Synergistic Collagenase Expression and Cartilage Collagenolysis Are Phosphatidylinositol 3-Kinase/Akt Signaling-dependent

Tim E. Cawston,Gary J. Litherland,Andrew D. Rowan,Timothy Robson,Rachel L. Lakey,Debra Jones,Craig Dixon,David A. Young

doi:10.1074/jbc.m710136200

Abstract

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has emerged as a major regulator of cellular functions and has been implicated in several pathologies involving remodeling of extracellular matrix (ECM). The end stage of inflammatory joint diseases is characterized by excessive ECM catabolism, and in this study we assess the role of PI3K signaling in the induction of collagenolytic matrix metalloproteinases (MMPs) in human chondrocytes. We used the most potent cytokine stimulus reported to promote cartilage ECM catabolism, namely interleukin-1 (IL-1) in combination with oncostatin M (OSM). Both OSM and IL-6 (in the presence of its soluble receptor), but not IL-1 nor leukemia inhibitory factor, induced Akt phosphorylation in human chondrocytes. Inhibition of PI3K signaling using LY294002 blocked IL-1+OSM-mediated Akt phosphorylation, induction of MMP-1 and MMP-13, and cartilage collagenolysis. To further explore the role of downstream substrates within the PI3K pathway, complementary use of small molecule inhibitors and specific small interfering RNAs demonstrated that the PI3K subunit p110alpha and Akt1 were required for MMP-1 mRNA induction. MMP-13 induction was also reduced by loss of function of these molecules and by a lack of p110delta, 3-phosphoinositide-dependent kinase-1 or Akt3. We therefore propose that the activities of specific elements of the PI3K signaling pathway, including Akt, are necessary for the synergistic induction of MMP-1 and MMP-13 and the cartilage breakdown stimulated by IL-1+OSM. Our data provide new insight into the mechanism of synergy between IL-1 and OSM and highlight new therapeutic targets for inflammatory joint diseases that aim to repress the expression of collagenases.

Highlights

Inflammation is a major characteristic of joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA)
Inflammatory mediators released by infiltrating immune cells as well as resident joint cells induce alterations in gene expression that can lead to extracellular matrix (ECM) degradaarthritis; OA, osteoarthritis; IL, interleukin; TNF␣, tumor necrosis factor ␣; OSM, oncostatin M; matrix metalloproteinases (MMPs), matrix metalloproteinase; STAT, signal transducers and activators of transcription; AP-1, activator protein-1; LIF, leukemia inhibitory factor; TIMP, tissue inhibitor of metalloproteinases; sIL-6R, soluble IL-6 receptor; LDH, lactate dehydrogenase; PDK1, phosphoinositidedependent kinase-1; RT, reverse transcription; ANOVA, analysis of variance
IL-6-family cytokines are known to be present in the rheumatoid joint [47, 60], as are elevated levels of both MMP-1 and MMP-13 [61], and our data strongly indicate a role for OSM-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling in the synergistic induction of these collagenase genes

Summary

Introduction

Inflammation is a major characteristic of joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). Because we failed to detect p110␥ transcripts in human chondrocytes, and AS-604850 did not affect Akt phosphorylation or collagenase induction at p110␥-selective concentrations, we chose the ␣, ␤, and ␦ isoforms for silencing, which was both effective (Ն75% mRNA reinduced IL-6 production [53] was seen in the presence of duction) and selective (data not shown).

Results

Conclusion