Pavalko Fredrick M, Gerard Rita L, Ponik Suzanne M, Gallagher Patricia J, Jin Yijun, Norvell Suzanne M
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
J Cell Physiol. 2003 Feb;194(2):194-205. doi: 10.1002/jcp.10221.
In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.
在骨骼中，很大一部分成骨细胞（负责新骨沉积的细胞）通常会经历程序性细胞死亡（凋亡）。由于骨骼的机械负荷会增加新骨形成的速率，我们推测对成骨细胞的机械刺激可能会提高其存活率。为了验证这一假设，我们使用三种成骨细胞类型研究了流体剪切应力（FSS）对成骨细胞凋亡的影响：原代大鼠颅骨成骨细胞（RCOB）、MC3T3-E1成骨细胞和UMR106骨肉瘤细胞。细胞在环己酰亚胺（CHX）存在的情况下用肿瘤坏死因子-α（TNF-α）处理以快速诱导凋亡。成骨细胞在暴露于TNF-α和CHX后4 - 6小时内显示出明显的凋亡迹象，而施加FSS（12达因/平方厘米）可显著减轻这种TNF-α诱导的凋亡。FSS激活磷脂酰肌醇-3激酶（PI3-激酶）信号传导，诱导Akt磷酸化，并抑制TNF-α诱导的半胱天冬酶-3激活。使用LY294002抑制PI3-激酶，阻断了FSS将成骨细胞从TNF-α诱导的凋亡中拯救出来的能力，并阻断了FSS诱导的对用TNF-α处理的成骨细胞中半胱天冬酶-3激活的抑制作用。然而，LY294002并未阻止FSS诱导的Akt磷酸化，这表明单独激活Akt不足以使细胞从凋亡中获救。该结果还表明FSS可以通过PI3-激酶非依赖性途径激活Akt。这些研究首次证明，在体外对成骨细胞施加FSS可通过涉及激活PI3-激酶信号传导和抑制半胱天冬酶的机制抑制TNF-α诱导的凋亡。FSS诱导的PI3-激酶激活可能通过一种不同于Akt介导的存活途径的机制促进细胞存活。
