Proteome characterization of human articular chondrocytes leads to novel insights in the function of small heat-shock proteins in chondrocyte homeostasis

Abstract

In recent years, studies have been initiated to disclose the proteome of human chondrocytes and cartilage. Despite these studies, comprehensive information of the chondrocyte proteome remains limited. This study aimed to further explore the proteome expressed by human knee chondrocytes, and to study the functional aspects of heat-shock protein 27 (HSP27), a protein related to the previously described alphaBcrystallin, in chondrocyte biology. Chondrocytes isolated from human knee articular cartilage were cultured in a three-dimensional alginate culture system. To simplify the protein mixtures, proteins extracted from chondrocyte cell lysates were fractionated based on hydrophobicity and molecular weight. Proteins were digested and the resulting peptides were separated and identified by an on-line two-dimensional (2-D) nanoliquid chromatography (nanoLC)-system coupled to a quadrupole time-of-flight (Qq-TOF) mass spectrometer. Differential expression analysis of HSP27 was performed by Western Blotting and quantitative polymerase chain reaction (QPCR). The effects of HSP27 on chondrocyte biology were explored by suppression of HSP27 expression induced by RNA interference (RNAi). In this study, we identified proteins with unknown functions together with membrane proteins, transcription factors and other low abundant proteins, which have not yet been described in chondrocytes. Based on previous knowledge on the related protein alphaBcrystallin, we selected HSP27 from the chondrocyte proteome database. Differential expression analysis revealed a decreased expression of HSP27 in Osteoarthritic (OA) chondrocytes. RNAi experiments revealed that HSP27 is involved in interleukin-1beta (IL-1beta) induced IL-6 secretion. These findings highlight that small HSPs, especially HSP27, play a prominent role in the maintenance of human articular chondrocyte homeostasis.