SAT0171 High oxidation status induced the rearrangement of F-ACTIN cytoskeleton of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus via downregulation of RHOA signaling pathway
Authors: D. Shi, X. Li, L. Sun
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Systemic lupus erythematosus (SLE) is an autoimmune disease which has been described as a hematopoietic stem cell (HSC) disorder. Besides HSCs, bone marrow contains mesenchymal stem cells (MSCs) which is a cell group owning the ability to self renew and differentiate into different cell lines. Previous studies have demonstrated that bone marrow-derived MSCs (BM-MSCs) from SLE patients had abnormal functions. F-actin cytoskeleton was associated with multiple cell vital processes including cell division, migration, apoptosis and differentiation. The polymerization and depolymerization of F-actin cytoskeleton are regulated by RhoA signaling pathway. <h3>Objectives</h3> This study was undertaken to determine the effect of oxidation status on F-actin cytoskeleton in BM-MSCs from SLE patients. <h3>Methods</h3> In order to find out the effect of oxidation status on MSCs, 20μM H<sub>2</sub>O<sub>2</sub> was added into the growth medium of normal MSCs 1h before testing and 10mM NAC, an antioxidant, was added into the growth medium of SLE MSCs. Serum level of SOD-1 was detected by ELISA and ROS level of MSCs was tested by flow cytometry. The F-actin cytoskeleton was observed by fluorescence microscopy after staining with Alexa Fluor 594 phalloidin. RT-PCR and western blot were carried out to detect changes of RhoA signaling pathway. MSCs treated with or without H<sub>2</sub>O<sub>2</sub>/NAC were cocultured with peripheral blood mononuclear cells (PBMCs) and the supernatants were collected to detect the level of IL-4 and IFN-γ. Cells were cultured in osteogenesis differentiation medium for 21 days and stained with alizarin red to evaluate the ability of differentiation of MSCs. <h3>Results</h3> F-actin cytoskeleton of MSCs was rearranged in SLE patients. The percentage of abnormal MSCs was significantly higher in SLE patients than normal controls and increased as they were passaged. Both of the mRNA level and protein expression of RhoA, which participates in a main regulatory signaling pathway of F-actin cytoskeleton, were downregulated in SLE MSCs. Reduced serum SOD-1 level and higher intracellular ROS level suggested high oxidation status in SLE patients. H<sub>2</sub>O<sub>2</sub>, as an exogenous oxidant, induced upregulation of ROS level in normal MSCs. As a result, the F-actin cytoskeleton was rearranged and the osteogenic differentiation capacity was impaired. NAC, which downregulated ROS level, reversed the rearrangement of F-actin cytoskeleton and the impairment of osteogenic differentiation capacity of SLE MSCs. While the levels of IL-4 and IFN-γ stayed same, suggesting there was no effect of high oxidation status on immunomodulatory property of MSCs. RhoA expression was downregulated by H<sub>2</sub>O<sub>2</sub> while upregulated by NAC. <h3>Conclusions</h3> These experimental findings suggest that the high oxidation status may contribute to the rearrangement of F-actin cytoskeleton of MSCs as well as their osteogenic differentiation ability in SLE patients via downregulation of RhoA. <h3>Disclosure of Interest</h3> None Declared