BACKGROUND Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. OBJECTIVES We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. METHODS Male wild-type (WT), global Nrf2-knockout (Nrf2-/-) and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or 100 ppm) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or 20 nM) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. RESULTS Compared with relevant control mice, global Nrf2-/- or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to 100 ppm Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to 20 nM) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants N-acetyl-l-cysteine (2 mM) and mitoquinone mesylate (MitoQ; 0.2μM) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. CONCLUSIONS This in vivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

中文翻译：

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长期镉暴露和破骨细胞生成：机制小鼠和体外研究。


背景技术镉（Cd）是一种剧毒且广泛存在的环境氧化应激源，会导致多种健康问题，包括骨质疏松症和骨损伤。尽管核因子红细胞 2 相关因子 2 (NRF2) 及其 Cap 'n' Collar 和碱性区域亮氨酸拉链 (CNC-bZIP) 家族成员核因子红细胞 2 相关因子 1 (NRF1) 通过调节转录来协调各种应激反应多种抗氧化和细胞保护基因，它们在骨代谢和重塑中发挥着独特的作用。然而，这两种转录因子在长期镉暴露引起的骨质流失中的确切作用仍不清楚。目的 我们旨在了解 Cd 引起的骨丢失的分子机制，主要关注 NRF2 和 NRF1 在 Cd 引发的破骨细胞生成中的作用。方法 通过饮用水给予雄性野生型 (WT)、整体 Nrf2 敲除 (Nrf2-/-) 和骨髓特异性 Nrf2 敲除 [Nrf2(M)-KO] 小鼠 Cd（50 或 100 ppm）8 或 16 次。一周，然后进行微型计算机断层扫描、组织学分析和血浆生化测试。在 Cd（10 或 20 nM）存在下，结合针对 NRF2 和 NRF1 的遗传和化学调节，使用骨髓源性破骨细胞祖细胞 (BM-OPC) 和 RAW 264.7 细胞评估破骨细胞生成。结果与相关对照小鼠相比，整体 Nrf2-/- 或 Nrf2(M)-KO 小鼠在暴露于含 100 ppm Cd 的饮用水长达 16 周后，骨质流失加剧，破骨细胞活性增强。体外破骨细胞生成分析表明，与 WT 细胞相比，Nrf2 缺陷的 BM-OPC 和 RAW 264.7 细胞对破骨细胞分化的低水平 Cd（高达 20 nM）反应更强烈。 进一步的机制研究支持 NRF1 长亚型 (L-NRF1) 的代偿性上调以及随后诱导活化 T 细胞核因子、细胞质、钙调神经磷酸酶依赖性 1 (NFATc1) 作为 Nrf2 缺乏恶化和恶化的关键分子事件。镉引起的破骨细胞生成。与单独暴露于 Cd 的 Nrf2-KD 细胞相比，L-Nrf1 沉默（通过慢病毒手段）Nrf2 敲低 (KD) RAW 细胞表现出显着不同的 NFATc1 和随后的破骨细胞生成结果，表明 Nrf1 沉默具有缓解作用。此外，外源性抗氧化剂 N-乙酰基-L-半胱氨酸 (2 mM) 和甲磺酸米托醌 (MitoQ；0.2μM) 抑制活性氧，减轻了 L-NRF1 对 Cd 暴露 Nrf2 中 NFATc1 驱动的破骨细胞生成结果的相关影响-KD细胞。结论 这项体内和体外研究支持了作者的假设，即镉暴露导致骨质流失，其中 NRF2 和 L-NRF1 以一种合作但矛盾的方式对镉和破骨细胞刺激作出反应，以协调 Nfatc1 表达、破骨细胞生成，从而协调骨体内平衡。我们的研究提出了一种针对 NRF2 和 L-NRF1 的新策略来预防和治疗镉的骨毒性。 https://doi.org/10.1289/EHP13849。