IL-1beta regulates FHL2 and other cytoskeleton-related genes in human chondrocytes

Abstract

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-1beta on components of the cytoskeleton in OA chondrocytes on gene expression level. Using a whole genome array, we found that IL-1beta 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-1beta. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 counteracted the influence of IL-1beta 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-1beta induced changes in the morphology of chondrocytes, the organization of the cytoskeleton, and the cellular distribution of FHL2. We conclude that L-1beta 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.

Figure 1

Preservation of the chondrogenic phenotype during the experiment. RT-PCR analysis for Collagen type II (COLII), Aggrecan (ACAN), and COMP is shown at different time points with GAPDH as housekeeping gene. M: Marker; A: directly after enzymatic preparation, passage 0 (P0); B: 1 day of monolayer culture (P0) prior to cryoconservation; C: 3 days of cultivation after cryoconservation (P1); D: 5 days of cultivation after cryoconservation (P1); E: 6 days of cultivation after cryoconservation (P1). The experiment was performed with three different donors and a representative example is shown.

Figure 2

Influence of cell treatment on gene expression. Quantitative real-time PCR analysis for levels of mRNA expression of cytoskeleton-related genes in IL-1β-stimulated chondrocytes. Values are the log₁₀-ratios of A: IL-1β stimulated to control gene expression levels and B: p38 MAPK-inhibited and IL-1β stimulated to IL-1β stimulated gene expression levels without inhibition. Six donors were analyzed. The stimulation times of 4 h and 24 h, respectively, are indicated. Significant changes are marked (*: P < 0.05, **: P < 0.001).

Figure 3

Influence of IL-1β on cell morphology. Chondrocytes with (B, D) and without (A, C) 24 h IL-1β stimulation (10 ng/mL). A + B: Phase contrast microscopy of chondrocytes grown on cell culture dishes (magnification 200x). C + D: Fluorescence microscopy of chondrocytes grown on Permanox slides and F-actin-stained with FITC-conjugated phalloidin.

Figure 4

Influence of IL-1β on FHL2 protein expression and distribution. A: Western blot analysis from treated and untreated chondrocytes and corresponding analysis of band intensity of four (IL-1β treatment) and two (SB203580 and IL-1β treatment) independent experiments, respectively. B + C: Chondrocytes with (C) and without (B) IL-1β stimulation stained with α FHL2 antibody.