7-Keto-Cholesterol and Cholestan-3beta, 5alpha, 6beta-Triol Induce Eryptosis through Distinct Pathways Leading to NADPH Oxidase and Nitric Oxide Synthase Activation

BACKGROUND/AIMS: We showed that patho-physiological concentrations of either 7-keto-cholesterol (7-KC), or cholestane-3beta, 5alpha, 6beta-triol (TRIOL) caused the eryptotic death of human red blood cells (RBC), strictly dependent on the early production of reactive oxygen species (ROS). The goal of the current study was to assess the contribution of the erythrocyte ROS-generating enzymes, NADPH oxidase (RBC-NOX), nitric oxide synthase (RBC-NOS) and xanthine oxido-reductase (XOR) to the oxysterol-dependent eryptosis and pertinent activation pathways. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, reactive oxygen/nitrogen species (RONS) and nitric oxide formation from 2',7'-dichloro-dihydrofluorescein (DCF-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) -dependent fluorescence, respectively; Akt1, phospho-NOS3 Ser1177, and PKCζ from Western blot analysis. The activity of individual 7-KC (7 μM) and TRIOL (2, μM) on ROS-generating enzymes and relevant activation pathways was assayed in the presence of Diphenylene iodonium chloride (DPI), N-nitro-L-arginine methyl ester (L-NAME), allopurinol, NSC23766 and LY294002, inhibitors in this order of RBC-NOX, RBC-NOS, XOR and upstream regulatory proteins Rac GTPase and phosphoinositide3 Kinase (PI3K); hemoglobin oxidation from spectrophotometric analysis. RESULTS: RBC-NOX was the target of 7-KC, through a signaling including Rac GTPase and PKCζ, whereas TRIOL caused activation of RBC-NOS according to the pathway PI3K/Akt, with the concurrent activity of a Rac-GTPase. In concomitance with the TRIOL-induced .NO production, formation of methemoglobin with global loss of heme were observed, ascribable to nitrosative stress. XOR, activated after modification of the redox environment by either RBC-NOX or RBC-NOS activity, concurred to the overall oxidative/nitrosative stress by either oxysterols. When 7-KC and TRIOL were combined, they acted independently and their effect on ROS/RONS production and PS exposure appeared the result of the effects of the oxysterols on RBC-NOX and RBC-NOS. CONCLUSION: Eryptosis of human RBCs may be caused by either 7-KC or TRIOL by oxidative/nitrosative stress through distinct signaling cascades activating RBC-NOX and RBC-NOS, respectively, with the complementary activity of XOR; when combined, the oxysterols act independently and both concur to the final eryptotic effect

Diverse effects of RacV12 on cell transformation by Raf: partial inhibition of morphological transformation versus deregulation of cell cycle control

AbstractActivated Raf kinases and Rac GTPases were shown to cooperate in the oncogenic transformation of fibroblasts, which is characterised by the disassembly of the cellular actin cytoskeleton, a nearly complete loss of focal adhesion complexes and deregulated cell proliferation. This is surprising since the Rac GTPase induces actin structures and the adhesion of suspended cells to extracellular matrix proteins. NIH 3T3 cells expressing a hydroxytamoxifen-inducible oncogenic c-Raf-1–oestrogen receptor fusion protein (c-Raf-1-BxB-ERTM, N-BxB-ERTM cells) undergo morphological transformation upon stimulation of the Raf kinase. We show that treatment with the Rac, Rho and Cdc42 activating Escherichia coli toxin CNF1 or coexpression of an activated RacV12 mutant partially inhibits and reverses the disassembly of cellular actin structures and focal adhesion complexes by oncogenic Raf. Activation of the Rac GTPase restores actin structures and focal adhesion complexes at the cellular boundary, leading to spreading of the otherwise spindle-shaped Raf-transformed cells. Actin stress fibres, however, which are regulated by the function of the Rho GTPase, are disassembled by oncogenic Raf even in the presence of activated Rac and Rho. With respect to the RacV12-mediated spreading of Raf-transformed cells, we postulate an anti-oncogenic function of the activated Rac. Another feature of cell transformation is the deregulation of cell cycle control. NIH 3T3 cells expressing high levels of the c-Raf-1-BxB-ERTM protein undergo a cell cycle arrest upon stimulation of the oncogenic Raf kinase. Our results show that in N-BxB-ERTM-RacV12 cells the expression of the activated RacV12 mediates cell proliferation in the presence of high-intensity Raf signals and high levels of the Cdk inhibitor p21Cip1. These results indicate a pro-oncogenic function of the Rac GTPase with respect to the deregulation of cell cycle control

Rac GTPase signaling in mechanotransduction during embryonic morphogenesis

How cells sense and respond to mechanical forces is attracting considerable attention. We recently demonstrated that mechanical tension originating from one tissue strongly influences the differentiation and morphogenesis of another tissue during C. elegans embryogenesis (Nature 471:99–103). Specifically, we found that the repeated contractions of muscle cells stimulate a signaling cascade involving the Rac GTPase within the epidermis. This pathway ultimately leads to strengthen hemidesmosome-like junctions and promote embryonic morphogenesis. Our work provides further evidence that mechanical inputs impact on development, much like inputs involving growth factors and morphogens. After briefly outlining the pioneering work that inspired us, I will present the mechanotransduction process underlying the response to tension and the key experiments supporting our conclusions

CCL2 induces prostate cancer transendothelial cell migration via activation of the small GTPase Rac

Nearly 85% of the men who will die of prostate cancer (PCa) have skeletal metastases present. The ability of PCa cells to interact with the microenvironment determines the success of the tumor cell to form metastatic lesions. The ability to bind to human bone marrow endothelial (HBME) cells and undergo transendothelial cell migration are key steps in allowing the PCa cell to extravasate from the bone microvasculature and invade the bone stroma. We have previously demonstrated that monoctyte chemoattractant protein 1 (MCP-1; CCL2) is expressed by HBME cells and promotes PCa proliferation and migration. In the current study, we demonstrate that the CCL2 stimulation of PCa cells activates the small GTPase, Rac through the actin-associated protein PCNT1. Activation of Rac GTPase is accompanied by morphologic changes and the ability of the cells to undergo diapedesis through HBME cells. These data suggest a role for HBME-secreted CCL2 in promoting PCa cell extravasation into the bone microenvironment. J. Cell. Biochem. 104: 1587–1597, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60467/1/21652_ftp.pd

Does Rac GTPase Play a Role in EphA4 Signaling in Xenopus Embroys?

In embryonic development, many cells migrate in order to correctly form new structures. One way that guidance of this migration occurs is via a repulsion mechanism involving the Eph family of receptors. Signaling through these receptors activates the repulsive mechanism that limits which tissues can interact with each other. The repulsion mechanism is not well understood, but involves the reorganization of the actin cytoskeleton and a loss of cell-cell adhesion. Activation of the EphA4 signaling pathway in Xenopus laevis embryos has been shown to cause the repulsion mechanism. the Rho family of GTPases consisting of Cdc42, Rho, and Rac, has been linked to the reorganization of the actin cytoskeleton and Rho has been demonstrated to be part of the EphA4 pathway. The hypothesis to be tested in this study is that activation of Rac is also part of the EphA4 pathway. To test this hypothesis, mutant Rac RNA coding for constitutively active Rac (caRac) was injected into Xenopus laevis embryos to determine if Rac activation can mimic EphA4 signaling. As the concentration of RNA coding for caRac increased, the degree of cellular dissociation also increased. While further experimentation must be doe to make a conclusive determination, it is possible that the activation of Rac is a part of EphA4 signaling

The Rac GTP Exchange Factor TIAM-1 Acts with CDC-42 and the Guidance Receptor UNC-40/DCC in Neuronal Protrusion and Axon Guidance

The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions

Rapid Activation of Rac GTPase in Living Cells by Force Is Independent of Src

It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET) method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor) and YPet (a variant yellow fluorescent protein as an acceptor) of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms) of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF) induced Rac activation at a much later time (>30 sec). There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17) was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src activation. These findings suggest that signaling pathways of mechanical forces via integrins might be fundamentally different from those of growth factors

Signal transducer and activator of transcription-5 mediates neuronal apoptosis induced by inhibition of Rac GTPase activity.

In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase

NEDD9 regulates 3D migratory activity independent of the Rac1 morphology switch in glioma and neuroblastoma.

Metastasizing tumor cells must transmigrate the dense extracellular matrix that surrounds most organs. The use of three-dimensional (3D) collagen gels has revealed that many cancer cells can switch between different modes of invasion that are characterized by distinct morphologies (e.g., rounded vs. elongated). The adhesion protein NEDD9 has the potential to regulate the switch between elongated and rounded morphologies; therefore, its role was interrogated in the invasion switch of glioblastoma and neuroblastoma tumors that similarly derive from populations of neural crest cells. Interestingly, siRNA-mediated depletion of NEDD9 failed to induce cell rounding in glioma or neuroblastoma cells, contrasting the effects that have been described in other tumor model systems. Given that Rac1 GTPase has been suggested to mediate the switch between elongated and rounded invasion, the functionality of the Rac1 morphology switch was evaluated in the glioma and neuroblastoma cells. Using both dominant-negative Rac1 and Rac1-specific siRNA, the presence of this morphologic switch was confirmed in the neuroblastoma, but not in the glioma cells. However, in the absence of a morphologic change following NEDD9 depletion, a significant decrease in the cellular migration rate was observed. Thus, the data reveal that NEDD9 can regulate 3D migration speed independent of the Rac1 morphology switch. IMPLICATIONS: NEDD9 targeting is therapeutically viable as it does not stimulate adaptive changes in glioma and neuroblastoma invasion.National Health and Medical Research Council (NHMRC) grant 63251

RACGAP1 (Rac GTPase activating protein 1)

Review on RACGAP1, with data on DNA, on the protein encoded, and where the gene is implicate