Biochemical and proteomic characterization of alkaptonuric chondrocytes

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin-like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub-populations were identified: cells coming from the black portion of the cartilage were referred to as ‘black’ AKU chondrocytes, while those coming from the white portion were referred to as ‘white’ AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release and levels of pro-inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both ‘white’ and ‘black’ AKU cells. We then undertook a proteomic and redox-proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding and cell organization. An increased post-translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in ‘black’ AKU chondrocytes

IL-1β Regulates FHL2 and Other Cytoskeleton-Related Genes in Human Chondrocytes

In osteoarthritis (OA), cartilage destruction is associated not only with an imbalance of anabolic and catabolic processes but also with alterations of the cytoskeletal organization in chondrocytes, although their pathogenetic origin is largely unknown so far. Therefore, we have studied possible effects of the proinflammatory cytokine IL-1β on components of the cytoskeleton in OA chondrocytes on gene expression level. Using a whole genome array, we found that IL-1β is involved in the regulation of many cytoskeleton-related genes. Apart from well-known cytoskeletal components, the expression and regulation of four genes coding for LIM proteins were shown. These four genes were previously undescribed in the chondrocyte context. Quantitative PCR analysis confirmed significant downregulation of Fhl1, Fhl2, Lasp1, and Pdlim1 as well as Tubb and Vim by IL-1β. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 counteracted the influence of IL-1β on Fhl2 and Tubb expression, indicating partial involvement of this signaling pathway. Downregulation of the LIM-only protein FHL2 was confirmed additionally on the protein level. In agreement with these results, IL-1β induced changes in the morphology of chondrocytes, the organization of the cytoskeleton, and the cellular distribution of FHL2. We conclude that L-1β is involved in the regulation of various cytoskeletal components in human chondrocytes including the multifunctional protein FHL2. This might be relevant for the pathogenesis of OA