Moderate alterations of the cytoskeleton in human chondrocytes after short‐term microgravity produced by parabolic flight maneuvers could be prevented by up‐regulation of BMP‐2 and SOX‐9

Abstract

Real and simulated microgravity induce a variety of changes in human cells. Most importantly, changes in the cytoskeleton have been noted, and studies on microtubules have shown that they are gravisensitive. This study focuses on the effects of short-term real microgravity on gene expression, protein content, and cytoskeletal structure of human chondrocytes. We cultivated human chondrocytes, took them along a parabolic flight during the 24th Deutsches Zentrum für Luft- und Raumfahrt Parabolic (DLR) Flight Campaign, and fixed them after the 1st and the 31st parabola. Immunofluorescence microscopy revealed no changes after the 1st parabola, but disruptions of β-tubulin, vimentin, and cytokeratin networks after the 31st parabola. No F-actin stress fibers were detected even after 31 parabolas. Furthermore, mRNA and protein quantifications after the 31st parabola showed a clear up-regulation of cytoskeletal genes and proteins. The mRNAs were significantly up-regulated as follows: TUBB, 2-fold; VIM, 1.3-fold; KRT8, 1.8-fold; ACTB, 1.9-fold; ICAM1, 4.8-fold; OPN, 7-fold; ITGA10, 1.5-fold; ITGB1, 1.2-fold; TGFB1, 1.5-fold; CAV1, 2.6-fold; SOX9, 1.7-fold; BMP-2, 5.3-fold. However, SOX5 (-25%) and SOX6 (-28%) gene expression was decreased. Contrary, no significant changes in gene expression levels were observed during vibration and hypergravity experiments. These data suggest that short-term microgravity affects the gene expression of distinct proteins. In contrast to poorly differentiated follicular thyroid cancer cells or human endothelial cells, chondrocytes only exert moderate cytoskeletal alterations. The up-regulation of BMP-2, TGF-β1, and SOX9 in chondrocytes may play a key role in preventing cytoskeletal alterations.

Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes

“…In fact, it has been proposed that mechanical stress may be a useful tool for tissue engineering of articular cartilage [36]. Here, we focused on vibrations, as we have already conducted short-term studies on chondrocytes earlier [9,10], which hinted towards a negative influence of vibration on cultured chondrocytes after 2 h.…”

“…The signal was generated with the software WaveLab 4.0 from Steinberg. The device was described in detail in [10].…”

“…F-actin staining F-actin was visualized by means of rhodamine-phalloidin staining (Molecular Probes) [10]. For this purpose, seeded cells were fixed for 30 min with 4% PFA (in DPBS), washed twice with DPBS, incubated with 5 mg/mL fluorescent phalloidin-conjugate solution in DPBS with 1% bovine serum albumin for 20 min at room temperature, and then washed 3 times with DPBS to remove unbound phalloidin conjugate.…”

“…As a control, a corresponding short-term, two-hour period of vibration as it occurs during the PF was affecting cartilage cells (upregulated inflammatory IL6 and CXCIL8 mRNAs, downregulated growth factor genes like EGF, VEGF, FGF17) [9]. However, no significant changes in morphology and gene expression levels of cytoskeletal genes, cell adhesion molecules, integrins, as well as of TGFB, CAV1 and SOX9 were observed during vibration [10].…”

Cytokine Release and Focal Adhesion Proteins in Normal Thyroid Cells Cultured on the Random Positioning Machine

“…Overexpression of β 1 -integrin has been demonstrated to improve the activities and functions of several benign cell types [66]. For example, r-µ g during a parabolic flight induced β 1 -integrin expression in human chondrocytes [67]. …”