Fluid Shear Stress and Osteoblast Apoptosis

描述(申请人提供)：众所周知，机械负荷增加了骨的形成和重塑。运动会导致骨量增加，而长期的骨骼卸载，如长时间卧床休息和太空飞行期间的微重力，会导致骨骼萎缩。我们研究的主要目的是了解调控机械诱导骨形成的细胞和分子机制。实验研究表明，流体切应力(FSS)诱导的成骨细胞活化可能刺激了机械诱导的骨形成。成骨细胞的机械激活被认为是由于间质液体通过骨内多孔间隙的运动导致成骨细胞在高冲击载荷下受到FSS的影响。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，骨改建部位的成骨细胞中有很大一部分注定要经历细胞程序性死亡(即细胞凋亡)。因此，在正常和疾病状态下，抑制成骨细胞凋亡的过程可能在促进骨形成和提高骨强度方面有效。我们的初步研究表明，体外对成骨细胞的机械刺激，通过暴露于稳定的流体剪应力，抑制了成骨细胞的凋亡。因此，在本申请中，我们提出了旨在研究FSS促进成骨细胞存活的信号机制的实验。我们将：(1)确定FSS调控细胞内信号通路的机制，以及(2)确定时间剪切梯度在成骨细胞对FSS的抗凋亡反应中的作用。这项研究的长期目标是通过更好地了解调控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。在这一应用中，我们建议使用体外细胞培养模型来检验这一假设，即细胞暴露在稳定或脉动的流体剪应力下，通过特定的细胞过程调节成骨细胞的存活，包括激活PI3-K/Akt和MAPK信号通路和抑制caspase的激活。我们将使用大鼠颅骨成骨细胞的原代培养，以及成骨细胞系，包括MC3T3-E1和UMR106.01细胞，来研究调控细胞凋亡的细胞机制。

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.