Abstract
IntroductionInsulin-like growth factor I (IGF-I) regulates articular cartilage homeostasis. During osteoarthritis (OA), the anabolic responses of chondrocytes to IGF-I are likely to be prevented by the enhanced production of IGF-binding proteins (IGFBPs), especially IGFBP-3. The aim of this study is to evaluate whether the architectural transcription factor high mobility group A1 (HMGA1) influences IGFBP-3 overexpression in vitro, in cultured chondrocytic cell lines, and ex vivo, in human osteoarthritic cartilage compared to healthy human cartilage controls.MethodsQuantitative real-time reverse transcription-PCR (qRT-PCR) was performed to assess the relative transcript levels of HMGA1 and IGFBP-3 in vitro, in the human chondrocytic cell lines T/C-28a4 and C-28/I2. An electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and transient transfection assays were performed to investigate the HMGA1-IGFBP-3 gene interaction. Samples of articular cartilage were harvested from osteoarthritic patients and controls and analyzed by qRT-PCR for HMGA1 and IGFBP-3 mRNA levels.ResultsA parallelism between HMGA1 protein levels and IGFBP-3 gene expression has been observed in T/C-28a4 and C-28/I2 cells. The interaction of HMGA1 with the IGFBP-3 gene promoter has been demonstrated by EMSA and ChIP. In transient transfections, IGFBP-3 promoter activity increased in cells overexpressing HMGA1 and decreased in cells pretreated with siRNA detected against HMGA1. IGFBP-3 mRNA expression was higher in cartilage from patients with OA, where the increased expression of IGFBP-3 closely paralleled the increased expression of HMGA1 mRNA.ConclusionsOur observations indicate that increased HMGA1 expression in human chondrocytes is associated with increased expression of IGFBP-3. It is tempting to speculate that, through the regulation of IGFBP3 expression, HMGA1 may act as a pathogenetic factor for OA.
Highlights
Insulin-like growth factor I (IGF-I) regulates articular cartilage homeostasis
high mobility group A1 (HMGA1) and insulin-like growth factor binding protein 3 (IGFBP-3) expression in T/C-28a4 and C-28/I2 cells As measured by qRT-polymerase chain reaction (PCR), IGFbinding proteins (IGFBPs)-3 and HMGA1 mRNA expression was observed in both T/C-28a4 and C-28/I2 cultured cell lines (Figure 1a)
To examine further the role of HMGA1 in this event, we carried out experiments in C-28/I2 cells treated with small interfering RNA (siRNA) directed against HMGA1
Summary
Insulin-like growth factor I (IGF-I) regulates articular cartilage homeostasis. During osteoarthritis (OA), the anabolic responses of chondrocytes to IGF-I are likely to be prevented by the enhanced production of IGFbinding proteins (IGFBPs), especially IGFBP-3. By interacting with AT-rich DNA binding sequences, HMGA1 is able to modify the structure of its target site and induce structural changes in the DNA to facilitate the progression of a wide range of DNA-dependent activities [2] This interaction enables HMGA1 to recruit additional transcription factors and alter chromatin structure, leading to the formation of higher-order transcriptional complexes called enhanceosomes [3,4,5]. In concert with other factors, HMGA1 modulates gene expression and determines whether the associated genes are switched on or off [6] Through such a mechanism, the transcription of cyclooxygenase-2, insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), and the insulin receptor is regulated by HMGA1 [4,5,7,8]. During osteoarthritis (OA), normal or increased amounts of IGF-I are produced, but osteoarthritic chondrocytes are hyporesponsive to IGF-I; this property has been attributed to increased IGFBP levels that may interfere with IGF-I functionality [16,17,18]
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