血液滤过联合灌流治疗对重症急性胰腺炎继发肺损伤患者血清TNF-α及血管内皮细胞RhoA磷酸化修饰的影响

目的探讨血液滤过联合血液灌流(HF+HP)治疗对重症急性胰腺炎(SAP)继发肺损伤患者血清中TNF-α的清除作用及对内皮细胞中RhoA的188位丝氨酸磷酸化修饰(p-RhoA)的影响。方法选取35例SAP患者纳入实验研究,其中SAP继发急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)28例,未继发ALI/ARDS 7例,同时设正常对照组20例。全部SAP患者收入中心ICU治疗,其中9例SAP继发ARDS患者给予连续性静脉-静脉血液滤过(CVVH)联合HP治疗(HF+HP组)。应用ELISA法检测不同程度肺损伤的SAP患者血清中TNF-α水平的变化,以及采用HF+HP治疗的9例SAP继发ARDS患者治疗不同时间点血清中TNF-α的浓度。体外实验采用HF+HP治疗不同时间点患者的血清分别刺激体外培养的人脐静脉内皮细胞(HUVECs),并以重组人TNF-α刺激细胞作为阳性对照,蛋白质印迹分析检测各组HUVECs中p-RhoA与总RhoA蛋白的表达,免疫荧光染色观察p-RhoA在细胞内的分布。结果与未继发ALI/ARDS患者比较,SAP继发ALI/ARDS的患者血清中TNF-α水平明显升高(P<0.05),尤以继发ARDS的患者升高最为明显(约为正常对照组的7倍)。采用HF+HP治疗的9例SAP继发ARDS患者治疗6h后,血清中TNF-α水平与HF+HP治疗前比较开始下降,至治疗20h下降最为明显,接近正常组水平;HF+HP治疗后,SAP继发ARDS患者动脉血氧分压(PaO2)、HCO3-水平、氧合指数(PaO2/FiO2)均较治疗前上升(P<0.05)。蛋白质印迹分析结果表明,HF+HP治疗后SAP继发ARDS患者血清诱导的HUVECs中p-RhoA水平逐渐升高(P<0.05),但各组RhoA总蛋白表达水平差异无统计学意义(P>0.05)。免疫荧光结果显示,在HUVEsC中,p-RhoA主要分布在细胞质,各组变化趋势与蛋白质印迹分析结果一致。结论 SAP继发ALI/ARDS患者血循环中存在高水平的TNF-α。HF+HP治疗可有效地清除SAP继发ALI/ARDS患者血液中大量生成的TNF-α,并抑制RhoA的活化,从而起到降低内皮细胞高通透性的作用。     

Molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteinechallenged rat aortic smooth muscle cells

Background and Purpose

Homocysteine is an intermediate product formed during the metabolism of methionine, and is increased in cells with folate deficiency. Patients with hyperhomocysteinemia tend to develop cardiovascular disease. Here, we have examined the molecular mechanisms underlying the anti-proliferative and anti-migratory effects of folate on homocysteine-challenged rat aortic smooth muscle cells (RASMCs).

Experimental Approach

Cultures of RASMC were challenged with homocysteine and then incubated with folate added. Changes in p21/p27, AKT and RhoA were followed by RT-PCR, Western blotting and immunocytochemistry. Transfection and anti-sense techniques were also used. Cell viability, growth and migration were measured.

Key Results

Folate up-regulated p21/p27 through a Src/ERK-dependent mechanism that accounted for its anti-proliferative effects on RASMC. Folate protected RASMC from the effects of homocysteine by reducing AKT1, focal adhesion kinase (FAK), paxillin, and p190RhoGAP activation/phosphorylation, along with cytosolic levels of p21 and p27, and increasing RhoA activation. Overexpression of AKT1, but not of AKT2, induced p21/p27 phosphorylation and increased cytosolic p21/p27 levels, as did homocysteine treatment. By contrast, and similarly to folate treatment, transfection with dominant negative (DN) AKT1 counteracted these effects. Additionally, AKT was shown to be an upstream target of FAK activation. In RASMC overexpressing constitutively active RhoA, activation of RhoA mediated the anti-migratory effects of folate. Addition of Y27632 (a RhoA inhibitor) and DNRhoA counteracted the anti-migratory effects, confirming RhoA involvement.

Conclusion and implications

Folate was anti-proliferative and anti-migratory in homocysteine-challenged RASMC. Mechanisms underlying folate-mediated protection against the proatherosclerotic effects of homocysteine have been delineated.     

癌基因RhoA对乳腺癌细胞血管内皮生长因子表达的调控机制研究

目的探讨乳腺癌细胞MDA-MB-231中癌基因Rho A对VEGF蛋白表达和胞外分泌的影响及可能的分子机制。方法将Rho A过表达质粒pc DNA3.0-V14Rho A、对照质粒pc DNA3.0、Rho A沉默质粒pc DNA3.0-shRho A转染到MDA-MB-231细胞中,通过Western blot和ELISA实验分别检测细胞内外VEGF蛋白的表达,通过Western blot和实时PCR方法分别检测Rho A对p53表达的影响及对VEGF的调控作用。均数比较用t检验;计量资料用x^-±s表示,采用方差分析。结果 MDA-MB-231细胞中上调Rho A表达后,胞内VEGF的蛋白表达水平增加;胞外分泌水平显著增加,与对照组相比差异具有统计学意义（F=4.020,P=0.032）;MDA-MB-231细胞中沉默Rho A表达后,胞内VEGF的蛋白表达水平降低;胞外分泌水平显著降低,与对照组相比差异具有统计学意义（F=5.131,P=0.001）;并且与0 h相比,在MDA-MB-231细胞中上调Rho A可以抑制p53的表达（48 h：F=3.231,P=0.043）,而p53表达的降低可以增加VEGF的表达水平（48 h：F=3.226,P=0.015）,均差异具有统计学意义。结论乳腺癌细胞MDA-MB-231中Rho A表达的变化可以引起胞内外VEGF水平的变化,并且Rho A可能是通过抑制p53的表达从而增加VEGF表达。     

Narciclasine as well as other Amaryllidaceae Isocarbostyrils are Promising GTP‐ase Targeting Agents against Brain Cancers

The anticancer activity of Amaryllidaceae isocarbostyrils is well documented. At pharmacological concentrations, that is, approximately 1 μM in vitro and approximately 10 mg/kg in vivo, narciclasine displays marked proapoptotic and cytotoxic activity, as does pancratistatin, and significant in vivo anticancer effects in various experimental models, but it is also associated with severe toxic side effects. At physiological doses, that is, approximately 50 nM in vitro and approximately 1 mg/kg in vivo, narciclasine is not cytotoxic but cytostatic and displays marked anticancer activity in vivo in experimental models of brain cancer (including gliomas and brain metastases), but it is not associated with toxic side effects. The cytostatic activity of narciclasine involves the impairment of actin cytoskeleton organization by targeting GTPases, including RhoA and the elongation factor eEF1A. We have demonstrated that chronic treatments of narciclasine (1 mg/kg) significantly increased the survival of immunodeficient mice orthotopically xenografted with highly invasive human glioblastomas and apoptosis‐resistant brain metastases, including melanoma‐ and non‐small‐cell‐lung cancer‐ (NSCLC) related brain metastases. Thus, narciclasine is a potentially promising agent for the treatment of primary brain cancers and various brain metastases. To date, efforts to develop synthetic analogs with anticancer properties superior to those of narciclasine have failed; thus, research efforts are now focused on narciclasine prodrugs.     

Rho GTPases in PC-3 prostate cancer cell morphology,invasion and tumor cell diapedesis

BACKGROUND: The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. RESULTS: Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. CONCLUSIONS: Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.     

In vivo BDNF modulation of adult functional and morphological synaptic plasticity at hippocampal mossy fibers

The Rho family of small GTPase proteins are involved in the formation and maintenance of neuronal dendrites. In this study, we show that Daam1, a member of the Diaphanous-related formin protein family and a downstream effector for RhoA, is localized to the dendrites of hippocampal neurons. Immunoblot analysis showed that Daam1 is enriched in the mouse hippocampus and co-fractionates in brain lysates with dendritic and synaptic proteins. Immunohistochemical analysis revealed that Daam1 protein distributes in a punctate pattern throughout the cell body and dendritic shafts of dissociated hippocampal neurons and organotypic hippocampal cultures. Although Daam1 is mostly expressed in the shaft of dendrites, co-stainings with SV2 or PSD95 revealed that Daam1 is also present at some synapses. In addition, viral directed expression of a fluorescently tagged Daam1 fusion protein in hippocampal slices resulted in targeted delivery to the dendrites of pyramidal neurons, leading to a reduction in the density of spines.     

Marked effect of RhoA-specific shRNA-producing plasmids on neurite growth in PC12 cells

In order to promote neurite outgrowth, we constructed a plasmid producing RhoA-specific small hairpin RNA (shRNA) to block RhoA expression and tested its actions in PC12 cells. Our results show that the shRNA-mediated RNA interference (RNAi) successfully suppressed RhoA expression. As a consequence of RhoA knockdown, F-actin levels were significantly reduced and processes were markedly induced. These processes express two neurite markers, neurofilament and betaIII tubulin. This study demonstrates that plasmid-producing shRNA specific for RhoA can act as an effective tool to induce neurite outgrowth and further confirms the neurite growth-inhibitory role of RhoA. This shRNA may thus be a useful tool in promoting neurite outgrowth and could be applicable in neural repair after CNS injury.     

Prognostic significance of immunohistochemical RhoA expression on survival in pancreatic ductal adenocarcinoma: a high-throughput analysis

Among all human carcinomas, pancreatic cancer has one of the worst survival rates. Most patients will die of this cancer shortly after diagnosis, and currently, surgery is the only potential cure. Ductal adenocarcinoma is the most common histologic type. The search for prognostic parameters has progressed from mere physical or histomorphological tumor properties to molecular parameters. These, in turn, might point toward new therapeutic strategies. The K-ras oncogene is known to play a role in early stages of ductal adenocarcinoma carcinogenesis, and ras homologues are differentially expressed in cancerous versus normal ductal cells. RhoA belongs to a family of ras homologues comprising RhoA, RhoB, and RhoC. It is a guanosine triphosphatase associated with the cytoskeleton that seems to be involved in epithelial mesenchymal transition, a process of dedifferentiation. Immunohistologic RhoA expression was studied in a tissue microarray of 94 pancreatic ductal adenocarcinomas and correlated with clinicopathologic parameters and follow-up. RhoA protein expression, measured as labeling intensity or evaluated as percentage of reactive tumor cells, correlated with overall survival. A multivariate analysis demonstrated that RhoA protein expression is independent from other known prognostic parameters such as tumor size or grade. Moreover, a score combining RhoA expression with tumor size and grade resulted in a highly significant increase in the prognostic value for the overall survival of patients with pancreatic ductal adenocarcinoma.     

Multi-layered hypertrophied MEE formation by microtubule disruption via GEF-H1/RhoA/ROCK signaling pathway

Background: Formation of the secondary palate is complex and disturbance during palatal fusion may result in cleft palate. The processes of adhesion, intercalation, and disappearance of medial edge epithelia (MEE) are characterized by morphological changes requiring dynamic cytoskeletal rearrangement. Microtubules are one of the cytoskeletal elements involved in maintenance of cell morphology. Microtubule‐disrupting drugs have been reported to cause craniofacial malformations including cleft palate. The mechanisms underlying the failure of palatal fusion remain poorly understood. We evaluated the effect of nocodazole (NDZ), a drug that disrupts microtubules, on palatal fusion in organ culture. Results: NDZ caused failure of palatal fusion due to the induction of a multi‐layered hypertrophied MEE in the mid‐region of the secondary palatal shelves. Microtubule disruption increased RhoA activity and stress fiber formation. Pharmacological inhibition of the RhoA/ROCK pathway blocked multi‐layered MEE formation. Partial prevention of hypertrophied MEE was observed with Y27632 and cytochalasin, but not with blebbistatin. NDZ induced re‐localization of GEF‐H1 into cytoplasm from cell–cell junctions. Conclusions: The present study provided evidence that the GEF‐H1/RhoA/ROCK pathway plays a pivotal role in linking microtubule disassembly to the remodeling of the actin cytoskeleton, which resulted in a multi‐layered hypertrophied MEE and failure of palatal fusion. Developmental Dynamics 241:1169–1182, 2012. © 2012 Wiley Periodicals, Inc.     

RhoA蛋白参与凝血酶对胎鼠大脑皮层神经元的损伤

目的 探讨凝血酶诱导的胎鼠大脑皮层神经元损伤与RhoA蛋白活化表达的关系.方法 体外培养胎鼠大脑皮层神经元8d后,采用Western blotting检测不同浓度凝血酶(0、1、10、30和100 U/mL)作用3h后及30 U/mL凝血酶作用不同时间(0、0.5、1、3和6h)对皮层神经元RhoA蛋白活化表达的影响;RhoA蛋白活化抑制剂Exoenzyme C3预处理0.5 h及30 U/mL凝血酶作用皮层神经元3h后,行Western blotting检测RhoA蛋白活化表达的情况,并采用Hoechst33258核染色及CCK-8法观察Exoenzyme C3预处理和未处理时凝血酶对皮层神经元的损伤情况. 结果 在浓度实验中,30 U/mL凝血酶作用皮层神经元3h后能明显增加RhoA膜蛋白表达,与0U/mL组相比差异有统计学意义(P＜0.05);而各浓度凝血酶对RhoA总蛋白表达无明显影响.在时程实验中,与其他时间点相比,30 U/mL凝血酶在3h时能明显增加RhoA膜蛋白表达,差异有统计学意义(P＜0.05),但对RhoA总蛋白表达也无明显影响.在Exoenzyme C3干预实验中,Exoenzyme C3预处理组与凝血酶组相比,RhoA膜蛋白表达明显降低,差异有统计学意义(P＜0.05);Hoechst3258核染色显示,与凝血酶组相比,Exoenzyme C3预处理组中核浓染细胞数量明显降低,差异有统计学意义(P＜0.05);CCK-8法检测细胞活性显示,凝血酶组细胞存活率明显低于Exoenzyme C3预处理组,差异有统计学意义(P＜0.05). 结论 凝血酶诱导的胎鼠大脑皮层神经元损伤与RhoA膜蛋白的高表达即RhoA蛋白活化表达有关.