Degeneration of neurons in Alzheimer's disease is mediated by ␤-amyloid peptide by diverse mechanisms, which include a putative apoptotic component stimulated by unidentified signaling events. This report describes a novel ␤-amyloid peptide-binding protein (denoted BBP) containing a G protein-coupling module. BBP is one member of a family of three proteins containing this conserved structure. The BBP subtype bound human ␤-amyloid peptide in vitro with high affinity and specificity. Expression of BBP in cell culture induced caspase-dependent vulnerability to ␤-amyloid peptide toxicity. Expression of a signaling-deficient dominant negative BBP mutant suppressed sensitivity of human Ntera-2 neurons to ␤-amyloid peptide mediated toxicity. These findings suggest that BBP is a target of neurotoxic ␤-amyloid peptide and provide new insight into the molecular pathophysiology of Alzheimer's disease.Genetic and biochemical data have coalesced to establish that ␤-amyloid peptide (A␤) 1 is a causative factor in neuron death and the consequent dimunition of cognitive abilities observed in Alzheimer's disease (1, 2). Plasma lipoproteins and their cell surface receptors influence sequestration and clearance of soluble A␤, contributing to the etiology of the disease (3-6). Inflammatory responses and oxidative damage also appear to contribute to the loss of neurons in Alzheimer's disease (7-10). Although the earliest cellular perturbations remain unclear, recent findings indicate that A␤ may act as an initiating factor in the death of neurons by inducing signaling pathways leading to apoptosis (11-17). However, the specific molecular target(s) transducing these A␤ effects has not been identified. The intracellular protein ERAB can bind A␤ in vitro, and neuroblastoma cells expressing recombinant ERAB undergo apoptosis when treated with exogenously added A␤ (18), but the mechanism by which ERAB may affect apoptotic signaling remains obscure. We identified a novel human ␤-amyloid peptide binding protein (BBP) utilizing yeast 2-hybrid technology. Analysis of the BBP amino acid sequence revealed the presence of a structural module related to that of the 7 transmembrane domain G protein-coupled receptor superfamily and known to be important in heterotrimeric G protein activation. Data suggest that BBP mediates cellular vulnerability to A␤ toxicity through a G protein-regulated program of cell death. Two related proteins (BLP1, BLP2; BBP-like proteins) were identified by sequence and structural similarities to BBP, but only the BBP subtype regulates a response to A␤. EXPERIMENTAL PROCEDURESYeast Two-hybrid Systems-Yeast 2-hybrid (Y2H) expression plasmids were constructed in the vectors pAS2 and pACT2 (19). Strain CY770 (20) served as host for Y2H assays. Sequences encoding A␤ 42 were amplified by PCR using primers incorporating restriction sites for subsequent ligation into pAS2, using a human APP (amyloid precursor protein) cDNA clone as template. A Y2H plasmid library consisting of cDNA fragments isolated from human fetal brain clone...

The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.

To further investigate the role of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue physiology, the production and regulation of PAI-1 was determined in primary cultures of human preadipocytes. When expressed as production per cell and cultured under identical conditions, human preadipocytes from both visceral (omental) and sc depots of lean and obese individuals released significant, yet similar, amounts of PAI-1 protein into the conditioned medium. High steady-state PAI-1 messenger RNA (mRNA) concentrations were observed in visceral and sc preadipocytes, with the relative level of expression equivalent to beta-actin mRNA. Tumor necrosis factor alpha significantly decreased PAI-1 production in a concentration-dependent manner in both visceral and sc cultures, whereas transforming growth factor beta significantly elevated PAI-1 production, but only in sc preadipocytes from obese individuals. Addition of insulin had no effect on antigen levels in conditioned medium of preadipocyte cultures. Stimulation of the preadipocyte cultures with a defined medium resulted in differentiation to the adipocyte phenotype, as determined by flow cytometric analysis, verifying the cultures as human preadipocyte. These studies are the first to observe significant PAI-1 mRNA expression and protein production in primary cultures of a human adipose tissue cellular component, and they suggest that nascent adipocytes contribute significantly to the elevated plasma PAI-1 observed in obesity.

One of the initial stages of adipogenesis is migration of preadipocytes of mesenchymal origin into cell clusters to form primitive fat organs. The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is synthesized and released from human adipose tissue ex vivo and regulates smooth muscle and endothelial cell migration in vitro, but its role in adipose tissue is not known. We investigated the role of PAI-1 in cultures of human preadipocytes from men and women of various ages and body mass indexes. Human preadipocytes expressed the messenger ribonucleic acid for PAI-1 and released significant quantities of PAI-1 protein into the medium. As PAI-1 regulates motility through the interaction of vitronectin with its receptor, the integrin alphaVbeta3, we identified this receptor in human preadipocytes. Flow cytometric analysis indicated that human preadipocytes express the vitronectin receptor alphaVbeta3 in a similar pattern as human umbilical vein endothelial cells. Functional studies indicated that active, but not latent, PAI-1 inhibited preadipocyte attachment to vitronectin with an IC(50) of 13.3 nmol/L, and preincubation of vitronectin-coated Transwells with active PAI-1 prevented preadipocyte migration. Vitronectin was identified in homogenates of the stromal-vascular fraction of human adipose tissue, but was absent from human adipocytes and cultured preadipocytes. These data indicate that human preadipocyte migration is regulated through the endogenous expression of PAI-1 and alphaVbeta3 integrin, a novel autocrine mechanism for potentially regulating cell cluster formation in adipogenesis.

BackgroundLow transient transfection efficiency limits the ability to characterize putative proapoptotic gene function in neurons. Laser scanning cytometry (LSC), with its high capacity, medium throughput means of collecting fluorescent emissions from cultured cells, offers an effective technology for scoring cell death in neuronal transfectants.MethodsCerebellar granule neurons (CGNs) were transfected with EGFP‐fusion constructs of Caspase‐3 and Caspase‐9 using a DNA‐calcium phosphate coprecipitation method. CGNs were fixed, permeablized, and stained with propidium iodide (PI) nuclear dye. An LSC method, based on a combination of Long Red Max Pixel, Long Red Integral, and Green Integral fluorescence parameters was validated for the scoring of apoptotic cell death in CGNs.ResultsIn Caspase‐3 and Caspase‐9 transfected CGNs, cell death was scored both in transfectants and nontransfected culture‐mates. The cell death phenotype was found to be independent of transfection efficiency. LSC scoring of Caspase‐9 transfectants was compared with visual scoring following Hoechst 33342 staining, yielding results that were similar qualitatively, but not quantitatively, likely owing to the greater sensitivity to green fluorescence of laser scanning compared to human vision.ConclusionLSC scoring of transiently transfected CGNs offers a rapid and reliable means of characterizing proapoptotic gene effects. © 2006 International Society for Analytical Cytology