BAG3 regulates bone marrow mesenchymal stem cell proliferation by targeting INTS7.

Abstract

BAG3 is an essential regulator of cell survival and has been investigated in the context of heart disease and cancer. Our previous study used immunoprecipitation-liquid chromatography-tandem mass spectrometry to show that BAG3 might directly interact with INTS7 and regulate bone marrow mesenchymal stem cell (BMMSCs) proliferation. However, whether BAG3 bound INTS7 directly and how it regulated BMMSCs expansion was unclear. BAG3 expression was detected by quantitative real-time PCR in BMMSCs after siRNA-mediated BAG3 knockdown. BMMSC proliferation was determined using the CCK-8 and colony formation assays. The transwell migration, flow cytometry and TUNEL assays were performed to measure BMMSC migration, cell cycle and apoptosis, respectively. Moreover, co-immunoprecipitation, protein half-life assay and western blotting analyses were used to determine the regulatory mechanism underlying the BAG3-mediated increase in BMMSC proliferation. The results showed that knocking down BAG3 in BMMSCs markedly decreased their proliferative activity, colony formation and migratory capacity, and induced cell apoptosis as well as cell cycle arrest. Meanwhile, overexpression of BAG3 had the opposite effect. Bioinformatics and BAG3-INTS7 co-immunoprecipitation analyses revealed that BAG3 directly interacted with INTS7. Moreover, the downregulation of BAG3 inhibited the expression of INTS7 and promoted its ubiquitination. We also observed that BAG3 knockdown increased the levels of reactive oxygen species and the extent of DNA damage in BMMSCs. Notably, the upregulation of INTS7 or the addition of an antioxidant scavenger could rescue the BMMSC phenotype induced by BAG3 downregulation. BAG3 directly interacts with INTS7 and promotes BMMSC expansion by reducing oxidative stress.