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The Rho GTPase RhoB has been shown to affect cell migration, but how it does this is not clear. Here we show that cells depleted of RhoB by RNAi are rounded and have defects in Rac-mediated spreading and lamellipodium extension, although they have active membrane ruffling around the periphery. Depletion of the exchange factor GEF-H1 induces a similar phenotype. RhoB-depleted cells migrate faster, but less persistently in a chemotactic gradient, and frequently round up during migration. RhoB-depleted cells have similar numbers of focal adhesions to control cells during spreading and migration, but show more diffuse and patchy contact with the substratum. They have lower levels of surface β1 integrin, and β1 integrin activity is reduced in actin-rich protrusions. We propose that RhoB contributes to directional cell migration by regulating β1 integrin surface levels and activity, thereby stabilizing lamellipodial protrusions.

Purpose: We investigated the action mechanism of a novel anticancer compound, KR28 (1-allyl-4-dodecanoyl-1-ethyl-piperazin-1-ium; bromide), to induce apoptosis of human prostate carcinoma PC-3 cells. Methods: To explore an apoptotic signaling of KR28, we used ROS assay, SRB assay, flow cytometry analysis, reporter assay, xenograft assay, Western blotting, and RT-PCR analysis. Results: The growth inhibitory action of KR28 is cell line specific, impeding the growth of prostate carcinoma PC-3 and stomach carcinoma NUGC-3 cells. KR28 showed strong antitumor activity in PC-3 mouse xenograft model. KR28 increased ROS production, leading to nuclear c-Abl expression, which in turn activated p38 mitogen-activated protein kinase (MAPK) to enhance the expression of RhoB, an apoptosis inducer. The KR28-induced apoptosis was abrogated by the ROS scavenger N-acetylcysteine and by knockdown of c-Abl, p38 MAPK, or ATF2. Moreover, the p300 binding site and two CCAAT boxes in the RhoB promoter appear to be involved in ROS-mediated RhoB expression in the presence of KR28. Conclusion: The antitumor agent KR28 induces apoptosis of PC-3 cells by ROS-mediated RhoB expression via c-Abl upregulation and activation of p38 MAPK/ATF-2. [ABSTRACT FROM AUTHOR]

Abstract: RhoB is expressed during tumor cell proliferation, survival, invasion, and metastasis. In malignant progression, the expression levels of RhoB are commonly attenuated. RhoB is known to be linked to the regulation of the PI3K/Akt survival pathways. Based on aliphatic amido-quaternary ammonium salts that induce apoptosis via up-regulation of RhoB, we synthesized novel aliphatic sulfonamido-quaternary ammonium salts. These new synthetic compounds were evaluated for their biological activities using an in vitro RhoB promoter assay in HeLa cells, and in a growth inhibition assay using human cancer cell lines including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. Compound 5b (ethyl-dimethyl-{3-[methyl-(tetradecane-1-sulfonyl)-amino]-propyl}-ammonium; iodide) was the most promising anticancer agent in the series, based upon the potency of growth inhibition and RhoB promotion. These new aliphatic sulfonamido-quaternary ammonium salts could be a valuable series for development of new anticancer chemotherapeutic agents. [Copyright &y& Elsevier]

Small GTPase RhoB has been well documented in regulating cell adhesion, motility, proliferation, and survival, but to date, there is little information about the relationship between RhoB and inflammation. In this study, the mRNA and protein levels of RhoB were induced by lipopolysaccharide (LPS) in RAW264.7 cells determined by real-time PCR and Western blot. The upregulation of RhoB by LPS was also observed in mouse peritoneal macrophages and in mouse lung, liver, and kidney. RhoB overexpression by transfecting with wild RhoB plasmid increased the secretion of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) in RAW264.7 cells, while RhoB knockdown by RNA interference decreased the secretion of TNF-α and NO in RAW264.7 cells. TNF-α and NO synthase are the target genes of nuclear factor-kappaB (NF-κB), and overexpression of RhoB increased, whereas inhibition of RhoB decreased the basal and LPS-activated transcriptional activity of NF-κB in the cells. These results demonstrated that LPS induced RhoB expression in mouse in vivo and in vitro and in RAW264.7 cells, and the role of RhoB on LPS-induced secretion of TNF-α and NO was at least partly mediated via NF-κB. These results indicated that RhoB was involved in LPS-induced inflammation in mouse in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Highlights: [•] Both the mucosal and serum levels of miR-21 were upregulated in UC. [•] Overexpression of miR-21 impaired the integrity of the tight junctions. [•] miR-21 induced the depletion of RhoB and the impairment of the tight junctions. [ABSTRACT FROM AUTHOR]

Abstract: A series of novel aliphatic amido-quaternary ammonium salts were synthesized and evaluated for their anticancer effects involving induction of RhoB. Most of these compounds, featuring open-ring forms of aliphatic amido-quaternary ammonium salts, exhibited potent anti-proliferative activities in human cancer cell lines, including PC-3, NUGC-3, MDA-MB-231, ACHN, HCT-15, and NCI-H23. In further evaluation, the representative compound N,N-diethyl-N-(2-(N-methyltetradecanamido)ethyl)prop-2-en-1-aminium bromide (3b) exhibited potent pro-apoptotic activity, through RhoB activation, in HeLa cells. [Copyright &y& Elsevier]

Abstract: Rapamycin suppresses endothelial proliferation and migration, which leads to delayed re-endothelialization in the rapamycin-eluted stents that are used in coronary heart disease patients. Because microRNAs (miRs) play important roles in endothelial angiogenesis, we tested the hypothesis that rapamycin induces endothelial suppression, partly through pathways that involve miRs. Rapamycin treatment increased the expression of miR-21 in HUVECs. The downregulation of miR-21 by inhibitors abolished the negative effects of rapamycin on endothelial cell growth and mobility. RhoB was confirmed as a direct target gene of miR-21. Knockdown of Raptor by siRNA mimicked the effects of rapamycin on miR-21 expression. Our study provides a new explanation of the mechanism of rapamycin-mediated inhibition of endothelial proliferation and migration. [Copyright &y& Elsevier]

Abstract: RhoB, one of the upstream signaling proteins of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway, is frequently mutated in human cancer. Based on a piperazine alkyl derivative that induced apoptosis via up-regulation of RhoB, we synthesized novel aliphatic amido/sulfonamido-quaternary ammonium salts and evaluated their biological activities using an in vitro growth inhibition assay and RhoB promoter assay in human cancer cells. Compound 3a was the most promising anticancer agent in the series, based upon its potent growth inhibition via RhoB-mediated signaling. These novel aliphatic amido/sulfonamido-quaternary ammonium salts may be useful as a platform for development of anticancer chemotherapeutic agents. [Copyright &y& Elsevier]

Abstract: Our previous study demonstrated the therapeutic potential of gallic acid (GA) for controlling tumor metastasis through its inhibitory effect on the motility of AGS cells. A noteworthy finding in our previous experiment was increased RhoB expression in GA-treated cells. The aim of this study was to evaluate the role of RhoB expression on the inhibitory effects of GA on AGS cells. By applying the transfection of RhoB siRNA into AGS cells and an animal model, we tested the effect of GA on inhibition of tumor growth and RhoB expression. The results confirmed that RhoB-siRNA transfection induced GA to inhibit AGS cells’ invasive growth involving blocking the AKT/small GTPase signals pathway and inhibition of NF-κB activity. Finally, we evaluated the effect of GA on AGS cell metastasis by colonization of tumor cells in nude mice. It showed GA inhibited tumor cells growth via the expression of RhoB. These data support the inhibitory effect of GA which was shown to inhibit gastric cancer cell metastasis and invasive growth via increased expression of RhoB, downregulation of AKT/small GTPase signals and inhibition of NF-κB activity. Thus, GA might be a potential agent in treating gastric cancer. [Copyright &y& Elsevier]

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies. [ABSTRACT FROM AUTHOR]

Abstract: Objective: The aim was to evaluate antitumor activity of the combination of ixabepilone and sunitinib in pre-clinical models of chemotherapy naïve and refractory epithelial ovarian tumors, and to investigate the mechanism of synergy of such drug combination. Methods: HOVTAX2 cell line was derived from a metastatic serous papillary epithelial ovarian tumor (EOC) and a paclitaxel-resistant derivative was established. Dose response curves for ixabepilone and sunitinib were generated and synergy was determined using combination indexes. The molecular mechanism of antitumor synergy was examined using shRNA silencing. Results: The combination of ixabepilone and sunitinib demonstrated robust antitumor synergy in naïve and paclitaxel-resistant HOVTAX2 cell lines due to increased apoptosis. The GTPase, RhoB, was synergistically upregulated in cells treated with ixabepilone and sunitinib. Using shRNA, RhoB was demonstrated to mediate antitumor synergy. These results were validated in two other EOC cell lines. Conclusions: Ixabepilone plus sunitinib demonstrated antitumor synergy via RhoB in naïve and paclitaxel-resistant cells resulting in apoptosis. This study demonstrates a novel mechanism of action leading to antitumor synergy and provides ‘proof-of-principle’ for combining molecular targeted agents with cytotoxic chemotherapy to improve antitumor efficacy. RhoB could be envisioned as an early biomarker of response to therapy in a planned Phase II clinical trial to assess the efficacy of ixabepilone combined with a receptor tyrosine kinase inhibitor such as sunitinib. To the best of our knowledge, this is the first demonstration of antitumor synergy between these two classes of drugs in EOC and the pivotal role of RhoB in this synergy. [Copyright &y& Elsevier]

Background: Dendritic cells are professional antigen presenting cells that perform antigen processing and antigen presentation functions and rely on the proper functioning and distribution of the endoplasmic reticulum (ER) and Golgi apparatus and of vesicular trafficking pathways. We previously developed a model system to study the mechanisms governing inhibition of chronic rejection of heart allografts. Methods: Heterotopic cardiac transplants were placed intra-abdominally and the major histocompatibility class (MHC) class I allochimeric molecule, [α1h1/u]-RT1.Aa, which contains donor-type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient-type (ACI, RT1a) sequences, was delivered by portal vein to the recipients of heterotopic hearts. Dendritic cells were isolated from the recipient bone marrow at 1 and 3 days after transplantation and were immunostained or processed for Western blotting with anti-RhoB, translationally controlled tumor protein (TCTP), Sprouty-related (Spred1) protein, ER, and Golgi antibodies. Results: Western blotting analyses showed the downregulation of RhoB GTPase, TCTP, and Spred1 in dendritic cells isolated from allochimeric molecule-treated rats. Immunostaining showed that in these cells, Spred 1 was shifted to the base of cellular processes, Rho B formed nonvesicular band in the cell equator, and TCTP was highly enriched in the cell nucleus. The Golgi apparatus was drastically reduced in size and formed a tiny nonvesicular aggregate, and the ER partially lost vesicular appearance. Conclusions: The function of allochimeric molecule in the abrogation of heart allograft rejection may rely on the downregulation of RhoB pathway components that regulate the structure and function of the ER/Golgi/vesicular trafficking pathways involved in antigen processing and presentation by dendritic cells. [Copyright &y& Elsevier]

RhoB, a member of the Rho subfamily of small GTPases, mediates diverse cellular functions, including cytoskeletal organization, cell transformation and vesicle trafficking. The thymus undergoes progressive decline in its structure and function after puberty. We found that RhoB was expressed in thymic medullary epithelium. To investigate a role of RhoB in the regulation of thymic epithelial organization or thymocyte development, we analyzed the thymi of RhoB-deficient mice. RhoB-deficient mice were found to display earlier thymic atrophy. RhoB deficiency showed significant reductions in thymus weight and cellularity, beginning as early as 5 weeks of age. The enhanced expression of TGF-β receptor type II (TGFβRII) in thymic medullary epithelium was observed in RhoB-null mice. In addition, the expression of fibronectin, which is shown to be regulated by TGF-β signaling, was accordingly increased in the mutant thymic medulla. Since there is no age-related change of RhoB expression in the thymus, it is unlikely that RhoB in thymic epithelium directly contributes to age-related thymic involution. Nevertheless, our findings strongly support a physiological role of RhoB in regulation of thymus development and maintenance through the inhibition of TGF-β signaling in thymic medullary epithelium. [ABSTRACT FROM AUTHOR]

Abstract: We synthesized novel aliphatic amido-quaternary ammonium salts in an effort to discover anticancer agents that increase Ras homolog gene family, member B, (RhoB) levels. These compounds exert anti-proliferative activities against several human cancer cell types. Seventeen compounds, varying in aliphatic carbon chain length and N-substituents, were synthesized and their biological activities were evaluated. Of these 17 compounds, compound 3i emerged as the most promising anticancer compound by promoting apoptosis through the RhoB mediated pathway. Potent biological activities observed for these novel aliphatic amido-quaternary ammonium salt analogues support their potential as anticancer, chemotherapeutic agents. [Copyright &y& Elsevier]

Abstract: The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (−23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (−72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in Jurkat cells. [Copyright &y& Elsevier]

Diarylsulfonylureas are potent antitumor agents that have been tested in clinical trials. However, detailed mechanisms of their apoptotic activity remain unclear. Here, we report a new diarylsulfonylurea derivative, LB2A, that upregulates RhoB, thereby inducing potent apoptosis in HCT-116 human colon cancer cells independently of p53 status. LB2A decreased procaspase-3, increased phospho-JNK, and cleaved PARP, leading to apoptosis of HCT-116 cells. Prior treatment of HCT-116 cells with the JNK inhibitor SP600125 and the RNA synthesis inhibitor DRB blocked apoptosis, implying that JNK activation and mRNA production are important for apoptosis by LB2A. Western blotting, RT-PCR, and RhoB-promoter luciferase reporter assays revealed that LB2A increased RhoB via JNK-mediated transcriptional activation. LB2A decreased HDAC1 and increased acetyl-H3, both of which activate the RhoB promoter and were blocked by SP600125. Ectopic expression of RhoB induced apoptosis of HCT-116 cells, suggesting that RhoB is critical for the anti-cancer activity of LB2A in human colon cancer cells. LB2A also exhibited potent tumor growth inhibition of HCT-116 cells in vivo using a mouse xenograft assay. Taken together, these results show that LB2A induces apoptosis of HCT-116 cells via JNK-mediated transcriptional upregulation of RhoB and may therefore provide a potential therapy for human colon cancer. [ABSTRACT FROM AUTHOR]

Abstract: Acquired resistance to cisplatin represents a major obstacle to an efficient chemotherapy. We found downregulation of RhoB expression in cisplatin-resistant tumor cell lines from different origin. In cisplatin-resistant laryngeal carcinoma subline overexpression of farnesylated or geranylgeranylated RhoB increased cisplatin-induced cell death, while silencing of RhoB expression diminished sensitivity of parental HEp-2 cells via decreased cellular accumulation of cisplatin. However, since RhoB silencing in additional tumor cell lines did not alter their sensitivity to cisplatin, we can assume that RhoB downregulation does not provide general protective role in cell response to cisplatin. Nevertheless, gene therapy involving restoration of RhoB expression might improve the efficiency of cisplatin treatment, especially in patients with laryngeal carcinoma that acquired resistance to this chemotherapeutic drug. [Copyright &y& Elsevier]

Rapid control of protein degradation is usually achieved through the ubiquitin-proteasome pathway. We recently found that the short-lived GTPase RhoB is degraded in lysosomes. Moreover, the fusion of the RhoB C-terminal sequence CINCCKVL, containing the isoprenylation and palmitoylation sites, to other proteins directs their sorting into multivesicular bodies (MVBs) and rapid lysosomal degradation. Here, we show that this process is highly specific for RhoB. Alteration of late endosome lipid dynamics produced the accumulation of RhoB, but not of other endosomal GTPases, including Rab5, Rab7, Rab9 or Rab11, into enlarged MVB. Other isoprenylated and bipalmitoylated GTPases, such as H-Ras, Rap2A, Rap2B and TC10, were not accumulated into MVB and were stable. Remarkably, although TC10, which is highly homologous to RhoB, was stable, a sequence derived from its C-terminus (CINCCLIT) elicited MVB sorting and degradation of a green fluorescent protein (GFP)-chimeric protein. This led us to identify a cluster of basic amino acids (KKH) in the TC10 hypervariable region, constituting a secondary signal potentially involved in electrostatic interactions with membrane lipids. Mutation of this cluster allowed TC10 MVB sorting and degradation, whereas inserting it into RhoB hypervariable region rescued this protein from its lysosomal degradation pathway. These findings define a highly specific structural module for entering the MVB pathway and rapid lysosomal degradation. [ABSTRACT FROM AUTHOR]

Rho family protein regulates variety of cellular functions as cytoskeletal organization, cell proliferation and apoptosis. In the present study, we demonstrate that RhoB-overexpressed prostate cancer cells showed an enhanced cell motility and the administration of the GSK-3 inhibitors inhibited this increase in migration. Among the extracellular matrix and adhesion-related molecules, MMP1 RNA expression was increased in RhoB-overexpressed cells, administration of MMP inhibitor suppressed the collagen gel invasion in these cells. This is the first report evaluating RhoB function and the downstream signaling events in prostate cancer cell. Our results indicate that RhoB promotes cell motility and invasion in a metastatic prostate cancer cell. [Copyright &y& Elsevier]

Abstract: The Ras-related small GTP-binding protein RhoB is known to be a pro-apoptotic protein and immediate-early inducible by genotoxic stresses. In addition, JNK activation is known to function in γ-radiation-induced apoptosis. However, it is unclear how JNK activation and γ-radiation-dependent RhoB induction are related. Here we verified the relationship between JNK activation and RhoB induction. RhoB induction by γ-radiation occurred at the transcriptional level and transcriptional activation of RhoB was concomitant with an increase in RhoB protein. γ-Radiation-induced RhoB expression was markedly attenuated by pretreatment with a JNK-specific inhibitor, SP600125, but not by a p38 MAPK inhibitor, SB203580. Inhibition of JNK caused a decrease in early apoptotic cell death that correlated with RhoB expression. However, PI3K inhibition had no significant effects, indicating that the AKT survival pathway was not involved. The siRNA knockdown of JNK resulted in a decrease in RhoB expression and the siRNA knockdown of RhoB restored cell growth even in the γ-irradiated cells. These results suggest that RhoB regulation involves the JNK pathway and contributes to the early apoptotic response of Jurkat T cells to γ-radiation. [Copyright &y& Elsevier]

Abstract: Epithelial to mesenchymal transition (EMT) is involved in embryological development, cancerous metastatic spread and organ fibrosis, including the kidney. This process is largely driven by transforming growth factor-beta and recent evidence has implicated Rho as a key intracellular signalling molecule. In this study we have used RNA interference to silence the genetically distinct Rho (A, B and C) isoforms to define their individual functions in human kidney epithelial cells undergoing EMT. We demonstrate that the downregulation of the epithelial cell marker E-cadherin is dependent upon the Rho effector, Rho-kinase. However, silencing RhoA or RhoC expression also results in E-cadherin loss, though each by different mechanisms. Loss of RhoA leads to an upregulation of Snail1 and a reduction in the transcription of E-cadherin whereas loss of RhoC upregulates its breakdown via proteasomal degradation. During EMT, the upregulation of alpha-smooth muscle actin can be blocked by inhibiting the expression of RhoA, but not by that of RhoB or RhoC. This effect is independent of Rho-kinase activity. RhoC is the isoform solely responsible for stress fibre formation and inhibiting its expression reduces EMT-induced migration by 50%. RhoB appears to play a role in cell survival as inhibiting its expression leads to >300% increase in cell apoptosis and a relocalization of focal adhesion kinase. We conclude that Rho is a key signalling molecule in the process of EMT but that each isoform has a distinct and specific role. [Copyright &y& Elsevier]

Abstract: Glucocorticoid (GC) hormones exert an antiproliferative effect on various cells. The effect is mainly mediated by glucocorticoid receptor (GR) which acts as a transcription factor. Ligand-bound GR translocates from the cytoplasm into the nucleus to modulate gene expression in a variety of ways. Although the framework of transcriptional regulation by the GC/GR has been described, the molecular mechanism of antiproliferative effect of GC is still largely unclear. In this article, we reviewed GC-induced changes in gene expression that are involved in GC-antiproliferative effect, and mainly focused on our recently identified glucocorticoid-responsive genes, TGF-β receptor type II (TβRII) and small GTP binding protein RhoB. We found that expressions of TβRII and RhoB were up-regulated by ligand-bound GR at mRNA and protein levels. Blocking the effect of TβRII by TβRII neutralizing antibody or reduction of RhoB mRNA expression by RNAi diminished dexamethasone-inhibitory effect on cell proliferation, thus confirming that these genes are involved in GC anti-proliferation effect. Collectively, GC up-regulating the expressions of RhoB and TβRII play an important role in GC anti-proliferation effect. [Copyright &y& Elsevier]

Acidic extracellular pH (pHe) is a common feature of the tumor microenvironment and has been implicated in tumor invasion through the induction of protease secretion. Since lysosomes constitute the major storehouse of cellular proteases, the trafficking of lysosomes to the cell periphery may be required in order to secrete proteases. We demonstrate that a pHe of 6.4-6.8 induced the trafficking of lysosomes to membrane protrusions in the cell periphery. This trafficking event depended upon the PI3K pathway, the GTPase RhoA and sodium-proton exchange activity, resulting in lysosomal exocytosis. Acidic pHe induced a cytoplasmic acidification (although cytoplasmic acidification was not sufficient for acidic pHe-induced lysosome trafficking and exocytosis) and inhibition of NHE activity with the amiloride derivative, EIPA or the anti-diabetic agent troglitazone prevented lysosome trafficking to the cell periphery. Interestingly, using the more specific NHE1 and NHE3 inhibitors, cariporide and s3226 respectively, we show that multiple NHE isoforms are involved in acidic pHe-induced lysosome trafficking and exocytosis. Moreover, in cells expressing NHE1 shRNA, although basal NHE activity was decreased, lysosomes still underwent acidic pHe-induced trafficking, suggesting compensation by other NHE family members. Together these data implicate proton exchangers, especially NHE1 and NHE3, in acidic pHe-induced lysosome trafficking and exocytosis. [ABSTRACT FROM AUTHOR]

Abstract: RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions. [Copyright &y& Elsevier]

The Rho family proteins Rac and Rho are believed to be key regulators of cell migration through their effects on the cytoskeleton and cell adhesion. However, recent studies in macrophages indicate that they are not always essential for migration, although they do affect cell shape and adhesion. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme is the most aggressive form of primary brain tumor and remains largely incurable, in large part, due to its highly invasive nature. The phosphoinositide (PI) 3-kinase pathway is often constitutively active in these tumors due to activating mutations in the epidermal growth factor receptor, or deletion/loss of function of the tumor suppressor PTEN. Protein kinase C type ι (PKCι), a member of the atypical protein kinase C family, is activated by the PI 3-kinase pathway and is an important downstream mediator. Here, we have assessed the role of PKCι in glioblastoma cell invasion. Depletion of PKCι with RNA interference caused an increase in actin stress fibers and a decrease in cell motility and invasion. Gene expression microarray analysis of U87MG cells showed that PKCι repressed expression of mRNA for RhoB, which has previously been shown to have a role in actin stress fiber formation. Western blot analysis showed that both PKCι depletion and pharmacological inhibition of PKCι caused an increase in the protein levels of RhoB, as did inhibition of PI 3-kinase. Expression of RhoB from a constitutive promoter caused changes in actin stress fibers and cell invasion that were similar to those seen with PKCι depletion. These data show that PKCι, activated as a consequence of aberrant upstream PI 3-kinase signaling, mediates glioblastoma cell motility and invasion, and that repression of RhoB is key downstream event in PKCι signaling leading to enhanced cell motility. In addition, constitutive expression of RhoB repressed PKCι activity, as assessed by its phosphorylation status on Thr555. PKCι and RhoB are, therefore, mutually antagonistic, potentially creating a sensitive switch between invasive and non-invasive phenotypes.Oncogene (2008) 27, 3587–3595; doi:10.1038/sj.onc.1211027; published online 21 January 2008 [ABSTRACT FROM AUTHOR]

The complete coding sequences of three porcine genes—Rho-related GTP-binding proteins RHOB and RHOG and Prenylated Rab acceptor protein 1 (PRAF1) were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR) based on the sequence information of the mouse or other mammals and referenced highly homologous pig ESTs. The nucleotide sequences of these three genes revealed that porcine RHOB gene encodes a protein of 196 amino acids that contains the conserved putative RhoA-like domain and has high homology with the RHOB precursor of three species—human, rat, and mouse (100%).The porcine RHOG gene encodes a protein of 191 amino acids that contains the conserved putative RhoG domain and has high homology with the RhoG precursor (RHOG) of three species—human, mouse, and Cricetus cricetus (98%). The porcine PRAF1 gene encodes a protein of 185 amino acids that contains the conserved putative PRA1 domain and has high homology with the PRAF1 of five species—dog (97%), cattle (97%), human (96%), rat (95%), and mouse (95%). The tissue expression analysis indicated swine RHOB gene was moderately expressed in lung; weakly in fat, spleen, and kidney; and almost not expressed in small intestine, large intestine, liver, and muscle. The swine RHOG gene was overexpressed in small intestine, large intestine, liver, and muscle; moderately expressed in kidney; weakly in spleen; and almost not expressed in fat and lung. The swine PRAF1 gene was overexpressed in fat and spleen, moderately in lung and kidney, weakly in small intestine and large intestine, and almost not expressed in liver and muscle. Our experiment established the primary foundation for further research on these three swine genes. [ABSTRACT FROM AUTHOR]

Tumor response to radiotherapy is influenced by many tumoral intracellular biological factors whose deregulation leads to the modulation of tumor sensitivity to radiotherapy. However, the pathways controlling intracellular radiation resistance are activated by or display common pathways with many factors also controlling the tumoral microenvironment, and particularly angiogenesis. One of the innovative strategies that could improve response to irradiation of aggressive or radiotherapy-insensitive tumors consists of combining radiotherapy with inhibitors of the angiogenesis pathways. This review details four main reasons for this: angiogenic factors control intracellular radioresistance, endothelial cell radiosensitivity controls tumor radiosensitivity, tumor angiogenesis is the cause of hypoxia, a major radiation resistance factor, and stem cells, known to be radiation resistant, are dependent on angiogenic factors. We describe the implication of different factors in the various mechanisms leading to radiation resistance. These factors include vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor 2 (FGF2), along with αvβ3 and αvβ5 integrins as well as their downstream cellular pathways including some small G proteins. The review also explains the radiation-sensitizing effect of the inhibition of these factors by targeted therapies. The optimal sequences of administration between antiangiogenics and radiotherapy are partially elucidated. The success of these combinations will depend on the specific study of the mechanisms of action of antiangiogenic agents and their interaction with ionizing radiations, and on the use in preclinical and clinical studies of metabolic and functional imaging. These techniques represent an essential tool for determining the optimal combination sequence followed by the assessment of these combinations. [ABSTRACT FROM AUTHOR]

Abstract: RhoB, a member of the Rho family of small GTPases, regulates the organization of the actin cytoskeleton, malignant transformation, and genotoxic stress-induced signaling. In order to characterize epigenetic regulation of RhoB transcription during aging, the mRNA levels of RhoB were investigated using various mouse tissues of different ages. Bisulfite sequencing revealed that the CpG dinucleotide methylation patterns of the RhoB promoter region were not altered in skeletal muscle and lung during aging. ChIP analysis showed that levels of histone H3 and H4 acetylation were reduced in a tissue-specific manner during aging due to direct HDAC1 binding. Histone H3 lysine 9 trimethylation level and deposition of HP1β increased in RhoB promoter during aging, whereas histone H3 lysine 4 dimethylation level gradually decreased. It was concluded that mouse RhoB transcription is epigenetically regulated in a tissue-specific manner during aging by histone modification, but not by CpG methylation. [Copyright &y& Elsevier]

Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca2+ entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca2+ and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca2+ release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca2+ entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca2+-dependent and Ca2+-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca2+. Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca2+ entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca2+ entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca2+ entry through reverse-mode Na+/Ca2+ exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia. [ABSTRACT FROM AUTHOR]

Abstract: Rho GTPases regulate multiple cellular responses, including cell motility and cell cycle progression. The Rho isoform RhoB represses transformation and affects endosomal trafficking, but its effects on cell adhesion and migration have not been investigated in detail. Here we show that RhoB-null macrophages are more rounded than wild-type macrophages on fibronectin and uncoated glass, and have reduced adhesion to ICAM-1 and glass but not fibronectin. This correlated with lower cell surface expression of β2 and β3 integrins but not β1 integrin. RhoB-null cells migrated faster than Wt cells on fibronectin, consistent with their smaller spread area, but slower than Wt cells on glass, reflecting their reduced adhesion. C3 transferase, which inhibits RhoA, RhoB and RhoC, induced cell spreading but this effect was reduced in RhoB-null cells. However, RhoB is not required for assembly of podosomes, which are integrin-based adhesion sites, whereas C3 transferase induced a decrease in podosomes and defects in tail retraction. Since macrophages do not express RhoC, these effects of C3 transferase are due to inhibition of RhoA rather than RhoB. Our results suggest that RhoB affects cell shape and migration by regulating surface integrin levels. [Copyright &y& Elsevier]

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system. [ABSTRACT FROM AUTHOR]

Recently, we have shown that RhoB suppresses EGFR-, ErbB2-, Ras- and Akt-mediated malignant transformation and metastasis. In this paper, we demonstrate that the novel antitumor agents farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase I inhibitors (GGTIs) upregulate RhoB expression in a wide spectrum of human cancer cells including those from pancreatic, breast, lung, colon, bladder and brain cancers. RhoB induction by FTI-277 and GGTI-298 occurs at the transcriptional level and is blocked by actinomycin D. Reverse transcription–PCR experiments documented that the increase in RhoB protein levels is due to an increase in RhoB transcription. Furthermore, treatment with FTIs and GGTIs of cancer cells results in HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter. Thus, promoter acetylation is a novel mechanism by which RhoB expression levels are regulated following treatment with the anticancer agents FTIs and GGTIs.Oncogene (2007) 26, 633–640. doi:10.1038/sj.onc.1209819; published online 14 August 2006 [ABSTRACT FROM AUTHOR]