Fei Sun

A chemokine receptor CXCR2 macromolecular complex regulates neutrophil functions in inflammatory diseases.

Background: CXCR2 plays an important role in various neutrophil-dominant inflammatory diseases. Results: A macromolecular signaling complex containing CXCR2, NHERF1, and phospholipase C (PLC)-β2 regulates neutrophil calcium mobilization, chemotaxis, and transepithelial migration. Conclusion: CXCR2·NHERF1·PLC-β2 macromolecular signaling complex is critical to neutrophil functions. Significance: CXCR2 macromolecular complex might be a potential therapeutic target for neutrophil infiltration-associated inflammatory diseases. Inflammation plays an important role in a wide range of human diseases such as ischemia-reperfusion injury, arteriosclerosis, cystic fibrosis, inflammatory bowel disease, etc. Neutrophilic accumulation in the inflamed tissues is an essential component of normal host defense against infection, but uncontrolled neutrophilic infiltration can cause progressive damage to the tissue epithelium. The CXC chemokine receptor CXCR2 and its specific ligands have been reported to play critical roles in the pathophysiology of various inflammatory diseases. However, it is unclear how CXCR2 is coupled specifically to its downstream signaling molecules and modulates cellular functions of neutrophils. Here we show that the PDZ scaffold protein NHERF1 couples CXCR2 to its downstream effector phospholipase C (PLC)-β2, forming a macromolecular complex, through a PDZ-based interaction. We assembled a macromolecular complex of CXCR2·NHERF1·PLC-β2 in vitro, and we also detected such a complex in neutrophils by co-immunoprecipitation. We further observed that the CXCR2-containing macromolecular complex is critical for the CXCR2-mediated intracellular calcium mobilization and the resultant migration and infiltration of neutrophils, as disrupting the complex with a cell permeant CXCR2-specific peptide (containing the PDZ motif) inhibited intracellular calcium mobilization, chemotaxis, and transepithelial migration of neutrophils. Taken together, our data demonstrate a critical role of the PDZ-dependent CXCR2 macromolecular signaling complex in regulating neutrophil functions and suggest that targeting the CXCR2 multiprotein complex may represent a novel therapeutic strategy for certain inflammatory diseases.

A critical role of CXCR2 PDZ-mediated interactions in endothelial progenitor cell homing and angiogenesis

Bone marrow-derived endothelial progenitor cells (EPCs) contribute to neovessel formation in response to growth factors, cytokines, and chemokines. Chemokine receptor CXCR2 and its cognate ligands are reported to mediate EPC recruitment and angiogenesis. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif which has been reported to modulate cellular signaling and functions. Here we examined the potential role of the PDZ motif in CXCR2-mediated EPC motility and angiogenesis. We observed that exogenous CXCR2 C-tail significantly inhibited in vitro EPC migratory responses and angiogenic activities, as well as in vivo EPC angiogenesis. However, the CXCR2 C-tail that lacks the PDZ motif (ΔTTL) did not cause any significant changes of these functions in EPCs. In addition, using biochemical assays, we demonstrated that the PDZ scaffold protein NHERF1 specifically interacted with CXCR2 and its downstream effector, PLC-β3, in EPCs. This suggests that NHERF1 might cluster CXCR2 and its relevant signaling molecules into a macromolecular signaling complex modulating EPC cellular functions. Taken together, our data revealed a critical role of a PDZ-based CXCR2 macromolecular complex in EPC homing and angiogenesis, suggesting that targeting this complex might be a novel and effective strategy to treat angiogenesis-dependent diseases.

Toll-like Receptor (TLR) Signaling Interacts with CREBH to Modulate High-density Lipoprotein (HDL) in Response to Bacterial Endotoxin

Bacterial endotoxin can induce inflammatory and metabolic changes in the host. In this study, we revealed a molecular mechanism by which a stress-inducible, liver-enriched transcription factor, cAMP-responsive element-binding protein hepatic-specific (CREBH), modulates lipid profiles to protect the liver from injuries upon the bacterial endotoxin lipopolysaccharide (LPS). LPS challenge can activate CREBH in mouse liver tissues in a toll-like receptor (TLR)/MyD88-dependent manner. Upon LPS challenge, CREBH interacts with TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase that functions as a key mediator of TLR signaling, and this interaction relies on MyD88. Further analysis demonstrated that TRAF6 mediates K63-linked ubiquitination of CREBH to facilitate CREBH cleavage and activation. CREBH directly activates expression of the gene encoding Apolipoprotein A4 (ApoA4) under LPS challenge, leading to modulation of high-density lipoprotein (HDL) in animals. CREBH deficiency led to reduced production of circulating HDL and increased liver damage upon high-dose LPS challenge. Therefore, TLR/MyD88-dependent, TRAF6-facilitated CREBH activation represents a mammalian hepatic defense response to bacterial endotoxin by modulating HDL.

Dysregulated Chemokine Signaling in Cystic Fibrosis Lung Disease: A Potential Therapeutic Target

CF lung disease is characterized by a chronic and non-resolving activation of the innate immune system with excessive release of chemokines/cytokines including IL-8 and persistent infiltration of immune cells, mainly neutrophils, into the airways. Chronic infection and impaired immune response eventually lead to pulmonary damage characterized by bronchiectasis, emphysema, and lung fibrosis. As a complete knowledge of the pathways responsible for the exaggerated inflammatory response in CF lung disease is lacking, understanding these pathways could reveal new therapeutic targets, and lead to novel treatments. Therefore, there is a strong rationale for the identification of mechanisms and pathways underlying the exaggerated inflammatory response in CF lung disease. This article reviews the role of inflammation in the pathogenesis of CF lung disease, with a focus on the dysregulated signaling involved in the overexpression of chemokine IL-8 and excessive recruitment of neutrophils in CF airways. The findings suggest that targeting the exaggerated IL-8/IL-8 receptor (mainly CXCR2) signaling pathway in immune cells (especially neutrophils) may represent a potential therapeutic strategy for CF lung disease.

A novel ER-microtubule-binding protein, ERLIN2, stabilizes Cyclin B1 and regulates cell cycle progression.

The gene encoding endoplasmic reticulum (ER) lipid raft-associated protein 2 (ERLIN2) is amplified in human breast cancers. ERLIN2 gene mutations were also found to be associated with human childhood progressive motor neuron diseases. Yet, an understanding of the physiological function and mechanism for ERLIN2 remains elusive. In this study, we reveal that ERLIN2 is a spatially and temporally regulated ER–microtubule-binding protein that has an important role in cell cycle progression by interacting with and stabilizing the mitosis-promoting factors. Whereas ERLIN2 is highly expressed in aggressive human breast cancers, during normal development ERLIN2 is expressed at the postnatal stage and becomes undetectable in adulthood. ERLIN2 interacts with the microtubule component α-tubulin, and this interaction is maximal during the cell cycle G2/M phase where ERLIN2 simultaneously interacts with the mitosis-promoting complex Cyclin B1/Cdk1. ERLIN2 facilitates K63-linked ubiquitination and stabilization of Cyclin B1 protein in G2/M phase. Downregulation of ERLIN2 results in cell cycle arrest, represses breast cancer proliferation and malignancy and increases sensitivity of breast cancer cells to anticancer drugs. In summary, our study revealed a novel ER–microtubule-binding protein, ERLIN2, which interacts with and stabilizes mitosis-promoting factors to regulate cell cycle progression associated with human breast cancer malignancy.

Dissection of the Role of VIMP in Endoplasmic Reticulum-Associated Degradation of CFTRΔF508

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is an important quality control mechanism that eliminates misfolded proteins from the ER. The Derlin-1/VCP/VIMP protein complex plays an essential role in ERAD. Although the roles of Derlin-1 and VCP are relatively clear, the functional activity of VIMP in ERAD remains to be understood. Here we investigate the role of VIMP in the degradation of CFTRΔF508, a cystic fibrosis transmembrane conductance regulator (CFTR) mutant known to be a substrate of ERAD. Overexpression of VIMP markedly enhances the degradation of CFTRΔF508, whereas knockdown of VIMP increases its half-life. We demonstrate that VIMP is associated with CFTRΔF508 and the RNF5 E3 ubiquitin ligase (also known as RMA1). Thus, VIMP not only forms a complex with Derlin-1 and VCP, but may also participate in recruiting substrates and E3 ubiquitin ligases. We further show that blocking CFTRΔF508 degradation by knockdown of VIMP substantially augments the effect of VX809, a drug that allows a fraction of CFTRΔF508 to fold properly and mobilize from ER to cell surface for normal functioning. This study provides insight into the role of VIMP in ERAD and presents a potential target for the treatment of cystic fibrosis patients carrying the CFTRΔF508 mutation.

Inhalation Exposure to PM 2.5 Counteracts Hepatic Steatosis in Mice Fed High-fat Diet by Stimulating Hepatic Autophagy

Air pollution is associated with the increased risk of metabolic syndrome. In this study, we performed inhalation exposure of mice fed normal chow or a high-fat diet to airborne fine particulate matters (PM2.5), and then investigated the complex effects and mechanisms of inhalation exposure to PM2.5 on hepatic steatosis, a precursor or manifestation of metabolic syndrome. Our studies demonstrated that inhalation exposure of mice fed normal chow to concentrated ambient PM2.5 repressed hepatic transcriptional regulators involved in fatty acid oxidation and lipolysis, and thus promoted hepatic steatosis. However, PM2.5 exposure relieved hepatic steatosis in high-fat diet-induced obese mice. Further investigation revealed that inhalation exposure to PM2.5 induced hepatic autophagy in mouse livers in a manner depending on the MyD88-mediated inflammatory pathway. The counteractive effect of PM2.5 exposure on high-fat diet-induced hepatic steatosis was mediated through PM2.5-induced hepatic autophagy. The findings from this study not only defined the effects and mechanisms of PM2.5 exposure in metabolic disorders, but also revealed the pleotrophic acts of an environmental stressor in a complex stress system relevant to public health.

SUMOylation represses the transcriptional activity of the Unfolded Protein Response transducer ATF6

The Unfolded Protein Response (UPR) is a cascade of intracellular stress signaling from the endoplasmic reticulum (ER) that protect the cells from the stress caused by accumulation of unfolded or misfolded proteins in the ER. Activating transcription factor 6 (ATF6) is one of primary UPR transducers that remodels the stressed cells through transcriptional regulation. Although the activation mechanism and biological roles of ATF6 have been well studied, the understanding of the negative or feedback regulation of ATF6 remains elusive. In this report, we showed that ATF6 protein can be modified by small ubiquitin-like modification (SUMOylation) and that the transcriptional activity of ATF6 is negatively regulated by SUMOylation. We identified that SUMOylation of ATF6 is significantly increased in the cells expressing misfolded cystic fibrosis transmembrane conductance regulator (CFTR) encoded by the mutant human CFTR gene (dF508CFTR). Further analyses revealed two highly conserved SUMOylation motifs within the trans-activation domain of ATF6 protein of human, mouse, or rat specie. The human ATF6 protein can be SUMOylated mediated through the small ubiquitin-like modifier protein 1 (SUMO-1) and E3 SUMO-protein ligase 1 (PIAS1) at the conserved sumoylation residue Lys149 that is located at the N-terminal of the activated form of ATF6 protein. Bimolecular fluorescence complementation (BiFC) analysis confirmed that the activated ATF6 protein can be SUMOylated and that the ATF6 sumoylation occurs in the nuclei. Moreover, trans-activation reporter analysis demonstrated that SUMOylation of the ATF6 protein at the conserved residue Lys149 represses the transcriptional activity of ATF6. In summary, our study revealed a negative regulation of the UPR transducer ATF6 through post-translational SUMOylation. The information from this study will not only increase our understanding of the fine-tuning regulation of the UPR signaling but will also be informative to the modulation of the UPR for therapeutic benefits.

Transcriptional signatures of unfolded protein response implicate the limitation of animal models in pathophysiological studies

Background The unfolded protein response (UPR) refers to intracellular stress signaling pathways that protect cells from the stress caused by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER). The UPR signaling is crucially involved in the initiation and progression of a variety of human diseases by modulating transcriptional and translational programs of the stressed cells. In this study, we analyzed the gene expression signatures of primary stress sensors and major mediators of UPR pathways in a variety of tissues/organs of human and murine species. Methods We first analyzed protein sequence similarities of major UPR transducers and mediators of human and murine species, and then examined their gene expression profiles in 26 human and mouse common tissues based on the microarray datasets of public domains. The differential expression patterns of the UPR genes in human diseases were delineated. The involvements of the UPR genes in mouse pathology were also analyzed with mouse gene knockout models. Results The results indicated that expression patterns and pathophysiologic involvements of the major UPR stress sensors and mediators significantly differ in 26 common tissues/organs of human and murine species. Gene expression profiles suggest that the IRE1α/XBP1-mediated UPR pathway is induced in secretory and metabolic tissues or organs. While deletion of the UPR trans-activator XBP1 leads to pathological phenotypes in mice, alteration in XBP1 is less associated with human disease conditions. Conclusions Expression signatures of the major UPR genes differ among tissues or organs and among human and mouse species. The differential induction of the UPR pathways reflects the pathophysiologic differences of tissues or organs. The difference in UPR induction between human and mouse suggests the limitation of using animal models to study human pathophysiology or drugology associated with environmental stress.