Cytosolic compartmentalization through liquid-liquid unmixing, such as the formation of RNA granules, is involved in many cellular processes and might be used to regulate signal transduction. However, specific molecular mechanisms by which liquid-liquid unmixing and signal transduction are coupled remain unknown. Here, we show that during cellular stress the dual specificity kinase DYRK3 regulates the stability of P-granule-like structures and mTORC1 signaling. DYRK3 displays a cyclic partitioning mechanism between stress granules and the cytosol via a low-complexity domain in its N terminus and its kinase activity. When DYRK3 is inactive, it prevents stress granule dissolution and the release of sequestered mTORC1. When DYRK3 is active, it allows stress granule dissolution, releasing mTORC1 for signaling and promoting its activity by directly phosphorylating the mTORC1 inhibitor PRAS40. This mechanism links cytoplasmic compartmentalization via liquid phase transitions with cellular signaling.

ObjectiveThe first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin.Approach and ResultsTo address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-α, VCAM, IL-6, and IL-1β) or cellular composition (smooth muscle cell, macrophage, and T-cell content).ConclusionsAortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.

Rationale:The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-B (NFB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention.Objective: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. Methods and Results: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2؊/؊ bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2 macrophages. Further analysis in Rip2؊/؊ macrophages showed that the lipid accumulation was scavengerreceptor independent and mediated by Toll-like receptor 4 (TLR4)-dependent lipid uptake. Ⅲ macrophages L ow-density lipoproteins (LDL) are the major extracellular carriers of cholesterol and, as such, play important physiological roles in cellular function and regulation of metabolic pathways. However, under pathological conditions of hyperlipidemia, cholesterol is diverted from its physiological targets and accumulates in lipid-loaded macrophages ("foam cells") in the vascular wall. [1][2][3][4] This pathological deposition of atherogenic lipoproteins activates the inflammatory response that characterizes atherosclerosis. Furthermore, by stimulating the synthesis and secretion of proteoglycans, 5,6 this inflammation further accelerates retention of atherogenic lipoproteins. Thus, lipid accumulation and inflammation are closely linked in atherogenesis. [7][8][9][10][11] The molecular mechanisms that link retention of atherogenic lipoproteins and activation of the inflammatory response are still unclear. Recent evidence implies a key role for the innate immune system and pathogen patternrecognition receptors, in particular the membrane-bound Toll-like receptors (TLRs). 12 Ligand binding to these receptors results in activation of the proinflammatory transcription factor nuclear factor-B (NF-B) and expression of proinflammatory molecules. 12,13 The receptor-interacting protein 2 (Rip2) is a serine/ threonine kinase that activates NF-B and is reported to mediate signaling through both TLRs and Nod-like receptors, although its involvement in TLR signaling has been questioned recently. 14 -18 Furthermore, it was demonstrated recently that the regulation of Rip2 involves a novel feedforward regulatory mechanism: Rip2 not only positively regulates NF-B activity, but inflammatory cytokines that activate the NF-B pathway induce increased Rip2 expression. 19 These studies thus suggest the therapeutic potential of inhibiting Rip2 to inhibit inflammation and...

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