Brazier Hélène, Stephens Sébastien, Ory Stéphane, Fort Philippe, Morrison Nigel, Blangy Anne
Centre de Recherches en Biochimie Macromoléculaire, CNRS FRE 2593, Montpellier, France.
J Bone Miner Res. 2006 Sep;21(9):1387-98. doi: 10.1359/jbmr.060613.
RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology.
During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis.
The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest.
Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts.
We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.
RhoGTP酶调节肌动蛋白细胞骨架动力学，这是破骨细胞生物学中的关键要素。我们在RANKL刺激的破骨细胞生成过程中，在RhoGTP酶及其交换因子中鉴定出三个在破骨细胞生物学中至关重要的新基因。
在分化、黏附于骨基质或骨吸收过程中，破骨细胞的肌动蛋白细胞骨架会发生深刻的重组。RhoGTP酶是肌动蛋白动力学的关键调节因子。它们通过作用于肌动蛋白细胞骨架来控制细胞黏附、迁移和形态。在小鼠中，有18种低分子量的RhoGTP酶。它们由鸟嘌呤核苷酸交换因子（RhoGEFs）激活。小鼠中有76种RhoGEFs：65种属于Dbl家族，11种属于CZH家族。为了在对破骨细胞重要的RhoGTP酶和RhoGEFs中鉴定新基因，我们建立了RANKL刺激的破骨细胞生成过程中RhoGTP酶和RhoGEFs完整家族的表达谱。
RAW264.7细胞系、小鼠骨髓巨噬细胞和造血干细胞用作RANKL诱导破骨细胞生成的前体细胞。进行基因芯片和实时定量PCR分析以建立分化过程中RhoGTP酶和RhoGEF基因的转录谱。使用小发夹RNA敲低感兴趣的基因。
在18种RhoGTP酶和76种RhoGEFs中，有三个基因的表达被RANKL上调：RhoGTP酶RhoU/Wrch1、Dbl家族交换因子Arhgef8/Net1和CZH家族交换因子Dock5。在RAW264.7细胞中进行的基因芯片和实时定量PCR实验中观察到了这种诱导。在骨髓巨噬细胞和造血干细胞中进一步得到证实。沉默Wrch1和Arhgef8的表达严重抑制分化并影响破骨细胞形态。抑制Dock5对破骨细胞前体细胞是致命的，而对成纤维细胞没有影响。
我们在RhoGTP酶信号通路中鉴定出三个在RANKL诱导的破骨细胞生成过程中上调的基因。这些基因是破骨细胞中的新的重要作用因子，很可能是通过控制肌动蛋白细胞骨架动力学来实现的。
