Co-treatment with TNF-α and atRA further reduces expression of extracellular matrix protein genes
We first investigated how expression of extracellular matrix genes responded to TNF-α and atRA. Chondrocytes were treated for 24 hours with TNF-α and/or increasing concentrations of atRA. Following treatment, transcript levels of type II collagen, aggrecan core protein and link protein were determined by Northern blot analysis and/or qPCR (Figure ). TNF-α significantly reduced levels of type II collagen, aggrecan core protein and link protein mRNA. Treatment of cells with atRA reduced mRNA levels of all three matrix genes in a concentration-dependent manner. Interestingly, co-treatment of cells with TNF-α and atRA decreased levels of these transcripts more than each factor alone. These results suggest that signalling from TNF-α and atRA converge to influence the activity of transcription factors, such as Sox9, that are necessary for the expression of cartilage matrix genes.
Figure 1 Effects of TNF-α and atRA on matrix gene expression. Chondrocytes were treated with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or all-trans retinoic acid (atRA; 1, 10, or 100 nmol/l) for 24 hours. Total RNA was evaluated (more ...)
Effects of TNF-α and atRA on Sox9 activity
We investigated the activity of Sox9 using a reporter construct based on the type II collagen minimal enhancer (Figure ). TNF-α significantly reduced Sox9 reporter activity (approximately 47%). Sox9 reporter activity also decreased with increasing concentrations of atRA. At 10-9 mol/l atRA, co-treatment with TNF-α resulted in a further decrease in Sox9 reporter activity, which is consistent with the observed changes in expression of cartilage matrix protein transcripts.
Figure 2 Effects of TNF-α and atRA on Sox9 activity. Chondrocytes were transfected with (a) the type II collagen enhancer luciferase reporter and some cultures were also co-transfected with (b) a phosphorylation site-deficient inhibitor of nuclear factor-κB (more ...)
Previously, we found that regulation of Sox9 activity at the type II collagen enhancer was dependent on NF-κB activation [13
]. To determine whether the effects of TNF-α and atRA were both mediated by NF-κB, we restricted NF-κB nuclear translocation by over-expression of IκB-2N – an IκBα that is resistant to phosphorylations required for NF-κB release (Figure ). IκB-2N did not significantly alter basal Sox9 activity or the reduction of Sox9 activity following atRA treatment alone. In contrast, IκB-2N did eliminate the further reduction observed in the presence of both TNF-α and atRA (10-9
mol/l). These results indicate that reduction of Sox9 activity by atRA is independent of NF-κB activation.
Binding of protein complexes to the Col2a1 48-bp minimal enhancer and nuclear Sox9 levels
Since Sox9 activity was decreased by TNF-α and atRA, we determined whether there were changes in protein complex binding to the Col2a1 48-bp minimal enhancer sequence or alterations in nuclear levels of Sox9. Chondrocytes were treated with TNF-α and nuclear extracts were analyzed by EMSA. TNF-α did not change the amount of protein complex bound to the 48-bp minimal enhancer sequence (Figure ). In addition, TNF-α and atRA (alone or in combination) did not change nuclear levels of Sox9 assessed by immunoblot (Figure ). Taken together, these findings indicate that the observed changes in Sox9 activity are independent of changes in DNA binding or nuclear protein levels.
Figure 3 Effect of TNF-α on Sox9-DNA binding and Sox9 nuclear protein levels. (a) Chondrocytes were treated for 24 hours with or without tumour necrosis factor (TNF)-α (30 ng/ml). Nuclear extracts (10 μg) were incubated with double-stranded (more ...)
atRA reduces NF-κB activity in a concentration-dependent manner
On their own, TNF-α (30 ng/ml) and atRA (10-9 mol/l) reduced Sox-9 activity by about 50%, and together activity was reduced by about 75% (Figure ). Because their effects were not completely additive, we determined whether there are interactions between atRA and TNF-α signalling that influence NF-κB or RAR activity. Chondrocytes were transfected with a κB luciferase reporter construct and were treated with TNF-α and/or atRA (Figure ). As expected, TNF-α induced NF-κB activity. atRA alone had no effect on basal NF-κB activity. However, atRA significantly inhibited TNF-α-activated NF-κB activity, suggesting that active RARs suppress NF-κB activity.
Figure 4 Effect of atRA on NF-κB and RAR activity. Chondrocytes were transfected with (a) a κB reporter or (b) a retinoic acid response element (RARE) reporter and treated with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or (more ...)
We next evaluated whether activation of NF-κB influences RAR function. Chondrocytes were transfected with a RARE reporter and treated with TNF-α and/or atRA (Figure ). As expected, atRA increased RAR activity in a concentration-dependent manner. TNF-α did not change basal or atRA-induced RAR activity. Thus, under these conditions, NF-κB had no inhibitory effect on RAR activity.
atRA inhibits binding of DNA by the TNF-α-activated complex
To define further the inhibitory effect of atRA on NF-κB activity, we examined the effects of atRA on nuclear localization of NF-κB and its affinity for DNA. The presence of NF-κB p65 in nuclear extracts from chondrocytes treated with TNF-α and/or atRA was analyzed by immunoblot (Figure ). TNF-α, but not atRA, induced nuclear localization of the NF-κB p65 isoform (Figure ). Furthermore, in the presence of TNF-α, treatment of cells with atRA did not significantly reduce the amount of nuclear p65. Moreover, no significant changes in nuclear levels of RARα protein were observed (Figure ). Thus, reduction in functional activity of NF-κB was not associated with changes in nuclear levels of NF-κB p65 or RARα.
Figure 5 Effects of TNF-α and atRA on nuclear levels of NF-κB and RARα. Chondrocytes were treated with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or all-trans retinoic acid (atRA; 1, 10, or 100 nmol/l) for 24 hours. (more ...)
We next evaluated the possibility that RAR activation changes the binding of NF-κB to DNA. Nuclear extracts of cells treated with TNF-α and/or atRA were analyzed by EMSA and supershift/antibody interference assays (Figure ). TNF-α, but not atRA, induced formation of a complex bound to the κB consensus site that contained the NF-κB p65 isoform. Interestingly, addition of anti-RARα antibody reduced the TNF-α-activated complex, indicating that RARα is a member of this complex (Figure ; compare lanes 2 and 4, and lanes 10 and 12). Furthermore, atRA decreased the intensity of TNF-α-activated complexes bound to the κB consensus site (Figure ; compare lanes 2 and 8 to 10). When chondrocytes were treated with both TNF-α and atRA, the complex that remained bound to DNA contained p65 and RARα (Figure ; compare lanes 10, 11 and 12). Taken together, we conclude that atRA binding to RARα decreases the affinity for DNA of the p65/RARα complex that is formed in response to TNF-α.
Figure 6 Effect of atRA on NF-κB/DNA binding. Chondrocytes were treated with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or all-trans retinoic acid (atRA; 1, 10, or 100 nmol/l) for 24 hours. Nuclear extracts were incubated with 32 (more ...)
MEKK1 inhibits the effect of atRA on NF-κB functional activity
Changes in transcription factor function can result from alterations in the level or activity of the transcription factor itself or of its required co-factors. Active MEKK1 induces NF-κB nuclear localization by promoting the degradation of IκB [32
]. In addition, MEKK1 phosphorylates p300, increasing its histone acetylase activity [33
]. Thus, the effect of active MEKK1 on NF-κB and RAR activity was investigated.
Chondrocytes were co-transfected with a caMEKK1 expression construct and either κB or RARE reporter constructs (Figure ). caMEKK1 dramatically increased basal NF-κB activity. For example, basal NF-κB activity was enhanced approximately 24-fold in the experiment shown (compare first columns in Figure and Figure from the same representative experiment; P < 0.001). Treatment of caMEKK1-transfected chondrocytes with TNF-α did not further increase NF-κB activity; however, activity levels were still 4-fold greater than those in cells transfected with reporter alone and treated with TNF-α (compare second columns in Figure and Figure ). Interestingly, treatment of caMEKK1-transfected chondrocytes with atRA, alone or in combination with TNF-α, did not reduce NF-κB activity (Figure ). Thus, NF-κB function was maximized by caMEKK1 and was also protected from inhibition by atRA.
Figure 7 Effect of caMEKK1 on NF-κB and RAR activities. Chondrocytes were co-transfected with a constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 expression vector and (a) nuclear factor-κB (NF-κB) or (b) retinoic (more ...)
RAR activity was not altered by expression of caMEKK1 in either the absence or presence of TNF-α (compare first two columns in Figure and Figure ). In contrast, atRA-induced RAR activity was increased approximately 11-fold in caMEKK1-transfected chondrocytes compared with cells transfected with reporter alone (compare Figure and Figure ; P < 0.001). To determine the effect of NF-κB activation by caMEKK1 on RAR function, cells were co-transfected with caMEKK1 and IκB-2N. IκB-2N inhibits NF-κB activation but should not affect other signalling events initiated by caMEKK1. As expected, IκB-2N inhibited NF-κB activity induced by caMEKK1 (Figure ). Surprisingly, IκB-2N dramatically increased atRA-induced RAR activity in MEKK1-transfected cells (Figure ).
In summary, caMEKK1 increases the functional activity of both NF-κB and atRA-induced RARs. Furthermore, in caMEKK1 expressing cells, atRA does not reduce NF-κB function. Finally, inhibition of NF-κB further enhances the effect of caMEKK1 on atRA-induced RAR function. It is likely that the caMEKK1-induced increases in NF-κB and RAR activity are mediated by hyperactivation of p300.
caMEKK1 attenuates TNF-α and atRA-induced decrease in Sox9 activity
To investigate the effect of caMEKK1 expression on Sox9 functional activity, cells were co-transfected with the caMEKK1 expression construct and the Sox9 reporter. Over-expression of caMEKK1 significantly increased basal Sox9 activity compared with cells lacking caMEKK1. A comparison of the non-normalized data from the first columns of Figure and Figure revealed that caMEKK1 increased Sox9 activity by 3.7 ± 0.7 fold (mean ± standard deviation; P < 0.01; data are from the same series of experiments). Moreover, in contrast to the reductions in Sox9 activity observed in cells lacking caMEKK1 (Figure ), TNF-α significantly increased Sox9 activity (Figure ). atRA significantly reduced Sox9 activity in cells expressing caMEKK1, but only at the highest concentration used (Figure ). In summary, caMEKK1 increased Sox9 functional activity, as it did NF-κB and RAR functional activity. caMEKK1 also reversed the inhibitory effect of TNF-α on Sox9 activity. Finally, the sensitivity of Sox9 to atRA was reduced by caMEKK1 expression. Thus, the activity state of p300, the common co-factor and MEKK1 target, probably plays a vital role in regulating Sox9, NF-κB and RAR function.
Figure 8 Effect of caMEKK1 and ectopic p300 on Sox9 activity. (a) Chondrocytes were co-transfected with the Sox9 reporter and constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 expression vector and treated for 24 hours with or without (more ...)
Sox9 functional activity is dependent on availability of p300
We next investigated directly whether availability of p300 contributes to the reduction in Sox9 activity induced by activation of NF-κB and RARs. Cells were co-transfected with the Sox9 reporter and a p300 expression construct. Cells over-expressing p300 exhibited significantly increased Sox9 activity compared with cells transfected with Sox9 reporter alone (Figure ). Ectopic p300 expression did not prevent reductions in Sox9 activity in response to TNF-α and atRA. However, under most conditions, over-expression of p300 maintained Sox9 activity at a level comparable to that observed in normal, untreated chondrocytes. Thus, increasing the availability of p300 increases Sox9 activity even when NF-κB and RARs are active.