Cellular and subcellular localization of the small G protein RhoA in the human and rat embryonic and adult kidney

Rho proteins, a subgroup of the Ras GTPase superfamily, control many cellular processes and morphogenetic events by acting as signaling molecules in the transduction pathways of various receptors. Among the "Rho-dependent" receptors are the extracellular matrix- and growth factor-binding sites; these are particularly involved in the modulation of renal development since they control the epithelial-mesenchymal interactions that drive kidney organogenesis. The present study has addressed the immunohistochemical localization of RhoA in developing and adult kidneys of rats and humans because: a) Rho proteins are known to have a morphogenetic role, b) data in the literature on expression of Rho GTPases during mammalian histogenesis and organogenesis are scarce, and c) their involvement in the transduction pathways of receptors is implicated in kidney development. In particular, RhoA peptide was found to be localized in the mesonephric duct and vesicles in both rats and humans; metanephric anlagen were mainly stained in ampullar-derived cells. Periglomerular tubules of fetal and adult kidneys as well as collecting ducts of adult kidneys showed intense staining. Therefore, the present study provides new information on the distribution patterns of RhoA during early stages of mammalian kidney development suggesting that this signaling molecule may take part in epithelial-mesenchymal induction processes that control kidney organogenesis. RhoA expression in adult structures may be linked with renewal of renal epithelial cells and the maintenance of their morphology and polarity.     

RhoA在子痫前期患者胎盘中的表达

目的:检测正常晚期妊娠和子痫前期患者胎盘RhoA的表达,探讨RhoA在子痫前期发病中的作用。方法:用免疫组织化学SP法及RT-PCR检测40例子痫前期和20例正常晚期妊娠胎盘RhoA蛋白及其mRNA的表达。结果:RhoA主要在滋养细胞表达。轻度子痫前期组和重度子痫前期组RhoA蛋白及mRNA均高于正常晚期妊娠组,差异有统计学意义(P<0.05)。结论:子痫前期患者胎盘RhoA的高表达可能与子痫前期的发病有一定的关系。     

急性肺损伤中炎症因子 IL-8与 RhoA 的相关性研究

目的：探讨油酸型急性肺损伤模型中IL‐8与RhoA的相关性。方法建立大鼠油酸急性肺损伤模型,检测各组大鼠肺湿／干重比及肺泡灌洗液（BALF）中中性粒细胞比例,比较肺组织病理学改变;采用聚合酶链反应检测肺组织匀浆RhoA表达,酶联免疫吸附法（ELISA ）测定大鼠IL‐8的变化。结果肺W／D损伤组在2、6、12、24 h均较正常组明显升高,并且损伤组2 h比值最高;损伤组各时间点BALF中中性粒细胞计数比例明显高于正常组,24 h中性粒细胞细胞比例达峰值;损伤组个时间点血浆及BALF中IL‐8较正常组明显升高,两者具有相关性;损伤组各时间点RhoA的表达明显高于正常组。结论油酸型急性肺损伤大鼠中存在RhoA的高表达,RhoA表达增加可致IL‐8的表达增加,进而引起炎症细胞浸润。     

基于RhoA/ROCK-1通路研究针刺干预SHR脑保护作用机制

[目的] 明确自发性高血压大鼠（SHR）脑损害与RhoA、ROCK-1的相关性,探讨针刺干预SHR脑保护的Rho通路机制。[方法] 将SHR随机分为模型对照组、太冲穴组,WKY大鼠设为正常对照组。太冲穴组针刺干预4周,其余两组在干预期内给予相同程度、相同时长的抓取。观测各组大鼠收缩压、舒张压、平均动脉压的改变;苏木精-伊红（HE）染色观察脑组织形态学改变;蛋白免疫印迹（Western blot）法测定各组大鼠脑组织中RhoA、ROCK-1的表达水平。[结果] 在干预周期内,正常对照组收缩压、舒张压、平均压均处于正常血压范围内。与正常对照组比较,模型对照组收缩压、舒张压、平均压均较高,且有随着周龄增加而增高的趋势。与模型对照组相比,太冲穴组可有效降低收缩压,有降低舒张压、平均压的趋势。模型对照组较正常对照组脑组织细胞排列紊乱,细胞坏死及间质水肿程度加重,RhoA、ROCK-1表达升高;太冲穴组与模型对照组比较,脑细胞排列规则,细胞坏死及间质水肿消失,RhoA、ROCK-1表达下降。[结论] 针刺太冲穴能有效降低SHR血压,改善脑损害,其机制可能与抑制RhoA/ROCK-1信号通路表达相关。     

C3 exoenzyme lacks effects on peripheral axon regeneration in vivo

Peripheral nerve injury triggers the activation of the small GTPase RhoA in spinal motor and peripheral sensory neurons. C3 transferase, an exoenzyme produced by Clostridium botulinum that inactivates RhoA by ADP‐ribosylation, has been successfully applied in central nervous system (CNS) lesion models to facilitate regeneration functionally and morphologically. Until now it has not been demonstrated if C3_bot exerts positive effects on peripheral axon regeneration as well. In organotypic spinal cord preparations, C3_bot reduced axonal growth of motoneurons, while no effect on sensory axon outgrowth from dorsal root ganglia (DRG) explants was observed. Enzymatically inactive C3_E174Q was ineffective in both culture models. Spinal cord slices exhibited a significant increase in microglia/macrophages after treatment with C3_bot suggesting an inflammatory component in the inhibition of axon growth. C3_bot or C3_E174Q were then applied into conduits implanted after transection of the sciatic nerve in rats. Functional evaluation by electrophysiology, nociception, and walking track tests did not show any significant difference between groups with active or mutant C3_E174Q. Transmission electron microscopy of the regenerated nerves revealed no significant differences in the number of myelinated and unmyelinated axons 6 weeks after surgery. Compared to the CNS, the functional significance of RhoA may be limited during nerve regeneration in a growth‐promoting environment.     

腺病毒siRNA抑制RhoA基因并联合TNF-α诱导肝癌细胞凋亡的研究

目的建立RhoA基因的腺病毒siRNA系统,并联合TNF-α诱导肝癌细胞凋亡,分析RhoA在肿瘤细胞中的功能和作用。方法用已构建的RhoA干扰质粒构建RhoA的腺病毒siRNA系统,筛选重组病毒并感染HepG2细胞,应用Western blot和RT-PCR检测RhoA蛋白表达和基因表达水平。感染Ad-siRNA-RhoA腺病毒的肝癌细胞用TNF-α诱导后进行MTT以及细胞内DNA片段化检测。结果成功构建RhoA基因的siRNA腺病毒系统。用重组病毒感染肝癌细胞,RhoA蛋白表达抑制率为76.48%;RhoA基因的mRNA转录水平降低74.46%。MTT检测显示,感染腺病毒Ad-U6-control对照组以及感染腺病毒Ad-siRNA-RhoA实验组的细胞A值差异无统计学意义(F=5.41,P>0.01),即利用siRNA抑制肝癌细胞中RhoA表达,肿瘤细胞凋亡不明显。TUNEL检测显示,RhoA腺病毒siRNA联合TNF-α致肿瘤细胞凋亡作用显著。结论构建的腺病毒siRNA载体系统能抑制目的基因RhoA的表达,联合TNF-α能抑制肿瘤细胞生长和增殖并诱导肿瘤细胞凋亡,为肿瘤基因功能的基础研究和肿瘤基因治疗打下了实验基础。     

α2β1 integrin and RhoA mediates electric field‐induced ligament fibroblast migration directionality

Guided cell migration is important in tissue development, repair, and engineering. We have previously demonstrated that applied electric fields (EFs) enhanced and directed ligament fibroblast migration and collagen production, depending on EF parameters. Electrical stimulation is widely used for the treatment of pain and to promote wound healing. In orthopaedic practices, applied EFs promote bone healing and ligament repair in vivo. In the current study, stimulation waveforms used in physical therapy for promoting tissue repair were adapted to examine their effects on ACL fibroblast migration. Using different waveform and field strengths, we discovered a decoupling of cell motility and directionality, which suggests disparate mechanisms. Integrin, a major extracellular matrix receptor, polarized in response to applied EFs and controlled cell directionality and signaling. Furthermore, we demonstrated that RhoA is a mediator between integrin aggregation and directed cell migration. Polarization is essential in directed cell migration and our study establishes an outside‐in signaling mechanism for EF‐induced cell directionality. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 322–327, 2013     

Nano-topography: Quicksand for cell cycle progression?

The 3-D spatial and mechanical features of nano-topography can create alternative environments, which influence cellular response. In this paper, murine fibroblast cells were grown on surfaces characterized by protruding nanotubes. Cells cultured on such nano-structured surface exhibit stronger cellular adhesion compared to control groups, but despite the fact that stronger adhesion is generally believed to promote cell cycle progression, the time cells spend in G1 phase is doubled. This apparent contradiction is solved by confocal microscopy analysis, which shows that the nano-topography inhibits actin stress fiber formation. In turn, this impairs RhoA activation, which is required to suppress the inhibition of cell cycle progression imposed by p21/p27. This finding suggests that the generation of stress fibers, required to impose the homeostatic intracellular tension, rather than cell adhesion/spreading is the limiting factor for cell cycle progression. Indeed, nano-topography could represent a unique tool to inhibit proliferation in adherent well-spread cells.     

β-Catenin Preserves the Stem State of Murine Bone Marrow Stromal Cells Through Activation of EZH2

During bone marrow stromal cell (BMSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin plays a critical role in the Wnt signaling pathway to facilitate downstream effects on gene expression. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chromatin immunoprecipitation (ChIP)-sequencing demonstrated binding of β-catenin to the promoter of enhancer of zeste homolog 2 (EZH2), a key component of the polycomb repressive complex 2 (PRC2) complex that catalyzes histone methylation. Knockdown of β-catenin reduced EZH2 protein levels and decreased methylated histone 3 (H3K27me3) at osteogenic loci. Further, when EZH2 was inhibited, β-catenin's anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin's effects on BMSCs to preserve multipotentiality. © 2020 American Society for Bone and Mineral Research.