A functional polymorphism in the promoter of RhoB is associated with susceptibility to Vibrio anguillarum in turbot (Scophthalmus maximus).

As an isoform of Rho family GTPases, RhoB plays a pivotal role in cytoskeletal organization, cell proliferation, apoptosis and immune response. However, the regulatory mechanisms of RhoB expression in aquatic animals are still unknown. In the present study, we first construct Vibrio anguillarum infection model in S. maximus, including susceptible and resistant individuals. Then the temporal expression of RhoB was detected after V. anguillarum challenge using qRT-PCR and found that RhoB transcripts were significantly induced in the liver, gill and blood despite of differential expression levels and responsive time points. In addition, the mRNA levels of RhoB in resistant individuals were significantly higher than in susceptible ones. The length of 2083 bp sequences of RhoB promoter was cloned and characterized. Moreover, DNA methylation of the RhoB promoter was measured by bisulfite sequencing (BSP) and hypo-methylated was detected in the CpG islands. Three SNPs (-1590, -1575 and -1449) and two haplotypes in the promoter region of RhoB were identified to be associated with V. anguillarum resistance in turbot by association analysis in group 17-R and 17-S. Deletion analysis indicated that these SNPs could negatively mediate the activity of RhoB promoter. Site-directed mutagenesis and qRT-PCR of individuals with different genotypes demonstrated that -1575 T/A polymorphism affected promoter activity. Further study showed that this mutation altered the binding site of the transcription factor CREB. Co-transfection of SmCREB and RhoB promoter was performed in HEK293T cells which confirmed the -1575 allelic differences on transcriptional activity, with the susceptibility allele showing reduced activity. Taken together, our findings implicate that losing of binding of CREB to SmRhoB promoter due to -1575T/A polymorphisms enhances SmRhoB expression in resistant turbot, which provide insights into the effect of SmRhoB expression in response to V. anguillarum infection.

RhoB antibody alters retinal vascularization in models of murine retinopathy.

Neovascularization in cancer or retinopathy is driven by pathological changes that foster abnormal sprouting of endothelial cells. Mouse genetic studies indicate that the stress-induced small GTPase RhoB is dispensable for normal physiology but required for pathogenic angiogenesis. In diabetic retinopathy, retinopathy of prematurity (ROP) or age-related wet macular degeneration (AMD), progressive pathologic anatomic changes and ischemia foster neovascularization are characterized by abnormal sprouting of endothelial cells. This process is driven by the angiogenic growth factor VEGF, which induces and supports the formation of new blood vessels. While injectable biologics targeting VEGF have been used to treat these pathological conditions, many patients respond poorly, prompting interest in other types of mechanism-based therapy. Here we report the preclinical efficacy of a monoclonal antibody that specifically targets RhoB, a signaling molecule that is genetically dispensable for normal physiology but required for pathogenic retinal angiogenesis. In murine models of proliferative retinal angiogenesis or oxygen-induced retinopathy, administering a monoclonal RhoB antibody (7F7) was sufficient to block neoangiogenesis or avascular pathology, respectively. Our findings offer preclinical proof of concept for antibody targeting of RhoB to limit diabetic retinopathy, ROP or wet AMD and perhaps other diseases of neovasculogenesis such as hemangioma or hemangiosarcoma nonresponsive to existing therapies.

Downregulated expression of miR-223 promotes Toll-like receptor-activated inflammatory responses in macrophages by targeting RhoB.

Toll-like receptors (TLRs) induced-inflammatory response must be tightly regulated to avoid impairment in host itself. Numerous factors have been identified in regulation of TLR-triggered inflammatory response. Among these, microRNAs (miRNAs) are small non-coding RNA molecules which have got much attention. MiR-223, which highly expresses in myeloid cells of the bone marrow, has reported to participate in kinds of inflammatory responses by targeting inflammasome sensor-NLRP3 to repress production of IL-6 and IL-1ß, and thus attenuate inflammatory response. However, the function of miR-223 in TLRs-activated inflammatory response of macrophages is not clear. Here we found miR-223 expression is dramatically reduced in macrophages by TLR ligand stimulation (e.g. LPS, CpG and poly (I:C)). The down-regulated miR-223 leads to the increase in the RhoB expression, which induce the activation of NF-κB and MAPK signaling, promoting TNF-α, IL-6 and IL-1ß production upon LPS stimulation. In addition, the histone deacetylase inhibitor trichostatin A increased miR-223 expression obviously in TLR-triggered macrophages, which in turn suppressed RhoB expression and downstream IL-6 production, suggesting that the inhibition of miR-223 by histone deacetylation may be involved in the regulation of TLR-activated inflammatory response. Herein, our findings suggest that miR-223-RhoB axis might be a novel target for the treatment of inflammatory diseases.

Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.

Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.

Genome-wide scan identifies TNIP1, PSORS1C1, and RHOB as novel risk loci for systemic sclerosis.

Systemic sclerosis (SSc) is an orphan, complex, inflammatory disease affecting the immune system and connective tissue. SSc stands out as a severely incapacitating and life-threatening inflammatory rheumatic disease, with a largely unknown pathogenesis. We have designed a two-stage genome-wide association study of SSc using case-control samples from France, Italy, Germany, and Northern Europe. The initial genome-wide scan was conducted in a French post quality-control sample of 564 cases and 1,776 controls, using almost 500 K SNPs. Two SNPs from the MHC region, together with the 6 loci outside MHC having at least one SNP with a P<10(-5) were selected for follow-up analysis. These markers were genotyped in a post-QC replication sample of 1,682 SSc cases and 3,926 controls. The three top SNPs are in strong linkage disequilibrium and located on 6p21, in the HLA-DQB1 gene: rs9275224, P = 9.18×10(-8), OR = 0.69, 95% CI [0.60-0.79]; rs6457617, P = 1.14×10(-7) and rs9275245, P = 1.39×10(-7). Within the MHC region, the next most associated SNP (rs3130573, P = 1.86×10(-5), OR = 1.36 [1.18-1.56]) is located in the PSORS1C1 gene. Outside the MHC region, our GWAS analysis revealed 7 top SNPs (P<10(-5)) that spanned 6 independent genomic regions. Follow-up of the 17 top SNPs in an independent sample of 1,682 SSc and 3,926 controls showed associations at PSORS1C1 (overall P = 5.70×10(-10), OR:1.25), TNIP1 (P = 4.68×10(-9), OR:1.31), and RHOB loci (P = 3.17×10(-6), OR:1.21). Because of its biological relevance, and previous reports of genetic association at this locus with connective tissue disorders, we investigated TNIP1 expression. A markedly reduced expression of the TNIP1 gene and also its protein product were observed both in lesional skin tissue and in cultured dermal fibroblasts from SSc patients. Furthermore, TNIP1 showed in vitro inhibitory effects on inflammatory cytokine-induced collagen production. The genetic signal of association with TNIP1 variants, together with tissular and cellular investigations, suggests that this pathway has a critical role in regulating autoimmunity and SSc pathogenesis.

Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

Effect of focal cerebral infarctions on lesional RhoA and RhoB expression.

BACKGROUND: Blockade of the small GTPase Rho (ras homology protein) or of its downstream target Rho-associated kinase has been shown to promote axon regeneration in vitro and in vivo and to improve functional recovery after experimental central nervous system lesions. OBJECTIVE: To determine the expression patterns of RhoA and RhoB after focal cerebral infarction (FCI) and to assess whether Rho is a possible target for pharmacologic intervention. METHODS: Expression patterns of RhoA and RhoB were investigated in brain tissue specimens from 22 patients who died after FCI-clinically appearing as stroke-and were compared with those in brain tissue specimens from 4 neuropathologically unaffected controls by immunohistochemical analysis. RESULTS: Compared with control brains, a significant lesional up-regulation of RhoA and RhoB was observed beginning 2 to 10 days after ischemia and continuing for 4 to 38 months after FCI (P<.001). The cellular sources of both molecules included polymorphonuclear granulocytes, monocytes/macrophages, and reactive astrocytes. Neuronal RhoB expression was detected in the very early stages after FCI and in some cases in the later stages adjacent to the lesion. CONCLUSIONS: Inhibition of Rho is a promising lead for the development of new pharmacologic interventions in FCI. Because the observed up-regulation of RhoA and RhoB was still detectable months after FCI, we speculate that even delayed treatment with Rho inhibitors might be a therapeutic option.