Sung Gil Ha

Retinoic acid-stimulated sequential phosphorylation, PML recruitment, and SUMOylation of nuclear receptor TR2 to suppress Oct4 expression

We previously reported an intricate mechanism underlying the homeostasis of Oct4 expression in normally proliferating stem cell culture of P19, mediated by SUMOylation of orphan nuclear receptor TR2. In the present study, we identify a signaling pathway initiated from the nongenomic activity of all-trans retinoic acid (atRA) to stimulate complex formation of extracellular signal-regulated kinase 2 (ERK2) with its upstream kinase, mitogen-activated protein kinase kinase (MEK). The activated ERK2 phosphorylates threonine-210 (Thr-210) of TR2, stimulating its subsequent SUMOylation. Dephosphorylated TR2 recruits coactivator PCAF and functions as an activator for its target gene Oct4. Upon phosphorylation at Thr-210, TR2 increasingly associates with promyelocytic leukemia (PML) nuclear bodies, becomes SUMOylated, and recruits corepressor RIP140 to act as a repressor for its target, Oct4. To normally proliferating P19 stem cell culture, exposure to a physiological concentration of atRA triggers a rapid nongenomic signaling cascade to suppress Oct4 gene and regulate cell proliferation.

Allergen-Induced Airway Remodeling Is Impaired in Galectin-3–Deficient Mice

The role played by the β-galactoside–binding lectin galectin-3 (Gal-3) in airway remodeling, a characteristic feature of asthma that leads to airway dysfunction and poor clinical outcome in humans, was investigated in a murine model of chronic allergic airway inflammation. Wild-type (WT) and Gal-3 knockout (KO) mice were subjected to repetitive allergen challenge with OVA up to 12 wk, and bronchoalveolar lavage fluid (BALF) and lung tissue collected after the last challenge were evaluated for cellular features associated with airway remodeling. Compared to WT mice, chronic OVA challenge in Gal-3 KO mice resulted in diminished remodeling of the airways with significantly reduced mucus secretion, subepithelial fibrosis, smooth muscle thickness, and peribronchial angiogenesis. The higher degree of airway remodeling in WT mice was associated with higher Gal-3 expression in the BALF as well as lung tissue. Cell counts in BALF and lung immunohistology demonstrated that eosinophil infiltration in OVA-challenged Gal-3 KO mice was significantly reduced compared with that WT mice. Evaluation of cellular mediators associated with eosinophil recruitment and airway remodeling revealed that levels of eotaxin-1, IL-5, IL-13, found in inflammatory zone 1, and TGF-β were substantially lower in Gal-3 KO mice. Finally, leukocytes from Gal-3 KO mice demonstrated decreased trafficking (rolling) on vascular endothelial adhesion molecules compared with that of WT cells. Overall, these studies demonstrate that Gal-3 is an important lectin that promotes airway remodeling via airway recruitment of inflammatory cells, specifically eosinophils, and the development of a Th2 phenotype as well as increased expression of eosinophil-specific chemokines and profibrogenic and angiogenic mediators.

ORMDL3 promotes eosinophil trafficking and activation via regulation of integrins and CD48

ORM (yeast)-Like protein isoform 3 (ORMDL3) has recently been identified as a candidate gene for susceptibility to asthma; however the mechanisms by which it contributes to asthma pathogenesis are not well understood. Here we demonstrate a functional role for ORMDL3 in eosinophils in the context of allergic inflammation. Eosinophils recruited to the airways of allergen-challenged mice express ORMDL3. ORMDL3 expression in bone marrow eosinophils is localized in the endoplasmic reticulum and is induced by IL-3 and eotaxin-1. Over-expression of ORMDL3 in eosinophils causes increased rolling, distinct cytoskeletal rearrangement, ERK (1/2) phosphorylation and nuclear translocation of NF-κB. Knock-down of ORMDL3 significantly inhibits activation-induced cell shape changes, adhesion and recruitment to sites of inflammation in vivo, combined with reduced expression of CD49d and CD18. Additionally, ORMDL3 regulates IL-3-induced expression of CD48 and CD48-mediated eosinophil degranulation. These studies show that ORMDL3 regulates eosinophil trafficking, recruitment and degranulation, further elucidating a role for this molecule in allergic asthma and potentially other eosinophilic disorders.

SUMOylation of Tr2 orphan receptor involves Pml and fine-tunes Oct4 expression in stem cells

The Tr2 orphan nuclear receptor can be SUMOylated, resulting in the replacement of coregulators recruited to the regulatory region of its endogenous target gene, Oct4. UnSUMOylated Tr2 activates Oct4, enhancing embryonal carcinoma-cell proliferation, and is localized to the promyelocytic leukemia (Pml) nuclear bodies. When its abundance is elevated, Tr2 is SUMOylated at Lys238 and seems to be released from the nuclear bodies to act as a repressor. SUMOylation of Tr2 induces an exchange of its coregulators: corepressor Rip140 replaces coactivator Pcaf, which switches Tr2 from an activator to a repressor. This involves dynamic partitioning of Tr2 into Pml-containing and Pml-free pools. These results support a model where SUMOylation-dependent partitioning and differential coregulator recruitment contribute to the maintenance of a homeostatic supply of activating, as opposed to repressive, Tr2, thus fine-tuning Oct4 expression and regulating stem-cell proliferation.

Modulation of lysine acetylation-stimulated repressive activity by Erk2-mediated phosphorylation of RIP140 in adipocyte differentiation

Receptor-interacting protein 140 is a co-regulator for many transcription factors. Previous mass spectrometry studies showed that either phosphorylation or lysine acetylation of RIP140 directly enhanced its trans-repressive activity. In this study, we first identified p300 as a specific lysine acetyltransferase, and extracellular-signal-related kinase 2 (Erk2) as a specific kinase for threonine phosphorylation, of RIP140 in vivo. We further determined two specific acetylated lysine residues (Lys(158)/Lys(287)) and phosphorylated threonine residues (Thr(202)/Thr(207)) that were critical for its gene-repressive activity. We then delineated signal transduction from Erk2-mediated phosphorylation of RIP140 that enhanced its recruiting p300 for subsequent lysine acetylation, and demonstrated the kinetics of activation of this signal transduction pathway in differentiating adipocytes. Finally, the physiological significance of this cell signal transduction pathway was illustrated in rescuing experiments where the defect in fat accumulation of RIP140-null cultures was rescued by re-expressing the wild type RIP140 or its phospho-mimetic mutant, but not its acetylation deficient mutant. These results demonstrate the signal transduction pathway, initiated from Erk2 activation for specific threonine phosphorylation, followed by p300 recruitment for lysine acetylation, which ultimately enhances the gene-repressive activity of RIP140 and its functional role in fat accumulation in differentiated adipocytes.

Regulation of Eosinophil Trafficking by SWAP-70 and Its Role in Allergic Airway Inflammation

Eosinophils are the predominant inflammatory cells recruited to allergic airways. In this article, we show that human and murine eosinophils express SWAP-70, an intracellular RAC-binding signaling protein, and examine its role in mediating eosinophil trafficking and pulmonary recruitment in a murine model of allergic airway inflammation. Compared with wild-type eosinophils, SWAP-70–deficient (Swap-70−/−) eosinophils revealed altered adhesive interactions within inflamed postcapillary venules under conditions of blood flow by intravital microscopy, exhibiting enhanced slow rolling but decreased firm adhesion. In static adhesion assays, Swap-70−/− eosinophils adhered poorly to VCAM-1 and ICAM-1 and exhibited inefficient leading edge and uropod formation. Adherent Swap-70−/− eosinophils failed to translocate RAC1 to leading edges and displayed aberrant cell surface localization/distribution of α4 and Mac-1. Chemokine-induced migration of Swap-70−/− eosinophils was significantly decreased, correlating with reduced intracellular calcium levels, defective actin polymerization/depolymerization, and altered cytoskeletal rearrangement. In vivo, recruitment of eosinophils to the lungs of allergen-challenged Swap-70−/− mice, compared with wild-type mice, was significantly reduced, along with considerable attenuation of airway inflammation, indicated by diminished IL-5, IL-13, and TNF-α levels; reduced mucus secretion; and improved airway function. These findings suggest that regulation of eosinophil trafficking and migration by SWAP-70 is important for the development of eosinophilic inflammation after allergen exposure.

Lysine Methylation of Nuclear Co-repressor Receptor Interacting Protein 140

Receptor interacting protein 140 (RIP140) undergoes extensive post-translational modifications (PTMs), including phosphorylation, acetylation, arginine methylation, and pyridoxylation. PTMs affect its subcellular distribution, protein-protein interaction, and biological activity in adipocyte differentiation. Arginine methylation on Arg(240), Arg(650), and Arg(948) suppresses the repressive activity of RIP140. Here, we find that endogenous RIP140 in differentiated 3T3-L1 cells is also modified by lysine methylation. Three lysine residues, Lys(591), Lys(653), and Lys(757), are mapped as potential methylation sites by mass spectrometry. Site-directed mutagenesis study shows that lysine methylation enhances its gene repressive activity. Mutation of lysine methylation sites enhances arginine methylation, while mutation on arginine methylation sites has little effect on its lysine methylation, suggesting a relationship between lysine methylation and arginine methylation. Kinetic analysis of PTMs of endogenous RIP140 in differentiated 3T3-L1 cells demonstrates sequential modifications on RIP140, initiated from constitutive lysine methylation, followed by increased arginine methylation later in differentiation. This study reveals a potential hierarchy of modifications, at least for lysine and arginine methylation, which bidirectionally regulate the functionality of a nonhistone protein.

The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.

Regulation of eosinophilia and allergic airway inflammation by the glycan-binding protein galectin-1

Significance Allergic asthma is a chronic airway disease, and the number of individuals with asthma continues to grow. Eosinophils recruited to allergic airways contribute significantly to airway inflammation via release of proinflammatory mediators that cause epithelial tissue damage, bronchoconstriction, and airway remodeling. Here we show that galectin-1 (Gal-1), an endogenous immunoregulatory lectin, binds to eosinophil-expressed surface glycans to inhibit cell migration and induce apoptosis. Using a mouse model of allergic asthma, we show that mice lacking Gal-1 exhibit increased airway eosinophils and airway hyperresponsiveness compared with wild-type mice. Because Gal-1 plays an important role in regulating airway inflammation, identifying pathways to induce Gal-1 synthesis and/or favor its biological activity might enable exploitation of its proresolving function to suppress allergic asthma. Galectin-1 (Gal-1), a glycan-binding protein with broad antiinflammatory activities, functions as a proresolving mediator in autoimmune and chronic inflammatory disorders. However, its role in allergic airway inflammation has not yet been elucidated. We evaluated the effects of Gal-1 on eosinophil function and its role in a mouse model of allergic asthma. Allergen exposure resulted in airway recruitment of Gal-1–expressing inflammatory cells, including eosinophils, as well as increased Gal-1 in extracellular spaces in the lungs. In vitro, extracellular Gal-1 exerted divergent effects on eosinophils that were N-glycan– and dose-dependent. At concentrations ≤0.25 µM, Gal-1 increased eosinophil adhesion to vascular cell adhesion molecule-1, caused redistribution of integrin CD49d to the periphery and cell clustering, but inhibited ERK(1/2) activation and eotaxin-1–induced migration. Exposure to concentrations ≥1 µM resulted in ERK(1/2)-dependent apoptosis and disruption of the F-actin cytoskeleton. At lower concentrations, Gal-1 did not alter expression of adhesion molecules (CD49d, CD18, CD11a, CD11b, L-selectin) or of the chemokine receptor CCR3, but decreased CD49d and CCR3 was observed in eosinophils treated with higher concentrations of this lectin. In vivo, allergen-challenged Gal-1–deficient mice exhibited increased recruitment of eosinophils and CD3+ T lymphocytes in the airways as well as elevated peripheral blood and bone marrow eosinophils relative to corresponding WT mice. Further, these mice had an increased propensity to develop airway hyperresponsiveness and displayed significantly elevated levels of TNF-α in lung tissue. This study suggests that Gal-1 can limit eosinophil recruitment to allergic airways and suppresses airway inflammation by inhibiting cell migration and promoting eosinophil apoptosis.

N-Glycans Differentially Regulate Eosinophil and Neutrophil Recruitment during Allergic Airway Inflammation

Background: Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment. Results: Deficiency of MGAT5 causes attenuation of allergen-induced eosinophilia and Th2 cytokines but increases neutrophilic inflammation and airway hyperreactivity. Conclusion: Recruitment of eosinophils and neutrophils is differentially regulated by MGAT5-modified N-glycans during airway inflammation. Significance: This study demonstrates a significant role for N-glycans in the development of allergic airway inflammation and asthma. Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-d-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5−/−) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5−/− eosinophils. Interestingly, allergen-challenged Mgat5−/− mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5−/− mice. Furthermore, there was significantly increased recruitment of infused Mgat5−/− neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5−/− neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.