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首页> 外文期刊>Bone >Rho GTPase signaling and PTH 3-34, but not PTH 1-34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells.
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Rho GTPase signaling and PTH 3-34, but not PTH 1-34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells.

机译:Rho GTPase信号传导和PTH 3-34(而非PTH 1-34)在小鼠成骨细胞MC3T3-E1细胞中维持肌动蛋白细胞骨架并拮抗双膦酸酯作用。

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摘要

Cytoskeletal elements are critical for cell morphology and signal transduction, and are involved in many cellular processes including motility, intracellular transport, and differentiation. Small GTP-binding proteins (G proteins) of the Ras family, such as RhoA, influence various elements of the cytoskeleton. RhoA stabilizes the actin cytoskeleton and promotes formation of focal adhesions. We found previously that RhoA is expressed in osteoblastic cells and is translocated to the plasma membrane and activated by PTH 1-34 as well as by Nleu(8,18) Tyr(34) PTH 3-34 amide, a PTH analog that does not increase cAMP. We therefore investigated effects of manipulating RhoA on the actin cytoskeleton of osteoblastic MC3T3-E1 cells. Three inhibitors were used: 1) GGTI-2166, a geranylgeranyl transferase I inhibitor that prevents the isoprenylation and membrane translocation of RhoA, 2) Y-27632, a Rho kinase inhibitor, and 3) alendronate, a nitrogen (N)-containing bisphosphonate that reduces intracellular geranylgeranylpyrophosphate through inhibiting farnesyl pyrophosphate synthase. To increase RhoA activity, we used the geranylgeranyl group donor geranylgeraniol (GGOH), and a constitutively active RhoA. The F-actin cytoskeleton and focal adhesions (FA) were visualized with rhodamine-phalloidin and fluorescent anti-vinculin antibodies, respectively. Cells were imaged with confocal microscopy. Actin stress fiber density, edge actin bundle density, focal adhesion density, cellular area and circularity (a morphological descriptor relating area and perimeter) were quantified by a program developed with Matlab software. GGTI-2166, Y-27632, and alendronate reduced actin stress fibers, FA density, and FA size, but had no effect on edge actin bundle density, cellular area, or circularity. GGOH completely antagonized the effects of alendronate, but did not significantly affect responses to GGTI-2166 or Y-27632. Constitutively active RhoA antagonized the effects of alendronate and GGTI-2166, but not those of Y-27632. The effects of alendronate were also antagonized by Nleu(8,18) Tyr(34) PTH 3-34 amide, but not by PTH 1-34. The results indicate that RhoA is involved in the maintenance of stress fibers and focal adhesions in osteoblastic cells, that PTH can affect this pathway independently of cAMP, and that a N-containing bisphosphonate can affect the actin cytoskeleton and focal adhesions through actions on geranylgeranyl groups and potentially through RhoA. In view of the importance of the actin cytoskeleton, the findings constitute evidence that N-containing bisphosphonates, when they attain certain concentrations, have effects on osteoblasts that could influence bone remodeling.
机译:细胞骨架元件对于细胞形态和信号转导至关重要,并参与许多细胞过程,包括运动性,细胞内转运和分化。 Ras家族的小GTP结合蛋白(G蛋白),例如RhoA,会影响细胞骨架的各种元素。 RhoA稳定肌动蛋白的细胞骨架,并促进粘着斑的形成。我们以前发现,RhoA在成骨细胞中表达,并易位至质膜,并由PTH 1-34以及Nleu(8,18)Tyr(34)PTH 3-34酰胺(一种没有这种作用的PTH类似物)激活增加cAMP。因此,我们研究了操纵RhoA对成骨细胞MC3T3-E1细胞肌动蛋白细胞骨架的影响。使用了三种抑制剂:1)GGTI-2166,一种防止RhoA异戊二烯化和膜移位的香叶基香叶基转移酶I抑制剂,2)Y-27632,一种Rho激酶抑制剂,和3)阿仑膦酸盐,一种含氮(N)的双膦酸酯通过抑制法呢基焦磷酸合酶减少细胞内的香叶基香叶基焦磷酸。为了增加RhoA活性,我们使用了香叶基香叶基供体香叶基香叶醇(GGOH)和功能性RhoA。 F-肌动蛋白的细胞骨架和粘着斑(FA)分别用若丹明-鬼笔环肽和荧光抗-vinculin抗体观察。用共聚焦显微镜对细胞成像。肌动蛋白应力纤维密度,边缘肌动蛋白束密度,粘着斑密度,细胞面积和圆形度(与面积和周长有关的形态学描述符)通过Matlab软件开发的程序进行定量。 GGTI-2166,Y-27632和阿仑膦酸盐降低了肌动蛋白应力纤维,FA密度和FA大小,但对边缘肌动蛋白束密度,细胞面积或圆形度没有影响。 GGOH完全拮抗了阿仑膦酸盐的作用,但并未显着影响对GGTI-2166或Y-27632的反应。组成型活性RhoA拮抗了阿仑膦酸盐和GGTI-2166的作用,但没有拮抗Y-27632的作用。阿仑膦酸的作用也被Nleu(8,18)Tyr(34)PTH 3-34酰胺拮抗,但不受PTH 1-34拮抗。结果表明,RhoA参与了成骨细胞中应力纤维和粘着斑的维持,PTH可以独立于cAMP来影响该途径,而含N的双膦酸酯可以通过对香叶基香叶基的作用来影响肌动蛋白的细胞骨架和粘着斑。并可能通过RhoA。考虑到肌动蛋白细胞骨架的重要性,这些发现构成了证据,即当含氮的双膦酸盐达到一定浓度时,它们会对成骨细胞产生影响,可能会影响骨骼的重塑。

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