IGF-1 does not moderate the time-dependent transcriptional patterns of key homeostatic genes induced by sustained compression of bovine cartilage

Abstract

Objective To determine changes in chondrocyte transcription of a range of anabolic, catabolic and signaling genes following simultaneous treatment of cartilage with IGF-1 and ramp-and-hold mechanical compression, and compare with effects on biosynthesis. Methods Explant disks of bovine calf cartilage were slowly compressed (unconfined) over 3-min to their 1mm cut-thickness (0%-compression) or to 50%-compression with or without 300 ng/ml IGF-1. Expression of 24 genes involved in cartilage homeostasis was measured using qPCR at 2, 8, 24, 32, 48 hours after compression ± IGF-1. Clustering analysis was used to identify groups of co-expressed genes to further elucidate mechanistic pathways. Results IGF-1 alone stimulated gene expression of aggrecan and collagen II, but simultaneous 24-hour compression suppressed this effect. Compression alone upregulated expression of MMP-3, MMP-13, ADAMTS-5 and TGF-β, an effect not reversed by simultaneous IGF-1 treatment. Temporal changes in expression following IGF-1 treatment were generally slower than that following compression. Clustering analysis revealed five distinct groups within which the pairings, TIMP-3 and ADAMTS-5, MMP-1 and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed, suggesting inherent regulation and feedback in chondrocyte gene expression. While aggrecan synthesis was transcriptionally regulated by IGF-1, inhibition of aggrecan synthesis by sustained compression appeared post-transcriptionally regulated. Conclusion Sustained compression markedly altered the effects of IGF-1 on expression of genes involved in cartilage homeostasis, while IGF-1 was largely unable to moderate the transcriptional effects of compression alone. The demonstrated co-expressed gene pairings suggest a balance of anabolic and catabolic activity following simultaneous mechanical and growth factor stimuli.

Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope formation during in vitro chondrogenesis of human mesenchymal stem cells

“…In your discussion you mention that ADAMTSs may be "stored in the ECM": is there any data if or where they may localise? Authors: There is evidence that expression and/or activity, of ADAMTS-4 and 5 can be regulated by mechanical loading in chondrocytes (Tetsunaga et al, 2011;Ding et al, 2010;Wheeler et al, 2009). Whether this is a direct effect through mechanosensitive elements in the upstream of the ADAMTS genes, or is secondary to mechanotransduction of other regulatory proteins such as IL-1, runx2, NF-kB or CITED2 (Leong et al, 2011) remains to be elucidated.…”

Effects of insulin-like growth factor-1 and dexamethasone on cytokine-challenged cartilage: relevance to post-traumatic osteoarthritis

“…Expression data for each gene were calculated from the threshold cycle (Ct) value, and normalized to the internal housekeeping gene 18S. (Our previous studies explored several housekeeping genes and normalization methods and, in the system used here, in the absence of impact mechanical injury to cartilage, normalization to 18s and G3PDH gave similar results 29,30 .…”

“…Using methods described in detail previously 29,30 , the 6 disks from each condition were pooled, pulverized, and homogenized in TRIzol reagent, and then separated using phase-gel tubes (Eppendorf, Hamburg, Germany). The supernatant was purified following the Qiagen RNeasy mini kit protocol (Qiagen, Chatsworth, CA).…”

“…Equal amounts of mRNA from each condition were reverse transcribed using the AmpliTaq-Gold Reverse Transcription kit (Applied Biosystems, Foster City, CA). Primer pairs used were previously reported 29,30 except for caspase-3: forward 5’-GAAGTCTGACTGGAAAACCC-3’, reverse 5’- GAAGTCTGCCTCAACTGGTA-3’. Real-time PCR were performed using the Applied Biosystems 7700HT instrument with SYBR Green Master Mix (Applied Biosystems).…”

Moderate dynamic compression inhibits pro-catabolic response of cartilage to mechanical injury, tumor necrosis factor-α and interleukin-6, but accentuates degradation above a strain threshold

“…On the extraction day, the 6 disks from each condition were pooled, pulverized, and lysed in TRIzol reagent (Invitrogen, Carlsbad, CA) with a homogenizer, as previously described in detail [28, 37]. The extract was then separated using phase-gel tubes (Eppendorf, Hamburg, Germany), and the supernatant was purified following the Qiagen RNeasy mini kit protocol (Qiagen, Chatsworth, CA).…”