We have shown here that mithramycin downregulates basal and proinflammatory cytokine-stimulated MMP-3 and MMP-13 gene expression in chondrocytes and cartilage. This inhibition might be via multiple mechanisms. Sp1 is a ubiquitous transcription factor generally associated with the constitutive expression of genes. However, serum and growth-promoting conditions can stimulate its phosphorylation at specific carboxy-terminal serine residues and can affect the expression of several genes [
15,
20,
26]. Mithramycin is a GC-specific DNA-binding drug, which prevents the binding of Sp1 to its cognate DNA [
19]. MMP-3 and MMP-13 induction by the three major inflammatory cytokines and inhibition by mithramycin imply that interference with Sp1 binding might be one of the possible mechanisms. The putative Sp1 site in the MMP-13 promoter [
16] might be the target of mithramycin. Because no obvious Sp1-binding site has been found in the MMP-3 promoter [
13], the mechanism of MMP-3 inhibition is not known. Suppression by mithramycin might also involve indirect mechanisms. These could include blocking the transcription of other Sp1-responsive MMP regulatory genes such as
ets-1, which has Sp1-binding sites in its promoter [
27]. Analogously to our results, a requirement for Sp1 activity was demonstrated for the induction of monocyte chemoattractant protein-1 (MCP-1) by TNF-α, and a possible interaction between Sp1 and NF-κB was suggested [
28]. Another possibility is that TNF-α-induced c-Jun (a component of AP-1) might superactivate Sp1, and their physical and functional interaction [
29] might upregulate MMP promoters. An interaction of Sp1 and c-Jun has also been observed in the gene encoding atrial natriuretic factor [
30]. ERK2 was shown to phosphorylate Sp1 [
31]. IL-1 can increase the phosphorylation and activity of Sp1 in synovial fibroblasts [
32]. However, in our experience, mithramycin had no effect on the IL-1-induced activation of ERK1/2, p38 and JNK MAPKs. Further, a calcium-influx-reducing agent (bis-(
o-aminophenoxy)ethane-
N,
N,
N',
N' -tetra-acetic acid acetoxymethyl ester (BAPTA-AM)) did not mimic the inhibition of MMP expression by mithramycin (results not shown). Thus, inhibition by mithramycin does not seem to involve MAPKs or a decrease in calcium concentration. Mithramycin might work through the aforementioned mechanisms or by interfering with Sp1/AP-1, ets-1/Sp1 and Sp1/NF-κB interactions, which are important regulators of MMPs. These hypotheses will be tested in future.
The inhibition of MMP gene expression by mithramycin is not unique to this antibiotic. Interestingly, a tetracycline analogue, doxycycline, downregulated the TNF-α-induced expression of MMP-13 RNA in human chondrocytes [
33]. Similarly, tetracycline also reduced the IL-1-induced accumulation of stromelysin mRNA [
34] as well as that of MMP-1 and MMP-3 in bovine chondrocytes [
35]. Subsequent studies revealed that inhibition occurred by decreasing IL-1 and increasing transforming growth factor-β and its receptors, which could downregulate MMP gene expression [
36]. It is not known whether mithramycin works through similar mechanisms. Mithramycin also has an interesting property of blocking bone resorption [
18], which could be through the suppression of MMP gene expression. Indeed, osteoblast-derived interstitial collagenase initiates bone resorption by the generation of collagen fragments, which in turn activate bone-resorbing osteoclasts [
37]. Thus, the ability of mithramycin to block the resorption of bone and cartilage (as implied here) can be advantageous in treating arthritis, in which both tissues are damaged by MMPs. Alternatively, it might work through multiple mechanisms attributed to bisphosphonates, which also prevent cartilage and bone loss and might have utility in treating arthritis [
38,
39]. Mithramycin is known to have several side effects in patients, including bleeding in the stomach [
17], so its benefits in arthritis
in vivo are questionable, requiring the development of safer and more specific analogues.