Targeting and activation of Rac1 are mediated by the exchange factor β-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator β-Pix (p21-activated kinase [Pak]–interacting exchange factor). The interaction with β-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1–β-Pix interaction is required for Rac1 activation by β-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the β-Pix–binding kinase Pak1, we show that Pak1 regulates the Rac1–β-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor β-Pix

Survival and cellular heterogeneity of epithelium in cultured mouse and rat precision-cut intestinal slices

Precision-cut intestinal slices (PCIS) are used to study intestinal (patho)physiology, drug efficacy, toxicity, transport and metabolism ex vivo. One of the factors that limit the use of PCIS is a relatively short life-span. Moreover, culture-induced changes in cellular composition of PCIS remain largely uncharacterized. In this study, we demonstrated the epithelial cell heterogeneity in mouse and rat PCIS and its alterations during culture. In addition, we evaluated whether the presence of niche growth factors impacts the survival of PCIS epithelial cells. We showed that freshly prepared PCIS retained the main epithelial cell types, namely absorptive enterocytes, goblet cells, enteroendocrine cells, stem cells, transit-amplifying cells and Paneth cells. Once placed in culture, PCIS displayed progressive epithelial damage, and loss of these epithelial cell types. Cells comprising the intestinal stem cell niche were especially sensitive to the damage, and the addition of niche growth factors beneficially affected the survival of stem cells and transit-amplifying cells in PCIS during culture. In conclusion, this study provides new insights into the dynamic changes in cellular composition of epithelium in cultured PCIS, paving the way to future toxicological and pharmacological studies in an informed and reliable ex vivo setting

Chartis Measurement of Collateral Ventilation:Conscious Sedation versus General Anesthesia

BACKGROUND: Absence of interlobar collateral ventilation using the Chartis measurement is the key predictor for successful endobronchial valve treatment in severe emphysema. Chartis was originally validated in spontaneous breathing patients under conscious sedation (CS); however, this can be challenging due to cough, mucus secretion, mucosal swelling, and bronchoconstriction. Performing Chartis under general anesthesia (GA) avoids these problems and may result in an easier procedure with a higher success rate. However, using Chartis under GA with positive pressure ventilation has not been validated. OBJECTIVES: In this study we investigated the impact of anesthesia technique, CS versus GA, on the feasibility and outcomes of Chartis measurement. METHODS: We retrospectively analyzed all Chartis measurements performed at our hospital from October 2010 until December 2017. RESULTS: We analyzed 250 emphysema patients (median forced expiratory volume in 1 s 26%, range 12-52% predicted). In 121 patients (48%) the measurement was performed using CS, in 124 (50%) using GA, and in 5 (2%) both anesthesia techniques were used. In total, 746 Chartis readings were analyzed (432 CS, 277 GA, and 37 combination). Testing under CS took significantly longer than GA (median 19 min [range 5-65] vs. 11 min [3-35], p < 0.001) and required more measurements (3 [1-13] vs. 2 [1-6], p < 0.001). There was no significant difference in target lobe volume reduction after treatment (-1,123 mL [-3,604 to 332] in CS vs. -1,251 mL [-3,333 to -1] in GA, p = 0.35). CONCLUSIONS: In conclusion, Chartis measurement under CS took significantly longer and required more measurements than under GA, without a difference in treatment outcome. We recommend a prospective trial comparing both techniques within the same patients to validate this approach

A New Oxygen Uptake Measurement Supporting Target Selection for Endobronchial Valve Treatment

BACKGROUND: Adequate target lobe selection for endobronchial valve (EBV) treatment in patients with severe emphysema is essential for treatment success and can be based on emphysema destruction, lobar perfusion, lobar volume, and collateral ventilation. As some patients have >1 target lobe for EBV treatment, we were interested whether we could identify the least functional lobe. OBJECTIVES: The objective of this study was to investigate the relationship between endoscopic lobar measurement of oxygen uptake, lobar destruction, and vascular volume, and whether this could help in identifying the least functional lobe and thus optimal target for EBV treatment. METHOD: We prospectively included patients who were scheduled for EBV treatment in our hospital. A customized gas analysis setup was used to measure lobar O2 uptake after lobar balloon occlusion. Quantitative CT analysis was performed to assess the degree of emphysematous destruction and lobar arterial and venous volumes. RESULTS: Twenty-one (5 male/16 female) patients with emphysema (median age 63 years, FEV1 25% of predicted, residual volume 234% of predicted) were included, and 49 endoscopic lobar measurements were performed. A lower O2 uptake significantly correlated with a higher degree of emphysematous lobar destruction (Spearman's ρ: 0.39, p < 0.01), and lower arterial and venous vascular volumes of the lobes (-0.46 and -0.47, respectively; both p < 0.001). CONCLUSIONS: Endoscopic measurement of lobar O2 uptake is feasible in patients with emphysema. Measurement of lobar O2 uptake helped to identify the least functional lobe and can be used as additional tool for EBV target lobe selection

A Rac1 inhibitory peptide suppresses antibody production and paw swelling in the murine collagen-induced arthritis model of rheumatoid arthritis

Introduction: The Rho family GTPase Rac1 regulates cytoskeletal rearrangements crucial for the recruitment, extravasation and activation of leukocytes at sites of inflammation. Rac1 signaling also promotes the activation and survival of lymphocytes and osteoclasts. Therefore, we assessed the ability of a cell-permeable Rac1 carboxy-terminal inhibitory peptide to modulate disease in mice with collagen-induced arthritis (CIA). Methods: CIA was induced in DBA/1 mice, and in either early or chronic disease, mice were treated three times per week by intraperitoneal injection with control peptide or Rac1 inhibitory peptide. Effects on disease progression were assessed by measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. Serum levels of anti-collagen type II antibodies were measured by enzyme-linked immunosorbent assay. T-cell phenotypes and activation were assessed by fluorescence-activated cell sorting analysis. Results were analyzed using Mann-Whitney U and unpaired Student t tests. Results: Treatment of mice with Rac1 inhibitory peptide resulted in a decrease in paw swelling in early disease and to a lesser extent in more chronic arthritis. Of interest, while joint destruction was unaffected by Rac1 inhibitory peptide, anti-collagen type II antibody production was significantly diminished in treated mice, in both early and chronic arthritis. Ex vivo, Rac1 inhibitory peptide suppressed T-cell receptor/CD28-dependent production of tumor necrosis factor a, interferon. and interleukin-17 by T cells from collagen-primed mice, and reduced induction of ICOS and CD154, T-cell costimulatory proteins important for B-cell help. Conclusions: The data suggest that targeting of Rac1 with the Rac1 carboxy-terminal inhibitory peptide may suppress T-cell activation and autoantibody production in autoimmune disease. Whether this could translate into clinically meaningful improvement remains to be show

Targeting and localized signalling by small GTPases

Polarized cellular responses, for example, cell migration, require the co-ordinated assembly of signalling complexes at a particular subcellular location, such as the leading edge of cells. Small GTPases of the Ras superfamily play central roles in many (polarized) responses to growth factors, chemokines or integrin ligands. These small GTPases are functionally distinct, yet remarkably homologous in their primary sequence and especially in their effector domains. Therefore it has long been unclear how GTPase signalling specificity is regulated. Small GTPases carry a lipid anchor, in the context of a hypervariable region, which mediates membrane association. However, whereas the lipid has long been proposed to be the critical regulator of subcellular GTPase targeting, there is now increasing evidence that specific protein-protein interactions are important as well. This review discusses recent findings on GTPase targeting and proposes a revised model for GTPase signalling. In this model, the hypervariable domain acts in conjunction with the lipid tail to target the GTPase to specific membrane-associated protein complexes. Here, local GTPase activation occurs, leading to subsequent exposure of the effector domain, binding to effector proteins and the initiation of downstream signallin

LKB1 and AMPK family signaling: the intimate link between cell polarity and energy metabolism.

Research on the LKB1 tumor suppressor protein mutated in cancer-prone Peutz-Jeghers patients has continued at a feverish pace following exciting developments linking energy metabolism and cancer development. This review summarizes the current state of research on the LKB1 tumor suppressor. The weight of the evidence currently indicates an evolutionary conserved role for the protein in the regulation of various aspects of cellular polarity and energy metabolism. We focus on studies examining the concept that both cellular polarity and energy metabolism are regulated through the conserved LKB1-AMPK signal transduction pathway. Recent studies from a variety of model organisms have given new insight into the mechanism of polyp development and cancer formation in Peutz-Jeghers patients and the role of LKB1 mutation in sporadic tumorigenesis. Conditional LKB1 mouse models have outlined a tissue-dependent context for pathway activation and suggest that LKB1 may affect different AMPK isoforms independently. Elucidation of the molecular mechanism responsible for Peutz-Jeghers syndrome will undoubtedly reveal important insight into cancer development in the larger population.

Type 2 diabetes-related proteins derived from an in vitro model of inflamed fat tissue

Currently, there is a worldwide increase of patients with type 2 diabetes (T2D). During the progression of healthy obese to T2D status, there is an influx of immune cells, in particular macrophages, into visceral adipose tissue, accompanied by an increase of inflammatory cytokines, such as, IL6, TNFα and Hp. To get a better insight in the underlying mechanisms, we performed a quantitative LCMS analysis on a modified in vitro assay, combining 3T3L1 adipocytes and activated RAW264.7 macrophages, thus mimicking inflamed adipose tissue. Clinically known proteins, e.g. IL6, TNFα AdipoQ, complement factor C3, B and D were identified, thus confirming the assay. In addition, we found 54 new proteins that can potentially be used for research into the mechanism of T2D. Comparison of our results to a study on human visceral fat of obese non-diabetic and obese diabetic subjects, indicated that AUH, NAGK, pCYT2, NNMT, STK39 and CSNK2A2 might indeed be linked to insulin resistance in humans. Moreover, the expression of some of these genes was also altered in human blood samples at early or later stages of insulin desensitization. Overall, we conclude that the direct contact co-culture of 3T3L1 adipocytes with activated macrophages could be a mechanistically relevant and partially translational model of inflamed visceral adipose tissue

Docosahexaenoyl Serotonin, an endogenously formed n-3 fatty acid-serotonin conjugate, has anti-inflammatory properties by attenuating IL23–IL17 signalling in macrophages

Conjugates of fatty acids and amines, including endocannabinoids, are known to play important roles as endogenous signalling molecules. Among these, the ethanolamine conjugate of the n-3 poly unsaturated long chain fatty acid (PUFA) docosahexaenoic acid (22:6n-3) (DHA) was shown to possess strong anti-inflammatory properties. Previously, we identified the serotonin conjugate of DHA, docosahexaenoyl serotonin (DHA-5-HT), in intestinal tissues and showed that its levels are markedly influenced by intake of n-3 PUFAs. However, its biological roles remain to be elucidated. Here, we show that DHA-5-HT possesses potent anti-inflammatory properties by attenuating the IL-23-IL-17 signalling cascade in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Transcriptome analysis revealed that DHA-5-HT down-regulates LPS-induced genes, particularly those involved in generating a CD4 + Th17 response. Hence, levels of PGE2, IL-6, IL-1β, and IL-23, all pivotal macrophage-produced mediators driving the activation of pathogenic Th17 cells in a concerted way, were found to be significantly suppressed by concentrations as low as 100–500 nM DHA-5-HT. Furthermore, DHA-5-HT inhibited the ability of RAW264.7 cells to migrate and downregulated chemokines like MCP-1, CCL-20, and gene-expression of CCL-22 and of several metalloproteinases. Gene set enrichment analysis (GSEA) suggested negative overlap with gene sets linked to inflammatory bowel disease (IBD) and positive overlap with gene sets related to the Nrf2 pathway. The specific formation of DHA-5-HT in the gut, combined with increasing data underlining the importance of the IL-23-IL-17 signalling pathway in the aetiology of many chronic inflammatory diseases merits further investigation into its potential as therapeutic compound in e.g. IBD or intestinal tumorigenesis