Inhibition of Rho and Rac Geranylgeranylation by Atorvastatin Is Critical for Preservation of Endothelial Junction Integrity

Background

Small GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.

Methods and Results

Confluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.

Conclusions

In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.     

GxcDD,a putative RacGEF,is involved in <Emphasis Type="Italic">Dictyostelium</Emphasis> development

Background  

Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.     

Angiopoietin-1 Requires Oxidant Signaling through p47phox to Promote Endothelial Barrier Defense

Background

Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.

Methods and Findings

Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47^−/−) littermates.

Conclusions

These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.     

GTPases and the origin of the ribosome

Background  

This paper is an attempt to trace the evolution of the ribosome through the evolution of the universal P-loop GTPases that are involved with the ribosome in translation and with the attachment of the ribosome to the membrane. The GTPases involved in translation in Bacteria/Archaea are the elongation factors EFTu/EF1, the initiation factors IF2/aeIF5b + aeIF2, and the elongation factors EFG/EF2. All of these GTPases also contain the OB fold also found in the non GTPase IF1 involved in initiation. The GTPase involved in the signal recognition particle in most Bacteria and Archaea is SRP54.     

On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF

Background  

The Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Gα_12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).     

Blockade of advanced glycation end product formation attenuates bleomycin-induced pulmonary fibrosis in rats

Background

Advanced glycation end products (AGEs) have been proposed to be involved in pulmonary fibrosis, but its role in this process has not been fully understood. To investigate the role of AGE formation in pulmonary fibrosis, we used a bleomycin (BLM)-stimulated rat model treated with aminoguanidine (AG), a crosslink inhibitor of AGE formation.

Methods

Rats were intratracheally instilled with BLM (5 mg/kg) and orally administered with AG (40, 80, 120 mg/kg) once daily for two weeks. AGEs level in lung tissue was determined by ELISA and pulmonary fibrosis was evaluated by Ashcroft score and hydroxyproline assay. The expression of heat shock protein 47 (HSP47), a collagen specific molecular chaperone, was measured with RT-PCR and Western blot. Moreover, TGFβ1 and its downstream Smad proteins were analyzed by Western blot.

Results

AGEs level in rat lungs, as well as lung hydroxyproline content and Ashcroft score, was significantly enhanced by BLM stimulation, which was abrogated by AG treatment. BLM significantly increased the expression of HSP47 mRNA and protein in lung tissues, and AG treatment markedly decreased BLM-induced HSP47 expression in a dose-dependent manner (p < 0.05). In addition, AG dose-dependently downregulated BLM-stimulated overexpressions of TGFβ1, phosphorylated (p)-Smad2 and p-Smad3 protein in lung tissues.

Conclusion

These findings suggest AGE formation may participate in the process of BLM-induced pulmonary fibrosis, and blockade of AGE formation by AG treatment attenuates BLM-induced pulmonary fibrosis in rats, which is implicated in inhibition of HSP47 expression and TGFβ/Smads signaling.     

Interacting with the biomolecular solvent accessible surface via a haptic feedback device

Background

Ras-like GTPases function as on-off switches in intracellular signalling pathways and include the Rab, Rho/Rac, Ran, Ras, Arf, Sar and Gα families. How these families have evolutionarily diverged from each other at the sequence level provides clues to underlying mechanisms associated with their functional specialization.

Results

Bayesian analysis of divergent patterns within a multiple alignment of Ras-like GTPase sequences identifies a structural component, termed here the glycine brace, as the feature that most distinguishes Rab, Rho/Rac, Ran and (to some degree) Ras family GTPases from other Ras-like GTPases. The glycine brace consists of four residues: An aromatic residue that forms a stabilizing CH-π interaction with a conserved glycine at the start of the guanine-binding loop; a second aromatic residue, which is nearly always a tryptophan, that likewise forms stabilizing CH-π and NH-π interactions with a glycine at the start of the phosphate-binding P-loop; and two other residues (typically an aspartate and a serine or threonine) that, together with a conserved buried water molecule, form a network of interactions connecting the two aromatic residues.

Conclusion

It is proposed that the two glycine residues function as hinges and that the glycine brace influences guanine nucleotide binding and release by interacting with these hinges.     

Clinical Relevance of Gain-Of-Function Mutations of p53 in High-Grade Serous Ovarian Carcinoma

Purpose

Inactivation of TP53, which occurs predominantly by missense mutations in exons 4–9, is a major genetic alteration in a subset of human cancer. In spite of growing evidence that gain-of-function (GOF) mutations of p53 also have oncogenic activity, little is known about the clinical relevance of these mutations.

Methods

The clinicopathological features of high-grade serous ovarian carcinoma (HGS-OvCa) patients with GOF p53 mutations were evaluated according to a comprehensive somatic mutation profile comprised of whole exome sequencing, mRNA expression, and protein expression profiles obtained from the Cancer Genome Atlas (TCGA).

Results

Patients with a mutant p53 protein (mutp53) with a GOF mutation showed higher p53 mRNA and protein expression levels than patients with p53 mutation with no evidence of GOF (NE-GOF). GOF mutations were more likely to occur within mutational hotspots, and at CpG sites, and resulted in mutp53 with higher functional severity (FS) scores. Clinically, patients with GOF mutations showed a higher frequency of platinum resistance (22/58, 37.9%) than patients with NE-GOF mutations (12/56, 21.4%) (p=0.054). Furthermore, patients with GOF mutations were more likely to develop distant metastasis (36/55, 65.5%) than local recurrence (19/55, 34.5%), whereas patients with NE-GOF mutations showed a higher frequency of locoregional recurrence (26/47, 55.3%) than distant metastasis (21/47, 44.7%) (p=0.035). There were no differences in overall or progression-free survival between patients with GOF or NE-GOF mutp53.

Conclusion

This study demonstrates that patient with GOF mutp53 is characterized by a greater likelihood of platinum treatment resistance and distant metastatic properties in HGS-OvCa.     

Identification of a novel germ-line mutation in the TP53 gene in a Mexican family with Li-Fraumeni syndrome

Background

Intra-peritoneal (i.p.) chemotherapy is an encouraging treatment option for ovarian cancer with peritoneum involvement in addition with intravenous (i.v.) chemotherapy. Intra-operative i.p. chemotherapy is an interesting method of administration by enhancing the diffusion of chemotherapy. This study had assessed the feasibility of intra-operative i.p. chemotherapy in patients with peritoneal carcinoma of ovarian cancer.

Methods

From January 2003 to February 2006, 47 patients with stage III ovarian cancer were treated with standard paclitaxel carboplatin intravenous chemotherapy and debulking surgery with intra-operative i.p. chemotherapy. After optimal cytoreductive surgery, defined by no unresectable residual disease > 1 cm, i.p. chemotherapy was performed during surgery. The peritoneal cavity was filled by 3 litres of isotonic saline pre-heated at 37 degrees and 90 mg of cisplatin. The sequence was repeated twice during 2 hours based on previous published studies which optimized the cisplatin dosage and exposure duration. Optimal diffusion was obtained by stirring by hands during the 2 hours.

Results

Median age was 59.6 years. No severe haematological or non-haematological toxicity induced by intra operative i.p. chemotherapy was reported. No patient died due to the complications of surgery or the i.p. chemotherapy. No neurotoxicity occurred, and one patients had renal impairment.

Conclusion

This study demonstrates the feasibility of intra-operative i.p. chemotherapy with cisplatin after optimal resection of peritoneal tumor nodules. Further randomized trials are planned to investigate the clinical benefit of this therapeutic modality.     

Direct Activation of RhoA by Reactive Oxygen Species Requires a Redox-Sensitive Motif

Background

Rho family GTPases are critical regulators of the cytoskeleton and affect cell migration, cell-cell adhesion, and cell-matrix adhesion. As with all GTPases, their activity is determined by their guanine nucleotide-bound state. Understanding how Rho proteins are activated and inactivated has largely focused on regulatory proteins such as guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, recent in vitro studies have indicated that GTPases may also be directly regulated by redox agents. We hypothesized that this redox-based mechanism occurs in cells and affects cytoskeletal dynamics, and in this report we conclude this is indeed a novel mechanism of regulating the GTPase RhoA.

Methodology/Principal Findings

In this report, we show that RhoA can be directly activated by reactive oxygen species (ROS) in cells, and that this requires two critical cysteine residues located in a unique redox-sensitive motif within the phosphoryl binding loop. First, we show that ROS can reversibly activate RhoA and induce stress fiber formation, a well characterized readout of RhoA activity. To determine the role of cysteine residues in this mechanism of regulation, we generated cysteine to alanine RhoA mutants. Mutation of these cysteines abolishes ROS-mediated activation and stress fiber formation, indicating that these residues are critical for redox-regulation of RhoA. Importantly, these mutants maintain the ability to be activated by GEFs.

Conclusions/Significance

Our findings identify a novel mechanism for the regulation of RhoA in cells by ROS, which is independent of classical regulatory proteins. This mechanism of regulation may be particularly relevant in pathological conditions where ROS are generated and the cellular redox-balance altered, such as in asthma and ischemia-reperfusion injury.     

A Theoretical Model for ROP Localisation by Auxin in Arabidopsis Root Hair Cells

Background

Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP) signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.

Methodology/Principal Findings

We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom ^eb.

Conclusions/Significance

We show how interactions between Rho GTPases (in this case ROPs) and regulatory molecules (in this case auxin) could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.     

Sustained delivery of activated Rho GTPases and BDNF promotes axon growth in CSPG-rich regions following spinal cord injury

Background

Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion.

Methodology/Principal Findings

We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site.

Conclusion

Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI.     

Control of IFN-γ-mediated host resistance to intracellular pathogens by immunity-related GTPases (p47 GTPases)

IRG proteins (also known as p47 GTPases) are key mediators of interferon-γ-induced resistance to pathogens. Absence of certain IRG proteins leads to profound susceptibility to protozoa and bacteria in mice. Underlying their roles in host resistance, IRG proteins regulate the processing of pathogen-containing vacuoles in host cells, and regulate hematopoiesis following infection.     

Generation and Characterisation of Mice Deficient in the Multi-GTPase Domain Containing Protein,GIMAP8

Background

GTPases of the immunity-associated protein family (GIMAPs) are predominantly expressed in mature lymphocytes. Studies of rodents deficient in GIMAP1, GIMAP4, or GIMAP5 have demonstrated that these GTPases regulate lymphocyte survival. In contrast to the other family members, GIMAP8 contains three potential GTP-binding domains (G-domains), a highly unusual feature suggesting a novel function for this protein. To examine a role for GIMAP8 in lymphocyte biology we examined GIMAP8 expression during lymphocyte development. We also generated a mouse deficient in GIMAP8 and examined lymphocyte development and function.

Principal Findings

We show that GIMAP8 is expressed in the very early and late stages of T cell development in the thymus, at late stages during B cell development, and peripheral T and B cells. We find no defects in T or B lymphocyte development in the absence of GIMAP8. A marginal decrease in the number of recirculating bone marrow B cells suggests that GIMAP8 is important for the survival of mature B cells within the bone marrow niche. We also show that deletion of GIMAP8 results in a delay in apoptotic death of mature T cell in vitro in response to dexamethasone or γ-irradiation. However, despite these findings we find that GIMAP8-deficient mice mount normal primary and secondary responses to a T cell dependent antigen.

Conclusions

Despite its unique structure, GIMAP8 is not required for lymphocyte development but appears to have a minor role in maintaining recirculating B cells in the bone marrow niche and a role in regulating apoptosis of mature T cells.     

The absence of reactive oxygen species production protects mice against bleomycin-induced pulmonary fibrosis

Background

Reactive oxygen species and tissue remodeling regulators, such as metalloproteinases (MMPs) and their inhibitors (TIMPs), are thought to be involved in the development of pulmonary fibrosis. We investigated these factors in the fibrotic response to bleomycin of p47^phox -/- (KO) mice, deficient for ROS production through the NADPH-oxidase pathway.

Methods

Mice are administered by intranasal instillation of 0.1 mg bleomycin. Either 24 h or 14 days after, mice were anesthetized and underwent either bronchoalveolar lavage (BAL) or lung removal.

Results

BAL cells from bleomycin treated WT mice showed enhanced ROS production after PMA stimulation, whereas no change was observed with BAL cells from p47^phox -/- mice. At day 1, the bleomycin-induced acute inflammatory response (increased neutrophil count and MMP-9 activity in the BAL fluid) was strikingly greater in KO than wild-type (WT) mice, while IL-6 levels increased significantly more in the latter. Hydroxyproline assays in the lung tissue 14 days after bleomycin administration revealed the absence of collagen deposition in the lungs of the KO mice, which had significantly lower hydroxyproline levels than the WT mice. The MMP-9/TIMP-1 ratio did not change at day 1 after bleomycin administration in WT mice, but increased significantly in the KO mice. By day 14, the ratio fell significantly from baseline in both strains, but more in the WT than KO strains.

Conclusions

These results suggest that NADPH-oxidase-derived ROS are essential to the development of pulmonary fibrosis. The absence of collagen deposition in KO mice seems to be associated with an elevated MMP-9/TIMP-1 ratio in the lungs. This finding highlights the importance of metalloproteinases and protease/anti-protease imbalances in pulmonary fibrosis.     

An MRI study on the relations between muscle atrophy, shoulder function and glenohumeral deformity in shoulders of children with obstetric brachial plexus injury

Objective

it has been established that chronic neck pain following whiplash is associated with the phenomenon of central sensitization, in which injured and uninjured parts of the body exhibit lowered pain thresholds due to an alteration in central pain processing. it has furthermore been hypothesized that peripheral sources of nociception in the muscles may perpetuate central sensitization in chronic whiplash. the hypothesis explored in the present study was whether myofascial trigger points serve as a modulator of central sensitization in subjects with chronic neck pain.

Design

controlled case series.

Setting

outpatient chronic pain clinic.

Subjects

seventeen patients with chronic and intractable neck pain and 10 healthy controls without complaints of neck pain.

Intervention

symptomatic subjects received anesthetic infiltration of myofascial trigger points in the upper trapezius muscles and controls received the anesthetic in the thigh. Outcome measures: pre and post injection cervical range of motion, pressure pain thresholds (ppt) over the infraspinatus, wrist extensor, and tibialis anterior muscles. sensitivity to light (photophobia) and subjects' perception of pain using a visual analog scale (vas) were also evaluated before and after injections. only the ppt was evaluated in the asymptomatic controls.

Results

immediate (within 1 minute) alterations in cervical range of motion and pressure pain thresholds were observed following an average of 3.8 injections with 1–2 cc of 1% lidocaine into carefully identified trigger points. cervical range of motion increased by an average of 49% (p = 0.000) in flexion and 44% (p = 0.001) in extension, 47% (p = 0.000) and 28% (p < 0.016) in right and left lateral flexion, and a 27% (p = 0.002) and 45% (p = 0.000) in right and left rotation. ppt were found increased by 68% over the infraspinatus (p = 0.000), by 78% over the wrist extensors (p = 0.000), and by 64% over the tibialis anterior (p = 0.002). among 11 subjects with photophobia, only 2 remained sensitive to light after the trigger point injections (p = 0.033). average vas dropped by 57%, from 6.1 to 2.6 (p = 0.000). no significant changes in ppt were observed in the control group following lidocaine infiltration of the thigh.

Conclusion

the present data suggest that myofascial trigger points serve to perpetuate lowered pain thresholds in uninjured tissues. additionally, it appears that lowered pain thresholds associated with central sensitization can be immediately reversed, even when associated with long standing chronic neck pain. although the effects resulting from anesthesia of trigger points in the present study were temporary, it is possible that surgical excision or ablation of the same trigger points may offer more permanent solutions for chronic neck pain patients. further study is needed to evaluate these and other options for such patients.     

Identification of HLA-DRPheβ47 as the susceptibility marker of hypersensitivity to beryllium in individuals lacking the berylliosis-associated supratypic marker HLA-DPGluβ69

Background

Susceptibility to beryllium (Be)-hypersensitivity (BH) has been associated with HLA-DP alleles carrying a glutamate at position 69 of the HLA-DP β-chain (HLA-DPGlu69) and with several HLA-DP, -DQ and -DR alleles and polymorphisms. However, no genetic associations have been found between BH affected subjects not carrying the HLA-DPGlu69 susceptibility marker.

Methods

In this report, we re-evaluated an already described patient populations after 7 years of follow-up including new 29 identified BH subjects. An overall population 36 berylliosis patients and 38 Be-sensitization without lung granulomas and 86 Be-exposed controls was analysed to assess the role of the individual HLA-class II polymorphisms associated with BH-susceptibility in HLA-DPGlu69 negative subjects by univariate and multivariate analysis.

Results

As previously observed in this population the HLA-DPGlu69 markers was present in higher frequency in berylliosis patients (31 out of 36, 86%) than in Be-sensitized (21 out of 38, 55%, p = 0.008 vs berylliosis) and 41 out of 86 (48%, p < 0.0001 vs berylliosis, p = 0.55 vs Be-sensitized) Be-exposed controls.However, 22 subjects presenting BH did not carry the HLA-DPGlu69 marker. We thus evaluated the contribution of all the HLA-DR, -DP and -DQ polymorphisms in determining BH susceptibility in this subgroup of HLA-Glu69 subjects. In HLA-DPGlu69-negatives a significant association with BH was found for the HLA-DQLeu26, for the HLA-DRB1 locus residues Ser13, Tyr26, His32, Asn37, Phe47 and Arg74 and for the HLA-DRB3 locus clusterized residues Arg11, Tyr26, Asp28, Leu38, Ser60 and Arg74. HLA-DRPhe47 (OR 2.956, p < 0.05) resulting independently associated with BH. Further, Be-stimulated T-cell proliferation in the HLA-DPGlu69-negative subjects (all carrying HLA-DRPhe47) was inhibited by the anti-HLA-DR antibody (range 70–92% inhibition) significantly more than by the anti-HLA-DP antibody (range: 6–29%; p < 0.02 compared to anti-HLA-DR) while it was not affected by the anti-HLA-DQ antibody.

Conclusion

We conclude that HLA-DPGlu69 is the primary marker of Be-hypersensitivity and HLA-DRPhe47 is associated with BH in Glu69-negative subjects, likely playing a role in Be-presentation and sensitization.