TPO Exerts a Protective Effect on Iron-Induced Apoptosis Via Erk1/2 Signaling in Human Mesenchymal Stem Cells.

Osteoporosis is a very common problem among adolescent and adult patients with thalassaemia major (TM). The pathogenesis of osteoporosis in TM is related to several factors including iron overload. Bone is derived from osteoblasts. And osteoblasts are differentiated from mesenchymal stem cells (MSC). Therefore, iron-overload induced MSC damage may contribute to osteopenia and osteoporosis. The effect of iron-overload on MSC has not been investigated previously. We hypothesize that iron-overload may induce apoptosis in MSC by caspase-dependent pathway, and haematopoietic growth factor thrombopoietin (TPO) and calcium channel blocker amlodipine may have a protective effect on iron-induced apoptosis in these cells. We have shown that iron (FeCl₃) reduced hMSCs viability in a dose-dependent manner (0–0.6 mM) (n=5). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM) for 72 hrs (n=4). The expression of active caspase-3 was significantly increased in iron-treated cells (0.15mM, 0.3mM) (n=5). Iron treatment also increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential. TPO exerted protective effect on iron-induced apoptosis in hMSCs. Human MSCs were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/ml) for 72 hrs (n=4). The cell viability was significantly increased with the treatment of TPO. Dot-plot analysis of annexin V/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells. Incubation with TPO also decreased the iron-induced caspase-3 expression. Flow cytometric dot-plot analysis of hMSCs also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO. The population of phospho-Erk1/2 was also significantly increased in TPO-treatment, and the increased phospho-Erk was significantly reversed by the upstream signaling inhibitor PD098059. Calcium channel blocker amlodipine (10⁻⁹M) also had a protective effect on iron-induced apoptosis in these cells. Our findings suggest that iron-overload induces apoptosis in hMSCs via the caspase-dependent pathway and that TPO and amlodipine might exert a protective effect on iron-induced apoptosis via the activation of Erk1/2 signaling. The use of either haematopoietic growth factor or calcium channel blocker for the protection of hMSCs from iron induced toxicity is a novel concept. Our study has the potential in minimizing the bone damage induced by iron-overload in patients with thalassaemia major.