Study of RhoGTPases in the regulation of Compression-induced Actin Protrusions(CAPs) in human mesenchymal stem cells(MSCs) embedded in collagen microspheres.

Abstract

Cell functions are regulated by an environmental change consisting of chemical and mechanical factors. To give an optimal response, cells swiftly generate protrusions at the leading edge for sensation and adaptation against a sudden change of niches. To date, several studies have demonstrated filopodial and lamellopodial protrusions induced by mechanical stress. Of these protrusions, they are highly dependent on the actin organization which are governed by small RhoGTPases such as CDC42, Rac1 and RhoA. Our previous study reported the formation of compression-induced actin protrusions(CAPs) in collagen-encapsulated human mesenchymal stem cells(MSCs) via a non-CDC42 RhoGTPase pathway. Although many studies have examined other RhoGTPases including Rac1 and RhoA, there is still lack of convincing evident on their regulatory roles for the mechanical induced protrusions. In this study, we aims to study RhoGTPases on the regulatory roles of CAPs in MSCs with collagen encapsulation. To examine the underlying mechanisms through which these RhoGTPases control CAPs formation in encapsulated MSCs, an inhibition treatment by special inhibitors and siRNAs for Rac1 and RhoA was performed in MSCs followed by a 9-hour cyclic compression, harvested and detected immediately after compression using stateof-the-art technologies including immunofluorescent staining, qPCR and protein analysis. The results of GLISA and qPCR analysis consistently showed more Rac1 expression in MSCs with compression. Despite the inhibition treatments revealed a modest suppression in the active Rac1 and RhoA of MSCs, more roundness of cells and reduced number of CAPs were concordantly obtained in those with the treatment of Rac1 inhibitor and compression together, possibly suggesting a dominant role of Rac1 on mediating CAP formation. These results suggest Rac1 as a key regulator, promoting the CAP formation and further studies on the downstream effectors along the pathway will be required to make a better understanding of the cytoskeletal mechanic and the functional significance of CAPs.