Li Qin Zhang

Neuronal protective role of PBEF in a mouse model of cerebral ischemia.

Pre-B-cell colony-enhancing factor (PBEF) (also known as nicotinamide phosphoribosyltransferase) is a rate-limiting enzyme in the salvage pathway for mammalian biosynthesis of nicotinamide adenine dinucleotide (NAD+). By synthesizing NAD+, PBEF functions to maintain an energy supply that has critical roles in cell survival. Cerebral ischemia is a major neural disorder with a high percentage of mortality and disability. Ischemia leads to energy depletion and eventually neuronal death and brain damage. This study investigated the role of PBEF in cerebral ischemia using a photothrombosis mouse model. Using immunostaining, we initially determined that PBEF is highly expressed in neurons, but not in glial cells in the mouse brain. To study the role of PBEF in ischemia in vivo, we used PBEF knockout heterozygous (Pbef+/−) mice. We showed that these mice have lower PBEF expression and NAD+ level than do wild-type (WT) mice. When subjected to photothrombosis, Pbef+/− mice have significantly larger infarct volume than do age-matched WT mice at 24 hours after ischemia. Higher density of degenerating neurons was detected in the penumbra of Pbef+/− mice than in WT mice using Fluoro-Jade B staining. Our study shows that PBEF has a neuronal protective role in cerebral ischemia presumably through enhanced energy metabolism.

Critical role of PBEF expression in pulmonary cell inflammation and permeability

Previous studies in our lab have identified pre‐B‐cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury. This study continues to elucidate the underlying molecular mechanism of PBEF in the pathogenesis of acute lung injury in pulmonary cell culture models. Our results revealed that IL‐1β induced PBEF expression in pulmonary vascular endothelial cells at the transcriptional level and a −1535 T‐variant in the human PBEF gene promoter significantly attenuated its binding to an IL‐1β‐induced unknown transcription factor. This may underlie the reduced expression of PBEF and thus the lower susceptibility to acute lung injury in −1535T carriers. Furthermore, overexpression of PBEF significantly augmented IL‐8 secretion and mRNA expression by more than 6‐fold and 2‐fold in A549 cells and HPAEC, respectively. It also significantly augmented IL‐1β‐mediated cell permeability by 44% in A549 cells and 65% in endothelial cells. The knockdown of PBEF expression significantly inhibited IL‐1β‐stimulated IL‐8 secretion and mRNA level by 60% and 70%, respectively, and the knockdown of PBEF expression also significantly attenuated IL‐1β‐induced cell permeability by 29% in epithelial cells and 24% in endothelial cells. PBEF expression also affected the expression of two other inflammatory cytokines (IL‐16 and CCR3 genes). These results suggest that PBEF is critically involved in pulmonary vascular and epithelial inflammation and permeability, which are hallmark features in the pathogenesis of acute lung injury. This study lends further support to our finding that PBEF is a potential new target in acute lung injury.

Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism

Although our previous studies found Pre-B-cell colony-enhancing factor (PBEF) as a highly up-regulated gene in acute lung injury that could stimulate expressions of other inflammatory cytokines, the underlying molecular mechanisms remain to be fully elucidated. Growing evidence indicates that PBEF is a nicotinamide phosphoribosyltransferase involved in the mammalian salvage pathway of NAD synthesis. This study was designed to determine whether the effect of PBEF to stimulate expressions of inflammatory cytokines depends on its enzymatic activity. We prepared two human PBEF mutant (H247E and H247A) recombinant proteins and overexpressing constructs for their overexpressions in A549 cells and confirmed that enzymatic activities of both mutants were nearly or completely abolished. Two mutants stimulated interleukin-8 (IL-8) expression at both the mRNA level and protein level just as equally effective as the wild-type PBEF did. These effects were due to the increased transcription, not the mRNA stability, of the IL-8 gene. Reporter gene assays and gel shift experiments indicated that AP-1 transcription factor is required to mediate these effects. SB203580, a p38 MAPK pathway inhibitor, and JNK inhibitor 1 can attenuate these effects. Both PBEF mutants similarly stimulated the expression of two other inflammatory cytokines: IL-16 and CCR3. These results indicate that PBEF stimulated expression of IL-8, IL-16, and CCR3 via its non-enzymatic activity. This effect is AP-1-dependent, in part via the p38 MAPK pathway and the JNK pathway. This finding reveals a new insight, which may manifest a novel role of PBEF in the pathogenesis of acute lung injury and other inflammatory disorders.

RNA-seq analysis of synovial fibroblasts brings new insights into rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune-disease of unknown origin that primarily affects the joints and ultimately leads to their destruction. Growing evidence suggests that synvovial fibroblasts play important roles in the initiation and the perpetuation of RA but underlying molecular mechanisms are not understood fully. In the present study, Illumina RNA sequencing was used to profile two human normal control and two rheumatoid arthritis synvovial fibroblasts (RASFs) transcriptomes to gain insights into the roles of synvovial fibroblasts in RA.ResultsWe found that besides known inflammatory and immune responses, other novel dysregulated networks and pathways such as Cell Morphology, Cell-To-Cell Signaling and Interaction, Cellular Movement, Cellular Growth and Proliferation, and Cellular Development, may all contribute to the pathogenesis of RA. Our study identified several new genes and isoforms not previously associated with rheumatoid arthritis. 122 genes were up-regulated and 155 genes were down-regulated by at least two-fold in RASFs compared to controls. Of note, 343 known isoforms and 561 novel isoforms were up-regulated and 262 known isoforms and 520 novel isoforms were down-regulated by at least two-fold. The magnitude of difference and the number of differentially expressed known and novel gene isoforms were not detected previously by DNA microarray.ConclusionsSince the activation and proliferation of RASFs has been implicated in the pathogenesis of rheumatoid arthritis, further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying synovial fibroblasts in arthritis and provide new leads of potential therapeutic targets.

Augmentation of Pulmonary Epithelial Cell IL-8 Expression and Permeability by Pre-B-cell Colony Enhancing Factor

BackgroundPrevious studies in our lab have identified Pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury (ALI). The molecular mechanism of PBEF involvement in the pathogenesis of ALI is still incompletely understood. This study examined the role of PBEF in regulating pulmonary alveolar epithelial cell IL-8 expression and permeability.MethodsHuman pulmonary alveolar epithelial cells (cell line and primary cells) were transfected with human PBEF cDNA or PBEF siRNA and then cultured in the presence or absence of TNFα. PBEF and IL-8 expression were analyzed by RT-PCR and Western blotting. In addition, changes in pulmonary alveolar epithelial and artery endothelial cell barrier regulation with altered PBEF expression was evaluated by an in vitro cell permeability assay.ResultsOur results demonstrated that, in human pulmonary alveolar epithelial cells, the overexpression of PBEF significantly augmented basal and TNFα-stimulated IL-8 secretion by more than 5 to 10-fold and increased cell permeability by >30%; the knockdown of PBEF expression with siRNA significantly inhibited basal and TNFα-stimulated IL-8 secretion by 70% and IL-8 mRNA levels by 74%. Further, the knockdown of PBEF expression also significantly attenuated TNFα-induced cell permeability by 43%. Similar result was observed in human pulmonary artery endothelial cells.ConclusionThese results suggest that PBEF may play a vital role in basal and TNFα-mediated pulmonary inflammation and pulmonary epithelial barrier dysfunction via its regulation of other inflammatory cytokines such as IL-8, which could in part explain the role of PBEF in the susceptibility and pathogenesis of ALI. These results lend further support to the potential of PBEF to serve as a diagnostic and therapeutic target to ALI.

Interactions between PBEF and oxidative stress proteins--a potential new mechanism underlying PBEF in the pathogenesis of acute lung injury.

MINT‐6538697: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with NADH1 (uniprotkb:P03886) by two hybrid (MI:0018) MINT‐6538811, MINT‐6538868: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with interferon‐induced transmembrane protein 3 (uniprotkb:Q01628) by anti bait coimmunoprecipitation (MI:0006) MINT‐6538787, MINT‐6538841: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with NADH1 (uniprotkb:P03886) by anti bait coimmunoprecipitation (MI:0006) MINT‐6538755: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with γ‐glutamyl‐transferase (uniprotkb:P19440) by two hybrid (MI:0018) MINT‐6538799, MINT‐6538862: PBEF (uniprotkb:P43490) physically interacts (MI:0218)with Ferritin light chain (uniprotkb:P02792) by anti bait coimmunoprecipitation (MI:0006) MINT‐6538769: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with E2L6 (uniprotkb:O14933) by two hybrid (MI:0018) MINT‐6538741: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with Adenosine A2aR (uniprotkb:P29274) by two hybrid (MI:0018) MINT‐6538727: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with interferon‐induced transmembrane protein 3 (uniprotkb:Q01628) by two hybrid (MI:0018) MINT‐6538712: PBEF (uniprotkb:P43490) physically interacts (MI:0218) with Ferritin light chain (uniprotkb:P02792) by two hybrid (MI:0018)

Nicotinamide Phosphoribosyltransferase in Rheumatoid Arthritis

Nicotinamide Phosphoribosyltransferase (Nampt) is a pleiotropic protein with multiple functions, including catalyzing a rate-limiting reaction of nicotinamide adenine dinucleotide (NAD) synthesis in a mammalian salvage pathway and mediating the innate immune system’s inflammatory response. The enzymatic activity of Nampt has been well characterized; however, the mechanism(s) by which Nampt regulates cytokine signaling have not been fully elucidated. Rheumatoid arthritis (RA) a chronic, systemic autoimmune disorder in which the pathological roles of proinflammatory cytokines such as Tumor Necrosis Factor (TNF), interleukin (IL)-1β, and IL- 6 have been clearly demonstrated. Recently, studies have shown that serum and synovial levels of Nampt are elevated in both RA patients and in mice with collagen induced arthritis (CIA), and that inhibition of Nampt activity decreases the expression of TNF, IL-1β, and IL-6. Thus, Nampt may represent a potential new therapeutic target in RA. This review will focus on Nampt’s role in NAD metabolism and cytokine signaling in the context of the pathophysiology and therapeutic potential of RA.

Nicotinamide Phosphoribosyltransferase in Sepsis

The word sepsis was derived from the Greek word: sepsis, which means the state of putrefaction or decay. Sepsis is a potentially deadly medical condition that is characterized by a whole-body inflammatory state, called a systemic inflammatory response syndrome, and the presence of a known or suspected infection. The more critical subsets of sepsis are severe sepsis with acute organ dysfunction, hypoperfusion, or hypotension and septic shock with refractory arterial hypotension despite adequate fluid resuscitation [1, 2]. Because the molecular pathogenesis of sepsis is incompletely understood and its specific and effective therapies are lacking, sepsis is still a major cause of death in intensive-care units worldwide, with mortality rates that range from 20% for sepsis, through 40% for severe sepsis, to over 60% for septic shock [3, 4]. More knowledge of the pathophysiology of sepsis is needed if we are to develop better, more effective interventions to sepsis. It has been increasingly recognized that genetic factors influence individual susceptibility, severity and outcome in sepsis [5, 6]. Identification of new genetic factors in sepsis may hold promise for new mechanistic insights and new therapeutic modalities.

Nicotinamide Phosphoribosyltransferase Inhibitors

Nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12) catalyzes the condensation of nicotinamide with 5-phosphoribosyl-1-pyrophosphate (PRPP) to yield nicotinamide mononucleotide (NMN), a rate limiting enzyme in a mammalian salvage pathway of nicotinamide adenine dinucleotide (NAD) synthesis. Human NAMPT consists of 491 amino acids with a molecular weight of 52 kDa (Samal et al., 1994). NAMPT was initially named pre-B-cell colony-enhancing factor (PBEF) for its growth factor like function on promoting pre-B-cell colony formation in the presence of stem cell factor plus interleukin 7 (Samal et al., 1994). Martin et al. (2001) found that the gene encoding the bacterial Haemophilus ducreyi nicotinamide phosphoribosyltransferase (nadV) had a significant homology to the mammalian PBEF gene. Since then, Rongvaux et al.(2002), Revollo et al. (2004) and others (van der Veer et al., 2005) have characterized the enzymological features of mammalian NAMPT. In 2005, NAMPT/PBEF was named visfatin, a “new visceral fat-derived hormone”, which is an adipocyte-derived adipokine that induces insulin mimetic effects (Fukuhara et al., 2005). To avoid the confusion, the name NAMPT will be used throughout this chapter since NAMPT was approved as the official name of this gene by the Human Genome Organization Gene Nomenclature Committee.