Transglutaminase 2 Prevents Premature Senescence and Promotes Osteoblastic Differentiation of Mesenchymal Stem Cells through NRF2 Activation

Figure 6

TG2 activates NRF2 in a transamidase activity-independent manner, and TG2 is required for MSCs proliferation under hypoxia conditions. (a) Wild-type and TG2-knockdowned MSCs were treated with tBHQ for 12 hr and then incubated in media containing 1 mM biotinylated pentylamine (BP) for 1 hr. BP-incorporated protein in the lysates was detected by immunoblotting with streptavidin-horseradish peroxidase (SA-HRP) to estimate intracellular transamidation activity. (b–d) HEK293 cells were transfected with expression vectors encoding wild-type TG2 (lane 2, 5, 100 ng), TG2^C277S, a transamidase active-site mutant (lane 3, 6, 100 ng), NRF2^Δ1−89, a constitutively active NRF2 mutant (caNRF2; lane 4–6, 100 ng), NRF2 reporter (8× ARE fuzed with firefly luciferase, 150 ng), and pRL-TK (50 ng). The empty pcDNA3.1 vector was used as the control (lane 1, 4, 100 ng). After treatment with tBHQ (25 μM) for 48 hr or transfection of caNRF2, intracellular transamidation activity was measured by BP-incorporation assay (b) or NRF2 reporter activity was monitored for 48 hr (c, d). Reporter activity was normalized with cotransfected Renilla luciferase activity. (e) Increase of TG2 protein levels under hypoxia. MSCs were cultured under hypoxic conditions, where the amount of oxygen was reduced to less than 1% or under normoxic conditions. The protein levels of TG2 and HIF-1α were analyzed by western blot with β-actin used as the loading control. (f) Cell proliferation of MSCs under hypoxia. MSCs were cultured under hypoxic conditions, where the amount of oxygen was reduced to less than 1% or under normoxic conditions. The trypan blue viability assay was performed on MSCs. The western blots are representative results from three independent experiments. The luciferase reporter assay and cell viability assay were performed in triplicate, with technical replicates within a single experiment, and the data are presented as the mean values ± SEM.

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