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 beta2 and beta3 integrins but not beta1 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.
"In the literature, there are a few reports connecting RhoB expression with the expression level of integrins. It has been shown that diminishing RhoB expression in different cell models causes reduction in the amount of integrins available on the cell surface, namely β1 in prostate cancer cell line  and β2 and β3 in macrophages derived from RhoB−/− mice . These data led us to examine whether silencing RhoB expression in HEp2 and CK2 cells may have increased Ad5-mediated transduction by influencing cell surface expression of Ad5 receptors. "
[Show abstract][Hide abstract] ABSTRACT: Adenovirus type 5 (Ad5) is a non-enveloped DNA virus frequently used as a gene transfer vector. Efficient Ad5 cell entry depends on the availability of its primary receptor, coxsackie and adenovirus receptor, which is responsible for attachment, and integrins, secondary receptors responsible for adenovirus internalization via clathrin-mediated endocytosis. However, efficacious adenovirus-mediated transgene expression also depends on successful trafficking of Ad5 particles to the nucleus of the target cell. It has been shown that changes occurring in tumor cells during development of resistance to anticancer drugs can be beneficial for adenovirus mediated transgene expression. In this study, using an in vitro model consisting of a parental cell line, human laryngeal carcinoma HEp2 cells, and a cisplatin-resistant clone CK2, we investigated the cause of increased Ad5-mediated transgene expression in CK2 as compared to HEp2 cells. We show that the primary cause of increased Ad5-mediated transgene expression in CK2 cells is not modulation of receptors on the cell surface or change in Ad5wt attachment and/or internalization, but is rather the consequence of decreased RhoB expression. We propose that RhoB plays an important role in Ad5 post-internalization events and more particularly in Ad5 intracellular trafficking. To the best of our knowledge, this is the first study showing changed Ad5 trafficking pattern between cells expressing different amount of RhoB, indicating the role of RhoB in Ad5 intracellular trafficking.
PLoS ONE 01/2014; 9(1):e86698. DOI:10.1371/journal.pone.0086698 · 3.23 Impact Factor
"Loss of RhoB is often found in human cancers during tumor progression. Wheeler AP et al. reported that deletion of RhoB affects the cell adhesion, spreading and migration speed, and that these effects are dependent on the substrate availability, correlating with reduced surface integrin levels . DeWard AD et al. show that the additional knockout of RhoB expression in Drf1-null mice accelerates the progression to myelodysplasia (MDS) . "
[Show abstract][Hide abstract] ABSTRACT: RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies.
PLoS ONE 11/2013; 8(11):e78417. DOI:10.1371/journal.pone.0078417 · 3.23 Impact Factor
"The formation of bipolar protrusion is incompatible with macrophage migration, as evidenced by migration assays. C3bot thus exhibit comparable biological activities in myeloid cells, which have already reported for tat- C3 (Park et al. 2003; Wheeler and Ridley 2007; Worthylake et al. 2001). C3bot and tat-C3 do thus not differ in their biological activity. "
[Show abstract][Hide abstract] ABSTRACT: C3-like exoenzymes are produced by various microorganism including Clostridium botulinum (C3bot), Bacillus cereus and Staphylococcus aureus. C3bot is the prototype of C3-like exoenzymes that specifically ADP-ribosylates and thereby inactivates Rho(A/B/C). C3-like exoenzymes are not yet regarded as virulence factors, as the lack of cell entry domains results in a poor accessibility of the C3-like exoenzymes to cells. In this study, the sensitivity of various cell lines to C3bot has been reinvestigated. Primary monocytes as well as cultured macrophage-like cells including J774A.1 cells and RAW macrophages exhibit a tenfold higher sensitivity to C3bot than fibroblasts and epithelial cells. RhoA ADP-ribosylation by C3bot resulted in the formation of pronounced bipolar protrusions based on defective tail retraction. The formation of bipolar protrusion resulted in inhibited macrophage migration. These findings suggested that macrophages appear to be target cells of C3bot. Migration of macrophage is a prerequiste for their recruitment to the site of pathogen invasion or tissue damage. Inhibition of macrophage migration likely preserves the survival of C3-producing microorganisms. The observations of this study reinforce the paradigm of a role of C3-like exoenzymes as virulence factors.
Archiv für Experimentelle Pathologie und Pharmakologie 05/2012; 385(9):883-90. DOI:10.1007/s00210-012-0764-9 · 2.47 Impact Factor
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