Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization.

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.

Cherubism allele heterozygosity amplifies microbe-induced inflammatory responses in murine macrophages.

Cherubism is a rare autoinflammatory bone disorder that is associated with point mutations in the SH3-domain binding protein 2 (SH3BP2) gene, which encodes the adapter protein 3BP2. Individuals with cherubism present with symmetrical fibro-osseous lesions of the jaw, which are attributed to exacerbated osteoclast activation and defective osteoblast differentiation. Although it is a dominant trait in humans, cherubism appears to be recessively transmitted in mice, suggesting the existence of additional factors in the pathogenesis of cherubism. Here, we report that macrophages from 3BP2-deficient mice exhibited dramatically reduced inflammatory responses to microbial challenge and reduced phagocytosis. 3BP2 was necessary for LPS-induced activation of signaling pathways involved in macrophage function, including SRC, VAV1, p38MAPK, IKKα/ß, RAC, and actin polymerization pathways. Conversely, we demonstrated that the presence of a single Sh3bp2 cherubic allele and pathogen-associated molecular pattern (PAMP) stimulation had a strong cooperative effect on macrophage activation and inflammatory responses in mice. Together, the results from our study in murine genetic models support the notion that infection may represent a driver event in the etiology of cherubism in humans and suggest limiting inflammation in affected individuals may reduce manifestation of cherubic lesions.

Technical advance: actin CytoFRET, a novel FRET flow cytometry method for detection of actin dynamics in resting and activated T cell.

Actin cytoskeleton plays a critical role in regulating T cell motility and activation. However, the lack of a real-time quantitative method to analyze actin assembly has limited the progress toward understanding actin regulation. Here, we describe a novel approach to probe actin dynamics on living T cells using FRET combined with flow cytometry. We have first generated a Jurkat T cell line stably coexpressing EGFP and mOrange FPs fused to actin. The real-time variation of actin monomer assembly or disassembly into filaments was quantified using a ratiometric flow cytometry method measuring changes in the mOrange/EGFP emission ratio. The method was validated on resting T cells by using chemical compounds with known effects on actin filaments and comparison with conventional microscopy imaging. Our method also detected the rapid and transient actin assembly in T cells stimulated by anti-CD3/CD28-coated beads, demonstrating its robustness and high sensitivity. Finally, we provide evidence that lentiviral-mediated transduction of shRNAs in engineered Jurkat cells could be used as a strategy to identify regulators of actin remodeling. In conclusion, the flow cytometric FRET analysis of actin polymerization represents a new technical advance to study the dynamics of actin regulation in intact cells.

Endothelial cell activation in patients with decompensated heart failure.

BACKGROUND: Vascular endothelial functions, other than nitric oxide (NO)-mediated control of vasomotor tone, are poorly characterized in patients with chronic heart failure (CHF). Veins and arteries are exposed to the same circulating proinflammatory mediators in patients with CHF. The present study tested whether endothelial cell activation occurs in veins of patients with decompensated CHF and whether activation, if present, subsides with return to a clinically compensated state. METHODS AND RESULTS: Fifteen patients with decompensated CHF requiring transient inotropic support and 6 age-matched, healthy controls were studied. Endothelial cells and blood were collected from a forearm vein, and brachial artery flow-mediated dilation (FMD) was measured before and 24 hours after discontinuation of short-term inotropic therapy, when patients had returned to a steady compensated state. Nitrotyrosine immunoreactivity (an intracellular marker of oxidative stress), cyclooxygenase-2 (COX-2), and inducible NO synthase (iNOS) expression were significantly higher in venous endothelial cells of patients in clinical decompensation when compared with healthy subjects. Return to a compensated state resulted in a significant reduction in nitrotyrosine immunoreactivity, COX-2, and iNOS expression. Concomitantly, a significant increase in FMD and a decline in plasma total 8-isoprostane and bicycloprostaglandin E2 levels were observed. Venous endothelial NOS expression was unaffected by clinical decompensation. CONCLUSIONS: Clinical decompensation in CHF is associated with activation of the venous endothelium. Return to a compensated state after short-term inotropic therapy results in a significant reduction in endothelial nitrotyrosine formation, COX-2, and iNOS expression.

Biopsy coupled to quantitative immunofluorescence: a new method to study the human vascular endothelium.

Limited availability of endothelial tissue is a major constraint when investigating the cellular mechanisms of endothelial dysfunction in patients with metabolic and cardiovascular diseases. We propose a novel approach that combines collection of 200-1,000 endothelial cells from a superficial forearm vein or the radial artery, with reliable measurements of protein expression by quantitative immunofluorescence analysis. This method was validated against immunoblot analysis in cultured endothelial cells. Levels of vascular endothelial cell activation, oxidative stress, and nitric oxide synthase expression were measured and compared in five patients with severe chronic heart failure and in four healthy age-matched subjects. In summary, vascular endothelial biopsy coupled with measurement of protein expression by quantitative immunofluorescence analysis provides a novel approach to the study of the vascular endothelium in humans.