Rac1, RhoA, and Cdc42 participate in HeLa cell invasion by group B streptococcus

The group B streptococcus (GBS) is an important human pathogen with the ability to cause invasive disease. To do so, the bacteria must invade host cells. It has been well documented that GBS are able to invade a variety of nonphagocytic host cell types, and this process is thought to involve a number of pathogen-host cell interactions. While some of the molecular aspects of the GBS-host cell invasion process have been characterized, many events still remain unclear. The objective of this investigation was to evaluate the role of the Rho-family GTPases Rac, Rho, and Cdc42 in GBS invasion into epithelial cells. The epithelial cell invasion process was modeled using HeLa 229 cell culture. Treatment of HeLa cells with 10 microM compactin, a pan-GTPase inhibitor, abolished GBS internalization, suggesting that GTPases are involved in the GBS invasion process. The addition of Toxin B or exoenzyme C3 to HeLa cells before GBS infection reduced invasion by 50%, further suggesting that the Rho-family GTPases are involved in GBS entry. Examining invasion of GBS into HeLa cells with altered genetic backgrounds was used to confirm these findings; GBS invasion into HeLa cells transiently transfected with dominant negative Rac1, Cdc42, or RhoA reduced invasion by 75%, 51%, and 42%, respectively. Results of this study suggest that the Rho-family GTPases are required for efficient invasion of HeLa cells by GBS.     

Adiponectin promotes migration activities of endothelial progenitor cells via Cdc42/Rac1

Adiponectin has anti-atherosclerotic effects through its direct actions on vascular cells. The present study investigates the molecular mechanisms of adiponectin in the migration of endothelial progenitor cells (EPCs) which play an important role in neovascularization and re-endothelization. The phosphorylation of Akt and the activations of Cdc42 and Rac1 were significantly increased by adiponectin. Adiponectin increased the migration activity of EPCs, which was completely inhibited by a PI3-kinase inhibitor. siRNA of Cdc42 or Rac1 completely inhibited the adiponectin-induced migration, but siRNA of Akt had no effects, indicating that adiponectin promotes the migration activities of EPCs mainly through PI3-kinase/Cdc42/Rac1.

Structured summary

MINT-7217629: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with CDC42 (uniprotkb:P60953) by pull down (MI:0096)MINT-7217644: PAK1 (uniprotkb:Q13153) physically interacts (MI:0914) with Rac1 (uniprotkb:P63000) by pull down (MI:0096)     

Regulation of anoikis by Cdc42 and Rac1

Ras family small GTPases play a critical role in malignant transformation, and Rho subfamily members contribute significantly to this process. Anchorage-independent growth and the ability to avoid detachment-induced apoptosis (anoikis) are hallmarks of transformed epithelial cells. In this study, we have demonstrated that constitutive activation of Cdc42 inhibits anoikis in Madin-Darby canine kidney (MDCK) epithelial cells. We showed that activated Cdc42 stimulates the ERK, JNK, and p38 MAPK pathways in suspension condition; however, inhibition of these signaling does not affect Cdc42-stimulated cell survival. However, we demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3K) pathway abolishes the protective effect of Cdc42 on anoikis. Taking advantage of a double regulatory expression system, we also showed that Cdc42-stimulated cell survival in suspension condition is, at least in part, mediated by Rac1. We also provide evidence for a positive feedback loop involving Rac1 and PI3K. In addition, we show that the survival functions of both constitutively active Cdc42 and Rac1 GTPases are abrogated by Latrunculin B, an actin filament-depolymerizing agent, implying an important role for the actin cytoskeleton in mediating survival signaling activated by Cdc42 and Rac1. Together, our results indicate a role for Cdc42 in anchorage-independent survival of epithelial cells. We also propose that this survival function depends on a positive feedback loop involving Rac1 and PI3K.     

Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles.     

Cdc42 and Rac1 are necessary for autotaxin-induced tumor cell motility in A2058 melanoma cells

Autotaxin (ATX) is a strong motogen that can increase invasiveness and angiogenesis. In the present study, we investigated the signal transduction mechanism of ATX-induced tumor cell motility. Unlike N19RhoA expressing cells, the cells expressing N17Cdc42 or N17Rac1 showed reduced motility against ATX. ATX activated Cdc42 and Rac1 and increased complex formation between these small G proteins and p21-activated kinase (PAK). Furthermore, ATX phosphorylated focal adhesion kinase (FAK) that was not shown in cells expressing dominant negative mutants of Cdc42 or Rac1. Collectively, these data strongly indicate that Cdc42 and Rac1 are essential for ATX-induced tumor cell motility in A2058 melanoma cells, and that PAK and FAK might be also involved in the process.     

RhoA modulates functional and physical interaction between ROCK1 and Erk1/2 in selenite-induced apoptosis of leukaemia cells

RhoA GTPase dysregulation is frequently reported in various tumours and haematologic malignancies. RhoA, regulating Rho-associated coiled-coil-forming kinase 1 (ROCK1), modulates multiple cell functions, including malignant transformation, metastasis and cell death. Therefore, RhoA/ROCK1 could be an ideal candidate target in cancer treatment. However, the roles of RhoA/ROCK1 axis in apoptosis of leukaemia cells remain elusive. In this study, we explored the effects of RhoA/ROCK1 cascade on selenite-induced apoptosis of leukaemia cells and the underlying mechanism. We found selenite deactivated RhoA/ROCK1 and decreased the association between RhoA and ROCK1 in leukaemia NB4 and Jurkat cells. The inhibited RhoA/ROCK1 signalling enhanced the phosphorylation of Erk1/2 in a Mek1/2-independent manner. Erk1/2 promoted apoptosis of leukaemia cells after it was activated. Intriguingly, it was shown that both RhoA and ROCK1 were present in the multimolecular complex containing Erk1/2. GST pull-down analysis showed ROCK1 had a direct interaction with GST-Erk2. In addition, selenite-induced apoptosis in an NB4 xenograft model was also found to be associated with the RhoA/ROCK1/Erk1/2 pathway. Our data demonstrate that the RhoA/ROCK1 signalling pathway has important roles in the determination of cell fates and the modulation of Erk1/2 activity at the Mek–Erk interplay level.     

Rac1和Cdc42在乳腺癌中的表达和临床意义

目的:研究Rac1和Cdc42在人乳腺癌中的表达及临床意义。方法:收集339例人乳腺癌组织样本,通过免疫组化的方法检测Rac1和Cdc42的表达情况,并分析其与乳腺癌临床病理学特征间的相关性。结果:Rac1和Cdc42在正常乳腺组织中几乎不表达,而在肿瘤组织的阳性表达率分别为35.9%和38.5%,均较正常乳腺组织显著升高,差异均具有统计学意义(P0.001和P0.05)。卡方检验分析表明,二者的表达与患者的年龄、肿瘤大小、组织分化程度、HER2状态无关(P0.05),而与TNM分期、淋巴结转移、肿瘤侵袭、ER状态和Ki-67表达有相(P0.05)。相关性分析表明,Rac1和Cdc42的表达与高TNM分期(r分别为0.443和0.295;P均0.001)、淋巴结转移阳性(r均为0.480和0.562;P均0.001)、肿瘤侵袭(r分别为0.412和0.440;P均0.001)、ER阴性表达(r分别为-0.517和-0.342;P均0.001)以及Ki-67高表达(r分别为0.338和0.454;P均0.001)呈正相关。结论:在乳腺癌组织中,Rac1和Cdc42作为癌基因表达增加,可能在乳腺癌恶性进程中发挥重要作用。     

RhoA and Rac1 contribute to type III group B streptococcal invasion of human brain microvascular endothelial cells

Type III group B streptococcus (GBS) has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying mechanisms remain incompletely understood. In the present study, we showed that the geranylgeranyl transferase I inhibitor, GGTI-298, not the farnesyltransferase inhibitor, FTI-277 inhibited type III GBS invasion of HBMEC. The substrates for GGTI-298 include Rho family GTPases, and we showed that RhoA and Rac1 are involved in type III GBS invasion of HBMEC. This was shown by the demonstration that infection with type III GBS strain K79 increased the levels of activated RhoA and Rac1 and GBS invasion was inhibited in HBMEC expressing dominant-negative RhoA and Rac1. Of interest, the level of activated Rac1 in response to type III GBS was decreased in HBMEC expressing dominant-negative RhoA, while the level of activated RhoA was not affected by dominant-negative Rac1. These findings indicate for the first time that activation of geranylgeranylated proteins including RhoA and Rac1 is involved in type III GBS invasion of HBMEC and RhoA is upstream of Rac1 in GBS invasion of HBMEC.     

NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways

Glioblastoma is the most common and lethal primary intracranial tumor. As the key regulator of tumor cell volume, sodium-potassium-chloride cotransporter 1 (NKCC1) expression increases along with the malignancy of the glioma, and NKCC1 has been implicated in glioblastoma invasion. However, little is known about the role of NKCC1 in the epithelial-mesenchymal transition-like process in gliomas. We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape, polarity, and adhesion of cells in glioma. Here, we investigated whether NKCC1 promotes an epithelial–mesenchymal transition (EMT)-like process in gliomas via the RhoA and Rac1 signaling pathways. Pharmacological inhibition and knockdown of NKCC1 both decrease the expressions of mesenchymal markers, such as N-cadherin, vimentin, and snail, whereas these treatments increase the expression of the epithelial marker E-cadherin. These findings indicate that NKCC1 promotes an EMT-like process in gliomas. The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1, which results in a significant enhancement of the EMT-like process. Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA, and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas. Furthermore, we illustrated that NKCC1 knockdown abolished the dissemination and spread of glioma cells in a nude mouse intracranial model. These findings suggest that elevated NKCC1 activity acts in the regulation of an EMT-like process in gliomas, and thus provides a novel therapeutic strategy for targeting the invasiveness of gliomas, which might help to inhibit the spread of malignant intracranial tumors.     

Rac and Cdc42-dependent regulation of c-Jun N-terminal kinases by the delta-opioid receptor

Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen-activated protein kinases (MAPKs). The c-Jun NH2-terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108-15 neuroblastoma x glioma hybrid cells that endogenously express delta-opioid receptors, delta-agonist dose-dependently stimulated JNK activity in a pertussis toxin-sensitive manner. By using COS-7 cells transiently transfected with the cDNAs of delta-opioid receptor and hemagglutinin (HA)-tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gbetagamma subunits by transducin suppressed the opioid-induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid-induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid-induced JNK activation. In addition, the receptor-mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol-3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the delta-opioid receptor, and this pathway requires Gbetagamma, Src kinases and the small GTPases Rac and Cdc42.     

The N-BAR domain protein,Bin3, regulates Rac1- and Cdc42-dependent processes in myogenesis

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.     

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.     

Rac1、Cdc42单分子探针在活细胞中表达差异的比较

利用构建的Rac1、Cdc42单分子探针,探索其在NIH3T3、Hela、COS7等活细胞中的最佳表达效果.结果表明在胞浆中表达的探针其荧光值在诱导5～10 min达到最大值,随着时间的推移,其荧光强度逐渐减弱;在细胞膜上表达的探针也是相似的.DNA/转染试剂为1:3时的表达效果最佳;每毫升转染培养基的质粒DNA含量为4或6 μg时,其转染表达可达到最佳;在不同种类的细胞中表达时其差异不明显;在细胞的不同部位表达差异不明显.     

MicroRNA-34a modulates cytoskeletal dynamics through regulating RhoA/Rac1 cross-talk in chondroblasts

MicroRNAs (miRNAs) have been implicated in various cellular processes, such as cell fate determination, cell death, and tumorigenesis. In the present study, we investigated the role of miRNA-34a (miR-34a) in the reorganization of the actin cytoskeleton, which is essential for chondrocyte differentiation. miRNA arrays to identify genes that appeared to be up-regulated or down-regulated during chondrogenesis were applied with chondrogenic progenitors treated with JNK inhibitor. PNA-based antisense oligonucleotides and miRNA precursor were used for investigation of the functional roles of miR-34a. We found that, in chick chondroprogenitors treated with JNK inhibitor, which suppresses chondrogenic differentiation, the expression levels of miR-34a and RhoA1 are up-regulated through modulation of Rac1 expression. Blockade of miR-34a via the use of PNA-based antisense oligonucleotides was associated with decreased protein expression of RhoA (a known modulator of stress fiber expression), down-regulation of stress fibers, up-regulation of Rac1, and recovery of protein level of type II collagen. miR-34a regulates RhoA/Rac1 cross-talk and negatively modulates reorganization of the actin cytoskeleton, which is one of the essential processes for establishing chondrocyte-specific morphology.     

Rac1 and RhoA GTPases have antagonistic functions during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts

Background information. N‐cadherin, a member of the Ca²⁺‐dependent cell—cell adhesion molecule family, plays an essential role in the induction of the skeletal muscle differentiation programme. However, the molecular mechanisms which govern the formation of N‐cadherin‐dependent cell—cell contacts in myoblasts remain unexplored. Results. In the present study, we show that N‐cadherin‐dependent cell contact formation in myoblasts is defined by two stages. In the first phase, N‐cadherin is highly mobile in the lamellipodia extensions between the contacting cells. The second stage corresponds to the formation of mature N‐cadherin‐dependent cell contacts, characterized by the immobilization of a pool of N‐cadherin which appears to be clustered in the interdigitated membrane structures that are also membrane attachment sites for F‐actin filaments. We also demonstrated that the formation of N‐cadherin‐dependent cell—cell contacts requires a co‐ordinated and sequential activity of Rac1 and RhoA. Rac1 is involved in the first stage and facilitates N‐cadherin‐dependent cell—cell contact formation, but it is not absolutely required. Conversely, RhoA is necessary for N‐cadherin‐dependent cell contact formation, since, via ROCK (Rho‐associated kinase) signalling and myosin 2 activation, it allows the stabilization of N‐cadherin at the cell—cell contact sites. Conclusions. We have shown that Rac1 and RhoA have opposite effects on N‐cadherin‐dependent cell—cell contact formation in C2C12 myoblasts and act sequentially to allow its formation.     

Expression of Rho-family GTPases (Rac, cdc42, RhoA) and their association with p-21 activated kinase in adult rat peripheral nerve

To clarify the presence of the Rho family of small GTPases p21-activated kinase (pak) signaling pathway in the PNS, we have examined their expression, the association between the small GTPases and pak and the pak kinase activity in the PNS using immunoblot analysis, immunohistochemistry, co-immunoprecipitation study, and in vitro kinase assay. Immunoblot analysis showed the expression of Rac, cdc42, RhoA and pak in the dorsal root ganglion (DRG) and sciatic nerve. The localization of these proteins in the DRG neurons and axons and Schwann cells of the sciatic nerve was confirmed by immunohistochemistry. Co-immunoprecipitation studies indicated the in vivo associations of pak with Rac and cdc42, but not with RhoA, in both the DRG and sciatic nerve. The autophosphorylation of pak and phosphorylation of histone H4 by pak were also found in the DRG and sciatic nerve as well as in the CNS. These results suggest that the Rac/cdc42-pak signaling pathway exists and functions in the PNS and may mediate some intracellular signals.     

RhoA/ROCK1 regulates the mitochondrial dysfunction through Drp1 induced by Porphyromonas gingivalis in endothelial cells

Porphyromonas gingivalis (P. gingivalis) is a pivotal pathogen of periodontitis. Our previous studies have confirmed that mitochondrial dysfunction in the endothelial cells caused by P. gingivalis was dependent on Drp1, which may be the mechanism of P. gingivalis causing endothelial dysfunction. Nevertheless, the signalling pathway induced the mitochondrial dysfunction remains unclear. The purpose of this study was to investigate the role of the RhoA/ROCK1 pathway in regulating mitochondrial dysfunction caused by P. gingivalis. P. gingivalis was used to infect EA.hy926 cells (endothelial cells). The expression and activation of RhoA and ROCK1 were assessed by western blotting and pull-down assay. The morphology of mitochondria was observed by mitochondrial staining and transmission electron microscopy. Mitochondrial function was measured by ATP content, mitochondrial DNA and mitochondrial permeability transition pore openness. The phosphorylation and translocation of Drp1 were evaluated using western blotting and immunofluorescence. The role of the RhoA/ROCK1 pathway in mitochondrial dysfunction was investigated using RhoA and ROCK1 inhibitors. The activation of RhoA/ROCK1 pathway and mitochondrial dysfunction were observed in P. gingivalis-infected endothelial cells. Furthermore, RhoA or ROCK1 inhibitors partly prevented mitochondrial dysfunction caused by P. gingivalis. The increased phosphorylation and mitochondrial translocation of Drp1 induced by P. gingivalis were both blocked by RhoA and ROCK1 inhibitors. In conclusion, we demonstrate that the RhoA/ROCK1 pathway was involved in mitochondrial dysfunction caused by P. gingivalis by regulating the phosphorylation and mitochondrial translocation of Drp1. Our research illuminated a possible new mechanism by which P. gingivalis promotes endothelial dysfunction.     

Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells

We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.