Pigment epithelium-derived factor (PEDF) is an extracellular multifunctional protein belonging to the serpin superfamily with demonstrable neurotrophic, gliastatic, neuronotrophic, antiangiogenic, and antitumorigenic properties. We have previously provided biochemical evidence for high affinity PEDFbinding sites and proteins in plasma membranes of retina, retinoblastoma, and CNS cells. This study was designed to reveal a receptor involved in the biological activities of PEDF. Using a yeast two-hybrid screening, we identified a novel gene from pigment epithelium of the human retina that codes for a PEDFbinding partner, which we term PEDF-R. The derived polypeptide has putative transmembrane, intracellular and extracellular regions, and a phospholipase domain. Recently, PEDF-R (TTS-2.2/independent phospholipase A 2 (PLA 2 ) and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA 2 /nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) family that possesses triglyceride lipase and acylglycerol transacylase activities. Here we describe the PEDF-R gene expression in the retina and its heterologous expression by bacterial and eukaryotic systems, and we demonstrate that its protein product has specific and high binding affinity for PEDF, has a potent phospholipase A 2 activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A 2 activity of PEDF-R. In conclusion, we have identified a novel PEDF-R gene in the retina for a phospholipase-linked membrane protein with high affinity for PEDF, suggesting a molecular pathway by which ligand/receptor interaction on the cell surface could generate a cellular signal.
Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-γ, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-γ, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.
Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-jB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-a and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-jB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-jB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IjB kinase a/b complex and the expression of some NF-jB family members. Treatment of macrophages with the c-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-jB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-a and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-jB signaling.Key words: Macrophages . NF-kB . Notch IntroductionMacrophages are essential cells for the innate immune response. They discriminate between pathogens and self through signals triggered by TLR, which recognize different pathogens' components, such as LPS, lipoproteins, or dsRNA, among others [1]. Activation of most TLR on the macrophage surface triggers a complex signaling pathway, which involves NF-kB activation (reviewed in [2]). In the classical NF-kB pathway, a ternary IkB kinase (IKK) complex, formed by IKK-a, IKK-b, and NF-kB essential modulator, is responsible for inducing IkB phosphorylation, allowing the release of sequestered cytoplasmic NF-kB from IkB and its translocation to the nucleus. Once in the nucleus, NF-kB controls the expression of multiple genes implicated in the inflammatory response, including cytokines, effector enzymes such as iNOS and COX-2, and adhesion molecules [2].Notch proteins encompass a family of transmembrane receptors composed of an extracellular subunit linked to a transmembrane and intracellular subunit via heterodimerization domains [3]. Ligand binding induces proteolytic cleavage of the transmembrane and intracellular receptor subunit by several proteases, including g-secretase [4], allowing the release of the intracellular domain of Notch (NIC), which then translocates to the nucleus and converts the CBF1 factor from a repressor to a transcriptional activator. Some NIC target genes have been characterized, including basic-helix-loop-helix transcription factors belonging to 2556the hairy/enhancer of split (HES) gene family [3]. Although some CBF1-independent Notch signaling can occur, its mechanism of action is not well characterized yet [5].Notch signaling is an evolutionarily conserved pathway that controls different aspects of tissue development and homeostasis [6]. In cells o...
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