In the present study, we investigated the effect of TGF-β1 on senescence of bmMSCs. Our results showed that treatments with TGF-β1 (1 ~ 10 ng/mL) increased SA-Gal activity and mtROS production in bmMSCs in a dose dependent manner. The aging promoter p16 and oxidative stress inducer 4-HNE were markedly increased; however, their opponents Id1 and SOD2 were significantly decreased in bmMSCs after exposure to TGF-β1. Application of mtROS inhibitor acetyl-L-carnitine significantly inhibited TGF-β1-induced mtROS production and bmMSC senescence. These findings demonstrate that TGF-β1 can cause senescence of bmMSCs, which involves mtROS production.
BmMSCs are the most promising sources of stem cells and have been widely applied to treat cardiac diseases . Transplantation of bmMSCs has the potential to reduce infarct size and improve ventricular compliance after myocardial infarction . BmMSCs are usually pre-treated with some biological reagents and chemicals, such as 5-azacytidine, TGF-β, angiotensin II, hypoxia-inducible factor-1 alpha (HIF-1α) and slingshot-1 L (SSH1L) for cardiomyocyte differentiation, prior to transplantation [6, 7, 31–33]. Among these reagents, TGF-β is the most commonly used one. Previous studies have shown that TGF-β1 and -β2 have the potential to induce senescence of tumor cells and other kinds of normal tissue cells [11–13]. Our results showed that treatments with TGF-β1 markedly increased SA-Gal activity in bmMSCs, which showed that these cells were undergoing senescence. Senescence would lead to phenotype changes and low proliferation of bmMSCs, which reduces the efficiency of bmMSC-based transplantation .
Our results showed that TGF-β1 induced expression of 4-HNE subunits. 4-HNE, a major lipid peroxidation (LPO) product, plays key roles in signal transduction pathways, and participates in cell cycle events. While lower levels of intracellular 4-HNE are beneficial to cells, possibly promoting cellular proliferation; however, higher levels cause toxic responses in cells, including cell senescence and apoptosis. Expression of 4-HNE has been proved to induce cell senescence and organ aging [23, 35, 36]. We found that p16, an important regulator of aging, was markedly upregulated in the TGF-β1-treated bmMSCs; however, Id1, a negative regulator of p16, was markedly downregulated in these cells. These data were consistent with previous reports from other groups, which showed that p16 protein was highly expressed, but Id1 protein was downregulated in the senescent cells and aged tissues [13, 26, 35].
More interestingly, mtROS production was also markedly increased in bmMSCs after exposure to TGF-β1. This data is also consistent with expression of 4-HNE that has been widely accepted as an inducer of cellular oxidative stress [23, 25]. Previous studies have shown that TGF-β1 can increase mtROS production in tumor cells . MtROS has also been known as key inducer of aging . We also observed that SOD2 was significantly downregulated in bmMSCs after exposure to TGF-β1. SOD2 is known to be a key enzyme that protects mitochondria from ROS insult [16, 38].
To further elucidate the role of mtROS in TGF-β1-induced bmMSC senescence, we treated bmMSCs with mtROS specific inhibitor acetyl-L-carnitine (ALCAR) when the cells were exposed to 5 ng/mL TGF-β1. Our results showed that ALCAR (0.1 mM) significantly inhibited TGF-β1-induced mtROS production and increase of SA-Gal activity. These data show that TGF-β1-induced senescence of bmMSCs at least partially depends on mtROS production.