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Mammalian Ste20-like kinase 1 inhibition as a cellular mediator of anoikis in mouse bone marrow mesenchymal stem cells

sarah Jonas

BACKGROUND

The low survival rate of mesenchymal stem cells (MSCs) caused by anoikis, a form of apoptosis, limits the therapeutic efficacy of MSCs. As a proapoptotic molecule, mammalian Ste20-like kinase 1 (Mst1) can increase the production of reactive oxygen species (ROS), thereby promoting anoikis. Recently, we found that Mst1 inhibition could protect mouse bone marrow MSCs (mBMSCs) from H2O2-induced cell apoptosis by inducing autophagy and reducing ROS production. However, the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.

AIM

To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.

METHODS

Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA (shRNA) adenovirus transfection. Integrin (ITGs) were tested by flow cytometry. Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA, respectively. The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays. The levels of the anoikis-related proteins ITGα5, ITGβ1, and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I, Beclin1 and p62 were detected by Western blotting.

RESULTS

In isolated mBMSCs, Mst1 expression was upregulated, and Mst1 inhibition significantly reduced cell apoptosis, induced autophagy and decreased ROS levels. Mechanistically, we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4, ITGαv, or ITGβ3 expression. Moreover, autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.

CONCLUSION

Mst1 inhibition ameliorated autophagy formation, increased ITGα5β1 expression, and decreased the excessive production of ROS, thereby reducing cell apoptosis in isolated mBMSCs. Based on these results, Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.

Key Words: Mouse bone marrow mesenchymal stem cell, Mammalian sterile 20-like kinase 1, Anoikis, Integrin, Autophagy, Reactive oxygen species

 

Core Tip: In isolated mouse bone marrow mesenchymal stem cell (mBMSCs), Mammalian sterile 20-like kinase 1 (Mst1) inhibition could ameliorate not only autophagy formation but also upregulate integrin (ITG) α5β1 expression (but not ITGα4, ITGαv, or ITGβ3). In addition, Mst1 inhibition-induced autophagy could scavenge the excessive production of ITGα5β1-triggered ROS. Therefore, Mst1 inhibition-based infusion may improve the survival of MSCs, thereby serving as an ideal candidate for clinical transplantation in pulmonary arterial hypertension.