RhoA、RhoC及其效应分子ROCK-1的表达与卵巢癌细胞系恶性行为相关性的体外研究

目的　研究RhoA、RhoC及其效应分子ROCK 1在卵巢癌细胞中的表达水平 ,探讨其与卵巢癌转移的相关性。方法　逆转录聚合酶链反应 (RT PCR)检测RhoA、RhoC及ROCK 1在SW6 2 6、Skov 3、A2 780和Caov 34种卵巢癌细胞中mRNA的表达水平 ;Westernblot法检测RhoA和ROCK 1蛋白质的表达情况 ;Boyden小室体外侵袭试验测定 4种卵巢癌细胞的体外迁移与侵袭能力。结果　RhoA、RhoC和ROCK 1在 4种卵巢癌细胞中呈不同程度表达 ,其中仅RhoC的表达水平与癌细胞侵袭力和迁移能力呈正相关 (r=0 .95 ,P <0 .0 1)。RhoA在转录水平和蛋白水平中呈不同步变化 ,且RhoA和RhoC的表达与ROCK 1的表达无相关性。 4种卵巢癌细胞的体外侵袭和迁移能力相一致 ,其中SW6 2 6最强 ,Caov 3最弱 ,Skov 3和A2 780居中。结论　RhoC的表达与卵巢癌细胞的体外侵袭和迁移能力相关 ,可能作为一个独立因素在卵巢癌的转移过程中起作用 ,有望成为阻断卵巢癌转移的新靶点。     

The effects of artificial E-cadherin matrix-induced embryonic stem cell scattering on paxillin and RhoA activation via α-catenin

Mechanical forces have been shown to affect stem cell behavior in a large array of ways. However, our understanding of how these mechanical cues may regulate the behavior of embryonic stem cells (ESCs) remains in its infancy. Here, we aim to clarify the effect of cell scattering on the regulation of Rho family GTPases Rac1 and RhoA as well as paxillin. Allowing ESCs to spread and scatter on a synthetically designed E-cadherin substratum causes phosphorylation of paxillin on consensus phosphorylation sites leading to activation of Rac1 and inactivation of RhoA. By culturing cells in presence of RhoA activator or growing cells to a highly confluent state reverses the effect of cell scattering phenotype. Knockdown of E-cadherin-adapter protein α-catenin revealed that it negatively affects paxillin phosphorylation and up-regulates RhoA activity in compact cellular aggregates. Collectively these results indicate that cell scattering might cause a conformational change of α-catenin limiting its capacity to inhibit paxillin phosphorylation that causes an increase in Rac1 activation and RhoA deactivation. Understanding how synthetically designed extracellular matrix affect ESC signaling through mechanical cues brings a new aspect for stem cell engineers to develop technologies for controlling cell function.     

Recent Advances in Pathology: the 2020 Annual Review Issue of The Journal of Pathology

This year's Annual Review Issue of The Journal of Pathology contains 18 invited reviews on current research areas in pathology. The subject areas reflect the broad range of topics covered by the journal and this year encompass the development and application of software in digital histopathology, implementation of biomarkers in pathology practice; genetics and epigenetics, and stromal influences in disease. The reviews are authored by experts in their field and provide comprehensive updates in the chosen areas, in which there has been considerable recent progress in our understanding of disease. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Xenopus Rnd1 and Rnd3 GTP‐binding proteins are expressed under the control of segmentation clock and required for somite formation

The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor‐8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP‐binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant‐negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation‐related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis. Developmental Dynamics 238:2867–2876, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of RhoGEF proteins by G12/13-coupled receptors

G protein-coupled receptors (GPCRs) represent a large family of seven transmembrane receptors, which communicate extracellular signals into the cellular lumen. The human genome contains 720–800 GPCRs, and their diverse signal characteristics are determined by their specific tissue and subcellular expression profiles, as well as their coupling profile to the various G protein families (G_s, G_i, G_q, G₁₂). The G protein coupling pattern links GPCR activation to the specific downstream effector pathways. G_12/13 signalling of GPCRs has been studied only recently in more detail, and involves activation of RhoGTPase nucleotide exchange factors (RhoGEFs). Four mammalian RhoGEFs regulated by G_12/13 proteins are known: p115-RhoGEF, PSD-95/Disc-large/ZO-1 homology-RhoGEF, leukemia-associated RhoGEF and lymphoid blast crisis-RhoGEF. These link GPCRs to activation of the small monomeric GTPase RhoA, and other downstream effectors. Misregulated G_12/13 signalling is involved in multiple pathophysiological conditions such as cancer, cardiovascular diseases, arterial and pulmonary hypertension, and bronchial asthma. Specific targeting of G_12/13 signalling-related diseases of GPCRs hence provides novel therapeutic approaches. Assays to quantitatively measure GPCR-mediated activation of G_12/13 are only emerging, and are required to understand the G_12/13-linked pharmacology. The review gives an overview of G_12/13 signalling of GPCRs with a focus on RhoGEF proteins as the immediate mediators of G_12/13 activation.     

Synaptic Plasticity,a Symphony in GEF

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RhoGTP酶对血吸虫病鼠肝窦毛细血管化的调控作用

目的探讨RhoGTP酶在肝窦内皮细胞转分化过程中的调控作用,及其对血吸虫病性鼠肝窦毛细血管化病变的影响和可能机制。方法采用腹部敷贴法感染血吸虫尾蚴建立血吸虫病性肝纤维化模型,于13周吡喹酮顿服杀虫,14周追加hydroxyfasudil。13周时正常对照组（A组,10只）与血吸虫病组（B组,6只）,16、19周和21周后（B组）、杀虫实验对照组（C组）、血吸虫病＋hydroxyfasudil组（D组）、杀虫＋hydroxyfasudil组（E组）各6只）剖腹取肝组织。分别作HE、Masson染色和透射电镜观察;免疫组化检测：p-膜联蛋白、CTGF、CD31、ColⅣ和LN;Western印迹检测：p-膜联蛋白、CTGF、RhoA、ColⅣ和LN蛋白表达;逆转录-聚合酶链反应（RT-PCR）检测：CTGF、RhoA、ROCKⅡ mRNA水平。结果与A组相比,动态观察B组肝组织RhoA、ROCKⅡ及CTGF mRNA表达水平明显上升,p-膜联蛋白、CTGF、RhoA、ColⅣ和LN蛋白表达量也增高。给予干预措施后,与C组及其它组相比,E组CTGF mRNA表达于16周显著下调,同时显示CTGF、LN、ColⅣ蛋白水平也降低。与B组相比,D组、E组p-膜联蛋白降低,16周时最为显著,但D组于19周开始p-膜联蛋白表达量渐恢复到原水平,其中E组明显低于D组。肝窦的超微结构观察,于21周时C组分别与B、D组相比,浸润的炎性细胞明显减少,Disse腔内胶原纤维有所减少,但肝窦内皮细胞窗孔、肝窦内皮下基底膜差异无统计学意义。E组与C组比较肝细胞器形态明显恢复,可见窗孔,未见基底膜。结论RhoGTP酶在调控血吸虫病性鼠肝窦内皮细胞转分化过程中,通过上调CTGF和p-膜联蛋白发挥作用,抑制RhoGTP酶信号通路有可能为防治肝窦毛细血管化提供新的有效靶点。     

Rho家族成员RhoC和Rac1在乳腺癌中的表达及其意义

目的检测RhoC和Rac1在人乳腺癌中表达的情况及意义。方法免疫组化S-P法检测60例乳腺浸润性导管癌组织以及20例乳腺良性疾病组织中RhoC和Rac1蛋白的表达情况,Western blot方法检测15例乳腺癌以及9例乳腺良性疾病组织中RhoC和Rac1蛋白的表达情况。结果 RhoC和Rac1蛋白在乳腺癌组织中高表达,并且与乳腺良性疾病组织中的表达相比,差异有统计学意义(P<0.05)。结论 RhoC和Rac1蛋白在乳腺癌中高表达,说明其与乳腺癌的发生、发展的临床效果关系密切,能通过其相应机制来参与乳腺癌侵袭和转移。提示RhoC和Rac1蛋白可能成为新的肿瘤标志物,在乳腺肿瘤的诊断及采取与之相对应的、切实有效的治疗方面具有十分重要的临床意义。