Effects of the mycotoxin nivalenol on bovine articular chondrocyte metabolism in vitro

Abstract

Objective: Kashin-Beck Disease (KBD) is an endemic, age-related degenerative osteoarthropathy and its cause is hypothesised to involve Fusarium mycotoxins. This study investigated the Fusarium mycotoxin Nivalenol (NIV) on the metabolism of bovine articular chondrocytes in vitro.

Design: The effect 0.0-0.5 µg/ml NIV on transcript levels of types I and II collagen, aggrecan, matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) and the tissue inhibitors of MMPs (TIMPs) was investigated using quantitative PCR. Amounts of sulphated glycosaminoglycans, MMPs and TIMPs were assessed using the Dimethylmethylene Blue assay, gelatin zymography and reverse gelatin zymography respectively. Cytoskeletal organisation was analysed using confocal microscopy and cytoskeletal gene and protein levels were measured by quantitative PCR and Western blot analysis, respectively.

Results: NIV caused a dose-dependent increase in aggrecan transcription with a concomitant retention of sGAG in the cell lysate. Furthermore, NIV significantly increased MMPs-2, -3 & -9, ADAMTS-4 and -5, and TIMP-2 and -3 transcript levels but inhibited type I collagen, MMP 1 and TIMP 1 mRNA levels. NIV promoted extensive cytoskeletal network remodelling, particularly with vimentin where a dose-dependent peri-nuclear aggregation occurred.

Conclusion: NIV exposure to chondrocytes decreased matrix deposition, whilst enhancing selective catabolic enzyme production, suggesting its potential for induction of cellular catabolism. This NIV-induced extracellular matrix remodelling may be due to extensive remodelling/disassembly of the cytoskeletal elements. Collectively, these findings support the hypothesis that trichothecene mycotoxins, and in particular NIV, have the potential to induce matrix catabolism and propagate the pathogenesis of KBD.

Figure 1. Nivalenol (NIV) decreases chondrocyte viability in a dose and duration-dependent manner.

Chondrocytes cultured as a high-density monolayer were treated with 0.1 0.2 or 0.5 µg/ml NIV for 1 & 3 days. Cell viability was determined using A. MTT assay, and B. LDH assay. Untreated cells served as controls and were assigned a viability of 100%; cell viability after NIV treatment is relative to the control. Representative data is presented as Mean ±95% CI (n = 6) [** p≤0.01, *** p≤0.001].

Figure 2. Differential effects of Nivalenol (NIV) on the expression of extracellular matrix components.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. Expression of A. Type I collagen, B. Type II collagen, and C. Aggrecan mRNAs were assessed using quantitative PCR. Data were normalised to the housekeeping gene GAPDH and are presented as fold change relative to the untreated cells. D. Total sGAG released into the culture media and sGAG levels in cell lysates (normalised to cell number) after NIV treatment for 1 day was determined using the DMMB assay. Representative data is presented as Mean ±95% CI (n = 6) [^* p≤0.05, ** p≤0.01, *** p≤0.001 when compared to untreated cells].

Figure 3. Nivalenol (NIV) induces ADAMTS-4 and -5 gene expression.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. Expression of A. ADAMTS-4 and B. ADAMTS-5 were assessed using quantitative PCR. Data were normalised to the housekeeping gene GAPDH and are presented as fold change relative to the untreated cells [refer to Figure 2 for data analysis and statistical representation].

Figure 4. Nivalenol (NIV) modulates MMP expression.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. Expression of A. MMP-1, B. MMP-2, C. MMP-3 and D. MMP-9 were assessed using quantitative PCR. Data were normalised to the housekeeping gene GAPDH and are presented as fold change relative to the untreated cells. E. Levels of MMP-2 released into the culture media, after 1 day of NIV treatment, was determined by gelatin zymography; data was normalised to protein content and presented as fold change relative to the untreated cells [refer to Figure 2 for data analysis and statistical representation].

Figure 5. Differential effects of Nivalenol (NIV) on TIMP expression.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. Expression levels of A. TIMP-2 and B. TIMP-3 were assessed using quantitative PCR. Data were normalised to the housekeeping gene GAPDH and are presented as fold change relative to the untreated cells. Levels of C. TIMP-1 and D. TIMP-2 released into the culture media, after 1 day of NIV treatment, was determined by reverse gelatin zymography; data was normalised to protein content and presented as fold change relative to the untreated cells [refer to Figure 2 for data analysis and statistical representation].

Figure 6. Nivalenol (NIV) disassembles the F-actin cytoskeleton and reduces β-actin expression.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. A. F-actin filament organisation as detected using Alexa488-phalloidin in conjunction with confocal microscopy; nuclei are counterstained with DAPI. Representative serial sections through the middle of the cell and 3D-reconstructions are presented [scale bar  = 2 µm]. B. β-actin mRNA levels were assessed using quantitative PCR. Data were normalised to the housekeeping gene GAPDH and are presented as fold change relative to the untreated cells. C. β-actin protein levels were determined by Western blotting (equivalent protein loading) and data presented as fold change relative to the untreated cells [refer to Figure 2 for data analysis and statistical representation].

Figure 7. Nivalenol (NIV) alters the organisation and expression of β-tubulin.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. A. Tubulin organisation as detected using anti-tubulin primary and TRITC-conjugated secondary antibodies in conjunction with confocal microscopy; nuclei are counterstained with DAPI. Representative serial sections through the middle of the cell and 3D-reconstructions are presented [scale bar  = 2 µm]. B. β-tubulin mRNA levels were assessed using quantitative PCR. Data were normalised to the housekeeping gene GAPDH and are presented as fold change relative to the untreated cells. C. β-tubulin protein levels were determined by Western blotting (equivalent protein loading) and data presented as fold change relative to the untreated cells [refer to Figure 2 for data analysis and statistical representation].

Figure 8. Nivalenol (NIV) induces peri-nuclear aggregation of the vimentin cytoskeleton.

Chondrocytes cultured as a high-density monolayer were treated with 0.1, 0.2 or 0.5 µg/ml NIV for 1 day. Untreated cells served as controls. Vimentin organisation was detected using anti-vimentin primary and TRITC-conjugated secondary antibodies in conjunction with confocal microscopy; nuclei are counterstained with DAPI. Representative serial sections through the middle of the cell and 3D-reconstructions are presented [scale bar  = 2 µm].