为了响应骨骼的机械载荷,骨细胞产生一氧化氮(NO•)和减少硬化蛋白的表达,导致骨量增加。然而,目前尚不清楚NO·产生和硬化蛋白损失是否在机械上相关,and,如果是,在已建立的机械转导途径中,它们的等级关系的性质。先前的工作表明,在流体剪切应力(FSS)之后,骨细胞产生NOX2衍生的活性氧,诱导钙(Ca2+)内流。细胞内Ca2+增加导致钙-钙调蛋白依赖性蛋白激酶II(CaMKII)活化,它调节硬化蛋白的溶酶体降解。这里,我们扩展我们的发现,鉴定NO•作为机械活化CaMKII下游硬化蛋白降解的调节剂。Ocy454骨细胞样细胞中一氧化氮合酶(NOS)活性的药理学抑制可防止FSS诱导的硬化蛋白蛋白丢失。相反,短期治疗与NO•供体在Ocy454细胞或分离的小鼠长骨足以诱导硬化蛋白丰度的快速降低,与Sost基因表达的变化无关。Ocy454细胞表达所有三种NOS基因,用靶向eNOS/Nos3的siRNA转染足以防止FSS诱导的硬化蛋白丢失,而针对iNOS/Nos2的siRNA轻度减弱了硬化蛋白的丢失,但未达到统计学意义。同样,靶向eNOS/Nos3和iNOS/Nos2的siRNA阻止FSS诱导的NO·产生。一起,这些数据显示iNOS/Nos2和eNOS/Nos3是FSS依赖性NO·的主要生产者,并且NO·对于硬化蛋白控制是必要和足够的。Further,该硬化蛋白控制机械转导途径中的元件的选择性抑制表明NO·产生发生在CaMKII激活的下游。在Ocy454细胞中用siRNA靶向Camk2d和Camk2g可防止FSS后NO的产生,表明NO·生产需要CaMKII。然而,NO•捐献(1min)导致CaMKII活化显著增加,表明NO·可能具有调节CaMKII反应的能力。一起,这些数据支持CaMKII是必要的,并且可以由NO·调制,这两个信号的相互作用涉及硬化蛋白丰度的控制,与骨合成代谢反应中的作用一致。
In response to mechanical loading of bone, osteocytes produce nitric oxide (NO•) and decrease
sclerostin protein expression, leading to an increase in bone mass. However, it is unclear whether NO• production and
sclerostin protein loss are mechanistically linked, and, if so, the nature of their hierarchical relationship within an established mechano-transduction pathway. Prior work showed that following fluid-shear stress (FSS), osteocytes produce NOX2-derived reactive oxygen species, inducing calcium (Ca2+) influx. Increased intracellular Ca2+ results in calcium-calmodulin dependent protein kinase II (CaMKII) activation, which regulates the lysosomal degradation of sclerostin protein. Here, we extend our discoveries, identifying NO• as a regulator of
sclerostin degradation downstream of mechano-activated CaMKII. Pharmacological inhibition of nitric oxide synthase (NOS) activity in Ocy454 osteocyte-like cells prevented FSS-induced sclerostin protein loss. Conversely, short-term treatment with a NO• donor in Ocy454 cells or isolated murine long bones was sufficient to induce the rapid decrease in
sclerostin protein abundance, independent of changes in Sost gene expression. Ocy454 cells express all three NOS genes, and transfection with siRNAs targeting eNOS/Nos3 was sufficient to prevent FSS-induced loss of sclerostin protein, while siRNAs targeting iNOS/Nos2 mildly blunted the loss of sclerostin but did not reach statistical significance. Similarly, siRNAs targeting both eNOS/Nos3 and iNOS/Nos2 prevented FSS-induced NO• production. Together, these data show iNOS/Nos2 and eNOS/Nos3 are the primary producers of FSS-dependent NO•, and that NO• is necessary and sufficient for sclerostin protein control. Further, selective inhibition of elements within this
sclerostin-controlling mechano-transduction pathway indicated that NO• production occurs downstream of CaMKII activation. Targeting Camk2d and Camk2g with siRNA in Ocy454 cells prevented NO• production following FSS, indicating that CaMKII is needed for NO• production. However, NO• donation (1min) resulted in a significant increase in CaMKII activation, suggesting that NO• may have the ability to tune CaMKII response. Together, these data support that CaMKII is necessary for, and may be modulated by NO•, and that the interaction of these two signals is involved in the control of sclerostin protein abundance, consistent with a role in bone anabolic responses.