Rac1 Knockdown Research Articles

Protein kinase Cα/β inhibitor Gö6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase Cδ

Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/β. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1μM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/β signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.

Angiotensin-II and MARCKS: A HYDROGEN PEROXIDE- AND RAC1-DEPENDENT SIGNALING PATHWAY IN VASCULAR ENDOTHELIUM

MARCKS is an actin-binding protein that modulates vascular endothelial cell migration and cytoskeleton signaling (Kalwa, H., and Michel, T. (2011) J. Biol. Chem. 286, 2320-2330). Angiotensin-II is a vasoactive peptide implicated in vascular physiology as well as pathophysiology; the pathways connecting angiotensin-II and cytoskeletal remodeling are incompletely understood. Here we show that MARCKS is expressed in intact arterial preparations, with prominent staining of the endothelium. In endothelial cells, angiotensin-II-promoted MARCKS phosphorylation is abrogated by PEG-catalase, implicating endogenous H(2)O(2) in the angiotensin-II response. Studies using the H(2)O(2) biosensor HyPer2 reveal that angiotensin-II promotes increases in intracellular H(2)O(2). We used a Rac1 FRET biosensor to show that angiotensin-II promotes Rac1 activation that is attenuated by PEG-catalase. siRNA-mediated Rac1 knockdown blocks angiotensin-II-stimulated MARCKS phosphorylation. Cell imaging studies using a phosphoinositide 4,5-bisphosphate (PIP(2)) biosensor revealed that angiotensin-II PIP(2) regulation depends on MARCKS and H(2)O(2). siRNA-mediated knockdown of MARCKS or Rac1 attenuates receptor-mediated activation of the tyrosine kinase c-Abl and disrupts actin fiber formation. These studies establish a critical role for H(2)O(2) in angiotensin-II signaling to the endothelial cytoskeleton in a novel pathway that is critically dependent on MARCKS, Rac1, and c-Abl.

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

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.

Abstract 2003: STAT3 is a critical mediator of the pro-survival effect of Bcl-2-Rac1 interaction in human cancer cells

Abstract We recently showed that Bcl-2 overexpression induced a pro-oxidant intracellular milieu resulting in an inhibition of apoptotic execution and that reversing the pro-oxidant milieu by inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We provide evidence that the small GTPase Rac1 is functionally involved since gene silencing and functional inhibition of Rac1 blocked Bcl-2-mediated increase in intracellular superoxide production in tumor cells. A co-localization and physical interaction between the two proteins were observed, which could be blocked by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides both in vitro and in vivo. The Bcl-2 BH3 peptides as well as silencing and functional inhibition of Rac1 reversed intracellular superoxide levels and overcame Bcl-2-mediated drug resistance in human leukemia and cervical cancer cells, demonstrating that this interaction is functionally relevant. Notably, the interaction was observed in primary cells from patients diagnosed with B-cell lymphoma with Bcl-2 overexpression but not in noncancerous tissue. Furthermore, computer simulation driven virtual predictive experiments based on the protein pathway dynamic network created by Cellworks Group Inc. were carried out to study the functional implication of this interaction. An HCT116 human colorectal cancer cell line with KRAS over-activation, PI3K overexpression, CDKN2A deletion, β-catenin overexpression and Bcl-2 overexpression was created as a base line. Phenotypic indices of angiogenesis, proliferation, viability, metastasis as well as tumor volume were all found to be amplified in a variant cell line when Rac1 was overexpressed based on the above computer modeled HCT116. Strikingly, the expression levels of both STAT3 and β-catenin were significantly upregulated in the variant cell line; however upon Rac1 or Bcl-2 knockdown in both the base and the variant cell lines, apoptotic markers such as Bax, caspase 3 and cleaved PARP1 were amplified while STAT3 and β-catenin were significantly down-regulated. Intrigued by these predictions, we set out to verify the correlation between STAT3 signaling and Bcl-2-Rac1 interaction. Interestingly, there is indeed a strong correlation between the activation status of STAT3 (pY705) and Bcl-2 expression as well as Rac1 activation status. Furthermore, STAT3 was found to be localized to mitochondria and our preliminary results suggest an association with Bcl-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2003. doi:1538-7445.AM2012-2003

ROS production as a common mechanism of ENaC regulation by EGF, insulin and IGF‐1

Epithelial Sodium Channel (ENaC) is a key transporter participating in the fine‐tuning of Na+ reabsorption in the nephron. ENaC activity is acutely upregulated by EGF, insulin and IGF‐1. It was also proposed that ROS, including H2O2, have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin and IGF‐1 correlate with ROS production in the mpkCCDc14 cells. Western Blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Short circuit current measurements showed an acute increase of ENaC‐mediated current through the mpkCCDc14 monolayer in response to 50 ng/ml EGF, 100 nM insulin or 100 ng/ml IGF‐1. Treatment of mpkCCDc14 cells with EGF, insulin or IGF‐1 also caused an increase in H2O2 production as measured by DCF fluorescence. Pretreatment with NADPH oxidase activity inhibitor apocynin blunted both H2O2 production and increase in ENaC‐mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we created a stable M‐1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride‐senstitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdowned cells. We hypothesize that EGF, insulin and IGF‐1 have a common stimulatory effect on ENaC mediated by ROS production.

Abstract C149: STAT3 is a critical mediator of the prosurvival effect of Bcl-2-Rac1 interaction in human cancer cells.

Abstract The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. Gene silencing and functional inhibition of Rac1 blocked Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. We provide evidence for a co-localization and physical interaction between the 2 proteins, which could be blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. In an attempt to study the functional implication of this interaction, we carried out a computer simulation driven virtual predictive experiments based on the protein pathway dynamic network created by Cellworks Group Inc. The base line used for the study was a KRAS over-activated, PI3K overexpressed, CDKN2A deleted, β-catenin overexpressed and Bcl-2 overexpressed system aligned to HCT116 human colorectal cancer cell line. When a variant of the above base line was created with Rac1 overexpression, phenotypic indices of angiogenesis, proliferation, viability, metastasis as well as tumor volume were all upregulated. Strikingly, the expression levels of STAT3 and β-catenin were significantly amplified in the computer modeled variant cell line; however upon Rac1 or Bcl-2 knockdown in both the base and the variant cell lines, apoptotic markers such as Bax, caspase 3 and cleaved PARP1 were increased while STAT3, β-catenin, NF-κB and AGER were significantly down-regulated. Intrigued by these predictions, we set out to verify the link between STAT3 signaling and Bcl-2-Rac1 interaction. Interestingly, the activation status of STAT3 (pY705) strongly correlated with Bcl-2 expression as well as Rac1 activation status. Furthermore, STAT3 was found localized to mitochondria and preliminary results suggest an interaction with Bcl-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C149.

Latent KSHV infection increases the vascular permeability of human endothelial cells

AbstractKaposi sarcoma–associated herpesvirus (KSHV) is associated with 3 different human malignancies: Kaposi sarcoma (KS), primary effusion lymphoma, and multicentric Castleman disease. The KS lesion is driven by KSHV-infected endothelial cells and is highly dependent on autocrine and paracrine factors for survival and growth. We report that latent KSHV infection increases the vascular permeability of endothelial cells. Endothelial cells with latent KSHV infection display increased Rac1 activation and activation of its downstream modulator, p21-activated kinase 1 (PAK1). The KSHV-infected cells also exhibit increases in tyrosine phosphorylation of vascular endothelial (VE)–cadherin and β-catenin, whereas total levels of these proteins remained unchanged, suggesting that latent infection disrupted endothelial cell junctions. Consistent with these findings, we found that KSHV-infected endothelial cells displayed increased permeability compared with uninfected endothelial cells. Knockdown of Rac1 and inhibition of reactive oxygen species (ROS) resulted in decreased permeability in the KSHV-infected endothelial cells. We further demonstrate that the KSHV K1 protein can activate Rac1. Rac1 was also highly activated in KSHV-infected endothelial cells and KS tumors. In conclusion, KSHV latent infection increases Rac1 and PAK1 activity in endothelial cells, resulting in the phosphorylation of VE-cadherin and β-catenin and leading to the disassembly of cell junctions and to increased vascular permeability of the infected endothelial cells.

A Role for Rac3 GTPase in the Regulation of Autophagy

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Abstract B19: Cigarette smoke promotes human airway epithelial migration through temporal coordination of p120-catenin-dependent and -independent pathways

Abstract Background: Cigarette smoking has been linked to almost all major types of cancer. Emerging evidence suggests that smoking initiates transformed cell growth and migration by disrupting cell-cell interactions in the polarized mucosal epithelium. Together with other adherens junction proteins, p120-catenin (p120ctn) maintains cell-cell adhesion through its direct interaction with E-cadherin. Mislocalization and/or loss of p120ctn have been reported in all lung cancer subtypes and are related to poor prognosis. Our purpose was to investigate the initiating (intact p120ctn) and late (loss of 120ctn) events of smoke-promoted lung carcinogenesis using primary human bronchial epithelial (HBE) cells. Methods: Wide-type and p120ctn-knockdown HBE cells exposed to cigarette smoke-conditioned medium were used to mimic the early and late progressive stages of lung cancer. Signaling cascades and cell migration under the two conditions were investigated. Results: Src and Jnk activation by smoke were delayed in p120ctn-knockdown cells and inhibition of migration through the chemical blockade of EGFR, Src, and/or Jnk was dependent on p120ctn. In contrast, chemical inhibition of signaling via Akt (cell survival) or Erk (cell proliferation) was sufficient to block smoke-induced migration independent of p120ctn. Also independent of p120ctn was smoke's effect on the RhoGTPase, Rac1. Rac1 knockdown abolished dephosphorylation of actin binding protein, cofilin, in the presence and absence of p120ctn, indicating its essential role in smoke-induced cell migration. Conclusions: These studies provide unprecedented insight regarding the multiple and interdependent molecular strategies employed by smoke to escape tightly regulated cellular processes such as cell migration, proliferation, and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B19.

Vasodilator-stimulated phosphoprotein regulates osteosarcoma cell migration

Vasodilator-stimulated phosphoprotein (VASP) has been reported to play an important role in the process of cell migration and tumor metastasis. However, to date, no study has examined VASP expression and its function in osteocarcoma cells. In this study, we analyzed the effect of VASP on osteosarcoma cell migration and the signal transduction pathways involved. We used two osteosarcoma cell strains (Mg-63 and Saos-2 cells) with different metastatic potential. Silencing of VASP gene expression was carried out using RNA interference in these cells. Knockdown of expression at the transcriptional or translational level was determined by RT-PCR or western blot analysis, respectively. The metastatic potential of the tumor cells was determined by a wound healing migration assay. VASP mRNA expression was also determined in 30 human osteosarcoma samples. Furthermore, Rac1 was determined as a regulator of VASP function. RT-PCR and western blotting showed that Mg-63 cells had a significantly higher VASP expression at both the transcriptional and translational levels compared to Saos-2 cells. The wound healing assay revealed that Mg-63 cells had more migratory potential compared to Saos-2 cells. The effect was found to be reversible when VASP was knocked down by siRNA in Mg-63 cells. Specimens from human patients with metastases had higher VASP expression compared to specimens of patients without metastases. Knockdown of Rac1 resulted in inhibition of VASP expression in sarcoma cells. These results suggest that VASP protein regulates osteosarcoma cell migration and metastasis. Rac1 and VASP interaction may be a potential target for osteo-sarcoma treatment.

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.

Pigment Epithelium-Derived Factor Blocks Tumor Extravasation by Suppressing Amoeboid Morphology and Mesenchymal Proteolysis

Metastatic melanoma cells are highly adaptable to their in vivo microenvironment and can switch between protease-dependent mesenchymal and protease-independent amoeboid invasion to facilitate metastasis. Such adaptability can be visualized in vitro, when cells are cultured in conditions that recapitulate three-dimensional microenvironments. Using thick collagen layers in cell culture and in vivo extravasation assays, we found that pigment epithelium-derived factor (PEDF) suppressed lung extravasation of aggressive melanoma by coordinated regulation of cell shape and proteolysis. In cells grown on a thick collagen bed, PEDF overexpression and exogenous PEDF blocked the rapidly invasive, rounded morphology, and promoted an elongated, mesenchymal-like phenotype associated with reduced invasion. These changes in cell shape depended on decreased RhoA and increased Rac1 activation and were mediated by the up-regulation of Rac1-GEF, DOCK3 and down-regulation of Rac1-GAP, ARHGAP22. Surprisingly, we found that PEDF overexpression also blocked the trafficking of membrane-tethered, MT1-MMP to the cell surface through RhoA inhibition and Rac1 activation. In vivo, knockdown of Rac1 and DOCK3 or overexpression of MT1-MMP was sufficient to reverse the inhibitory effect of PEDF on extravasation. Using functional studies, we demonstrated that PEDF suppressed the rounded morphology and MT1-MMP surface localization through its antiangiongenic, 34-mer epitope and the recently identified PEDF receptor candidate, PNPLA2. Our findings unveil the coordinated regulation of cell shape and proteolysis and identify an unknown mechanism for PEDF's antimetastatic activity.

Rac1/osmosensing scaffold for MEKK3 contributes via phospholipase C-γ1 to activation of the osmoprotective transcription factor NFAT5

Separate reports that hypertonicity activates p38 via a Rac1-OSM-MEKK3-MKK3-p38 pathway and that p38α contributes to activation of TonEBP/OREBP led us to the hypothesis that Rac1 might activate TonEBP/OREBP via p38. The present studies examine that possibility. High NaCl is hypertonic. We find that siRNA knockdown of Rac1 reduces high NaCl-induced increase of TonEBP/OREBP transcriptional activity (by reducing its transactivating activity but not its nuclear localization). Similarly, siRNA knockdown of osmosensing scaffold for MEKK3 (OSM) also reduces high NaCl-dependent TonEBP/OREBP transcriptional and transactivating activities. Simultaneous siRNA knockdown of Rac1 and OSM is not additive in reduction of TonEBP/OREBP transcriptional activity, indicating a common pathway. However, siRNA knockdown of MKK3 does not reduce TonEBP/OREBP transcriptional activity, although siRNA knockdown of MKK6 does. Nevertheless, the effect of Rac1 on TonEBP/OREBP is also independent of MKK6 because it occurs in MKK6-null cells. Furthermore, we find that siRNA knockdown of Rac1 or OSM actually increases activity (phosphorylation) of p38, rather than decreasing it, as previously reported. Thus, the effect of Rac1 on TonEBP/OREBP is independent of p38. We find instead that phospholipase C-γ1 (PLC-γ1) is involved. When transfected into PLC-γ1-null mouse embryonic fibroblast cells, catalytically active Rac1 does not increase TonEBP/OREBP transcriptional activity unless PLC-γ1 is reconstituted. Similarly, dominant-negative Rac1 also does not inhibit TonEBP/OREBP in PLC-γ1-null cells unless PLC-γ1 is reconstituted. We conclude that Rac1/OSM supports TonEBP/OREBP activity and that this activity is mediated via PLC-γ1, not p38.

The small GTPase Rac1 is involved in the maintenance of stemness and malignancies in glioma stem-like cells

A subpopulation of cancer cells with stem cell properties is responsible for tumor formation, maintenance, and malignant progression; however, the molecular mechanisms underlying the maintenance of cancer stem-like cell properties have remained unclear. Here, we show that the Rho family GTPase Rac1 is involved in the glioma stem-like cell (GSLC) maintenance and tumorigenicity in human glioma. The Rac1-Pak signaling was markedly activated in GSLCs. Knockdown of Rac1 caused reduction of expression of GSLC markers, self-renewal-related proteins and neurosphere formation. Moreover, down-regulation of Rac1 suppressed the migration, invasion, and malignant transformation in GSLCs. Furthermore, inhibition of Rac1 enhanced radiation sensitivity of GSLCs. These results indicate that the small GTPase Rac1 is involved in the maintenance of stemness and malignancies in GSLCs.

Role of Rac1 in regulation of NOX5-S function in Barrett's esophageal adenocarcinoma cells

We have shown that a novel NADPH oxidase isoform, NOX5-S, is the major isoform of NADPH oxidases in an esophageal adenocarcinoma (EA) cell line, FLO, and is overexpressed in Barrett's mucosa with high-grade dysplasia. NOX5-S is responsible for acid-induced reactive oxygen species production. In this study, we found that mRNA levels of NOX5-S were significantly higher in FLO EA cells than in the normal human esophageal squamous cell line HET-1A or in a Barrett cell line, BAR-T. The mRNA levels of NOX5-S were also significantly increased in EA tissues. The data suggest that NOX5-S may be important in the development of EA. Mechanisms of functional regulation of NOX5-S are not fully understood. We show that small G protein Rac1 was present in HET-1A cells, BAR-T cells, and EA cell lines FLO and OE33. Rac1 protein levels were significantly higher in FLO and OE33 cells than in HET-1A or BAR-T cells. Knockdown of Rac1 with Rac1 small interfering RNA significantly decreased acid-induced increase in H(2)O(2) production in FLO EA cells. Overexpression of constitutively active Rac1 significantly increased H(2)O(2) production, an increase that was blocked by knockdown of NOX5-S. By immunofluorescence staining and immunoprecipitation, we found that NOX5-S was present in the cytosol of FLO EA cells and colocalized with Rac1 and SERCA1/2 Ca(2+)-ATPase which is located in the endoplasmic reticulum membrane. We conclude that Rac1 may be important in activation of NOX5-S in FLO EA cells.

H-Ras-specific upregulation of granulocyte colony-stimulating factor promotes human breast cell invasion via matrix metalloproteinase-2

Ras expression has been suggested to be a marker for tumor aggressiveness of breast cancer. We previously showed that H-Ras, but not N-Ras, induced invasive/migratory phenotypes in MCF10A human breast epithelial cells. The present study aimed to determine the role of granulocyte colony-stimulating factor in H-Ras-induced malignant progression of human breast epithelial cells. Here, we show that G-CSF plays a crucial role in H-Ras-induced MCF10A cell invasion and migration. The siRNA-mediated knockdown of G-CSF significantly reduced H-Ras-induced matrix metalloproteinase (MMP)-2 expression, as well as invasion/migration, suggesting the functional significance of G-CSF in the invasive phenotype of human breast cells. Importantly, the induction of G-CSF expression conferred the invasive/migratory phenotypes to MCF10A cells with up-regulation of MMP-2 and activation of Rac1, MKK3/6, p38 MAPK, Akt, and ERKs. Knockdown of Rac1 by siRNA significantly inhibited MMP-2 upregulation and invasiveness of G-CSF MCF10A cells, demonstrating that G-CSF-induced MMP-2 upregulation and invasive phenotype is mediated by Rac1. Using human breast tissues and sera from breast cancer patients, we further demonstrate that the expression level of G-CSF is strongly correlated with pathologically-diagnosed breast cancer. These data provide a molecular basis for the crucial role of G-CSF in promoting invasiveness of human breast epithelial cells.

Abstract 3478: Latent KSHV infection increases the vascular permeability of endothelial cells

Abstract Infection with Kaposi's sarcoma-associated herpesvirus (KSHV) is closely linked to the development of Kaposi's sarcoma (KS). The KS lesion is driven by KSHV-infected endothelial cells and is highly dependent on autocrine and paracrine factors for survival and growth. We report that latent KSHV infection increases the vascular permeability of endothelial cells. Latent KSHV-infected endothelial cells display increased Src kinase activity, increased Rac1 activation and activation of its downstream modulator, p21 activated kinase (PAK1). The KSHV-infected endothelial cells exhibited increases in tyrosine phosphorylation of VE-cadherin and β-catenin, while total levels of these proteins remained unchanged, suggesting that latent infection disrupted endothelial cell junctions. Consistent with these findings, we found that latent KSHV-infected endothelial cells were more permeable than uninfected endothelial cells. Knockdown of Rac1, as well as inhibition of ROS, resulted in decreased permeability of the KSHV-infected endothelial cells. In sum, KSHV latent infection increases the vascular permeability of infected endothelial cells thereby contributing to the pathogenesis of Kaposi's sarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3478. doi:10.1158/1538-7445.AM2011-3478

Novel Role of Rac1/WAVE Signaling Mechanism in Regulation of the Epithelial Na + Channel

The epithelial Na(+) channel (ENaC) is an essential channel responsible for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Consequently, ENaC is a major effector impacting systemic blood volume and pressure. We have shown recently that Rac1 increases ENaC activity, whereas Cdc42 fails to change channel activity. Here we tested whether Rac1 signaling plays a physiological role in modulating ENaC in native tissue and polarized epithelial cells. We found that Rac1 inhibitor NSC23766 markedly decreased ENaC activity in freshly isolated collecting ducts. Knockdown of Rac1 in native principal cells decreased ENaC-mediated sodium reabsorption and the number of channels at the apical plasma membrane. Members of the Wiskott-Aldrich syndrome protein (WASP) family play a central role in the control of the actin cytoskeleton. N-WASP functions downstream of Cdc42, whereas WAVEs are effectors of Rac1 activity. N-WASP and all 3 isoforms of WAVE significantly increased ENaC activity when coexpressed in Chinese hamster ovary cells. However, wiskostatin, an inhibitor of N-WASP, had no effect on ENaC activity. Immunoblotting demonstrated the presence of WAVE1 and WAVE2 and absence of N-WASP and WAVE3 in mpkCCD(c14) and M-1 principal cells. Immunohistochemistry analysis also revealed localization of WAVE1 and WAVE2 but not N-WASP in the cortical collecting duct of Sprague-Dawley rat kidneys. Moreover, patch clamp analysis revealed that Rac1 and WAVE1/2 are parts of the same signaling pathway with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.

Novel role of Rac1/WAVE signaling mechanism in regulation of the epithelial Na + channel (ENaC)

ENaC is an essential channel responsible for Na+ reabsorption in the ASDN. We have shown recently that small GTPase Rac1 increases ENaC activity, whereas Cdc42 fails to change channel activity. Here we tested whether Rac1 signaling plays a physiologic role in modulating ENaC in native tissue and polarized epithelial cells. We have found that Rac1 inhibitor NSC23766 markedly decreased ENaC activity in freshly isolated collecting ducts. Knockdown of Rac1 in native principal cells decreased ENaC-mediated sodium reabsorption and the number of channels at the apical plasma membrane. Members of the Wiskott-Aldrich syndrome protein (WASP) family play central roles in control of the actin cytoskeleton. N-WASP binds Arp2/3 in response to Cdc42, whereas WAVE proteins are effectors of Rac1 activity. Using electrophysiological approaches we have shown that WAVE proteins significantly increase ENaC activity. Immunoblotting demonstrated the presence of WAVE1 and WAVE2 and absence of N-WASP and WAVE3 in mpkCCDc14 and M-1 principal cells. Immunohistochemistry analysis also detected only expression of WAVE1 and WAVE2 in the cortical collecting ducts in Sprague-Dawley rat kidneys. Patch clamp analysis revealed that Rac1 and WAVE1/2 are parts of the same signaling with respect to activation of ENaC. Thus, our findings suggest that Rac1 is essential for ENaC activity and regulates the channel via WAVE proteins.

Rac1/OSM and PLC‐gamma1 contribute in combination to high NaCl‐induced activation of the osmoprotective transcription factor TonEBP/OREBP

Separate reports that hypertonicity activates p38 via a Rac1/OSM ->MEKK3 ->MKK3 -> p38pathway and that p38 contributes to activation of TonEBP/OREBP led us to hypothesize that Rac1 might activate TonEBP/OREBP via p38. The present studies examine that possibility. We find that siRNA knockdown of Rac1 or OSM reduces high NaCl-induced increase of TonEBP transcriptional activity (by reducing transactivating activity, but not nuclear localization). siRNA knock down of MKK3 does not reduce TonEBP/OREBP transcriptional activity, but siRNA knockdown of MKK6 does. Nevertheless, the effect of Rac1 on TonEBP/OREBP is independent of MKK6 (and MKK3) since it occurs in MKK3/MKK6 null cells. Further, we find that siRNA knock down of Rac1 or OSM actually increases activity (phosphorylation) of p38 (both with and without elevation of NaCl), rather than decreasing it, as previously reported. Thus, the effect of Rac1/OSM on TonEBP/OREBP is independent of p38. It has a different explanation involving PLC-γ1. When transfected into PLC-γ1 null MEF cells, catalytically active Rac1 does not increase TonEBP/OREBP transcriptional activity, but it does when PLC-γ1 is reconstituted. Similarly, dominant negative Rac1 does not inhibit TonEBP/OREBP in PLC-γ1 null cells, unless they are reconstituted. We conclude that Rac1/OSM supports TonEBP/OREBP activity, but that this is mediated byPLC-γ1, not p38. Supported by DIR NHLBI and USUHS