Rationale: Inflammation impairs macrophage cholesterol clearance from vascular tissues and promotes atherosclerosis. Inflammatory macrophages suppress expression of the transcription cofactor interferon regulatory factor 2–binding protein 2 (IRF2BP2), and genetic variants near IRF2BP2 associate with ischemic heart disease progression in humans. Objectives: To test whether IRF2BP2 in macrophages affects atherosclerosis in mice and humans. Methods and Results: We generated mice that delete IRF2BP2 in macrophages. IRF2BP2-deficient macrophages worsened atherosclerosis in irradiated low-density lipoprotein receptor null-recipient mice and in apolipoprotein E null mice. IRF2BP2-deficient macrophages were inflammatory and had impaired cholesterol efflux because of their inability to activate the cholesterol transporter ABCA1 in response to cholesterol loading. Their expression of the anti-inflammatory transcription factor Krüppel-like factor 2 was markedly reduced. Promoter studies revealed that IRF2BP2 is required for MEF2-dependent activation of Krüppel-like factor 2. Importantly, restoring Krüppel-like factor 2 in IRF2BP2-deficient macrophages attenuated M1 inflammatory and rescued M2 anti-inflammatory gene activation and improved the cholesterol efflux deficit by restoring ABCA1 activation in response to cholesterol loading. In a cohort of 1066 angiographic cases and 1011 controls, homozygous carriers of a deletion polymorphism (rs3045215) in the 3′ untranslated region sequence of human IRF2BP2 mRNA had a higher risk of coronary artery disease (recessive model, odds ratio [95% confidence interval]=1.560 [1.179–2.065], P =1.73E-03) and had lower IRF2BP2 (and Krüppel-like factor 2) protein levels in peripheral blood mononuclear cells. The effect of this deletion polymorphism to suppress protein expression was confirmed in luciferase reporter studies. Conclusion: Ablation of IRF2BP2 in macrophages worsens atherosclerosis in mice, and a deletion variant that lowers IRF2BP2 expression predisposes to coronary artery disease in humans.

ϩ -ATPase rich (HR) cells in zebrafish skin and gills are also responsible for Na ϩ uptake and acid secretion functions. However, the basolateral transport pathways in HR cells are still unclear. In the present study, we tested the hypothesis if there are specific slc4 members involved in basolateral ion transport pathways in HR cells. Fourteen isoforms were identified in the zebrafish(z) slc4 family, and the full-length cDNAs of two novel isoforms, zslc4a1b (anion exchanger, zAE1b) and zslc4a4b (Na ϩ /HCO 3 Ϫ cotransporter, zNBCe1b), were sequenced. mRNA signals of zslc4a1b and zslc4a4b were mainly detected in certain groups of ionocytes in zebrafish skin/gills. Further double immunocytochemistry or in situ hybridization demonstrated that zAE1b, but not zNBCe1b, was localized to basolateral membranes of HR cells. Acclimation to low-Na ϩ or acidic environments stimulated the mRNA expression of zslc4a1b in zebrafish gills, and loss-of-function of zslc4a1b with specific morpholinos caused significant decreases in both the whole body Na ϩ content and the skin H ϩ activity in the morphants. On the basis of these results, it was concluded that zAE1b, but not zNBCe1b, is involved in the basolateral transport pathways in Na ϩ uptake/acid secretion mechanisms in zebrafish HR cells.acid-base regulation; fish; ion regulation; mitochondrion-rich cell; Na uptake BICARBONATE (HCO 3 Ϫ ) is a single-carbon molecule that plays crucial roles in diverse animal physiological processes, including whole body and cellular pH regulation, cellular volume mediation, and NaCl absorption (1,4,34). The solute carrier family 4 (SLC4) was identified as the transporters responsible for the transmembrane movement of HCO 3 Ϫ , and the family contains 10 members in mammals (34). In mammalian nephrons, about 70 -80% of filtered sodium and bicarbonate is reabsorbed in proximal tubules (43), and the electrogenic Na ϩ -HCO 3 Ϫ cotransporter (NBCe1/SLC4A4) is present in the basolateral membrane of proximal tubular cells for the epithelial transport of bicarbonate from the lumen to the blood (34). Human mutations in NBCe1 are associated with proximal renal tubular acidosis (pRTA), which results in increased renal HCO 3Ϫ levels due to a failure of bicarbonate reabsorption (4). On the other hand, in ␣-intercalated cells of the collecting duct, 5% filtered bicarbonate is reabsorbed, and the major basolateral bicarbonate transporter is the kidney form of the anion exchanger (kAE1/SLC4A1) (1). Different sets of AE1 mutations, such as AE1R589H (21) and AE1901X (6, 40), result in distal RTA, which is due to the failure of acid secretion by ␣-intercalated cells of the cortical collecting duct in the kidney. In proximal tubular cells and collecting duct ␣-intercalated cells, cytosolic carbonic anhydrase generates protons and bicarbonate, protons are excreted by the apical Na ϩ /H ϩ exchanger (NHE) and/or H ϩ -ATPase (HA), and the generated HCO 3 Ϫ is reabsorbed into the blood by the basolateral NBCe1 (proximal tubular cells) or kAE1 (␣-intercalated cells) (1,4...

Cortisol is the major endogenous glucocorticoid (GC) both in human and fish, mediated by corticosteroid receptors. Due to the absence of aldosterone production in teleost fish, cortisol is also traditionally accepted to function as mineralocorticoid (MC); but whether it acts through the glucocorticoid receptor (GR) or the mineralocorticoid receptor (MR) remains a subject of debate. Here, we used loss-of-function and rescue assays to determine whether cortisol affects zebrafish epidermal ionocyte development and function via the GR and/or the MR. GR knockdown morphants displayed a significant decrease in the major ionocytes, namely Na+-K+-ATPase-rich cells (NaRCs) and H+-ATPase-rich cells (HRCs), as well as other cells, including epidermal stem cells (ESCs), keratinocytes, and mucus cells; conversely, cell numbers were unaffected in MR knockdown morphants. In agreement, GR morphants, but not MR morphants, exhibited decreased NaRC-mediated Ca2+ uptake and HRC-mediated H+ secretion. Rescue via GR capped mRNA injection or exogenous cortisol incubation normalized the number of epidermal ionocytes in GR morphants. We also provide evidence for GR localization in epidermal cells. At the transcript level, GR mRNA is ubiquitously expressed in gill sections and present in both NaRCs and HRCs, supporting the knockdown and functional assay results in embryo. Altogether, we have provided solid molecular evidence that GR is indeed present on ionocytes, where it mediates the effects of cortisol on ionocyte development and function. Hence, cortisol-GR axis performs the roles of both GC and MC in zebrafish skin and gills.

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