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Sample records for rac1 activity regulates

  1. Rac1 activity regulates proliferation of aggressive metastatic melanoma

    SciTech Connect

    Bauer, Natalie N. Chen Yihwen; Samant, Rajeev S.; Shevde, Lalita A.; Fodstad, Oystein

    2007-11-01

    Molecular mechanisms underlying the different capacity of two in vivo selected human melanoma cell variants to form experimental metastases were studied. The doubling times of the FEMX-I and FEMX-V cell sublines in vitro were 15 and 25 h, respectively. The invasive capacity of FEMX-I cells was 8-fold higher than FEMX-V cells, and the time to form approximately 10 mm s.c. tumors in nude mice was 21 versus 35 days. FEMX-I displayed a spindle-like formation in vitro, whereas FEMX-V cells had a rounded shape. Hence, we examined known determinants of cell shape and proliferation, the small GTPases. The four studied showed equal expression in both cell types, but Rac1 activity was significantly decreased in FEMX-V cells. Rac1 stimulates NF{kappa}B, and we found that endogenous NF{kappa}B activity of FEMX-V cells was 2% of that of FEMX-I cells. Inhibition of Rac1 resulted in blocked NF{kappa}B activity. Specific inhibition of either Rac1 or NF{kappa}B significantly reduced proliferation and invasion of FEMX-I cells, the more pronounced effects observed with Rac1 inhibition. These data indicate that Rac1 activity in FEMX cells regulates cell proliferation and invasion, in part via its effect on NF{kappa}B, signifying Rac1 as a key molecule in melanoma progression and metastasis.

  2. TIPE1 induces apoptosis by negatively regulating Rac1 activation in hepatocellular carcinoma cells.

    PubMed

    Zhang, Z; Liang, X; Gao, L; Ma, H; Liu, X; Pan, Y; Yan, W; Shan, H; Wang, Z; Chen, Y H; Ma, C

    2015-05-14

    TIPE1 (tumor necrosis factor-α-induced protein 8-like 1 or TNFAIP8L1) is a newly identified member of the TIPE (TNFAIP8) family, which play roles in regulating cell death. However, the biologic functions of TIPE1 in physiologic and pathologic conditions are largely unknown. Here, we report the roles of TIPE1 in hepatocellular carcinoma (HCC). Evaluated by immunohistochemical staining, HCC tissues showed significantly downregulated TIPE1 expression compared with adjacent non-tumor tissues, which positively correlated with tumor pathologic grades and patient survival. Using a homograft tumor model in Balb/c mice, we discovered that TIPE1 significantly diminished the growth and tumor weight of murine liver cancer homografts. Consistently, TIPE1 inhibited both cell growth and colony formation ability of cultured HCC cell lines, which was further identified to be due to TIPE1-inducing apoptosis in a caspase-independent, necrostatin-1 (Nec-1)-insensitive manner. Furthermore, mechanistic investigations revealed that TIPE1 interacted with Rac1, and inhibited the activation of Rac1 and its downstream p65 and c-Jun N-terminal kinase pathway. Moreover, overexpression of constitutively active Rac1 partially rescued the apoptosis induced by TIPE1, and Rac1 knockdown significantly restored the deregulated cell growth induced by TIPE1 small interfering RNA. Our findings revealed that TIPE1 induced apoptosis in HCC cells by negatively regulating Rac1 pathway, and loss of TIPE1 might be a new prognostic indicator for HCC patients. PMID:25043299

  3. Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis.

    PubMed

    Chen, J-Y; Lin, Y-Y; Jou, T-S

    2012-06-01

    We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and β-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. PMID:22301917

  4. Hippocampal Activation of Rac1 Regulates the Forgetting of Object Recognition Memory.

    PubMed

    Liu, Yunlong; Du, Shuwen; Lv, Li; Lei, Bo; Shi, Wei; Tang, Yikai; Wang, Lianzhang; Zhong, Yi

    2016-09-12

    Forgetting is a universal feature for most types of memories. The best-defined and extensively characterized behaviors that depict forgetting are natural memory decay and interference-based forgetting [1, 2]. Molecular mechanisms underlying the active forgetting remain to be determined for memories in vertebrates. Recent progress has begun to unravel such mechanisms underlying the active forgetting [3-11] that is induced through the behavior-dependent activation of intracellular signaling pathways. In Drosophila, training-induced activation of the small G protein Rac1 mediates natural memory decay and interference-based forgetting of aversive conditioning memory [3]. In mice, the activation of photoactivable-Rac1 in recently potentiated spines in a motor learning task erases the motor memory [12]. These lines of evidence prompted us to investigate a role for Rac1 in time-based natural memory decay and interference-based forgetting in mice. The inhibition of Rac1 activity in hippocampal neurons through targeted expression of a dominant-negative Rac1 form extended object recognition memory from less than 72 hr to over 72 hr, whereas Rac1 activation accelerated memory decay within 24 hr. Interference-induced forgetting of this memory was correlated with Rac1 activation and was completely blocked by inhibition of Rac1 activity. Electrophysiological recordings of long-term potentiation provided independent evidence that further supported a role for Rac1 activation in forgetting. Thus, Rac1-dependent forgetting is evolutionarily conserved from invertebrates to vertebrates. PMID:27593377

  5. Redox regulation of Rac1 by thiol oxidation.

    PubMed

    Hobbs, G Aaron; Mitchell, Lauren E; Arrington, Megan E; Gunawardena, Harsha P; DeCristo, Molly J; Loeser, Richard F; Chen, Xian; Cox, Adrienne D; Campbell, Sharon L

    2015-02-01

    The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and posttranslational modifications. Whereas much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys(18)) that can be selectively oxidized at physiological pH because of its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys(18) by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1(C18D). We also evaluated Rac1(C18S) as a redox-insensitive variant and found that it retains structural and biochemical properties similar to those of Rac1(WT) but is resistant to thiol oxidation. In addition, Rac1(C18D), but not Rac1(C18S), shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1(C18D) in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1(C18D) in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1(WT) and the redox-insensitive Rac1(C18S) variant. Moreover, expression of Rac1(C18D) in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys(18) is a novel

  6. Corticosterone regulates fear memory via Rac1 activity in the hippocampus.

    PubMed

    Gan, Ping; Ding, Ze-Yang; Gan, Cheng; Mao, Rong-Rong; Zhou, Heng; Xu, Lin; Zhou, Qi-Xin

    2016-09-01

    Stressful events can generate enduring memories, which may induce certain psychiatric disorders such as post-traumatic stress disorder (PTSD). However, the underlying molecular mechanisms in these processes remain unclear. In this study, we examined whether the active form of the small G protein Rac1, Rac1-GTP, is involved in fear memory. Firstly, we detected the time course changes of Rac1-GTP after foot shocks (a strong stressor) and exogenous corticosterone (CORT) treatment. The data showed that stress and CORT induced the downregulation of Rac1-GTP in the hippocampus. Changes in the serum CORT level were negatively correlated with the level of Rac1-GTP. Additionally, a glucocorticoid receptor antagonist, RU38486, not only recovered the expression of Rac1-GTP but also impaired fear memory. Furthermore, systemic administration of NSC23766, an inhibitor of Rac1-GTP, improved fear memory at 1.5 and 24h. Therefore, Rac1 activity plays a critical role in stress-related cognition and may be a potential target in stress-related disorders. PMID:27249795

  7. Regulation of Rac1 translocation and activation by membrane domains and their boundaries

    PubMed Central

    Moissoglu, Konstadinos; Kiessling, Volker; Wan, Chen; Hoffman, Brenton D.; Norambuena, Andres; Tamm, Lukas K.; Schwartz, Martin Alexander

    2014-01-01

    ABSTRACT The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo–ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling. PMID:24695858

  8. Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation*

    PubMed Central

    Arrizabalaga, Onetsine; Lacerda, Hadriano M.; Zubiaga, Ana M.; Zugaza, José L.

    2012-01-01

    Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191–270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity. PMID:22337875

  9. Activation of Rac1 by RhoG regulates cell migration.

    PubMed

    Katoh, Hironori; Hiramoto, Kiyo; Negishi, Manabu

    2006-01-01

    Cell migration is essential for normal development and many pathological processes. Rho-family small GTPases play important roles in this event. In particular, Rac regulates lamellipodia formation at the leading edge during migration. The small GTPase RhoG activates Rac through its effector ELMO and the ELMO-binding protein Dock180, which functions as a Rac-specific guanine nucleotide exchange factor. Here we investigated the role of RhoG in cell migration. RNA interference-mediated knockdown of RhoG in HeLa cells reduced cell migration in Transwell and scratch-wound migration assays. In RhoG-knockdown cells, activation of Rac1 and formation of lamellipodia at the leading edge in response to wounding were attenuated. By contrast, expression of active RhoG promoted cell migration through ELMO and Dock180. However, the interaction of Dock180 with Crk was dispensable for the activation of Rac1 and promotion of cell migration by RhoG. Taken together, these results suggest that RhoG contributes to the regulation of Rac activity in migrating cells. PMID:16339170

  10. Geometry sensing through POR1 regulates Rac1 activity controlling early osteoblast differentiation in response to nanofiber diameter.

    PubMed

    Higgins, A M; Banik, B L; Brown, J L

    2015-02-01

    Bone grafting procedures in the United States rely heavily upon autografts and allografts, which are donor-dependent, cause donor site pain, and can transmit disease. Synthetic bone grafts can reduce these risks; however, synthetics lack the bone differentiating (osteoinductive) abilities of auto- and allografts. Achieving innate osteoinductive properties of synthetics through surface modifications is currently under investigation. This study focuses on nanofibers, with emphasis on how fiber diameter and the potential curvature sensor POR1 affect the activation of the signaling molecules Rac1 and Arf1, and leading to expression of alkaline phosphatase (ALP), an osteoinductive marker. Diameters of 0.1, 0.3, and 1.0 μm were compared against a flat control. The highest level of Rac1 activation was achieved on the smallest fibers (0.1 μm), a trend that was lost in POR1 knockdowns. This supports the hypothesis that on small nanofibers, POR1 favorably binds to highly curved cell membranes, which allows Rac1 to subsequently dissociate and activate. When the curvature is insufficient to bind POR1, POR1 binds to inactive Rac1 and competitively inhibits its activation. Arf1 activation followed an opposite trend, with the largest nanofibers exhibiting the highest activity. This trend reinforces the known interaction between Rac1 and Arf1 through the GIT-PIX complex, an Arf1 GAP and Rac1 GEF, respectively. Large, (1.0 μm), nanofibers demonstrated the highest ALP activity, indicating that ALP expression is inversely dependent on Rac1 activation. Knockdown of POR1 resulted in increased ALP activity across the substrates but without regard to the curvature sensing trend seen previously. Thus, POR1 senses curvature and increases Rac1 activity, which negatively regulates bone differentiation. PMID:25539497

  11. Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

    PubMed Central

    Marei, Hadir; Carpy, Alejandro; Woroniuk, Anna; Vennin, Claire; White, Gavin; Timpson, Paul; Macek, Boris; Malliri, Angeliki

    2016-01-01

    The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner. PMID:26887924

  12. The role of Rac1 in the regulation of NF-kB activity, cell proliferation, and cell migration in non-small cell lung carcinoma

    PubMed Central

    Gastonguay, Adam; Berg, Tracy; Hauser, Andrew D.; Schuld, Nathan; Lorimer, Ellen; Williams, Carol L.

    2012-01-01

    The small GTPase Rac1 regulates many cellular processes, including cytoskeletal reorganization, cell migration, proliferation, and survival. Additionally, Rac1 plays a major role in activating NF-κB-mediated transcription. Both Rac1 and NF-κB regulate many properties of the malignant phenotype, including anchorage-independent proliferation and survival, metastasis, and angiogenesis. Despite these findings, the roles of Rac1and NF-κB in non-small cell lung carcinoma, a leading cause of cancer deaths, have not been thoroughly investigated. Here, we compared the effects of Rac1 siRNA to that of the Rac1 inhibitor NSC23766 on multiple features of the NSCLC malignant phenotype, including NF-κB activity. We show that the siRNA-mediated silencing of Rac1 in lung cancer cells results in decreased cell proliferation and migration. The decrease in proliferation was observed in both anchorage-dependent and anchorage-independent assays. Furthermore, cells with decreased Rac1 expression have a slowed progression through the G1 phase of the cell cycle. These effects induced by Rac1 siRNA correlated with a decrease in NF-κB transcriptional activity. Additionally, inhibition of NF-κB signaling with BAY 11–7082 inhibited proliferation; indicating that the loss of cell proliferation and migration induced by the silencing of Rac1 expression may be attributed in part to loss of NF-κB activity. Interestingly, treatment with the Rac1 inhibitor NSC23766 strongly inhibits cell proliferation, cell cycle progression, and NF-κB activity in lung cancer cells, to an even greater extent than the inhibition induced by Rac1 siRNA. These findings indicate that Rac1 plays an important role in lung cancer cell proliferation and migration, most likely through its ability to promote NF-κB activity, and highlight Rac1 pathways as therapeutic targets for the treatment of lung cancer. PMID:22549160

  13. Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation.

    PubMed

    Reyes-Reyes, E Merit; Šalipur, Francesca R; Shams, Mitra; Forsthoefel, Matthew K; Bates, Paula J

    2015-08-01

    AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that

  14. Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

    PubMed

    Davila, Juanmahel; Laws, Mary J; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N; Bagchi, Milan K; Bagchi, Indrani C

    2015-08-01

    During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

  15. Rac1 Regulates Endometrial Secretory Function to Control Placental Development

    PubMed Central

    Davila, Juanmahel; Laws, Mary J.; Kannan, Athilakshmi; Li, Quanxi; Taylor, Robert N.; Bagchi, Milan K.; Bagchi, Indrani C.

    2015-01-01

    During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1-null decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1-null decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal

  16. Rac1 promotes chondrogenesis by regulating STAT3 signaling pathway.

    PubMed

    Kim, Hyoin; Sonn, Jong Kyung

    2016-09-01

    The small GTPase protein Rac1 is involved in a wide range of biological processes including cell differentiation. Previously, Rac1 was shown to promote chondrogenesis in micromass cultures of limb mesenchyme. However, the pathways mediating Rac1's role in chondrogenesis are not fully understood. This study aimed to explore the molecular mechanisms by which Rac1 regulates chondrogenic differentiation. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was increased as chondrogenesis proceeded in micromass cultures of chick wing bud mesenchyme. Inhibition of Rac1 with NSC23766, janus kinase 2 (JAK2) with AG490, or STAT3 with stattic inhibited chondrogenesis and reduced phosphorylation of STAT3. Conversely, overexpression of constitutively active Rac1 (Rac L61) increased phosphorylation of STAT3. Rac L61 expression resulted in increased expression of interleukin 6 (IL-6), and treatment with IL-6 increased phosphorylation of STAT3. NSC23766, AG490, and stattic prohibited cell aggregation, whereas expression of Rac L61 increased cell aggregation, which was reduced by stattic treatment. Our studies indicate that Rac1 induces STAT3 activation through expression and action of IL-6. Overexpression of Rac L61 increased expression of bone morphogenic protein 4 (BMP4). BMP4 promoted chondrogenesis, which was inhibited by K02288, an activin receptor-like kinase-2 inhibitor, and increased phosphorylation of p38 MAP kinase. Overexpression of Rac L61 also increased phosphorylation of p38 MAPK, which was reduced by K02288. These results suggest that Rac1 activates STAT3 by expression of IL-6, which in turn increases expression and activity of BMP4, leading to the promotion of chondrogenesis. PMID:27306109

  17. Non-prenylatable, cytosolic Rac1 alters neurite outgrowth while retaining the ability to be activated.

    PubMed

    Reddy, Jairus M; Samuel, Filsy G; McConnell, Jordan A; Reddy, Cristina P; Beck, Brian W; Hynds, DiAnna L

    2015-03-01

    Rac1 is an important regulator of axon extension, cell migration and actin reorganization. Like all Rho guanine triphosphatases (GTPases), Rac1 is targeted to the membrane by the addition of a geranylgeranyl moiety, an action thought to result in Rac1 guanosine triphosphate (GTP) binding. However, the role that Rac1 localization plays in its activation (GTP loading) and subsequent activation of effectors is not completely clear. To address this, we developed a non-prenylatable emerald green fluorescent protein (EmGFP)-Rac1 fusion protein (EmGFP-Rac1(C189A)) and assessed how expressing this construct affected neurite outgrowth, Rac1 localization and activation in neuroblastoma cells. Expression of EmGFP-Rac1(C189A) increased localization to the cytosol and induced cell clustering while increasing neurite initiation. EmGFP-Rac1(C189A) expression also increased Rac1 activation in the cytosol, compared to cells expressing wild-type Rac1 (EmGFP-Rac1). These results suggest that activation of Rac1 may not require plasma membrane localization, potentially leading to differential activation of cytosolic signaling pathways that alter cell morphology. Understanding the consequences of differential localization and activation of Rho GTPases, including Rac1, could lead to new therapeutic targets for treating neurological disorders. PMID:25479592

  18. A novel interaction between the SH2 domain of signaling adaptor protein Nck-1 and the upstream regulator of the Rho family GTPase Rac1 engulfment and cell motility 1 (ELMO1) promotes Rac1 activation and cell motility.

    PubMed

    Zhang, Guo; Chen, Xia; Qiu, Fanghua; Zhu, Fengxin; Lei, Wenjing; Nie, Jing

    2014-08-15

    Nck family proteins function as adaptors to couple tyrosine phosphorylation signals to actin cytoskeleton reorganization. Several lines of evidence indicate that Nck family proteins involve in regulating the activity of Rho family GTPases. In the present study, we characterized a novel interaction between Nck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that the Nck-1-ELMO1 interaction is mediated by the SH2 domain of Nck-1 and the phosphotyrosine residues at position 18, 216, 395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which the SH2 domain was inactive), or a 4YF mutant of ELMO1 lacking these four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitor treatment and overexpression of Src family kinase Hck significantly enhanced Nck-1-ELMO1 interaction. Moreover, wild type Nck-1, but not R308K mutant, significantly augmented the interaction between ELMO1 and constitutively active RhoG (RhoG(V12A)), thus promoted Rac1 activation and cell motility. Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulating Rac1 activity. PMID:24928514

  19. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

    PubMed

    Marei, Hadir; Carpy, Alejandro; Macek, Boris; Malliri, Angeliki

    2016-08-01

    The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects. PMID:27152953

  20. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors

    PubMed Central

    Marei, Hadir; Carpy, Alejandro; Macek, Boris; Malliri, Angeliki

    2016-01-01

    ABSTRACT The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects. PMID:27152953

  1. PIKfyve, MTMR3 and their product PtdIns5P regulate cancer cell migration and invasion through activation of Rac1.

    PubMed

    Oppelt, Angela; Haugsten, Ellen M; Zech, Tobias; Danielsen, Håvard E; Sveen, Anita; Lobert, Viola H; Skotheim, Rolf I; Wesche, Jørgen

    2014-08-01

    Previously, we have shown that the phosphoinositide metabolizing enzymes PIKfyve (phosphoinositide 5-kinase, FYVE finger containing) and MTMR3 (myotubularin-related protein 3), together with their lipid product PtdIns5P, are important for migration of normal human fibroblasts. As these proteins are a kinase and a phosphatase respectively, and thereby considered druggable, we wanted to test their involvement in cancer cell migration and invasion. First, we showed that PIKfyve and MTMR3 are expressed in most cancer cells. Next, we demonstrated that depletion of PIKfyve or MTMR3 resulted in decreased velocity in three different cancer cell lines by using new software for cell tracking. Inhibition of the enzymatic activity of PIKfyve by the inhibitor YM201636 also led to a strong reduction in cell velocity. Mechanistically, we show that PIKfyve and MTMR3 regulate the activation of the Rho family GTPase Rac1. Further experiments also implicated PtdIns5P in the activation of Rac1. The results suggest a model for the activation of Rac1 in cell migration where PIKfyve and MTMR3 produce PtdIns5P on cellular membranes which may then serve to recruit effectors to activate Rac1. Finally, in an invasion assay, we demonstrate that both PIKfyve and MTMR3 are implicated in invasive behaviour of cancer cells. Thus PIKfyve and MTMR3 could represent novel therapeutic targets in metastatic cancer. PMID:24840251

  2. The P2Y2 Receptor Interacts with VE-Cadherin and VEGF Receptor-2 to Regulate Rac1 Activity in Endothelial Cells

    PubMed Central

    Liao, Zhongji; Cao, Chen; Wang, Jianjie; Huxley, Virginia H.; Baker, Olga; Weisman, Gary A.

    2015-01-01

    Vascular endothelial cadherin (VE-cadherin) mediates homophylic adhesion between endothelial cells and is an important regulator of angiogenesis, blood vessel permeability and leukocyte trafficking. Rac1, a member of the Rho family of GTPases, controls VE-cadherin adhesion by acting downstream of several growth factors, including angiopoietin-1 and vascular endothelial growth factor (VEGF). Here we show that UTP-induced activation of the Gq protein-coupled P2Y2 nucleotide receptor (P2Y2R) in human coronary artery endothelial cells (HCAECs) activated Rac1 and caused a transient complex to form between P2Y2R, VE-cadherin and VEGF receptor-2 (VEGFR-2). Knockdown of VE-cadherin expression with siRNA did not affect UTP-induced activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but led to a loss of UTP-induced Rac1 activation and tyrosine phosphorylation of p120 catenin, a cytoplasmic protein known to interact with VE-cadherin. Activation of the P2Y2R by UTP also caused a prolonged interaction between p120 catenin and vav2 (a guanine nucleotide exchange factor for Rac) that correlated with the kinetics of UTP-induced tyrosine phosphorylation of p120 catenin and VE-cadherin. Inhibitors of VEGFR-2 (SU1498) or Src (PP2) significantly diminished UTP-induced Rac1 activation, tyrosine phosphorylation of p120 catenin and VE-cadherin, and association of the P2Y2R with VE-cadherin and p120 catenin with vav2. These findings suggest that the P2Y2R uses Src and VEGFR-2 to mediate association of the P2Y2R with VE-cadherin complexes in endothelial adherens junctions to activate Rac1. PMID:25657827

  3. Fluctuation of Rac1 activity is associated with the phenotypic and transcriptional heterogeneity of glioma cells.

    PubMed

    Yukinaga, Hiroko; Shionyu, Clara; Hirata, Eishu; Ui-Tei, Kumiko; Nagashima, Takeshi; Kondo, Shinji; Okada-Hatakeyama, Mariko; Naoki, Honda; Matsuda, Michiyuki

    2014-04-15

    Phenotypic heterogeneity of cancer cells is caused not only by genetic and epigenetic alterations but also by stochastic variation of intracellular signaling molecules. Using cells that stably express Förster resonance energy transfer (FRET) biosensors, we show here a correlation between a temporal fluctuation in the activity of Rac1 and the invasive properties of C6 glioma cells. By using long-term time-lapse imaging, we found that Rac1 activity in C6 glioma cells fluctuated over a timescale that was substantially longer than that of the replication cycle. Because the relative level of Rac1 activity in each cell was unaffected by a suspension-adhesion procedure, we were able to sort C6 glioma cells according to the levels of Rac1 activity, yielding Rac1(high) and Rac1(low) cells. The Rac1(high) cells invaded more efficiently than did Rac1(low) cells in a Matrigel invasion assay. We assessed the transcriptional profiles of Rac1(high) and Rac1(low) cells and performed gene ontology analysis. Among the 14 genes that were most associated with the term 'membrane' (membrane-related genes) in Rac1(high) cells, we identified four genes that were associated with glioma invasion and Rac1 activity by using siRNA knockdown experiments. Among the transcription factors upregulated in Rac1(high) cells, Egr2 was found to positively regulate expression of the four membrane-related invasion-associated genes. The identified signaling network might cause the fluctuations in Rac1 activity and the heterogeneity in the invasive capacity of glioma cells. PMID:24522191

  4. Inhibition of Rac1 Activity in the Hippocampus Impairs the Forgetting of Contextual Fear Memory.

    PubMed

    Jiang, Lizhu; Mao, Rongrong; Zhou, Qixin; Yang, Yuexiong; Cao, Jun; Ding, Yuqiang; Yang, Yuan; Zhang, Xia; Li, Lingjiang; Xu, Lin

    2016-03-01

    Fear is crucial for survival, whereas hypermnesia of fear can be detrimental. Inhibition of the Rac GTPase is recently reported to impair the forgetting of initially acquired memory in Drosophila. Here, we investigated whether inhibition of Rac1 activity in rat hippocampus could contribute to the hypermnesia of contextual fear. We found that spaced but not massed training of contextual fear conditioning caused inhibition of Rac1 activity in the hippocampus and heightened contextual fear. Furthermore, intrahippocampal injection of the Rac1 inhibitor NSC23766 heightened contextual fear in massed training, while Rac1 activator CN04-A weakened contextual fear in spaced training rats. Our study firstly demonstrates that contextual fear memory in rats is actively regulated by Rac1 activity in the hippocampus, which suggests that the forgetting impairment of traumatic events in posttraumatic stress disorder may be contributed to the pathological inhibition of Rac1 activity in the hippocampus. PMID:25613020

  5. Rac1 is a novel regulator of contraction-stimulated glucose uptake in skeletal muscle.

    PubMed

    Sylow, Lykke; Jensen, Thomas E; Kleinert, Maximilian; Mouatt, Joshua R; Maarbjerg, Stine J; Jeppesen, Jacob; Prats, Clara; Chiu, Tim T; Boguslavsky, Shlomit; Klip, Amira; Schjerling, Peter; Richter, Erik A

    2013-04-01

    In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (~60-100%) and humans (~40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20-58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake. PMID:23274900

  6. Rac1 Is a Novel Regulator of Contraction-Stimulated Glucose Uptake in Skeletal Muscle

    PubMed Central

    Sylow, Lykke; Jensen, Thomas E.; Kleinert, Maximilian; Mouatt, Joshua R.; Maarbjerg, Stine J.; Jeppesen, Jacob; Prats, Clara; Chiu, Tim T.; Boguslavsky, Shlomit; Klip, Amira; Schjerling, Peter; Richter, Erik A.

    2013-01-01

    In skeletal muscle, the actin cytoskeleton-regulating GTPase, Rac1, is necessary for insulin-dependent GLUT4 translocation. Muscle contraction increases glucose transport and represents an alternative signaling pathway to insulin. Whether Rac1 is activated by muscle contraction and regulates contraction-induced glucose uptake is unknown. Therefore, we studied the effects of in vivo exercise and ex vivo muscle contractions on Rac1 signaling and its regulatory role in glucose uptake in mice and humans. Muscle Rac1-GTP binding was increased after exercise in mice (∼60–100%) and humans (∼40%), and this activation was AMP-activated protein kinase independent. Rac1 inhibition reduced contraction-stimulated glucose uptake in mouse muscle by 55% in soleus and by 20–58% in extensor digitorum longus (EDL; P < 0.01). In agreement, the contraction-stimulated increment in glucose uptake was decreased by 27% (P = 0.1) and 40% (P < 0.05) in soleus and EDL muscles, respectively, of muscle-specific inducible Rac1 knockout mice. Furthermore, depolymerization of the actin cytoskeleton decreased contraction-stimulated glucose uptake by 100% and 62% (P < 0.01) in soleus and EDL muscles, respectively. These are the first data to show that Rac1 is activated during muscle contraction in murine and human skeletal muscle and suggest that Rac1 and possibly the actin cytoskeleton are novel regulators of contraction-stimulated glucose uptake. PMID:23274900

  7. Activated Rac1 regulates the degradation of IκBα and the nuclear translocation of STAT3–NFκB complexes in starved cancer cells

    PubMed Central

    Kim, Sung Joo; Yoon, Sarah

    2016-01-01

    In several human tumors, signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB (NFκB) are activated and interact; how these STAT3–NFκB complexes are transported to the nucleus is not fully understood. In this study, we found that Rac1 was activated in starved cancer cells and that activated Rac1 coexisted with STAT3 and NFκB. Rac1 knockdown and overexpression of the dominant-negative mutant Rac1N19 inhibited the degradation of IκBα, an inhibitor of NFκB. MG132, an inhibitor of the ubiquitin proteasome pathway, increased the amount of non-phosphorylated IκBα, but not serine-phosphorylated IκBα, indicating that IκBα degradation by Rac1 in starved cancer cells is independent of IκBα serine phosphorylation by IKK. Rac1 knockdown also inhibited the nuclear translocation of STAT3–NFκB complexes, indicating that this translocation requires activated Rac1. We also demonstrated that the mutant STAT3 Y705F could form complexes with NFκB, and these unphosphorylated STAT3–NFκB complexes translocated into the nucleus and upregulated the activity of NFκB in starved cancer cells, suggesting that phosphorylation of STAT3 is not essential for its translocation. To our knowledge, this is the first study demonstrating the crucial role of Rac1 in the function of STAT3–NFκB complexes in starved cancer cells and implies that targeting Rac1 may have future therapeutic significance in cancer therapy. PMID:27151455

  8. Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promoters

    PubMed Central

    MacKay, Joanna L.

    2014-01-01

    The GTPases RhoA and Rac1 are key regulators of cell spreading, adhesion, and migration, and they exert distinct effects on the actin cytoskeleton. While RhoA classically stimulates stress fiber assembly and contraction, Rac1 promotes branched actin polymerization and membrane protrusion. These competing influences are reinforced by antagonistic crosstalk between RhoA and Rac1, which has complicated efforts to identify the specific mechanisms by which each GTPase regulates cell behavior. We therefore wondered whether RhoA and Rac1 are intrinsically coupled or whether they can be manipulated independently. To address this question, we placed constitutively active (CA) RhoA under a doxycycline-inducible promoter and CA Rac1 under an orthogonal cumate-inducible promoter, and we stably introduced both constructs into glioblastoma cells. We found that doxycycline addition increased RhoA activity without altering Rac1 and similarly cumate addition increased Rac1 activity without altering RhoA. Furthermore, co-expression of both mutants enabled high activation of RhoA and Rac1 simultaneously. When cells were cultured on collagen hydrogels, RhoA activation prevented cell spreading and motility, whereas Rac1 activation stimulated migration and dynamic cell protrusions. Interestingly, high activation of both GTPases induced a third phenotype, in which cells migrated at intermediate speeds similar to control cells but also aggregated into large, contractile clusters. In addition, we demonstrate dynamic and reversible switching between high RhoA and high Rac1 phenotypes. Overall, this approach represents a unique way to access different combinations of RhoA and Rac1 activity levels in a single cell and may serve as a valuable tool for multiplexed dissection and control of mechanobiological signals. PMID:25044255

  9. CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

    PubMed Central

    Ghosh, Manik C.; Makena, Patrudu S.; Gorantla, Vijay; Sinclair, Scott E.

    2012-01-01

    Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury. PMID:22345572

  10. A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

    PubMed Central

    Liu, Yunhao; Collins, Caitlin; Kiosses, William B.; Murray, Ann M.; Joshi, Monika; Shepherd, Tyson R.; Fuentes, Ernesto J.

    2013-01-01

    Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase. PMID:23733346

  11. Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia

    PubMed Central

    Ablack, Amber; Hall, Emily H.; Butcher, Lindsay D.; Bhattacharyya, Asima; Eckmann, Lars; Harris, Paul R.; Das, Soumita; Ernst, Peter B.; Crowe, Sheila E.

    2016-01-01

    Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections. PMID:26761793

  12. Supervillin Binds the Rac/Rho-GEF Trio and Increases Trio-mediated Rac1 Activation

    PubMed Central

    Son, Kyonghee; Smith, Tara C.; Luna, Elizabeth J.

    2015-01-01

    We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange (GEF) activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4–7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276 – 669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4–7, amino acids 531 – 1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771 – 1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension. PMID:25655724

  13. Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases.

    PubMed

    Jou, T S; Schneeberger, E E; Nelson, W J

    1998-07-13

    Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane. PMID:9660866

  14. The NKD1/Rac1 feedback loop regulates the invasion and migration ability of hepatocarcinoma cells.

    PubMed

    Li, Jie; Zhang, Sheng; Hu, Qing; Zhang, Kang; Jin, Jianbin; Zheng, Xuqing; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatocellular carcinoma (HCC) is complicated by aggressive migration and invasion, which contribute to the increased mortality of HCC patients. The NKD1 protein is abnormally expressed in many neoplasms and plays an important role in tumor progression. However, the regulation and underlying molecular mechanisms of NKD1 in HCC cell invasion and migration remain poorly understood. In the present study, ectopic expression of NKD1 in HCC cells attenuated migration and invasion in vitro and in vivo by down-regulating Rac1 expression level and activity, which affected the HCC cell cytoskeleton and E-cadherin expression. Mechanistic studies showed that NKD1 interacted with Rac1 in the cytoplasm and promoted its degradation by the ubiquitin-proteasome pathway. Over-expression of Rac1 enhanced the transcription of the NKD1 gene and protein expression conversely owing to its negative regulation of EZH2. Analysis of clinical samples showed that abnormal expression of NKD1 and Rac1 was associated with the poor prognosis of HCC patients. In summary, our data indicate a new role for NKD1 as a regulator of HCC cell invasion and migration via a feedback loop involving Rac1. PMID:27231134

  15. The NKD1/Rac1 feedback loop regulates the invasion and migration ability of hepatocarcinoma cells

    PubMed Central

    Li, Jie; Zhang, Sheng; Hu, Qing; Zhang, Kang; Jin, Jianbin; Zheng, Xuqing; Yin, Zhenyu; Wang, Xiaomin

    2016-01-01

    Hepatocellular carcinoma (HCC) is complicated by aggressive migration and invasion, which contribute to the increased mortality of HCC patients. The NKD1 protein is abnormally expressed in many neoplasms and plays an important role in tumor progression. However, the regulation and underlying molecular mechanisms of NKD1 in HCC cell invasion and migration remain poorly understood. In the present study, ectopic expression of NKD1 in HCC cells attenuated migration and invasion in vitro and in vivo by down-regulating Rac1 expression level and activity, which affected the HCC cell cytoskeleton and E-cadherin expression. Mechanistic studies showed that NKD1 interacted with Rac1 in the cytoplasm and promoted its degradation by the ubiquitin-proteasome pathway. Over-expression of Rac1 enhanced the transcription of the NKD1 gene and protein expression conversely owing to its negative regulation of EZH2. Analysis of clinical samples showed that abnormal expression of NKD1 and Rac1 was associated with the poor prognosis of HCC patients. In summary, our data indicate a new role for NKD1 as a regulator of HCC cell invasion and migration via a feedback loop involving Rac1. PMID:27231134

  16. Regulation of Pathogenic Spore Germination by CgRac1 in the Fungal Plant Pathogen Colletotrichum gloeosporioides ▿ ‡

    PubMed Central

    Nesher, Iris; Minz, Anna; Kokkelink, Leonie; Tudzynski, Paul; Sharon, Amir

    2011-01-01

    Colletotrichum gloeosporioides is a facultative plant pathogen: it can live as a saprophyte on dead organic matter or as a pathogen on a host plant. Different patterns of conidial germination have been recognized under saprophytic and pathogenic conditions, which also determine later development. Here we describe the role of CgRac1 in regulating pathogenic germination. The hallmark of pathogenic germination is unilateral formation of a single germ tube following the first cell division. However, transgenic strains expressing a constitutively active CgRac1 (CA-CgRac1) displayed simultaneous formation of two germ tubes, with nuclei continuing to divide in both cells after the first cell division. CA-CgRac1 also caused various other abnormalities, including difficulties in establishing and maintaining cell polarity, reduced conidial and hyphal adhesion, and formation of immature appressoria. Consequently, CA-CgRac1 isolates were completely nonpathogenic. Localization studies with cyan fluorescent protein (CFP)-CgRac1 fusion protein showed that the CgRac1 protein is abundant in conidia and in hyphal tips. Although the CFP signal was equally distributed in both cells of a germinating conidium, reactive oxygen species accumulated only in the cell that produced a germ tube, indicating that CgRac1 was active only in the germinating cell. Collectively, our results show that CgRac1 is a major regulator of asymmetric development and that it is involved in the regulation of both morphogenesis and nuclear division. Modification of CgRac1 activity disrupts the morphogenetic program and prevents fungal infection. PMID:21460190

  17. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

    PubMed Central

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M.; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  18. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

    PubMed

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2016-06-01

    The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation. PMID:27154205

  19. Expression and activity of Rac1 is negatively affected in the dehydroepiandrosterone induced polycystic ovary of mouse

    PubMed Central

    2014-01-01

    Background Polycystic ovarian syndrome (PCOS) is characterized by the presence of multiple follicular cysts, giving rise to infertility due to anovulation. This syndrome affects about 10% of women, worldwide. The exact molecular mechanism leading to PCOS remains obscure. RhoGTPase has been associated with oogenesis, but its role in PCOS remains unexplored. Therefore, we attempted to elucidate the Vav-Rac1 signaling in PCOS mice model. Methods We generated a PCOS mice model by injecting dehydroepiandrosterone (DHEA) for a period of 20 days. The expression levels of Rac1, pRac1, Vav, pVav and Caveolin1 were analyzed by employing immuno-blotting and densitometry. The association between Vav and Rac1 proteins were studied by immuno-precipitation. Furthermore, we analyzed the activity of Rac1 and levels of inhibin B and 17β-estradiol in ovary using biochemical assays. Results The presence of multiple follicular cysts in ovary were confirmed by histology. The activity of Rac1 (GTP bound state) was significantly reduced in the PCOS ovary. Similarly, the expression levels of Rac1 and its phosphorylated form (pRac1) were decreased in PCOS in comparison to the sham ovary. The expression level and activity (phosphorylated form) of guanine nucleotide exchanger of Rac1, Vav, was moderately down-regulated. We observed comparatively increased expressions of Caveolin1, 17β-estradiol, and inhibin B in the polycystic ovary. Conclusion We conclude that hyperandrogenization (PCOS) by DHEA diminishes ovarian Rac1 and Vav expression and activity along with an increase in expression of Caveolin1. This is accompanied by an increase in the intra-ovarian level of '17 β-estradiol and inhibin B. PMID:24628852

  20. MT1-MMP is required for myeloid cell fusion via regulation of Rac1 signaling

    PubMed Central

    Gonzalo, Pilar; Guadamillas, Marta C.; Hernández-Riquer, Mª Victoria; Pollán, Ángela; Grande-García, Araceli; Bartolomé, Rubén A.; Vasanji, Amit; Ambrogio, Chiara; Chiarle, Roberto; Teixidó, Joaquín; Risteli, Juha; Apte, Suneel S.; del Pozo, Miguel A.; Arroyo, Alicia G.

    2009-01-01

    SUMMARY Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse in various circumstances but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP-null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas. PMID:20152179

  1. Structural details of light activation of the LOV2-based photoswitch PA-Rac1.

    PubMed

    Winkler, Andreas; Barends, Thomas R M; Udvarhelyi, Anikó; Lenherr-Frey, Daniel; Lomb, Lukas; Menzel, Andreas; Schlichting, Ilme

    2015-02-20

    Optical control of cellular processes is an emerging approach for studying biological systems, affording control with high spatial and temporal resolution. Specifically designed artificial photoswitches add an interesting extension to naturally occurring light-regulated functionalities. However, despite a great deal of structural information, the generation of new tools cannot be based fully on rational design yet; in many cases design is limited by our understanding of molecular details of light activation and signal transduction. Our biochemical and biophysical studies on the established optogenetic tool PA-Rac1, the photoactivatable small GTPase Rac1, reveal how unexpected details of the sensor-effector interface, such as metal coordination, significantly affect functionally important structural elements of this photoswitch. Together with solution scattering experiments, our results favor differences in the population of pre-existing conformations as the underlying allosteric activation mechanism of PA-Rac1, rather than the assumed release of the Rac1 domain from the caging photoreceptor domain. These results have implications for the design of new optogenetic tools and highlight the importance of including molecular details of the sensor-effector interface, which is however difficult to assess during the initial design of novel artificial photoswitches. PMID:25368973

  2. IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation.

    PubMed

    Oberoi, Tripat Kaur; Dogan, Taner; Hocking, Jennifer C; Scholz, Rolf-Peter; Mooz, Juliane; Anderson, Carrie L; Karreman, Christiaan; Meyer zu Heringdorf, Dagmar; Schmidt, Gudula; Ruonala, Mika; Namikawa, Kazuhiko; Harms, Gregory S; Carpy, Alejandro; Macek, Boris; Köster, Reinhard W; Rajalingam, Krishnaraj

    2012-01-01

    Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis. PMID:22117219

  3. IbeA and OmpA of Escherichia coli K1 exploit Rac1 activation for invasion of human brain microvascular endothelial cells.

    PubMed

    Maruvada, Ravi; Kim, Kwang Sik

    2012-06-01

    Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC. PMID:22451524

  4. RAC1 activation drives pathologic interactions between the epidermis and immune cells.

    PubMed

    Winge, Mårten C G; Ohyama, Bungo; Dey, Clara N; Boxer, Lisa M; Li, Wei; Ehsani-Chimeh, Nazanin; Truong, Allison K; Wu, Diane; Armstrong, April W; Makino, Teruhiko; Davidson, Matthew; Starcevic, Daniela; Kislat, Andreas; Nguyen, Ngon T; Hashimoto, Takashi; Homey, Bernard; Khavari, Paul A; Bradley, Maria; Waterman, Elizabeth A; Marinkovich, M Peter

    2016-07-01

    Interactions between the epidermis and the immune system govern epidermal tissue homeostasis. These epidermis-immune interactions are altered in the inflammatory disease psoriasis; however, the pathways that underlie this aberrant immune response are not well understood. Here, we determined that Ras-related C3 botulinum toxin substrate 1 (RAC1) is a key mediator of epidermal dysfunction. RAC1 activation was consistently elevated in psoriatic epidermis and primary psoriatic human keratinocytes (PHKCs) exposed to psoriasis-related stimuli, but not in skin from patients with basal or squamous cell carcinoma. Expression of a constitutively active form of RAC1 (RACV12) in mice resulted in the development of lesions similar to those of human psoriasis that required the presence of an intact immune system. RAC1V12-expressing mice and human psoriatic skin showed similar RAC1-dependent signaling as well as transcriptional overlap of differentially expressed epidermal and immune pathways. Coculture of PHKCs with immunocytes resulted in the upregulation of RAC1-dependent proinflammatory cytokines, an effect that was reproduced by overexpressing RAC1 in normal human keratinocytes. In keratinocytes, modulating RAC1 activity altered differentiation, proliferation, and inflammatory pathways, including STAT3, NFκB, and zinc finger protein 750 (ZNF750). Finally, RAC1 inhibition in xenografts composed of human PHKCs and immunocytes abolished psoriasiform hyperplasia and inflammation in vivo. These studies implicate RAC1 as a potential therapeutic target for psoriasis and as a key orchestrator of pathologic epidermis-immune interactions. PMID:27294528

  5. Rab5 is required in metastatic cancer cells for Caveolin-1-enhanced Rac1 activation, migration and invasion.

    PubMed

    Díaz, Jorge; Mendoza, Pablo; Ortiz, Rina; Díaz, Natalia; Leyton, Lisette; Stupack, Dwayne; Quest, Andrew F G; Torres, Vicente A

    2014-06-01

    Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion. PMID:24659799

  6. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

    PubMed

    Aranda, Juan F; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A

    2011-04-15

    Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes. PMID:21325632

  7. CD81 is essential for the formation of membrane protrusions and regulates Rac1-activation in adhesion-dependent immune cell migration.

    PubMed

    Quast, Thomas; Eppler, Felix; Semmling, Verena; Schild, Cora; Homsi, Yahya; Levy, Shoshana; Lang, Thorsten; Kurts, Christian; Kolanus, Waldemar

    2011-08-18

    CD81 (TAPA-1) is a member of the widely expressed and evolutionary conserved tetraspanin family that forms complexes with a variety of other cell surface receptors and facilitates hepatitis C virus entry. Here, we show that CD81 is specifically required for the formation of lamellipodia in migrating dendritic cells (DCs). Mouse CD81(-/-) DCs, or murine and human CD81 RNA interference knockdown DCs lacked the ability to form actin protrusions, thereby impairing their motility dramatically. Moreover, we observed a selective loss of Rac1 activity in the absence of CD81, the latter of which is exclusively required for integrin-dependent migration on 2-dimensional substrates. Neither integrin affinity for substrate nor the size of basal integrin clusters was affected by CD81 deficiency in adherent DCs. However, the use of total internal reflection fluorescence microscopy revealed an accumulation of integrin clusters above the basal layer in CD81 knockdown cells. Furthermore, β1- or β2-integrins, actin, and Rac are strongly colocalized at the leading edge of DCs, but the very fronts of these cells protrude CD81-containing membranes that project outward from the actin-integrin area. Taken together, these data suggest a thus far unappreciated role for CD81 in the mobilization of preformed integrin clusters into the leading edge of migratory DCs on 2-dimensional surfaces. PMID:21677313

  8. Melatonin suppresses hypoxia-induced migration of HUVECs via inhibition of ERK/Rac1 activation.

    PubMed

    Yang, Ling; Zheng, Jianchao; Xu, Rui; Zhang, Yujie; Gu, Luo; Dong, Jing; Zhu, Yichao; Zhou, Ruijue; Zheng, Lu; Zhang, Xiaoying; Du, Jun

    2014-01-01

    Melatonin, a naturally-occurring hormone, possesses antioxidant properties and ameliorates vascular endothelial dysfunction. In this study, we evaluate the impact of melatonin on the migratory capability of human umbilical vein endothelial cells (HUVECs) to hypoxia and further investigate whether ERK/Rac1 signaling is involved in this process. Here, we found that melatonin inhibited hypoxia-stimulated hypoxia-inducible factor-1α (HIF-1α) expression and cell migration in a dose-dependent manner. Mechanistically, melatonin inhibited Rac1 activation and suppressed the co-localized Rac1 and F-actin on the membrane of HUVECs under hypoxic condition. In addition, the blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1-T17N suppressed HIF-1α expression and cell migration in response to hypoxia, as well, but constitutive activation of Rac1 mutant Rac1-V12 restored HIF-1α expression, preventing the inhibition of melatonin on cell migration. Furthermore, the anti-Rac1 effect of melatonin in HUVECs appeared to be associated with its inhibition of ERK phosphorylation, but not that of the PI3k/Akt signaling pathway. Taken together, our work indicates that melatonin exerts an anti-migratory effect on hypoxic HUVECs by blocking ERK/Rac1 activation and subsequent HIF-1α upregulation. PMID:25123138

  9. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    PubMed Central

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2013-01-01

    Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm2) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate that both Rac1 and Cdc42 GTPases are critical regulators in shear stress-driven β-catenin signaling in osteoblasts. PMID:23524265

  10. Rac1 and Cdc42 GTPases regulate shear stress-driven β-catenin signaling in osteoblasts

    SciTech Connect

    Wan, Qiaoqiao; Cho, Eunhye; Yokota, Hiroki; Na, Sungsoo

    2013-04-19

    Highlights: •Shear stress increased TCF/LEF activity and stimulated β-catenin nuclear localization. •Rac1, Cdc42, and RhoA displayed distinct dynamic activity patterns under flow. •Rac1 and Cdc42, but not RhoA, regulate shear stress-driven TCF/LEF activation. •Cytoskeleton did not significantly affect shear stress-induced TCF/LEF activation. -- Abstract: Beta-catenin-dependent TCF/LEF (T-cell factor/lymphocyte enhancing factor) is known to be mechanosensitive and an important regulator for promoting bone formation. However, the functional connection between TCF/LEF activity and Rho family GTPases is not well understood in osteoblasts. Herein we investigated the molecular mechanisms underlying oscillatory shear stress-induced TCF/LEF activity in MC3T3-E1 osteoblast cells using live cell imaging. We employed fluorescence resonance energy transfer (FRET)-based and green fluorescent protein (GFP)-based biosensors, which allowed us to monitor signal transduction in living cells in real time. Oscillatory (1 Hz) shear stress (10 dynes/cm{sup 2}) increased TCF/LEF activity and stimulated translocation of β-catenin to the nucleus with the distinct activity patterns of Rac1 and Cdc42. The shear stress-induced TCF/LEF activity was blocked by the inhibition of Rac1 and Cdc42 with their dominant negative mutants or selective drugs, but not by a dominant negative mutant of RhoA. In contrast, constitutively active Rac1 and Cdc42 mutants caused a significant enhancement of TCF/LEF activity. Moreover, activation of Rac1 and Cdc42 increased the basal level of TCF/LEF activity, while their inhibition decreased the basal level. Interestingly, disruption of cytoskeletal structures or inhibition of myosin activity did not significantly affect shear stress-induced TCF/LEF activity. Although Rac1 is reported to be involved in β-catenin in cancer cells, the involvement of Cdc42 in β-catenin signaling in osteoblasts has not been identified. Our findings in this study demonstrate

  11. Glutaminase 2 is a novel negative regulator of small GTPase Rac1 and mediates p53 function in suppressing metastasis

    PubMed Central

    Zhang, Cen; Liu, Juan; Zhao, Yuhan; Yue, Xuetian; Zhu, Yu; Wang, Xiaolong; Wu, Hao; Blanco, Felix; Li, Shaohua; Bhanot, Gyan; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

    2016-01-01

    Glutaminase (GLS) isoenzymes GLS1 and GLS2 are key enzymes for glutamine metabolism. Interestingly, GLS1 and GLS2 display contrasting functions in tumorigenesis with elusive mechanism; GLS1 promotes tumorigenesis, whereas GLS2 exhibits a tumor-suppressive function. In this study, we found that GLS2 but not GLS1 binds to small GTPase Rac1 and inhibits its interaction with Rac1 activators guanine-nucleotide exchange factors, which in turn inhibits Rac1 to suppress cancer metastasis. This function of GLS2 is independent of GLS2 glutaminase activity. Furthermore, decreased GLS2 expression is associated with enhanced metastasis in human cancer. As a p53 target, GLS2 mediates p53’s function in metastasis suppression through inhibiting Rac1. In summary, our results reveal that GLS2 is a novel negative regulator of Rac1, and uncover a novel function and mechanism whereby GLS2 suppresses metastasis. Our results also elucidate a novel mechanism that contributes to the contrasting functions of GLS1 and GLS2 in tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.10727.001 PMID:26751560

  12. Inhibition of Rac1 activity in the hippocampus impaired extinction of contextual fear.

    PubMed

    Jiang, Lizhu; Mao, Rongrong; Tong, Jianbin; Li, Jinnan; Chai, Anping; Zhou, Qixin; Yang, Yuexiong; Wang, Liping; Li, Lingjiang; Xu, Lin

    2016-10-01

    Promoting extinction of fear memory is the main treatment of fear disorders, especially post-traumatic stress disorder (PTSD). However, fear extinction is often incomplete in these patients. Our previous study had shown that Rac1 activity in hippocampus plays a crucial role in the learning of contextual fear memory in rats. Here, we further investigated whether Rac1 activity also modulated the extinction of contextual fear memory. We found that massed extinction obviously upregulated hippocampal Rac1 activity and induced long-term extinction of contextual fear in rats. Intrahippocampal injection of the Rac1 inhibitor NSC23766 prevents extinction of contextual fear in massed extinction training rats. In contrast, long-spaced extinction downregulated Rac1 activity and caused less extinction. And Rac1 activator CN04-A promotes extinction of contextual fear in long-spaced extinction rats. Our study demonstrates that inhibition of Rac1 activity in the hippocampus impaired extinction of contextual fear, suggesting that modulating Rac1 activity of the hippocampus may be promising therapy of fear disorders. PMID:27329554

  13. The Small GTPases RhoA and Rac1 Regulate Cerebellar Development by Controlling Cell Morphogenesis, Migration and Foliation

    PubMed Central

    Mulherkar, Shalaka; Uddin, Mohammad Danish; Couvillon, Anthony D.; Sillitoe, Roy V.; Tolias, Kimberley F.

    2014-01-01

    The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology. PMID:25128586

  14. Raft endocytosis of AMF regulates mitochondrial dynamics through Rac1 signaling and the Gp78 ubiquitin ligase.

    PubMed

    Shankar, Jay; Kojic, Liliana D; St-Pierre, Pascal; Wang, Peter T C; Fu, Min; Joshi, Bharat; Nabi, Ivan R

    2013-08-01

    Gp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (PGI; also called autocrine motility factor, AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized through a PI3K- and dynamin-dependent raft endocytic pathway to the smooth ER; however, the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, is reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent and is inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and is therefore distinct from Cdc42- and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, but this is reduced by dynasore treatment and is absent in Gp78-knockdown cells; therefore, AMF activities require Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, mitofusin 1 and 2 in a dynamin-, Rac1- and phosphoinositide 3-kinase (PI3K)-dependent manner. Gp78 induces mitochondrial clustering and fission in a manner dependent on GP78 ubiquitin ligase activity, and this is also reversed by uptake of AMF. The raft-dependent endocytosis of AMF, therefore, promotes Rac1-PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics. PMID:23690547

  15. CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

    PubMed

    Lichius, Alexander; Goryachev, Andrew B; Fricker, Mark D; Obara, Boguslaw; Castro-Longoria, Ernestina; Read, Nick D

    2014-05-01

    Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms. We demonstrate that RAC-1 is essential for CAT formation and cell fusion, whereas CDC-42 is necessary and sufficient for normal germ tube development. Cdc42-Rac-interactive-binding (CRIB) reporters were constructed to exclusively label locally activated GTP-bound GTPases. Time course analyses showed that repositioning of these activated GTPase clusters within germ tube and CAT tip apices controls directional growth in the absence of a tip-localized vesicle supply center (Spitzenkörper). We propose a model in which the local assembly of a plasma-membrane-associated GTPase-PAK-MAPK signaling platform regulates chemoattractant perception and secretion in order to synchronize oscillatory cell-cell communication and directional CAT tip growth. PMID:24790223

  16. Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

    PubMed Central

    Housley, Michael P.; Weiner, Orion D.

    2012-01-01

    Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions. PMID:22945937

  17. Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

    SciTech Connect

    Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene; Raptis, Leda

    2010-03-10

    Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

  18. CCK activates RhoA and Rac1 differentially through Gα13 and Gαq in mouse pancreatic acini

    PubMed Central

    Bi, Yan; Ji, Baoan; Ernst, Stephen A.; Williams, John A.

    2010-01-01

    Cholecystokinin (CCK) has been shown to activate RhoA and Rac1, as well as reorganize the actin cytoskeleton and, thereby, modify acinar morphology and amylase secretion in mouse pancreatic acini. The aim of the present study was to determine which heterotrimeric G proteins activate RhoA and Rac1 upon CCK stimulation. Gα13, but not Gα12, was identified in mouse pancreatic acini by RT-PCR and Western blotting. Using specific assays for RhoA and Rac1 activation, we showed that only active Gα13 activated RhoA. By contrast, active Gα13 and Gαq, but not Gαs, slightly increased GTP-bound Rac1 levels. A greater increase in Rac1 activation was observed when active Gα13 and active Gαq were coexpressed. Gαi was not required for CCK-induced RhoA or Rac1 activation. The regulator of G protein signaling (RGS) domain of p115-Rho guanine nucleotide exchange factor (p115-RGS), a specific inhibitor of Gα12/13-mediated signaling, abolished CCK-stimulated RhoA activation. By contrast, both RGS-2, an inhibitor of Gαq, and p115-RGS abolished CCK-induced Rac1 activation, which was PLC pathway-independent. Active Gαq and Gα13, but not Gαs, induced morphological changes and actin redistribution similar to 1 nM CCK. CCK-induced actin cytoskeletal reorganization was inhibited by RGS-2, but not by p115-RGS, whereas CCK-induced amylase secretion was blocked by both inhibitors. Together, these findings indicate that, in mouse pancreatic acini, Gα13 links CCK stimulation to the activation of RhoA, whereas both Gα13 and Gαq link CCK stimulation to the activation of Rac1. CCK-induced actin cytoskeletal reorganization is mainly mediated by Gαq. By contrast, Gα13 and Gαq signaling are required for CCK-induced amylase secretion. PMID:19940064

  19. Inhibition of Rac1 activity by controlled release of NSC23766 from chitosan microspheres effectively ameliorates osteoarthritis development in vivo

    PubMed Central

    Zhu, Shouan; Lu, Ping; Liu, Huanhuan; Chen, Pengfei; Wu, Yan; Wang, Yanyan; Sun, Heng; Zhang, Xiaolei; Xia, Qingqing; Heng, Boon Chin; Zhou, Yiting; Ouyang, Hong Wei

    2015-01-01

    Background Osteoarthritis (OA) is a degenerative joint disease characterised by cartilage degradation and chondrocyte hypertrophy. A recent study showed that Rac1 promoted expression of MMP13 and chondrocyte hypertrophy within the growth plate. These findings warrant further investigations on the roles of Rac1 in OA development and therapy in animal models. Objective To investigate the role and mechanistic pathway of Rac1 involvement in pathological changes of OA chondrocytes in vitro and OA development in vivo, as well as to develop a strategy of modulating Rac1 activity for OA treatment. Material and methods OA and normal cartilage from human or mice were used for immunohistochemical study and Rac1 activity assay. Chondrocytes treated with IL1β and the untreated control were subjected to the Rac1 activity assay. Chondrocytes transfected with CA-Rac1, DN-Rac1 or GFP were cultured under conditions for inducing calcification. To evaluate the effect of Rac1 in OA development, an OA model was created by anterior cruciate ligament transection in mice. CA-Rac1, DN-Rac1 and GFP lentivirus, or NSC23766, were injected intra-articularly. Joints were subjected to histological analysis. Results It was found that there is aberrant Rac1 activation in human OA cartilage. Rac1 activity could also be elevated by IL1β. Additionally, activated Rac1 promoted expression of MMP13, ADAMTS-5 and COLX by chondrocytes, partially through the β-catenin pathway. Moreover, activation of Rac1 in knee joints by CA-Rac1 lentivirus accelerated OA progression, while inhibition of Rac1 activity by DN-Rac1 lentivirus or Rac1 inhibitor NSC23766 delayed OA development. Therefore, we developed a strategy of controlled release of NSC23766 from chitosan microspheres to OA joints, which effectively protected cartilage from destruction. Conclusions These findings demonstrated that Rac1 activity is implicated in OA development. Also, controlled release of Rac1 inhibitor is a promising strategy for OA

  20. Central Role of Protein Kinase Cε in Constitutive Activation of ERK1/2 and Rac1 in the Malignant Cells of Hairy Cell Leukemia

    PubMed Central

    Slupsky, Joseph R.; Kamiguti, Aura S.; Harris, Robert J.; Cawley, John C.; Zuzel, Mirko

    2007-01-01

    We have previously identified the presence of Ras/Raf-independent constitutive activation of extracellular signal-regulated kinase (ERK) in the hairy cells (HCs) of hairy cell leukemia. The aim of the present study was to characterize the signaling components involved in this activation and their relationship to the reported activation of Rac1. We found that both Rac1 and ERK activation in HCs are downstream of active Src and protein kinase C (PKC). Inhibition with toxin B showed that Rac1 plays no role in ERK activation in HCs. However, toxin B inhibited p60src and the Rac1-GEF Vav, demonstrating a positive feedback/activation of p60src by Rac1. Treatment with specific small interfering RNA for various PKC isoforms, or with PKC isoform-specific inhibitors, demonstrated a central role for PKCε in the constitutive activation of Rac1 and ERK in HCs. PKCε and active ERK were mutually associated and co-localized with mitochondria in HCs. Furthermore, active PKCε was nitrated on tyrosine, pointing to a reactive oxygen species-dependent mechanism of activation. By being involved in activation of ERK and Rac1, PKCε plays roles in both the survival of HCs and in the cytoskeletal dynamics responsible for the distinctive morphology and tissue homing of these cells. Our study therefore describes novel aspects of signaling important for the pathogenesis of hairy cell leukemia. PMID:17255340

  1. Cellular inhibitor of apoptosis protein 2 controls human colonic epithelial restitution, migration, and Rac1 activation.

    PubMed

    Seidelin, Jakob Benedict; Larsen, Sylvester; Linnemann, Dorte; Vainer, Ben; Coskun, Mehmet; Troelsen, Jesper Thorvald; Nielsen, Ole Haagen

    2015-01-15

    Identification of pathways involved in wound healing is important for understanding the pathogenesis of various intestinal diseases. Cellular inhibitor of apoptosis protein 2 (cIAP2) regulates proliferation and migration in nonepithelial cells and is expressed in human colonocytes. The aim of the study was to investigate the role of cIAP2 for wound healing in the normal human colon. Wound tissue was generated by taking rectosigmoidal biopsies across an experimental ulcer in healthy subjects after 5, 24, and 48 h. In experimental ulcers, the expression of cIAP2 in regenerating intestinal epithelial cells (IECs) was increased at the wound edge after 24 h (P < 0.05), returned to normal after reepithelialization, and correlated with the inflammatory reaction in the experimental wounds (P < 0.001). cIAP2 was induced in vitro in regenerating Caco2 IECs after wound infliction (P < 0.01). Knockdown of cIAP2 caused a substantial impairment of the IEC regeneration through inhibition of migration (P < 0.005). cIAP2 overexpression lead to formation of migrating IECs and upregulation of expression of RhoA and Rac1 as well as GTP-activation of Rac1. Transforming growth factor-β1 enhanced the expression of cIAP2 but was not upregulated in wounds in vivo and in vitro. NF-κB and MAPK pathways did not affect cIAP2 expression. cIAP2 is in conclusion a regulator of human intestinal wound healing through enhanced migration along with activation of Rac1, and the findings suggest that cIAP2 could be a future therapeutic target to improve intestinal wound healing. PMID:25394657

  2. Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells

    PubMed Central

    Cardama, Georgina A; Gonzalez, Nazareno; Ciarlantini, Matias; Gandolfi Donadío, Lucia; Comin, María Julieta; Alonso, Daniel F; Menna, Pablo Lorenzano; Gomez, Daniel E

    2014-01-01

    Malignant gliomas are characterized by an intrinsic ability to invade diffusely throughout the normal brain tissue. This feature contributes mainly to the failure of existing therapies. Deregulation of small GTPases signaling, in particular Rac1 activity, plays a key role in the invasive phenotype of gliomas. Here we report the effect of ZINC69391, a specific Rac1 inhibitor developed by our group, on human glioma cell lines LN229 and U-87 MG. ZINC69391 is able to interfere with the interaction of Rac1 with Dock180, a relevant Rac1 activator in glioma invasion, and to reduce Rac1-GTP levels. The kinase Pak1, a downstream effector of Dock180–Rac1 signaling, was also downregulated upon ZINC69391 treatment. ZINC69391 reduced cell proliferation, arrested cells in G1 phase, and triggered apoptosis in glioma cells. Importantly, ZINC69391 dramatically affected cell migration and invasion in vitro, interfering with actin cytoskeleton dynamics. We also evaluated the effect of analog 1A-116, a compound derived from ZINC69391 structure. 1A-116 showed an improved antiproliferative and antiinvasive activity on glioma cells. These findings encourage further preclinical testing in clinically relevant animal models. PMID:25378937

  3. Rac1/Pak1/p38/MMP-2 axis regulates angiogenesis in ovarian cancer

    PubMed Central

    Gonzalez-Villasana, Vianey; Fuentes-Mattei, Enrique; Ivan, Cristina; Dalton, Heather J.; Rodriguez-Aguayo, Cristian; Fernandez-de Thomas, Ricardo J.; Aslan, Burcu; Monroig, Paloma del C.; Velazquez-Torres, Guermarie; Previs, Rebecca A.; Pradeep, Sunila; Kahraman, Nermin; Wang, Huamin; Kanlikilicer, Pinar; Ozpolat, Bulent; Calin, George; Sood, Anil K.; Lopez-Berestein, Gabriel

    2015-01-01

    Purpose Zoledronic acid (ZA) is being increasingly recognized for its anti-tumor properties, but the underlying functions are not well understood. In this study, we hypothesized that ZA inhibits ovarian cancer (OC) angiogenesis preventing Rac1 activation. Experimental Design The biological effects of ZA were examined using a series of in vitro (cell invasion, cytokine production, Rac1 activation, reverse-phase protein array and in vivo (orthotopic mouse models) experiments. Results There was significant inhibition of OC (HeyA8-MDR and OVCAR-5) cell invasion as well as reduced production of pro-angiogenic cytokines in response to ZA treatment. Furthermore, ZA inactivated Rac1 and decreased the levels of Pak1/p-38/matrix metalloproteinase-2 in OC cells. In vivo, ZA reduced tumor growth, angiogenesis and cell proliferation and inactivated Rac1 in both HeyA8-MDR and OVCAR-5 models. These in vivo antitumor effects were enhanced in both models when ZA was combined with nab-paclitaxel. Conclusion ZA has robust anti-tumor and anti-angiogenic activity and merits further clinical development as OC treatment. PMID:25595279

  4. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle

    PubMed Central

    Takenaka, Nobuyuki; Nihata, Yuma; Satoh, Takaya

    2016-01-01

    Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling. PMID:27163697

  5. Cla4, but not Rac1, regulates the filamentous response of Ustilago maydis to low ammonium conditions

    PubMed Central

    Lovely, C. Ben

    2011-01-01

    Ustilago maydis, the fungal pathogen of maize, undergoes a dimorphic transition from budding yeast-like growth to filamentous growth, both as part of its program for pathogenesis and distinctly, in response to environmental cues, such as acid pH or low nitrogen availability. Smu1 is a p21-activated protein kinase (PAK) with roles in both the mating response required for the former function, as well as for the nutrient response. Hsl7 may be a negative regulator of Smu1 and appears to play a role in cell length and cell cycle.  Additional proteins that participate in cell polarity and filamentation pathways include the small G protein, Rac1, and its effector PAK kinase, Cla4. Here we describe further experiments that explore the roles of Cla4 and Rac1 in the response to nitrogen availability. While deletion of rac1severely delays filamentous growth on solid media low in ammonium (SLAD), we found that deletion of cla4 does not abolish filamentous cell morphology on solid SLAD. Unexpectedly, however, the Dcla4 mutants also filament in liquid SLAD. The filamentous cell morphology of the cla4 mutant in liquid SLAD has only been seen previously for one other mutant, a strain deleted for hsl7 that simultaneously over-expresses smu1.  PMID:22446524

  6. Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

    PubMed Central

    Jay, Steven M.; Skokos, Eleni; Laiwalla, Farah; Krady, Marie-Marthe; Kyriakides, Themis R.

    2007-01-01

    Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can recapitulate the fusion process in vitro. In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo. These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites. PMID:17556592

  7. Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity

    PubMed Central

    Chen, Lihua; DeWispelaere, Allison; Dastvan, Frank; Osborne, William R. A.; Blechner, Christine; Windhorst, Sabine; Daum, Guenter

    2016-01-01

    Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration. PMID:27176050

  8. Narrow-band UVB radiation promotes dendrite formation by activating Rac1 in B16 melanoma cells.

    PubMed

    Wang, Wu-Qing; Wu, Jin-Feng; Xiao, Xiao-Qing; Xiao, Qin; Wang, Jing; Zuo, Fu-Guo

    2013-09-01

    Melanocytes are found scattered throughout the basal layer of the epidermis. Following hormone or ultraviolet (UV) light stimulation, the melanin pigments contained in melanocytes are transferred through the dendrites to the surrounding keratinocytes to protect against UV light damage or carcinogenesis. This has been considered as a morphological indicator of melanocytes and melanoma cells. Small GTPases of the Rho family have been implicated in the regulation of actin reorganization underlying dendrite formation in melanocytes and melanoma cells. It has been proven that ultraviolet light plays a pivotal role in melanocyte dendrite formation; however, the molecular mechanism underlying this process has not been fully elucidated. The effect of small GTPases, such as Rac1 and RhoA, on the morphology of B16 melanoma cells treated with narrow-band UVB radiation was investigated. The morphological changes were observed under a phase contrast microscope and the F-actin microfilament of the cytoskeleton was observed under a laser scanning confocal microscope. The pull-down assay was performed to detect the activity of the small GTPases Rac1 and RhoA. The morphological changes were evident, with globular cell bodies and increased numbers of tree branch-like dendrites. The cytoskeletal F-actin appeared disassembled following narrow-band UVB irradiation of B16 melanoma cells. Treatment of B16 melanoma cells with narrow-band UVB radiation resulted in the activation of Rac1 in a time-dependent manner. In conclusion, the present study may provide a novel method through which narrow-band UVB radiation may be used to promote dendrite formation by activating the Rac1 signaling pathway, resulting in F-actin rearrangement in B16 melanoma cells. PMID:24649261

  9. The N-BAR Domain Protein, Bin3, Regulates Rac1- and Cdc42-Dependent Processes in Myogenesis

    PubMed Central

    Simionescu-Bankston, Adriana; Leoni, Giovanna; Wang, Yanru; Pham, Peter P.; Ramalingam, Arivudainambi; DuHadaway, James B.; Faundez, Victor; Nusrat, Asma; Prendergast, George C.; Pavlath, Grace K.

    2013-01-01

    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. PMID:23872330

  10. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  11. Regulation of hyphal morphogenesis by cdc42 and rac1 homologues in Aspergillus nidulans.

    PubMed

    Virag, Aleksandra; Lee, Maurice P; Si, Haoyu; Harris, Steven D

    2007-12-01

    The ability of filamentous fungi to form hyphae requires the establishment and maintenance of a stable polarity axis. Based on studies in yeasts and animals, the GTPases Cdc42 and Rac1 are presumed to play a central role in organizing the morphogenetic machinery to enable axis formation and stabilization. Here, we report that Cdc42 (ModA) and Rac1 (RacA) share an overlapping function required for polarity establishment in Aspergillus nidulans. Nevertheless, Cdc42 appears to have a more important role in hyphal morphogenesis in that it alone is required for the timely formation of lateral branches. In addition, we provide genetic evidence suggesting that the polarisome components SepA and SpaA function downstream of Cdc42 in a pathway that may regulate microfilament formation. Finally, we show that microtubules become essential for the establishment of hyphal polarity when the function of either Cdc42 or SepA is compromised. Our results are consistent with the action of parallel Cdc42 and microtubule-based pathways in regulating the formation of a stable axis of hyphal polarity in A. nidulans. PMID:18005099

  12. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2

    PubMed Central

    Grassart, Alexandre; Dujeancourt, Annick; Lazarow, Paul B; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2008-01-01

    There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism—clathrin- and caveolae-independent endocytosis—used by the interleukin-2 receptor β (IL-2Rβ). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2Rβ entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2Rβ uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin—two essential endocytic factors—is required for IL-2Rβ uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2Rβ internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway. PMID:18344974

  13. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2.

    PubMed

    Grassart, Alexandre; Dujeancourt, Annick; Lazarow, Paul B; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2008-04-01

    There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway. PMID:18344974

  14. Cdc42 and Rac1 activity is reduced in human pheochromocytoma and correlates with FARP1 and ARHGEF1 expression.

    PubMed

    Croisé, Pauline; Houy, Sébastien; Gand, Mathieu; Lanoix, Joël; Calco, Valérie; Tóth, Petra; Brunaud, Laurent; Lomazzi, Sandra; Paramithiotis, Eustache; Chelsky, Daniel; Ory, Stéphane; Gasman, Stéphane

    2016-04-01

    Among small GTPases from the Rho family, Cdc42, RAC, and Rho are well known to mediate a large variety of cellular processes linked with cancer biology through their ability to cycle between an inactive (GDP-bound) and an active (GTP-bound) state. Guanine nucleotide exchange factors (GEFs) stimulate the exchange of GDP for GTP to generate the activated form, whereas the GTPase-activating proteins (GAPs) catalyze GTP hydrolysis, leading to the inactivated form. Modulation of Rho GTPase activity following altered expression of RHO-GEFs and/or RHO-GAPs has already been reported in various human tumors. However, nothing is known about the Rho GTPase activity or the expression of their regulators in human pheochromocytomas, a neuroendocrine tumor (NET) arising from chromaffin cells of the adrenal medulla. In this study, we demonstrate, through an ELISA-based activity assay, that Rac1 and Cdc42 activities decrease in human pheochromocytomas (PCCs) compared with the matched adjacent non-tumor tissue. Furthermore, through quantitative mass spectrometry (MS) approaches, we show that the expression of two RHO-GEF proteins, namely ARHGEF1 and FARP1, is significantly reduced in tumors compared with matched non-tumor tissue, whereas ARHGAP36 expression is increased. Moreover, siRNA-based knockdown of ARHGEF1 and FARP1 in PC12 cells leads to a significant inhibition of Rac1 and Cdc42 activities, respectively. Finally, a principal component analysis (PCA) of our dataset was able to discriminate PCC from non-tumor tissue and indicates a close correlation between Cdc42/Rac1 activity and FARP1/ARHGEF1 expression. Altogether, our findings reveal for the first time the importance of modulation of Rho GTPase activities and expression of their regulators in human PCCs. PMID:26911374

  15. Serine-71 phosphorylation of Rac1 modulates downstream signaling.

    PubMed

    Schwarz, Janett; Proff, Julia; Hävemeier, Anika; Ladwein, Markus; Rottner, Klemens; Barlag, Britta; Pich, Andreas; Tatge, Helma; Just, Ingo; Gerhard, Ralf

    2012-01-01

    The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways. PMID:22970203

  16. Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans

    PubMed Central

    Shen, Gui; Zhou, Erxun; Alspaugh, J. Andrew

    2012-01-01

    Human Wiskott-Aldrich syndrome protein (WASP) is a scaffold linking upstream signals to the actin cytoskeleton. In response to intersectin ITSN1 and Rho GTPase Cdc42, WASP activates the Arp2/3 complex to promote actin polymerization. The human pathogen Cryptococcus neoformans contains the ITSN1 homolog Cin1 and the WASP homolog Wsp1, which share more homology with human proteins than those of other fungi. Here we demonstrate that Cin1, Cdc42/Rac1, and Wsp1 function in an effector pathway similar to that of mammalian models. In the cin1 mutant, expression of the autoactivated Wsp1-B-GBD allele partially suppressed the mutant defect in endocytosis, and expression of the constitutively active CDC42Q61L allele restored normal actin cytoskeleton structures. Similar phenotypic suppression can be obtained by the expression of a Cdc42-green fluorescent protein (GFP)-Wsp1 fusion protein. In addition, Rac1, which was found to exhibit a role in early endocytosis, activates Wsp1 to regulate vacuole fusion. Rac1 interacted with Wsp1 and depended on Wsp1 for its vacuolar membrane localization. Expression of the Wsp1-B-GBD allele restored vacuolar membrane fusion in the rac1 mutant. Collectively, our studies suggest novel ways in which this pathogenic fungus has adapted conserved signaling pathways to control vesicle transport and actin organization, likely benefiting survival within infected hosts. PMID:22327008

  17. Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases.

    PubMed

    Oprea, Tudor I; Sklar, Larry A; Agola, Jacob O; Guo, Yuna; Silberberg, Melina; Roxby, Joshua; Vestling, Anna; Romero, Elsa; Surviladze, Zurab; Murray-Krezan, Cristina; Waller, Anna; Ursu, Oleg; Hudson, Laurie G; Wandinger-Ness, Angela

    2015-01-01

    Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses-using the rotationally constrained carboxylate in R-naproxen-led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and

  18. Novel Activities of Select NSAID R-Enantiomers against Rac1 and Cdc42 GTPases

    PubMed Central

    Oprea, Tudor I.; Sklar, Larry A.; Agola, Jacob O.; Guo, Yuna; Silberberg, Melina; Roxby, Joshua; Vestling, Anna; Romero, Elsa; Surviladze, Zurab; Murray-Krezan, Cristina; Waller, Anna; Ursu, Oleg; Hudson, Laurie G.; Wandinger-Ness, Angela

    2015-01-01

    Rho family GTPases (including Rac, Rho and Cdc42) collectively control cell proliferation, adhesion and migration and are of interest as functional therapeutic targets in numerous epithelial cancers. Based on high throughput screening of the Prestwick Chemical Library® and cheminformatics we identified the R-enantiomers of two approved drugs (naproxen and ketorolac) as inhibitors of Rac1 and Cdc42. The corresponding S-enantiomers are considered the active component in racemic drug formulations, acting as non-steroidal anti-inflammatory drugs (NSAIDs) with selective activity against cyclooxygenases. Here, we show that the S-enantiomers of naproxen and ketorolac are inactive against the GTPases. Additionally, more than twenty other NSAIDs lacked inhibitory action against the GTPases, establishing the selectivity of the two identified NSAIDs. R-naproxen was first identified as a lead compound and tested in parallel with its S-enantiomer and the non-chiral 6-methoxy-naphthalene acetic acid (active metabolite of nabumetone, another NSAID) as a structural series. Cheminformatics-based substructure analyses—using the rotationally constrained carboxylate in R-naproxen—led to identification of racemic [R/S] ketorolac as a suitable FDA-approved candidate. Cell based measurement of GTPase activity (in animal and human cell lines) demonstrated that the R-enantiomers specifically inhibit epidermal growth factor stimulated Rac1 and Cdc42 activation. The GTPase inhibitory effects of the R-enantiomers in cells largely mimic those of established Rac1 (NSC23766) and Cdc42 (CID2950007/ML141) specific inhibitors. Docking predicts that rotational constraints position the carboxylate moieties of the R-enantiomers to preferentially coordinate the magnesium ion, thereby destabilizing nucleotide binding to Rac1 and Cdc42. The S-enantiomers can be docked but are less favorably positioned in proximity to the magnesium. R-naproxen and R-ketorolac have potential for rapid translation and

  19. GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis.

    PubMed

    Chang, Jeng-Shou; Su, Chia-Yi; Yu, Wen-Hsuan; Lee, Wei-Jiunn; Liu, Yu-Peng; Lai, Tsung-Ching; Jan, Yi-Hua; Yang, Yi-Fang; Shen, Chia-Ning; Shew, Jin-Yuh; Lu, Jean; Yang, Chih-Jen; Huang, Ming-Shyan; Lu, Pei-Jung; Lin, Yuan-Feng; Kuo, Min-Liang; Hua, Kuo-Tai; Hsiao, Michael

    2015-11-01

    G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42. PMID:26462147

  20. GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis

    PubMed Central

    Chang, Jeng-Shou; Su, Chia-Yi; Yu, Wen-Hsuan; Lee, Wei-Jiunn; Liu, Yu-Peng; Lai, Tsung-Ching; Jan, Yi-Hua; Yang, Yi-Fang; Shen, Chia-Ning; Shew, Jin-Yuh; Lu, Jean; Yang, Chih-Jen; Huang, Ming-Shyan; Lu, Pei-Jung; Lin, Yuan-Feng; Kuo, Min-Liang; Hua, Kuo-Tai; Hsiao, Michael

    2015-01-01

    G-protein-coupled receptor kinase interacting protein 1 (GIT1) is participated in cell movement activation, which is a fundamental process during tissue development and cancer progression. GIT1/PIX forming a functional protein complex that contributes to Rac1/Cdc42 activation, resulting in increasing cell mobility. Although the importance of Rac1/Cdc42 activation is well documented in cancer aggressiveness, the clinical importance of GIT1 remains largely unknown. Here, we investigated the clinical significance of GIT1 expression in non-small-cell lung cancer (NSCLC) and also verified the importance of GIT1-Rac1/Cdc42 axis in stimulating NSCLC cell mobility. The result indicated higher GIT1 expression patients had significantly poorer prognoses in disease-free survival (DFS) and overall survival (OS) compared with lower GIT1 expression patients. Higher GIT1 expression was an independent prognostic factor by multivariate analysis and associated with migration/invasion of NSCLC cells in transwell assay. In vivo studies indicated that GIT1 promotes metastasis of NSCLC cells. Finally, GIT1 was found to stimulate migration/invasion by altering the activity of Rac1/Cdc42 in NSCLC cells. Together, the GIT1 expression is associated with poor prognosis in patients with NSCLC. GIT1 is critical for the invasiveness of NSCLC cells through stimulating the activity of Rac1/Cdc42. PMID:26462147

  1. Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement.

    PubMed

    Zhang, Xun E; Adderley, Shaquria P; Breslin, Jerome W

    2016-01-01

    Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process. PMID:27187066

  2. Activation of RhoA, but Not Rac1, Mediates Early Stages of S1P-Induced Endothelial Barrier Enhancement

    PubMed Central

    Zhang, Xun E.; Adderley, Shaquria P.

    2016-01-01

    Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine-1-phosphate (S1P)-induced endothelial barrier enhancement, remain elusive. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell-substrate impedance sensor (ECIS). The roles of Rac1 and RhoA were tested in S1P-induced barrier enhancement. The results show that pharmacologic inhibition of Rac1 with Z62954982 failed to block S1P-induced barrier enhancement. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P-induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 significantly reduced S1P-induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain-2 phosphorylation at cell edges, and increased F-actin and vinculin near intercellular junctions, which could all be blocked with pharmacologic inhibition of RhoA. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local activation of the actin cytoskeleton and focal adhesions, and resulting in endothelial barrier enhancement. S1P-induced Rac1 activation, however, does not appear to have a significant role in this process. PMID:27187066

  3. PTEN inhibits PREX2-catalyzed activation of RAC1 to restrain tumor cell invasion

    PubMed Central

    Mense, Sarah M.; Barrows, Douglas; Hodakoski, Cindy; Steinbach, Nicole; Schoenfeld, David; Su, William; Hopkins, Benjamin D.; Su, Tao; Fine, Barry; Hibshoosh, Hanina; Parsons, Ramon

    2016-01-01

    The tumor suppressor PTEN restrains cell migration and invasion by a mechanism that is independent of inhibition of the PI3K pathway and decreased activation of the kinase AKT. PREX2, a widely distributed GEF that activates the GTPase RAC1, binds to and inhibits PTEN. We used mouse embryonic fibroblasts and breast cancer cell lines to show that PTEN suppresses cell migration and invasion by blocking PREX2 activity. In addition to metabolizing the phosphoinositide PIP3, PTEN inhibited PREX2-induced invasion by a mechanism that required the tail domain of PTEN, but not its lipid phosphatase activity. Fluorescent nucleotide exchange assays revealed that PTEN inhibited the GEF activity of PREX2 toward RAC1. PREX2 is a frequently mutated GEF in cancer, and examination of human tumor data showed that PREX2 mutation was associated with high PTEN expression. Therefore, we tested whether cancer-derived somatic PREX2 mutants, which accelerate tumor formation of immortalized melanocytes, were inhibited by PTEN. The three stably expressed, somatic PREX2 cancer mutants that we tested were resistant to PTEN-mediated inhibition of invasion but retained the ability to inhibit the lipid phosphatase activity of PTEN. In vitro analysis showed that PTEN did not block the GEF activity of two PREX2 cancer mutants and had a reduced binding affinity for the third. Thus, PTEN antagonized migration and invasion by restraining PREX2 GEF activity, and PREX2 mutants are likely selected in cancer to escape PTEN-mediated inhibition of invasion. PMID:25829446

  4. Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

    PubMed Central

    Mohan, Vijay; Pendyala, Srikanth; He, Donghong; Ebenezer, David L.; Harijith, Anantha; Fu, Panfeng; Huang, Long Shuang; Bear, James E.; Garcia, Joe G. N.; Natarajan, Viswanathan

    2013-01-01

    Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis. PMID:23667561

  5. EspT triggers formation of lamellipodia and membrane ruffles through activation of Rac-1 and Cdc42

    PubMed Central

    Bulgin, Richard R; Arbeloa, Ana; Chung, Jade C S; Frankel, Gad

    2009-01-01

    Subversion of the eukaryotic cell cytoskeleton is a virulence strategy employed by many bacterial pathogens. Due to the pivotal role of Rho GTPases in actin dynamics they are common targets of bacterial effector proteins and toxins. IpgB1, IpgB2 (Shigella), SifA, SifB (Salmonella) and Map and EspM (attaching and effacing pathogens) constitute a family of type III secretion system effectors that subverts small GTPase signalling pathways. In this study we identified and characterized EspT from Citrobacter rodentium that triggers formation of lamellipodia on Swiss 3T3 and membrane ruffles on HeLa cells, which are reminiscent of the membrane ruffles induced by IpgB1. Ectopic expression of EspT and IpgB1, but not EspM, resulted in a mitochondrial localization. Using dominant negative constructs we found that EspT-induced actin remodelling is dependent on GTP-bound Rac-1 and Cdc42 but not ELMO or Dock180, which are hijacked by IpgB1 in order to form a Rac-1 specific guanine nucleotide exchange factor. Using pull-down assays with the Rac-1 and Cdc42 binding domains of Pak and WASP we demonstrate that EspT is capable of activating both Rac-1 and Cdc42. These results suggest that EspT modulates the host cell cytoskeleton through coactivation of Rac-1 and Cdc42 by a distinct mechanism. PMID:19016787

  6. Helicobacter pylori-elicited induction in gastric mucosal matrix metalloproteinase-9 (MMP-9) release involves ERK-dependent cPLA2 activation and its recruitment to the membrane-localized Rac1/p38 complex.

    PubMed

    Slomiany, B L; Slomiany, A

    2016-06-01

    Matrix metalloproteinases (MMPs) are a family of endopeptidases implicated in a wide rage of degenerative and inflammatory diseases, including Helicobacter pylori-associated gastritis, and gastric and duodenal ulcer. As gastric mucosal inflammatory responses to H. pylori are characterized by the rise in MMP-9 production, as well as the induction in mitogen-activated protein kinase (MAPK) and Rac1 activation, we investigated the role of Rac1/MAPK in the processes associated with the release of MMP-9. We show that H. pylori LPS-elicited induction in gastric mucosal MMP-9 release is associated with MAPK, ERK and p38 activation, and occurs with the involvement of Rac1 and cytosolic phospholipase A2 (cPLA2). Further, we demonstrate that the LPS-induced MMP-9 release requires ERK-mediated phosphorylation of cPLA2 on Ser(505) that is essential for its membrane localization with Rac1, and that this process necessitates p38 participation. Moreover, we reveal that the activation and membrane translocation of p38 to the Rac1-GTP complex plays a pivotal role in cPLA2-dependent enhancement in MMP-9 release. Hence, our findings provide a strong evidence for the role of ERK/cPLA2 and Rac1/p38/cPLA2 cascade in H. pylori LPS-induced up-regulation in gastric mucosal MMP-9 release. PMID:26886372

  7. Actin depolymerization mediated loss of SNTA1 phosphorylation and Rac1 activity has implications on ROS production, cell migration and apoptosis.

    PubMed

    Bhat, Sehar Saleem; Parray, Arif Ali; Mushtaq, Umar; Fazili, Khalid Majid; Khanday, Firdous Ahmad

    2016-06-01

    Alpha-1-syntrophin (SNTA1) and Rac1 are part of a signaling pathway via the dystrophin glycoprotein complex (DGC). Both SNTA1 and Rac1 proteins are over-expressed in various carcinomas. It is through the DGC signaling pathway that SNTA1 has been shown to act as a link between the extra cellular matrix, the internal cell signaling apparatus and the actin cytoskeleton. SNTA1 is involved in the modulation of the actin cytoskeleton and actin reorganization. Rac1 also controls actin cytoskeletal organization in the cell. In this study, we present the interplay between f-actin, SNTA1 and Rac1. We analyzed the effect of actin depolymerization on SNTA1 tyrosine phosphorylation and Rac1 activity using actin depolymerizing drugs, cytochalasin D and latrunculin A. Our results indicate a marked decrease in the tyrosine phosphorylation of SNTA1 upon actin depolymerization. Results suggest that actin depolymerization mediated loss of SNTA1 phosphorylation leads to loss of interaction between SNTA1 and Rac1, with a concomitant loss of Rac1 activation. The loss of SNTA1tyrosine phosphorylation and Rac1 activity by actin depolymerization results in increased apoptosis, decreased cell migration and decreased reactive oxygen species (ROS) levels in breast carcinoma cells. Collectively, our results present a possible role of f-actin in the SNTA1-Rac1 signaling pathway and implications of actin depolymerization on cell migration, ROS production and apoptosis. PMID:27048259

  8. Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

    PubMed

    Lv, Qi; Zhu, Xian-Yang; Xia, Yu-Feng; Dai, Yue; Wei, Zhi-Feng

    2015-11-01

    Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 μmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK. PMID:26614458

  9. Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

    PubMed

    Alvarez Juliá, Anabel; Frasch, Alberto C; Fuchsova, Beata

    2016-04-01

    Stress-responsive neuronal membrane glycoprotein M6a (Gpm6a) functions in neurite extension, filopodium and spine formation and synaptogenesis. The mechanisms of Gpm6a action in these processes are incompletely understood. Previously, we identified the actin regulator coronin-1a (Coro1a) as a putative Gpm6a interacting partner. Here, we used co-immunoprecipitation assays with the anti-Coro1a antibody to show that Coro1a associates with Gpm6a in rat hippocampal neurons. By immunofluorescence microscopy, we demonstrated that in hippocampal neurons Coro1a localizes in F-actin-enriched regions and some of Coro1a spots co-localize with Gpm6a labeling. Notably, the over-expression of a dominant-negative form of Coro1a as well as its down-regulation by siRNA interfered with Gpm6a-induced filopodium formation. Coro1a is known to regulate the plasma membrane translocation and activation of small GTPase Rac1. We show that Coro1a co-immunoprecipitates with Rac1 together with Gpm6a. Pharmacological inhibition of Rac1 resulted in a significant decrease in filopodium formation by Gpm6a. The same was observed upon the co-expression of Gpm6a with the inactive GDP-bound form of Rac1. In this case, the elevated membrane recruitment of GDP-bound Rac1 was detected as well. Moreover, the kinase activity of the p21-activated kinase 1 (Pak1), a main downstream effector of Rac1 that acts downstream of Coro1a, was required for Gpm6a-induced filopodium formation. Taken together, our results provide evidence that a signaling pathway including Coro1a, Rac1, and Pak1 facilitates Gpm6a-induced filopodium formation. Formation of filopodia by membrane glycoprotein M6a (Gpm6a) requires actin regulator coronin-1a (Coro1a), known to regulate plasma membrane localization and activation of Rac1 and its downstream effector Pak1. Coro1a associates with Gpm6a. Blockage of Coro1a, Rac1, or Pak1 interferes with Gpm6a-induced filopodium formation. Moreover, Gpm6a facilitates Rac1 membrane recruitment

  10. ELMO1 Directly Interacts with Gβγ Subunit to Transduce GPCR Signaling to Rac1 Activation in Chemotaxis

    PubMed Central

    Wang, Youhong; Xu, Xuehua; Pan, Miao; Jin, Tian

    2016-01-01

    Diverse chemokines bind to G protein-coupled receptors (GPCRs) to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. ELMO and Dock proteins form complexes that function as guanine nucleotide exchange factors (GEFs) for Rac activation. However, the linkage between GPCR activation and the ELMO/Dock-mediated Rac activation is not fully understood. In the present study, we show that chemoattractants induce dynamic membrane translocation of ELMO1 in mammalian cells. ELMO1 plays an important role in GPCR-mediated chemotaxis. We also reveal that ELMO1 and Dock1 form a stable complex. Importantly, activation of chemokine GPCR promotes the interaction between ELMO1 and Gβγ. The ELMO1-Gβγ interaction is through the N-terminus of ELMO1 protein and is important for the membrane translocation of ELMO1. ELMO1 is required for Rac1 activation upon chemoattractant stimulation. Our results suggest that chemokine GPCR-mediated interaction between Gβγ and ELMO1/Dock1 complex might serve as an evolutionarily conserved mechanism for Rac activation to regulate actin cytoskeleton for chemotaxis of human cells. PMID:27313788

  11. PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway

    PubMed Central

    Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

    2016-01-01

    Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised. PMID:27052191

  12. Astrocyte Stellation, a Process Dependent on Rac1 Is Sustained by the Regulated Exocytosis of Enlargeosomes

    PubMed Central

    Racchetti, Gabriella; D'Alessandro, Rosalba; Meldolesi, Jacopo

    2012-01-01

    Cultured astrocytes exhibit a flat/epitelioid phenotype much different from the star-like phenotype of tissue astrocytes. Upon exposure to treatments that affect the small GTPase Rho and/or its effector ROCK, however, flat astrocytes undergo stellation, with restructuring of cytoskeleton and outgrowth of processes with lamellipodia, assuming a phenotype closer to that exhibited in situ. The mechanisms of this change are known only in part. Using the ROCK blocker drug Y27632, which induces rapid (tens of min), dose-dependent and reversible stellations, we focused on two specific aspects of the process: its dependence on small GTPases and the large surface expansion of the cells. Contrary to previous reports, we found stellation to be governed by the small G protein Rac1, up to disappearance of the process when Rac1 was downregulated or blocked by a specific drug. In contrast cdc42, the other G-protein often involved in phenotype changes, appeared not involved. The surface expansion concomitant to cytoskeleton restructuring, also dependent on Rac1, was found to be at least partially sustained by the exocytosis of enlargeosomes, small vesicles distinct from classical cell organelles, which are abundant in astrocytes. Exhaustion of stellation induced by repeated administrations of Y27632 correlated with the decrease of the enlargeosome pool. A whole-cell process like stellation of cultured astrocytes might be irrelevant in the brain tissue. However, local restructuring of the cytoskeleton coordinate with surface expansion, occurring at critical cell sites and sustained by mechanisms analogous to those of stellation, might be of importance in both astrocyte physiology and pathology. © 2011 Wiley Periodicals, Inc. PMID:22144092

  13. The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module

    PubMed Central

    van Rahden, Vanessa A.; Brand, Kristina; Najm, Juliane; Heeren, Joerg; Pfeffer, Suzanne R.; Braulke, Thomas; Kutsche, Kerstin

    2012-01-01

    Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P2 modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P2 dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN. PMID:22907655

  14. Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics

    PubMed Central

    Martin, Katrin; Reimann, Andreas; Fritz, Rafael D.; Ryu, Hyunryul; Jeon, Noo Li; Pertz, Olivier

    2016-01-01

    The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility. PMID:26912264

  15. Merlin/NF2 Regulates Angiogenesis in Schwannomas through a Rac1/Semaphorin 3F-Dependent Mechanism1

    PubMed Central

    Wong, Hon-Kit; Shimizu, Akio; Kirkpatrick, Nathaniel D; Garkavtsev, Igor; Chan, Annie W; di Tomaso, Emmanuelle; Klagsbrun, Michael; Jain, Rakesh K

    2012-01-01

    Neurofibromatosis type 2 (NF2) is an autosomal-dominant multiple neoplasia syndrome that results from mutations in the NF2 tumor suppressor gene. Patients with NF2 develop hallmark schwannomas that require surgery or radiation, both of which have significant adverse effects. Recent studies have indicated that the tumor microenvironment—in particular, tumor blood vessels—of schwannomas may be an important therapeutic target. Furthermore, although much has been done to understand how merlin, the NF2 gene product, functions as a tumor suppressor gene in schwannoma cells, the functional role of merlin in the tumor microenvironment and the mechanism(s) by which merlin regulates angiogenesis to support schwannoma growth is largely unexplored. Here we report that the expression of semaphorin 3F (SEMA3F) was specifically downregulated in schwannoma cells lacking merlin/NF2. When we reintroduced SEMA3F in schwannoma cells, we observed normalized tumor blood vessels, reduced tumor burden, and extended survival in nude mice bearing merlin-deficient brain tumors. Next, using chemical inhibitors and gene knockdown with RNA interference, we found that merlin regulated expression of SEMA3F through Rho GTPase family member Rac1. This study shows that, in addition to the tumor-suppressing activity of merlin, it also functions to maintain physiological angiogenesis in the nervous system by regulating antiangiogenic factors such as SEMA3F. Restoring the relative balance of proangiogenic and antiangiogenic factors, such as increases in SEMA3F, in schwannoma microenvironment may represent a novel strategy to alleviate the clinical symptoms of NF2-related schwannomas. PMID:22431917

  16. Burn injury-induced mechanical allodynia is maintained by Rac1-regulated dendritic spine dysgenesis.

    PubMed

    Tan, Andrew M; Samad, Omar A; Liu, Shujun; Bandaru, Samira; Zhao, Peng; Waxman, Stephen G

    2013-10-01

    Although nearly 11 million individuals yearly require medical treatment due to burn injuries and develop clinically intractable pain, burn injury-induced pain is poorly understood, with relatively few studies in preclinical models. To elucidate mechanisms of burn injury-induced chronic pain, we utilized a second-degree burn model, which produces a persistent neuropathic pain phenotype. Rats with burn injury exhibited reduced mechanical pain thresholds ipsilateral to the burn injury. Ipsilateral WDR neurons in the spinal cord dorsal horn exhibited hyperexcitability in response to a range of stimuli applied to their hindpaw receptive fields. Because dendritic spine morphology is strongly associated with synaptic function and transmission, we profiled dendritic spine shape, density, and distribution of WDR neurons. Dendritic spine dysgenesis was observed on ipsilateral WDR neurons in burn-injured animals exhibiting behavioral and electrophysiological evidence of neuropathic pain. Heat hyperalgesia testing produced variable results, as expected from previous studies of this model of second-degree burn injury in rats. Administration of Rac1-inhibitor, NSC23766, attenuated dendritic spine dysgenesis, decreased mechanical allodynia and electrophysiological signs of burn-induced neuropathic pain. These results support two related implications: that the presence of abnormal dendritic spines contributes to the maintenance of neuropathic pain, and that therapeutic targeting of Rac1 signaling merits further investigation as a novel strategy for pain management after burn injury. PMID:23933578

  17. IAPs as E3 ligases of Rac1: shaping the move.

    PubMed

    Oberoi-Khanuja, Tripat Kaur; Rajalingam, Krishnaraj

    2012-01-01

    Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination-dependent inactivation of Rac1 signaling. PMID:22790203

  18. miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

    PubMed Central

    Franke, Kristin; Otto, Wolfgang; Johannes, Sascha; Baumgart, Jan; Nitsch, Robert; Schumacher, Stefan

    2012-01-01

    The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching. PMID:22588079

  19. miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling.

    PubMed

    Franke, Kristin; Otto, Wolfgang; Johannes, Sascha; Baumgart, Jan; Nitsch, Robert; Schumacher, Stefan

    2012-06-29

    The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching. PMID:22588079

  20. Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

    PubMed Central

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea; Gálvez, Patricia; Lacerda, Hadriano M.; Parada, Luis A.; Zugaza, José L.

    2015-01-01

    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation. PMID:25694429

  1. Ultrasonic Stimulation of Mouse Skin Reverses the Healing Delays in Diabetes and Aging by Activation of Rac1.

    PubMed

    Roper, James A; Williamson, Rosalind C; Bally, Blandine; Cowell, Christopher A M; Brooks, Rebecca; Stephens, Phil; Harrison, Andrew J; Bass, Mark D

    2015-11-01

    Chronic skin-healing defects are one of the leading challenges to lifelong well-being, affecting 2-5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed, and driving wound contraction. We discover that mechanical stimulation of the skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in the skin, we identify future opportunities for management of chronic wounds. PMID:26079528

  2. Ultrasonic stimulation of mouse skin reverses the healing delays in diabetes and aging by activation of Rac1

    PubMed Central

    Roper, James A; Williamson, Rosalind C; Bally, Blandine; Cowell, Christopher AM; Brooks, Rebecca; Stephens, Phil; Harrison, Andrew J; Bass, Mark D

    2015-01-01

    Chronic skin healing defects are one of the leading challenges to lifelong wellbeing, affecting 2-5% of populations. Chronic wound formation is linked to age and diabetes and frequently leads to major limb amputation. Here we identify a strategy to reverse fibroblast senescence and improve healing rates. In healthy skin, fibronectin activates Rac1 in fibroblasts, causing migration into the wound bed and driving wound contraction. We discover that mechanical stimulation of skin with ultrasound can overturn healing defects by activating a calcium/CamKinaseII/Tiam1/Rac1 pathway that substitutes for fibronectin-dependent signaling and promotes fibroblast migration. Treatment of diabetic and aged mice recruits fibroblasts to the wound bed and reduces healing times by 30%, restoring healing rates to those observed in young, healthy animals. Ultrasound treatment is equally effective in rescuing the healing defects of animals lacking fibronectin receptors, and can be blocked by pharmacological inhibition of the CamKinaseII pathway. Finally, we discover that the migration defects of fibroblasts from human venous leg ulcer patients can be reversed by ultrasound, demonstrating that the approach is applicable to human chronic samples. By demonstrating that this alternative Rac1 pathway can substitute for that normally operating in skin, we identify future opportunities for management of chronic wounds. PMID:26079528

  3. The Rac1 regulator ELMO controls basal body migration and docking in multiciliated cells through interaction with Ezrin.

    PubMed

    Epting, Daniel; Slanchev, Krasimir; Boehlke, Christopher; Hoff, Sylvia; Loges, Niki T; Yasunaga, Takayuki; Indorf, Lara; Nestel, Sigrun; Lienkamp, Soeren S; Omran, Heymut; Kuehn, E Wolfgang; Ronneberger, Olaf; Walz, Gerd; Kramer-Zucker, Albrecht

    2015-01-01

    Cilia are microtubule-based organelles that are present on most cells and are required for normal tissue development and function. Defective cilia cause complex syndromes with multiple organ manifestations termed ciliopathies. A crucial step during ciliogenesis in multiciliated cells (MCCs) is the association of future basal bodies with the apical plasma membrane, followed by their correct spacing and planar orientation. Here, we report a novel role for ELMO-DOCK1, which is a bipartite guanine nucleotide exchange factor complex for the small GTPase Rac1, and for the membrane-cytoskeletal linker Ezrin, in regulating centriole/basal body migration, docking and spacing. Downregulation of each component results in ciliopathy-related phenotypes in zebrafish and disrupted ciliogenesis in Xenopus epidermal MCCs. Subcellular analysis revealed a striking impairment of basal body docking and spacing, which is likely to account for the observed phenotypes. These results are substantiated by showing a genetic interaction between elmo1 and ezrin b. Finally, we provide biochemical evidence that the ELMO-DOCK1-Rac1 complex influences Ezrin phosphorylation and thereby probably serves as an important molecular switch. Collectively, we demonstrate that the ELMO-Ezrin complex orchestrates ciliary basal body migration, docking and positioning in vivo. PMID:25516973

  4. Post-training activation of Rac1 in the basolateral amygdala is required for the formation of both short-term and long-term auditory fear memory

    PubMed Central

    Gao, Qinqin; Yao, Wenqing; Wang, Junjun; Yang, Tong; Liu, Cao; Tao, Yezheng; Chen, Yuejun; Liu, Xing; Ma, Lan

    2015-01-01

    Rac1, a member of the Rho family of small GTPases, is crucial for morphological changes of the mature neuronal synapse including spine formation and activity-dependent spine enlargement, while its role in the formation of associated memories, such as conditioned fear memory, is not clear. Here, we report that selective deletion of Rac1 in excitatory neurons, but not in parvalbumin inhibitory neurons, impaired short- and long-term memories (STM and LTM) of fear conditioning. Conditional knockout of Rac1 before associative fear training in the basolateral amygdala (BLA), a key area for fear memory acquisition and storage, impaired fear memory. The expression of dominant-negative mutant of Rac1, or infusion of Rac1 inhibitor NSC23766 into BLA blocked both STM and LTM of fear conditioning. Furthermore, selective inhibition of Rac1 activation in BLA immediately following fear conditioning impaired STM and LTM, demonstrating that fear conditioning-induced Rac1 activation in BLA plays a critical role in the formation of both STM and LTM of conditioned fear. PMID:26582975

  5. ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

    PubMed Central

    2014-01-01

    Background Endothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis. Results In human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls

  6. Deficiency of Rac1 Blocks NADPH Oxidase Activation, Inhibits Endoplasmic Reticulum Stress, and Reduces Myocardial Remodeling in a Mouse Model of Type 1 Diabetes

    PubMed Central

    Li, Jianmin; Zhu, Huaqing; Shen, E; Wan, Li; Arnold, J. Malcolm O.; Peng, Tianqing

    2010-01-01

    OBJECTIVE Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes. RESEARCH DESIGN AND METHODS Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose. RESULTS In diabetic hearts, NADPH oxidase activation, its subunits' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose–induced ER stress was inhibited by blocking Rac1 or NADPH oxidase. CONCLUSIONS Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy. PMID:20522592

  7. MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell–cell junctions in epithelial cells

    PubMed Central

    Breznau, Elaina B.; Semack, Ansley C.; Higashi, Tomohito; Miller, Ann L.

    2015-01-01

    Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell–cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc's GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc's function at cell–cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc's role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc's GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells—RhoA at the cleavage furrow and RhoA and Rac1 at cell–cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc's GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc's GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell–cell junction structure. PMID:25947135

  8. RAC1 regulate tumor necrosis factor-α-mediated impaired osteogenic differentiation of dental pulp stem cells.

    PubMed

    Feng, Guijuan; Shen, Qijie; Lian, Min; Gu, Zhifeng; Xing, Jing; Lu, Xiaohui; Huang, Dan; Li, Liren; Huang, Shen; Wang, Yi; Zhang, Jinlong; Shi, Jiahai; Zhang, Dongmei; Feng, Xingmei

    2015-09-01

    Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self-renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor-α (TNF-α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor-κB (NF-κB) signaling pathway. While previous studies showed that cells treated with TNF-α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF-α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF-α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2). Further, we revealed that TNF-α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β-catenin signaling pathway and liberate β-catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments. PMID:26219349

  9. Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities

    PubMed Central

    Schmidt, Tracy Thennes; Tauseef, Mohammad; Yue, Lili; Bonini, Marcelo G.; Gothert, Joachim; Shen, Tang-Long; Guan, Jun-Lin; Predescu, Sanda; Sadikot, Ruxana

    2013-01-01

    Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoA's interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK−/− ECs and lung vascular integrity in EC-FAK−/− mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities. PMID:23771883

  10. Conditional deletion of FAK in mice endothelium disrupts lung vascular barrier function due to destabilization of RhoA and Rac1 activities.

    PubMed

    Schmidt, Tracy Thennes; Tauseef, Mohammad; Yue, Lili; Bonini, Marcelo G; Gothert, Joachim; Shen, Tang-Long; Guan, Jun-Lin; Predescu, Sanda; Sadikot, Ruxana; Mehta, Dolly

    2013-08-15

    Loss of lung-fluid homeostasis is the hallmark of acute lung injury (ALI). Association of catenins and actin cytoskeleton with vascular endothelial (VE)-cadherin is generally considered the main mechanism for stabilizing adherens junctions (AJs), thereby preventing disruption of lung vascular barrier function. The present study identifies endothelial focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that canonically regulates focal adhesion turnover, as a novel AJ-stabilizing mechanism. In wild-type mice, induction of ALI by intraperitoneal administration of lipopolysaccharide or cecal ligation and puncture markedly decreased FAK expression in lungs. Using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), we show that loss of EC-FAK mimicked key features of ALI (diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung). EC-FAK deletion disrupted AJs due to impairment of the fine balance between the activities of RhoA and Rac1 GTPases. Deletion of EC-FAK facilitated RhoA's interaction with p115-RhoA guanine exchange factor, leading to activation of RhoA. Activated RhoA antagonized Rac1 activity, destabilizing AJs. Inhibition of Rho kinase, a downstream effector of RhoA, reinstated normal endothelial barrier function in FAK-/- ECs and lung vascular integrity in EC-FAK-/- mice. Our findings demonstrate that EC-FAK plays an essential role in maintaining AJs and thereby lung vascular barrier function by establishing the normal balance between RhoA and Rac1 activities. PMID:23771883

  11. Identification, characterization and immunological analysis of Ras related C3 botulinum toxin substrate 1 (Rac1) from grass carp Ctenopharyngodon idella.

    PubMed

    Hu, Mo-Yan; Shen, Yu-Bang; Xu, Xiao-Yan; Yu, Hong-Yan; Zhang, Meng; Dang, Yun-Fei; Li, Jia-Le

    2016-01-01

    Rac1, a Rho GTPase, serves critical immunological functions in mammals. Here, a Rac1 homolog (gcRac1) was identified in grass carp (Ctenopharyngodon idella). The full-length 2023-base pair gcRac1 cDNA contained a 579-bp open reading frame encoding a 192-residue protein, including a conserved RHO domain and nuclear localization signal. The gcRac1 protein shares high identity with other Rac1 counterparts and phylogenetically clustered with Danio rerio Rac1. The gcRac1 transcript showed wide tissue distribution and was inducible by Aeromonas hydrophila in vivo and in vitro; its expression also fluctuated with LPS or flagellin stimulation in vitro. With gcRac1 over-expression, gcPAK1, gcIL1-β, gcTNF-α and gcIFN were basically up-regulated by A. hydrophila and bacterial PAMPs induction, while gcRac1 knockdown decreased these transcripts after A. hydrophila challenge. Over-expression of gcRac1 reduced, while its suppression facilitated, bacterial invasion. Moreover, gcRac1 could activate NF-κB signaling. These findings implicate the vital role of gcRac1 in grass carp innate immunity. PMID:26315145

  12. Neurexin-Neuroligin Synaptic Complex Regulates Schizophrenia-Related DISC1/Kal-7/Rac1 "Signalosome".

    PubMed

    Owczarek, Sylwia; Bang, Marie Louise; Berezin, Vladimir

    2015-01-01

    Neurexins (NXs) and neuroligins (NLs) are cell adhesion molecules that are localized at opposite sites of synaptic membranes. They interact with each other to promote the assembly, maintenance, and function of synapses in the central nervous system. Both NX and NL are cleaved from a membrane-attached intracellular domain in an activity-dependent manner, generating the soluble ectodomain of NX or NL. Expression of the NX1 and NX3 genes in the brain appears to be regulated by a schizophrenia-related protein, DISC1. Here, we show that soluble ecto-NX1β can regulate the expression of DISC1 and induce signaling downstream of DISC1. We also show that NL1 binds to a well-characterized DISC1 interaction partner, Kal-7, and this interaction can be compromised by DISC1. Our results indicate that the NX/NL synaptic complex is intrinsically involved in the regulation of DISC1 function, thus contributing to a better understanding of the pathology of schizophrenia. PMID:26078884

  13. Differential Tiam1/Rac1 activation in hippocampal and cortical neurons mediates differential spine shrinkage in response to oxygen/glucose deprivation

    PubMed Central

    Blanco-Suárez, Elena; Fiuza, Maria; Liu, Xun; Chakkarapani, Elavazhagan; Hanley, Jonathan G

    2014-01-01

    Distinct neuronal populations show differential sensitivity to global ischemia, with hippocampal CA1 neurons showing greater vulnerability compared to cortical neurons. The mechanisms that underlie differential vulnerability are unclear, and we hypothesize that intrinsic differences in neuronal cell biology are involved. Dendritic spine morphology changes in response to ischemic insults in vivo, but cell type-specific differences and the molecular mechanisms leading to such morphologic changes are unexplored. To directly compare changes in spine size in response to oxygen/glucose deprivation (OGD) in cortical and hippocampal neurons, we used separate and equivalent cultures of each cell type. We show that cortical neurons exhibit significantly greater spine shrinkage compared to hippocampal neurons. Rac1 is a Rho-family GTPase that regulates the actin cytoskeleton and is involved in spine dynamics. We show that Rac1 and the Rac guanine nucleotide exchange factor (GEF) Tiam1 are differentially activated by OGD in hippocampal and cortical neurons. Hippocampal neurons express more Tiam1 than cortical neurons, and reducing Tiam1 expression in hippocampal neurons by shRNA enhances OGD-induced spine shrinkage. Tiam1 knockdown also reduces hippocampal neuronal vulnerability to OGD. This work defines fundamental differences in signalling pathways that regulate spine morphology in distinct neuronal populations that may have a role in the differential vulnerability to ischemia. PMID:25248834

  14. Angiotensin Receptor Blockers and Statins Could Alleviate Atrial Fibrosis via Regulating Platelet-Derived Growth Factor/Rac1 /Nuclear Factor-Kappa B Axis

    PubMed Central

    Yang, Dongfang; Yuan, Jia; Liu, Gan; Ling, Zhiyu; Zeng, Haiyan; Chen, Yunqing; Zhang, Yue; She, Qiang; Zhou, Xue

    2013-01-01

    Aims: To investigate whether the administration of renin-angiotensin system (RAS) inhibitors and statins could alleviate atrial fibrosis via platelet-derived growth factor (PDGF)/Rac1 /nuclear factor-kappa B (NF-κB) axis. Methods and Results: In human left atrium, the degree of atrial fibrosis, as well as the expression levels of PDGF, Rac1 and NF-κB increased 1.5 to 2.9 folds in patients with atrial fibrillation compared to that with sinus rhythm, (P<0.0001). There were strongly positive correlations between angiotensin II (Ang II) or procollagen type III-alpha-1 (COL3A1) with PDGF, Rac1, NF-κB, and among PDGF, Rac1 and NF-κB (all P<0.05). At 3 weeks after the transverse aorta constriction (TAC) operation in rat model and with intervention of irbesartan or/and simvastatin, the collagen volume fraction (CVF) and atrial natriuretic peptide (ANP) values respectively increased 6-folds and 3.5-folds in the TAC group compared to SHAM group (P<0.0001), but these levels decreased by 16% to 63% with following drug intervention (all P<0.0001), the combined treatment was the lowest. Accordingly, the expression levels of PDGF (3-folds), Rac1 (1.6-folds), NF-κB (7-folds) and AngII (12-folds) significantly increased in the TAC group compared to the SHAM group, and these levels were also reduced by 25% to 64% with following drug intervention. The highest reduction could be seen after treatment with irbesartan and simvastatin in combination (all P<0.001).There were strongly positive correlations between AngII or CVF with PDGF, Rac1, NF-κB, and among PDGF, Rac1 and NF-κB (all P<0.05). Conclusions: Irbesartan or/and simvastatin can improve atrial fibrosis by regulating PDGF/Rac1/NF-κB axis. PMID:23794945

  15. Inability to activate Rac1-dependent forgetting contributes to behavioral inflexibility in mutants of multiple autism-risk genes.

    PubMed

    Dong, Tao; He, Jing; Wang, Shiqing; Wang, Lianzhang; Cheng, Yuqi; Zhong, Yi

    2016-07-01

    The etiology of autism is so complicated because it involves the effects of variants of several hundred risk genes along with the contribution of environmental factors. Therefore, it has been challenging to identify the causal paths that lead to the core autistic symptoms such as social deficit, repetitive behaviors, and behavioral inflexibility. As an alternative approach, extensive efforts have been devoted to identifying the convergence of the targets and functions of the autism-risk genes to facilitate mapping out causal paths. In this study, we used a reversal-learning task to measure behavioral flexibility in Drosophila and determined the effects of loss-of-function mutations in multiple autism-risk gene homologs in flies. Mutations of five autism-risk genes with diversified molecular functions all led to a similar phenotype of behavioral inflexibility indicated by impaired reversal-learning. These reversal-learning defects resulted from the inability to forget or rather, specifically, to activate Rac1 (Ras-related C3 botulinum toxin substrate 1)-dependent forgetting. Thus, behavior-evoked activation of Rac1-dependent forgetting has a converging function for autism-risk genes. PMID:27335463

  16. Spatial regulation of CLASP affinity for microtubules by Rac1 and GSK3β in migrating epithelial cells

    PubMed Central

    Wittmann, Torsten; Waterman-Storer, Clare M.

    2005-01-01

    Proteins that in cells specifically bind to growing microtubule plus ends (+TIPs) are thought to play important roles in polarization of the cytoskeleton. However, most +TIPs do not show a bias of their microtubule-binding behavior toward different subcellular regions. Here, we examine the dynamics of the +TIP CLASP in migrating PtK1 epithelial cells. We find that, although CLASPs track microtubule plus ends in the cell body, they dynamically decorate the entire microtubule lattice in the leading edge lamella and lamellipodium. Microtubule lattice binding is mediated by the COOH-terminal region of the CLASP microtubule-binding domain and is regulated downstream of Rac1. Phosphorylation of sites in the NH2-terminal part of the microtubule-binding domain by glycogen synthase kinase 3β likely regulates the affinity of CLASPs for microtubule lattices. These results demonstrate the striking difference of the microtubule cytoskeleton in the lamella as compared with the cell body and provide the first direct observation of subcellular regulation of a microtubule-associated protein in migrating cells. PMID:15955847

  17. Tiam1 and Rac1 are required for platelet-activating factor-induced endothelial junctional disassembly and increase in vascular permeability.

    PubMed

    Knezevic, Ivana I; Predescu, Sanda A; Neamu, Radu F; Gorovoy, Matvey S; Knezevic, Nebojsa M; Easington, Cordus; Malik, Asrar B; Predescu, Dan N

    2009-02-20

    It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases. PMID:19095647

  18. Tiam1 and Rac1 Are Required for Platelet-activating Factor-induced Endothelial Junctional Disassembly and Increase in Vascular Permeability*

    PubMed Central

    Knezevic, Ivana I.; Predescu, Sanda A.; Neamu, Radu F.; Gorovoy, Matvey S.; Knezevic, Nebojsa M.; Easington, Cordus; Malik, Asrar B.; Predescu, Dan N.

    2009-01-01

    It is known that platelet-activating factor (PAF) induces severe endothelial barrier leakiness, but the signaling mechanisms remain unclear. Here, using a wide range of biochemical and morphological approaches applied in both mouse models and cultured endothelial cells, we addressed the mechanisms of PAF-induced disruption of interendothelial junctions (IEJs) and of increased endothelial permeability. The formation of interendothelial gaps filled with filopodia and lamellipodia is the cellular event responsible for the disruption of endothelial barrier. We observed that PAF ligation of its receptor induced the activation of the Rho GTPase Rac1. Following PAF exposure, both Rac1 and its guanine nucleotide exchange factor Tiam1 were found associated with a membrane fraction from which they co-immunoprecipitated with PAF receptor. In the same time frame with Tiam1-Rac1 translocation, the junctional proteins ZO-1 and VE-cadherin were relocated from the IEJs, and formation of numerous interendothelial gaps was recorded. Notably, the response was independent of myosin light chain phosphorylation and thus distinct from other mediators, such as histamine and thrombin. The changes in actin status are driven by the PAF-induced localized actin polymerization as a consequence of Rac1 translocation and activation. Tiam1 was required for the activation of Rac1, actin polymerization, relocation of junctional associated proteins, and disruption of IEJs. Thus, PAF-induced IEJ disruption and increased endothelial permeability requires the activation of a Tiam1-Rac1 signaling module, suggesting a novel therapeutic target against increased vascular permeability associated with inflammatory diseases. PMID:19095647

  19. Eiger-induced cell death relies on Rac1-dependent endocytosis.

    PubMed

    Ruan, W; Srinivasan, A; Lin, S; Kara, K-I; Barker, P A

    2016-01-01

    Signaling via tumor necrosis factor receptor (TNFR) superfamily members regulates cellular life and death decisions. A subset of mammalian TNFR proteins, most notably the p75 neurotrophin receptor (p75NTR), induces cell death through a pathway that requires activation of c-Jun N-terminal kinases (JNKs). However the receptor-proximal signaling events that mediate this remain unclear. Drosophila express a single tumor necrosis factor (TNF) ligand termed Eiger (Egr) that activates JNK-dependent cell death. We have exploited this model to identify phylogenetically conserved signaling events that allow Egr to induce JNK activation and cell death in vivo. Here we report that Rac1, a small GTPase, is specifically required in Egr-mediated cell death. rac1 loss of function blocks Egr-induced cell death, whereas Rac1 overexpression enhances Egr-induced killing. We identify Vav as a GEF for Rac1 in this pathway and demonstrate that dLRRK functions as a negative regulator of Rac1 that normally acts to constrain Egr-induced death. Thus dLRRK loss of function increases Egr-induced cell death in the fly. We further show that Rac1-dependent entry of Egr into early endosomes is a crucial prerequisite for JNK activation and for cell death and show that this entry requires the activity of Rab21 and Rab7. These findings reveal novel regulatory mechanisms that allow Rac1 to contribute to Egr-induced JNK activation and cell death. PMID:27054336

  20. Eiger-induced cell death relies on Rac1-dependent endocytosis

    PubMed Central

    Ruan, W; Srinivasan, A; Lin, S; Kara, k-I; Barker, P A

    2016-01-01

    Signaling via tumor necrosis factor receptor (TNFR) superfamily members regulates cellular life and death decisions. A subset of mammalian TNFR proteins, most notably the p75 neurotrophin receptor (p75NTR), induces cell death through a pathway that requires activation of c-Jun N-terminal kinases (JNKs). However the receptor-proximal signaling events that mediate this remain unclear. Drosophila express a single tumor necrosis factor (TNF) ligand termed Eiger (Egr) that activates JNK-dependent cell death. We have exploited this model to identify phylogenetically conserved signaling events that allow Egr to induce JNK activation and cell death in vivo. Here we report that Rac1, a small GTPase, is specifically required in Egr-mediated cell death. rac1 loss of function blocks Egr-induced cell death, whereas Rac1 overexpression enhances Egr-induced killing. We identify Vav as a GEF for Rac1 in this pathway and demonstrate that dLRRK functions as a negative regulator of Rac1 that normally acts to constrain Egr-induced death. Thus dLRRK loss of function increases Egr-induced cell death in the fly. We further show that Rac1-dependent entry of Egr into early endosomes is a crucial prerequisite for JNK activation and for cell death and show that this entry requires the activity of Rab21 and Rab7. These findings reveal novel regulatory mechanisms that allow Rac1 to contribute to Egr-induced JNK activation and cell death. PMID:27054336

  1. SPARC Controls Melanoma Cell Plasticity through Rac1

    PubMed Central

    Salvatierra, Edgardo; Alvarez, Mariano J.; Leishman, Claudia C.; Rivas Baquero, Elvia; Lutzky, Viviana P.; Chuluyan, H. Eduardo; Podhajcer, Osvaldo L.

    2015-01-01

    Cell transition to a more aggressive mesenchymal-like phenotype is a hallmark of cancer progression that involves different steps and requires tightly regulated cell plasticity. SPARC (Secreted Protein Acidic and Rich in Cysteine) is a matricellular protein that promotes this transition in various malignant cell types, including melanoma cells. We found that suppression of SPARC expression in human melanoma cells compromised cell migration, adhesion, cytoskeleton structure, and cell size. These changes involved the Akt/mTOR pathway. Re-expression of SPARC or protein addition restored all the cell features. Suppression of SPARC expression was associated with increased Rac1-GTP levels and its membrane localization. Expression of the dominant negative mutant of Rac1 counteracted almost all the changes observed in SPARC-deficient cells. Overall, these data suggest that most of the SPARC-mediated effects occurred mainly through the blockade of Rac1 activity. PMID:26248315

  2. Morphological changes and spatial regulation of diacylglycerol kinase-zeta, syntrophins, and Rac1 during myoblast fusion.

    PubMed

    Abramovici, Hanan; Gee, Stephen H

    2007-07-01

    The fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well-defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase-zeta (DGK-zeta), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK-zeta has a role in the fusion of cultured C2C12 myoblasts. We show that DGK-zeta and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK-zeta, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK-zeta codistributed with the Ca(2+)-dependent cell adhesion molecule N-cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell-cell junctions by N-cadherin-containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell-cell junctions. Reconstruction of confocal optical sections showed these form dome-like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK-zeta and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion. PMID:17410543

  3. Rac1/p21-activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes.

    PubMed

    Zaldua, Natalia; Llavero, Francisco; Artaso, Alain; Gálvez, Patricia; Lacerda, Hadriano M; Parada, Luis A; Zugaza, José L

    2016-02-01

    Small GTPases of the Ras superfamily are capable of activating E2F-dependent transcription leading to cell proliferation, but the molecular mechanisms are poorly understood. In this study, using immortalized chicken DT40 B cell lines to investigate the role of the Vav/Rac signalling cascade on B cell proliferation, it is shown that the proliferative response triggered by B cell receptor activation is dramatically reduced in the absence of Vav3 expression. Analysis of this proliferative defect shows that in the absence of Vav3 expression, retinoblastoma protein (RB) phosphorylation and the subsequent E2F activation do not take place. By combining pharmacological and genetic approaches, phosphatidylinositol-3-kinase and phospholipase Cγ2 (PLCγ2) were identified as the key regulatory signalling molecules upstream of the Vav3/Rac pathway leading to RB phosphorylation and E2F transcription factor activation. Additionally, vav3(-/-) and plcγ2(-/-) DT40 B cells were not able to activate the RB-E2F complex wild-type phenotype when these genetically modified cells were transfected with constitutively active forms of RhoA or Cdc42. However, when these knockout cells were transfected with different constitutively active versions of PLCγ, Vav or Rac1, not only activation of the RB-E2F complex wild-type phenotype was recovered but also the cellular proliferation. Furthermore, by evaluating the effect of two known effector mutants of Rac1 (Rac1(Q61L/F37A) and Rac1(Q61L/Y40C) ), the RB-E2F complex activation dependency on p21-activated kinase (PAK) and protein kinase Cε (PKCε) activities was established, being independent of both actin cytoskeleton reorganization and Ras activity. These results suggest that PAK1 and PKCε may be potential therapeutic targets to stop uncontrolled B cell proliferation mediated by the Vav/Rac pathway. PMID:26663827

  4. The involvement of mutant Rac1 in the formation of invadopodia in cultured melanoma cells

    PubMed Central

    Revach, Or-Yam; Winograd-Katz, Sabina E; Samuels, Yardena; Geiger, Benjamin

    2016-01-01

    In this article, we discuss the complex involvement of a Rho-family GTPase, Rac1, in cell migration and in invadopodia-mediated matrix degradation. We discuss the involvement of invadopodia in invasive cell migration, and their capacity to promote cancer metastasis. Considering the regulation of invadopodia formation, we describe studies that demonstrate the role of Rac1 in the metastatic process, and the suggestion that this effect is attributable to the capacity of Rac1 to promote invadopodia formation. This notion is demonstrated here by showing that knockdown of Rac1 in melanoma cells expressing a wild-type form of this GTPase, reduces invadopodia-dependent matrix degradation. Interestingly, we also show that excessive activity of Rac1, displayed by the P29S, hyperactive, “fast cycling” mutant of Rac1, which is present in 5–10% of melanoma tumors, inhibits invadopodia function. Moreover, knockdown of this hyperactive mutant enhanced matrix degradation, indicating that excessive Rac1 activity by this mutant can negatively regulate invadopodia formation and function. PMID:26873115

  5. Temporal Rac1 - HIF-1 crosstalk modulates hypoxic survival of aged neurons.

    PubMed

    Güntert, Tanja; Gassmann, Max; Ogunshola, Omolara O

    2016-07-01

    Neurodegenerative diseases are frequently associated with hypoxic conditions. During hypoxia the neuronal cytoskeleton is rapidly reorganized and such abnormalities are directly linked to adverse outcomes. Besides their roles as master regulators of the cytoskeleton, the Rho GTPases are also involved in cellular processes stimulated by hypoxic stress. We investigated the contribution of Rac1-mediated signaling to hypoxic responses of mature neurons using primary cortical cells cultured for 17 days in vitro. We show Rac1 is both upregulated and activated during hypoxia. Pharmacological inhibition of Rac1, but not RhoA, completely abrogated hypoxic HIF-1α stabilization and expression of the HIF-1 targets VEGF and GLUT1. Furthermore activity of JNK and GSK3β were also highly dependent on Rac1 activity and biphasic effects were observed after 6 and 24h of exposure. Notably, inhibition of either pathway suppressed HIF-1α accumulation. Although inhibition of Rac1 did not affect neuronal viability during acute exposure cell death was strongly induced after 24h revealing a time-dependent effect of Rac1 signaling on survival. Thus hypoxia-activated Rac1 is critical for neuronal HIF-1α stabilization and survival during oxygen deprivation via integration of complex signaling cascades. PMID:27018294

  6. Rap1 GTPase Inhibits Tumor Necrosis Factor-α-Induced Choroidal Endothelial Migration via NADPH Oxidase- and NF-κB-Dependent Activation of Rac1.

    PubMed

    Wang, Haibo; Fotheringham, Lori; Wittchen, Erika S; Hartnett, M Elizabeth

    2015-12-01

    Macrophage-derived tumor necrosis factor (TNF)-α has been found in choroidal neovascularization (CNV) surgically removed from patients with age-related macular degeneration. However, the role of TNF-α in CNV development remains unclear. In a murine laser-induced CNV model, compared with un-lasered controls, TNF-α mRNA was increased in retinal pigment epithelial and choroidal tissue, and TNF-α colocalized with lectin-stained migrating choroidal endothelial cells (CECs). Inhibition of TNF-α with a neutralizing antibody reduced CNV volume and reactive oxygen species (ROS) level around CNV. In CECs, pretreatment with the antioxidant apocynin or knockdown of p22phox, a subunit of NADPH oxidase, inhibited TNF-α-induced ROS generation. Apocynin reduced TNF-α-induced NF-κB and Rac1 activation, and inhibited TNF-α-induced CEC migration. TNF-α-induced Rac1 activation and CEC migration were inhibited by NF-κB inhibitor Bay11-7082. Overexpression of Rap1a prevented TNF-α-induced ROS generation and reduced NF-κB and Rac1 activation. Activation of Rap1 by 8-(4-chlorophenylthio)adenosine-2'-O-Me-cAMP prevented TNF-α-induced CEC migration and reduced laser-induced CNV volume, ROS generation, and activation of NF-κB and Rac1. These findings provide evidence that active Rap1a inhibits TNF-α-induced CEC migration by inhibiting NADPH oxidase-dependent NF-κB and Rac1 activation and suggests that Rap1a de-escalates CNV development by interfering with ROS-dependent signaling in several steps of the pathogenic process. PMID:26476350

  7. Rac1 augments Wnt signaling by stimulating β-catenin-lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import.

    PubMed

    Jamieson, Cara; Lui, Christina; Brocardo, Mariana G; Martino-Echarri, Estefania; Henderson, Beric R

    2015-11-01

    β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1-β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin-lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin-LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. PMID:26403202

  8. Rac1 augments Wnt signaling by stimulating β-catenin–lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import

    PubMed Central

    Jamieson, Cara; Lui, Christina; Brocardo, Mariana G.; Martino-Echarri, Estefania; Henderson, Beric R.

    2015-01-01

    ABSTRACT β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1–β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin–lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin–LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. PMID:26403202

  9. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation

    PubMed Central

    Sabbir, Mohammad G.; Dillon, Rachelle; Mowat, Michael R. A.

    2016-01-01

    ABSTRACT The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

  10. Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation.

    PubMed

    Sabbir, Mohammad G; Dillon, Rachelle; Mowat, Michael R A

    2016-01-01

    The Deleted in liver cancer 1 (Dlc1) gene codes for a Rho GTPase-activating protein that also acts as a tumour suppressor gene. Several studies have consistently found that overexpression leads to excessive cell elongation, cytoskeleton changes and subsequent cell death. However, none of these studies have been able to satisfactorily explain the Dlc1-induced cell morphological phenotypes and the function of the different Dlc1 isoforms. Therefore, we have studied the interacting proteins associated with the three major Dlc1 transcriptional isoforms using a mass spectrometric approach in Dlc1 overexpressing cells. We have found and validated novel interacting partners in constitutive Dlc1-expressing cells. Our study has shown that Dlc1 interacts with non-muscle myosin heavy chain II-A (Myh9), plectin and spectrin proteins in different multiprotein complexes. Overexpression of Dlc1 led to increased phosphorylation of Myh9 protein and activation of Rac1 GTPase. These data support a role for Dlc1 in induced cell elongation morphology and provide some molecular targets for further analysis of this phenotype. PMID:26977077

  11. Aegeline from Aegle marmelos stimulates glucose transport via Akt and Rac1 signaling, and contributes to a cytoskeletal rearrangement through PI3K/Rac1.

    PubMed

    Gautam, Sudeep; Ishrat, Nayab; Singh, Rohit; Narender, Tadigoppula; Srivastava, Arvind K

    2015-09-01

    Aegeline is an alkaloidal-amide, isolated from the leaves of Aegle marmelos and have shown antihyperglycemic as well as antidyslipidemic activities in the validated animal models of type 2 diabetes mellitus. Here we delineate, aegeline enhanced GLUT4 translocation mediated 2-deoxy-glucose uptake in both time and concentration-dependent manner. 2-deoxy-glucose uptake was completely stymied by the transport inhibitors (wortmannin and genistein) in C2C12 myotubes. Pharmacological inhibition of Akt (also known as protein kinase B) and Ras-related C3 botulinum toxin substrate 1 (Rac1) suggest that both Akt and Rac1 operate aegeline-stimulated glucose transport via distinct parallel pathways. Moreover, aegeline activates p21 protein-activated kinase 1 (PAK1) and cofilin (an actin polymerization regulator). Rac1 inhibitor (Rac1 inhib II) and PAK1 inhibitor (IPA-3) completely blocked aegeline-induced phosphorylation of cofilin and p21 protein-activated kinase 1 (PAK1). In summary, these findings suggest that aegeline stimulates the glucose transport through Akt and Rac1 dependent distinct parallel pathways and have cytoskeletal roles via stimulation of the PI3-kinase-Rac1-PAK1-cofilin pathway in the skeletal muscle cells. Therefore, multiple targets of aegeline in the improvement of insulin sensitivity of the skeletal muscle cells may be suggested. PMID:26102565

  12. Cucurbitacin I Inhibits Rac1 Activation in Breast Cancer Cells by a Reactive Oxygen Species-Mediated Mechanism and Independently of Janus Tyrosine Kinase 2 and P-Rex1

    PubMed Central

    Lopez-Haber, Cynthia

    2013-01-01

    The small GTPase Rac1 has been widely implicated in mammary tumorigenesis and metastasis. Previous studies established that stimulation of ErbB receptors in breast cancer cells activates Rac1 and enhances motility via the Rac-guanine nucleotide exchange factor P-Rex1. As the Janus tyrosine kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) pathway has been shown to be functionally associated with ErbB receptors, we asked if this pathway could mediate P-Rex1/Rac1 activation in response to ErbB ligands. Here we found that the anticancer agent cucurbitacin I, a Jak2 inhibitor, reduced the activation of Rac1 and motility in response to the ErbB3 ligand heregulin in breast cancer cells. However, Rac1 activation was not affected by Jak2 or Stat3 RNA interference, suggesting that the effect of cucurbitacin I occurs through a Jak2-independent mechanism. Cucurbitacin I also failed to affect the activation of P-Rex1 by heregulin. Subsequent analysis revealed that cucurbitacin I strongly activates RhoA and the Rho effector Rho kinase (ROCK) in breast cancer cells and induces the formation of stress fibers. Interestingly, disruption of the RhoA-ROCK pathway prevented the inhibitory effect of cucurbitacin I on Rac1 activation by heregulin. Lastly, we found that RhoA activation by cucurbitacin I is mediated by reactive oxygen species (ROS). The ROS scavenger N-acetyl l-cysteine and the mitochondrial antioxidant Mito-TEMPO rescued the inhibitory effect of cucurbitacin I on Rac1 activation. In conclusion, these results indicate that ErbB-driven Rac1 activation in breast cancer cells proceeds independently of the Jak2 pathway. Moreover, they established that the inhibitory effect of cucurbitacin I on Rac1 activity involves the alteration of the balance between Rho and Rac. PMID:23478800

  13. The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines.

    PubMed

    Tolias, Kimberley F; Bikoff, Jay B; Burette, Alain; Paradis, Suzanne; Harrar, Dana; Tavazoie, Sohail; Weinberg, Richard J; Greenberg, Michael E

    2005-02-17

    NMDA-type glutamate receptors play a critical role in the activity-dependent development and structural remodeling of dendritic arbors and spines. However, the molecular mechanisms that link NMDA receptor activation to changes in dendritic morphology remain unclear. We report that the Rac1-GEF Tiam1 is present in dendrites and spines and is required for their development. Tiam1 interacts with the NMDA receptor and is phosphorylated in a calcium-dependent manner in response to NMDA receptor stimulation. Blockade of Tiam1 function with RNAi and dominant interfering mutants of Tiam1 suggests that Tiam1 mediates effects of the NMDA receptor on dendritic development by inducing Rac1-dependent actin remodeling and protein synthesis. Taken together, these findings define a molecular mechanism by which NMDA receptor signaling controls the growth and morphology of dendritic arbors and spines. PMID:15721239

  14. Rac1 is required for Prkar1a-mediated Nf2 suppression in Schwann cell tumors

    PubMed Central

    Manchanda, Parmeet K.; Jones, Georgette N.; Lee, Audrey A.; Pringle, Daphne R.; Zhang, Mei; Yu, Lianbo; La Perle, Krista M. D.; Kirschner, Lawrence S.

    2012-01-01

    Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including Neurofibromatosis Types 1 (NF1) and 2 (NF2), Familial Schwannomatosis (FS) and Carney Complex (CNC). Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to play a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout of the CNC gene Prkar1a, which encodes the type 1A regulatory subunit of Protein Kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double knockout of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation.. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicates that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA. PMID:23045281

  15. Rac1 is required for Prkar1a-mediated Nf2 suppression in Schwann cell tumors.

    PubMed

    Manchanda, P K; Jones, G N; Lee, A A; Pringle, D R; Zhang, M; Yu, L; La Perle, K M D; Kirschner, L S

    2013-07-25

    Schwannomas are peripheral nerve sheath tumors that often occur in the setting of an inherited tumor predisposition syndrome, including neurofibromatosis types 1 (NF1) and 2 (NF2), familial schwannomatosis and Carney complex. Loss of the NF2 tumor suppressor (encoding NF2, or Merlin) is associated with upregulation of the Rac1 small GTPase, which is thought to have a key role in mediating tumor formation. In prior studies, we generated a mouse model of schwannomas by performing tissue-specific knockout (KO) of the Carney complex gene Prkar1a, which encodes the type 1A regulatory subunit of protein kinase A. These tumors exhibited down-regulation of Nf2 protein and an increase in activated Rac1. To assess the requirement for Rac1 in schwannoma formation, we generated a double KO (DKO) of Prkar1a and Rac1 in Schwann cells and monitored tumor formation. Loss of Rac1 reduced tumor formation by reducing proliferation and enhancing apoptosis. Surprisingly, the reduction of tumor formation was accompanied by re-expression of the Nf2 protein. Furthermore, activated Rac1 was able to downregulate Nf2 in vitro in a Pak-dependent manner. These in vivo data indicate that activation of Rac1 is responsible for suppression of Nf2 protein production; deficiency of Nf2 in Schwann cells leads to loss of cellular growth control and tumor formation. Further, PKA activation through mutation in Prkar1a is sufficient to initiate Rac1 signaling, with subsequent reduction of Nf2 and schwannomagenesis. Although in vitro evidence has shown that loss of Nf2 activates Rac1, our data indicate that signaling between Nf2 and Rac1 occurs in a bidirectional fashion, and these interactions are modulated by PKA. PMID:23045281

  16. Growth arrest of lung carcinoma cells (A549) by polyacrylate-anchored peroxovanadate by activating Rac1-NADPH oxidase signalling axis.

    PubMed

    Chatterjee, Nirupama; Anwar, Tarique; Islam, Nashreen S; Ramasarma, T; Ramakrishna, Gayatri

    2016-09-01

    Hydrogen peroxide is often required in sublethal, millimolar concentrations to show its oxidant effects on cells in culture as it is easily destroyed by cellular catalase. Previously, we had shown that diperoxovanadate, a physiologically stable peroxovanadium compound, can substitute H2O2 effectively in peroxidation reactions. We report here that peroxovanadate when anchored to polyacrylic acid (PAPV) becomes a highly potent inhibitor of growth of lung carcinoma cells (A549). The early events associated with PAPV treatment included cytoskeletal modifications, increase in GTPase activity of Rac1, accumulation of the reactive oxygen species, and also increase in phosphorylation of H2AX (γH2AX), a marker of DNA damage. These effects persisted even at 24 h after removal of the compound and culminated in increased levels of p53 and p21 together with growth arrest. The PAPV-mediated growth arrest was significantly abrogated in cells pre-treated with the N-acetylcysteine, Rac1 knocked down by siRNA and DPI an inhibitor of NADPH oxidase. In conclusion, our results show that polyacrylate derivative of peroxovanadate efficiently arrests growth of A549 cancerous cells by activating the axis of Rac1-NADPH oxidase leading to oxidative stress and DNA damage. PMID:27435854

  17. Rac1 Participates in Thermally Induced Alterations of the Cytoskeleton, Cell Morphology and Lipid Rafts, and Regulates the Expression of Heat Shock Proteins in B16F10 Melanoma Cells

    PubMed Central

    Gungor, Burcin; Gombos, Imre; Crul, Tim; Ayaydin, Ferhan; Szabó, László; Török, Zsolt; Mátés, Lajos; Vígh, László; Horváth, Ibolya

    2014-01-01

    Eukaryotic cells exhibit a characteristic response to hyperthermic treatment, involving morphological and cytoskeletal alterations and the induction of heat shock protein synthesis. Small GTPases of the Ras superfamily are known to serve as molecular switches which mediate responses to extracellular stimuli. We addressed here how small GTPase Rac1 integrates signals from heat stress and simultaneously induces various cellular changes in mammalian cells. As evidence that Rac1 is implicated in the heat shock response, we first demonstrated that both mild (41.5°C) and severe (43°C) heat shock induced membrane translocation of Rac1. Following inhibition of the activation or palmitoylation of Rac1, the size of its plasma membrane-bound pool was significantly decreased while the heat shock-induced alterations in the cytoskeleton and cell morphology were prevented. We earlier documented that the size distribution pattern of cholesterol-rich rafts is temperature dependent and hypothesized that this is coupled to the triggering mechanism of stress sensing and signaling. Interestingly, when plasma membrane localization of Rac1 was inhibited, a different and temperature independent average domain size was detected. In addition, inhibition of the activation or palmitoylation of Rac1 resulted in a strongly decreased expression of the genes of major heat shock proteins hsp25 and hsp70 under both mild and severe heat stress conditions. PMID:24586549

  18. RhoGDI2 is expressed in human trophoblasts and involved in their migration by inhibiting the activation of RAC1.

    PubMed

    Liu, Sishi; Cui, Hong; Li, Qiuling; Zhang, Lijuan; Na, Quan; Liu, Caixia

    2014-04-01

    The invasion of placental trophoblast cells into the maternal decidua is an essential aspect of placental embedment. The process of placentation bears several striking similarities to tumor cell metastasis. However, trophoblastic migration during implantation and placentation is stringently controlled both in space and time. RhoGDI2 belongs to a family of Rho guanosine diphosphate dissociation inhibitors (RhoGDIs), and RhoGDI2 is a metastasis suppressor gene and a marker of aggressive human cancer. We evaluated whether RhoGDI2 has a physiological role in embryo implantation, particularly trophoblast migration. The mRNA and protein expression levels of RhoGDI2 were higher in term placentas compared with first-trimester placentas as detected by real-time PCR and Western blot. Immunohistochemical studies indicated that RhoGDI2 localized to the cytotrophoblast layer and extravillous trophoblast in first-trimester placentas and was distributed in the syncytiotrophoblast layers of term placentas. Overexpression of RhoGDI2 in HTR-8/SVneo cells was associated with reduced RAC1-guanosine triphosphate (GTP) levels and inhibited cell migration. Conversely, small interfering RNA-mediated downregulation of RhoGDI2 resulted in significantly increased RAC1-GTP levels. Altered RhoGDI2 expression had no significant effects on cell proliferation. In conclusion, RhoGDI2 inhibits trophoblast cell migration, and this function may involve suppression of RAC1 activation. PMID:24554735

  19. Preclinical Development of Novel Rac1-GEF Signaling Inhibitors using a Rational Design Approach in Highly Aggressive Breast Cancer Cell Lines

    PubMed Central

    Cardama, Georgina A; Comin, Maria J; Hornos, Leandro; Gonzalez, Nazareno; Defelipe, Lucas; Turjanski, Adrian G; Alonso, Daniel F; Gomez, Daniel E; Menna, Pablo Lorenzano

    2014-01-01

    Rho GTPases play a key role in the regulation of multiple essential cellular processes, including actin dynamics, gene transcription and cell cycle progression. Aberrant activation of Rac1, a member of Rho family of small GTPases, is associated with tumorigenesis, cancer progression, invasion and metastasis. Particularly, Rac1 is overexpressed and hyperactivated in highly aggressive breast cancer. Thus, Rac1 appears to be a promising and relevant target for the development of novel anticancer drugs. We identified the novel Rac1 inhibitor ZINC69391 through a docking-based virtual library screening targeting Rac1 activation by GEFs. This compound was able to block Rac1 interaction with its GEF Tiam1, prevented EGF-induced Rac1 activation and inhibited cell proliferation, cell migration and cell cycle progression in highly aggressive breast cancer cell lines. Moreover, ZINC69391 showed an in vivo antimetastatic effect in a syngeneic animal model. We further developed the novel analog 1A-116 by rational design and showed to be specific and more potent than the parental compound in vitro and interfered Rac1-P-Rex1 interaction. We also showed an enhanced in vivo potency of 1A-116 analog. These results show that we have developed novel Rac1 inhibitors that may be used as a novel anticancer therapy. PMID:24066799

  20. CdGAP/ARHGAP31, a Cdc42/Rac1 GTPase regulator, is critical for vascular development and VEGF-mediated angiogenesis.

    PubMed

    Caron, Christine; DeGeer, Jonathan; Fournier, Patrick; Duquette, Philippe M; Luangrath, Vilayphone; Ishii, Hidetaka; Karimzadeh, Fereshteh; Lamarche-Vane, Nathalie; Royal, Isabelle

    2016-01-01

    Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP(-/-) mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction. PMID:27270835

  1. CdGAP/ARHGAP31, a Cdc42/Rac1 GTPase regulator, is critical for vascular development and VEGF-mediated angiogenesis

    PubMed Central

    Caron, Christine; DeGeer, Jonathan; Fournier, Patrick; Duquette, Philippe M.; Luangrath, Vilayphone; Ishii, Hidetaka; Karimzadeh, Fereshteh; Lamarche-Vane, Nathalie; Royal, Isabelle

    2016-01-01

    Mutations in the CdGAP/ARHGAP31 gene, which encodes a GTPase-activating protein for Rac1 and Cdc42, have been reported causative in the Adams-Oliver developmental syndrome often associated with vascular defects. However, despite its abundant expression in endothelial cells, CdGAP function in the vasculature remains unknown. Here, we show that vascular development is impaired in CdGAP-deficient mouse embryos at E15.5. This is associated with superficial vessel defects and subcutaneous edema, resulting in 44% embryonic/perinatal lethality. VEGF-driven angiogenesis is defective in CdGAP−/− mice, showing reduced capillary sprouting from aortic ring explants. Similarly, VEGF-dependent endothelial cell migration and capillary formation are inhibited upon CdGAP knockdown. Mechanistically, CdGAP associates with VEGF receptor-2 and controls VEGF-dependent signaling. Consequently, CdGAP depletion results in impaired VEGF-mediated Rac1 activation and reduced phosphorylation of critical intracellular mediators including Gab1, Akt, PLCγ and SHP2. These findings are the first to demonstrate the importance of CdGAP in embryonic vascular development and VEGF-induced signaling, and highlight CdGAP as a potential therapeutic target to treat pathological angiogenesis and vascular dysfunction. PMID:27270835

  2. Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein, BCR, and Regulates Levels of Rac1-GTP*

    PubMed Central

    Gray, Elizabeth J.; Petsalaki, Evangelia; James, D. Andrew; Bagshaw, Richard D.; Stacey, Melissa M.; Rocks, Oliver; Gingras, Anne-Claude; Pawson, Tony

    2014-01-01

    SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein. PMID:25331951

  3. Gnb isoforms control a signaling pathway comprising Rac1, Plcβ2, and Plcβ3 leading to LFA-1 activation and neutrophil arrest in vivo.

    PubMed

    Block, Helena; Stadtmann, Anika; Riad, Daniel; Rossaint, Jan; Sohlbach, Charlotte; Germena, Giulia; Wu, Dianqing; Simon, Scott I; Ley, Klaus; Zarbock, Alexander

    2016-01-21

    Chemokines are required for leukocyte recruitment and appropriate host defense and act through G protein-coupled receptors (GPCRs), which induce downstream signaling leading to integrin activation. Although the α and β subunits of the GPCRs are the first intracellular molecules that transduce signals after ligand binding and are therefore indispensable for downstream signaling, relatively little is known about their contribution to lymphocyte function-associated antigen 1 (LFA-1) activation and leukocyte recruitment. We used knockout mice and short hairpin RNA to knock down guanine nucleotide binding protein (GNB) isoforms (GNB1, GNB2, GNB4, and GNB5) in HL60 cells and primary murine hematopoietic cells. Neutrophil function was assessed by using intravital microscopy, flow chamber assays, and chemotaxis and biochemistry studies. We unexpectedly discovered that all expressed GNB isoforms are required for LFA-1 activation. Their downregulation led to a significant impairment of LFA-1 activation, which was demonstrated in vitro and in vivo. Furthermore, we showed that GPCR activation leads to Ras-related C3 botulinum toxin substrate 1 (Rac1)-dependent activation of both phospholipase C β2 (Plcβ2) and Plcβ3. They act nonredundantly to produce inositol triphosphate-mediated intracellular Ca(2+) flux and LFA-1 activation that support chemokine-induced arrest in vivo. In a complex inflammatory disease model, Plcβ2-, Plcβ3-, or Rac1-deficient mice were protected from lipopolysaccharide-induced lung injury. Taken together, we demonstrated that all Gnb isoforms are required for chemokine-induced downstream signaling, and Rac1, Plcβ2, and Plcβ3 are critically involved in integrin activation and leukocyte arrest. PMID:26468229

  4. Rac1 expression in epithelial ovarian cancer: effect on cell EMT and clinical outcome.

    PubMed

    Leng, Ruobing; Liao, Gang; Wang, Haixia; Kuang, Jun; Tang, Liangdan

    2015-02-01

    Ras-related C3 botulinum toxin substrate 1 (rac1) has been implicated in tumor epithelial-mesenchymal transition (EMT); however, limited information is available regarding the role of rac1 in epithelial ovarian cancer (EOC). This study aimed to evaluate the correlation of rac1 expression with EMT and EOC prognosis. Rac1 protein levels of 150 EOC specimens were evaluated by immunohistochemical staining. Survival analysis was performed to determine the correlation between rac1 expression and survival. Cellular and molecular changes were also examined after rac1 in ovarian cancer cells was silenced in vitro and in vivo. The mechanism of rac1 on EMT was investigated by Western blot analysis. Rac1 was highly expressed in EOC. Rac1 overexpression was closely associated with advanced stage based on International Federation of Gynecology and Obstetrics, poor grade, serum Ca-125, and residual tumor size. Survival analyses demonstrated that patients with high rac1 expression levels were more susceptible to early tumor recurrence with very poor prognosis. This study revealed that rac1 downregulation decreased cell EMT and proliferation capability in vitro and in vivo. Rac1 expression possibly altered cell EMT by interacting with p21-activated kinase 1 and p38 mitogen-activated protein kinase signaling pathways. The present study showed that rac1 overexpression is associated with cell EMT and poor EOC prognosis. Rac1 possibly plays an important role in predicting EOC metastasis. PMID:25585684

  5. TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA.

    PubMed

    Varon, Christine; Basoni, Caroline; Reuzeau, Edith; Moreau, Violaine; Kramer, I Jsbrand; Génot, Elisabeth

    2006-11-01

    TGFbeta is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFbeta1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFbeta1 on the activation state of members of the RhoGTPase family. TGFbeta1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFbeta1-mediated change of phenotype. Although TGFbeta1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFbeta1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFbeta1 in the process of endothelial tube formation in collagen I gels. PMID:16978608

  6. Inhibition of Rac1 reduces store overload-induced calcium release and protects against ventricular arrhythmia.

    PubMed

    Zhang, Lili; Lu, Xiangru; Gui, Le; Wu, Yan; Sims, Stephen M; Wang, Guoping; Feng, Qingping

    2016-08-01

    Rac1 is a small GTPase and plays key roles in multiple cellular processes including the production of reactive oxygen species (ROS). However, whether Rac1 activation during myocardial ischaemia and reperfusion (I/R) contributes to arrhythmogenesis is not fully understood. We aimed to study the effects of Rac1 inhibition on store overload-induced Ca(2+) release (SOICR) and ventricular arrhythmia during myocardial I/R. Adult Rac1(f/f) and cardiac-specific Rac1 knockdown (Rac1(ckd) ) mice were subjected to myocardial I/R and their electrocardiograms (ECGs) were monitored for ventricular arrhythmia. Myocardial Rac1 activity was increased and ventricular arrhythmia was induced during I/R in Rac1(f/f) mice. Remarkably, I/R-induced ventricular arrhythmia was significantly decreased in Rac1(ckd) compared to Rac1(f/f) mice. Furthermore, treatment with Rac1 inhibitor NSC23766 decreased I/R-induced ventricular arrhythmia. Ca(2+) imaging analysis showed that in response to a 6 mM external Ca(2+) concentration challenge, SOICR was induced with characteristic spontaneous intracellular Ca(2+) waves in Rac1(f/f) cardiomyocytes. Notably, SOICR was diminished by pharmacological and genetic inhibition of Rac1 in adult cardiomyocytes. Moreover, I/R-induced ROS production and ryanodine receptor 2 (RyR2) oxidation were significantly inhibited in the myocardium of Rac1(ckd) mice. We conclude that Rac1 activation induces ventricular arrhythmia during myocardial I/R. Inhibition of Rac1 suppresses SOICR and protects against ventricular arrhythmia. Blockade of Rac1 activation may represent a new paradigm for the treatment of cardiac arrhythmia in ischaemic heart disease. PMID:27222313

  7. RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border.

    PubMed

    Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C; Alonso, Miguel A; Correas, Isabel; Cox, Susan; Ridley, Anne J; Millán, Jaime

    2016-05-01

    Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

  8. Loss of beta1-integrin enhances TGF-beta1-induced collagen expression in epithelial cells via increased alphavbeta3-integrin and Rac1 activity.

    PubMed

    Hayashida, Tomoko; Jones, Jonathan C R; Lee, Carrie K; Schnaper, H William

    2010-10-01

    Transforming growth factor β (TGF-β) promotes tissue fibrosis via the receptor-specific Smad pathway and non-canonical pathways. We recently reported that TGF-β1-stimulated collagen expression by cultured kidney cells requires integrin-dependent activation of focal adhesion kinase (FAK) and consequent ERK MAP kinase activity leading to Smad3 linker region phosphorylation. Here, we defined a role for αvβ3-integrin in this non-canonical pathway. A human kidney tubular cell line in which β1-integrin was knocked down (β1-k/d) demonstrated enhanced type I collagen mRNA expression and promoter activity. A second shRNA to either αv-integrin or β3-integrin, but not to another αv-binding partner, β6-integrin, abrogated the enhanced COL1A2 promoter activity in β1-k/d cells. Although αvβ3-integrin surface expression levels were not different, αvβ3-integrins colocalized with sites of focal adhesion significantly more in β1-k/d cells, and activated αvβ3-integrin was detected only in β1-k/d cells. Further, the collagen response was decreased by a function-blocking antibody or a peptide inhibitor of αvβ3-integrin. In cells lacking αvβ3-integrin, the responses were attenuated, whereas the response was enhanced in αvβ3-overexpressing cells. Rac1 and ERK, previously defined mediators for this non-canonical pathway, showed increased activities in β1-k/d cells. Finally, inhibition of αvβ3-integrin decreased Rac1 activity and COL1A2 promoter activity in β1-k/d cells. Together, our results indicate that decreasing β1 chain causes αvβ3-integrin to become functionally dominant and promotes renal cell fibrogenesis via Rac1-mediated ERK activity. PMID:20650890

  9. Rac1 at the crossroad of actin dynamics and neuroinflammation in Amyotrophic Lateral Sclerosis

    PubMed Central

    D’Ambrosi, Nadia; Rossi, Simona; Gerbino, Valeria; Cozzolino, Mauro

    2014-01-01

    Rac1 is a major player of the Rho family of small GTPases that controls multiple cell signaling pathways, such as the organization of cytoskeleton (including adhesion and motility), cell proliferation, apoptosis and activation of immune cells. In the nervous system, in particular, Rac1 GTPase plays a key regulatory function of both actin and microtubule cytoskeletal dynamics and thus it is central to axonal growth and stability, as well as dendrite and spine structural plasticity. Rac1 is also a crucial regulator of NADPH-dependent membrane oxidase (NOX), a prominent source of reactive oxygen species (ROS), thus having a central role in the inflammatory response and neurotoxicity mediated by microglia cells in the nervous system. As such, alterations in Rac1 activity might well be involved in the processes that give rise to Amyotrophic Lateral Sclerosis (ALS), a complex syndrome where cytoskeletal disturbances in motor neurons and redox alterations in the inflammatory compartment play pivotal and synergic roles in the final disease outcomes. Here we will discuss the genetic and mechanistic evidence indicating the relevance of Rac1 dysregulation in the pathogenesis of ALS. PMID:25249940

  10. Overexpression of Rac1 in leukemia patients and its role in leukemia cell migration and growth

    SciTech Connect

    Wang, Jiying; Rao, Qing; Wang, Min; Wei, Hui; Xing, Haiyan; Liu, Hang; Wang, Yanzhong; Tang, Kejing; Peng, Leiwen; Tian, Zheng; Wang, Jianxiang

    2009-09-04

    Rac1 belongs to the Rho family that act as critical mediators of signaling pathways controlling cell migration and proliferation and contributes to the interactions of hematopoietic stem cells with their microenvironment. Alteration of Rac1 might result in unbalanced interactions and ultimately lead to leukemogenesis. In this study, we analyze the expression of Rac1 protein in leukemia patients and determine its role in the abnormal behaviours of leukemic cells. Rac1 protein is overexpressed in primary acute myeloid leukemia cells as compared to normal bone marrow mononuclear cells. siRNA-mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation, and colony formation. Additionally, blocking Rac1 activity by an inhibitor of Rac1-GTPase, NSC23766, suppressed cell migration and growth. We conclude that overexpression of Rac1 contributes to the accelerated migration and high proliferation potential of leukemia cells, which could be implicated in leukemia development and progression.

  11. Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α.

    PubMed

    Lopez-Haber, Cynthia; Barrio-Real, Laura; Casado-Medrano, Victoria; Kazanietz, Marcelo G

    2016-08-01

    The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions -1376 to -1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4. PMID:27185877

  12. Simvastatin induces the apoptosis of normal vascular smooth muscle through the disruption of actin integrity via the impairment of RhoA/Rac-1 activity.

    PubMed

    Kang, Seojin; Kim, Keunyoung; Noh, Ji-Yoon; Jung, Yeryeon; Bae, Ok-Nam; Lim, Kyung-Min; Chung, Jin-Ho

    2016-08-30

    Statins, lipid-lowering agents for the prevention of atherosclerosis and fatal coronary heart diseases, have pleiotropic modalities on the function and physiology of vascular smooth muscle that include anti-contractile and pro-apoptotic effects. These effects were suggested to stem from the inhibition of small GTPase Rho A, but they are largely regarded as distinct and unrelated. Recently, we discovered that simvastatin causes both contractile dysfunction and apoptosis of vascular smooth muscle cells (VSMCs), reflecting that they may be closely related, yet their connecting link remains unexplained. Here, we elaborated the mechanism underlying simvastatin-induced apoptosis of normal VSMCs in connection with contractile dysfunction. Repeated oral administration of simvastatin to rats in vivo resulted in contractile dysfunction and apoptosis of vascular smooth muscle, of which pattern was well reproduced in rat VSMCs in vitro. Of note, contractile dysfunction and apoptosis occurred in concerted manners both in vivo and in vitro in the aspects of time course and dose of exposure. In rat VSMCs, simvastatin impaired the activation of small GTPases, RhoA along with Rac-1, which resulted in the disruption of actin integrity, a pivotal factor both for the generation of contractile force and survival of VSMCs. In line with the disruption of actin integrity, Bmf, a pro-apoptotic factor bound to intact actin, dissociated and translocated into mitochondria, which corresponded well with the dissipation of mitochondrial membrane potential, caspase-3 activation and ultimately apoptosis. These events were all rescued by an actin stabilisation agent, jasplakinolide as well as geranylgeraniol, indicating that damages of the actin integrity from disrupted activation of RhoA/Rac-1 lies at the center of simvastatin-induced contractile dysfunction and apoptosis in vascular smooth muscle. PMID:27306926

  13. The Candida albicans ELMO homologue functions together with Rac1 and Dck1, upstream of the MAP Kinase Cek1, in invasive filamentous growth.

    PubMed

    Hope, Hannah; Schmauch, Christian; Arkowitz, Robert A; Bassilana, Martine

    2010-06-01

    Regulation of Rho G-proteins is critical for cytoskeletal organization and cell morphology in all eukaryotes. In the human opportunistic pathogen Candida albicans, Rac1 and its activator Dck1, a member of the CED5, Dock180, myoblast city family of guanine nucleotide exchange factors, are required for the budding to filamentous transition during invasive growth. We show that Lmo1, a protein with similarity to human ELMO1, is necessary for invasive filamentous growth, similar to Rac1 and Dck1. Furthermore, Rac1, Dck1 and Lmo1 are required for cell wall integrity, as the deletion mutants are sensitive to cell wall perturbing agents, but not to oxidative or osmotic stresses. The region of Lmo1 encompassing the ELMO and PH-like domains is sufficient for its function. Both Rac1 and Dck1 can bind Lmo1. Overexpression of a number of protein kinases in the rac1, dck1 and lmo1 deletion mutants indicates that Rac1, Dck1 and Lmo1 function upstream of the mitogen-activated protein kinases Cek1 and Mkc1, linking invasive filamentous growth to cell wall integrity. We conclude that the requirement of ELMO/CED12 family members for Rac1 function is conserved from fungi to humans. PMID:20444104

  14. Modulation of dendritic spines and synaptic function by Rac1: A possible link to Fragile X syndrome pathology

    PubMed Central

    Bongmba, Odelia Y. N.; Martinez, Luis A.; Elhardt, Mary E.; Butler, Karlis; Tejada-Simon, Maria V.

    2011-01-01

    Rac1, a protein of the Rho GTPase subfamily, has been implicated in neuronal and spine development as well as the formation of synapses with appropriate partners. Dendrite and spine abnormalities have been implicated in several psychiatric disorders such as Fragile-X syndrome, where neurons show a high density of long, thin, and immature dendritic spines. Although abnormalities in dendrites and spines have been correlated with impaired cognitive abilities in mental retardation, the causes of these malformations are not yet well understood. Fragile X syndrome is the most common type of inherited mental retardation caused by the absence of FMRP protein, a RNA-binding protein implicated in the regulation of mRNA translation and transport, leading to protein synthesis. We suggest that FMRP might act as a negative regulator on the synthesis of Rac1. Maintaining an optimal level of Rac1 and facilitating the reorganization of the cytoskeleton likely leads to normal neuronal morphology during activity-dependent plasticity. In our study, we first demonstrated that Rac1 is not only associated but necessary for normal spine development and long-term synaptic plasticity. We further showed that, in Fmr1 knockout mice, lack of FMRP induces an overactivation of Rac1 in the mouse brain and other organs that have been shown to be altered in Fragile X syndrome. In those animals, pharmacological manipulation of Rac1 partially reverses their altered long-term plasticity. Thus, regulation of Rac1 may provide a functional link among deficient neuronal morphology, aberrant synaptic plasticity and cognition impairment in Fragile X syndrome. PMID:21645877

  15. The Rac1 Inhibitor NSC23766 Suppresses CREB Signaling by Targeting NMDA Receptor Function

    PubMed Central

    Hou, Hailong; Chávez, Andrés E.; Wang, Chih-Chieh; Yang, Hongtian; Gu, Hua; Siddoway, Benjamin A.; Hall, Benjamin J.; Castillo, Pablo E.

    2014-01-01

    NMDA receptor signaling plays a complex role in CREB activation and CREB-mediated gene transcription, depending on the subcellular location of NMDA receptors, as well as how strongly they are activated. However, it is not known whether Rac1, the prototype of Rac GTPase, plays a role in neuronal CREB activation induced by NMDA receptor signaling. Here, we report that NSC23766, a widely used specific Rac1 inhibitor, inhibits basal CREB phosphorylation at S133 (pCREB) and antagonizes changes in pCREB levels induced by NMDA bath application in rat cortical neurons. Unexpectedly, we found that NSC23766 affects the levels of neuronal pCREB in a Rac1-independent manner. Instead, our results indicate that NSC23766 can directly regulate NMDA receptors as indicated by their strong effects on both exogenous and synaptically evoked NMDA receptor-mediated currents in mouse and rat neurons, respectively. Our findings strongly suggest that Rac1 does not affect pCREB signaling in cortical neurons and reveal that NSC23766 could be a novel NMDA receptor antagonist. PMID:25319697

  16. Inhibition of the GTPase Rac1 mediates the antimigratory effects of metformin in prostate cancer cells.

    PubMed

    Dirat, Béatrice; Ader, Isabelle; Golzio, Muriel; Massa, Fabienne; Mettouchi, Amel; Laurent, Kathiane; Larbret, Frédéric; Malavaud, Bernard; Cormont, Mireille; Lemichez, Emmanuel; Cuvillier, Olivier; Tanti, Jean François; Bost, Frédéric

    2015-02-01

    Cell migration is a critical step in the progression of prostate cancer to the metastatic state, the lethal form of the disease. The antidiabetic drug metformin has been shown to display antitumoral properties in prostate cancer cell and animal models; however, its role in the formation of metastases remains poorly documented. Here, we show that metformin reduces the formation of metastases to fewer solid organs in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin leads to a major inhibition of Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cAMP, and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1, or P-Rex, as well as the inhibition of the adenylate cyclase, was able to reverse the antimigratory effects of metformin. These results establish a novel mechanism of action for metformin and highlight its potential antimetastatic properties in prostate cancer. PMID:25527635

  17. Rac1 modulates the formation of primordial follicles by facilitating STAT3-directed Jagged1, GDF9 and BMP15 transcription in mice

    PubMed Central

    Zhao, Lihua; Du, Xinhua; Huang, Kun; Zhang, Tuo; Teng, Zhen; Niu, Wanbao; Wang, Chao; Xia, Guoliang

    2016-01-01

    The size of the primordial follicle pool determines the reproductive potential of mammalian females, and establishment of the pool is highly dependent on specific genes expression. However, the molecular mechanisms by which the essential genes are regulated coordinately to ensure primordial follicle assembly remain a mystery. Here, we show that the small GTPase Rac1 plays an indispensable role in controlling the formation of primordial follicles in mouse ovary. Employing fetal mouse ovary organ culture system, we demonstrate that disruption of Rac1 retarded the breakdown of germline cell cysts while Rac1 overexpression accelerated the formation of primordial follicles. In addition, in vivo inhibitor injection resulted in the formation of multi-oocyte follicles. Subsequent investigation showed that Rac1 induced nuclear import of STAT3 by physical binding. In turn, nuclear STAT3 directly activated the transcription of essential oocyte-specific genes, including Jagged1, GDF9, BMP15 and Nobox. Further, GDF9 and BMP15 regulated the translation of Notch2 via mTORC1 activation in pregranulosa cells. Overexression or addition of Jagged1, GDF9 and BMP15 not only reversed the effect of Rac1 disruption, but also accelerated primordial follicle formation via Notch2 signaling activation. Collectively, these results indicate that Rac1 plays important roles as a key regulator in follicular assembly. PMID:27050391

  18. Rac1 Modulates Stimulus-evoked Ca2+ Release in Neuronal Growth Cones via Parallel Effects on Microtubule/Endoplasmic Reticulum Dynamics and Reactive Oxygen Species Production

    PubMed Central

    Zhang, Xiao-Feng

    2009-01-01

    The small G protein Rac regulates cytoskeletal protein dynamics in neuronal growth cones and has been implicated in axon growth, guidance, and branching. Intracellular Ca2+ is another well known regulator of growth cone function; however, effects of Rac activity on intracellular Ca2+ metabolism have not been well characterized. Here, we investigate how Rac1 activity affects release of Ca2+ from intracellular endoplasmic reticulum (ER) stores stimulated by application of serotonin (5-hydroxytriptamine). We also address how Rac1 effects on microtubule assembly dynamics affect distribution of Ca2+ release sites. Multimode fluorescent microscopy was used to correlate microtubule and ER behavior, and ratiometric imaging was used to assess intracellular Ca2+ dynamics. We report that Rac1 activity both promotes Ca2+ release and affects its spatial distribution in neuronal growth cones. The underlying mechanism involves synergistic Rac1 effects on microtubule assembly and reactive oxygen species (ROS) production. Rac1 activity modulates Ca2+ by 1) enhancing microtubule assembly which in turn promotes spread of the ER-based Ca2+ release machinery into the growth cone periphery, and 2) by increasing ROS production which facilitated inositol 1,4,5-trisphosphate-dependent Ca2+ release. These results cast Rac1 as a key modulator of intracellular Ca2+ function in the neuronal growth cone. PMID:19570918

  19. Association of syntenin-1 with M-RIP polarizes Rac-1 activation during chemotaxis and immune interactions.

    PubMed

    Sala-Valdés, Mónica; Gordón-Alonso, Mónica; Tejera, Emilio; Ibáñez, Anna; Cabrero, J Román; Ursa, Angeles; Mittelbrunn, María; Lozano, Francisco; Sánchez-Madrid, Francisco; Yáñez-Mó, María

    2012-03-01

    In this study, we describe that the PDZ protein syntenin-1 is a crucial element for the generation of signaling asymmetry during the cellular response to polarized extracellular cues. We analyze the role of syntenin-1 in the control of asymmetry in two independent models of T cell polarization--the migratory response to chemoattractants and the establishment of cognate interactions between T cells and antigen-presenting cells (APCs). A combination of mutant, biochemical and siRNA approaches demonstrate that syntenin-1 is vital for the generation of polarized actin structures such as the leading edge and the contact zone with APCs. We found that the mechanism by which syntenin-1 controls actin polymerization relies on its mandatory role for activation of the small GTPase Rac. Syntenin-1 controls Rac through a specific association with the myosin phosphatase Rho interacting protein (M-RIP), which occurs in response to phosphorylation of syntenin-1 by Src at Tyr4. Our data indicate the key role of syntenin-1 in the generation of functional asymmetry in T cells and provide a novel mechanistic link between receptor activation and actin polymerization and accumulation in response to extracellular stimulation. PMID:22349701

  20. Epithelial-specific knockout of the Rac1 gene leads to enamel defects

    PubMed Central

    Huang, Zhan; Kim, Jieun; Lacruz, Rodrigo; Bringas, Pablo; Kaartinen, Vesa M.; Snead, Malcolm L.

    2015-01-01

    Rac1 encodes a 21kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction; activities with consequences to cell growth, adhesion, migration, and differentiation. To examine the role(s) played by Rac1 protein in cell-to-matrix interaction and in enamel matrix biomineralization we used the Cre/loxP binary recombination system to characterize enamel matrix proteins expression and enamel formation in Rac1 knockout mice. Mating between mice bearing the floxed Rac1 allele with mice bearing a keratin14-Cre transgene generate animals in which Rac1 is absent from epithelial organs. The enamel of Rac1 conditional knockout mouse was characterized by computerized tomography (microCT), light microscopy, histochemistry, and back-scatter electron microscopy. Enamel matrix protein expression was analyzed by Western blotting. Major findings showed that the Tomes’ processes of Rac1−/− ameloblasts loose contact with the forming enamel matrix in un-erupted teeth. The abundance of amelogenin and ameloblastin was reduced in the Rac1−/− ameloblasts. After eruption, the enamel from the Rac1−/− mice displayed severe structural defects with the complete loss of enamel. These results support an essential role for Rac1 function in the dental epithelium involving cell-matrix interaction and matrix biomineralization. PMID:22243243

  1. Rac1 recruitment to the archipelago structure of the focal adhesion through the fluid membrane as revealed by single-molecule analysis.

    PubMed

    Shibata, Akihiro C E; Chen, Limin H; Nagai, Rie; Ishidate, Fumiyoshi; Chadda, Rahul; Miwa, Yoshihiro; Naruse, Keiji; Shirai, Yuki M; Fujiwara, Takahiro K; Kusumi, Akihiro

    2013-03-01

    The focal adhesion (FA) is an integrin-based structure built in/on the plasma membrane (PM), linking the extracellular matrix to the actin stress-fibers, working as cell migration scaffolds. Previously, we proposed the archipelago architecture of the FA, in which FA largely consists of fluid membrane, dotted with small islands accumulating FA proteins: membrane molecules enter the inter-island channels in the FA zone rather freely, and the integrins in the FA-protein islands rapidly exchanges with those in the bulk membrane. Here, we examined how Rac1, a small G-protein regulating FA formation, and its activators αPIX and βPIX, are recruited to the FA zones. PIX molecules are recruited from the cytoplasm to the FA zones directly. In contrast, majorities of Rac1 molecules first arrive from the cytoplasm on the general inner PM surface, and then enter the FA zones via lateral diffusion on the PM, which is possible due to rapid Rac1 diffusion even within the FA zones, slowed only by a factor of two to four compared with that outside. The constitutively-active Rac1 mutant exhibited temporary and all-time immobilizations in the FA zone, suggesting that upon PIX-induced Rac1 activation at the FA-protein islands, Rac1 tends to be immobilized at the FA-protein islands. PMID:23341328

  2. Liposome reconstitution and modulation of recombinant prenylated human Rac1 by GEFs, GDI1 and Pak1.

    PubMed

    Zhang, Si-Cai; Gremer, Lothar; Heise, Henrike; Janning, Petra; Shymanets, Aliaksei; Cirstea, Ion C; Krause, Eberhard; Nürnberg, Bernd; Ahmadian, Mohammad Reza

    2014-01-01

    Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes. PMID:25014207

  3. Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs, GDI1 and Pak1

    PubMed Central

    Zhang, Si-Cai; Gremer, Lothar; Heise, Henrike; Janning, Petra; Shymanets, Aliaksei; Cirstea, Ion C.; Krause, Eberhard; Nürnberg, Bernd; Ahmadian, Mohammad Reza

    2014-01-01

    Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes. PMID:25014207

  4. Arf nucleotide binding site opener [ARNO] promotes sequential activation of Arf6, Cdc42 and Rac1 and insulin secretion in INS 832/13 β-cells and rat islets

    PubMed Central

    Jayaram, Bhavaani; Syed, Ismail; Kyathanahalli, Chandrashekara N.; Rhodes, Christopher J.; Kowluru, Anjaneyulu

    2011-01-01

    Glucose-stimulated insulin secretion [GSIS] involves interplay between small G-proteins and their regulatory factors. Herein, we tested the hypothesis that Arf nucleotide binding site opener [ARNO], a guanine nucleotide exchange factor [GEF] for the small G-protein Arf6, mediates the functional activation of Arf6, and that ARNO/Arf6 signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have been implicated in GSIS. Molecular biological [i.e., expression of inactive mutants or siRNA] and pharmacological approaches were employed to assess the roles for ARNO/Arf6 signaling pathway in insulin secretion in normal rat islets and INS 832/13 cells. Degrees of activation of Arf6 and Cdc42/Rac1 were quantitated by GST-GGA3 and PAK-1 kinase pull-down assays, respectively. ARNO is expressed in INS 832/13 cells, rat islets and human islets. Expression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] or siRNA-ARNO markedly reduced GSIS in isolated β-cells. secinH3, a selective inhibitor of ARNO/Arf6 signaling axis, also inhibited GSIS in INS 832/13 cells and rat islets. Stimulatory concentrations of glucose promoted Arf6 activation, which was inhibited by secinH3 or siRNA-ARNO, suggesting that ARNO/Arf6 signaling cascade is necessary for GSIS. secinH3 or siRNA-ARNO also inhibited glucose-induced activation of Cdc42 and Rac1 suggesting that ARNO/Arf6 might be upstream to Cdc42 and Rac1 activation steps, which are necessary for GSIS. Lastly, co-immunoprecipitation and confocal microscopic studies suggested increased association between Arf6 and ARNO in glucose-stimulated β-cells. These findings provide the first evidence to implicate ARNO in the sequential activation of Arf6, Cdc42 and Rac1 culminating in GSIS. PMID:21276423

  5. Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling

    PubMed Central

    Sabbatini, Maria Eugenia; Williams, John A.

    2013-01-01

    RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling. PMID:23776598

  6. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma

    SciTech Connect

    Krauthammer, Michael; Kong, Yong; Ha, Byung Hak; Evans, Perry; Bacchiocchi, Antonella; McCusker, James P.; Cheng, Elaine; Davis, Matthew J.; Goh, Gerald; Choi, Murim; Ariyan, Stephan; Narayan, Deepak; Dutton-Regester, Ken; Capatana, Ana; Holman, Edna C.; Bosenberg, Marcus; Sznol, Mario; Kluger, Harriet M.; Brash, Douglas E.; Stern, David F.; Materin, Miguel A.; Lo, Roger S.; Mane, Shrikant; Ma, Shuangge; Kidd, Kenneth K.; Hayward, Nicholas K.; Lifton, Richard P.; Schlessinger, Joseph; Boggon, Titus J.; Halaban, Ruth

    2012-10-11

    We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1{sup P29S}) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1{sup P29S} showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

  7. Carbon-Ion Irradiation Suppresses Migration and Invasiveness of Human Pancreatic Carcinoma Cells MIAPaCa-2 via Rac1 and RhoA Degradation

    SciTech Connect

    Fujita, Mayumi; Imadome, Kaori; Shoji, Yoshimi; Isozaki, Tetsurou; Endo, Satoshi; Yamada, Shigeru; Imai, Takashi

    2015-09-01

    Purpose: To investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation. Methods and Materials: Human pancreatic cancer cells MIAPaCa-2, AsPC-1, and BxPC-3 were treated by x-ray (4 Gy) or C-ion (0.5, 1, 2, or 4 Gy) irradiation, and their migration and invasion were assessed 2 days later. The levels of guanosine triphosphate (GTP)-bound Rac1 and RhoA were determined by the active GTPase pull-down assay with or without a proteasome inhibitor, and the binding of E3 ubiquitin ligase to GTP-bound Rac1 was examined by immunoprecipitation. Results: Carbon-ion irradiation reduced the levels of GTP-bound Rac1 and RhoA, 2 major regulators of cell motility, in MIAPaCa-2 cells and GTP-bound Rac1 in AsPC-1 and BxPC-3 cells. Proteasome inhibition reversed the effect, indicating that C-ion irradiation induced Rac1 and RhoA degradation via the ubiquitin (Ub)-proteasome pathway. E3 Ub ligase X-linked inhibitor of apoptosis protein (XIAP), which directly targets Rac1, was selectively induced in C-ion–irradiated MIAPaCa-2 cells and coprecipitated with GTP-bound Rac1 in C-ion–irradiated cells, which was associated with Rac1 ubiquitination. Cell migration and invasion reduced by C-ion radiation were restored by short interfering RNA–mediated XIAP knockdown, indicating that XIAP is involved in C-ion–induced inhibition of cell motility. Conclusion: In contrast to x-ray irradiation, C-ion treatment inhibited the activity of Rac1 and RhoA in MIAPaCa-2 cells and Rac1 in AsPC-1 and BxPC-3 cells via Ub-mediated proteasomal degradation, thereby blocking the motility of these pancreatic cancer cells.

  8. Endothelial Rac1 is essential for hematogenous metastasis to the lung.

    PubMed

    Yao, Hongyi; Shi, Wei; Wu, Junsong; Xu, Chengyun; Wang, Jirong; Shao, Yanan; Wu, Ximei; Zhang, Zhongmiao

    2015-07-10

    A variety of vasoactive stimuli induce endothelial permeability through Rac1, a membrane of Rho small GTPases. Here, we determine whether tumor-secreted vasoactive stimulant through Rac1 inducing permeability contributes to hematogenous metastasis. Activation of Rac1 was assayed in human umbilical vein endothelial cells (HUVEC), transendothelial passages were measured by Transwell chambers, and hematogenously metastatic mouse model was generated by intravenous injection with Lewis lung carcinoma cells (LLC). LLC secreted abundant vascular endothelial growth factor (VEGF) in the culture media and sera of mice bearing LLC xenografts or metastatic LLC, and VEGF activated Rac1 through VEGF receptors/PI3Kβ signaling cascade, resulting in hyperoxidative stress and consequent hyperpermeability in HUVEC. Moreover, in co-culture of LLC and HUVEC, significant increases in endothelial permeability and transendothelial migration of LLC were robustly attenuated by either anti-VEGF neutralizing antibody or Rac1 knockdown in HUVEC. Finally, in metastatic mouse model, deletion of one copy of Rac1 in endothelium not only significantly attenuated LLC-induced vascular permeability, but robustly reduced the metastasis of LLC to lungs. This study supports that tumor-secreted vasoactive stimuli activate Rac1 to induce permeability and consequent transendothelial migration of tumor cells, and that loss of Rac1 function in endothelium is an effective therapeutic intervention for hematogenous metastasis. PMID:25991673

  9. Endothelial Rac1 is essential for hematogenous metastasis to the lung

    PubMed Central

    Wu, Junsong; Xu, Chengyun; Wang, Jirong; Shao, Yanan; Wu, Ximei; Zhang, Zhongmiao

    2015-01-01

    A variety of vasoactive stimuli induce endothelial permeability through Rac1, a membrane of Rho small GTPases. Here, we determine whether tumor-secreted vasoactive stimulant through Rac1 inducing permeability contributes to hematogenous metastasis. Activation of Rac1 was assayed in human umbilical vein endothelial cells (HUVEC), transendothelial passages were measured by Transwell chambers, and hematogenously metastatic mouse model was generated by intravenous injection with Lewis lung carcinoma cells (LLC). LLC secreted abundant vascular endothelial growth factor (VEGF) in the culture media and sera of mice bearing LLC xenografts or metastatic LLC, and VEGF activated Rac1 through VEGF receptors/PI3Kβ signaling cascade, resulting in hyperoxidative stress and consequent hyperpermeability in HUVEC. Moreover, in co-culture of LLC and HUVEC, significant increases in endothelial permeability and transendothelial migration of LLC were robustly attenuated by either anti-VEGF neutralizing antibody or Rac1 knockdown in HUVEC. Finally, in metastatic mouse model, deletion of one copy of Rac1 in endothelium not only significantly attenuated LLC-induced vascular permeability, but robustly reduced the metastasis of LLC to lungs. This study supports that tumor-secreted vasoactive stimuli activate Rac1 to induce permeability and consequent transendothelial migration of tumor cells, and that loss of Rac1 function in endothelium is an effective therapeutic intervention for hematogenous metastasis. PMID:25991673

  10. The Rac1 hypervariable region in targeting and signaling: a tail of many stories.

    PubMed

    Lam, B Daniel; Hordijk, Peter L

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control subcellullar localization. The HVR encodes in many small GTPases a polybasic region (PBR) that permits charge-mediated association to the inner leaflet of the plasma membrane or to intracellular organelles. Over the past 15-20 years, evidence has accumulated for specific protein-protein interactions, mediated by the HVR, that control both targeting and signaling specificity of small GTPases. Using the RhoGTPase Rac1 as a paradigm we here review a series of protein partners that require the Rac1 HVR for association and that control various aspects of localized Rac1 signaling. Some of these proteins represent Rac1 activators, whereas others mediate Rac1 inactivation and degradation and yet others potentiate Rac1 downstream signaling. Finally, evidence is discussed which shows that the HVR of Rac1 also contributes to effector interactions, co-operating with the N-terminal effector domain. The complexity of localized Rac1 signaling, reviewed here, is most likely exemplary for many other small GTPases as well, representing a challenge to identify and define similar mechanisms controlling the specific signaling induced by small GTPases. PMID:23354415

  11. Rac1-mediated indentation of resting neurons promotes the chain migration of new neurons in the rostral migratory stream of post-natal mouse brain.

    PubMed

    Hikita, Takao; Ohno, Akihisa; Sawada, Masato; Ota, Haruko; Sawamoto, Kazunobu

    2014-03-01

    New neurons generated in the ventricular-subventricular zone in the post-natal brain travel toward the olfactory bulb by using a collective cell migration process called 'chain migration.' These new neurons show a saltatory movement of their soma, suggesting that each neuron cycles through periods of 'rest' during migration. Here, we investigated the role of the resting neurons in chain migration using post-natal mouse brain, and found that they undergo a dynamic morphological change, in which a deep indentation forms in the cell body. Inhibition of Rac1 activity resulted in less indentation of the new neurons in vivo. Live cell imaging using a Förster resonance energy transfer biosensor revealed that Rac1 was activated at the sites of contact between actively migrating and resting new neurons. On the cell surface of resting neurons, Rac1 activation coincided with the formation of the indentation. Furthermore, Rac1 knockdown prevented the indentation from forming and impaired migration along the resting neurons. These results suggest that Rac1 regulates a morphological change in the resting neurons, which allows them to serve as a migratory scaffold, and thereby non-cell-autonomously promotes chain migration. PMID:24188721

  12. Impaired cell death and mammary gland involution in the absence of Dock1 and Rac1 signaling

    PubMed Central

    Bagci, H; Laurin, M; Huber, J; Muller, W J; Côté, J-F

    2014-01-01

    Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) in Caenorhabditis elegans are part of a signaling module in phagocytes that is linking apoptotic cell recognition to cytoskeletal reorganization required for engulfment. In mammals, Elmo1 was shown to interact with the phosphatidylserine receptor Bai1 and relay signals to promote phagocytosis of apoptotic cells. Still, the role of the RacGEF Dock1 in the clearance of dying cells in mammals was never directly addressed. We generated two mouse models with conditional inactivation of Dock1 and Rac1 and revealed that the expression of these genes is not essential in the mammary gland during puberty, pregnancy and lactation. We induced mammary gland involution in these mice to investigate the role of Dock1/Rac1 signaling in the engulfment of cell corpses. Unpredictably, activation of Stat3 (signal transducer and activator of transcription 3), a key regulator of mammary gland involution, was impaired in the absence of Rac1 and Dock1 expression. Likewise, failure to activate properly Stat3 was coinciding with a significant delay in the initiation and progression of mammary gland involution in mutant animals. By using an in vitro phagocytosis assay, we observed that Dock1 and Rac1 are essential to mediate engulfment in epithelial phagocytes. In vivo, cell corpses accumulated at late time points of involution in Dock1 and Rac1 mutant mammary glands. Overall, our study demonstrated an unsuspected role for Dock1/Rac1 signaling in the initiation of mammary gland involution, and also

  13. Impaired cell death and mammary gland involution in the absence of Dock1 and Rac1 signaling.

    PubMed

    Bagci, H; Laurin, M; Huber, J; Muller, W J; Côté, J-F

    2014-01-01

    Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) in Caenorhabditis elegans are part of a signaling module in phagocytes that is linking apoptotic cell recognition to cytoskeletal reorganization required for engulfment. In mammals, Elmo1 was shown to interact with the phosphatidylserine receptor Bai1 and relay signals to promote phagocytosis of apoptotic cells. Still, the role of the RacGEF Dock1 in the clearance of dying cells in mammals was never directly addressed. We generated two mouse models with conditional inactivation of Dock1 and Rac1 and revealed that the expression of these genes is not essential in the mammary gland during puberty, pregnancy and lactation. We induced mammary gland involution in these mice to investigate the role of Dock1/Rac1 signaling in the engulfment of cell corpses. Unpredictably, activation of Stat3 (signal transducer and activator of transcription 3), a key regulator of mammary gland involution, was impaired in the absence of Rac1 and Dock1 expression. Likewise, failure to activate properly Stat3 was coinciding with a significant delay in the initiation and progression of mammary gland involution in mutant animals. By using an in vitro phagocytosis assay, we observed that Dock1 and Rac1 are essential to mediate engulfment in epithelial phagocytes. In vivo, cell corpses accumulated at late time points of involution in Dock1 and Rac1 mutant mammary glands. Overall, our study demonstrated an unsuspected role for Dock1/Rac1 signaling in the initiation of mammary gland involution, and also

  14. The Role of Rac1 in the Growth Cone Dynamics and Force Generation of DRG Neurons

    PubMed Central

    Sayyad, Wasim A.; Fabris, Paolo; Torre, Vincent

    2016-01-01

    We used optical tweezers, video imaging, immunocytochemistry and a variety of inhibitors to analyze the role of Rac1 in the motility and force generation of lamellipodia and filopodia from developing growth cones of isolated Dorsal Root Ganglia neurons. When the activity of Rac1 was inhibited by the drug EHop-016, the period of lamellipodia protrusion/retraction cycles increased and the lamellipodia retrograde flow rate decreased; moreover, the axial force exerted by lamellipodia was reduced dramatically. Inhibition of Arp2/3 by a moderate amount of the drug CK-548 caused a transient retraction of lamellipodia followed by a complete recovery of their usual motility. This recovery was abolished by the concomitant inhibition of Rac1. The filopodia length increased upon inhibition of both Rac1 and Arp2/3, but the speed of filopodia protrusion increased when Rac1 was inhibited and decreased instead when Arp2/3 was inhibited. These results suggest that Rac1 acts as a switch that activates upon inhibition of Arp2/3. Rac1 also controls the filopodia dynamics necessary to explore the environment. PMID:26766136

  15. Deletion of Rac1GTPase in the Myeloid Lineage Protects against Inflammation-Mediated Kidney Injury in Mice

    PubMed Central

    Nagase, Miki; Kurihara, Hidetake; Aiba, Atsu; Young, Morag J.; Sakai, Tatsuo

    2016-01-01

    Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing

  16. Deletion of Rac1GTPase in the Myeloid Lineage Protects against Inflammation-Mediated Kidney Injury in Mice.

    PubMed

    Nagase, Miki; Kurihara, Hidetake; Aiba, Atsu; Young, Morag J; Sakai, Tatsuo

    2016-01-01

    Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing

  17. GABARAP proteins as scaffolds in localized TIAM1-RAC1 signaling

    PubMed Central

    Genau, Heide Marika; Behrends, Christian

    2016-01-01

    Spatially restricted signaling is a hallmark of RAC1 signaling. Recent work has uncovered a novel role of gamma-aminobutyric acid receptor-associated proteins (GABARAPs), a subfamily of human ATG8 ubiquitin-like modifiers, in providing a scaffold for recruitment of an ubiquitin E3 ligase complex to its substrate, T-lymphoma invasion and metastasis-inducing protein 1 (TIAM1), to enable ubiquitylation and thereby local control of RAC1 activity. PMID:27308540

  18. Remedial Strategies in Structural Proteomics: Expression, Purification, And Crystallization of the Vav1/Rac1 Complex

    SciTech Connect

    Brooun, A.; Foster, S.A.; Chrencik, H.E.; Chien, E.Y.T.; Kolatkar, A.R.; Streiff, M.; Ramage, P.; Widmer, H.; Weckbecker, G.; Kuhn, P.

    2007-07-03

    The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.

  19. Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

    PubMed Central

    Thomas, Audrey; Mariani-Floderer, Charlotte; López-Huertas, Maria Rosa; Gros, Nathalie; Hamard-Péron, Elise; Favard, Cyril; Ohlmann, Theophile; Alcamí, José

    2015-01-01

    ABSTRACT During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCE During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in

  20. Rac1 functions as a reversible tension modulator to stabilize VE-cadherin trans-interaction.

    PubMed

    Daneshjou, Nazila; Sieracki, Nathan; van Nieuw Amerongen, Geerten P; Schwartz, Martin A; Komarova, Yulia A; Malik, Asrar B; Conway, Daniel E

    2015-01-01

    The role of the RhoGTPase Rac1 in stabilizing mature endothelial adherens junctions (AJs) is not well understood. In this paper, using a photoactivatable probe to control Rac1 activity at AJs, we addressed the relationship between Rac1 and the dynamics of vascular endothelial cadherin (VE-cadherin). We demonstrated that Rac1 activation reduced the rate of VE-cadherin dissociation, leading to increased density of VE-cadherin at AJs. This response was coupled to a reduction in actomyosin-dependent tension across VE-cadherin adhesion sites. We observed that inhibiting myosin II directly or through photo-release of the caged Rho kinase inhibitor also reduced the rate of VE-cadherin dissociation. Thus, Rac1 functions by stabilizing VE-cadherin trans-dimers in mature AJs by counteracting the actomyosin tension. The results suggest a new model of VE-cadherin adhesive interaction mediated by Rac1-induced reduction of mechanical tension at AJs, resulting in the stabilization of VE-cadherin adhesions. PMID:25559184

  1. Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

    PubMed

    Eller-Borges, Roberta; Batista, Wagner L; da Costa, Paulo E; Tokikawa, Rita; Curcio, Marli F; Strumillo, Scheilla T; Sartori, Adriano; Moraes, Miriam S; de Oliveira, Graciele A; Taha, Murched O; Fonseca, Fábio V; Stern, Arnold; Monteiro, Hugo P

    2015-05-01

    The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK. PMID:25819133

  2. ARF6 promotes the formation of Rac1 and WAVE-dependent ventral F-actin rosettes in breast cancer cells in response to epidermal growth factor.

    PubMed

    Marchesin, Valentina; Montagnac, Guillaume; Chavrier, Philippe

    2015-01-01

    Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration. PMID:25799492

  3. mTOR Directs Breast Morphogenesis through the PKC-alpha-Rac1 Signaling Axis

    PubMed Central

    Morrison, Meghan M.; Young, Christian D.; Wang, Shan; Sobolik, Tammy; Sanchez, Violeta M.; Hicks, Donna J.; Cook, Rebecca S.; Brantley-Sieders, Dana M.

    2015-01-01

    Akt phosphorylation is a major driver of cell survival, motility, and proliferation in development and disease, causing increased interest in upstream regulators of Akt like mTOR complex 2 (mTORC2). We used genetic disruption of Rictor to impair mTORC2 activity in mouse mammary epithelia, which decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival. These effects were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival or invasion, and modestly increased branching of mTORC2-impaired mammary epithelial cells (MECs) in culture and in vivo. However, another mTORC2 substrate, protein kinase C (PKC)-alpha, fully rescued mTORC2-impaired MEC branching, invasion, and survival, as well as branching morphogenesis in vivo. PKC-alpha-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor/mTORC2 in MEC survival and motility during branching morphogenesis through a PKC-alpha/Rac1-dependent mechanism. PMID:26132202

  4. Arhgef16, a novel Elmo1 binding partner, promotes clearance of apoptotic cells via RhoG-dependent Rac1 activation.

    PubMed

    Lee, Juyeon; Park, Boyeon; Kim, Gayoung; Kim, Kwangwoo; Pak, Jeongjun; Kim, Kwanhyeong; Ye, Michael B; Park, Sung-Gyoo; Park, Daeho

    2014-11-01

    Elmo is an evolutionarily conserved mammalian ortholog of Caenorhabditis elegans CED-12 with proposed roles during the removal of apoptotic cells, cell migration, neurite outgrowth, and myoblast fusion (Katoh and Negishi (2003) [1], Park and Tosello (2007) [2], Grimsley et al. (2004) [3], Hamoud et al. (2014) [4]). Elmo mediates these cellular processes by interacting with various proteins located in the plasma membrane, cytoplasm and nucleus, and by modulating their activities although it has no intrinsic catalytic activity (Park and Tosello (2007) [2], Hamoud et al. (2014) [4], Li et al. (2013) [5], Margaron, Fradet and Cote (2013) [6], and Mauldin et al. (2013)[7]). Because there are a limited number of proteins known to interact with Elmo, we performed a yeast two-hybrid screen using Elmo1 as bait to identify Elmo1-interacting proteins and to evaluate their mode of regulation. Arhgef16 was one of the proteins identified through the screen and subsequent analyses revealed that Arhgef16 interacted with Elmo1 in mammalian cells as well. Expression of Arhgef16 in phagocytes promoted engulfment of apoptotic cells, and engulfment mediated by Arhgef16 increased synergistically in the presence of Elmo1 but was abrogated in the absence of Elmo1. In addition, Arhgef16-mediated removal of apoptotic cells was dependent on RhoG, but independent of Dock1. Taken together, this study suggests that the newly identified Elmo1-interacting protein, Arhgef16, functions synergistically with Elmo1 to promote clearance of apoptotic cells in a RhoG-dependent and Dock1-independent manner. PMID:25063526

  5. MicroRNA-224 promotes the sensitivity of osteosarcoma cells to cisplatin by targeting Rac1.

    PubMed

    Geng, Shuo; Gu, Lina; Ju, Fang; Zhang, Hepeng; Wang, Yiwen; Tang, Han; Bi, ZhengGang; Yang, Chenglin

    2016-09-01

    Osteosarcoma is the most common primary bone tumour in children and adolescents. Accumulating evidence has shown that microRNAs (miRNAs) participate in the development of almost all types of cancer. Here, we investigated the role of miR-224 in the development and progression of osteosarcoma. We demonstrated that miR-224 was down-regulated in osteosarcoma cell lines and tissues. Lower miR-224 levels were correlated with shorter survivalin osteosarcoma patients. Furthermore, overexpression of miR-224 suppressed osteosarcoma cell proliferation, migration and invasion and contributed to the increased sensitivity of MG-63 cells to cisplatin. We identified Rac1 as a direct target gene of miR-224 in osteosarcoma. Rac1 expression was up-regulated in the osteosarcoma cell lines and tissues, and there was an inverse correlation between Rac1 and miR-224 expression in osteosarcoma tissues. Furthermore, rescuing Rac1 expression decreased the sensitivity of miR-224-overexpressing MG-63 cells to cisplatin. We also demonstrated that ectopic expression of Rac1 promoted the proliferation, migration and invasion of miR-224-overexpressing MG-63 cells. These data suggest that miR-224 plays a tumour suppressor role in the development of osteosarcoma and is related to the sensitivity of osteosarcoma to cisplatin. PMID:27222381

  6. Distinct predictive performance of Rac1 and Cdc42 in cell migration

    PubMed Central

    Yamao, Masataka; Naoki, Honda; Kunida, Katsuyuki; Aoki, Kazuhiro; Matsuda, Michiyuki; Ishii, Shin

    2015-01-01

    We propose a new computation-based approach for elucidating how signaling molecules are decoded in cell migration. In this approach, we performed FRET time-lapse imaging of Rac1 and Cdc42, members of Rho GTPases which are responsible for cell motility, and quantitatively identified the response functions that describe the conversion from the molecular activities to the morphological changes. Based on the identified response functions, we clarified the profiles of how the morphology spatiotemporally changes in response to local and transient activation of Rac1 and Cdc42, and found that Rac1 and Cdc42 activation triggers laterally propagating membrane protrusion. The response functions were also endowed with property of differentiator, which is beneficial for maintaining sensitivity under adaptation to the mean level of input. Using the response function, we could predict the morphological change from molecular activity, and its predictive performance provides a new quantitative measure of how much the Rho GTPases participate in the cell migration. Interestingly, we discovered distinct predictive performance of Rac1 and Cdc42 depending on the migration modes, indicating that Rac1 and Cdc42 contribute to persistent and random migration, respectively. Thus, our proposed predictive approach enabled us to uncover the hidden information processing rules of Rho GTPases in the cell migration. PMID:26634649

  7. Resveratrol Inhibition of Rac1-Derived Reactive Oxygen Species by AMPK Decreases Blood Pressure in a Fructose-Induced Rat Model of Hypertension.

    PubMed

    Cheng, Pei-Wen; Lee, Hui-Chieh; Lu, Pei-Jung; Chen, Hsin-Hung; Lai, Chi-Cheng; Sun, Gwo-Ching; Yeh, Tung-Chen; Hsiao, Michael; Lin, Yu-Te; Liu, Chun-Peng; Tseng, Ching-Jiunn

    2016-01-01

    Recent studies have reported that the activation of AMP-activated protein kinase (AMPK) suppressed oxidative stress. The aim of this study was to examine whether the activation of AMPK in the brain decreased Rac1-induced ROS generation, thereby reducing blood pressure (BP) in rats with fructose-induced hypertension. The inhibition of ROS by treatment with an AMPK activator (oral resveratrol, 10 mg/kg/day) for 1 week decreased the BP and increased the NO production in the rostral ventrolateral medulla (RVLM) of fructose-fed rats but not in control Wistar-Kyoto (WKY) rats. In addition, resveratrol treatment abolished the Rac1-induced increases in the activity of the NADPH oxidase subunits p22-phox and reduced the activity of SOD2, while treatment with an AMPK inhibitor (compound C, 40 μM/day) had the opposite effect, in the fructose-fed rats. Interestingly, the activation of AMPK abolished Rac1 activation and decreased BP by inducing the activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and ribosomal protein S6 kinase (RSK) and nNOS phosphorylation in the fructose-fed rats. We conclude that the activation of AMPK decreased BP, abolished ROS generation, and enhanced ERK1/2-RSK-nNOS pathway activity by negatively regulating Racl-induced NADPH oxidase levels in the RVLM during oxidative stress-associated hypertension. PMID:27138844

  8. Resveratrol Inhibition of Rac1-Derived Reactive Oxygen Species by AMPK Decreases Blood Pressure in a Fructose-Induced Rat Model of Hypertension

    PubMed Central

    Cheng, Pei-Wen; Lee, Hui-Chieh; Lu, Pei-Jung; Chen, Hsin-Hung; Lai, Chi-Cheng; Sun, Gwo-Ching; Yeh, Tung-Chen; Hsiao, Michael; Lin, Yu-Te; Liu, Chun-Peng; Tseng, Ching-Jiunn

    2016-01-01

    Recent studies have reported that the activation of AMP-activated protein kinase (AMPK) suppressed oxidative stress. The aim of this study was to examine whether the activation of AMPK in the brain decreased Rac1-induced ROS generation, thereby reducing blood pressure (BP) in rats with fructose-induced hypertension. The inhibition of ROS by treatment with an AMPK activator (oral resveratrol, 10 mg/kg/day) for 1 week decreased the BP and increased the NO production in the rostral ventrolateral medulla (RVLM) of fructose-fed rats but not in control Wistar-Kyoto (WKY) rats. In addition, resveratrol treatment abolished the Rac1-induced increases in the activity of the NADPH oxidase subunits p22-phox and reduced the activity of SOD2, while treatment with an AMPK inhibitor (compound C, 40 μM/day) had the opposite effect, in the fructose-fed rats. Interestingly, the activation of AMPK abolished Rac1 activation and decreased BP by inducing the activities of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and ribosomal protein S6 kinase (RSK) and nNOS phosphorylation in the fructose-fed rats. We conclude that the activation of AMPK decreased BP, abolished ROS generation, and enhanced ERK1/2-RSK-nNOS pathway activity by negatively regulating Racl-induced NADPH oxidase levels in the RVLM during oxidative stress–associated hypertension. PMID:27138844

  9. R-Ketorolac Targets Cdc42 and Rac1 and Alters Ovarian Cancer Cell Behaviors Critical for Invasion and Metastasis.

    PubMed

    Guo, Yuna; Kenney, S Ray; Muller, Carolyn Y; Adams, Sarah; Rutledge, Teresa; Romero, Elsa; Murray-Krezan, Cristina; Prekeris, Rytis; Sklar, Larry A; Hudson, Laurie G; Wandinger-Ness, Angela

    2015-10-01

    Cdc42 (cell division control protein 42) and Rac1 (Ras-related C3 botulinum toxin substrate 1) are attractive therapeutic targets in ovarian cancer based on established importance in tumor cell migration, adhesion, and invasion. Despite a predicted benefit, targeting GTPases has not yet been translated to clinical practice. We previously established that Cdc42 and constitutively active Rac1b are overexpressed in primary ovarian tumor tissues. Through high-throughput screening and computational shape homology approaches, we identified R-ketorolac as a Cdc42 and Rac1 inhibitor, distinct from the anti-inflammatory, cyclooxygenase inhibitory activity of S-ketorolac. In the present study, we establish R-ketorolac as an allosteric inhibitor of Cdc42 and Rac1. Cell-based assays validate R-ketorolac activity against Cdc42 and Rac1. Studies on immortalized human ovarian adenocarcinoma cells (SKOV3ip) and primary patient-derived ovarian cancer cells show that R-ketorolac is a robust inhibitor of growth factor or serum-dependent Cdc42 and Rac1 activation with a potency and cellular efficacy similar to small-molecule inhibitors of Cdc42 (CID2950007/ML141) and Rac1 (NSC23766). Furthermore, GTPase inhibition by R-ketorolac reduces downstream p21-activated kinases (PAK1/PAK2) effector activation by >80%. Multiple assays of cell behavior using SKOV3ip and primary patient-derived ovarian cancer cells show that R-ketorolac significantly inhibits cell adhesion, migration, and invasion. In summary, we provide evidence for R-ketorolac as a direct inhibitor of Cdc42 and Rac1 that is capable of modulating downstream GTPase-dependent, physiologic responses, which are critical to tumor metastasis. Our findings demonstrate the selective inhibition of Cdc42 and Rac1 GTPases by an FDA-approved drug, racemic ketorolac, that can be used in humans. PMID:26206334

  10. RAC1 inhibition as a therapeutic target for gefitinib-resistant non-small-cell lung cancer

    PubMed Central

    Kaneto, Naoki; Yokoyama, Satoru; Hayakawa, Yoshihiro; Kato, Shinichiro; Sakurai, Hiroaki; Saiki, Ikuo

    2014-01-01

    Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKI), including gefitinib, provide a significant clinical benefit in non-small-cell lung cancer (NSCLC) patients, the acquisition of drug resistance has been known to limit the efficacy of EGFR-TKI therapy. In this study, we demonstrated the involvement of EGF-EGFR signaling in NSCLC cell migration and the requirement of RAC1 in EGFR-mediated progression of NSCLC. We showed the significant role of RAC1 pathway in the cell migration or lamellipodia formation by using gene silencing of RAC1 or induction of constitutive active RAC1 in EGFR-mutant NSCLC cells. Importantly, the RAC1 inhibition suppressed EGFR-mutant NSCLC cell migration and growth in vitro, and growth in vivo even in the gefitinib-resistant cells. In addition, these suppressions by RAC1 inhibition were mediated through MEK or PI3K independent mechanisms. Collectively, these results open up a new opportunity to control the cancer progression by targeting the RAC1 pathway to overcome the resistance to EGFR-TKI in NSCLC patients. PMID:24750242

  11. Stability and cellular studies of [rac-1,2-bis(4-fluorophenyl)-ethylenediamine][cyclobutane-1,1- dicarboxylato]platinum(II), a novel, highly active carboplatin derivative.

    PubMed

    Gust, R; Schnurr, B; Krauser, R; Bernhardt, G; Koch, M; Schmid, B; Hummel, E; Schönenberger, H

    1998-01-01

    The synthesis of the diastereomeric [1,2-bis(4-fluorophenyl)ethylenediamine][cyclobutane-1, 1-dicarboxylato]platinum(II) complexes, rac- and meso-4F-Pt(CBDC), the evaluation of their structures, their tumor-inhibiting properties and their stability in physiological environment are described (reference complexes: the dichloro- and sulfatoplatinum(II) analogues, carboplatin and cisplatin). The most interesting diastereomer, rac-4F-Pt(CBDC), equals cisplatin and surpasses carboplatin in its effect on human breast cancer cell lines (MCF-7 and MDA-MB-231). Rac-4F-Pt(CBDC) is largely insensitive against attack of nucleophiles e.g. Cl-, a prerequisite for sufficient stability in vivo and for fewer side effects. In accordance with this, in vitro studies on the binding of rac-4F-Pt(CBDC) to albumin, the main plasma protein, show that the free, non-protein-bound fraction is relatively high, coming close to that of carboplatin. These properties are of importance for the transferability of the promising effects found in the cell culture experiments to in vivo conditions. The distinctly better anti-breast cancer activity of rac-4F-Pt(CBDC) than of carboplatin has been attributed to its ability to accumulate in the tumor cells. The human ovarian cancer cell line NIH-OVCAR-3 is also strongly inhibited by rac-4F-Pt(CBDC). PMID:9860287

  12. Actin-Mediated Gene Expression Depends on RhoA and Rac1 Signaling in Proximal Tubular Epithelial Cells

    PubMed Central

    Giehl, Klaudia; Keller, Christof; Muehlich, Susanne; Goppelt-Struebe, Margarete

    2015-01-01

    Morphological alterations of cells can lead to modulation of gene expression. An essential link is the MKL1-dependent activation of serum response factor (SRF), which translates changes in the ratio of G- and F-actin into mRNA transcription. SRF activation is only partially characterized in non-transformed epithelial cells. Therefore, the impact of GTPases of the Rho family and changes in F-actin structures were analyzed in renal proximal tubular epithelial cells. Activation of SRF signaling was compared to the regulation of a known MKL1/SRF target gene, connective tissue growth factor (CTGF). In the human proximal tubular cell line HKC-8 overexpression of two actin mutants either favoring or preventing the formation of F-actin fibers regulated SRF-mediated transcription as well as CTGF expression. Only overexpression of constitutively active RhoA activated SRF-dependent gene expression whereas no effect was detected upon overexpression of Rac1 mutants. To elucidate the functional role of Rho kinases as downstream mediators of RhoA, pharmacological inhibition and genetic inhibition by transient siRNA knock down were compared. Upon stimulation with lysophosphatidic acid (LPA) Rho kinase inhibitors partially suppressed SRF-mediated transcription, whereas interference with Rho kinase expression by siRNA reduced activation of SRF, but barely affected CTGF expression. Together with the partial inhibition of CTGF expression by the pharmacological inhibitors Y27432 and H1154, Rho kinases seem to be less important in mediating RhoA signaling related to CTGF expression in HKC-8 epithelial cells. Short term pharmacological inhibition of Rac1 activity by EHT1864 reduced SRF-dependent CTGF expression in HKC-8 cells, but was overcome by a stimulatory effect after prolonged incubation after 4-6 h. Similarly, human primary cells of proximal but not of distal tubular origin showed inhibitory as well as stimulatory effects of Rac1 inhibition. Thus, RhoA signaling activates MKL1-SRF

  13. The bipartite rac1 Guanine nucleotide exchange factor engulfment and cell motility 1/dedicator of cytokinesis 180 (elmo1/dock180) protects endothelial cells from apoptosis in blood vessel development.

    PubMed

    Schäker, Kathrin; Bartsch, Susanne; Patry, Christian; Stoll, Sandra J; Hillebrands, Jan-Luuk; Wieland, Thomas; Kroll, Jens

    2015-03-01

    Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). Elmo1/Dock180 regulates Rac1 activity in a specific spatiotemporal manner in endothelial cells (ECs) during zebrafish development and acts downstream of the Netrin-1/Unc5-homolog B (Unc5B) signaling cascade. However, mechanistic details on the pathways by which Elmo1/Dock180 regulates endothelial function and vascular development remained elusive. In this study, we aimed to analyze the vascular function of Elmo1 and Dock180 in human ECs and during vascular development in zebrafish embryos. In vitro overexpression of Elmo1 and Dock180 in ECs reduced caspase-3/7 activity and annexin V-positive cell number upon induction of apoptosis. This protective effect of Elmo1 and Dock180 is mediated by activation of Rac1, p21-activated kinase (PAK) and AKT/protein kinase B (AKT) signaling. In zebrafish, Elmo1 and Dock180 overexpression reduced the total apoptotic cell and apoptotic EC number and promoted the formation of blood vessels during embryogenesis. In conclusion, Elmo1 and Dock180 protect ECs from apoptosis by the activation of the Rac1/PAK/AKT signaling cascade in vitro and in vivo. Thus, Elmo1 and Dock180 facilitate blood vessel formation by stabilization of the endothelium during angiogenesis. PMID:25586182

  14. The Bipartite Rac1 Guanine Nucleotide Exchange Factor Engulfment and Cell Motility 1/Dedicator of Cytokinesis 180 (Elmo1/Dock180) Protects Endothelial Cells from Apoptosis in Blood Vessel Development*

    PubMed Central

    Schäker, Kathrin; Bartsch, Susanne; Patry, Christian; Stoll, Sandra J.; Hillebrands, Jan-Luuk; Wieland, Thomas; Kroll, Jens

    2015-01-01

    Engulfment and cell motility 1/dedicator of cytokinesis 180 (Elmo1/Dock180) is a bipartite guanine nucleotide exchange factor for the monomeric GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). Elmo1/Dock180 regulates Rac1 activity in a specific spatiotemporal manner in endothelial cells (ECs) during zebrafish development and acts downstream of the Netrin-1/Unc5-homolog B (Unc5B) signaling cascade. However, mechanistic details on the pathways by which Elmo1/Dock180 regulates endothelial function and vascular development remained elusive. In this study, we aimed to analyze the vascular function of Elmo1 and Dock180 in human ECs and during vascular development in zebrafish embryos. In vitro overexpression of Elmo1 and Dock180 in ECs reduced caspase-3/7 activity and annexin V-positive cell number upon induction of apoptosis. This protective effect of Elmo1 and Dock180 is mediated by activation of Rac1, p21-activated kinase (PAK) and AKT/protein kinase B (AKT) signaling. In zebrafish, Elmo1 and Dock180 overexpression reduced the total apoptotic cell and apoptotic EC number and promoted the formation of blood vessels during embryogenesis. In conclusion, Elmo1 and Dock180 protect ECs from apoptosis by the activation of the Rac1/PAK/AKT signaling cascade in vitro and in vivo. Thus, Elmo1 and Dock180 facilitate blood vessel formation by stabilization of the endothelium during angiogenesis. PMID:25586182

  15. Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway

    PubMed Central

    Reinardy, Jessica L.; Corey, Daniel M.; Golzio, Christelle; Mueller, Sarah B.; Katsanis, Nicholas; Kontos, Christopher D.

    2015-01-01

    The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis. PMID:26436659

  16. Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer.

    PubMed

    Zhu, Xingli; Gao, Guanghui; Chu, Kaili; Yang, Xiufang; Ren, Shengxiang; Li, Yao; Wu, Hai; Huang, Yan; Zhou, Caicun

    2015-04-01

    MicroRNAs are a class of small non-coding RNAs regulating gene expression. In this study, we demonstrated that retinoic acid (RA) treatment increases the expression of miR-512-3p. Overexpression of miR-512-3p inhibited cell adhesion, migration, and invasion in non-small cell lung cancer (NSCLC) cell lines A549 and H1299. miR-512-3p inhibitor partially reversed these effects in H1299 cells stably expressing miR-512. We identified DOCK3, a RAC1-GEF (guanine nucleotide exchange factor), as a target gene of miR-512-3p. Overexpression of miR-512-3p led to the decrease of DOCK3 protein but not its mRNA. Knockdown of DOCK3 resulted in similar effects on adhesion, migration, and invasion as observed of miR-512-3p overexpression. Active RAC1 pull-down assay indicated that overexpression of miR-512-3p could decrease the activity of RAC1 with a higher efficiency than that of DOCK3 knockdown. Furthermore, expression of miR-512-3p was suppressed in most NSCLC patient tumor samples compared to its paired normal controls, suggesting that miR-512-3p might play a crucial role in lung cancer development. In conclusion, our results supported that miR-512-3p could inhibit tumor cell adhesion, migration, and invasion by regulating the RAC1 activity via DOCK3 in NSCLC A549 and H1299 cell lines. PMID:25687035

  17. Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

    SciTech Connect

    Abu-Issa, Radwan

    2015-01-24

    Highlights: • Conditional knockout of Rac1 using Nkx2.5 Cre line is lethal at E13.5. • The myocardium of the mutant is thin and disorganized. • The phenotype is not due to cardiomyocyte low proliferation or apoptosis. • The phenotype is due to specific defect in cardiomyocyte adhesion. - Abstract: Rac1, a member of the Rho subfamily of small GTPases, is involved in morphogenesis and differentiation of many cell types. Here we define a role of Rac1 in cardiac development by specifically deleting Rac1 in the pre-cardiac mesoderm using the Nkx2.5-Cre transgenic driver line. Rac1-conditional knockout embryos initiate heart development normally until embryonic day 11.5 (E11.5); their cardiac mesoderm is specified, and the heart tube is formed and looped. However, by E12.5-E13.5 the mutant hearts start failing and embryos develop edema and hemorrhage which is probably the cause for the lethality observed soon after. The hearts of Rac1-cKO embryos exhibit disorganized and thin myocardial walls and defects in outflow tract alignment. No significant differences of cardiomyocyte death or proliferation were found between developing control and mutant embryos. To uncover the role of Rac1 in the heart, E11.5 primary heart cells were cultured and analyzed in vitro. Rac1-deficient cardiomyocytes were less spread, round and loosely attached to the substrate and to each other implying that Rac1-mediated signaling is required for appropriate cell–cell and/or cellmatrix adhesion during cardiac development.

  18. The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

    SciTech Connect

    Nikolova, Ekaterina; Mitev, Vanio; Zhelev, Nikolai; Deroanne, Christophe F. . E-mail: yves.poumay@fundp.ac.be

    2007-08-03

    Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity.

  19. Rac1 drives intestinal stem cell proliferation and regeneration

    PubMed Central

    Myant, Kevin B; Scopelliti, Alessandro; Haque, Sara; Vidal, Marcos; Sansom, Owen J; Cordero, Julia B

    2013-01-01

    Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology. PMID:23974108

  20. The role of Crk/Dock180/Rac1 pathway in the malignant behavior of human ovarian cancer cell SKOV3.

    PubMed

    Wang, Hui; Linghu, Hua; Wang, Jin; Che, Ya-ling; Xiang, Ting-xiu; Tang, Wei-xue; Yao, Zhen-wei

    2010-01-01

    Small GTPases, particularly the Rho family, are key regulators of cell motility and migration. Dock180 was well known for the main target of signal adaptor protein Crk and acted as a guanine-nucleotide exchange factor for small GTPase Rac1. In the present study, Dock180 was found to combine primarily with CrkI other than CrkII, and its association with Elmo1 was also demonstrated in ovarian cancer cell SKOV3. To evaluate the role of Dock180 in human ovarian cancer cell, we performed RNAi-mediated knockdown of Dock180 in SKOV3 cells using small interfering RNA expression vector. In Dock180 knockdown cells, we found that Elmo1 expression and Rac1 activity were decreased simultaneously. By contrast, the expressions of both another Crk-combining molecule C3G and Rap1 activity were observed to increase obviously. Accordingly, all Dock180 knockdown cells present with evident change in cell morphology, reduced cell proliferation, and attenuated cell migration. Taken together, these results suggest that signal transfer of Crk/Dock180/Rac1 is implicated in actin cytoskeleton reorganization and thus in the cell proliferation, motility, invasion, and of human ovarian cancer cell line SKOV3. PMID:20237902

  1. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

    PubMed Central

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying

    2015-01-01

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

  2. Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1.

    PubMed

    Grikscheit, Katharina; Frank, Tanja; Wang, Ying; Grosse, Robert

    2015-05-11

    Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell-cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell-cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell-cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell-cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1. PMID:25963818

  3. Staphylococcus aureus keratinocyte invasion is mediated by integrin-linked kinase and Rac1.

    PubMed

    Sayedyahossein, Samar; Xu, Stacey X; Rudkouskaya, Alena; McGavin, Martin J; McCormick, John K; Dagnino, Lina

    2015-02-01

    Staphylococcus aureus is a major component of the skin microbiota and causes a large number of serious infections. S. aureus first interacts with epidermal keratinocytes to breach the epidermal barrier through mechanisms not fully understood. By use of primary keratinocytes from mice with epidermis-restricted Ilk gene inactivation and control integrin-linked kinase (ILK)-expressing littermates, we investigated the role of ILK in epidermal S. aureus invasion. Heat-killed, but not live, bacteria were internalized to Rab5- and Rab7-positive phagosomes, and incubation with keratinocyte growth factor increased their uptake 2.5-fold. ILK-deficient mouse keratinocytes internalized bacteria 2- to 4-fold less efficiently than normal cells. The reduced invasion by live S. aureus of ILK-deficient cells was restored in the presence of exogenous, constitutively active Rac1. Thus, Rac1 functions downstream from ILK during invasion. Further, invasion by S. aureus of Rac1-deficient cells was 2.5-fold lower than in normal cells. Paradoxically, staphylococcal cutaneous penetration of mouse skin explants with ILK-deficient epidermis was 35-fold higher than that of normal skin, indicating defects in epidermal barrier function in the absence of ILK. Thus, we identified an ILK-Rac1 pathway essential for bacterial invasion of keratinocytes, and established ILK as a key contributor to prevent invasive staphylococcal cutaneous infection. PMID:25416549

  4. Opposing effects of TIGAR- and RAC1-derived ROS on Wnt-driven proliferation in the mouse intestine

    PubMed Central

    Cheung, Eric C.; Lee, Pearl; Ceteci, Fatih; Nixon, Colin; Blyth, Karen; Sansom, Owen J.; Vousden, Karen H.

    2016-01-01

    Reactive oxygen species (ROS) participate in numerous cell responses, including proliferation, DNA damage, and cell death. Based on these disparate activities, both promotion and inhibition of ROS have been proposed for cancer therapy. However, how the ROS response is determined is not clear. We examined the activities of ROS in a model of Apc deletion, where loss of the Wnt target gene Myc both rescues APC loss and prevents ROS accumulation. Following APC loss, Myc has been shown to up-regulate RAC1 to promote proliferative ROS through NADPH oxidase (NOX). However, APC loss also increased the expression of TIGAR, which functions to limit ROS. To explore this paradox, we used three-dimensional (3D) cultures and in vivo models to show that deletion of TIGAR increased ROS damage and inhibited proliferation. These responses were suppressed by limiting damaging ROS but enhanced by lowering proproliferative NOX-derived ROS. Despite having opposing effects on ROS levels, loss of TIGAR and RAC1 cooperated to suppress intestinal proliferation following APC loss. Our results indicate that the pro- and anti-proliferative effects of ROS can be independently modulated in the same cell, with two key targets in the Wnt pathway functioning to integrate the different ROS signals for optimal cell proliferation. PMID:26679840

  5. Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks

    PubMed Central

    Pennucci, Roberta; Talpo, Francesca; Astro, Veronica; Montinaro, Valentina; Morè, Lorenzo; Cursi, Marco; Castoldi, Valerio; Chiaretti, Sara; Bianchi, Veronica; Marenna, Silvia; Cambiaghi, Marco; Tonoli, Diletta; Leocani, Letizia; Biella, Gerardo; D'Adamo, Patrizia; de Curtis, Ivan

    2016-01-01

    Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons. PMID:26582364

  6. Programmed Application of Transforming Growth Factor β3 and Rac1 Inhibitor NSC23766 Committed Hyaline Cartilage Differentiation of Adipose-Derived Stem Cells for Osteochondral Defect Repair

    PubMed Central

    Zhu, Shouan; Chen, Pengfei; Wu, Yan; Xiong, Si; Sun, Heng; Xia, Qingqing; Shi, Libing

    2014-01-01

    Hyaline cartilage differentiation is always the challenge with application of stem cells for joint repair. Transforming growth factors (TGFs) and bone morphogenetic proteins can initiate cartilage differentiation but often lead to hypertrophy and calcification, related to abnormal Rac1 activity. In this study, we developed a strategy of programmed application of TGFβ3 and Rac1 inhibitor NSC23766 to commit the hyaline cartilage differentiation of adipose-derived stem cells (ADSCs) for joint cartilage repair. ADSCs were isolated and cultured in a micromass and pellet culture model to evaluate chondrogenic and hypertrophic differentiation. The function of Rac1 was investigated with constitutively active Rac1 mutant and dominant negative Rac1 mutant. The efficacy of ADSCs with programmed application of TGFβ3 and Rac1 inhibitor for cartilage repair was studied in a rat model of osteochondral defects. The results showed that TGFβ3 promoted ADSCs chondro-lineage differentiation and that NSC23766 prevented ADSC-derived chondrocytes from hypertrophy in vitro. The combination of ADSCs, TGFβ3, and NSC23766 promoted quality osteochondral defect repair in rats with much less chondrocytes hypertrophy and significantly higher International Cartilage Repair Society macroscopic and microscopic scores. The findings have illustrated that programmed application of TGFβ3 and Rac1 inhibitor NSC23766 can commit ADSCs to chondro-lineage differentiation and improve the efficacy of ADSCs for cartilage defect repair. These findings suggest a promising stem cell-based strategy for articular cartilage repair. PMID:25154784

  7. Rac1 inactivation by lethal toxin from Clostridium sordellii modifies focal adhesions upstream of actin depolymerization.

    PubMed

    Geny, Blandine; Grassart, Alexandre; Manich, Maria; Chicanne, Gaëtan; Payrastre, Bernard; Sauvonnet, Nathalie; Popoff, Michel R

    2010-02-01

    Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT-82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT-82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1(V12). Rac1 inactivation by LT-82 modifies interactions between proteins from AJ and FA complexes as shown by pull-down assays. We showed that in Triton X-100-insoluble membrane proteins from these complexes, namely E-cadherin, beta-catenin, p120-catenin and talin, are decreased upon LT-82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT-82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization. PMID:19840028

  8. Cyfip1 Regulates Presynaptic Activity during Development

    PubMed Central

    Hsiao, Kuangfu; Harony-Nicolas, Hala; Buxbaum, Joseph D.

    2016-01-01

    Copy number variations encompassing the gene encoding Cyfip1 have been associated with a variety of human diseases, including autism and schizophrenia. Here we show that juvenile mice hemizygous for Cyfip1 have altered presynaptic function, enhanced protein translation, and increased levels of F-actin. In developing hippocampus, reduced Cyfip1 levels serve to decrease paired pulse facilitation and increase miniature EPSC frequency without a change in amplitude. Higher-resolution examination shows these changes to be caused primarily by an increase in presynaptic terminal size and enhanced vesicle release probability. Short hairpin-mediated knockdown of Cyfip1 coupled with expression of mutant Cyfip1 proteins indicates that the presynaptic alterations are caused by dysregulation of the WAVE regulatory complex. Such dysregulation occurs downstream of Rac1 as acute exposure to Rac1 inhibitors rescues presynaptic responses in culture and in hippocampal slices. The data serve to highlight an early and essential role for Cyfip1 in the generation of normally functioning synapses and suggest a means by which changes in Cyfip1 levels could impact the generation of neural networks and contribute to abnormal and maladaptive behaviors. SIGNIFICANCE STATEMENT Several developmental brain disorders have been associated with gene duplications and deletions that serve to increase or decrease levels of encoded proteins. Cyfip1 is one such protein, but the role it plays in brain development is poorly understood. We asked whether decreased Cyfip1 levels altered the function of developing synapses. The data show that synapses with reduced Cyfip1 are larger and release neurotransmitter more rapidly. These effects are due to Cyfip1's role in actin polymerization and are reversed by expression of a Cyfip1 mutant protein retaining actin regulatory function or by inhibiting Rac1. Thus, Cyfip1 has a more prominent early role regulating presynaptic activity during a stage of development when

  9. Rac1 and Cdc42 Play Important Roles in Arsenic Neurotoxicity in Primary Cultured Rat Cerebellar Astrocytes.

    PubMed

    An, Yuan; Liu, Tingting; Liu, Xiaona; Zhao, Lijun; Wang, Jing

    2016-03-01

    This study aimed to explore whether Rac1 and Cdc42, representative members of Ras homologue guanosine triphosphatases (Rho GTPases), are involved in neurotoxicity induced by arsenic exposure in rat nervous system. Expressions of Rac1 and Cdc42 in rat cerebellum and cerebrum exposed to different doses of NaAsO2 (Wistar rats drank 0, 2, 10, and 50 mg/L NaAsO2 water for 3 months) were examined. Both Rac1 and Cdc42 expressions increased significantly in a dose-dependent manner in cerebellum (P < 0.01) by Western blot and immunohistochemistry assay, but in cerebrum, Rac1 and Cdc42 expressions only in 2 mg/L exposure groups were significantly higher than those in control groups (P < 0.01). Five to 50 μM NaAsO2 decreased cell viability in a dose-dependent manner in primary cultured rat astrocytes, whereas 1 μM NaAsO2 increased the cell viability in these cells. Rac1 inhibitor, NSC23766, decreased NaAsO2-induced apoptosis and increased the cell viability in primary cultured rat cerebellar astrocytes exposed to 30 μM NaAsO2. Cdc42 inhibitor, ZCL278, increased cell viability in the cells exposed to 30 μM NaAsO2. Taken together, our current studies in vivo and in vitro indicate that activations of Rac1 and Cdc42 play a very important role in arsenic neurotoxicity in rat cerebellum, providing a new insight into arsenic neurotoxicity. PMID:26231544

  10. Dock10, a Cdc42 and Rac1 GEF, induces loss of elongation, filopodia, and ruffles in cervical cancer epithelial HeLa cells

    PubMed Central

    Ruiz-Lafuente, Natalia; Alcaraz-García, María-José; García-Serna, Azahara-María; Sebastián-Ruiz, Silvia; Moya-Quiles, María-Rosa; García-Alonso, Ana-María; Parrado, Antonio

    2015-01-01

    Dock10 is one of the three members of the Dock-D family of Dock proteins, a class of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Its homologs Dock9 and Dock11 are Cdc42 GEFs. Dock10 is required for maintenance of rounded morphology and amoeboid-type movement. Full-length isoforms of Dock10 have been recently cloned. Here, we address GTPase specificity and GEF activity of Dock10. In order of decreasing intensity, Dock10 interacted with nucleotide-free Rac1, Cdc42, and Rac3, and more weakly with Rac2, RhoF, and RhoG. Inducible expression of Dock10 in HeLa epithelial cells promoted GEF activity on Cdc42 and Rac1, and a morphologic change in two-dimensional culture consisting in loss of cell elongation, increase of filopodia, and ruffles. Area in contact with the substrate of cells that spread with non-elongated morphology was larger in cells expressing Dock10. Inducible expression of constitutively active mutants of Cdc42 and Rac1 in HeLa cells also induced loss of elongation. However, Cdc42 induced filopodia and contraction, and Rac1 induced membrane ruffles and flattening. When co-expressed with Dock10, Cdc42 potentiated filopodia, and Rac1 potentiated ruffles. These results suggest that Dock10 functions as a dual GEF for Cdc42 and Rac1, affecting cell morphology, spreading and actin cytoskeleton protrusions of adherent HeLa cells. PMID:25862245

  11. Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches

    PubMed Central

    Byrne, Kate M.; Monsefi, Naser; Dawson, John C.; Degasperi, Andrea; Bukowski-Wills, Jimi-Carlo; Volinsky, Natalia; Dobrzyński, Maciej; Birtwistle, Marc R.; Tsyganov, Mikhail A.; Kiyatkin, Anatoly; Kida, Katarzyna; Finch, Andrew J.; Carragher, Neil O.; Kolch, Walter; Nguyen, Lan K.; von Kriegsheim, Alex; Kholodenko, Boris N.

    2016-01-01

    Summary Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches. PMID:27136688

  12. Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches.

    PubMed

    Byrne, Kate M; Monsefi, Naser; Dawson, John C; Degasperi, Andrea; Bukowski-Wills, Jimi-Carlo; Volinsky, Natalia; Dobrzyński, Maciej; Birtwistle, Marc R; Tsyganov, Mikhail A; Kiyatkin, Anatoly; Kida, Katarzyna; Finch, Andrew J; Carragher, Neil O; Kolch, Walter; Nguyen, Lan K; von Kriegsheim, Alex; Kholodenko, Boris N

    2016-01-27

    Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches. PMID:27136688

  13. Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells.

    PubMed

    Zhou, Hui-Chao; Fang, Jian-Hong; Luo, Xu; Zhang, Lei; Yang, Jine; Zhang, Chong; Zhuang, Shi-Mei

    2014-12-15

    Metastasis is responsible for rapid recurrence of hepatocellular carcinoma (HCC) and poor survival of HCC patients. Here we showed that miR-100 downregulation in HCC tissues was significantly associated with venous invasion, advanced TNM stage, tumor nodule without complete capsule, poorer cell differentiation, and shorter recurrence-free survival. Both gain- and loss-of-function studies showed that miR-100 dramatically suppressed the ability of HCC cells to migrate and to invade through Matrigel in vitro. Analyses using mouse orthotopic xenograft model further revealed that xenografts of miR-100-stable-expressing HCC cells displayed a significant reduction in pulmonary metastasis, compared with control group. Subsequent investigations revealed that miR-100 directly inhibited the expression of isoprenylcysteine carboxyl methyltransferase (ICMT) and ras-related C3 botulinum toxin substrate 1 (Rac1) by binding to their 3'-UTRs, and in turn suppressed lamellipodia formation and matrix metallopeptidase 2 (MMP2) activation. Furthermore, knockdown of ICMT and Rac1 phenocopied the anti-metastasis effect of miR-100, whereas overexpression of the constitutively active Rac1 (Q61L) antagonized the function of miR-100. Taken together, miR-100 represses metastasis of HCC cells by abrogating the ICMT-Rac1 signaling. Downregulation of miR-100 contributes to HCC metastasis and the restoration of miR-100 is a potential strategy for cancer therapy. PMID:25361001

  14. MST3 promotes proliferation and tumorigenicity through the VAV2/Rac1 signal axis in breast cancer

    PubMed Central

    Cho, Chien-Yu; Lee, Kuo-Ting; Chen, Wei-Ching; Wang, Chih-Yang; Chang, Yung-Sheng; Huang, Hau-Lun; Hsu, Hui-Ping; Yen, Meng-Chi; Lai, Ming-Zong; Lai, Ming-Derg

    2016-01-01

    MST3 (mammalian STE20-like kinase 3) belongs to the Ste20 serine/threonine protein kinase family. The role of MST3 in tumor growth is less studied; therefore, we investigates the function of MST3 in breast cancer. Here, we demonstrate that MST3 is overexpressed in human breast tumors. Online Kaplan-Meier plotter analysis reveals that overexpression of MST3 predicts poor prognosis in breast cancer patients. Knockdown of MST3 with shRNA inhibits proliferation and anchorage-independent growth in vitro. Downregulation of MST3 in triple-negative MDA-MB-231 and MDA-MB-468 breast cancer cells decreases tumor formation in NOD/SCID mice. MST3 interacts with VAV2, but not VAV3, as demonstrated by co-immunoprecipitation and confocal microscopy. By domain mapping of MST3, we determine that the proline-rich region of MST3 (353KDIPKRP359) interacts with the SH3 domain of VAV2. Mutation of the two proline residues in this domain significantly attenuates the interaction between MST3 and VAV2. Overexpression of wild-type MST3 (WT-MST3), but not proline-rich-deleted MST3 (ΔP-MST3), enhances the proliferation rate and anchorage-independent growth of MDA-MB-468 cells. Overexpression of MST3 increases VAV2 phosphorylation and GTP-Rac1, whereas downregulation of MST3 or delivery of ΔP-MST3 results in a reduction of VAV2 and Rac1 activation. Knockdown of MST3 inhibits cyclin D1 protein expression. The Rac1 inhibitor EHop-016 attenuates cell proliferation induced by WT-MST3. Finally, Knockdown of MST3 or Rac1 inhibitor decreases cyclin D protein expression, which is important for tumor growth. These results indicate that MST3 interacts with VAV2 to activate Rac1 and promote the tumorigenicity of breast cancer. PMID:26910843

  15. Scrib:Rac1 interactions are required for the morphogenesis of the ventricular myocardium

    PubMed Central

    Boczonadi, Veronika; Gillespie, Rachel; Keenan, Iain; Ramsbottom, Simon A.; Donald-Wilson, Charlotte; Al Nazer, Mariana; Humbert, Patrick; Schwarz, Robert J.; Chaudhry, Bill; Henderson, Deborah J.

    2014-01-01

    Aims The organization and maturation of ventricular cardiomyocytes from the embryonic to the adult form is crucial for normal cardiac function. We have shown that a polarity protein, Scrib, may be involved in regulating the early stages of this process. Our goal was to establish whether Scrib plays a cell autonomous role in the ventricular myocardium, and whether this involves well-known polarity pathways. Methods and results Deletion of Scrib in cardiac precursors utilizing Scribflox mice together with the Nkx2.5-Cre driver resulted in disruption of the cytoarchitecture of the forming trabeculae and ventricular septal defects. Although the majority of mice lacking Scrib in the myocardium survived to adulthood, they developed marked cardiac fibrosis. Scrib did not physically interact with the planar cell polarity (PCP) protein, Vangl2, in early cardiomyocytes as it does in other tissues, suggesting that the anomalies did not result from disruption of PCP signalling. However, Scrib interacted with Rac1 physically in embryonic cardiomyocytes and genetically to result in ventricular abnormalities, suggesting that this interaction is crucial for the development of the early myocardium. Conclusions The Scrib–Rac1 interaction plays a crucial role in the organization of developing cardiomyocytes and formation of the ventricular myocardium. Thus, we have identified a novel signalling pathway in the early, functioning, heart muscle. These data also show that the foetus can recover from relatively severe abnormalities in prenatal ventricular development, although cardiac fibrosis can be a long-term consequence. PMID:25139745

  16. Ofloxacin induces apoptosis via β1 integrin-EGFR-Rac1-Nox2 pathway in microencapsulated chondrocytes

    SciTech Connect

    Sheng, Zhi-Guo; Huang, Wei; Liu, Yu-Xiang; Yuan, Ye; Zhu, Ben-Zhan

    2013-02-15

    Quinolones (QNs)-induced arthropathy is an important toxic side-effect in immature animals leading to the restriction of their therapeutic use in pediatrics. Ofloxacin, a typical QN, was found to induce the chondrocytes apoptosis in the early phase (12–48 h) of arthropathy in our previous study. However, the exact mechanism(s) is unclear. Microencapsulated juvenile rabbit joint chondrocytes, a three-dimensional culture system, is utilized to perform the present study. Ofloxacin, at a therapeutically relevant concentration (10 μg/ml), disturbs the interaction between β1 integrin and activated intracellular signaling proteins at 12 h, which is inhibited when supplementing Mg{sup 2+}. Intracellular reactive oxygen species (ROS) significantly increases in a time-dependent manner after exposure to ofloxacin for 12–48 h. Furthermore, ofloxacin markedly enhances the level of activated Rac1 and epidermal growth factor receptor (EGFR) phosphorylation, and its inhibition in turn reduces the ROS production, apoptosis and Rac1 activation. Silencing Nox2, Rac1 or supplementing Mg{sup 2+} inhibits ROS accumulation, apoptosis occurrence and EGFR phosphorylation induced by ofloxacin. However, depletion of Nox2, Rac1 and inhibition of EGFR do not affect ofloxacin-mediated loss of interaction between β1 integrin and activated intracellular signaling proteins. In addition, ofloxacin also induces Vav2 phosphorylation, which is markedly suppressed after inactivating EGFR or supplementing Mg{sup 2+}. These results suggest that ofloxacin causes Nox2-mediated intracellular ROS production by disrupting the β1 integrin function and then activating the EGFR-Vav2-Rac1 pathway, finally resulting in apoptosis within 12–48 h exposure. The present study provides a novel insight regarding the potential role of Nox-driven ROS in QNs-induced arthropathy. - Highlights: ► Ofloxacin induces Nox2-driven ROS in encapsulated chondrocyte at 12–48 h. ► Ofloxacin stimulates ROS production via

  17. Lck/PLCγ control migration and proliferation of interleukin (IL)-2-stimulated T cells via the Rac1 GTPase/glycogen phosphorylase pathway.

    PubMed

    Llavero, Francisco; Artaso, Alain; Lacerda, Hadriano M; Parada, Luis A; Zugaza, José L

    2016-11-01

    Recently, we have reported that the IL-2-stimulated T cells activate PKCθ in order to phosphorylate the serine residues of αPIX-RhoGEF, and to switch on the Rac1/PYGM pathway resulting in T cell migration and proliferation. However, the molecular mechanism connecting the activated IL-2-R with the PKCθ/αPIX/Rac1/PYGM pathway is still unknown. In this study, the use of a combined pharmacological and genetic approach identified Lck, a Src family member, as the tyrosine kinase phosphorylating PLCγ leading to Rac1 and PYGM activation in the IL-2-stimulated Kit 225 T cells via the PKCθ/αPIX pathway. The PLCγ tyrosine phosphorylation was required to activate first PKCθ, and then αPIX and Rac1/PYGM. The results presented here delineate a novel signalling pathway ranking equally in importance to the three major pathways controlled by the IL-2-R, i.e. PI3K, Ras/MAPK and JAK/STAT pathways. The overall evidence strongly indicates that the central biological role of the novel IL-2-R/Lck/PLCγ/PKCθ/αPIX/Rac1/PYGM signalling pathway is directly related to the control of fundamental cellular processes such as T cell migration and proliferation. PMID:27519475

  18. Epidermal growth factor attenuates blood-spinal cord barrier disruption via PI3K/Akt/Rac1 pathway after acute spinal cord injury.

    PubMed

    Zheng, Binbin; Ye, Libing; Zhou, Yulong; Zhu, Sipin; Wang, Qingqing; Shi, Hongxue; Chen, Daqing; Wei, Xiaojie; Wang, Zhouguang; Li, Xiaokun; Xiao, Jian; Xu, Huazi; Zhang, Hongyu

    2016-06-01

    After spinal cord injury (SCI), disruption of blood-spinal cord barrier (BSCB) elicits blood cell infiltration such as neutrophils and macrophages, contributing to permanent neurological disability. Previous studies show that epidermal growth factor (EGF) produces potent neuroprotective effects in SCI models. However, little is known that whether EGF contributes to the integrity of BSCB. The present study is performed to explore the mechanism of BSCB permeability changes which are induced by EGF treatment after SCI in rats. In this study, we demonstrate that EGF administration inhibits the disruption of BSCB permeability and improves the locomotor activity in SCI model rats. Inhibition of the PI3K/Akt pathways by a specific inhibitor, LY294002, suppresses EGF-induced Rac1 activation as well as tight junction (TJ) and adherens junction (AJ) expression. Furthermore, the protective effect of EGF on BSCB is related to the activation of Rac1 both in vivo and in vitro. Blockade of Rac1 activation with Rac1 siRNA downregulates EGF-induced TJ and AJ proteins expression in endothelial cells. Taken together, our results indicate that EGF treatment preserves BSCB integrity and improves functional recovery after SCI via PI3K-Akt-Rac1 signalling pathway. PMID:26769343

  19. Roles of Rac1 and Rac3 GTPases during the development of cortical and hippocampal GABAergic interneurons

    PubMed Central

    de Curtis, Ivan

    2014-01-01

    Rac GTPases are regulators of the cytoskeleton that play an important role in several aspects of neuronal and brain development. Two distinct Rac GTPases are expressed in the developing nervous system, the widely expressed Rac1 and the neural-specific Rac3 proteins. Recent experimental evidence supports a central role of these two Rac proteins in the development of inhibitory GABAergic interneurons, important modulatory elements of the brain circuitry. The combined inactivation of the genes for the two Rac proteins has profound effects on distinct aspects of interneuron development, and has highlighted a synergistic contribution of the two proteins to the postmitotic maturation of specific populations of cortical and hippocampal interneurons. Rac function is modulated by different types of regulators, and can influence the activity of specific effectors. Some of these proteins have been associated to the development and maturation of interneurons. Cortical interneuron dysfunction is implicated in several neurological and psychiatric diseases characterized by cognitive impairment. Therefore the description of the cellular processes regulated by the Rac GTPases, and the identification of the molecular networks underlying these processes during interneuron development is relevant to the understanding of the role of GABAergic interneurons in cognitive functions. PMID:25309333

  20. Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas.

    PubMed

    Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

    2016-01-01

    RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

  1. Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas

    PubMed Central

    Faria, Márcia; Capinha, Liliana; Simões-Pereira, Joana; Bugalho, Maria João; Silva, Ana Luísa

    2016-01-01

    RAC1b is a hyperactive variant of the small GTPase RAC1 known to be a relevant molecular player in different cancers. Previous studies from our group lead to the evidence that its overexpression in papillary thyroid carcinoma (PTC) is associated with an unfavorable prognosis. In the present study, we intended to extend the analysis of RAC1b expression to thyroid follicular neoplasms and to seek for clinical correlations. RAC1b expression levels were determined by RT-qPCR in thyroid follicular tumor samples comprising 23 follicular thyroid carcinomas (FTCs) and 33 follicular thyroid adenomas (FTAs). RAC1b was found to be overexpressed in 33% of carcinomas while no RAC1b overexpression was documented among follicular adenomas. Patients with a diagnosis of FTC were divided into two groups based on longitudinal evolution and final outcome. RAC1b overexpression was significantly associated with both the presence of distant metastases (P = 0.01) and poorer clinical outcome (P = 0.01) suggesting that, similarly to that previously found in PTCs, RAC1b overexpression in FTCs is also associated with worse outcomes. Furthermore, the absence of RAC1b overexpression in follicular adenomas hints its potential as a molecular marker likely to contribute, in conjunction with other putative markers, to the preoperative differential diagnosis of thyroid follicular lesions. PMID:27127508

  2. Rac1 signaling in the establishment of the fucoid algal body plan.

    PubMed

    Hable, Whitney E

    2014-01-01

    Fucoid zygotes use environmental vectors, including sunlight, to initiate a growth axis a few hours after fertilization. The first division is then transversely oriented by the growth axis, producing daughter cells of distinct fates. The tip growing rhizoid cell gives rise to the holdfast, anchoring the alga to the intertidal substratum, while the opposite thallus cell mainly generates the photosynthetic and reproductive stipe and fronds. Elaboration of this simple growth axis thus establishes the basic body plan of the adult; and elucidating the mechanisms responsible for formation of the growth axis is paramount to understanding fucoid morphogenesis. Recent studies have culminated in a model whereby sunlight, and perhaps other environmental cues, activate the signaling protein Rac1 at the rhizoid pole. Here it sets in motion nucleation of a patch of actin filaments that in turn, targets ions, proteins, and cellular processes to the future growth site. At germination, Rac1 initiates morphogenesis by inducing transformation of the patch of actin filaments to a structure that delivers vesicles to the growing tip, and a few hours later orients the spindle and cytokinetic plate. PMID:25540648

  3. Rac1 and ROCK are implicated in the cell surface delivery of GLUT4 under the control of the insulin signal mimetic diDCP-LA-PE.

    PubMed

    Tsuchiya, Ayako; Kanno, Takeshi; Shimizu, Tadashi; Tanaka, Akito; Nishizaki, Tomoyuki

    2015-08-01

    The phosphatidylethanolamine derivative 1,2-O-bis-[8-{2-(2-pentyl-cyclopropylmethyl)-cyclopropyl}-octanoyl]-sn-glycero-3-phosphatidylethanolamine (diDCP-LA-PE) promoted GLUT4 translocation to the cell surface in differentiated 3T3-L1-GLUT4myc adipocytes through a pathway along a phosphatidylinositol 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt axis, that mimics insulin signaling. Moreover, diDCP-LA-PE-induced GLUT4 translocation was suppressed by inhibitors of the Rho GTPase Rac1 and Rho-associated coiled-coil-containing protein kinase (ROCK) or knocking-down Rac1 and ROCK1. The results of the present study show that Rac1 and ROCK are critical for regulation of GLUT4 trafficking by diDCP-LA-PE as well as insulin. PMID:26238253

  4. IRGM1 enhances B16 melanoma cell metastasis through PI3K-Rac1 mediated epithelial mesenchymal transition

    PubMed Central

    Tian, Linlu; Li, Lixian; Xing, Wenjing; Li, Rui; Pei, Chunying; Dong, Xiao; Fu, Yanran; Gu, Changcong; Guo, Xize; Jia, Yulong; Wang, Guangyou; Wang, Jinghua; Li, Bo; Ren, Huan; Xu, Hongwei

    2015-01-01

    Melanoma is one of the most aggressive skin cancers and is well known for its high metastatic rate. Studies have shown that epithelial mesenchymal transition (EMT) is essential for melanoma cell metastasis. However, the molecular mechanisms underlying EMT are still not fully understood. We have shown that IRGM1, a member of immunity-related GTPase family that regulates immune cell motility, is highly expressed by melanoma cells. The current study aimed to explore whether and how IRGM1 may regulate melanoma cell metastasis. To test this, we modified IRGM1 expression in B16 melanoma cells. We found that over-expression of IRGM1 substantially enhanced pulmonary metastasis in vivo. In keeping with that, knocking-in IRGM1 strongly enhanced while knocking-down IRGM1 impaired B16 cell migration and invasion ability in vitro. Interestingly, we observed that IRGM1 enhanced F-actin polymerization and triggers epithelial mesenchymal transition (EMT) through a mechanism involved in PIK3CA mediated Rac1 activation. Together, these data reveals a novel molecular mechanism that involved in melanoma metastasis. PMID:26202910

  5. CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways.

    PubMed

    Lin, Chien-Huang; Shih, Chung-Huang; Tseng, Chih-Chieh; Yu, Chung-Chi; Tsai, Yuan-Jhih; Bien, Mauo-Ying; Chen, Bing-Chang

    2014-01-01

    CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression. PMID:25121739

  6. Heterotypic RPE-choroidal endothelial cell contact increases choroidal endothelial cell transmigration via PI 3-kinase and Rac1.

    PubMed

    Peterson, Lynda J; Wittchen, Erika S; Geisen, Pete; Burridge, Keith; Hartnett, M Elizabeth

    2007-04-01

    Age-related macular degeneration (AMD) is the major cause of non-preventable blindness. Severe forms of AMD involve breaching of the retinal pigment epithelial (RPE) barrier by underlying choroidal endothelial cells (CECs), followed by migration into, and subsequent neovascularization of the neurosensory retina. However, little is known about the interactions between RPE and CECs and the signaling events leading to CEC transmigration. While soluble chemotactic factors secreted from RPE can contribute to inappropriate CEC transmigration, other unidentified stimuli may play an additional role. Using a coculture model that maintains the natural structural orientation of CECs to the basal aspect of RPE, we show that "contact" with RPE and/or RPE extracellular matrix increases CEC transmigration of the RPE barrier. From a biochemical standpoint, contact between CECs and RPE results in an increase in the activity of the GTPase Rac1 within the CECs; this increase is dependent on upstream activation of PI 3-K and Akt1. To confirm a link between these signaling molecules and increased CEC transmigration, we performed transmigration assays while inhibiting both PI 3-K and Rac1 activity, and observed that both decreased CEC transmigration. We hypothesize that contact between CECs and RPE stimulates a signaling pathway involving PI 3-K, Akt1, and Rac1 that facilitates CEC transmigration across the RPE barrier, an important step in the development of neovascular AMD. PMID:17292356

  7. Heterotypic RPE-choroidal endothelial cell contact increases choroidal endothelial cell transmigration via PI 3-kinase and Rac1

    PubMed Central

    Peterson, Lynda J.; Wittchen, Erika S.; Geisen, Pete; Burridge, Keith; Hartnett, M. Elizabeth

    2008-01-01

    Age-related macular degeneration (AMD) is the major cause of non-preventable blindness. Severe forms of AMD involve breaching of the retinal pigment epithelial (RPE) barrier by underlying choroidal endothelial cells (CECs), followed by migration into, and subsequent neovascularization of the neurosensory retina. However, little is known about the interactions between RPE and CECs and the signaling events leading to CEC transmigration. While soluble chemotactic factors secreted from RPE can contribute to inappropriate CEC transmigration, other unidentified stimuli may play an additional role. Using a coculture model that maintains the natural structural orientation of CECs to the basal aspect of RPE, we show that “contact” with RPE and/or RPE extracellular matrix increases CEC transmigration of the RPE barrier. From a biochemical standpoint, contact between CECs and RPE results in an increase in the activity of the GTPase Rac1 within the CECs; this increase is dependent on upstream activation of PI 3-K and Akt1. To confirm a link between these signaling molecules and increased CEC transmigration, we performed transmigration assays while inhibiting both PI 3-K and Rac1 activity, and observed that both decreased CEC transmigration. We hypothesize that contact between CECs and RPE stimulates a signaling pathway involving PI 3-K, Akt1, and Rac1 that facilitates CEC transmigration across the RPE barrier, an important step in the development of neovascular AMD. PMID:17292356

  8. The Rac-GAP alpha2-chimaerin regulates hippocampal dendrite and spine morphogenesis.

    PubMed

    Valdez, Chris M; Murphy, Geoffrey G; Beg, Asim A

    2016-09-01

    Dendritic spines are fine neuronal processes where spatially restricted input can induce activity-dependent changes in one spine, while leaving neighboring spines unmodified. Morphological spine plasticity is critical for synaptic transmission and is thought to underlie processes like learning and memory. Significantly, defects in dendritic spine stability and morphology are common pathogenic features found in several neurodevelopmental and neuropsychiatric disorders. The remodeling of spines relies on proteins that modulate the underlying cytoskeleton, which is primarily composed of filamentous (F)-actin. The Rho-GTPase Rac1 is a major regulator of F-actin and is essential for the development and plasticity of dendrites and spines. However, the key molecules and mechanisms that regulate Rac1-dependent pathways at spines and synapses are not well understood. We have identified the Rac1-GTPase activating protein, α2-chimaerin, as a critical negative regulator of Rac1 in hippocampal neurons. The loss of α2-chimaerin significantly increases the levels of active Rac1 and induces the formation of aberrant polymorphic dendritic spines. Further, disruption of α2-chimaerin signaling simplifies dendritic arbor complexity and increases the presence of dendritic spines that appear poly-innervated. Our data suggests that α2-chimaerin serves as a "brake" to constrain Rac1-dependent signaling to ensure that the mature morphology of spines is maintained in response to network activity. PMID:27297944

  9. Nuclear expression of Rac1 in cervical premalignant lesions and cervical cancer cells

    PubMed Central

    2012-01-01

    Background Abnormal expression of Rho-GTPases has been reported in several human cancers. However, the expression of these proteins in cervical cancer has been poorly investigated. In this study we analyzed the expression of the GTPases Rac1, RhoA, Cdc42, and the Rho-GEFs, Tiam1 and beta-Pix, in cervical pre-malignant lesions and cervical cancer cell lines. Methods Protein expression was analyzed by immunochemistry on 102 cervical paraffin-embedded biopsies: 20 without Squamous Intraepithelial Lesions (SIL), 51 Low- grade SIL, and 31 High-grade SIL; and in cervical cancer cell lines C33A and SiHa, and non-tumorigenic HaCat cells. Nuclear localization of Rac1 in HaCat, C33A and SiHa cells was assessed by cellular fractionation and Western blotting, in the presence or not of a chemical Rac1 inhibitor (NSC23766). Results Immunoreacivity for Rac1, RhoA, Tiam1 and beta-Pix was stronger in L-SIL and H-SIL, compared to samples without SIL, and it was significantly associated with the histological diagnosis. Nuclear expression of Rac1 was observed in 52.9% L-SIL and 48.4% H-SIL, but not in samples without SIL. Rac1 was found in the nucleus of C33A and SiHa cells but not in HaCat cells. Chemical inhibition of Rac1 resulted in reduced cell proliferation in HaCat, C33A and SiHa cells. Conclusion Rac1 is expressed in the nucleus of epithelial cells in SILs and cervical cancer cell lines, and chemical inhibition of Rac1 reduces cellular proliferation. Further studies are needed to better understand the role of Rho-GTPases in cervical cancer progression. PMID:22443139

  10. Single Nucleotide Polymorphisms that Increase Expression of the GTPase RAC1 are Associated with Ulcerative Colitis

    PubMed Central

    Muise, Aleixo M; Walters, Thomas; Xu, Wei; Shen-Tu, Grace; Guo, Cong-Hui; Fattouh, Ramzi; Lam, Grace Y; Wolters, Victorien M; Bennitz, Joshua; Van Limbergen, Johan; Renbaum, Paul; Kasirer, Yair; Ngan, Bo-Yee; Turner, Dan; Denson, Lee A; Sherman, Philip M; Duerr, Richard H; Cho, Judy; Lees, Charlie W; Satsangi, Jack; Wilson, David C; Paterson, Andrew D; Griffiths, Anne M; Glogauer, Michael; Silverberg, Mark S; Brumell, John H

    2011-01-01

    Background & Aims RAC1 is a GTPase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases (IBD) are associated with dysregulation of immune defenses. We studied the role of RAC1 in IBD using human genetic and functional studies and animal models of colitis. Methods We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms (SNPs) in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 mRNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulphate sodium (DSS). Results We observed a genetic association between RAC1 with ulcerative colitis (UC) in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the SNPs rs10951982 (Pcombined UC = 3.3 × 10–8, odds ratio [OR]=1.43 [1.26–1.63]) and rs4720672 (Pcombined UC=4.7 × 10–6, OR=1.36 [1.19–1.58]). Patients with IBD who had the rs10951982 risk allele had increased expression of RAC1, compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected them against DSS-induced colitis. Conclusion Studies of human tissue samples and knockout mice demonstrated a role for the GTPase RAC1 in the development of UC; increased expression of RAC1 was associated with susceptibility to colitis. PMID:21684284

  11. Rac1 controls epithelial tube length through the apical secretion and polarity pathways

    PubMed Central

    Sollier, Kévin; Gaudé, Helori-Mael; Chartier, François J.-M.; Laprise, Patrick

    2016-01-01

    ABSTRACT The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis. PMID:26700724

  12. Rac1 controls epithelial tube length through the apical secretion and polarity pathways.

    PubMed

    Sollier, Kévin; Gaudé, Helori-Mael; Chartier, François J-M; Laprise, Patrick

    2015-01-01

    The morphometric parameters of epithelial tubes are critical to the physiology and homeostasis of most organs. In addition, many human diseases are associated with tube-size defects. Here, we show that Rac1 limits epithelial tube elongation in the developing fly trachea by promoting Rab5-dependent endocytosis of the apical determinant Crumbs. Rac1 is also involved in a positive feedback loop with the septate junction protein Coracle. Thereby, Rac1 precludes paracellular diffusion and contributes to the septate junction-dependent secretion of the chitin-modifying enzymes Vermiform and Serpentine, which restrict epithelial tube length independently of Crumbs. Thus, Rac1 is a critical component of two important pathways controlling epithelial tube morphogenesis. PMID:26700724

  13. Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

    PubMed

    Guignandon, Alain; Faure, Céline; Neutelings, Thibaut; Rattner, Aline; Mineur, Pierre; Linossier, Marie-Thérèse; Laroche, Norbert; Lambert, Charles; Deroanne, Christophe; Nusgens, Betty; Demets, René; Colige, Alain; Vico, Laurence

    2014-09-01

    Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects. PMID:24903274

  14. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

    SciTech Connect

    Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  15. Local extracellular matrix alignment directs cellular protrusion dynamics and migration through Rac1 and FAK.

    PubMed

    Carey, Shawn P; Goldblatt, Zachary E; Martin, Karen E; Romero, Bethsabe; Williams, Rebecca M; Reinhart-King, Cynthia A

    2016-08-01

    Cell migration within 3D interstitial microenvironments is sensitive to extracellular matrix (ECM) properties, but the mechanisms that regulate migration guidance by 3D matrix features remain unclear. To examine the mechanisms underlying the cell migration response to aligned ECM, which is prevalent at the tumor-stroma interface, we utilized time-lapse microscopy to compare the behavior of MDA-MB-231 breast adenocarcinoma cells within randomly organized and well-aligned 3D collagen ECM. We developed a novel experimental system in which cellular morphodynamics during initial 3D cell spreading served as a reductionist model for the complex process of matrix-directed 3D cell migration. Using this approach, we found that ECM alignment induced spatial anisotropy of cells' matrix probing by promoting protrusion frequency, persistence, and lengthening along the alignment axis and suppressing protrusion dynamics orthogonal to alignment. Preference for on-axis behaviors was dependent upon FAK and Rac1 signaling and translated across length and time scales such that cells within aligned ECM exhibited accelerated elongation, front-rear polarization, and migration relative to cells in random ECM. Together, these findings indicate that adhesive and protrusive signaling allow cells to respond to coordinated physical cues in the ECM, promoting migration efficiency and cell migration guidance by 3D matrix structure. PMID:27384462

  16. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ

    PubMed Central

    Pomoransky, Julia L.; Parks, Robin J.; Trinkle-Mulcahy, Laura; Bell, John C.; Gee, Stephen H.

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis. PMID:26701304

  17. ELMO1 and Dock180, a bipartite Rac1 guanine nucleotide exchange factor, promote human glioma cell invasion.

    PubMed

    Jarzynka, Michael J; Hu, Bo; Hui, Kwok-Min; Bar-Joseph, Ifat; Gu, Weisong; Hirose, Takanori; Haney, Lisa B; Ravichandran, Kodi S; Nishikawa, Ryo; Cheng, Shi-Yuan

    2007-08-01

    A distinct feature of malignant gliomas is the intrinsic ability of single tumor cells to disperse throughout the brain, contributing to the failure of existing therapies to alter the progression and recurrence of these deadly brain tumors. Regrettably, the mechanisms underlying the inherent invasiveness of glioma cells are poorly understood. Here, we report for the first time that engulfment and cell motility 1 (ELMO1) and dedicator of cytokinesis 1 (Dock180), a bipartite Rac1 guanine nucleotide exchange factor (GEF), are evidently linked to the invasive phenotype of glioma cells. Immunohistochemical analysis of primary human glioma specimens showed high expression levels of ELMO1 and Dock180 in actively invading tumor cells in the invasive areas, but not in the central regions of these tumors. Elevated expression of ELMO1 and Dock180 was also found in various human glioma cell lines compared with normal human astrocytes. Inhibition of endogenous ELMO1 and Dock180 expression significantly impeded glioma cell invasion in vitro and in brain tissue slices with a concomitant reduction in Rac1 activation. Conversely, exogenous expression of ELMO1 and Dock180 in glioma cells with low level endogenous expression increased their migratory and invasive capacity in vitro and in brain tissue. These data suggest that the bipartite GEF, ELMO1 and Dock180, play an important role in promoting cancer cell invasion and could be potential therapeutic targets for the treatment of diffuse malignant gliomas. PMID:17671188

  18. TGF-{beta}1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-{kappa}B/IL-6/MMP-2

    SciTech Connect

    Binker, Marcelo G.; Binker-Cosen, Andres A.; Gaisano, Herbert Y.; Cosen, Rodica H. de; Cosen-Binker, Laura I.

    2011-02-04

    Research highlights: {yields} Rac1 mediates TGF-{beta}1-induced SW1990 invasion through MMP-2 secretion and activation. {yields} NADPH-generated ROS act downstream of Rac1 in TGF-{beta}1-challenged SW1990 cells. {yields} TGF-{beta}1-stimulated ROS activate NF-{kappa}B in SW1990 cells. {yields} NF{kappa}B-induced IL-6 release is required for secretion and activation of MMP-2 in SW1990 cells. -- Abstract: Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-{beta}1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-{beta}1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-{beta}1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-{beta}1-stimulated invasion. Our results also indicate that signaling events involved in TGF-{beta}1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.

  19. miR-101 inhibits cell proliferation by targeting Rac1 in papillary thyroid carcinoma

    PubMed Central

    LIN, XIAOJIE; GUAN, HONGYU; LI, HAI; LIU, LIEHUA; LIU, JUAN; WEI, GUOHONG; HUANG, ZHIMIN; LIAO, ZHIHONG; LI, YANBING

    2014-01-01

    Accumulating evidence suggests that some microRNAs (miRNAs) are involved in papillary thyroid carcinoma (PTC) progression. However, it remains necessary to elucidate the underlying molecular mechanisms involved. In the present study, we investigated the role of microRNA-101 (miR-101) in PTC via targeting of Ras-related C3 botulinum toxin substrate 1 (Rac1). The results showed that miR-101 was significantly downregulated in PTC tissues compared with adjacent normal tissues. Restoration of miR-101 expression significantly inhibited cell proliferation in the K1 PTC cell line. Moreover, algorithm-based and experimental strategies verified Rac1 as a direct target of miR-101 in the K1 cell line. Taken together, these findings suggest that miR-101 inhibited PTC growth via the downregulation of Rac1 expression, providing a better understanding of miRNA-modulated signaling networks for future cancer therapeutics. PMID:24649082

  20. Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1

    PubMed Central

    DeGeer, Jonathan; Kaplan, Andrew; Mattar, Pierre; Morabito, Morgane; Stochaj, Ursula; Kennedy, Timothy E.; Debant, Anne; Cayouette, Michel; Fournier, Alyson E.

    2015-01-01

    During development, netrin-1 is both an attractive and repulsive axon guidance cue and mediates its attractive function through the receptor Deleted in Colorectal Cancer (DCC). The activation of Rho guanosine triphosphatases within the extending growth cone facilitates the dynamic reorganization of the cytoskeleton required to drive axon extension. The Rac1 guanine nucleotide exchange factor (GEF) Trio is essential for netrin-1–induced axon outgrowth and guidance. Here, we identify the molecular chaperone heat shock cognate protein 70 (Hsc70) as a novel Trio regulator. Hsc70 dynamically associated with the N-terminal region and Rac1 GEF domain of Trio. Whereas Hsc70 expression supported Trio-dependent Rac1 activation, adenosine triphosphatase–deficient Hsc70 (D10N) abrogated Trio Rac1 GEF activity and netrin-1–induced Rac1 activation. Hsc70 was required for netrin-1–mediated axon growth and attraction in vitro, whereas Hsc70 activity supported callosal projections and radial neuronal migration in the embryonic neocortex. These findings demonstrate that Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance. PMID:26323693

  1. The PERK pathway independently triggers apoptosis and a Rac1/Slpr/JNK/Dilp8 signaling favoring tissue homeostasis in a chronic ER stress Drosophila model

    PubMed Central

    Demay, Y; Perochon, J; Szuplewski, S; Mignotte, B; Gaumer, S

    2014-01-01

    The endoplasmic reticulum (ER) has a major role in protein folding. The accumulation of unfolded proteins in the ER induces a stress, which can be resolved by the unfolded protein response (UPR). Chronicity of ER stress leads to UPR-induced apoptosis and in turn to an unbalance of tissue homeostasis. Although ER stress-dependent apoptosis is observed in a great number of devastating human diseases, how cells activate apoptosis and promote tissue homeostasis after chronic ER stress remains poorly understood. Here, using the Drosophila wing imaginal disc as a model system, we validated that Presenilin overexpression induces chronic ER stress in vivo. We observed, in this novel model of chronic ER-stress, a PERK/ATF4-dependent apoptosis requiring downregulation of the antiapoptotic diap1 gene. PERK/ATF4 also activated the JNK pathway through Rac1 and Slpr activation in apoptotic cells, leading to the expression of Dilp8. This insulin-like peptide caused a developmental delay, which partially allowed the replacement of apoptotic cells. Thanks to a novel chronic ER stress model, these results establish a new pathway that both participates in tissue homeostasis and triggers apoptosis through an original regulation. PMID:25299777

  2. p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing

    PubMed Central

    Posada-Duque, Rafael Andres; López-Tobón, Alejandro; Piedrahita, Diego; González-Billault, Christian; Cardona-Gomez, Gloria Patricia

    2015-01-01

    CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles and prevented neuronal loss in triple transgenic Alzheimer’s mice. Here, we report that CDK5 RNAi protected against glutamate-mediated excitotoxicity using primary hippocampal neurons transduced with AAV2.5 viral vector eGFP-tagged SCR or CDK5 shRNA-miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down-stream Rho GTPase activity. Furthermore, p35 overexpression and constitutively active Rac1 mimicked CDK5 silencing-induced neuroprotection. In addition, 3xTg-AD mice (24 months old) were injected in the hippocampus with SCR or CDK5 shRNA-miR, and spatial learning and memory were performed three weeks post injection using “Morris” water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer’s mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi. PMID:25864429

  3. p35 and Rac1 underlie the neuroprotection and cognitive improvement induced by CDK5 silencing.

    PubMed

    Posada-Duque, Rafael Andres; López-Tobón, Alejandro; Piedrahita, Diego; González-Billault, Christian; Cardona-Gomez, Gloria Patricia

    2015-07-01

    CDK5 plays an important role in neurotransmission and synaptic plasticity in the normal function of the adult brain, and dysregulation can lead to Tau hyperphosphorylation and cognitive impairment. In a previous study, we demonstrated that RNAi knock down of CDK5 reduced the formation of neurofibrillary tangles (NFT) and prevented neuronal loss in triple transgenic Alzheimer's mice. Here, we report that CDK5 RNAi protected against glutamate-mediated excitotoxicity using primary hippocampal neurons transduced with adeno-associated virus 2.5 viral vector eGFP-tagged scrambled or CDK5 shRNA-miR during 12 days. Protection was dependent on a concomitant increase in p35 and was reversed using p35 RNAi, which affected the down-stream Rho GTPase activity. Furthermore, p35 over-expression and constitutively active Rac1 mimicked CDK5 silencing-induced neuroprotection. In addition, 3xTg-Alzheimer's disease mice (24 months old) were injected in the hippocampus with scrambled or CDK5 shRNA-miR, and spatial learning and memory were performed 3 weeks post-injection using 'Morris' water maze test. Our data showed that CDK5 knock down induced an increase in p35 protein levels and Rac activity in triple transgenic Alzheimer's mice, which correlated with the recovery of cognitive function; these findings confirm that increased p35 and active Rac are involved in neuroprotection. In summary, our data suggest that p35 acts as a mediator of Rho GTPase activity and contributes to the neuroprotection induced by CDK5 RNAi. PMID:25864429

  4. P21-activated kinase 2 (PAK2) regulates glucose uptake and insulin sensitivity in neuronal cells.

    PubMed

    Varshney, Pallavi; Dey, Chinmoy Sankar

    2016-07-01

    P21-activated kinases (PAKs) are recently reported as important players of insulin signaling and glucose homeostasis in tissues like muscle, pancreas and liver. However, their role in neuronal insulin signaling is still unknown. Present study reports the involvement of PAK2 in neuronal insulin signaling, glucose uptake and insulin resistance. Irrespective of insulin sensitivity, insulin stimulation decreased PAK2 activity. PAK2 downregulation displayed marked enhancement of GLUT4 translocation with increase in glucose uptake whereas PAK2 over-expression showed its reduction. Treatment with Akti-1/2 and wortmannin suggested that Akt and PI3K are mediators of insulin effect on PAK2 and glucose uptake. Rac1 inhibition demonstrated decreased PAK2 activity while inhibition of PP2A resulted in increased PAK2 activity, with corresponding changes in glucose uptake. Taken together, present study demonstrates an inhibitory role of insulin signaling (via PI3K-Akt) and PP2A on PAK2 activity and establishes PAK2 as a Rac1-dependent negative regulator of neuronal glucose uptake and insulin sensitivity. PMID:27040307

  5. Oncogenic epithelial cell-derived exosomes containing Rac1 and PAK2 induce angiogenesis in recipient endothelial cells

    PubMed Central

    Gopal, Shashi K.; Greening, David W.; Hanssen, Eric G.; Zhu, Hong-Jian; Simpson, Richard J.; Mathias, Rommel A.

    2016-01-01

    The metastatic cascade describes the escape of primary tumour cells to distant secondary sites. Cells at the leading tumour edge are thought to undergo epithelial-mesenchymal transition (EMT), to enhance their motility and invasion for spreading. Whether EMT cells directly promote tumour angiogenesis, and the role of exosomes (30-150 nm extracellular vesicles) remains largely unknown. We examined the functional effects of exosomes from MDCK cells, MDCK cells stably expressing YBX1 (MDCKYBX1, intermediate EMT), and Ras-transformed MDCK cells (21D1 cells, complete EMT). 2F-2B cell motility and tube formation (length and branching) was significantly increased following supplementation with MDCKYBX1 or 21D1 exosomes, but not MDCK exosomes. Next, Matrigel™ plugs containing exosome-supplemented 2F-2B cells were subcutaneously injected into mice. Systemic perfusion was only observed for plugs supplemented with MDCKYBX1 or 21D1 exosomes. Comparative proteomics revealed that 21D1 exosomes contained VEGF-associated proteins, while MDCKYBX1 exosomes were enriched with activated Rac1 and PAK2. To validate, 2F-2B cells and HUVECs were pre-treated with PAK inhibitors prior to exosome supplementation. PAK inhibition nullified the effects of MDCKYBX1 exosomes by reducing the tube length and branching to baseline levels. By contrast, the effects of 21D1 exosomes were not significantly decreased. Our results demonstrate for the first time that oncogenic cells undergoing EMT can communicate with endothelial cells via exosomes, and establish exosomal Rac1/PAK2 as angiogenic promoters that may function from early stages of the metastatic cascade. PMID:26919098

  6. FilGAP and its close relatives: a mediator of Rho-Rac antagonism that regulates cell morphology and migration.

    PubMed

    Nakamura, Fumihiko

    2013-07-01

    Cell migration, phagocytosis and cytokinesis are mechanically intensive cellular processes that are mediated by the dynamic assembly and contractility of the actin cytoskeleton. GAPs (GTPase-activating proteins) control activities of the Rho family proteins including Cdc42, Rac1 and RhoA, which are prominent upstream regulators of the actin cytoskeleton. The present review concerns a class of Rho GAPs, FilGAP (ARHGAP24 gene product) and its close relatives (ARHGAP22 and AHRGAP25 gene products). FilGAP is a GAP for Rac1 and a binding partner of FLNa (filamin A), a widely expressed F-actin (filamentous actin)-cross-linking protein that binds many different proteins that are important in cell regulation. Phosphorylation of FilGAP serine/threonine residues and binding to FLNa modulate FilGAP's GAP activity and, as a result, its ability to regulate cell protrusion and spreading. FLNa binds to FilGAP at F-actin-enriched sites, such as at the leading edge of the cell where Rac1 activity is controlled to inhibit actin assembly. FilGAP then dissociates from FLNa in actin networks by myosin-dependent mechanical deformation of FLNa's FilGAP-binding site to relocate at the plasma membrane by binding to polyphosphoinositides. Since actomyosin contraction is activated downstream of RhoA-ROCK (Rho-kinase), RhoA activity regulates Rac1 through FilGAP by signalling to the force-generating system. FilGAP and the ARHGAP22 gene product also act as mediators between RhoA and Rac1 pathways, which lead to amoeboid and mesenchymal modes of cell movements respectively. Therefore FilGAP and its close relatives are key regulators that promote the reciprocal inhibitory relationship between RhoA and Rac1 in cell shape changes and the mesenchymal-amoeboid transition in tumour cells. PMID:23763313

  7. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    PubMed Central

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

  8. RabGEF1/Rabex-5 Regulates TrkA-Mediated Neurite Outgrowth and NMDA-Induced Signaling Activation in NGF-Differentiated PC12 Cells

    PubMed Central

    Tam, See-Ying; Lilla, Jennifer N.; Chen, Ching-Cheng; Kalesnikoff, Janet; Tsai, Mindy

    2015-01-01

    Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells. PMID:26588713

  9. Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells.

    PubMed

    Henriques, Andreia F A; Barros, Patrícia; Moyer, Mary Pat; Matos, Paulo; Jordan, Peter

    2015-12-28

    Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that around 80% of colorectal tumors carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and then analyzed the effect on expression of senescence markers. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence-associated β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21 whereas proliferation marker Ki67 was suppressed. Upon co-expression of splice variant Rac1b, but not of Rac1, the B-Raf-induced senescence phenotype was reverted and expression of the cell-cycle inhibitors downregulated in a reactive oxygen-species dependent manner. We thus provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence, indicating the selection for increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS. PMID:26341689

  10. ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration.

    PubMed

    Yuan, Liangping; Santi, Mariarita; Rushing, Elisabeth J; Cornelison, Robert; MacDonald, Tobey J

    2010-10-01

    We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity. PMID:20526801

  11. ERK activation of p21 activated kinase-1 (Pak1) is critical for medulloblastoma cell migration

    PubMed Central

    Yuan, Liangping; Santi, Mariarita; Rushing, Elisabeth J.; Cornelison, Robert

    2010-01-01

    We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity. PMID:20526801

  12. Lateral Mobility of E-cadherin Enhances Rac1 Response in Epithelial Cells

    PubMed Central

    Tsai, J.; Kam, L.C.

    2010-01-01

    The fluidity of cellular membranes imparts lateral mobility of proteins across the cell surface. To understand the impact of lateral mobility on cell-cell communication, a protein consisting of the extracellular recognition domains of E-cadherin was associated with the surface of silica beads by either tethering to a bead-supported lipid bilayer or direct adsorption, resulting in laterally mobile and immobile presentations of this protein. These beads were then seeded onto the upper surface of MDCK cells. Functional engagement of these beads was compared by measurement of Rac1 recruitment around the bead. Lateral mobility enhanced recognition of E-cadherin, promoting cell response to the beads at lower per-area concentrations than their immobilized counterparts. A more complete understanding of how lateral mobility of membrane-associated proteins influences molecular recognition, and potentially other downstream responses, could provide new strategies for the design of materials and devices intended to capture the architecture of natural tissues. PMID:20368760

  13. TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function.

    PubMed

    Lee, Guan-Lin; Wu, Jing-Yiing; Tsai, Chien-Sung; Lin, Chih-Yuan; Tsai, Yi-Ting; Lin, Chin-Sheng; Wang, Yi-Fu; Yet, Shaw-Fang; Hsu, Yu-Juei; Kuo, Cheng-Chin

    2016-01-01

    Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration. PMID:27563891

  14. Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation.

    PubMed

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara; Wang, Zhixiang

    2016-01-01

    The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity. PMID:26816343

  15. Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

    PubMed Central

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara; Wang, Zhixiang

    2016-01-01

    The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity. PMID:26816343

  16. A novel KLF6-Rho GTPase axis regulates hepatocellular carcinoma cell migration and dissemination

    PubMed Central

    Ahronian, Leanne G.; Zhu, Lihua Julie; Chen, Ya-Wen; Chu, Hsiao-Chien; Klimstra, David S.; Lewis, Brian C.

    2016-01-01

    The presence of invasion into the extra-hepatic portion of the portal vein or the development of distant metastases renders hepatocellular carcinoma (HCC) patients ineligible for the only potential curative options for this malignancy - tumor resection or organ transplantation. Gene expression profiling of murine HCC cell lines identified KLF6 as a potential regulator of HCC cell migration. KLF6 knockdown increases cell migration, consistent with the correlation between decreased KLF6 mRNA levels and the presence of vascular invasion in human HCC. Concordantly, single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both HCC development and metastasis. By combining gene expression profiling and chromatin immunoprecipitation coupled to deep sequencing, we identified novel transcriptional targets of KLF6 in HCC cells including VAV3, a known activator of the RAC1 small GTPase. Indeed, RAC1 activity is increased in KLF6 knockdown cells in a VAV3-dependent manner, and knockdown of either RAC1 or VAV3 impairs HCC cell migration. Together, our data demonstrate a novel function for KLF6 in constraining HCC dissemination through the regulation of a VAV3-RAC1 signaling axis. PMID:26876204

  17. Exploration of supramolecular assemblies of rac-1,3-cyclohexanedicarboxylic acid

    NASA Astrophysics Data System (ADS)

    Giri, Lopamudra; Pedireddi, V. R.

    2015-11-01

    Supramolecular assemblies of rac-1,3-cyclohexanedicarboxylic acid (1) with melamine (a), 1,2-bis(4-pyridyl)ethene (b); 1, 2-bis(4pyridyl)ethane (c) in the presence of Co(II), and 1,10-phenanthroline (d) along with Cu(II) and Ni(II), respectively 1a - 1d and 1d', have been reported. All the assemblies were prepared by crystallization method, through slow-evaporation process, at ambient conditions. All the complexes yield sheet structures that are stacked in three-dimensional arrangement, however, each structure is unique within three-dimensional networks with varied arrangements of either organic entities or coordinated ensembles. For instance, while a host-guest type assembly was observed in 1a, only crinkled tapes are observed in 1b. Among coordination complexes, 1c has an interpenetrated cubic network, whereas 1d and 1d' form host-guest networks. A noteworthy feature to highlight is that the water molecules in the channels of 1d organize in the form of pentamers, which are further held together through tetrameric network, with the aid of hydrogen bonds. A further interesting feature is the presence of acid 1 in different conformations in the complexes as cis form in 1b, 1c and 1d and trans form in 1d'. However, in 1a both cis and trans conformers are observed.

  18. Drug design for cardiovascular disease: the effect of solvation energy on Rac1-ligand interactions.

    PubMed

    Maggi, Norbert; Arrigo, Patrizio; Ruggiero, Carmelina

    2011-01-01

    'OMICS' techniques have deeply changed the drug discovery process. The availability of many different potential druggable genes, generated by these new techniques, have exploited the complexity of new lead compounds screening. 'Virtual screening', based on the integration of different analytical tools on high performance hardware platforms, has speeded up the search for new chemical entities suitable for experimental validation. Docking is a key step in the screening process. The aim of this paper is the evaluation of binding differences due to solvation. We have compared two commonly used software, one of which takes into account solvation, on a set of small molecules (Morpholines, flavonoids and imidazoles) which are able to target the RAC1 protein--a cardiovascular target. We have evaluated the degree of agreement between the two different programs using a machine learning approach combined with statistical test. Our analysis, on a sample of small molecules, has pointed out that 35% of the molecules seem to be sensitive to solvation. This result, even though quite preliminary, stresses the need to combine different algorithms to obtain a more reliable filtered set of ligands. PMID:22255029

  19. Tumor cell migration and invasion are enhanced by depletion of Rap1 GTPase-activating protein (Rap1GAP).

    PubMed

    Tsygankova, Oxana M; Wang, Hongbin; Meinkoth, Judy L

    2013-08-23

    The functional significance of the widespread down-regulation of Rap1 GTPase-activating protein (Rap1GAP), a negative regulator of Rap activity, in human tumors is unknown. Here we show that human colon cancer cells depleted of Rap1GAP are endowed with more aggressive migratory and invasive properties. Silencing Rap1GAP enhanced the migration of confluent and single cells. In the latter, migration distance, velocity, and directionality were increased. Enhanced migration was a consequence of increased endogenous Rap activity as silencing Rap expression selectively abolished the migration of Rap1GAP-depleted cells. ROCK-mediated cell contractility was suppressed in Rap1GAP-depleted cells, which exhibited a spindle-shaped morphology and abundant membrane protrusions. Tumor cells can switch between Rho/ROCK-mediated contractility-based migration and Rac1-mediated mesenchymal motility. Strikingly, the migration of Rap1GAP-depleted, but not control cells required Rac1 activity, suggesting that loss of Rap1GAP alters migratory mechanisms. Inhibition of Rac1 activity restored membrane blebbing and increased ROCK activity in Rap1GAP-depleted cells, suggesting that Rac1 contributes to the suppression of contractility. Collectively, these findings identify Rap1GAP as a critical regulator of aggressive tumor cell behavior and suggest that the level of Rap1GAP expression influences the migratory mechanisms that are operative in tumor cells. PMID:23864657

  20. Living cell imaging and Rac1-GTP levels of CXCL12-treated migrating neural progenitor cells in stripe assay.

    PubMed

    Zhang, Min; Song, Aihong; Lai, Siqiang; Qiu, Lisha; Huang, Yunlong; Chen, Qiang; Zhu, Bing; Xu, Dongsheng; Zheng, Jialin C

    2015-12-01

    This data article contains three figures and three videos related to the research article entitled "Applications of Stripe Assay in the Study of CXCL12-mediated Neural Progenitor Cell Migration and Polarization" Zhang et al. (2015) [1], which uses stripe assay to study mouse neural progenitor cell (NPC) migration and polarization. The current article describes the neurosphere method used to culture NPCs. NPCs in neurospheres and monolayer were characterized using immunocytochemistry method with antibodies against two classic NPC markers: nestin and SOX2. The article also describes method to obtain sufficient protein lysates from NPCs in the stripe assay. When protein lysates were subjected to Rac1 affinity precipitation, Rac1-GTP was detected in the pull-down samples. In addition, the articles provides live cell imaging data to better understand CXCL12-mediated cellular migration and polarization. PMID:26693502

  1. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model

    PubMed Central

    Harraz, Maged M.; Marden, Jennifer J.; Zhou, Weihong; Zhang, Yulong; Williams, Aislinn; Sharov, Victor S.; Nelson, Kathryn; Luo, Meihui; Paulson, Henry; Schöneich, Christian; Engelhardt, John F.

    2008-01-01

    Neurodegeneration in familial amyotrophic lateral sclerosis (ALS) is associated with enhanced redox stress caused by dominant mutations in superoxide dismutase–1 (SOD1). SOD1 is a cytosolic enzyme that facilitates the conversion of superoxide (O2•–) to H2O2. Here we demonstrate that SOD1 is not just a catabolic enzyme, but can also directly regulate NADPH oxidase–dependent (Nox-dependent) O2•– production by binding Rac1 and inhibiting its GTPase activity. Oxidation of Rac1 by H2O2 uncoupled SOD1 binding in a reversible fashion, producing a self-regulating redox sensor for Nox-derived O2•– production. This process of redox-sensitive uncoupling of SOD1 from Rac1 was defective in SOD1 ALS mutants, leading to enhanced Rac1/Nox activation in transgenic mouse tissues and cell lines expressing ALS SOD1 mutants. Glial cell toxicity associated with expression of SOD1 mutants in culture was significantly attenuated by treatment with the Nox inhibitor apocynin. Treatment of ALS mice with apocynin also significantly increased their average life span. This redox sensor mechanism may explain the gain-of-function seen with certain SOD1 mutations associated with ALS and defines new therapeutic targets. PMID:18219391

  2. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    PubMed

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  3. Small rho GTPases mediate tumor-induced inhibition of endocytic activity of dendritic cells.

    PubMed

    Tourkova, Irina L; Shurin, Galina V; Wei, Sheng; Shurin, Michael R

    2007-06-15

    The generation, maturation, and function of dendritic cells (DC) have been shown to be markedly compromised in the tumor microenvironment in animals and humans. However, the molecular mechanisms and intracellular pathways involved in the regulation of the DC system in cancer are not yet fully understood. Recently, we have reported on the role of the small Rho GTPase family members Cdc42, Rac1, and RhoA in regulating DC adherence, motility, and Ag presentation. To investigate involvement of small Rho GTPases in dysregulation of DC function by tumors, we next evaluated how Cdc42, Rac1, and RhoA regulated endocytic activity of DC in the tumor microenvironment. We revealed a decreased uptake of dextran 40 and polystyrene beads by DC generated in the presence of different tumor cell lines, including RM1 prostate, MC38 colon, 3LL lung, and B7E3 oral squamous cell carcinomas in vitro and by DC prepared from tumor-bearing mice ex vivo. Impaired endocytic activity of DC cocultured with tumor cells was associated with decreased levels of active Cdc42 and Rac1. Transduction of DC with the dominant negative Cdc42 and Rac1 genes also led to reduced phagocytosis and receptor-mediated endocytosis. Furthermore, transduction of DC with the constitutively active Cdc42 and Rac1 genes restored endocytic activity of DC that was inhibited by the tumors. Thus, our results suggest that tumor-induced dysregulation of endocytic activity of DC is mediated by reduced activity of several members of the small Rho GTPase family, which might serve as new targets for improving the efficacy of DC vaccines. PMID:17548616

  4. α2-Chimaerin interacts with EphA4 and regulates EphA4-dependent growth cone collapse

    PubMed Central

    Shi, Lei; Fu, Wing-Yu; Hung, Kwok-Wang; Porchetta, Cassandra; Hall, Christine; Fu, Amy K. Y.; Ip, Nancy Y.

    2007-01-01

    EphA4-dependent growth cone collapse requires reorganization of actin cytoskeleton through coordinated activation of Rho family GTPases. Whereas various guanine exchange factors have recently been identified to be involved in EphA4-mediated regulation of Rho GTPases and growth cone collapse, the functional roles of GTPase-activating proteins in the process are largely unknown. Here we report that EphA4 interacts with α2-chimaerin through its Src homology 2 domain. Activated EphA4 induces a rapid increase of tyrosine phosphorylation of α2-chimaerin and enhances its GTPase-activating protein activity toward Rac1. More importantly, α2-chimaerin regulates the action of EphA4 in growth cone collapse through modulation of Rac1 activity. Our findings have therefore identified a new α2-chimaerin-dependent signaling mechanism through which EphA4 transduces its signals to the actin cytoskeleton and modulates growth cone morphology. PMID:17911252

  5. RhoGDI-1 modulation of the activity of monomeric RhoGTPase RhoA regulates endothelial barrier function in mouse lungs.

    PubMed

    Gorovoy, Matvey; Neamu, Radu; Niu, Jiaxin; Vogel, Stephen; Predescu, Dan; Miyoshi, Jun; Takai, Yoshimi; Kini, Vidisha; Mehta, Dolly; Malik, Asrar B; Voyno-Yasenetskaya, Tatyana

    2007-07-01

    Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1(-/-) mice. We observed that basal endothelial permeability in lungs of RhoGDI-1(-/-) mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1(-/-) lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1-mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome. PMID:17525371

  6. Cytoskeletal regulation by AUTS2 in neuronal migration and neuritogenesis.

    PubMed

    Hori, Kei; Nagai, Taku; Shan, Wei; Sakamoto, Asami; Taya, Shinichiro; Hashimoto, Ryoya; Hayashi, Takashi; Abe, Manabu; Yamazaki, Maya; Nakao, Keiko; Nishioka, Tomoki; Sakimura, Kenji; Yamada, Kiyofumi; Kaibuchi, Kozo; Hoshino, Mikio

    2014-12-24

    Mutations in the Autism susceptibility candidate 2 gene (AUTS2), whose protein is believed to act in neuronal cell nuclei, have been associated with multiple psychiatric illnesses, including autism spectrum disorders, intellectual disability, and schizophrenia. Here we show that cytoplasmic AUTS2 is involved in the regulation of the cytoskeleton and neural development. Immunohistochemistry and fractionation studies show that AUTS2 localizes not only in nuclei, but also in the cytoplasm, including in the growth cones in the developing brain. AUTS2 activates Rac1 to induce lamellipodia but downregulates Cdc42 to suppress filopodia. Our loss-of-function and rescue experiments show that a cytoplasmic AUTS2-Rac1 pathway is involved in cortical neuronal migration and neuritogenesis in the developing brain. These findings suggest that cytoplasmic AUTS2 acts as a regulator of Rho family GTPases to contribute to brain development and give insight into the pathology of human psychiatric disorders with AUTS2 mutations. PMID:25533347

  7. Factors regulating microglia activation

    PubMed Central

    Kierdorf, Katrin; Prinz, Marco

    2013-01-01

    Microglia are resident macrophages of the central nervous system (CNS) that display high functional similarities to other tissue macrophages. However, it is especially important to create and maintain an intact tissue homeostasis to support the neuronal cells, which are very sensitive even to minor changes in their environment. The transition from the “resting” but surveying microglial phenotype to an activated stage is tightly regulated by several intrinsic (e.g., Runx-1, Irf8, and Pu.1) and extrinsic factors (e.g., CD200, CX3CR1, and TREM2). Under physiological conditions, minor changes of those factors are sufficient to cause fatal dysregulation of microglial cell homeostasis and result in severe CNS pathologies. In this review, we discuss recent achievements that gave new insights into mechanisms that ensure microglia quiescence. PMID:23630462

  8. DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis.

    PubMed

    Kunisaki, Yuya; Nishikimi, Akihiko; Tanaka, Yoshihiko; Takii, Ryosuke; Noda, Mayuko; Inayoshi, Ayumi; Watanabe, Ken-ichi; Sanematsu, Fumiyuki; Sasazuki, Takehiko; Sasaki, Takehiko; Fukui, Yoshinori

    2006-08-28

    Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation. PMID:16943182

  9. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    PubMed

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization. PMID:24268197

  10. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    PubMed Central

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Kim, Hyo Jeong; Ko, Myoung Seok; Seol, Wongi; Joe, Yeonsoo; Chung, Hun Taeg; Lee, Byung Ju; Moon, Chang Hoon; Cho, Wha Ja; Park, Jeong Woo

    2016-01-01

    The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. PMID:26582392

  11. Dock4 is regulated by RhoG and promotes Rac-dependent cell migration.

    PubMed

    Hiramoto, Kiyo; Negishi, Manabu; Katoh, Hironori

    2006-12-10

    Cell migration is essential for normal development and many pathological processes including tumor metastasis. Rho family GTPases play important roles in this event. In particular, Rac is required for lamellipodia formation at the leading edge during migration. Dock4 is a member of the Dock180 family proteins, and Dock4 mutations are present in a subset of human cancer cell lines. However, the function and the regulatory mechanism of Dock4 remain unclear. Here we show that Dock4 is regulated by the small GTPase RhoG and its effector ELMO and promotes cell migration by activating Rac1. Dock4 formed a complex with ELMO, and expression of active RhoG induced translocation of the Dock4-ELMO complex from the cytoplasm to the plasma membrane and enhanced the Dock4- and ELMO-dependent Rac1 activation and cell migration. On the other hand, RNA interference-mediated knockdown of Dock4 in NIH3T3 cells reduced cell migration. Taken together, these results suggest that Dock4 plays an important role in the regulation of cell migration through activation of Rac1, and that RhoG is a key upstream regulator for Dock4. PMID:17027967

  12. Actin cytoskeleton organization regulated by the PAK family of protein kinases.

    PubMed

    Eby, J J; Holly, S P; van Drogen, F; Grishin, A V; Peter, M; Drubin, D G; Blumer, K J

    1998-08-27

    Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton. PMID:9742399

  13. An evolutionarily conserved autoinhibitory molecular switch in ELMO proteins regulates Rac signaling.

    PubMed

    Patel, Manishha; Margaron, Yoran; Fradet, Nadine; Yang, Qi; Wilkes, Brian; Bouvier, Michel; Hofmann, Kay; Côté, Jean-François

    2010-11-23

    Dedicator of cytokinesis (DOCK) proteins are guanine nucleotide exchange factors (GEFs) controlling the activity of Rac1/Cdc42 during migration, phagocytosis, and myoblast fusion [1-4]. Engulfment and cell motility (ELMO) proteins bind a subset of DOCK members and are emerging as critical regulators of Rac signaling [5-10]. Although formation of a DOCK180/ELMO complex is not essential for Rac1 activation, ELMO mutants deficient in binding to DOCK180 are unable to promote cytoskeleton remodeling [11]. How ELMO regulates signaling through DOCK GEFs is poorly understood. Here, we identify an autoinhibitory switch in ELMO presenting homology to a regulatory unit described for Dia formins. One part of the switch, composed of a Ras-binding domain (RBD) and Armadillo repeats, is positioned N-terminally while the other is housed in the C terminus. We demonstrate interaction between these fragments, suggesting autoinhibition of ELMO. Using a bioluminescence resonance energy transfer biosensor, we establish that ELMO undergoes conformational changes upon disruption of autoinhibition. We found that engagement of ELMO to RhoG, or with DOCK180, promotes the relief of autoinhibition in ELMO. Functionally, we found that ELMO mutants with impaired autoregulatory activity promote cell elongation. These results demonstrate an unsuspected level of regulation for Rac1 signaling via autoinhibition of ELMO. PMID:21035343

  14. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    SciTech Connect

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori . E-mail: hirokato@pharm.kyoto-u.ac.jp

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.

  15. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism.

    PubMed

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85alpha and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1. PMID:17570359

  16. Regio- and Enantioselective Sequential Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB.

    PubMed

    Gross, Johannes; Prokop, Zbyněk; Janssen, Dick; Faber, Kurt; Hall, Mélanie

    2016-08-01

    The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87 %). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product. PMID:27223496

  17. Scribble Scaffolds a Signalosome for Active Forgetting.

    PubMed

    Cervantes-Sandoval, Isaac; Chakraborty, Molee; MacMullen, Courtney; Davis, Ronald L

    2016-06-15

    Forgetting, one part of the brain's memory management system, provides balance to the encoding and consolidation of new information by removing unused or unwanted memories or by suppressing their expression. Recent studies identified the small G protein, Rac1, as a key player in the Drosophila mushroom bodies neurons (MBn) for active forgetting. We subsequently discovered that a few dopaminergic neurons (DAn) that innervate the MBn mediate forgetting. Here we show that Scribble, a scaffolding protein known primarily for its role as a cell polarity determinant, orchestrates the intracellular signaling for normal forgetting. Knocking down scribble expression in either MBn or DAn impairs normal memory loss. Scribble interacts physically and genetically with Rac1, Pak3, and Cofilin within MBn, nucleating a forgetting signalosome that is downstream of dopaminergic inputs that regulate forgetting. These results bind disparate molecular players in active forgetting into a single signaling pathway: Dopamine→ Dopamine Receptor→ Scribble→ Rac→ Cofilin. PMID:27263975

  18. Regulation of inflammasome activation.

    PubMed

    Man, Si Ming; Kanneganti, Thirumala-Devi

    2015-05-01

    Inflammasome biology is one of the most exciting and rapidly growing areas in immunology. Over the past 10 years, inflammasomes have been recognized for their roles in the host defense against invading pathogens and in the development of cancer, auto-inflammatory, metabolic, and neurodegenerative diseases. Assembly of an inflammasome complex requires cytosolic sensing of pathogen-associated molecular patterns or danger-associated molecular patterns by a nucleotide-binding domain and leucine-rich repeat receptor (NLR) or absent in melanoma 2 (AIM2)-like receptors (ALR). NLRs and ALRs engage caspase-1, in most cases requiring the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC), to catalyze proteolytic cleavage of pro-interleukin-1β (pro-IL-1β) and pro-IL-18 and drive pyroptosis. Recent studies indicate that caspase-8, caspase-11, IL-1R-associated kinases (IRAK), and receptor-interacting protein (RIP) kinases contribute to inflammasome functions. In addition, post-translational modifications, including ubiquitination, deubiquitination, phosphorylation, and degradation control almost every aspect of inflammasome activities. Genetic studies indicate that mutations in NLRP1, NLRP3, NLRC4, and AIM2 are linked with the development of auto-inflammatory diseases, enterocolitis, and cancer. Overall, these findings transform our understanding of the basic biology and clinical relevance of inflammasomes. In this review, we provide an overview of the latest development of inflammasome research and discuss how inflammasome activities govern health and disease. PMID:25879280

  19. Regulation of inflammasome activation

    PubMed Central

    Man, Si Ming; Kanneganti, Thirumala-Devi

    2015-01-01

    Summary Inflammasome biology is one of the most exciting and rapidly growing areas in immunology. Over the past 10 years, inflammasomes have been recognized for their roles in the host defense against invading pathogens and in the development of cancer, autoinflammatory, metabolic, and neurodegenerative diseases. Assembly of an inflammasome complex requires cytosolic sensing of pathogen-associated molecular patterns or danger-associated molecular patterns by a nucleotide-binding domain and leucine-rich repeat receptor (NLR) or absent in melanoma 2-like receptor (ALR). NLRs and ALRs engage caspase-1, in most cases requiring the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC), to catalyze proteolytic cleavage of pro-interleukin-1β (pro-IL-1β) and pro-IL-18 and drive pyroptosis. Recent studies indicate that caspase-8, caspase-11, IL-1R–associated kinases (IRAK), and receptor-interacting protein (RIP) kinases contribute to inflammasome functions. In addition, post-translational modifications, including ubiquitination, deubiquitination, phosphorylation, and degradation, control almost every aspect of inflammasome activities. Genetic studies indicate that mutations in NLRP1, NLRP3, NLRC4, and AIM2 are linked to the development of autoinflammatory diseases, enterocolitis, and cancer. Overall, these findings transform our understanding of the basic biology and clinical relevance of inflammasomes. In this review, we provide an overview of the latest development of inflammasome research and discuss how inflammasome activities govern health and disease. PMID:25879280

  20. ARMS/Kidins220 and synembryn-B levels regulate NGF-mediated secretion.

    PubMed

    López-Benito, Saray; Lillo, Concepción; Hernández-Hernández, Ángel; Chao, Moses V; Arévalo, Juan C

    2016-05-01

    Proper development of the nervous system requires a temporally and spatially orchestrated set of events including differentiation, synapse formation and neurotransmission. Nerve growth factor (NGF) acting through the TrkA neurotrophin receptor (also known as NTRK1) regulates many of these events. However, the molecular mechanisms responsible for NGF-regulated secretion are not completely understood. Here, we describe a new signaling pathway involving TrkA, ARMS (also known as Kidins220), synembryn-B and Rac1 in NGF-mediated secretion in PC12 cells. Whereas overexpression of ARMS blocked NGF-mediated secretion, without affecting basal secretion, a decrease in ARMS resulted in potentiation. Similar effects were observed with synembryn-B, a protein that interacts directly with ARMS. Downstream of ARMS and synembryn-B are Gαq and Trio proteins, which modulate the activity of Rac1 in response to NGF. Expression of dominant-negative Rac1 rescued the secretion defects of cells overexpressing ARMS or synembryn-B. Thus, this neurotrophin pathway represents a new mechanism responsible for NGF-regulated secretion. PMID:26966186

  1. The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

    PubMed Central

    Colombatti, Marco; Fracasso, Giulio; Scupoli, Maria Teresa; Cingarlini, Sara; Poffe, Ornella; Naim, Hassan Y.; Heine, Martin; Tridente, Giuseppe; Mainiero, Fabrizio; Ramarli, Dunia

    2009-01-01

    The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis. PMID:19242540

  2. Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells.

    PubMed

    Bekhite, Mohamed M; Müller, Veronika; Tröger, Sebastian H; Müller, Jörg P; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2016-04-01

    The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration. PMID:26553657

  3. Cloning and characterization of bovine low molecular weight GTPases (Rac1 and Rac2) and rho GDP-dissociation inhibitor 2 (D4-GDI).

    PubMed

    Davis, A R; Clements, M K; Bunger, P L; Siemsen, D W; Quinn, M T

    2000-05-23

    GTPases of the Rho family play important roles in human leukocyte signal transduction pathways; however, little is known about the function of these proteins in bovine cells. In the present studies, we isolated molecular clones of bovine Rac1, Rac2, and the Rac/Rho GTPase regulatory protein D4-GDP dissociation inhibitor (D4-GDI) from a bovine bone marrow cDNA library. These clones contained complete open reading frames, encoding 192, 192, and 200 amino acids, respectively. Comparison of the bovine amino acid sequences with those of other species demonstrated a high degree of identity of these proteins across all species, suggesting that these proteins likely play conserved functional roles in bovine leukocyte signal transduction pathways. Comparative Western blotting of these proteins in human and bovine neutrophil cytosol demonstrated that Rac2 was the predominant Rac species and that D4-GDI was the predominant GDI species in bovine neutrophil cytosol. Despite the high degree of homology between human and bovine Rac2, some of the anti-peptide antibody probes prepared against human Rac2 failed to recognize the bovine homologue. We also showed by subcellular fractionation techniques that Rac2 is localized primarily to the cytosolic compartment of resting bovine neutrophils, but is translocated to the plasma membrane after stimulation with PMA. These findings suggest that Rac2 does play a role in bovine neutrophil activation. In addition, these data will be helpful in developing more specific probes for investigating the role of these proteins in bovine leukocyte signal transduction pathways and for studying various inflammatory diseases in cattle. PMID:10802295

  4. Molecular regulation of osteoclast activity.

    PubMed

    Bruzzaniti, Angela; Baron, Roland

    2006-06-01

    Osteoclasts are multinucleated cells derived from hematopoietic precursors that are primarily responsible for the degradation of mineralized bone during bone development, homeostasis and repair. In various skeletal disorders such as osteoporosis, hypercalcemia of malignancy, tumor metastases and Paget's disease, bone resorption by osteoclasts exceeds bone formation by osteoblasts leading to decreased bone mass, skeletal fragility and bone fracture. The overall rate of osteoclastic bone resorption is regulated either at the level of differentiation of osteoclasts from their monocytic/macrophage precursor pool or through the regulation of key functional proteins whose specific activities in the mature osteoclast control its attachment, migration and resorption. Thus, reducing osteoclast numbers and/or decreasing the bone resorbing activity of osteoclasts are two common therapeutic approaches for the treatment of hyper-resorptive skeletal diseases. In this review, several of the key functional players involved in the regulation of osteoclast activity will be discussed. PMID:16951988

  5. Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

    PubMed Central

    Dios-Esponera, Ana; Isern de Val, Soledad; Sevilla-Movilla, Silvia; García-Verdugo, Rosa; García-Bernal, David; Arellano-Sánchez, Nohemí; Cabañas, Carlos; Teixidó, Joaquin

    2015-01-01

    Stimulation by chemokines of integrin α4β1–dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase–inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76–, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation. PMID:26202465

  6. Loss-of-Function Mutations in ELMO2 Cause Intraosseous Vascular Malformation by Impeding RAC1 Signaling.

    PubMed

    Cetinkaya, Arda; Xiong, Jingwei Rachel; Vargel, İbrahim; Kösemehmetoğlu, Kemal; Canter, Halil İbrahim; Gerdan, Ömer Faruk; Longo, Nicola; Alzahrani, Ahmad; Camps, Mireia Perez; Taskiran, Ekim Zihni; Laupheimer, Simone; Botto, Lorenzo D; Paramalingam, Eeswari; Gormez, Zeliha; Uz, Elif; Yuksel, Bayram; Ruacan, Şevket; Sağıroğlu, Mahmut Şamil; Takahashi, Tokiharu; Reversade, Bruno; Akarsu, Nurten Ayse

    2016-08-01

    Vascular malformations are non-neoplastic expansions of blood vessels that arise due to errors during angiogenesis. They are a heterogeneous group of sporadic or inherited vascular disorders characterized by localized lesions of arteriovenous, capillary, or lymphatic origin. Vascular malformations that occur inside bone tissue are rare. Herein, we report loss-of-function mutations in ELMO2 (which translates extracellular signals into cellular movements) that are causative for autosomal-recessive intraosseous vascular malformation (VMOS) in five different families. Individuals with VMOS suffer from life-threatening progressive expansion of the jaw, craniofacial, and other intramembranous bones caused by malformed blood vessels that lack a mature vascular smooth muscle layer. Analysis of primary fibroblasts from an affected individual showed that absence of ELMO2 correlated with a significant downregulation of binding partner DOCK1, resulting in deficient RAC1-dependent cell migration. Unexpectedly, elmo2-knockout zebrafish appeared phenotypically normal, suggesting that there might be human-specific ELMO2 requirements in bone vasculature homeostasis or genetic compensation by related genes. Comparative phylogenetic analysis indicated that elmo2 originated upon the appearance of intramembranous bones and the jaw in ancestral vertebrates, implying that elmo2 might have been involved in the evolution of these novel traits. The present findings highlight the necessity of ELMO2 for maintaining vascular integrity, specifically in intramembranous bones. PMID:27476657

  7. The Rac-GAP Bcr is a novel regulator of the Par complex that controls cell polarity

    PubMed Central

    Narayanan, Anjana S.; Reyes, Steve B.; Um, Kyongmi; McCarty, Joseph H.; Tolias, Kimberley F.

    2013-01-01

    Cell polarization is essential for many biological processes, including directed cell migration, and loss of polarity contributes to pathological conditions such as cancer. The Par complex (Par3, Par6, and PKCζ) controls cell polarity in part by recruiting the Rac-specific guanine nucleotide exchange factor T-lymphoma invasion and metastasis 1 (Tiam1) to specialized cellular sites, where Tiam1 promotes local Rac1 activation and cytoskeletal remodeling. However, the mechanisms that restrict Par-Tiam1 complex activity to the leading edge to maintain cell polarity during migration remain unclear. We identify the Rac-specific GTPase-activating protein (GAP) breakpoint cluster region protein (Bcr) as a novel regulator of the Par-Tiam1 complex. We show that Bcr interacts with members of the Par complex and inhibits both Rac1 and PKCζ signaling. Loss of Bcr results in faster, more random migration and striking polarity defects in astrocytes. These polarity defects are rescued by reducing PKCζ activity or by expressing full-length Bcr, but not an N-terminal deletion mutant or the homologous Rac-GAP, Abr, both of which fail to associate with the Par complex. These results demonstrate that Bcr is an integral member of the Par-Tiam1 complex that controls polarized cell migration by locally restricting both Rac1 and PKCζ function. PMID:24152735

  8. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family.

    PubMed Central

    Chuang, T H; Xu, X; Kaartinen, V; Heisterkamp, N; Groffen, J; Bokoch, G M

    1995-01-01

    Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling. Images Fig. 3 PMID:7479768

  9. Phosphatase regulation of macrophage activation.

    PubMed

    Kozicky, Lisa K; Sly, Laura M

    2015-08-01

    Macrophages are innate immune cells that play critical roles in tissue homeostasis and the immune response to invading pathogens or tumor cells. A hallmark of macrophages is their "plasticity," that is, their ability to respond to cues in their local microenvironment and adapt their activation state or phenotype to mount an appropriate response. During the inflammatory response, macrophages may be required to mount a profound anti-bacterial or anti-tumor response, an anti-inflammatory response, an anti-parasitic response, or a wound healing response. To do so, macrophages express cell surface receptors for growth factors, chemokines and cytokines, as well pathogen and danger associated molecular patterns. Downstream of these cell surface receptors, cell signalling cascades are activated and deactivated by reversible and competing activities of lipid and protein kinases and phosphatases. While kinases drive the activation of cell signalling pathways critical for macrophage activation, the strength and duration of the signalling is regulated by phosphatases. Hence, gene knockout mouse models have revealed critical roles for lipid and protein phosphatases in macrophage activation. Herein, we describe our current understanding and the key roles of specific cellular phosphatases in the regulation of the quality of macrophage polarization as well as the quantity of cytokines produced by activated macrophages. PMID:26216598

  10. Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases Activation

    PubMed Central

    Ambriz-Peña, Xochitl; García-Zepeda, Eduardo Alberto; Meza, Isaura; Soldevila, Gloria

    2014-01-01

    We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3−/− lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3−/− lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines. PMID:24498424

  11. Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1*

    PubMed Central

    Pan, Dingxin; Barber, Mark A.; Hornigold, Kirsti; Baker, Martin J.; Toth, Judit M.; Oxley, David; Welch, Heidi C. E.

    2016-01-01

    P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gβγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gβγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1. PMID:26792863

  12. RhoA activation promotes transendothelial migration of monocytes via ROCK.

    PubMed

    Honing, Henk; van den Berg, Timo K; van der Pol, Susanne M A; Dijkstra, Christine D; van der Kammen, Rob A; Collard, John G; de Vries, Helga E

    2004-03-01

    Monocyte infiltration into inflamed tissue requires the initial arrest of the cells on the endothelium followed by firm adhesion and their subsequent migration. Migration of monocytes and other leukocytes is believed to involve a coordinated remodeling of the actin cytoskeleton. The small GTPases RhoA, Rac1, and Cdc42 are critical regulators of actin reorganization. In this study, we have investigated the role of Rho-like GTPases RhoA, Rac1, and Cdc42 in the adhesion and migration of monocytes across brain endothelial cells by expressing their constitutively active or dominant-negative constructs in NR8383 rat monocytic cells. Monocytes expressing the active form of Cdc42 show a reduced migration, whereas Rac1 expression did not affect adhesion or migration. In contrast, expression of the active form of RhoA in monocytes leads to a dramatic increase in their adhesion and migration across endothelial cells. The effect of RhoA was found to be mediated by its down-stream effector Rho kinase (ROCK), as pretreatment with the selective ROCK inhibitor Y-27632 prevented this enhanced adhesion and migration. These results demonstrate that RhoA activation in monocytes is sufficient to enhance adhesion and migration across monolayers of endothelial cells. PMID:14634067

  13. Cdc42 and p190RhoGAP activation by CCN2 regulates cell spreading and polarity and induces actin disassembly in migrating keratinocytes.

    PubMed

    Kiwanuka, Elizabeth; Lee, Cameron Cy; Hackl, Florian; Caterson, Edward J; Junker, Johan Pe; Gerdin, Bengt; Eriksson, Elof

    2016-06-01

    Cell migration requires spatiotemporal integration of signals that regulate cytoskeletal dynamics. In response to a migration-promoting agent, cells begin to polarise and extend protrusions in the direction of migration. These cytoskeletal rearrangements are orchestrated by a variety of proteins, including focal adhesion kinase (FAK) and the Rho family of GTPases. CCN2, also known as connective tissue growth factor, has emerged as a regulator of cell migration but the mechanism by which CCN2 regulates keratinocyte function is not well understood. In this article, we sought to elucidate the basic mechanism of CCN2-induced cell migration in human keratinocytes. Immunohistochemical staining was used to demonstrate that treatment with CCN2 induces a migratory phenotype through actin disassembly, spreading of lamellipodia and re-orientation of the Golgi. In vitro assays were used to show that CCN2-induced cell migration is dependent on FAK, RhoA and Cdc42, but independent of Rac1. CCN2-treated keratinocytes displayed increased Cdc42 activity and decreased RhoA activity up to 12 hours post-treatment, with upregulation of p190RhoGAP. An improved understanding of how CCN2 regulates cell migration may establish the foundation for future therapeutics in fibrotic and neoplastic diseases. PMID:25185742

  14. Knee loading reduces MMP13 activity in the mouse cartilage

    PubMed Central

    2013-01-01

    Background Moderate loads with knee loading enhance bone formation, but its effects on the maintenance of the knee are not well understood. In this study, we examined the effects of knee loading on the activity of matrix metalloproteinase13 (MMP13) and evaluated the role of p38 MAPK and Rac1 GTPase in the regulation of MMP13. Methods Knee loading (0.5–3 N for 5 min) was applied to the right knee of surgically-induced osteoarthritis (OA) mice as well as normal (non-OA) mice, and MMP13 activity in the femoral cartilage was examined. The sham-loaded knee was used as a non-loading control. We also employed primary non-OA and OA human chondrocytes as well as C28/I2 chondrocyte cells, and examined MMP13 activity and molecular signaling in response to shear at 2–20 dyn/cm2. Results Daily knee loading at 1 N for 2 weeks suppressed cartilage destruction in the knee of OA mice. Induction of OA elevated MMP13 activity and knee loading at 1 N suppressed this elevation. MMP13 activity was also increased in primary OA chondrocytes, and this increase was attenuated by applying shear at 10 dyn/cm2. Load-driven reduction in MMP13 was associated with a decrease in the phosphorylation level of p38 MAPK (p-p38) and NFκB (p-NFκB). Molecular imaging using a fluorescence resonance energy transfer (FRET) technique showed that Rac1 activity was reduced by shear at 10 dyn/cm2 and elevated by it at 20 dyn/cm2. Silencing Rac1 GTPase significantly reduced MMP13 expression and p-p38 but not p-NFκB. Transfection of a constitutively active Rac1 GTPase mutant increased MMP13 activity, while a dominant negative mutant decreased it. Conclusions Knee loading reduces MMP13 activity at least in part through Rac1-mediated p38 MAPK signaling. This study suggests the possibility of knee loading as a therapy not only for strengthening bone but also preventing tissue degradation of the femoral cartilage. PMID:24180431

  15. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

    PubMed

    Nguyen, Trang Thi Thu; Park, Wei Sun; Park, Byung Ouk; Kim, Cha Yeon; Oh, Yohan; Kim, Jin Man; Choi, Hana; Kyung, Taeyoon; Kim, Cheol-Hee; Lee, Gabsang; Hahn, Klaus M; Meyer, Tobias; Heo, Won Do

    2016-09-01

    Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration. PMID:27555588

  16. Filamin A regulates monocyte migration through Rho small GTPases during osteoclastogenesis.

    PubMed

    Leung, Roland; Wang, Yongqiang; Cuddy, Karl; Sun, Chunxiang; Magalhaes, Joyce; Grynpas, Marc; Glogauer, Michael

    2010-05-01

    Osteoclastogenesis (OCG) results from the fusion of monocytes after stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). Migration of monocytes into close proximity precedes critical fusion events that are required for osteoclast formation. Cellular migration requires leading-edge actin cytoskeleton assembly that drives cellular locomotion. Filamin A (FLNa) cross-links F-actin filaments in the leading edge of migrating cells and also has been shown to regulate signal transduction during cell migration. However, little is known about the possible role of FLNa in osteoclastogenesis. Our objective in this study was to investigate the role of FLNa in osteoclastogenesis. Bone marrow monocytes isolated from the tibiae and femora of wild type (WT) and Flna-null mice were cultured for 6 days with M-CSF and RANKL, and osteoclasts were identified by tartrate-resistant acid phosphatase (TRACP) staining. The Flna-null mouse skeletal phenotype was characterized using dual-energy X-ray absorptiometry (DXA) to analyze the skeleton, as well as tests on blood chemistry. Osteoclast levels in vivo were quantified by counting of TRACP-stained histologic sections of distal femora. To elucidate the mechanisms by which Flna regulates osteoclastogenesis, migration, actin polymerization, and activation of Rho GTPases, Rac1, Cdc42, and RhoA were assessed in monocytes during in vitro OCG. Deficiencies in migration were rescued using constitutively active Rac1 and Cdc42 TAT fusion proteins. The RANKL signaling pathway was evaluated for activation by monitoring nuclear translocation of NF kappaB and c-jun and expression of key osteoclast genes using quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that Flna-null monocytes formed fewer osteoclasts in vitro, and those that were formed were smaller with fewer nuclei. Decreased OCG was reflected in vivo in TRACP-stained histologic bone sections. Flna

  17. Dual 4- and 5-phosphatase activities regulate SopB-dependent phosphoinositide dynamics to promote bacterial entry.

    PubMed

    Piscatelli, Heather L; Li, Menghan; Zhou, Daoguo

    2016-05-01

    Salmonella are able to invade non-phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4-phosphatase and 5-phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4-phosphatase activity of SopB is required for PtdIns-3-phosphate (PtdIns(3)P) accumulation and SopB-mediated invasion. We show here that both the 4-phosphatase as well as the 5-phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5-phosphatase activity of SopB is likely responsible for generating PtdIns-3,4-bisphosphate (PtdIns(3,4)P2 ) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4-phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P2 into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4-phosphatase and 5-phosphatase activities led to SNX9-mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4- and 5-phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry. PMID:26537021

  18. p21-activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes

    PubMed Central

    DeSantiago, Jaime; Bare, Dan J; Xiao, Lei; Ke, Yunbo; Solaro, R. John; Banach, Kathrin

    2014-01-01

    Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca2+ handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1-/-) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca2+ transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1-/- VMs during 15 min of simulated ischemia. However, Pak1-/- VMs exhibited an exaggerated increase in [Ca2+]i, which resulted in spontaneous Ca2+ release events and waves. The Ca2+ overload in Pak1-/- VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1-/- VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47phox-/-) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1-/- VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca2+ overload in Pak1-/- VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca2+ overload under conditions where no significant changes in excitation-contraction coupling are yet evident. PMID:24380729

  19. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800–1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction. PMID:26198629

  20. Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration.

    PubMed

    Jones, Matthew C; Machida, Kazuya; Mayer, Bruce J; Turner, Christopher E

    2013-06-01

    The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF β-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and β-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/β-PIX signaling during cell migration. PMID:23615439

  1. Regulating Rac in the Nervous System: Molecular Function and Disease Implication of Rac GEFs and GAPs

    PubMed Central

    Bai, Yanyang; Xiang, Xiaoliang; Liang, Chunmei

    2015-01-01

    Rho family GTPases, including RhoA, Rac1, and Cdc42 as the most studied members, are master regulators of actin cytoskeletal organization. Rho GTPases control various aspects of the nervous system and are associated with a number of neuropsychiatric and neurodegenerative diseases. The activity of Rho GTPases is controlled by two families of regulators, guanine nucleotide exchange factors (GEFs) as the activators and GTPase-activating proteins (GAPs) as the inhibitors. Through coordinated regulation by GEFs and GAPs, Rho GTPases act as converging signaling molecules that convey different upstream signals in the nervous system. So far, more than 70 members of either GEFs or GAPs of Rho GTPases have been identified in mammals, but only a small subset of them have well-known functions. Thus, characterization of important GEFs and GAPs in the nervous system is crucial for the understanding of spatiotemporal dynamics of Rho GTPase activity in different neuronal functions. In this review, we summarize the current understanding of GEFs and GAPs for Rac1, with emphasis on the molecular function and disease implication of these regulators in the nervous system. PMID:25879033

  2. Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

    PubMed Central

    Lee, Hye Shin; Cheerathodi, Mujeeburahiman; Chaki, Sankar P.; Reyes, Steve B.; Zheng, Yanhua; Lu, Zhimin; Paidassi, Helena; DerMardirossian, Celine; Lacy-Hulbert, Adam; Rivera, Gonzalo M.

    2015-01-01

    Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells. PMID:25666508

  3. Rab11-FIP3 is a Rab11-binding protein that regulates breast cancer cell motility by modulating the actin cytoskeleton

    PubMed Central

    Jing, Jian; Tarbutton, Elizabeth; Wilson, Gayle; Prekeris, Rytis

    2009-01-01

    Cell adhesion and motility are very dynamic processes that require the temporal and spatial coordination of many cellular structures. ADP-ribosylation factor 6 (Arf6) has emerged as master regulator of endocytic membrane traffic and cytoskeletal dynamics during cell movement. Recently, a novel Arf6-binding protein known as FIP3/arfophilin/eferin has been identified. In addition to Arf6, FIP3 also interacts with Rab11, a small monomeric GTPase that regulates endocytic membrane transport. Both Arf6 and Rab11 GTPases have been implicated in regulation of cell motility. Here we test the role of FIP3 in breast carcinoma cell motility. First, we demonstrate that FIP3 is associated with recycling endosomes that are present at the leading edge of motile cells. Second, we show that FIP3 is required for the motility of MDA-MB-231 breast carcinoma cells. Third, we demonstrate that FIP3 regulates Rac1-dependent actin cytoskeleton dynamics and modulates the formation and ruffling of lamellipodia. Finally, we demonstrate that FIP3 regulates the localization of Arf6 at the plasma membrane of MDA-MB-231 cells. Based on our data we propose that FIP3 affects cell motility by regulating Arf6 localization to the plasma membrane of the leading edge, thus regulating polarized Rac1 activation and actin dynamics. PMID:19327867

  4. S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release.

    PubMed

    Bianchi, Roberta; Kastrisianaki, Eirini; Giambanco, Ileana; Donato, Rosario

    2011-03-01

    The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia. PMID:21209080

  5. Pak1 Regulates the Orientation of Apical Polarization and Lumen Formation by Distinct Pathways

    PubMed Central

    Smits, Jos; ter Beest, Martin B.; Zegers, Mirjam M.

    2012-01-01

    The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways. PMID:22815903

  6. Regulation of lamellipodia formation and cell invasion by CLIP-170 in invasive human breast cancer cells.

    PubMed

    Suzuki, Katsuo; Takahashi, Kazuhide

    2008-04-01

    Lamellipodia formation necessary for cell invasion is regulated by Rac1. We report here that lamellipodia formation and three-dimensional invasion were significantly promoted by HGF and serum, respectively, in invasive human breast cancer cells. Rac1 formed a complex with CLIP-170, IQGAP1, and kinesin in serum-starved cells, and stimulation of the cells with HGF and serum caused the partial release of IQGAP1 and kinesin from Rac1-CLIP-170 complex. The HGF-induced release of the proteins and promotion of lamellipodia formation were inhibited by an inhibitor of PI3K. Moreover, downregulation of CLIP-170 by siRNA released IQGAP1 and kinesin from Rac1 and promoted lamellipodia formation and invasion, independent of HGF and serum. The results suggest that promotion of lamellipodia formation and invasion by HGF or serum requires PI3K-dependent release of IQGAP1 and kinesin from Rac1-CLIP-170 complex and that CLIP-170 prevents cells from the extracellular stimulus-independent lamellipodia formation and invasion by tethering IQGAP1 and kinesin to Rac1. PMID:18237546

  7. Rac1 mediates cytokine-stimulated hemocyte spreading via prostaglandin biosynthesis in the beet armyworm, Spodoptera exigua

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell spreading is an integral component of insect hemocytic immune reactions to infections and invasions. Cell spreading is accomplished by cytoskeleton rearrangement, which is activated by three major immune mediators, biogenic monoamines, plasmatocyte-spreading peptide (PSP), and eicosanoids, part...

  8. Rsu1 regulates ethanol consumption in Drosophila and humans

    PubMed Central

    Ojelade, Shamsideen A.; Jia, Tianye; Rodan, Aylin R.; Chenyang, Tao; Kadrmas, Julie L.; Cattrell, Anna; Ruggeri, Barbara; Charoen, Pimphen; Lemaitre, Hervé; Banaschewski, Tobias; Büchel, Christian; Bokde, Arun L. W.; Carvalho, Fabiana; Conrod, Patricia J.; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny A.; Heinz, Andreas; Ittermann, Bernd; Lathrop, Mark; Lubbe, Steven; Martinot, Jean-Luc; Paus, Tomás; Smolka, Michael N.; Spanagel, Rainer; O’Reilly, Paul F.; Laitinen, Jaana; Veijola, Juha M.; Feng, Jianfeng; Desrivières, Sylvane; Jarvelin, Marjo-Riitta; Schumann, Gunter; Rothenfluh, Adrian

    2015-01-01

    Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla. PMID:26170296

  9. Rsu1 regulates ethanol consumption in Drosophila and humans.

    PubMed

    Ojelade, Shamsideen A; Jia, Tianye; Rodan, Aylin R; Chenyang, Tao; Kadrmas, Julie L; Cattrell, Anna; Ruggeri, Barbara; Charoen, Pimphen; Lemaitre, Hervé; Banaschewski, Tobias; Büchel, Christian; Bokde, Arun L W; Carvalho, Fabiana; Conrod, Patricia J; Flor, Herta; Frouin, Vincent; Gallinat, Jürgen; Garavan, Hugh; Gowland, Penny A; Heinz, Andreas; Ittermann, Bernd; Lathrop, Mark; Lubbe, Steven; Martinot, Jean-Luc; Paus, Tomás; Smolka, Michael N; Spanagel, Rainer; O'Reilly, Paul F; Laitinen, Jaana; Veijola, Juha M; Feng, Jianfeng; Desrivières, Sylvane; Jarvelin, Marjo-Riitta; Schumann, Gunter; Rothenfluh, Adrian

    2015-07-28

    Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla. PMID:26170296

  10. Regulation of Epithelial-Mesenchymal Transition in Breast Cancer Cells by Cell Contact and Adhesion

    PubMed Central

    Cichon, Magdalena A; Nelson, Celeste M; Radisky, Derek C

    2015-01-01

    Epithelial-mesenchymal transition (EMT) is a physiological program that is activated during cancer cell invasion and metastasis. We show here that EMT-related processes are linked to a broad and conserved program of transcriptional alterations that are influenced by cell contact and adhesion. Using cultured human breast cancer and mouse mammary epithelial cells, we find that reduced cell density, conditions under which cell contact is reduced, leads to reduced expression of genes associated with mammary epithelial cell differentiation and increased expression of genes associated with breast cancer. We further find that treatment of cells with matrix metalloproteinase-3 (MMP-3), an inducer of EMT, interrupts a defined subset of cell contact-regulated genes, including genes encoding a variety of RNA splicing proteins known to regulate the expression of Rac1b, an activated splice isoform of Rac1 known to be a key mediator of MMP-3-induced EMT in breast, lung, and pancreas. These results provide new insights into how MMPs act in cancer progression and how loss of cell–cell interactions is a key step in the earliest stages of cancer development. PMID:25698877

  11. Drosophila DOCK family protein sponge regulates the JNK pathway during thorax development.

    PubMed

    Morishita, Kazushige; Ozasa, Fumito; Eguchi, Koichi; Yoshioka, Yasuhide; Yoshida, Hideki; Hiai, Hiroshi; Yamaguchi, Masamitsu

    2014-01-01

    The dedicator of cytokinesis (DOCK) family proteins that are conserved in a wide variety of species are known as DOCK1-DOCK11 in mammals. The Sponge (Spg) is a Drosophila counterpart to the mammalian DOCK3. Specific knockdown of spg by pannir-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Conversely, overexpression of bsk suppressed the split thorax phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK (anti-pJNK) antibody together with examination of lacZ expression in a puckered-lacZ enhancer trap line revealed the strong reduction of the JNK activity in the spg knockdown clones. This was further confirmed by Western immunoblot analysis of extracts from wing discs of spg knockdown fly with anti-pJNK antibody. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rac1 in the wing discs. Taken together, these results indicate Spg positively regulates JNK pathway that is required for thorax development and the regulation is mediated by interaction with Rac1. PMID:25311449

  12. Roles of Aspergillus nidulans Cdc42/Rho GTPase regulators in hyphal morphogenesis and development.

    PubMed

    Si, Haoyu; Rittenour, William R; Harris, Steven D

    2016-01-01

    The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A nidulans. PMID:26932184

  13. Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line.

    PubMed

    Mascia, Anna; Gentile, Flaviana; Izzo, Antonella; Mollo, Nunzia; De Luca, Maria; Bucci, Cecilia; Nitsch, Lucio; Calì, Gaetano

    2016-08-01

    Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over-expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over-expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over-expressing cells, Rab7 and LC3B-II co-localized and co-localization in large circular structures occurred only in Rab7 over-expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co-localization of Rab7 with cortactin and f-actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over-expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP-stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over-expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695-1708, 2016. © 2015 Wiley Periodicals, Inc. PMID:26599499

  14. Hypoxia Inducible Factor-1α Regulates the Migration of Bone Marrow Mesenchymal Stem Cells via Integrin α4

    PubMed Central

    Choi, Jong Ho; Lee, Yun Bin; Jung, Jieun; Hwang, Seong Gyu; Oh, IL-Hoan; Kim, Gi Jin

    2016-01-01

    Although hypoxic environments have been known to regulate the migratory ability of bone marrow-derived mesenchymal stem cells (BM-MSCs), which is a critical factor for maximizing the therapeutic effect, the underlying mechanisms remain unclear. Therefore, we aimed to confirm the effect of hypoxia-inducible factor-1α (HIF-1α) on the migration of BM-MSCs and to analyze the interaction between HIF-1α and integrin-mediated signals. Hypoxia-activated HIF-1α significantly increased BM-MSC migration. The expression of integrin α4 was decreased in BM-MSCs by increased HIF-1α under hypoxia, whereas the expression of Rho-associated kinase 1 (ROCK1) and Rac1/2/3 was increased. After downregulation of HIF-1α by YC-1, which is an inhibitor of HIF-1α, BM-MSC migration was decreased via upregulation of integrin α4 and downregulation of ROCK1 and Rac1/2/3. Knockdown of integrin α4 by integrin α4 siRNA (siITGA4) treatment increased BM-MSC migration by upregulation of ROCK1, Rac1/2/3, and matrix metalloproteinase-2 regardless of oxygen tension. Moreover, siITGA4 treatment increased HIF-1α expression and augmented the translocation of HIF-1α into the nucleus under hypoxia. Taken together, the alternative expression of HIF-1α induced by microenvironment factors, such as hypoxia and integrin α4, may regulate the migration of BM-MSCs. These findings may provide insights to the underlying mechanisms of BM-MSC migration for successful stem cell-based therapy. PMID:26880989

  15. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    SciTech Connect

    Krauss, Robert S.

    2010-11-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre and Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed.

  16. Regulation of promyogenic signal transduction by cell-cell contact and adhesion

    PubMed Central

    Krauss, Robert S.

    2010-01-01

    Skeletal myoblast differentiation involves acquisition of the muscle-specific transcriptional program and morphological changes, including fusion into multinucleated myofibers. Differentiation is regulated by extracellular signaling cues, including cell-cell contact and adhesion. Cadherin and Ig adhesion receptors have been implicated in distinct but overlapping stages of myogenesis. N-cadherin signals through the Ig receptor Cdo to activate p38 MAP kinase, while the Ig receptor neogenin signals to activate FAK; both processes promote muscle-specific gene expression and myoblast fusion. M-cadherin activates Rac1 to enhance fusion. Specific Ig receptors (Kirre, Sns) are essential for myoblast fusion in Drosophila, also signaling through Rac, and vertebrate orthologs of Kirre and Sns have partially conserved function. Mice lacking specific cytoplasmic signaling factors activated by multiple receptors (e.g., Rac1) have strong muscle phenotypes in vivo. In contrast, mice lacking individual adhesion receptors that lie upstream of these factors have modest phenotypes. Redundancy among receptors may account for this. Many of the mammalian Ig receptors and cadherins associate with each other, and multivalent interactions within these complexes may require removal of multiple components to reveal dramatic defects in vivo. Nevertheless, it is possible that the murine adhesion receptors rate-limiting in vivo have not yet been identified or fully assessed. PMID:20471976

  17. Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells

    PubMed Central

    Lee, Hye-Jung; Bae, Song Yi; Jung, Cholomi; Park, Hyen Joo; Lee, Sang Kook

    2015-01-01

    Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2-mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of p-AMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization. PMID:26656173

  18. Transglutaminase 2 Regulates the GTPase-activating Activity of Bcr*

    PubMed Central

    Yi, Sun-Ju; Groffen, John; Heisterkamp, Nora

    2009-01-01

    Transglutaminase 2 (TG2) is a multifunctional protein that has been implicated in numerous pathologies including that of neurodegeneration and celiac disease, but the molecular interactions that mediate its diverse activities are largely unknown. Bcr and the closely related Abr negatively regulate the small G-protein Rac: loss of their combined function in vivo results in increased reactivity of innate immune cells. Bcr and Abr are GTPase-activating proteins that catalyze the hydrolysis of the GTP bound to Rac. However, how the Bcr and Abr GTPase-activating activity is regulated is not precisely understood. We here report a novel mechanism of regulation through direct protein-protein interaction with TG2. TG2 bound to the Rac-binding pocket in the GTPase-activating domains of Bcr and Abr, blocked Bcr activity and, through this mechanism, increased levels of active GTP-bound Rac and EGF-stimulated membrane ruffling. TG2 exists in at least two different conformations. Interestingly, experiments using TG2 mutants showed that Bcr exhibits preferential binding to the non-compacted conformation of TG2, in which its catalytic domain is exposed, but transamidation is not needed for the interaction. Thus, TG2 regulates levels of cellular GTP-bound Rac and actin cytoskeletal reorganization through a new mechanism involving direct inhibition of Bcr GTPase-activating activity. PMID:19840940

  19. 50 CFR 665.964 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Regulated activities. 665.964 Section 665.964 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Rose Atoll Marine...

  20. 50 CFR 665.964 - Regulated activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Regulated activities. 665.964 Section 665.964 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Rose Atoll Marine...

  1. Regulation of ROCK activity in cancer.

    PubMed

    Morgan-Fisher, Marie; Wewer, Ulla M; Yoneda, Atsuko

    2013-03-01

    Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)-loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer. PMID:23204112

  2. Regulation of brain aromatase activity in rats

    SciTech Connect

    Roselli, C.E.; Ellinwood, W.E.; Resko, J.A.

    1984-01-01

    The distribution and regulation of aromatase activity in the adult rat brain with a sensitive in vitro assay that measures the amount of /sup 3/H/sub 2/O formed during the conversion of (1 beta-/sup 3/H)androstenedione to estrone. The rate of aromatase activity in the hypothalamus-preoptic area (HPOA) was linear with time up to 1 h, and with tissue concentrations up to 5 mgeq/200 microliters incubation mixture. The enzyme demonstrated a pH optimum of 7.4 and an apparent Michaelis-Menten constant (Km) of 0.04 microns. The greatest amount of aromatase activity was found in amygdala and HPOA from intact male rats. The hippocampus, midbrain tegmentum, cerebral cortex, cerebellum, and anterior pituitary all contained negligible enzymatic activity. Castration produced a significant decrease in aromatase activity in the HPOA, but not in the amygdala or cerebral cortex. The HPOAs of male rats contained significantly greater aromatase activity than the HPOAs of female rats. In females, this enzyme activity did not change during the estrous cycle or after ovariectomy. Administration of testosterone to gonadectomized male and female rats significantly enhanced HPOA aromatase activities to levels approximating those found in HPOA from intact males. Therefore, the results suggest that testosterone, or one of its metabolites, is a major steroidal regulator of HPOA aromatase activity in rats.

  3. Regulating the regulators: modulators of transcription factor activity.

    PubMed

    Everett, Logan; Hansen, Matthew; Hannenhalli, Sridhar

    2010-01-01

    Gene transcription is largely regulated by DNA-binding transcription factors (TFs). However, the TF activity itself is modulated via, among other things, post-translational modifications (PTMs) by specific modification enzymes in response to cellular stimuli. TF-PTMs thus serve as "molecular switchboards" that map upstream signaling events to the downstream transcriptional events. An important long-term goal is to obtain a genome-wide map of "regulatory triplets" consisting of a TF, target gene, and a modulator gene that specifically modulates the regulation of the target gene by the TF. A variety of genome-wide data sets can be exploited by computational methods to obtain a rough map of regulatory triplets, which can guide directed experiments. However, a prerequisite to developing such computational tools is a systematic catalog of known instances of regulatory triplets. We first describe PTM-Switchboard, a recent database that stores triplets of genes such that the ability of one gene (the TF) to regulate a target gene is dependent on one or more PTMs catalyzed by a third gene, the modifying enzyme. We also review current computational approaches to infer regulatory triplets from genome-wide data sets and conclude with a discussion of potential future research. PTM-Switchboard is accessible at http://cagr.pcbi.upenn.edu/PTMswitchboard / PMID:20827600

  4. Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation

    PubMed Central

    Hsu, Jinn-Yuan; Chang, Kwang-Yu; Chen, Shang-Hung; Lee, Chung-Ta; Chang, Sheng-Tsung; Cheng, Hung-Chi; Chang, Wen-Chang; Chen, Ben-Kuen

    2015-01-01

    Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed by COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF enhancement of cell migration and invasion, but the addition of PGE2 compensated for this inhibitory effect in COX-2-knockdown cells. COX-2 depletion inhibited EGF-induced matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. The inhibitory effect of COX-2 depletion on MMPs and the fibronectin/Rac1/cdc42 axis were reversed by co-treatment with PGE2. Furthermore, depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results demonstrate that EGF-induced COX-2 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis. PMID:25595899

  5. The adhesion-GPCR BAI3, a gene linked to psychiatric disorders, regulates dendrite morphogenesis in neurons.

    PubMed

    Lanoue, V; Usardi, A; Sigoillot, S M; Talleur, M; Iyer, K; Mariani, J; Isope, P; Vodjdani, G; Heintz, N; Selimi, F

    2013-08-01

    Adhesion-G protein-coupled receptors (GPCRs) are a poorly studied subgroup of the GPCRs, which have diverse biological roles and are major targets for therapeutic intervention. Among them, the Brain Angiogenesis Inhibitor (BAI) family has been linked to several psychiatric disorders, but despite their very high neuronal expression, the function of these receptors in the central nervous system has barely been analyzed. Our results, obtained using expression knockdown and overexpression experiments, reveal that the BAI3 receptor controls dendritic arborization growth and branching in cultured neurons. This role is confirmed in Purkinje cells in vivo using specific expression of a deficient BAI3 protein in transgenic mice, as well as lentivirus driven knockdown of BAI3 expression. Regulation of dendrite morphogenesis by BAI3 involves activation of the RhoGTPase Rac1 and the binding to a functional ELMO1, a critical Rac1 regulator. Thus, activation of the BAI3 signaling pathway could lead to direct reorganization of the actin cytoskeleton through RhoGTPase signaling in neurons. Given the direct link between RhoGTPase/actin signaling pathways, neuronal morphogenesis and psychiatric disorders, our mechanistic data show the importance of further studying the role of the BAI adhesion-GPCRs to understand the pathophysiology of such brain diseases. PMID:23628982

  6. The adhesion-GPCR BAI3, a gene linked to psychiatric disorders, regulates dendrite morphogenesis in neurons

    PubMed Central

    Lanoue, V; Usardi, A; Sigoillot, S M; Talleur, M; Iyer, K; Mariani, J; Isope, P; Vodjdani, G; Heintz, N; Selimi, F

    2013-01-01

    Adhesion-G protein-coupled receptors (GPCRs) are a poorly studied subgroup of the GPCRs, which have diverse biological roles and are major targets for therapeutic intervention. Among them, the Brain Angiogenesis Inhibitor (BAI) family has been linked to several psychiatric disorders, but despite their very high neuronal expression, the function of these receptors in the central nervous system has barely been analyzed. Our results, obtained using expression knockdown and overexpression experiments, reveal that the BAI3 receptor controls dendritic arborization growth and branching in cultured neurons. This role is confirmed in Purkinje cells in vivo using specific expression of a deficient BAI3 protein in transgenic mice, as well as lentivirus driven knockdown of BAI3 expression. Regulation of dendrite morphogenesis by BAI3 involves activation of the RhoGTPase Rac1 and the binding to a functional ELMO1, a critical Rac1 regulator. Thus, activation of the BAI3 signaling pathway could lead to direct reorganization of the actin cytoskeleton through RhoGTPase signaling in neurons. Given the direct link between RhoGTPase/actin signaling pathways, neuronal morphogenesis and psychiatric disorders, our mechanistic data show the importance of further studying the role of the BAI adhesion-GPCRs to understand the pathophysiology of such brain diseases. PMID:23628982

  7. Src regulates the activity of SIRT2

    SciTech Connect

    Choi, You Hee; Kim, Hangun; Lee, Sung Ho; Jin, Yun-Hye; Lee, Kwang Youl

    2014-07-25

    Highlights: • Src decreases the protein levels of Sirt2. • Src inhibitor and knockdown of Src increase the protein levels of Sirt2. • Src interacts with and phosphorylates Sirt2. • Src regulate the activity of Sirt2. - Abstract: SIRT2 is a mammalian member of the Sirtuin family of NAD{sup +}-dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation.

  8. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin.

    PubMed

    Fusté, Noel P; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  9. Fc-receptor-mediated phagocytosis is regulated by mechanical properties of the target

    NASA Technical Reports Server (NTRS)

    Beningo, Karen A.; Wang, Yu-li

    2002-01-01

    Phagocytosis is an actin-based process used by macrophages to clear particles greater than 0.5 microm in diameter. In addition to its role in immunological responses, phagocytosis is also necessary for tissue remodeling and repair. To prevent catastrophic autoimmune reactions, phagocytosis must be tightly regulated. It is commonly assumed that the recognition/selection of phagocytic targets is based solely upon receptor-ligand binding. Here we report an important new criterion, that mechanical parameters of the target can dramatically affect the efficiency of phagocytosis. When presented with particles of identical chemical properties but different rigidity, macrophages showed a strong preference to engulf rigid objects. Furthermore, phagocytosis of soft particles can be stimulated with the microinjection of constitutively active Rac1 but not RhoA, and with lysophosphatidic acid, an agent known to activate the small GTP-binding proteins of the Rho family. These data suggest a Rac1-dependent mechanosensory mechanism for phagocytosis, which probably plays an important role in a number of physiological and pathological processes from embryonic development to autoimmune diseases.

  10. Cytoplasmic cyclin D1 regulates cell invasion and metastasis through the phosphorylation of paxillin

    PubMed Central

    Fusté, Noel P.; Fernández-Hernández, Rita; Cemeli, Tània; Mirantes, Cristina; Pedraza, Neus; Rafel, Marta; Torres-Rosell, Jordi; Colomina, Neus; Ferrezuelo, Francisco; Dolcet, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis. PMID:27181366

  11. The Focal Adhesion-Localized CdGAP Regulates Matrix Rigidity Sensing and Durotaxis

    PubMed Central

    Wormer, Duncan B.; Davis, Kevin A.; Henderson, James H.; Turner, Christopher E.

    2014-01-01

    Motile cells are capable of sensing the stiffness of the surrounding extracellular matrix through integrin-mediated focal adhesions and migrate towards regions of higher rigidity in a process known as durotaxis. Durotaxis plays an important role in normal development and disease progression, including tumor invasion and metastasis. However, the signaling mechanisms underlying focal adhesion-mediated rigidity sensing and durotaxis are poorly understood. Utilizing matrix-coated polydimethylsiloxane gels to manipulate substrate compliance, we show that cdGAP, an adhesion-localized Rac1 and Cdc42 specific GTPase activating protein, is necessary for U2OS osteosarcoma cells to coordinate cell shape changes and migration as a function of extracellular matrix stiffness. CdGAP regulated rigidity-dependent motility by controlling membrane protrusion and adhesion dynamics, as well as by modulating Rac1 activity. CdGAP was also found to be necessary for U2OS cell durotaxis. Taken together, these data identify cdGAP as an important component of an integrin-mediated signaling pathway that senses and responds to mechanical cues in the extracellular matrix in order to coordinate directed cell motility. PMID:24632816

  12. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    SciTech Connect

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  13. The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation

    PubMed Central

    Huff, Lauren P.; DeCristo, Molly J.; Trembath, Dimitri; Kuan, Pei Fen; Yim, Margaret; Liu, Jinsong; Cook, Danielle R.; Miller, C. Ryan; Der, Channing J.

    2013-01-01

    Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation. PMID:24386507

  14. Regulators of Slc4 bicarbonate transporter activity

    PubMed Central

    Thornell, Ian M.; Bevensee, Mark O.

    2015-01-01

    The Slc4 family of transporters is comprised of anion exchangers (AE1-4), Na+-coupled bicarbonate transporters (NCBTs) including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2), electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2), and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE), as well as a borate transporter (BTR1). These transporters regulate intracellular pH (pHi) and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO−3 either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO−3 transporter contributes to a cell's ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s) (e.g., Na+ or Cl−). In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both well-known and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family. PMID:26124722

  15. Team Regulation, Regulation of Social Activities or Co-Regulation: Different Labels for Effective Regulation of Learning in CSCL

    ERIC Educational Resources Information Center

    Saab, Nadira

    2012-01-01

    Computer-supported collaborative learning (CSCL) is an approach to learning in which learners can actively and collaboratively construct knowledge by means of interaction and joint problem solving. Regulation of learning is especially important in the domain of CSCL. Next to the regulation of task performance, the interaction between learners who…

  16. Activities and regulation of peptidoglycan synthases

    PubMed Central

    Egan, Alexander J. F.; Biboy, Jacob; van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-01-01

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN. PMID:26370943

  17. Molecular mechanisms regulating NLRP3 inflammasome activation

    PubMed Central

    Jo, Eun-Kyeong; Kim, Jin Kyung; Shin, Dong-Min; Sasakawa, Chihiro

    2016-01-01

    Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inflammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome. PMID:26549800

  18. Regulation of Trichophyton rubrum proteolytic activity.

    PubMed Central

    Apodaca, G; McKerrow, J H

    1989-01-01

    Trichophyton rubrum is the most common dermatophyte of humans and normally colonizes the superficial layers of the epidermis (stratum corneum). Several proteinases with a possible role in the metabolism of host proteins have been purified from this fungus. The regulation of these enzymes and their role in fungal metabolism were studied at the biochemical level. General proteolytic (azocollytic) activity was repressed when log-phase cultures of T. rubrum were grown in a minimal medium that contained readily metabolized sources of carbon, nitrogen, sulfur, and phosphorus. When either carbon, nitrogen, or sulfur was deleted from this minimal medium, azocollytic activity was derepressed. In all cases a high-molecular-weight activity (Mr, greater than 200,000) was expressed. A 71,000-Mr proteinase was observed in nitrogen-depleted cultures, and proteolytic species of Mr 124,000 and 27,000 were secreted in sulfur-depleted cultures. The addition of either inorganic (MgSO4, Na2SO3, NaS2O3) or organic (methionine, cysteine) sulfur to the sulfur-depleted medium repressed the expression of azocollytic activity. In contrast, keratinolytic activity was not repressed by carbon, nitrogen, or sulfur but instead was induced when a protein source was included in the minimal medium. Stationary-phase cultures of T. rubrum secreted all proteolytic activities constitutively. Unlike log-phase cultures, the stationary-phase cultures secreted azocollytic, elastinolytic, and keratinolytic activity in minimal medium. These activities fell in the carbon-, nitrogen-, and phosphorous-depleted media but remained high in sulfur-depleted medium. The following model is proposed for the regulation of T. rubrum proteolytic activity. In the initial stages of infection, T. rubrum grows logarithmically. In this state, proteolytic activity is derepressed whenever carbon, nitrogen, or sulfur is lacking in the fungal milieu. The general proteinases produced would act on the nonkeratinous proteins in the

  19. Magnetoreception Regulates Male Courtship Activity in Drosophila

    PubMed Central

    Wu, Chia-Lin; Fu, Tsai-Feng; Chiang, Meng-Hsuan; Chang, Yu-Wei; Her, Jim-Long; Wu, Tony

    2016-01-01

    The possible neurological and biophysical effects of magnetic fields on animals is an area of active study. Here, we report that courtship activity of male Drosophila increases in a magnetic field and that this effect is regulated by the blue light-dependent photoreceptor cryptochrome (CRY). Naïve male flies exhibited significantly increased courtship activities when they were exposed to a ≥ 20-Gauss static magnetic field, compared with their behavior in the natural environment (0 Gauss). CRY-deficient flies, cryb and crym, did not show an increased courtship index in a magnetic field. RNAi-mediated knockdown of cry in cry-GAL4-positive neurons disrupted the increased male courtship activity in a magnetic field. Genetically expressing cry under the control of cry-GAL4 in the CRY-deficient flies restored the increase in male courtship index that occurred in a magnetic field. Interestingly, artificially activating cry-GAL4-expressing neurons, which include large ventral lateral neurons and small ventral lateral neurons, via expression of thermosensitive cation channel dTrpA1, also increased the male courtship index. This enhancement was abolished by the addition of the cry-GAL80 transgene. Our results highlight the phenomenon of increased male courtship activity caused by a magnetic field through CRY-dependent magnetic sensation in CRY expression neurons in Drosophila. PMID:27195955

  20. Regulation of pokemon 1 activity by sumoylation.

    PubMed

    Roh, Hee-Eun; Lee, Min-Nyung; Jeon, Bu-Nam; Choi, Won-Il; Kim, Yoo-Jin; Yu, Mi-Young; Hur, Man-Wook

    2007-01-01

    Pokemon 1 is a proto-oncogenic transcriptional regulator that contains a POZ domain at the N-terminus and four Kruppel-like zinc fingers at the C-terminus. Pokemon 1 plays an important role in adipogenesis, osteogenesis, oncogenesis, and transcription of NF-kB responsive genes. Recent reports have shown that biological activities of transcription factors are regulated by sumolylation. We investigated whether Pokemon 1 is post-translationally modified by sumoylation and whether the modification affects Pokemon 1's transcriptional properties. We found that Pokemon 1 is sumoylated in vitro and in vivo. Upon careful analysis of the amino acid sequence of Pokemon 1, we found ten potential sumoylation sites located at lysines 61, 354, 371, 379, 383, 396, 486, 487, 536 and 539. We mutated each of these amino acids into arginine and tested whether the mutation could affect the transcriptional properties of Pokemon 1 on the Pokemon 1 responsive genes, such as ADH5/FDH and pG5-FRE-Luc. Wild-type Pokemon 1 potently represses transcription of ADH5/FDH. Most of the mutants, however, were weaker transcription repressors and repressed transcription 1.3-3.3 fold less effective. Although potential sumoylation sites were located close to the DNA binding domain or the nuclear localization sequence, the mutations did not alter nuclear localization or DNA binding activity. In addition, on the pG5-FRE-Luc test promoter construct, ectopic SUMO-1 repressed transcription in the presence of Pokemon 1. The sumoylation target lysine residue at amino acid 61, which is located in the middle of the POZ-domain, is important because K61R mutation resulted in a much weaker molecular interaction with corepressors. Our data suggest that Pokemon 1's activity as a transcription factor may involve sumoylation, and that sumoylation might be important in the regulation of transcription by Pokemon 1. PMID:17595526

  1. Regulation of Aicda expression and AID activity.

    PubMed

    Zan, Hong; Casali, Paolo

    2013-03-01

    Activation-induced cytidine deaminase (AID) is expressed in a B cell differentiation stage-specific fashion and is essential for immunoglobulin (Ig) gene class switch DNA recombination (CSR) and somatic hypermutation (SHM). CSR and SHM play a central role in the maturation of antibody and autoantibody responses. AID displays a mutagenic activity by catalyzing targeted deamination of deoxycytidine (dC) residues in DNA resulting in dU:dG mismatches, which are processed into point-mutations in SHM or double-strand breaks (DSBs) in CSR. Although AID specifically targets the Ig gene loci (IgH, Igκ and Igλ), it can also home into a wide array of non-Ig genes in B-and non-B-cell backgrounds. Aberrant expression of AID is associated with multiple diseases such as allergy, inflammation, autoimmunity and cancer. In autoimmune systemic lupus erythematosus, dysregulated AID expression underpins increased CSR, SHM and autoantibody production. As a potent mutator, AID is under stringent transcriptional, post-transcriptional and post-translational regulation. AID is also regulated in its targeting and enzymatic function. In resting naïve or memory B cells, AID transcripts and protein are undetectable. These, however, are readily and significantly up-regulated in B cells induced to undergo CSR and/or SHM. Transcription factors, such as HoxC4 and NF-κB, which are up-regulated in a B cell lineage-and/or differentiation stage-specific manner, regulate the induction of AID. HoxC4 induces AID expression by directly binding to the AID gene promoter through an evolutionarily conserved 5'-ATTT-3' motif. HoxC4 is induced by the same stimuli that induce AID and CSR. It is further up-regulated by estrogen through three estrogen responsive elements in its promoter region. The targeting of AID to switch (S) regions is mediated by 14-3-3 adaptor proteins, which specifically bind to 5'-AGCT-3' repeats that are exist at high frequency in S region cores. Like HoxC4, 14-3-3 adaptors are induced

  2. Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets.

    PubMed

    Beck, S; Fotinos, A; Lang, F; Gawaz, M; Elvers, M

    2013-01-01

    Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that - beside the GAP domain - the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation. PMID:22975681

  3. Endoglin regulates cyclooxygenase-2 expression and activity.

    PubMed

    Jerkic, Mirjana; Rivas-Elena, Juan V; Santibanez, Juan F; Prieto, Marta; Rodríguez-Barbero, Alicia; Perez-Barriocanal, Fernando; Pericacho, Miguel; Arévalo, Miguel; Vary, Calvin P H; Letarte, Michelle; Bernabeu, Carmelo; López-Novoa, Jose M

    2006-08-01

    The endoglin heterozygous (Eng(+/-)) mouse, which serves as a model of hereditary hemorrhagic telangiectasia (HHT), was shown to express reduced levels of endothelial NO synthase (eNOS) with impaired activity. Because of intricate changes in vasomotor function in the Eng(+/-) mice and the potential interactions between the NO- and prostaglandin-producing pathways, we assessed the expression and function of cyclooxygenase (COX) isoforms. A specific upregulation of COX-2 in the vascular endothelium and increased urinary excretion of prostaglandin E(2) were observed in the Eng(+/-) mice. Specific COX-2 inhibition with parecoxib transiently increased arterial pressure in Eng(+/-) but not in Eng(+/+) mice. Transfection of endoglin in L6E9 myoblasts, shown previously to stimulate eNOS expression, led to downregulation of COX-2 with no change in COX-1. In addition, COX-2 promoter activity and protein levels were inversely correlated with endoglin levels, in doxycyclin-inducible endothelial cells. Chronic NO synthesis inhibition with N(omega)-nitro-l-arginine methyl ester induced a marked increase in COX-2 only in the normal Eng(+/+) mice. N(omega)-nitro-l-arginine methyl ester also increased COX-2 expression and promoter activity in doxycyclin-inducible endoglin expressing endothelial cells, but not in control cells. The level of COX-2 expression following transforming growth factor-beta1 treatment was less in endoglin than in mock transfected L6E9 myoblasts and was higher in human endothelial cells silenced for endoglin expression. Our results indicate that endoglin is involved in the regulation of COX-2 activity. Furthermore, reduced endoglin levels and associated impaired NO production may be responsible, at least in part, for augmented COX-2 expression and activity in the Eng(+/-) mice. PMID:16840721

  4. Structural Basis for Plexin Activation and Regulation.

    PubMed

    Kong, Youxin; Janssen, Bert J C; Malinauskas, Tomas; Vangoor, Vamshidhar R; Coles, Charlotte H; Kaufmann, Rainer; Ni, Tao; Gilbert, Robert J C; Padilla-Parra, Sergi; Pasterkamp, R Jeroen; Jones, E Yvonne

    2016-08-01

    Class A plexins (PlxnAs) act as semaphorin receptors and control diverse aspects of nervous system development and plasticity, ranging from axon guidance and neuron migration to synaptic organization. PlxnA signaling requires cytoplasmic domain dimerization, but extracellular regulation and activation mechanisms remain unclear. Here we present crystal structures of PlxnA (PlxnA1, PlxnA2, and PlxnA4) full ectodomains. Domains 1-9 form a ring-like conformation from which the C-terminal domain 10 points away. All our PlxnA ectodomain structures show autoinhibitory, intermolecular "head-to-stalk" (domain 1 to domain 4-5) interactions, which are confirmed by biophysical assays, live cell fluorescence microscopy, and cell-based and neuronal growth cone collapse assays. This work reveals a 2-fold role of the PlxnA ectodomains: imposing a pre-signaling autoinhibitory separation for the cytoplasmic domains via intermolecular head-to-stalk interactions and supporting dimerization-based PlxnA activation upon ligand binding. More generally, our data identify a novel molecular mechanism for preventing premature activation of axon guidance receptors. PMID:27397516

  5. δ-Catenin Activates Rho GTPase, Promotes Lymphangiogenesis and Growth of Tumor Metastases

    PubMed Central

    Lin, P. Charles

    2015-01-01

    δ-catenin, an adherens junctions protein, is not only involved in early development, cell-cell adhesion and cell motility in neuronal cells, but it also plays an important role in vascular endothelial cell motility and pathological angiogenesis. In this study, we report a new function of δ-catenin in lymphangiogenesis. Consistent with expression of δ-catenin in vascular endothelial cells, we detected expression of the gene in lymphatic endothelial cells (LECs). Ectopic expression of δ-catenin in LECs increased cell motility and lymphatic vascular network formation in vitro and lymphangiogenesis in vivo in a Matrigel plug assay. Conversely, knockdown of δ-catenin in LECs impaired lymphangiogenesis in vitro and in vivo. Biochemical analysis shows that δ-catenin regulates activation of Rho family small GTPases, key mediators in cell motility. δ-catenin activates Rac1 and Cdc42 but inhibits RhoA in LECs. Notably, blocking of Rac1 activation impaired δ-catenin mediated lymphangiogenesis in a Matrigel assay. Consistently, loss of δ-catenin in mice inhibited the growth of tumor metastases. Taken together, these findings identify a new function of δ-catenin in lymphangiogenesis and tumor growth/metastasis, likely through modulation of small Rho GTPase activation. Targeting δ-catenin may offer a new way to control tumor metastasis. PMID:25635825

  6. Low-level laser therapy regulates microglial function through Src-mediated signaling pathways: implications for neurodegenerative diseases

    PubMed Central

    2012-01-01

    Background Activated microglial cells are an important pathological component in brains of patients with neurodegenerative diseases. The purpose of this study was to investigate the effect of He-Ne (632.8 nm, 64.6 mW/cm2) low-level laser therapy (LLLT), a non-damaging physical therapy, on activated microglia, and the subsequent signaling events of LLLT-induced neuroprotective effects and phagocytic responses. Methods To model microglial activation, we treated the microglial BV2 cells with lipopolysaccharide (LPS). For the LLLT-induced neuroprotective study, neuronal cells with activated microglial cells in a Transwell™ cell-culture system were used. For the phagocytosis study, fluorescence-labeled microspheres were added into the treated microglial cells to confirm the role of LLLT. Results Our results showed that LLLT (20 J/cm2) could attenuate toll-like receptor (TLR)-mediated proinflammatory responses in microglia, characterized by down-regulation of proinflammatory cytokine expression and nitric oxide (NO) production. LLLT-triggered TLR signaling inhibition was achieved by activating tyrosine kinases Src and Syk, which led to MyD88 tyrosine phosphorylation, thus impairing MyD88-dependent proinflammatory signaling cascade. In addition, we found that Src activation could enhance Rac1 activity and F-actin accumulation that typify microglial phagocytic activity. We also found that Src/PI3K/Akt inhibitors prevented LLLT-stimulated Akt (Ser473 and Thr308) phosphorylation and blocked Rac1 activity and actin-based microglial phagocytosis, indicating the activation of Src/PI3K/Akt/Rac1 signaling pathway. Conclusions The present study underlines the importance of Src in suppressing inflammation and enhancing microglial phagocytic function in activated microglia during LLLT stimulation. We have identified a new and important neuroprotective signaling pathway that consists of regulation of microglial phagocytosis and inflammation under LLLT treatment. Our research

  7. Discoidin domain receptor 1 activation suppresses alpha2beta1 integrin-dependent cell spreading through inhibition of Cdc42 activity.

    PubMed

    Yeh, Yi-Chun; Wang, Chau-Zen; Tang, Ming-Jer

    2009-01-01

    Upregulation and overexpression of discoidin domain receptor 1 (DDR1) have been implied in the regulation of kidney development and progression of cancers. Our previous studies with Mardin-Darby canine kidney (MDCK) cells showed that overexpression of DDR1 inhibited cell spreading, whereas dominant negative DDR1 promoted cell spreading on collagen-coated dish. Cell spreading is an important characteristic for cell differentiation and survival. However, little is known about the molecular mechanisms underlying the role of DDR1 in cell spreading. We have found here a novel signaling pathway of DDR1 consisting of Cdc42 that regulates the assembly and disassembly of cytoskeleton and cell spreading in MDCK cells. Cell spreading involves the organization of cytoskeleton that is mainly regulated by Rho-family GTPases. We assessed the activity of Rho-family GTPases and transfected MDCK cells with constitutively active or dominant negative GTPases, and quantified the extent of cell spreading. These results showed that DDR1 decreased the filamentous actin ratio and Rac1/Cdc42 activities, but had no effects on RhoA activity. Neither constitutively active nor dominant negative Rac1 altered DDR1-inhibited cell spreading. Constitutively active Cdc42 could rescue the DDR1-inhibited cell spreading, whereas dominant negative Cdc42 inhibited cell spreading, indicating that DDR1-inhibited cell spreading is Cdc42 dependent. With the use of alpha(2)beta(1) integrin blocking antibody, we showed that collagen-induced Cdc42 activation was mediated by alpha(2)beta(1) integrin. Moreover, ectopic FAK expression enhanced the Cdc42 activity. Reducing FAK activity by dominant negative FAK (FRNK) markedly abolished the Cdc42 activity. These findings show that DDR1a/b activation inhibits cell spreading through suppressing alpha(2)beta(1) integrin-mediated Cdc42 activation. PMID:18780290

  8. Association between Gαi2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis.

    PubMed

    Li, Hongyan; Yang, Lei; Fu, Hui; Yan, Jianshe; Wang, Ying; Guo, Hua; Hao, Xishan; Xu, Xuehua; Jin, Tian; Zhang, Ning

    2013-01-01

    The chemokine CXCL12 and its G-protein-coupled receptor CXCR4 control the migration, invasiveness and metastasis of breast cancer cells. Binding of CXCL12 to CXCR4 triggers activation of heterotrimeric Gi proteins that regulate actin polymerization and migration. However, the pathways linking chemokine G-protein-coupled receptor/Gi signalling to actin polymerization and cancer cell migration are not known. Here we show that CXCL12 stimulation promotes interaction between Gαi2 and ELMO1. Gi signalling and ELMO1 are both required for CXCL12-mediated actin polymerization, migration and invasion of breast cancer cells. CXCL12 triggers a Gαi2-dependent membrane translocation of ELMO1, which associates with Dock180 to activate small G-proteins Rac1 and Rac2. In vivo, ELMO1 expression is associated with lymph node and distant metastasis, and knocking down ELMO1 impairs metastasis to the lung. Our findings indicate that a chemokine-controlled pathway, consisting of Gαi2, ELMO1/Dock180, Rac1 and Rac2, regulates the actin cytoskeleton during breast cancer metastasis. PMID:23591873

  9. Endothelial cell migration during murine yolk sac vascular remodeling occurs by means of a Rac1 and FAK activation pathway in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The molecular mechanism(s) controlling cell migration during vascular morphogenesis in vivo remain largely undefined. To address this within a physiological context, we used retinaldehyde dehydrogenase-2 (Raldh2) null mouse embryos and demonstrate that retinoic acid (RA) deficiency results in abnorm...

  10. Activity and Regulation of Archaeal DNA Alkyltransferase

    PubMed Central

    Perugino, Giuseppe; Vettone, Antonella; Illiano, Giuseppina; Valenti, Anna; Ferrara, Maria C.; Rossi, Mosè; Ciaramella, Maria

    2012-01-01

    Agents that form methylation adducts in DNA are highly mutagenic and carcinogenic, and organisms have evolved specialized cellular pathways devoted to their repair, including DNA alkyltransferases. These are proteins conserved in eucarya, bacteria and archaea, acting by a unique reaction mechanism, which leads to direct repair of DNA alkylation damage and irreversible protein alkylation. The alkylated form of DNA alkyltransferases is inactive, and in eukaryotes, it is rapidly directed to degradation. We report here in vitro and in vivo studies on the DNA alkyltransferase from the thermophilic archaeon Sulfolobus solfataricus (SsOGT). The development of a novel, simple, and sensitive fluorescence-based assay allowed a careful characterization of the SsOGT biochemical and DNA binding activities. In addition, transcriptional and post-translational regulation of SsOGT by DNA damage was studied. We show that although the gene transcription is induced by alkylating agent treatment, the protein is degraded in vivo by an alkylation-dependent mechanism. These experiments suggest a striking conservation, from archaea to humans, of this important pathway safeguarding genome stability. PMID:22167184

  11. 76 FR 12364 - Agency Information Collection Activities: Bonded Warehouse Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-07

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Bonded Warehouse... Bonded Warehouse Regulations. This request for comment is being made pursuant to the Paperwork Reduction... concerning the following information collection: Title: Bonded Warehouse Regulations. OMB Number:...

  12. Salmonella enterica Serovar Typhi conceals the invasion-associated type three secretion system from the innate immune system by gene regulation.

    PubMed

    Winter, Sebastian E; Winter, Maria G; Poon, Victor; Keestra, A Marijke; Sterzenbach, Torsten; Faber, Franziska; Costa, Luciana F; Cassou, Fabiane; Costa, Erica A; Alves, Geraldo E S; Paixão, Tatiane A; Santos, Renato L; Bäumler, Andreas J

    2014-07-01

    Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression. PMID:24992093

  13. Salmonella enterica Serovar Typhi Conceals the Invasion-Associated Type Three Secretion System from the Innate Immune System by Gene Regulation

    PubMed Central

    Winter, Sebastian E.; Winter, Maria G.; Poon, Victor; Keestra, A. Marijke; Sterzenbach, Torsten; Faber, Franziska; Costa, Luciana F.; Cassou, Fabiane; Costa, Erica A.; Alves, Geraldo E. S.; Paixão, Tatiane A.; Santos, Renato L.; Bäumler, Andreas J.

    2014-01-01

    Delivery of microbial products into the mammalian cell cytosol by bacterial secretion systems is a strong stimulus for triggering pro-inflammatory host responses. Here we show that Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, tightly regulates expression of the invasion-associated type III secretion system (T3SS-1) and thus fails to activate these innate immune signaling pathways. The S. Typhi regulatory protein TviA rapidly repressed T3SS-1 expression, thereby preventing RAC1-dependent, RIP2-dependent activation of NF-κB in epithelial cells. Heterologous expression of TviA in S. enterica serovar Typhimurium (S. Typhimurium) suppressed T3SS-1-dependent inflammatory responses generated early after infection in animal models of gastroenteritis. These results suggest that S. Typhi reduces intestinal inflammation by limiting the induction of pathogen-induced processes through regulation of virulence gene expression. PMID:24992093

  14. KCC2 Gates Activity-Driven AMPA Receptor Traffic through Cofilin Phosphorylation.

    PubMed

    Chevy, Quentin; Heubl, Martin; Goutierre, Marie; Backer, Stéphanie; Moutkine, Imane; Eugène, Emmanuel; Bloch-Gallego, Evelyne; Lévi, Sabine; Poncer, Jean Christophe

    2015-12-01

    Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons. PMID:26631461

  15. Mechanisms of specificity in neuronal activity-regulated gene transcription

    PubMed Central

    Lyons, Michelle R.; West, Anne E.

    2011-01-01

    The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. PMID:21620929

  16. Cysteine Cathepsin Activity Regulation by Glycosaminoglycans

    PubMed Central

    Lenarčič, Brigita

    2014-01-01

    Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail. PMID:25587532

  17. [Regulation of peptide hydrolase activity in psoriasis].

    PubMed

    Suworow, A P

    1990-01-01

    Clinico-biological examination of 154 patients with psoriasis resulted in data showing high activity of endo- and exopeptidases in efflorescences of that dermatosis. This was accompanied by depressed activity of trypsin inhibitor. At the same time magnesium deficiency, polysaccharide decrease and leucocyte increase were stated to be in the focus of skin damage. That character of interrelation, which play an important role in the pathogenesis of this widespread skin disease, is demonstrated. PMID:2257941

  18. Desmethylanhydroicaritin isolated from Sophora flavescens, shows antitumor activities in U87MG cells via inhibiting the proliferation, migration and invasion.

    PubMed

    Kang, Chang-Won; Kim, Nan-Hee; Jung, Huyn Ah; Choi, Hyung-Wook; Kang, Min-Jae; Choi, Jae-Sue; Kim, Gun-Do

    2016-04-01

    This study is the first report of the antitumor activities of desmethylanhydroicaritin (DMAI) isolated from Sophora flavescens on U87MG cells. Human glioblastoma is one of the most aggressive malignant type of brain tumors and highly diffuses to around normal brain tissues. DMAI showed anti-proliferation effects on U87MG cells at the concentration of 30μM, however did not affect to HEK-293 cells. DMAI induced anti-proliferation effects via ERK/MAPK, PI3K/Akt/mTOR signal pathway and G2/M phase cell cycle arrest. DMAI led to morphological change and inhibition of filapodia formation through regulation of Rac 1 and Cdc 42. In addition, migration and invasion of U87MG cells were inhibited by DMAI via down-regulation of matrix metalloproteinase (MMP) -2 and MMP -9 expressions and activities. Our results suggest that DMAI has a potential as a therapeutic agent against glioblastoma cells. PMID:26991848

  19. Dietary methanol regulates human gene activity.

    PubMed

    Shindyapina, Anastasia V; Petrunia, Igor V; Komarova, Tatiana V; Sheshukova, Ekaterina V; Kosorukov, Vyacheslav S; Kiryanov, Gleb I; Dorokhov, Yuri L

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  20. Endogenous Methanol Regulates Mammalian Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Petrunia, Igor V.; Shindyapina, Anastasia V.; Silachev, Denis N.; Sheshukova, Ekaterina V.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis. PMID:24587296

  1. Dietary Methanol Regulates Human Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Sheshukova, Ekaterina V.; Kosorukov, Vyacheslav S.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  2. 15 CFR 922.102 - Prohibited or otherwise regulated activities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 3 2011-01-01 2011-01-01 false Prohibited or otherwise regulated activities. 922.102 Section 922.102 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT NATIONAL...

  3. Physical Activity and Self-Regulation Strategy Use in Adolescents

    ERIC Educational Resources Information Center

    Matthews, James; Moran, Aidan

    2011-01-01

    Objective: To examine the degree to which the use of selected theoretically derived self-regulation strategies (eg, goal setting) could predict adolescents' self-reported leisure-time physical activity behavior. Method: Two hundred thirty-three (M age = 15.88) high school students completed measures assessing their self-regulation strategy use and…

  4. 15 CFR 922.152 - Prohibited or otherwise regulated activities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 3 2011-01-01 2011-01-01 false Prohibited or otherwise regulated activities. 922.152 Section 922.152 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE OCEAN AND COASTAL RESOURCE MANAGEMENT NATIONAL...

  5. Renalase regulates peripheral and central dopaminergic activities

    PubMed Central

    Serrão, Maria Paula; Soares-Silva, Isabel; Fernandes-Cerqueira, Cátia; Simões-Silva, Liliana; Pinho, Maria João; Remião, Fernando; Sampaio-Maia, Benedita; Desir, Gary V.; Pestana, Manuel

    2014-01-01

    Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency. PMID:25411385

  6. Antiviral activity of squalamine: Role of electrostatic membrane binding

    NASA Astrophysics Data System (ADS)

    Beckerman, Bernard; Qu, Wei; Mishra, Abhijit; Zasloff, Michael; Wong, Gerard; Luijten, Erik

    2012-02-01

    Recent workootnotetextM. Zasloff et al., Proc. Nat. Acad. Sci. (USA) 108, 15978 (2011). has demonstrated that squalamine, a molecule found in the liver of sharks, exhibits broad-spectrum antiviral properties. It has been proposed that this activity results from the charge-density matching of squalamine and phospholipid membranes, causing squalamine to bind to membranes and displace proteins such as Rac1 that are crucial for the viral replication cycle. Here we investigate this hypothesis by numerical simulation of a coarse-grained model for the competition between Rac1 and squalamine in binding affinity to a flat lipid bilayer. We perform free-energy calculations to test the ability of squalamine to condense stacked bilayer systems and thereby displace bulkier Rac1 molecules. We directly compare our findings to small-angle x-ray scattering results for the same setup.

  7. Regulation of Hippo signalling by p38 signalling.

    PubMed

    Huang, Dashun; Li, Xiaojiao; Sun, Li; Huang, Ping; Ying, Hao; Wang, Hui; Wu, Jiarui; Song, Haiyun

    2016-08-01

    The Hippo signalling pathway has a crucial role in growth control during development, and its dysregulation contributes to tumorigenesis. Recent studies uncover multiple upstream regulatory inputs into Hippo signalling, which affects phosphorylation of the transcriptional coactivator Yki/YAP/TAZ by Wts/Lats. Here we identify the p38 mitogen-activated protein kinase (MAPK) pathway as a new upstream branch of the Hippo pathway. In Drosophila, overexpression of MAPKK gene licorne (lic), or MAPKKK gene Mekk1, promotes Yki activity and induces Hippo target gene expression. Loss-of-function studies show that lic regulates Hippo signalling in ovary follicle cells and in the wing disc. Epistasis analysis indicates that Mekk1 and lic affect Hippo signalling via p38b and wts We further demonstrate that the Mekk1-Lic-p38b cascade inhibits Hippo signalling by promoting F-actin accumulation and Jub phosphorylation. In addition, p38 signalling modulates actin filaments and Hippo signalling in parallel to small GTPases Ras, Rac1, and Rho1. Lastly, we show that p38 signalling regulates Hippo signalling in mammalian cell lines. The Lic homologue MKK3 promotes nuclear localization of YAP via the actin cytoskeleton. Upregulation or downregulation of the p38 pathway regulates YAP-mediated transcription. Our work thus reveals a conserved crosstalk between the p38 MAPK pathway and the Hippo pathway in growth regulation. PMID:27402810

  8. The Dioxin Receptor Regulates the Constitutive Expression of the Vav3 Proto-Oncogene and Modulates Cell Shape and Adhesion

    PubMed Central

    Carvajal-Gonzalez, Jose M.; Mulero-Navarro, Sonia; Roman, Angel Carlos; Sauzeau, Vincent; Merino, Jaime M.; Bustelo, Xose R.

    2009-01-01

    The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR−/−) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR−/− cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR−/− fibroblasts had reduced Rac1 activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Rac1 shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knockdown of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3−/− MEFs, as AhR−/− mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states. PMID:19158396

  9. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  10. Regulation of Adherens Junctions in Trabecular Meshwork Cells by Rac GTPase and their influence on Intraocular Pressure

    PubMed Central

    Pattabiraman, Padmanabhan P; Epstein, David L; Rao, Ponugoti Vasantha

    2013-01-01

    Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits. PMID:24932460

  11. Regulation of Adherens Junctions in Trabecular Meshwork Cells by Rac GTPase and their influence on Intraocular Pressure.

    PubMed

    Pattabiraman, Padmanabhan P; Epstein, David L; Rao, Ponugoti Vasantha

    2013-06-01

    Intercellular adherens junctions and cell-extracellular matrix interactions are presumed to influence aqueous humor (AH) drainage via the conventional route, however, their direct role in modulation of intraocular pressure (IOP) is not well understood. Here, we investigated the role of Rac GTPase signaling in basal and growth factor-induced formation of adherens junctions in human trabecular meshwork (HTM) cells as compared to human umbilical vascular endothelial cells, and evaluated the effects of inhibition of Rac GTPase activity on IOP in rabbits. Expression of a constitutively active Rac1 GTPase or treatment with platelet derived growth factor (PDGF), a known activator of Rac GTPase, induced formation of β-catenin-based adherens junctions, actin cytoskeletal reorganization and membrane ruffle in HTM cells. In contrast, treatment of HTM cells with inhibitors of Rac GTPase caused cell-cell separation, a decrease in adherens junctions, and reorganization of actin stress fibers to the cell cortical regions and focal adhesion to the cell leading edges. Both, constitutively active Rac1 and PDGF stimulated generation of Reactive Oxygen Species (ROS) in HTM cells, and ROS were found to increase adherens junction formation and transendothelial electrical resistance (TEER) in HTM cells. Topical application of Rac GTPase inhibitors (EHT1864 and NSC23766), however, only marginally influenced IOP in rabbit eyes. Taken together, these data reveal that while Rac GTPase signaling plays a significant role in regulation of adherens junctions, ROS production and TEER in cells of the AH outflow pathway, Rac inhibitors showed only a marginal influence on IOP in live rabbits. PMID:24932460

  12. The epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells

    PubMed Central

    Hu, Weiwei; Zhu, Liqi; Yang, Xing; Lin, Jian; Yang, Qian

    2016-01-01

    Transmissible gastroenteritis virus (TGEV), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. The porcine intestinal epithelium is the target of TGEV infection, but the mechanisms that TGEV disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. We not only found that TGEV infection stimulates F-actin to gather at the cell membrane but the disruption of F-actin inhibits TGEV entry as well. Cofilin is involved in F-actin reorganization and TGEV entry. The TGEV spike protein is capable of binding with EGFR, activating the downstream phosphoinositide-3 kinase (PI3K), then causing the phosphorylation of cofilin and F-actin polymerization via Rac1/Cdc42 GTPases. Inhibition of EGFR and PI3K decreases the entry of TGEV. EGFR is also the upstream activator of mitogen-activated protein kinase (MAPK) signaling pathways that is involved in F-actin reorganization. Additionally, lipid rafts act as signal platforms for the EGFR-associated signaling cascade and correlate with the adhesion of TGEV. In conlusion, these results provide valuable data of the mechanisms which are responsible for the TGEV pathogenesis and may lead to the development of new methods about controlling TGEV. PMID:26933809

  13. Absence of canonical active chromatin marks in developmentally regulated genes

    PubMed Central

    Ruiz-Romero, Marina; Corominas, Montserrat; Guigó, Roderic

    2015-01-01

    The interplay of active and repressive histone modifications is assumed to play a key role in the regulation of gene expression. In contrast to this generally accepted view, we show that transcription of genes temporally regulated during fly and worm development occurs in the absence of canonically active histone modifications. Conversely, strong chromatin marking is related to transcriptional and post-transcriptional stability, an association that we also observe in mammals. Our results support a model in which chromatin marking is associated to stable production of RNA, while unmarked chromatin would permit rapid gene activation and de-activation during development. In this case, regulation by transcription factors would play a comparatively more important regulatory role. PMID:26280901

  14. Chromatin Remodeling Inactivates Activity Genes and Regulates Neural Coding

    PubMed Central

    Hill, Kelly K.; Hemberg, Martin; Reddy, Naveen C.; Cho, Ha Y.; Guthrie, Arden N.; Oldenborg, Anna; Heiney, Shane A.; Ohmae, Shogo; Medina, Javier F.; Holy, Timothy E.; Bonni, Azad

    2016-01-01

    Activity-dependent transcription influences neuronal connectivity, but the roles and mechanisms of inactivation of activity-dependent genes have remained poorly understood. Genome-wide analyses in the mouse cerebellum revealed that the nucleosome remodeling and deacetylase (NuRD) complex deposits the histone variant H2A.z at promoters of activity-dependent genes, thereby triggering their inactivation. Purification of translating mRNAs from synchronously developing granule neurons (Sync-TRAP) showed that conditional knockout of the core NuRD subunit Chd4 impairs inactivation of activity-dependent genes when neurons undergo dendrite pruning. Chd4 knockout or expression of NuRD-regulated activity genes impairs dendrite pruning. Imaging of behaving mice revealed hyperresponsivity of granule neurons to sensorimotor stimuli upon Chd4 knockout. Our findings define an epigenetic mechanism that inactivates activity-dependent transcription and regulates dendrite patterning and sensorimotor encoding in the brain. PMID:27418512

  15. Developmental regulation of aromatase activity in the rat hypothalamus

    SciTech Connect

    Lephart, E.D.

    1989-01-01

    The brain of all mammalian species studied thus far contain an enzymatic activity (aromatase) that catalyzes the conversion of androgens to estrogens. The activity is highest during prenatal development and contributes to the establishment of sex differences which determine adult gonadotropin secretion patterns and reproductive behavior. The studies presented in this dissertation represent a systematic effort to elucidate the mechanism(s) that control the initiation of and contribute to maintaining rat hypothalamic aromatase activity during pre- and postnatal development. Aromatase enzyme activity was measured by the {sup 3}H{sub 2}O release assay or by traditional estrogen product isolation. Brain aromatase mRNA was detected by hybridization to a cDNA encoding rat aromatase cytochrome P-450. In both males and females the time of puberty was associated with a decline in hypothalamic aromatase activity. This decline may represent a factor underlying the peri-pubertal decrease in the sensitivity to gonadal steroid feedback that accompanies completion of puberty. The results also indicate that androgens regulate brain aromatase levels during both the prepubertal and peri-pubertal stages of sexual development and that this regulation is transiently lost in young adults. Utilizing a hypothalamic organotypic culture system, aromatase activity in vitro was maintained for as long as two days. The results of studies of a variety of hormonal and metabolic regulators suggest that prenatal aromatase activity is regulated by factor(s) that function independently from the classical cyclic AMP and protein kinase C trans-membrane signaling pathways.

  16. Bacterial differentiation via gradual activation of global regulators.

    PubMed

    Kovács, Ákos T

    2016-02-01

    Bacteria have evolved to adapt to various conditions and respond to certain stress conditions. The ability to sense and efficiently reply to these environmental effects involve versatile array of sensors and global or specific regulators. Interestingly, modulation of the levels of active global regulators enables bacteria to respond to diverse signals via a single central transcriptional regulator and to activate or repress certain differentiation pathways at a spatio-temporal manner. The Gram-positive Bacillus subtilis is an ideal bacterium to study how membrane bound and cytoplasmic sensor kinases affect the level of phosphorylated global regulator, Spo0A which in response activates genes related to sliding, biofilm formation, and sporulation. In addition, other global regulators, including the two-component system DegS-DegU, modulate overlapping and complementary genes in B. subtilis related to surface colonization and biofilm formation. The intertwinement of global regulatory systems also allows the accurate modulation of differentiation pathways. Studies in the last decade enable us to get a deeper insight into the role of global regulators on the smooth transition of developmental processes in B. subtilis. PMID:26458398

  17. Epigenetic regulator Lid maintains germline stem cells through regulating JAK-STAT signaling pathway activity

    PubMed Central

    Tarayrah, Lama; Li, Yuping; Gan, Qiang; Chen, Xin

    2015-01-01

    ABSTRACT Signaling pathways and epigenetic mechanisms have both been shown to play essential roles in regulating stem cell activity. While the role of either mechanism in this regulation is well established in multiple stem cell lineages, how the two mechanisms interact to regulate stem cell activity is not as well understood. Here we report that in the Drosophila testis, an H3K4me3-specific histone demethylase encoded by little imaginal discs (lid) maintains germline stem cell (GSC) mitotic index and prevents GSC premature differentiation. Lid is required in germ cells for proper expression of the Stat92E transcription factor, the downstream effector of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Our findings support a germ cell autonomous role for the JAK-STAT pathway in maintaining GSCs and place Lid as an upstream regulator of this pathway. Our study provides new insights into the biological functions of a histone demethylase in vivo and sheds light on the interaction between epigenetic mechanisms and signaling pathways in regulating stem cell activities. PMID:26490676

  18. 34 CFR 110.2 - To what programs or activities do these regulations apply?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... labor-management joint apprenticeship training program, except any program or activity receiving Federal... regulations apply? (a) These regulations apply to any program or activity receiving Federal...

  19. 34 CFR 110.2 - To what programs or activities do these regulations apply?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... labor-management joint apprenticeship training program, except any program or activity receiving Federal... regulations apply? (a) These regulations apply to any program or activity receiving Federal...

  20. 34 CFR 110.2 - To what programs or activities do these regulations apply?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... labor-management joint apprenticeship training program, except any program or activity receiving Federal... regulations apply? (a) These regulations apply to any program or activity receiving Federal...

  1. 34 CFR 110.2 - To what programs or activities do these regulations apply?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... labor-management joint apprenticeship training program, except any program or activity receiving Federal... regulations apply? (a) These regulations apply to any program or activity receiving Federal...

  2. 34 CFR 110.2 - To what programs or activities do these regulations apply?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... labor-management joint apprenticeship training program, except any program or activity receiving Federal... regulations apply? (a) These regulations apply to any program or activity receiving Federal...

  3. Minimal regulation of platelet activity by PECAM-1.

    PubMed

    Dhanjal, Tarvinder S; Ross, Ewan A; Auger, Jocelyn M; McCarty, Owen J T; Hughes, Craig E; Senis, Yotis A; Buckley, Chris D; Watson, Steve P

    2007-02-01

    PECAM-1 is a member of the superfamily of immunoglobulins (Ig) and is expressed on platelets at moderate level. PECAM-1 has been reported to have contrasting effects on platelet activation by the collagen receptor GPVI and the integrin, alphaIIbbeta3, even though both receptors signal through Src-kinase regulation of PLCgamma2. The present study compares the role of PECAM-1 on platelet activation by these two receptors and by the lectin receptor, CLEC-2, which also signals via PLCgamma2. Studies using PECAM-1 knockout-mice and cross-linking of PECAM-1 using specific antibodies demonstrated a minor inhibitory role on platelet responses to the above three receptors and also under some conditions to the G-protein agonist thrombin. The degree of inhibition was considerably less than that produced by PGI2, which elevates cAMP. There was no significant difference in thrombus formation on collagen in PECAM-1-/- platelets relative to litter-matched controls. The very weak inhibitory effect of PECAM-1 on platelet activation relative to that of PGI2 indicate that the Ig-receptor is not a major regulator of platelet activation. PECAM-1 has been reported to have contrasting effects on platelet activation. The present study demonstrates a very mild or negligible effect on platelet activation in response to stimulation by a variety of agonists, thereby questioning the physiological role of the immunoglobulin receptor as a major regulator of platelet activation. PMID:17365855

  4. Platelet activating factor: regulation by mast cells and aspirin.

    PubMed

    Denburg, J A; Williams, D B; Kinlough-Rathbone, R L; Cazenave, J P; Bienenstock, J

    1984-02-01

    We have investigated some aspects of the regulation of production of rat platelet activating factor (PAF)2 in vitro. Suspensions of unseparated (PLC1), mast cell-depleted (PLC2), or mast cell (MC)-enriched rat peritoneal lavage cells (PLC) were analyzed for PAF content by extraction at alkaline pH. PAF activity extracted from PLC1 varied inversely with viable cell concentration: at 1 X 10(6) cells/ml, 32 +/- 9.3 PAF units, decreasing to 11.2 +/- 9.5 units at 10 X 10(6) cells/ml, and no activity at higher concentrations. Incubation of PLC1 in Tyrode's buffer or acetylsalicylic acid (ASA), but not salicylate, resulted in a time-dependent loss of PAF activity. Mean PAF activity of PLC2 was similar to that in PLC1, while no PAF activity was extractable from MC. Co-incubation with MC extracts inhibited PAF activity of PLC1 extracts in a dose-dependent fashion. Ultracentrifugation of PAF-containing samples led to a loss of all PAF activity in PLC1 extracts, suggesting the association of PAF activity with subcellular components. PAF appears to be derived from a non-MC population of rat PLC, is not extractable from rat PLC in the presence of ASA and is inhibited by MC extracts. These studies suggest that ASA regulates PAF availability unrelated to its effect on cyclooxygenase and that MC membrane products directly inhibit PAF activity from rat PLC. PMID:6711391

  5. Intracerebroventricular injection of adiponectin regulates locomotor activity in rats.

    PubMed

    Miyatake, Yumiko; Shiuchi, Tetsuya; Ueta, Tomoyo; Taniguchi, Yasuko; Futami, Akari; Sato, Fukiko; Kitamura, Tadahiro; Tsutsumi, Rie; Harada, Nagakatsu; Nakaya, Yutaka; Sakaue, Hiroshi

    2015-01-01

    Enhancing exercise motivation is the best way to prevent obesity and diabetes. In this study, we examined whether adiponectin affects locomotion activity in Wister and Spontaneously-Running Tokushima-Shikoku (SPORTS) rats using two types of behavioral assays: home cage and wheel running activity. SPORTS rats were established from an original line from Wister strain that had shown high level of wheel running activity in our laboratory. Injection of adiponectin into the lateral ventricle of Wister rats and SPORTS rats decreased home cage activity, but no change was observed in the food intake and oxygen consumption. This result indicates the possibility that adiponectin can reduce non-exercise activity thermogenesis (NEAT) and physical activity via the central nervous system. In contrast, injection of adiponectin did not change wheel running activity in SPORTS rats. We produced hypothalamus-destructed model rat using monosodium glutamate (MSG) to elucidate the regulation site of adiponectin. Injection of adiponectin into MSG-treated SPORTS rats did not change amount of home cage activity and food intake, suggesting that adiponectin action on home cage activity was in the hypothalamic area. These results suggest that adiponectin regulates locomotion activity through mediobasal hypothalamus. PMID:26399348

  6. Active Inference, homeostatic regulation and adaptive behavioural control

    PubMed Central

    Pezzulo, Giovanni; Rigoli, Francesco; Friston, Karl

    2015-01-01

    We review a theory of homeostatic regulation and adaptive behavioural control within the Active Inference framework. Our aim is to connect two research streams that are usually considered independently; namely, Active Inference and associative learning theories of animal behaviour. The former uses a probabilistic (Bayesian) formulation of perception and action, while the latter calls on multiple (Pavlovian, habitual, goal-directed) processes for homeostatic and behavioural control. We offer a synthesis these classical processes and cast them as successive hierarchical contextualisations of sensorimotor constructs, using the generative models that underpin Active Inference. This dissolves any apparent mechanistic distinction between the optimization processes that mediate classical control or learning. Furthermore, we generalize the scope of Active Inference by emphasizing interoceptive inference and homeostatic regulation. The ensuing homeostatic (or allostatic) perspective provides an intuitive explanation for how priors act as drives or goals to enslave action, and emphasises the embodied nature of inference. PMID:26365173

  7. Active Inference, homeostatic regulation and adaptive behavioural control.

    PubMed

    Pezzulo, Giovanni; Rigoli, Francesco; Friston, Karl

    2015-11-01

    We review a theory of homeostatic regulation and adaptive behavioural control within the Active Inference framework. Our aim is to connect two research streams that are usually considered independently; namely, Active Inference and associative learning theories of animal behaviour. The former uses a probabilistic (Bayesian) formulation of perception and action, while the latter calls on multiple (Pavlovian, habitual, goal-directed) processes for homeostatic and behavioural control. We offer a synthesis these classical processes and cast them as successive hierarchical contextualisations of sensorimotor constructs, using the generative models that underpin Active Inference. This dissolves any apparent mechanistic distinction between the optimization processes that mediate classical control or learning. Furthermore, we generalize the scope of Active Inference by emphasizing interoceptive inference and homeostatic regulation. The ensuing homeostatic (or allostatic) perspective provides an intuitive explanation for how priors act as drives or goals to enslave action, and emphasises the embodied nature of inference. PMID:26365173

  8. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

    PubMed

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-05-23

    RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs. PMID:23706742

  9. Regulation of the extracellular ligand binding activity of integrins.

    PubMed

    Fernandez, C; Clark, K; Burrows, L; Schofield, N R; Humphries, M J

    1998-07-01

    Integrins are a large heterodimeric family of cell surface adhesion receptors that bind extracellular matrix and cell surface ligands. The extracellular ligand binding activity of integrins is a dynamic and highly regulated event involving the induction of conformational changes within the integrin structure. The adhesive properties of integrins can be controlled by altering the activation state of the integrin, either through conformational change or receptor clustering, using mechanisms that are regulated by intracellular proteins. In this review, we will discuss what is currently known about integrin structure and the ligand binding sites present within the receptor. In addition, the mechanisms by which the ligand binding event is regulated through conformational change will be addressed, and the potential role of intracellular cytoplasmic proteins will be discussed. PMID:9637803

  10. Signal integration by Ca2+ regulates intestinal stem cell activity

    PubMed Central

    Deng, Hansong; Gerencser, Akos A.; Jasper, Heinrich

    2015-01-01

    Summary Somatic stem cells (SCs) maintain tissue homeostasis by dynamically adjusting proliferation and differentiation in response to stress and metabolic cues. Here, we identify Ca2+ signaling as a central regulator of intestinal SC (ISC) activity in Drosophila. We find that dietary L-glutamate stimulates ISC division and gut growth. The metabotropic glutamate receptor (mGluR) is required in ISCs for this response and for an associated modulation of cytosolic Ca2+ oscillations that results in sustained high cytosolic Ca2+ concentrations. High cytosolic Ca2+ induces ISC proliferation by regulating Calcineurin and CREB - regulated transcriptional co-activator (CRTC). In response to a wide range of dietary and stress stimuli, ISCs reversibly transition between Ca2+ oscillation states that represent poised or activated modes of proliferation, respectively. We propose that the dynamic regulation of intracellular Ca2+ levels allows effective integration of diverse mitogenic signals in ISCs to tailor their proliferative activity to the needs of the tissue. PMID:26633624

  11. Primary cilia and Gli3 activity regulate cerebral cortical size

    PubMed Central

    Wilson, Sandra L.; Wilson, John P.; Wang, Chengbing; Wang, Baolin; McConnell, Susan K.

    2012-01-01

    During neural development, patterning, neurogenesis and overall growth are highly regulated and coordinated between different brain regions. Here, we show that primary cilia and the regulation of Gli activity, are necessary for the normal expansion of the cerebral cortex. We show that loss of Kif3a, an important functional component of primary cilia, leads to the degeneration of primary cilia, marked overgrowth of the cortex, and altered cell cycle kinetics within cortical progenitors. The G1 phase of the cell cycle is shortened through a mechanism likely involving reduced Gli3 activity and a resulting increase in expression of cyclin D1 and Fgf15. The defects in Gli3 activity alone are sufficient to accelerate cell cycle kinetics and cause the molecular changes seen in brains that lack cilia. Finally, we show that levels of full-length and repressor Gli3 proteins are tightly regulated during normal development and correlate with changes in expression of two known Shh-target genes, CyclinD1 and Fgf15, and with the normal lengthening of the cell cycle during corticogenesis. These data suggest that Gli3 activity is regulated through the primary cilium to control cell cycle length in the cortex and thus determine cortical size. PMID:21976438

  12. 76 FR 44350 - Agency Information Collection Activities: Drawback Process Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-25

    ... SECURITY U.S. CUSTOMS AND BORDER PROTECTION Agency Information Collection Activities: Drawback Process... approval in accordance with the Paperwork Reduction Act: Drawback Process Regulations (CBP Forms 7551, 7552... collection was previously published in the Federal Register (76 FR 19120) on April 6, 2011, allowing for a...

  13. 76 FR 19120 - Agency Information Collection Activities: Drawback Process Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-06

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Drawback Process... Drawback Process Regulations (CBP Forms 7551, 7552 and 7553). This request for comment is being made... CBP is soliciting comments concerning the following information collection: Title: Drawback...

  14. 76 FR 28801 - Agency Information Collection Activities: Bonded Warehouse Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-18

    ... Federal Register (76 FR 11254) on March 1, 2011, allowing for a 60-day comment period. This notice allows... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Bonded Warehouse... approval in accordance with the Paperwork Reduction Act: Bonded Warehouse Regulations. This is a...

  15. Structural basis of AMPK regulation by small molecule activators

    NASA Astrophysics Data System (ADS)

    Xiao, Bing; Sanders, Matthew J.; Carmena, David; Bright, Nicola J.; Haire, Lesley F.; Underwood, Elizabeth; Patel, Bhakti R.; Heath, Richard B.; Walker, Philip A.; Hallen, Stefan; Giordanetto, Fabrizio; Martin, Stephen R.; Carling, David; Gamblin, Steven J.

    2013-12-01

    AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.

  16. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis

    PubMed Central

    Muthukumar, Allie K.; Stork, Tobias; Freeman, Marc R.

    2014-01-01

    Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system, however mechanisms by which astrocytes regulate GAT levels are undefined. Here we show at mid-pupal stages the Drosophila CNS neuropil is devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrate the neuropil coordinate with synaptogenesis and a strocyte ablation reduces synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses form in earnest, the GABA transporter, GAT, is up-regulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor (GABABR1/2) signaling in astrocytes leads to decreased astrocytic GAT levels. Interestingly, developmental depletion of astrocytic GABABR1/2 signaling suppresses mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling, and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity. PMID:25151265

  17. Rho GTPase protein expression and activation in murine monocytes/macrophages is not modulated by model biomaterial surfaces in serum-containing in vitro cultures.

    PubMed

    Godek, M L; Sampson, J A; Duchsherer, N L; McElwee, Q; Grainger, D W

    2006-01-01

    The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-)macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue culture polystyrene, polystyrene, poly-l-lactide and Teflon(®) AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue culture polystyrene and poly

  18. Rho GTPase protein expression and activation in murine monocytes/macrophages is not modulated by model biomaterial surfaces in serum-containing in vitro cultures

    PubMed Central

    GODEK, M. L.; SAMPSON, J. A.; DUCHSHERER, N. L.; McELWEE, Q.; GRAINGER, D. W.

    2006-01-01

    The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-)macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue culture polystyrene, polystyrene, poly-l-lactide and Teflon® AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue culture polystyrene and poly

  19. Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

    SciTech Connect

    Yi, TacGhee; Kim, Hye-Jin; Cho, Je-Yoel; Woo, Kyung Mi; Ryoo, Hyun-Mo; Kim, Gwan-Shik; Baek, Jeong-Hwa . E-mail: baekjh@snu.ac.kr

    2006-08-18

    Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.

  20. KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX

    PubMed Central

    Llano, Olaya; Smirnov, Sergey; Soni, Shetal; Golubtsov, Andrey; Guillemin, Isabelle; Hotulainen, Pirta; Medina, Igor; Nothwang, Hans Gerd

    2015-01-01

    Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses. PMID:26056138

  1. KCC2 regulates actin dynamics in dendritic spines via interaction with β-PIX.

    PubMed

    Llano, Olaya; Smirnov, Sergey; Soni, Shetal; Golubtsov, Andrey; Guillemin, Isabelle; Hotulainen, Pirta; Medina, Igor; Nothwang, Hans Gerd; Rivera, Claudio; Ludwig, Anastasia

    2015-06-01

    Chloride extrusion in mature neurons is largely mediated by the neuron-specific potassium-chloride cotransporter KCC2. In addition, independently of its chloride transport function, KCC2 regulates the development and morphology of dendritic spines through structural interactions with the actin cytoskeleton. The mechanism of this effect remains largely unknown. In this paper, we show a novel pathway for KCC2-mediated regulation of the actin cytoskeleton in neurons. We found that KCC2, through interaction with the b isoform of Rac/Cdc42 guanine nucleotide exchange factor β-PIX, regulates the activity of Rac1 GTPase and the phosphorylation of one of the major actin-regulating proteins, cofilin-1. KCC2-deficient neurons had abnormally high levels of phosphorylated cofilin-1. Consistently, dendritic spines of these neurons exhibited a large pool of stable actin, resulting in reduced spine motility and diminished density of functional synapses. In conclusion, we describe a novel signaling pathway that couples KCC2 to the cytoskeleton and regulates the formation of glutamatergic synapses. PMID:26056138

  2. Tbx16 regulates hox gene activation in mesodermal progenitor cells.

    PubMed

    Payumo, Alexander Y; McQuade, Lindsey E; Walker, Whitney J; Yamazoe, Sayumi; Chen, James K

    2016-09-01

    The transcription factor T-box 16 (Tbx16, or Spadetail) is an essential regulator of paraxial mesoderm development in zebrafish (Danio rerio). Mesodermal progenitor cells (MPCs) fail to differentiate into trunk somites in tbx16 mutants and instead accumulate within the tailbud in an immature state. However, the mechanisms by which Tbx16 controls mesoderm patterning have remained enigmatic. We describe here the use of photoactivatable morpholino oligonucleotides to determine the Tbx16 transcriptome in MPCs. We identified 124 Tbx16-regulated genes that were expressed in zebrafish gastrulae, including several developmental signaling proteins and regulators of gastrulation, myogenesis and somitogenesis. Unexpectedly, we observed that a loss of Tbx16 function precociously activated posterior hox genes in MPCs, and overexpression of a single posterior hox gene was sufficient to disrupt MPC migration. Our studies support a model in which Tbx16 regulates the timing of collinear hox gene activation to coordinate the anterior-posterior fates and positions of paraxial MPCs. PMID:27376691

  3. Activation and Regulation of DNA-Driven Immune Responses

    PubMed Central

    2015-01-01

    SUMMARY The innate immune system provides early defense against infections and also plays a key role in monitoring alterations of homeostasis in the body. DNA is highly immunostimulatory, and recent advances in this field have led to the identification of the innate immune sensors responsible for the recognition of DNA as well as the downstream pathways that are activated. Moreover, information on how cells regulate DNA-driven immune responses to avoid excessive inflammation is now emerging. Finally, several reports have demonstrated how defects in DNA sensing, signaling, and regulation are associated with susceptibility to infections or inflammatory diseases in humans and model organisms. In this review, the current literature on DNA-stimulated innate immune activation is discussed, and important new questions facing this field are proposed. PMID:25926682

  4. Flavonoids as dietary regulators of nuclear receptor activity

    PubMed Central

    Avior, Yishai; Bomze, David; Ramon, Ory

    2013-01-01

    Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds. PMID:23598551

  5. Harvester ants use interactions to regulate forager activation and availability

    PubMed Central

    Pinter-Wollman, Noa; Bala, Ashwin; Merrell, Andrew; Queirolo, Jovel; Stumpe, Martin C.; Holmes, Susan; Gordon, Deborah M.

    2013-01-01

    Social groups balance flexibility and robustness in their collective response to environmental changes using feedback between behavioural processes that operate at different timescales. Here we examine how behavioural processes operating at two timescales regulate the foraging activity of colonies of the harvester ant, Pogonomyrmex barbatus, allowing them to balance their response to food availability and predation. Previous work showed that the rate at which foragers return to the nest with food influences the rate at which foragers leave the nest. To investigate how interactions inside the nest link the rates of returning and outgoing foragers, we observed outgoing foragers inside the nest in field colonies using a novel observation method. We found that the interaction rate experienced by outgoing foragers inside the nest corresponded to forager return rate, and that the interactions of outgoing foragers were spatially clustered. Activation of a forager occurred on the timescale of seconds: a forager left the nest 3–8 s after a substantial increase in interactions with returning foragers. The availability of outgoing foragers to become activated was adjusted on the timescale of minutes: when forager return was interrupted for more than 4–5 min, available foragers waiting near the nest entrance went deeper into the nest. Thus, forager activation and forager availability both increased with the rate at which foragers returned to the nest. This process was checked by negative feedback between forager activation and forager availability. Regulation of foraging activation on the timescale of seconds provides flexibility in response to fluctuations in food abundance, whereas regulation of forager availability on the timescale of minutes provides robustness in response to sustained disturbance such as predation. PMID:24031094

  6. Distinct roles for paxillin and Hic-5 in regulating breast cancer cell morphology, invasion, and metastasis

    PubMed Central

    Deakin, Nicholas O.; Turner, Christopher E.

    2011-01-01

    Individual metastatic tumor cells exhibit two interconvertible modes of cell motility during tissue invasion that are classified as either mesenchymal or amoeboid. The molecular mechanisms by which invasive breast cancer cells regulate this migratory plasticity have yet to be fully elucidated. Herein we show that the focal adhesion adaptor protein, paxillin, and the closely related Hic-5 have distinct and unique roles in the regulation of breast cancer cell lung metastasis by modulating cell morphology and cell invasion through three-dimensional extracellular matrices (3D ECMs). Cells depleted of paxillin by RNA interference displayed a highly elongated mesenchymal morphology, whereas Hic-5 knockdown induced an amoeboid phenotype with both cell populations exhibiting reduced plasticity, migration persistence, and velocity through 3D ECM environments. In evaluating associated signaling pathways, we determined that Rac1 activity was increased in cells devoid of paxillin whereas Hic-5 silencing resulted in elevated RhoA activity and associated Rho kinase–induced nonmuscle myosin II activity. Hic-5 was essential for adhesion formation in 3D ECMs, and analysis of adhesion dynamics and lifetime identified paxillin as a key regulator of 3D adhesion assembly, stabilization, and disassembly. PMID:21148292

  7. Disorders of regulation of cognitive activity in autistic children.

    PubMed

    Adrien, J L; Martineau, J; Barthélémy, C; Bruneau, N; Garreau, B; Sauvage, D

    1995-06-01

    Infantile autism is a pervasive developmental disorder characterized by disturbances concerning not only the areas of socialization and communication ("aloneness") but also the ability to modify and change behavior ("need for sameness"). In most recent studies, various abnormal and deviant cognitive activities, such as the ability to regulate one's behavior, were considered as accounting for these signs. In this report, we examined the regulation of cognitive activity, from a developmental perspective in comparing autistic with mentally retarded children matched in a pairwise manner by global, verbal, and nonverbal developmental ages. All children were tested with tasks adapted from the Object Permanence Test which corresponds to Piaget's sensorimotor development Stages IV to VI. Results showed that autistic children had a pervasive difficulty in maintenance set, made more perseverative errors when the abstraction degree of task was higher, and were more variable in their behavioral strategies. Discussion is focused on the interests and limits of these tasks for the examination of regulation activity from diagnostic and developmental perspectives. Finally, interpretations about recent neuropsychological and neurophysiological works, and additional interdisciplinary studies are suggested. PMID:7559291

  8. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity

    PubMed Central

    Semenkov, Illya; Molinski, Steven; Pasyk, Stan; Ahmadi, Saumel; Bui, Hai H.; Bear, Christine E.; Lidington, Darcy; Bolz, Steffen-Sebastian

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking. PMID:26079370

  9. Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity.

    PubMed

    Malik, Firhan A; Meissner, Anja; Semenkov, Illya; Molinski, Steven; Pasyk, Stan; Ahmadi, Saumel; Bui, Hai H; Bear, Christine E; Lidington, Darcy; Bolz, Steffen-Sebastian

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking. PMID:26079370

  10. The immune system as a regulator of thyroid hormone activity.

    PubMed

    Klein, John R

    2006-03-01

    It has been known for decades that the neuroendocrine system can both directly and indirectly influence the developmental and functional activity of the immune system. In contrast, far less is known about the extent to which the immune system collaborates in the regulation of endocrine activity. This is particularly true for immune-endocrine interactions of the hypothalamus-pituitary-thyroid axis. Although thyroid-stimulating hormone (TSH) can be produced by many types of extra-pituitary cells--including T cells, B cells, splenic dendritic cells, bone marrow hematopoietic cells, intestinal epithelial cells, and lymphocytes--the functional significance of those TSH pathways remains elusive and historically has been largely ignored from a research perspective. There is now, however, evidence linking cells of the immune system to the regulation of thyroid hormone activity in normal physiological conditions as well as during times of immunological stress. Although the mechanisms behind this are poorly understood, they appear to reflect a process of local intrathyroidal synthesis of TSH mediated by a population of bone marrow cells that traffic to the thyroid. This hitherto undescribed cell population has the potential to microregulate thyroid hormone secretion leading to critical alterations in metabolic activity independent of pituitary TSH output, and it has expansive implications for understanding mechanisms by which the immune system may act to modulate neuroendocrine function during times of host stress. In this article, the basic underpinnings of the hematopoietic-thyroid connection are described, and a model is presented in which the immune system participates in the regulation of thyroid hormone activity during acute infection. PMID:16514168

  11. Regulation of maternal transcript destabilization during egg activation in Drosophila.

    PubMed Central

    Tadros, Wael; Houston, Simon A; Bashirullah, Arash; Cooperstock, Ramona L; Semotok, Jennifer L; Reed, Bruce H; Lipshitz, Howard D

    2003-01-01

    In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation. PMID:12871909

  12. Antiviral Regulation in Porcine Monocytic Cells at Different Activation States

    PubMed Central

    Rowland, Raymond R. R.

    2014-01-01

    ABSTRACT Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii) in situ PRRSV replication-competent expression of interferon alpha (IFN-α). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity. IMPORTANCE Activation statuses of monocytic cells, including monocytes, macrophages (Mϕs), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status

  13. Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity

    PubMed Central

    2013-01-01

    Background Rho GTPases play important roles in cytoskeleton organization, cell cycle progression and are key regulators of tumor progression. Strategies to modulate increased Rho GTPase activities during cancer progression could have therapeutic potential. Methods We report here the characterization of a Cdc42-selective small-molecule inhibitor AZA197 for the treatment of colon cancer that was developed based on structural information known from previously developed compounds affecting Rho GTPase activation. We investigated the effects of AZA197 treatment on RhoA, Rac1 and Cdc42 activities and associated molecular mechanisms in colon cancer cells in vitro. Therapeutic effects of AZA197 were examined in vivo using a xenograft mouse model of SW620 human colon cancer cells. After treatment, tumors were excised and processed for Ki-67 staining, TUNEL assays and Western blotting to evaluate proliferative and apoptotic effects induced by AZA197. Results In SW620 and HT-29 human colon cancer cells, AZA197 demonstrated selectivity for Cdc42 without inhibition of Rac1 or RhoA GTPases from the same family. AZA197 suppressed colon cancer cell proliferation, cell migration and invasion and increased apoptosis associated with down-regulation of the PAK1 and ERK signaling pathways in vitro. Furthermore, systemic AZA197 treatment reduced tumor growth in vivo and significantly increased mouse survival in SW620 tumor xenografts. Ki-67 staining and tissue TUNEL assays showed that both inhibition of cell proliferation and induction of apoptosis associated with reduced PAK/ERK activation contributed to the AZA197-induced therapeutic effects in vivo. Conclusions These data indicate the therapeutic potential of the small-molecule inhibitor AZA197 based on targeting Cdc42 GTPase activity to modulate colorectal cancer growth. PMID:24279335

  14. The molecular regulation of Janus kinase (JAK) activation.

    PubMed

    Babon, Jeffrey J; Lucet, Isabelle S; Murphy, James M; Nicola, Nicos A; Varghese, Leila N

    2014-08-15

    The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  15. Oxidative Regulation of Large Conductance Calcium-Activated Potassium Channels

    PubMed Central

    Tang, Xiang D.; Daggett, Heather; Hanner, Markus; Garcia, Maria L.; McManus, Owen B.; Brot, Nathan; Weissbach, Herbert; Heinemann, Stefan H.; Hoshi, Toshinori

    2001-01-01

    Reactive oxygen/nitrogen species are readily generated in vivo, playing roles in many physiological and pathological conditions, such as Alzheimer's disease and Parkinson's disease, by oxidatively modifying various proteins. Previous studies indicate that large conductance Ca2+-activated K+ channels (BKCa or Slo) are subject to redox regulation. However, conflicting results exist whether oxidation increases or decreases the channel activity. We used chloramine-T, which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation in the cloned human Slo (hSlo) channel expressed in mammalian cells. In the virtual absence of Ca2+, the oxidant shifted the steady-state macroscopic conductance to a more negative direction and slowed deactivation. The results obtained suggest that oxidation enhances specific voltage-dependent opening transitions and slows the rate-limiting closing transition. Enhancement of the hSlo activity was partially reversed by the enzyme peptide methionine sulfoxide reductase, suggesting that the upregulation is mediated by methionine oxidation. In contrast, hydrogen peroxide and cysteine-specific reagents, DTNB, MTSEA, and PCMB, decreased the channel activity. Chloramine-T was much less effective when concurrently applied with the K+ channel blocker TEA, which is consistent with the possibility that the target methionine lies within the channel pore. Regulation of the Slo channel by methionine oxidation may represent an important link between cellular electrical excitability and metabolism. PMID:11222629

  16. The molecular regulation of Janus kinase (JAK) activation

    PubMed Central

    2014-01-01

    The Janus Kinase (JAK) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2, was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haematopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organisation of the component FERM-SH2, pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain containing proteins, SOCS (Suppressors of Cytokine Signalling) proteins and Lnk. These recent advances highlight the diversity of regulatory mechanisms utilised by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  17. Tax-interacting protein 1 coordinates the spatiotemporal activation of Rho GTPases and regulates the infiltrative growth of human glioblastoma

    PubMed Central

    Wang, Hailun; Han, Miaojun; Whetsell, William; Wang, Jialiang; Rich, Jeremy; Hallahan, Dennis; Han, Zhaozhong

    2014-01-01

    PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. TIP-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as one novel TIP-1 interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector, and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas. PMID:23563176

  18. Synapsins are late activity-induced genes regulated by birdsong

    PubMed Central

    Velho, Tarciso A. F.; Mello, Claudio V.

    2008-01-01

    The consolidation of long-lasting sensory memories requires the activation of gene expression programs in the brain. In spite of considerable knowledge about the early components of this response, little is known about late components (i.e. genes regulated 2-6 hr after stimulation) and the relationship between early and late genes. Birdsong represents one of the best natural behaviors to study sensory-induced gene expression in awake, freely behaving animals. Here we show that the expression of several isoforms of synapsins, a group of phosphoproteins thought to regulate the dynamics of synaptic vesicle storage and release, is induced by auditory stimulation with birdsong in the caudomedial nidopallium (NCM) of the zebra finch (Taeniopygia guttata) brain. This induction occurs mainly in excitatory (non-GABAergic) neurons and is modulated (suppressed) by early song-inducible proteins. We also show that ZENK, an early song-inducible transcription factor, interacts with the syn3 promoter in vivo, consistent with a direct regulatory effect and an emerging novel view of ZENK action. These results demonstrate that synapsins are a late component of the genomic response to neuronal activation and that their expression depends on a complex set of regulatory interactions between early and late regulated genes. PMID:19005052

  19. Regulation of signaling events involved in the pathophysiology of neovascular AMD

    PubMed Central

    Wang, Haibo

    2016-01-01

    include the importance of feed-forward loops that overwhelm mechanisms that seek to restore homeostasis in cells and the importance of regulating, instead of abolishing, signaling events in a chronic, complex disease, such as neovascular AMD. These concepts are important as we move to the next stages in developing treatments for neovascular AMD. A novel therapeutic strategy that will be discussed is activating an isoform of the GTPase, Rap1, which can regulate downstream signaling and a pathologic feed-forward loop leading to Rac1 activation and migration of CECs. PMID:27013848

  20. Calcium and cargoes as regulators of myosin 5a activity

    SciTech Connect

    Sellers, James R. Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-04-25

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

  1. S100B/RAGE-dependent activation of microglia via NF-kappaB and AP-1 Co-regulation of COX-2 expression by S100B, IL-1beta and TNF-alpha.

    PubMed

    Bianchi, Roberta; Giambanco, Ileana; Donato, Rosario

    2010-04-01

    Extracellular S100B is known to affect astrocytic, neuronal and microglial activities, with different effects depending on its concentration. Whereas at relatively low concentrations S100B exerts trophic effects on neurons and astrocytes, at relatively high concentrations the protein causes neuronal apoptosis and activates astrocytes and microglia, thus potentially representing an endogenous factor implicated in neuroinflammation. We have reported that RAGE ligation by S100B in BV-2 microglia results in the upregulation of expression of the pro-inflammatory cyclo-oxygenase 2 (COX-2) via parallel Ras-Cdc42-Rac1-dependent activation of c-Jun NH(2) terminal protein kinase (JNK) and Ras-Rac1-dependent stimulation of NF-kappaB transcriptional activity. We show here that: (1) S100B also stimulates AP-1 transcriptional activity in microglia via RAGE-dependent activation of JNK; (2) S100B upregulates IL-1beta and TNF-alpha expression in microglia via RAGE engagement; and (3) S100B/RAGE-induced upregulation of COX-2, IL-1beta and TNF-alpha expression requires the concurrent activation of NF-kappaB and AP-1. We also show that S100B synergizes with IL-1beta and TNF-alpha to upregulate on COX-2 expression in microglia. Given the crucial roles of COX-2, IL-1beta and TNF-alpha in the inflammatory response, we propose that, by engaging RAGE, S100B might play an important role in microglia activation in the course of brain damage. PMID:18599158