The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin‐1 and oncostatin M: A time‐course study using real‐time quantitative reverse transcription–polymerase chain reaction

Objective

Previous studies have reported elevated levels of interleukin‐1 (IL‐1) and oncostatin M (OSM) in rheumatoid joints, as well as the synergistic degradation of human articular cartilage by this cytokine combination. The present study was undertaken to investigate the ability of IL‐1 and OSM to modulate gene expression of matrix metalloproteinase (MMP), ADAM, and ADAM‐TS (ADAM with thrombospondin motifs) family members in human chondrocytes.

Methods

T/C28a4 human chondrocytes were stimulated for 2–48 hours with IL‐1 and/or OSM. Total RNA was harvested, reverse transcribed, and assessed by real‐time polymerase chain reaction for the expression of various MMP, ADAM, and ADAM‐TS messenger RNAs (mRNA). Results were normalized to 18S ribosomal RNA.

Results

IL‐1 and OSM synergized to markedly induce the expression of the collagenases MMP‐1, MMP‐8, and MMP‐13 as well as MMP‐3, an activator of proMMPs. Expression of mRNA for MMPs 1, 3, and 13 was induced early, whereas that of MMP‐8 mRNA occurred late. Gene expression of MMP‐14, an MMP that degrades collagen and activates proMMP‐13, was elevated by this combination. IL‐1 and OSM also synergized to induce gene expression of the aggrecanase ADAM‐TS4, but not ADAM‐TS5.

Conclusion

These data indicate that the potent cartilage‐degrading properties of the combination of IL‐1 and OSM are potentially mediated by a synergistic induction of the aggrecan‐degrading enzyme ADAM‐TS4 and the collagen‐degrading enzymes MMP‐1, MMP‐8, MMP‐13, and MMP‐14, although differences in the magnitude of response and in the time course of induction were observed. A role for MMPs 3 and 14 in the activation of proMMPs may also be implicated.

     

Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of phosphatidylinositol 3-kinase and S6K1 kinase

Phosphorylation of insulin receptor substrate-1 (IRS-1) on serine residues has been recognized as a mechanism responsible for a diminution of insulin action and insulin resistance. Potential approaches to improve insulin sensitivity may include interference with and/or reduction in expression of certain signaling intermediates that participate in the pathogenesis of insulin resistance. In this study, we transduced fully differentiated 3T3-L1 adipocytes with a constitutively active myristoylated Akt that led to hyperactivation of mammalian target of rapamycin and p70 S6 kinase (S6K1), increased serine phosphorylation of IRS-1, and reduction in insulin-stimulated phosphatidylinositol (PI) 3-kinase activity and glucose transport. We then reduced expression of the PI 3-kinase regulatory subunit, p85alpha, or expression of S6K1 kinase using small interfering RNA transfections, which led to a reduction in p85alpha expression of 70% at 48 h (P < 0.05) and S6K1 of 49% (P < 0.05). Reduction in expression of either p85alpha or S6K1 achieved with small interfering RNA in the presence of myristoylated Akt rescued 3T3-L1 adipocytes from the insulin resistance induced by serine phosphorylation of IRS-1 and completely restored insulin-stimulated activation of PI 3-kinase and glucose uptake. We conclude that reduction in expression of p85alpha or S6K1 could represent therapeutic targets to mitigate insulin resistance.     

Interleukin‐17 receptor deficiency results in impaired synovial expression of interleukin‐1 and matrix metalloproteinases 3, 9, and 13 and prevents cartilage destruction during chronic reactivated streptococcal cell wall–induced arthritis

Objective

To examine the role of interleukin‐17 receptor (IL‐17R) signaling in cartilage destruction and its interrelationship with synovial IL‐1 expression during chronic reactivated streptococcal cell wall (SCW)–induced arthritis.

Methods

SCW arthritis was repeatedly induced in wild‐type (WT) and IL‐17R–deficient (IL‐17R^–/–) mice. At different time points, joint inflammation was assessed by using calipers to measure joint swelling. On day 42, mice were killed, and knee joints were removed for histologic analysis. Quantitative polymerase chain reaction (PCR) analyses for different proinflammatory mediators and matrix metalloproteinases (MMPs) were performed on inflamed synovium from WT and IL‐17R^–/– mice after 5 repeated injections of SCW fragments.

Results

IL‐17R signaling did not play a significant role in acute joint swelling induced by a single injection of SCW fragments directly into the joint. However, repeated local injections of SCW fragments into the knee joints of IL‐17R^–/– mice resulted in fewer infiltrating cells in the joint compared with WT mice. Moreover, histologic analysis on day 42 revealed a significant suppression of the degree of chondrocyte death and an absence of cartilage surface erosion in IL‐17R^–/– mice. Quantitative PCR analysis revealed impaired synovial expression of IL‐1, IL‐6, cyclooxygenase 2, stromelysin (MMP‐3), gelatinase B (MMP‐9), and collagenase 3 (MMP‐13) in IL‐17R^–/– mice.

Conclusion

These data show a critical role of IL‐17R signaling in driving the synovial expression of proinflammatory and catabolic mediators, such as IL‐1 and different MMPs, during progression from an acute, macrophage‐driven joint inflammation to a chronic, cartilage‐destructive, T cell–mediated synovitis. Prevention of IL‐17R signaling warrants consideration as a therapeutic target in chronic destructive arthritis.