Mechanisms of Cadmium-induced Osteotoxicity

描述（由申请人提供）：重金属镉是一种广泛存在的环境污染物，在体内积累，对人体健康构成威胁。骨是镉的关键靶部位。人体接触镉与骨质疏松和骨质减少等骨骼疾病有关。2005年，美国疾病控制与预防中心的一份报告将镉列为一种值得监测的毒素。本报告结合骨质疏松症医疗保健费用的不断上升，强调了促进研究以破译镉诱导骨毒性的潜在机制的重要性。尽管镉被认为是一种重要的环境毒素，但人们对镉如何直接影响骨细胞，特别是成骨细胞知之甚少。我们之前报道过镉诱导人成骨细胞样细胞（Saos-2）凋亡，即程序性细胞死亡。本研究的目的是利用体外成骨细胞模型确定镉诱导细胞凋亡的细胞内机制。有证据表明，氧化应激促进活性氧（ROS）的生成，在镉中毒和骨质疏松的发病机制中起作用。具体目的1)是研究氧化应激（如ROS形成、谷胱甘肽耗竭和脂质过氧化）在镉诱导的成骨细胞凋亡中的作用。转录因子RUNX2是已知的成骨细胞表型的关键介质，研究表明RUNX2对绝经后妇女骨质疏松症起保护作用。因此，具体目的2)是确定镉诱导的氧化应激是否导致RUNX2表达降低。采用RT-PCR和Western blot检测RUNX2 mRNA和蛋白的表达变化。用抗氧化剂处理细胞，以防止镉诱导的细胞凋亡和RUNX2表达的降低。最后，了解镉诱导细胞凋亡机制的关键是破译所涉及的信号通路。具体目的3)是探讨蛋白激酶C （PKC）通路在镉诱导的成骨细胞凋亡中的作用。使用的方法包括使用已知的PKC抑制剂来阻断镉诱导的RUNX2表达下降和细胞凋亡。PKC参与的确认将通过Western blot分析评估PKC活性磷酸化形式来完成。本研究将为镉诱导骨细胞凋亡信号中断的机制提供深入的见解。反过来，这种镉骨毒性模型可能有助于确定治疗和预防骨质疏松症的靶点。长期目标是利用成年斑马鱼建立实验动物模型来验证这些体外研究，并将镉诱导的成骨细胞凋亡与净骨质流失联系起来。公共卫生相关性：重金属镉是一种广泛存在的环境污染物和香烟成分，在体内积累，对人体健康构成威胁。骨骼是镉的一个重要靶点，人类接触镉与骨质疏松症等骨骼疾病有关。这项研究是相关的，因为它提供了镉如何直接影响骨骼的见解，并可能有助于确定治疗和预防骨质疏松症的目标。

DESCRIPTION (provided by applicant): The heavy metal cadmium is a widespread environmental contaminant that accumulates in the body and poses a threat to human health. Bone is a critical target site for cadmium. Human exposure to cadmium is linked to bone diseases, including osteoporosis and osteopenia. In 2005, a CDC report listed cadmium as a toxin that merits monitoring. This report, combined with the escalating cost of healthcare for osteoporosis, emphasizes the importance of promoting research to decipher the underlying mechanisms of cadmium-induced osteotoxicity. Despite its recognized importance as an environmental toxin, little is known about how cadmium directly impacts bone cells, in particular the bone-forming osteoblasts. We previously reported that cadmium induces apoptosis, programmed cell death, in human osteoblast-like cells (Saos-2). The goal of this research is to determine the intracellular mechanisms involved in cadmium-induced apoptosis using an in vitro osteoblast cell model. Evidence indicates that oxidative stress, enhanced generation of reactive oxygen species (ROS), plays a role in cadmium toxicity and the pathogenesis of osteoporosis. Specific aim 1) is to investigate the role of oxidative stress (e.g., ROS formation, glutathione depletion, and lipid peroxidation) in cadmium-induced osteoblast apoptosis. The transcriptional factor RUNX2 is a known critical mediator of the osteoblast phenotype and research indicates RUNX2 plays a protective role against osteoporosis in postmenopausal women. Therefore, specific aim 2) is to determine if cadmium-induced oxidative stress leads to a decrease in RUNX2 expression. The methods employed include RT-PCR and Western blot analysis to detect changes in RUNX2 mRNA and protein expression, respectively. Cells will be treated with antioxidants to prevent cadmium-induced apoptosis and decrease in RUNX2 expression. Lastly, key to understanding the mechanisms involved in cadmium-induced apoptosis is deciphering the signaling pathways involved. Specific aim 3) is to explore the role of the protein kinase C (PKC) pathway in cadmium-induced osteoblast apoptosis. The methods used include using known PKC inhibitors to block cadmium-induced decrease in RUNX2 expression and apoptosis. Confirmation of PKC involvement will be done by assessing active phosphorylated forms of PKC by Western blot analysis. This research will provide insight into mechanism underlying cadmium-induced disruption of apoptotic signaling in bone. In turn, this cadmium osteotoxicity model may help identify targets for treatment and prevention of osteoporosis. The long term goal is to develop an experimental animal model using adult zebrafish to validate these in vitro studies and link cadmium-induced osteoblast apoptosis to net bone loss. PUBLIC HEALTH RELEVANCE: The heavy metal cadmium, a widespread environmental contaminant and component in cigarettes, accumulates in the body and poses a threat to human health. Bone is a critical target site for cadmium and human exposure to cadmium is linked to bone disease, including osteoporosis. This research is relevant because it provides insight into how cadmium directly impacts bone, and may help identify targets for treatment and prevention of osteoporosis.