Fluid Shear Stress and Osteoblast Apoptosis

描述（由申请人提供）：众所周知，机械载荷会增加骨的形成和重塑。骨量随着运动而增加，而长期的骨卸载，如长时间卧床休息期间和空间飞行期间的微重力状态下发生的骨卸载，导致骨萎缩。我们研究的主要目的是了解调节机械诱导骨形成的细胞和分子机制。实验研究表明，机械诱导的骨形成可能是由流体剪切应力（FSS）诱导的成骨细胞激活刺激的。成骨细胞的机械活化被认为是由于间质液通过骨内的多孔空间的运动，在高冲击载荷期间使成骨细胞受到FSS。然而，FSS促进成骨细胞合成代谢反应的细胞机制尚不清楚。有趣的是，骨重建部位的大部分成骨细胞注定要经历程序性细胞死亡（凋亡）。因此，抑制成骨细胞凋亡的过程可能在正常和疾病状态下有效增加骨形成和改善骨强度。我们的初步研究表明，机械刺激成骨细胞在体外，通过暴露于稳定的流体剪切应力，抑制成骨细胞凋亡。因此，在这个应用程序中，我们提出的实验，旨在调查的信号转导机制，通过FSS促进成骨细胞的生存。我们将：（1）确定FSS调节细胞内信号通路的机制，这些信号通路参与细胞凋亡的控制，以及（2）确定时间剪切梯度在成骨细胞对FSS的抗细胞凋亡反应中的作用。这项研究的长期目标是通过更好地理解调节成骨细胞存活的细胞和分子机制来确定改善骨骼健康的策略。在本申请中，我们建议使用体外细胞培养模型来测试这一假设，即细胞暴露于稳定或脉动的流体剪切应力通过特定的细胞过程调节成骨细胞的存活，包括PI 3-激酶/Akt和MAPK信号通路的激活和caspase激活的抑制。我们将使用原代培养的大鼠颅骨成骨细胞，成骨细胞系，包括MC 3 T3-E1和UMR 106.01细胞，研究细胞凋亡的细胞调节机制。

DESCRIPTION (provided by applicant): It is well known that mechanical loading increases formation and remodeling of bone. Bone mass is increased in response to exercise, while chronic unloading of bone, such as occurs during prolonged bed rest and in microgravity during space flight, results in atrophy of bone. The broad aim of our research is to understand the cellular and molecular mechanisms that regulate mechanically-induced bone formation. Experimental studies suggest that mechanically-induced bone formation may be stimulated by fluid shear stress (FSS)-induced activation of osteoblasts. Mechanical activation of osteoblasts is thought to result from the movement of interstitial fluid through the porous spaces inside bone that subjects osteoblasts to FSS during high impact loading. However, the cellular mechanisms through which FSS promotes an anabolic response in osteoblasts are not clearly understood. Interestingly, a large proportion of osteoblasts at sites of bone remodeling are destined to undergo programmed cell death (apoptosis). Therefore, processes that inhibit osteoblast apoptosis may be effective in increasing bone formation and improving bone strength in normal and disease states. Our preliminary studies indicate that mechanical stimulation of osteoblasts in vitro, by exposure to steady fluid shear stress, inhibits osteoblast apoptosis. Therefore, in this application we propose experiments that are designed to investigate the signaling mechanisms through which FSS promotes the survival of osteoblasts. We will: (1) determine the mechanisms through which FSS regulates intracellular signaling pathways involved in control of apoptosis, and (2) determine the role of temporal shear gradients in the anti-apoptotic response of osteoblasts to FSS. The long-term goal of this research is to identify strategies for improving bone health by better understanding the cellular and molecular mechanisms that regulate osteoblast survival. In this application, we propose to use an in vitro cell culture model to test the hypothesis that exposure of cells to either steady or pulsatile fluid shear stress regulates osteoblast survival through specific cellular processes, including activation of the PI3-kinase/Akt and MAPK signaling pathways and inhibition of caspase activation. We will use primary cultures of rat calvarial osteoblasts, and osteoblast cell lines, including MC3T3-E1 and UMR106.01 cells, to investigate the cellular mechanisms that regulate apoptosis.