Pre-B Cell Colony-enhancing Factor Expression Research Articles

Inhibition of Pre-B Cell Colony Enhancing Factor Reduces Lung Injury in Rats Receiving Cardiopulmonary Bypass.

ObjectivePre-B cell colony enhancing factor (PBEF) is an important proinflammatory cytokine involved in acute lung injury. However, whether PBEF participates in lung injury caused by cardiopulmonary bypass (CPB) is still unknown. This study aimed to investigate the effects of silencing PBEF on lung injury and the sodium and water transport system in rats receiving CPB.MethodsMorphological changes in lung tissues were evaluated using hematoxylin and eosin (H&E) staining. PBEF was detected using immunohistochemistry. The sodium and water transport system-related proteins and cellular signaling pathways were detected by Western blotting.ResultsRats receiving CPB (model group) had more severe alveolar wall damage and higher expression of PBEF in free form than the control rats. Western blotting showed that the expression of PBEF, surfactant protein D (SP), aquaporin (AQP) 1, AQP5, and epithelial sodium channel (ENaC) was significantly higher in the lung tissue of CPB rats than control rats. By contrast, adenovirus-encoding sh-PBEF significantly reduced the expression of PBEF, SP, AQP1, AQP5, and ENaC in the lung tissues of rats treated with CPB. The phosphorylation levels of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase B (AKT), and p38 mitogen-activated protein kinase (MAPK) were significantly increased in the lung tissue of rats that received CPB, and were downregulated by adenovirus-encoding sh-PBEF.ConclusionAdenovirus-encoding sh-PBEF could reduce lung injury and repair the sodium–water transport system in rats receiving CPB, likely through reducing MAPK, ERK1/2, and Akt signaling pathways.

Emodin alleviates severe acute pancreatitis-associated acute lung injury by decreasing pre-B-cell colony-enhancing factor expression and promoting polymorphonuclear neutrophil apoptosis.

The present study aimed to evaluate the protective effects of emodin on severe acute pancreatitis (SAP)-associated acute lung injury (ALI), and investigated the possible mechanism involved. SAP was induced in Sprague-Dawley rats by retrograde infusion of 5% sodium taurocholate (1 ml/kg), after which, rats were divided into various groups and were administered emodin, FK866 [a competitive inhibitor of pre-B-cell colony-enhancing factor (PBEF)] or dexamethasone (DEX). DEX was used as a positive control. Subsequently, PBEF expression was detected in polymorphonuclear neutrophils (PMNs) isolated from rat peripheral blood by reverse transcription-quantitative polymerase chain reaction and western blotting. In addition, histological alterations, apoptosis in lung/pancreatic tissues, apoptosis of peripheral blood PMNs and alterations in the expression of apoptosis-associated proteins were examined by hematoxylin and eosin staining, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assay, Annexin V/propidium iodide (PI) assay and western blotting, respectively. Serum amylase activity and wet/dry (W/D) weight ratios were also measured. An in vitro study was also conducted, in which PMNs were obtained from normal Sprague-Dawley rats and were incubated with emodin, FK866 or DEX in the presence of lipopolysaccharide (LPS). Apoptosis of PMNs and the expression levels of apoptosis-associated proteins were examined in cultured PMNs in vitro by Annexin V/PI assay and western blotting, respectively. The results demonstrated that emodin, FK866 and DEX significantly downregulated PBEF expression in peripheral blood PMNs. In addition, emodin, FK866 and DEX reduced serum amylase activity, decreased lung and pancreas W/D weight ratios, alleviated lung and pancreatic injuries, and promoted PMN apoptosis by regulating the expression of apoptosis-associated proteins: Fas, Fas ligand, B-cell lymphoma (Bcl)-2-associated X protein, cleaved caspase-3 and Bcl-extra-large. In addition, the in vitro study demonstrated that emodin, FK866 and DEX significantly reversed the LPS-induced decrease of apoptosis in PMNs by regulating the expression of apoptosis-associated proteins. In conclusion, the present study demonstrated that emodin may protect against SAP-associated ALI by decreasing PBEF expression, and promoting PMN apoptosis via the mitochondrial and death receptor apoptotic pathways.

Pre-B-cell colony enhancing factor expression and its clinical significance in colorectal cancer

Objective To explore the expression of pre-B-cell colony enhancing factor (PBEF) in colorectal cancer, and analyze the relationship between PBEF expression and the clinical pathological characte-ristics and its diagnostic and prognostic value. Methods Sixty-eight human colorectal tumor and normal colo-rectal tissues were collected, and the expression of PBEF was detected by immunohistochemical method. The relationship between the expression level of PBEF and the characteristics of tumors and prognosis was analyzed. Results The positive expression rate of PBEF in colorectal cancer tissues was 80.9%, higher than that in normal colorectal tissues, which was 29.4% (χ2=36.41, P 0.05), sex (χ2=0.39, P>0.05), histology type (χ2=0.54, P>0.05) or penetrating serosa (χ2=1.55, P>0.05), but significantly associated with the degree of differentiation (χ2=6.29, P 0.05). Conclusion The abnormal high expression of PBEF may be closely related to the occurrence and development of colorectal cancer. Key words: Colorectal neoplasms; Immunohistochemistry; Pre-B-cell colony enhancing factor

PBEF promotes the apoptosis of pulmonary microvascular endothelial cells and regulates the expression of inflammatory factors and AQP1 through the MAPK pathways.

Pre-B cell colony-enhancing factor (PBEF) has been shown to have a variety of biological functions. Studies have proven that PBEF plays a functional role in acute lung injury (ALI). Therefore, in this study, we aimed to confirm the importance of PBEF in ALI. The effects of PBEF overexpression on the apoptosis of human pulmonary microvascular endothelial cells (HPMECs) were analyzed by flow cytometry, and the results indicated that PBEF promoted the apoptosis of HPMECs, which aggravated the development of ALI. Comparative experiments involving increasing and decreasing PBEF expression demonstrated that PBEF promoted the expression of inflammatory factors, such as interleukin (IL)‑1β, IL‑6 and IL‑8 in the HPMECs , thus intensifying the inflammatory response. PBEF also inhibited the expression of aquaporin 1 (AQP1), which caused a dysfunction and imbalance in water transport. Moreover, we also found that tumor necrosis factor (TNF)‑α promoted the expression of PBEF in the HPMECs. After blocking the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways, we found that PBEF regulated the expression of inflammatory factors and AQP1, mainly through the MAPK pathways. Taken together, these results demonstrate that the increase in intracellular PBEF expression promoted the apoptosis of HPMECs and the expression of inflammatory factors and thus enhanced the inflammatory response and inhibited the expression of AQP1, which resulted in abnormal water transport, diminishing the regulatory effects of AQP1 on water transport.

Abstract 19769: Cardiac Injury and Stunning after Ischemia is Related to Loss of PBEF/Nampt Function and is Rescued by PTEN Inhibition

Introduction: Therapeutic hypothermia protection during cardiovascular resuscitation has been associated by us with preservation of heart tissue Pre-B-cell colony-enhancing factor (PBEF)/nicotinamide phosphoribosyltransferase (Nampt) level. This finding was associated with increased heart Akt activity that is negatively regulated by the phosphatase PTEN. We hypothesized that the PTEN inhibitor, VO-OHpic (VO), can improve heart cell survival and contractility via its role in energetic recovery following ischemia. Methods: Primary neonatal heart cells were exposed to 90 min ischemia and 180 min reperfusion to evaluate viability by propidium iodide uptake. Contractility was assessed in a stunning model of 30 min ischemia and 90 min reperfusion. VO (1μM) was given at reperfusion and PBEF/Nampt inhibitor FK866 (100 nM) was administered 1 h prior to ischemia. Akt phosphorylation and PBEF expression was detected by Western blot and ELISA assay, respectively. ATP content was measured by a fluorescent assay kit. Results: Compared to control cells, VO improved cell survival (56.3 ± 7.3 vs. 32.5 ± 7.6%, p < 0.05), a protective effect blocked by FK866. VO also rapidly improved contractile recovery following 30 min ischemia and decreased PBEF/Nampt release from heart cells, protective effects blocked by FK866. Improved contraction was related to increased Akt Thr308 and Ser473 phosphorylation and total ATP content. Conclusions: PTEN inhibition improves heart cell survival, ATP content and contractile recovery after ischemia. Protection is associated with attenuation of PBEF/Nampt cell release and is blocked by PBEF/Nampt inhibitors.

A key role for Pre–B cell colony–enhancing factor in experimental hepatitis

Pre-B cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase or visfatin, plays an important role in metabolic, inflammatory, and malignant diseases. Recent evidence suggests that blocking its enzymatic activity using a specific small-molecule inhibitor (FK866) might be beneficial in acute experimental inflammation. We investigated the role of PBEF in human liver disease and experimental hepatitis. PBEF serum levels and hepatic expression were determined in patients with chronic liver diseases. These studies were followed by in vivo experiments using concanavalin A (ConA) and D-galactosamine/lipopolysaccharide (LPS) models of experimental hepatitis. PBEF was either overexpressed by hydrodynamic perfusion or inhibited by FK866. In vivo findings were corroborated studying inflammatory responses of lentivirally PBEF-silenced or control FL83B mouse hepatocytes. Here, we demonstrate that PBEF serum levels were increased in patients with chronic liver diseases irrespective of disease stage and etiology. In particular, we observed enhanced PBEF expression in hepatocytes. Liver-targeted overexpression of PBEF rendered mice more susceptible to ConA- and D-galactosamine/LPS-induced hepatitis compared with control animals. In contrast, inhibition of PBEF using FK866 protected mice from ConA-induced liver damage and apoptosis. Administration of FK866 resulted in depletion of liver nicotinamide adenine dinucleotide+ levels and reduced proinflammatory cytokine expression. Additionally, FK866 protected mice in the D-galactosamine/LPS model of acute hepatitis. In vitro, PBEF-silenced mouse hepatocytes showed decreased responses after stimulation with LPS, lipoteichoic acid, and tumor necrosis factor α. In primary murine Kupffer cells, FK866 suppressed LPS-induced interleukin (IL)-6 production, whereas incubation with recombinant PBEF resulted in increased IL-6 release. Our data suggest that PBEF is of key importance in experimental hepatitis. Its specific inhibition might be considered a novel treatment option for inflammatory liver diseases.

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.

Novel role of Pre‐B‐Cell Colony Enhancing Factor (PBEF) in pulmonary arterial hypertension (PAH)

The discovery of novel mediators in PAH allows for novel insights into PAH pathobiology and identification of therapeutic targets. PBMC expression profiles from 7 PAH patients (49±7 years, mPAP 55±18 mmHg, PVR 14±11 U) and 10 healthy controls (45±7 years) identified 224 differentially expressed genes including PBEF as an upregulated gene in PAH. We previously identified PBEF as a mediator in lung injury, with extracellular proinflammatory cytokine‐like activity and intracellular enzymatic activity regulating NAD levels, cell redox state, histone deacetylases and inhibiting apoptosis. Plasma PBEF levels were higher in PAH patients (n=10, 46±17 years, mPAP 56±18 mmHg, PVR 10±4 U) than controls (n=9, 33±7 years), (5000±2000 vs. 7500 ± 3000 pg/ml; P=0.04) and trended with BNP levels (R=0.6, P=0.08) and inversely with 6‐minute walk distance (R=−0.57, P=0.08). Immunohistochemistry in explanted PAH lungs (n=3) revealed significant PBEF expression in plexiform lesions and in remodeled PAs, without PBEF staining in PAs of controls. Finally, compared to WT control mice, PBEF heterozygous mice (PBEF+/−) developed less severe PH (FIO2=10% and SU5416 for 3 weeks) with less significant elevations in RVSP (PBEF+/+ 19 ± 2 vs. PBEF+/− 12 ± 4 mmHg, P=0.03) and less RVH (RV/LV+S: PBEF+/+ 0.44 ± 0.05 vs. PBEF+/− 0.34 ± 0.08, P=0.06). Our results implicate PBEF as a novel biomarker and therapeutic target in PAH.

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-1beta 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-1beta-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-1beta-mediated cell permeability by 44% in A549 cells and 65% in endothelial cells. The knockdown of PBEF expression significantly inhibited IL-1beta-stimulated IL-8 secretion and mRNA level by 60% and 70%, respectively, and the knockdown of PBEF expression also significantly attenuated IL-1beta-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.

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.

Chronic Stretching of Amniotic Epithelial Cells Increases Pre-B Cell Colony-Enhancing Factor (PBEF/Visfatin) Expression and Protects Them from Apoptosis

In normal pregnancy, the fetal membranes become increasingly distended towards term and in multifetal gestations they become over-distended. Apoptosis of the amniotic epithelium increases with advancing gestation and may contribute to fetal membrane weakening and rupture. The effects of chronic static stretching for 36 h have been investigated using primary amniotic epithelial cells. Pre-B cell colony-enhancing factor (PBEF) is a stretch-responsive cytokine and expression of its gene, intracellular and secreted protein were all significantly increased by 4 h and its secretion sustained over 36 h, contrasting with the rapid increase and decline in expression of IL-8. Increased expression of SIRT1 and decreased p53 paralleled the changes in PBEF, are known to be responsive to PBEF, and contribute to cell survival. Distension had no effects on proliferation or necrosis but protected the cells from apoptosis, knocking-down PBEF with antisense probes abrogated this protective effect. There was increased immunostaining of PBEF in the compact layer of the amnion in multifetal tissues and significantly fewer apoptotic amniotic epithelial cells. These results show that chronic stretching of the amniotic epithelial cells increases PBEF expression, which protects them from apoptosis.

Pre-B cell colony-enhancing factor (PBEF)/visfatin: a novel mediator of innate immunity

Pre-B cell colony-enhancing factor (PBEF), also known as visfatin, is a highly conserved, 52-kDa protein found in living species from bacteria to humans. Originally a curiosity identified serendipitously in microarray studies but having no obvious functional importance, PBEF has now been shown to exert three distinct activities of central importance to cellular energetics and innate immunity. Within the cell, PBEF functions as a nicotinamide phosphoribosyl transferase, the rate-limiting step in a salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. By virtue of this role, it can regulate cellular levels of NAD and so impact not only cellular energetics but also NAD-dependent enzymes such as sirtuins. Although it lacks a signal peptide, PBEF is released by a variety of cells, and elevated levels can be found in the systemic circulation of patients with a variety of inflammatory diseases. As an extracellular cytokine, PBEF can induce the cellular expression of inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6. Finally, PBEF has been shown to be an adipokine expressed by fat cells that exerts a number of insulin mimetic and antagonistic effects. PBEF expression is up-regulated in a variety of acute and chronic inflammatory diseases including sepsis, acute lung injury, rheumatoid arthritis, inflammatory bowel disease, and myocardial infarction and plays a key role in the persistence of inflammation through its capacity to inhibit neutrophil apoptosis. This review summarizes the admittedly incomplete body of emerging knowledge about a remarkable new mediator of innate immunity.

Pre–B cell colony‐enhancing factor/visfatin, a new marker of inflammation in rheumatoid arthritis with proinflammatory and matrix‐degrading activities

To study possible mechanisms that mediate induction of the recently described adipocytokine pre-B cell colony-enhancing factor (PBEF) in joints of patients with rheumatoid arthritis (RA), and to analyze whether levels of PBEF correlate with disease severity and whether PBEF itself has the potential to act as a proinflammatory and destructive mediator in RA. RA synovial fibroblasts (RASFs) and monocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and recombinant human PBEF or were transfected with PBEF expression constructs or with PBEF-specific small interfering RNA. Production of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNFalpha) was measured by enzyme-linked immunosorbent assay, and expression of matrix metalloproteinases (MMPs) was assessed by real-time polymerase chain reaction. PBEF expression in synovial tissue, synovial fluid, serum, and SFs was assessed by immunohistochemistry, in situ hybridization, Western blotting, and enzyme immunoassays. In RASFs, PBEF was up-regulated by TLR ligands and cytokines that are characteristically present in the joints of patients with RA. In synovial tissue, RASFs were the major PBEF-expressing cells. A predominance of PBEF was found in the synovial lining layer and at sites of invasion into cartilage. Levels of PBEF in serum and synovial fluid correlated with the degree of inflammation and clinical disease activity. Moreover, PBEF itself activated the transcription factors NF-kB and activator protein 1 and induced IL-6, IL-8, MMP-1, and MMP-3 in RASFs as well as IL-6 and TNFalpha in monocytes. PBEF knockdown in RASFs significantly inhibited basal and TLR ligand-induced production of IL-6, IL-8, MMP-1, and MMP-3. Our findings establish PBEF as a proinflammatory and destructive mediator of joint inflammation in RA and identify PBEF as a potential therapeutic target.

Regulation of pre–B cell colony‐enhancing factor by STAT‐3–dependent interleukin‐6 trans‐signaling: Implications in the pathogenesis of rheumatoid arthritis

To determine whether interleukin-6 (IL-6) trans-signaling directs the expression of pre-B cell colony-enhancing factor (PBEF) in vitro and in vivo. Complementary DNA from rheumatoid arthritis (RA) synovial fibroblasts treated with IL-6 and soluble IL-6 receptor (sIL-6R) was used to probe a cytokine microarray. PBEF regulation by the IL-6-related cytokines, IL-6, sIL-6R, oncostatin M (OSM), IL-11, and leukemia inhibitory factor (LIF) was determined by reverse transcription-polymerase chain reaction analysis. IL-6-mediated STAT-3 regulation of PBEF was determined using a cell-permeable STAT-3 inhibitor peptide. Antigen-induced arthritis (AIA) was induced in wild-type (IL-6(+/+)) and IL-6-deficient (IL-6(-/-)) mice. PBEF and STAT were detected by immunohistochemistry, immunoblotting, and electrophoretic mobility shift assay. Synovial levels of PBEF were quantified by enzyme immunoassay. IL-6 trans-signaling regulated PBEF in a STAT-3-dependent manner. In addition, PBEF was regulated by the IL-6-related cytokine OSM, but not IL-11 or LIF. Flow cytometric analysis of the IL-6-related cognate receptors suggested that OSM regulates PBEF via its OSM receptor beta and not its LIF receptor. The involvement of PBEF in arthritis progression was confirmed in vivo, where induction of AIA resulted in a 4-fold increase in the synovial expression of PBEF. In contrast, little or no change was observed in IL-6(-/-) mice, in which the inflammatory infiltrate was markedly reduced and synovial STAT-1/3 activity was also impaired. Analysis of human RA synovial tissue confirmed that PBEF immunolocalized in apical synovial membrane cells, endothelial cells, adipocytes, and lymphoid aggregates. Synovial fluid levels of PBEF were significantly higher in RA patients than in osteoarthritis patients. Experiments presented herein demonstrate that PBEF is regulated via IL-6 trans-signaling and the IL-6-related cytokine OSM. PBEF is also actively expressed during arthritis. Although these data confirm an involvement of PBEF in disease progression, the consequence of its action remains to be determined.

Pre-B-cell Colony-enhancing Factor (PBEF/Visfatin) Gene Expression is Modulated by NF-κB and AP-1 in Human Amniotic Epithelial Cells

A localized intrauterine inflammatory response is often associated with the initiation of normal human parturition, whereas infection causes a similar but more florid response initiating preterm labor. Pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes and is up-regulated by labor, severe infection and inflammatory stimuli. The aim of this study was to determine the involvement of the transcription factors NF-κB and AP-1 in the response of PBEF to an inflammatory stimulus and compare it with IL-8. The results showed that this treatment of amniotic epithelial-like cells (WISH) and primary amniotic epithelial cells increased expression of PBEF and IL-8, but IL-8 responded 100-fold more than PBEF. IL-1β treatment together with a panel of NF-κB and AP-1 inhibitors demonstrated the involvement of these transcription factors in the up-regulation of PBEF. These data show that an inflammatory stimulus in the fetal membranes inducing NF-κB and AP-1 would up-regulate PBEF as well as IL-8.

Pre-B-Cell Colony Enhancing Factor (PBEF) Is a New Cytokine Regulating Myeloid Differentiation in Healthy Individuals and Patients with Severe Congenital Neutropenia (CN).

Pre-B-cell colony-enhancing factor (PBEF) was first isolated from blood lymphocytes and was found to be involved in the maturation of B-cell precursors. Recently, PBEF was described as cytosolic enzyme nicotinamide phosphoribosyltranserase (NAmPRTase), promoted NAD+ biosynthesis. In this study we tested the ability of PBEF to induce myelopoiesis. Primary CD34+ cells treated with 50 ng/ml of PBEF in the serum-free RPMI medium without any additional cytokines, led to differentiation into mature granulocytes and macrophages, as assessed by FACS analysis (expression of CD11b, CD14, CD16), and by morphology. The same results were observed after lentiviral transduction of CD34+ progenitors with PBEF-GFP reporter. PBEF-dependent maturation was accompanied by increased mRNA expression of hematopoietic transcription factors such as C/EBPα , C/EBPε , PU.1, and ELA2. Moreover, PBEF had synergistic effect on G-CSF-triggered myeloid differentiation of CD34+ cells. Interestingly, G-CSF-treated differentiated CD34+ cells produced increased amounts of PBEF mRNA, as well as of intracellular and secreted PBEF protein in a time-dependent manner, as measured by real-time RT-PCR, ELISA, FACS analysis, and confocal microscopy. Increased synthesis of PBEF was in line with high NAD+ levels in G-CSF-treated cells. Expression of PBEF mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR analysis of different myeloid precursor cells (myeloblasts, promyelocytes, myelocytes, metamyelocytes, mature granulocytes and monocytes) from bone marrow smears of healthy individuals. PBEF mRNA expression was continually increased during myeloid differentiation with highest levels in mature granulocytes and monocytes.In addition, we compared expression patterns of PBEF mRNA/protein in CD33+ progenitors and PMNs of healthy individuals after in vivo and in vitro treatment with G-CSF. We found PBEF mRNA/protein expression to be markedly increased in both cell types after G-CSF simulation (12 times for granulocytes, and 10 times for monocytes). Intriguingly, PBEF mRNA/protein expression in CD33+ cells and PMNs from G-CSF-treated patients with severe congenital neutropenia (CN) was also up-regulated and was significantly higher, as in G-CSF-treated healthy donors, suggesting that myeloid cells from CN patients try to overcome the differentiation arrest by upregulating PBEF. This was also supported by the fact that CN patients had at least 20 times higher PBEF plasma levels, in comparison to control group. G-CSF-dependent increase of PBEF expression was in line with increased NAD+ levels in G-CSF-treated cells. Indeed, in vitro PBEF treatment as well as lentiviral transduction of CD34+ cells from one CN patient with PBEF-GFP reporter gene resulted in partial restoration of myelopoiesis in vitro. Taken together, PBEF promotes maturation of myeloid cells by NAD+ dependent pathway. Moreover, increased expression of PBEF with subsequent up-regulation of NAD+ synthesis in CD33+ cells and neutrophils from CN patients could reveal mechanisms of G-CSF-dependent restoration of defective myelopoiesis in these patients.rPBEF protein and anti-PBEF antibody were provided by Amgen, USA

Pre–B-Cell Colony–Enhancing Factor Regulates NAD + -Dependent Protein Deacetylase Activity and Promotes Vascular Smooth Muscle Cell Maturation

Conversion of vascular smooth muscle cells (SMCs) from a proliferative state to a nonproliferative, contractile state confers vasomotor function to developing and remodeling blood vessels. Using a maturation-competent human SMC line, we determined that this shift in phenotype was accompanied by upregulation of pre-B-cell colony-enhancing factor (PBEF), a protein proposed to be a cytokine. Knockdown of endogenous PBEF increased SMC apoptosis and reduced the capacity of synthetic SMCs to mature to a contractile state. In keeping with these findings, human SMCs transduced with the PBEF gene had enhanced survival, an elongated bipolar morphology, and increased levels of h-caldesmon, smoothelin-A, smoothelin-B, and metavinculin. Notwithstanding some prior reports, PBEF did not have attributes of a cytokine but instead imparted the cell with increased nicotinamide phosphoribosyltransferase activity. Intracellular nicotinamide adenine dinucleotide (NAD+) content was increased in PBEF-overexpressing SMCs and decreased in PBEF-knockdown SMCs. Furthermore, NAD+-dependent protein deacetylase activity was found to be essential for SMC maturation and was increased by PBEF. Xenotransplantation of human SMCs into immunodeficient mice revealed an increased capacity for PBEF-overexpressing SMCs to mature and intimately invest nascent endothelial channels. This microvessel chimerism and maturation process was perturbed when SMC PBEF expression was lowered. These findings identify PBEF as a regulator of NAD+-dependent reactions in SMCs, reactions that promote, among other potential processes, the acquisition of a mature SMC phenotype.

Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes.

Pre-B-cell colony enhancing factor (PBEF) was first isolated from an activated peripheral blood lymphocyte cDNA library and was found to be involved in the maturation of B-cell precursors. It was subsequently identified as one of the genes upregulated by distending the human fetal membranes in vitro. Here we report on the genomic organization of this gene, which is composed of 11 exons and 10 introns, spanning 34.7 kb of genomic DNA. Neither the gene nor the protein has any homology with other cytokines in any currently available database. The use of two promoters (proximal and distal) may result in differential, tissue specific expression of the PBEF transcripts. The 5'-flanking region lacks the classical sequence motif that would place it with the hematopoietic cytokines; however, it has several putative regulatory elements, suggesting that this gene may be chemically and mechanically responsive to inducers of transcription. The three PBEF mRNA transcripts were observed in both normal and infected human fetal membranes but were significantly upregulated (P<0.05) in severe infection. The PBEF protein was immunolocalized, in both normal and infected tissues, to both the normal fetal cells of the amnion and chorion and the maternal decidua of the membranes, and to the invading neutrophils. These stained strongly and were likely to contribute to the increased expression in infection. The amniotic epithelial cell line (WISH cells) has been used as a model to study PBEF gene modulation. Lipopolysaccharide, interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha and IL-6 all significantly increased the expression of PBEF in 4 h of treatment. The addition of dexamethasone to IL-1beta and TNFalpha significantly reduced the response of PBEF to these cytokines. IL-8 treatment failed to alter PBEF gene expression. Thus PBEF is a cytokine expressed in the normal fetal membranes and upregulated when they are infected. It is likely to have a central role in the mechanism of infection-induced preterm birth.