Abstract
Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.
Highlights
Iron, an essential micronutrient, plays a crucial role in a wide scale of biological processes like DNA synthesis, energy metabolism, and oxygen transport; excess iron is toxic to cells as leading to organ dysfunction and diseases [1, 2]
The results showed the dose-dependent cytotoxicity as evidenced by the decrease of absorbance in the osteoblastic cells with increasing Ferric ammonium citrate (FAC) treatment for 72 h and 120 h (Figure 1)
Iron gradually accumulates in the course of aging, and excess iron could accelerate bone loss in physically fit postmenopausal women and old men [35]
Summary
An essential micronutrient, plays a crucial role in a wide scale of biological processes like DNA synthesis, energy metabolism, and oxygen transport; excess iron is toxic to cells as leading to organ dysfunction and diseases [1, 2]. Patients with iron overload associated diseases like hemochromatosis, thalassemia, and sickle cell disease are much more prone to suffer from osteoporosis [4, 5]. The fundamental mechanisms by which iron overload causes osteoporosis remain poorly understood. Substantial evidence has accumulated to demonstrate that oxidative stress caused by iron overload is the major contributor to the pathogenesis of osteoporosis [6,7,8]. We have demonstrated that reactive oxygen species was essential for iron overloadinduced apoptosis in the osteoblastic cells [9]. We noticed that osteoblastic cell death was only partially mediated by apoptosis under iron overload conditions [9].
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