutathione peroxidase was more effective than MnSOD overexpression at inhibiting FN-f induced MMP-13 production.
The present results are consistent with previous work in synovial fibroblasts which demonstrated a requirement for ROS production during α5β1 stimulation of MMP-1 production [12
] and a study in bovine cartilage explants where N- acetylcysteine (NAC) was shown to block MMP-3 production induced by the 29kD FN-f [17
]. However, since we only studied the 110kD FN-f, we do not know if the requirement for ROS in the signaling induced by the 29kD FN-f follows a similar mechanism. But similar to our findings in chondrocytes, the studies in synovial fibroblasts found ROS were required for activation of NFκB and the increase in MMP-1 was inhibited with NAC but not with NO synthase inhibitors.
However, in synovial fibroblasts the increase in MMP-1 also required expression and release of IL-1 which acted in an autocrine manner to stimulate MMP-1 production while we have shown IL-1 is not required for FN-f stimulation of MMP-13 or cytokine production by chondrocytes [13
]. Another difference between α5β1 signaling in synovial fibroblasts and in chondrocytes is that rotenone, which inhibits mitochondrial ROS production by blocking complex I in the electron transport chain, completely inhibited MMP-1 production in fibroblasts [35
] while in chondrocytes rotenone showed partial but not complete inhibition. Also, in chondrocytes Tiron was less effective than NAC or MnTBAP. These findings suggest differences in sources and types of ROS in chondrocytes in response to integrin signaling compared to fibroblasts or differences in their matrices and/or matrix receptors that affect ROS signaling. Partial inhibition by NDGA in chondrocytes indicates that 5-lipoxygenase may be another source of ROS which would be consistent with integrin signaling studies in NIH-3T3 fibroblasts where ROS production was blocked by NDGA but not rotenone [29
]. However, because of the lack of specificity of NDGA these results should be interpreted with caution. It is also not clear why ETYA was effective at inhibiting FN-f induced JNK and p65 phosphorylation but did not appear to reduce MMP-13 production. Together, the findings in different cell types indicate that there are likely multiple pathways for ROS generation downstream from integrin activation.
Regulation of MAP kinase activation has been shown to be redox-sensitive and we found that anti-oxidants blocked FN-f stimulated phosphorylation of all 3 MAP kinases with JNK phosphorylation being particularly sensitive. ROS have been shown to activate JNK through the oxidative inactivation of endogenous JNK inhibitors such as the JNK phosphatases [36
]. Treatment of chondrocytes with MnTBAP appeared to cause a modest increase in ERK phosphorylation in the absence of FN-f and rotenone had much less inhibitory affect on ERK phosphorylation as it did on JNK and p38. These findings suggest redox regulation of the ERK pathway may be different than JNK and p38.
Consistent with our previous results using chemical MAP kinase inhibitors [13
] and overexpression of dominant negative MAP kinase constructs [14
], we found that anti-oxidant inhibition of MAP kinase phosphorylation was associated with inhibition of MMP-13 production. Using an MMP array, we also found that the anti-oxidant MnTBAP inhibited FN-f stimulation of MMP-1 and MMP-10 as well. TIMP-1 levels in the media were also increased by FN-f and were reduced to basal control levels by MnTBAP whereas TIMP-2 did not change with MnTBAP and/or FN-f. The MMP-13 ELISA assay results demonstrated that FN-f was able to stimulate production of active MMP-13, which was consistent with our previous work [13
], and MnTBAP inhibited production of both total and active enzyme.
MMP-13 is increased in OA cartilage and is thought to play a key role in the degradation of type II collagen [37
]. For this reason targeting signaling pathways which regulate MMP-13 expression would represent a potential therapeutic approach for slowing or stopping cartilage destruction in arthritis. The results from the present study suggest that specific anti-oxidants may be useful in this regard. Because of additional evidence that excessive ROS levels may contribute to cartilage loss by other mechanisms as well [3
], further studies of these compounds for their ability to block cartilage destruction are warranted.