Meyers Valerie E, Zayzafoon Majd, Douglas Joanne T, McDonald Jay M
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0007, USA.
J Bone Miner Res. 2005 Oct;20(10):1858-66. doi: 10.1359/JBMR.050611. Epub 2005 Jun 27.
Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity.
Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects.
hMSCs were seeded onto plastic microcarrier beads at a density of 10(6) and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM beta-glycerol phosphate, and 50 muM ascorbic acid 2-phosphate.
F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88+/-2% and 77+/-9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG.
Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.
太空飞行、衰老和废用会导致骨密度降低。本研究表明，组成型活性RhoA的过表达可恢复肌动蛋白细胞骨架排列，增强成骨细胞表型，并抑制在模拟微重力条件下培养的人间充质干细胞的脂肪细胞表型。
太空飞行期间骨密度降低部分是由于骨形成减少。然而，导致这种骨质流失的机制仍不清楚。我们之前已经表明，在模拟微重力（MMG）条件下培养的人间充质干细胞（hMSCs）的成骨细胞生成减少而脂肪生成增强。小GTP酶RhoA调节肌动蛋白应力纤维的形成，并与hMSCs的谱系定向有关。我们研究了MMG对肌动蛋白细胞骨架组织和RhoA活性的影响，以及组成型活性RhoA逆转这些影响的能力。
将hMSCs以10⁶的密度接种到塑料微载体珠上，并在含有10%胎牛血清的DMEM中形成聚集体7天。聚集体在含有2%胎牛血清的DMEM中孵育6小时，加入或不加入含有组成型活性RhoA的腺病毒载体，感染复数（moi）为500，然后在10%胎牛血清中恢复24小时。将细胞转移到旋转细胞培养系统中模拟微重力，或在DMEM、10%胎牛血清、10 nM地塞米松、10 mMβ-甘油磷酸和50 μM抗坏血酸2-磷酸中在正常重力下维持7天。
在MMG开始后3小时内，hMSCs中的F-肌动蛋白应力纤维被破坏，到7天时完全消失，而单体G-肌动蛋白增加。由于G-肌动蛋白与脂肪细胞中的脂滴相关，在MMG中培养的hMSCs中观察到的细胞内脂质积累增加310%也就不足为奇了。与这些细胞形态变化一致，7天的MMG分别显著降低RhoA活性以及随后的丝切蛋白磷酸化88±2%和77±9%。重要的是，引入表达组成型活性RhoA的腺病毒构建体可逆转应力纤维的消除，显著增加在MMG中培养的hMSCs中I型胶原蛋白、碱性磷酸酶和与矮小相关转录因子2的成骨细胞基因表达，并抑制瘦素和葡萄糖转运蛋白4的脂肪细胞基因表达。
MMG期间RhoA活性的抑制代表了hMSCs成骨细胞生成减少和脂肪生成增强的一种新机制。
