Current paradigms suggest that two macrophage subsets, termed M1 and M2, are involved in inflammation and host defense. While the distinct functions of M1 and M2 macrophages have been intensively studied -the former are considered proinflammatory and the latter antiinflammatory -the determinants of their speciation are incompletely understood. Here we report our studies that identify Krüppel-like factor 4 (KLF4) as a critical regulator of macrophage polarization. Macrophage KLF4 expression was robustly induced in M2 macrophages and strongly reduced in M1 macrophages, observations that were recapitulated in human inflammatory paradigms in vivo. Mechanistically, KLF4 was found to cooperate with Stat6 to induce an M2 genetic program and inhibit M1 targets via sequestration of coactivators required for NF-κB activation. KLF4-deficient macrophages demonstrated increased proinflammatory gene expression, enhanced bactericidal activity, and altered metabolism. Furthermore, mice bearing myeloid-specific deletion of KLF4 exhibited delayed wound healing and were predisposed to developing diet-induced obesity, glucose intolerance, and insulin resistance. Collectively, these data identify KLF4 as what we believe to be a novel regulator of macrophage polarization.

Primary cilia are implicated in the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD), which results from defects in polycystin-1 (PC1), but the function of PC1 remains poorly understood. Here, we show that PC1 undergoes proteolytic cleavage that results in nuclear translocation of its cytoplasmic tail. The PC1 tail interacts with the transcription factor STAT6 and the coactivator P100, and it stimulates STAT6-dependent gene expression. Under normal conditions, STAT6 localizes to primary cilia of renal epithelial cells. Cessation of apical fluid flow results in nuclear translocation of STAT6. Cyst-lining cells in ADPKD exhibit elevated levels of nuclear STAT6, P100, and the PC1 tail. Exogenous expression of the human PC1 tail results in renal cyst formation in zebrafish embryos. These results identify a novel mechanism of cilia function in the transduction of a mechanical signal to changes of gene expression involving PC1 and show that this pathway is inappropriately activated in ADPKD.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease 1, 2, but the molecular basis is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (e.g. short or long QT syndromes, heart failure) 3-5 or pattern (e.g. Brugada syndrome) 6 of myocardial repolarization. Here we provide the first molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion channel expression and QT interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a novel clock-dependent oscillator, Krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito). 7 Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. In sum, these findings identify circadian transcription of ion channels as a novel mechanism for cardiac arrhythmogenesis.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal. IntroductionThrough the elaboration of numerous biological substances, ECs actively regulate fundamental physiological processes, such as regulation of blood coagulation, homing of immune cells, and barrier function. Studies over the past several decades have also identified key physiologic and pathologic phenotypic modulators of ECs. For example, stimulation of the endothelium with proinflammatory cytokines renders the endothelium dysfunctional, inducing a proadhesive and prothrombotic phenotype. In contrast, laminar flow induces critical genes that confer potent antithrombotic, antiadhesive, and antiinflammatory properties. The significance of fluid shear stress is evidenced by the observation that segments of the arterial tree exposed to laminar flow (e.g., straight regions of the vasculature) are less prone to the development of atherosclerotic lesions than are regions exposed to nonlaminar/disturbed flow (e.g., branch points). These observations have led to the current view that the balance of biochemical and biomechanical stimuli is the central determinant of vascular function under physiologic and pathologic conditions. Given the importance of the endothelium in vessel homeostasis, there is great interest in identifying molecular pathways that mediate the effects of both biochemical and biomechanical stimuli. Prior studies from our group and others have identified 2 members of the Kruppel-like factor (KLF) family of transcription factors, KLF2 and KLF4, as being of particular interest. Both KLF2 and KLF4 are induced by laminar flow and in in vitro stud-

SUMMARY Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia indusable factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NFκB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

Syntaxins 3 and 4 localize to the apical and basolateral plasma membrane, respectively, of epithelial cells where they mediate vesicle fusion. Here, we report that before establishment of cell polarity, syntaxins 3 and 4 are confined to mutually exclusive, submicron-sized clusters. Syntaxin clusters are remarkably uniform in size, independent of expression levels, and are distinct from caveolae and clathrin-coated pits. SNAP-23 partially colocalizes with both syntaxin 3 and 4 clusters. Deletion of the apical targeting signal of syntaxin 3 does not prevent sorting into clusters away from syntaxin 4. Syntaxin 3 and 4 cluster formation depends on different mechanisms because the integrity of syntaxin 3 clusters depends on intact microtubules, whereas syntaxin 4 clusters depend on intact actin filaments. Cholesterol depletion causes dispersion of syntaxin 3 but not syntaxin 4 clusters. In migrating cells, syntaxin clusters polarize to the leading edge, suggesting a role in polarized exocytosis. These results suggest that exocytosis occurs at small fusion sites exhibiting high local concentrations of SNARE proteins that may be required for efficient membrane fusion. The establishment of separate clusters for each syntaxin suggests that the plasma membrane is inherently polarized on an ultrastructural level even before the establishment of true cell polarity.

Objective To investigate the role of Krüppel-like factor 4 (KLF4), an essential transcriptional regulator of macrophage polarization (M1/M2), in the pathogenesis of atherosclerosis. Methods and Results Despite the acknowledged importance of macrophages in atherosclerosis, the role of M1 (classically activated or pro-inflammatory) versus M2 (alternatively activated or anti-inflammatory) macrophages in this process remains incompletely understood. We recently identified KLF4 as a regulator of macrophage subset specification i.e. KLF4 promotes the M2 and inhibits the M1 phenotype. Here, we provide evidence that KLF4 deficient macrophages exhibit enhanced pro-inflammatory activation and foam cell formation in response to oxidized lipids. In vivo, myeloid KLF4 deficient mice (ApoE-/- background) develop significantly more vascular inflammation and atherosclerotic lesion formation. Conclusion Our findings identify myeloid KLF4 as an essential regulator of vascular inflammation and experimental atherogenesis.