S. Patricia Becerra

Identification of a Lipase-linked Cell Membrane Receptor for Pigment Epithelium-derived Factor

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 PEDF-binding 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 PEDF-binding 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 A2 (PLA2)ζ and mouse desnutrin/ATGL) has been described in adipose cells as a member of the new calcium-independent PLA2/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 A2 activity that liberates fatty acids, and is associated with eukaryotic cell membranes. Most importantly, PEDF binding stimulates the enzymatic phospholipase A2 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.

Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture

Abstract: Pigment epithelium‐derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y‐79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose‐response relationship, with a larger effect in chemically defined medium than in serum‐containing medium [ED50 = 30 ng/ml (0.70 nM) in chemically defined medium and 100 ng/ml (2.3 nM) in serum‐containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme‐linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.

Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death.

We have shown previously that pigment epithelium‐derived factor (PEDF) acts as a survival factor for cerebellar granule cell neurons in culture, as well as protecting them against glutamate toxicity. In this study we have examined effects of PEDF on apoptotic cell death. We find that the granule cells die of apoptosis throughout the culture period, what we have termed “natural” apoptosis. PEDF prevents this natural apoptosis if added to immature cells, within the first 2 days in vitro (DIV), and the effect is maintained for up to DIV12. However, PEDF has no effect if added to mature cells at DIV5. Similar results are obtained when apoptosis is induced by shifting the cells from a serum‐ and 25 mM KCl‐containing medium to serum‐free medium with 5 mM KCl. PEDF most effectively blocks induced apoptosis in immature cells (DIV2) when added 24 hr prior to the change of medium, but still provides some protection when added simultaneously. However, 24 hr pretreatment with PEDF has a minimal effect when apoptosis is induced in mature DIV6 cells; addition at the same time is completely ineffective. Two polypeptide fragments of PEDF, only one of which contains the serine‐protease inhibitory site, are equally active, supporting previous results which suggest that the neurotrophic effects of PEDF are not mediated by protease inhibition. We conclude that PEDF protects immature but not mature granule cells against both natural and induced apoptosis. J. Neurosci. Res. 53:7–15, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Pigment epithelium-derived factor promotes the survival and differentiation of developing spinal motor neurons.

Pigment epithelium‐derived factor (PEDF) is a member of the serine protease inhibitor (serpin) superfamily that has been shown previously to promote the survival and/or differentiation of rat cerebellar granule neurons and human retinoblastoma cells in vitro. However, in contrast to most serpins, PEDF has no inhibitory activity against any known proteases, and its described biological activities do not appear to require the serpin‐reactive loop located toward the carboxy end of the polypeptide. Because another serpin, protease nexin‐1, has been shown to promote the in vivo survival and growth of motor neurons, the authors investigated the potential neurotrophic effects of PEDF on spinal cord motor neurons in highly enriched cultures and in vivo after injury. Here, it is shown that native bovine and recombinant human PEDF promoted the survival and differentiation (neurite outgrowth) of embryonic chick spinal cord motor neurons in vitro in a dose‐dependent manner. A truncated form of PEDF that lacks ≈62% of the carboxy end of the polypeptide comprising the homologous serpin‐reactive loop also exhibited neurotrophic activities similar to those of the full‐length protein. Furthermore, the data here showed that PEDF was transported retrogradely and prevented the death and atrophy of spinal motor neurons in the developing neonatal mouse after axotomy. These results indicate that PEDF exerts trophic effects on motor neurons, and, together with previous reports, these findings suggest that this protein may be useful as a pharmacologic agent to promote the development and maintenance of motor neurons. J. Comp. Neurol. 412:506–514, 1999. Published 1999 Wiley‐Liss, Inc.

Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity.

Abstract: Pigment epithelium‐derived factor (PEDF) is a survival factor for cerebellar granule cells in culture. In the present study, we have investigated the ability of a recombinant form of PEDF (rPEDF) to protect against glutamate neurotoxicity. When rPEDF was added to cerebellar granule cell cultures 30 min before addition of 100 µM glutamate, glutamate‐induced neuronal death was significantly reduced. The protective effect of rPEDF was dose‐dependent in the range from 0.023 to 7.0 nM (1–500 ng/ml), with a half‐maximal dose of 0.47 nM. An antibody to rPEDF blocked this protective effect. Measurement of intraneuronal free calcium levels demonstrated that rPEDF raised the basal calcium content. However, after the elevation of intracellular calcium in response to administration of glutamate, rPEDF reduced the plateau level seen in the presence of glutamate. These data show that PEDF can protect neurons against glutamate‐induced neurotoxicity, possibly via a calcium‐related pathway. The finding that only 30 min of preincubation is required for the neuroprotective effect, significantly faster than other known neurotrophic factors, suggests that PEDF may be useful clinically as a neuroprotective agent in the CNS.

The effects of PEDF on cancer biology: mechanisms of action and therapeutic potential

The potent actions of pigment epithelium-derived factor (PEDF) on tumour-associated cells, and its extracellular localization and secretion, stimulated research on this multifunctional serpin. Such studies have identified several PEDF receptors and downstream signalling pathways. Known cellular PEDF responses have expanded from the initial discovery that PEDF induces retinoblastoma cell differentiation to its anti-angiogenic, antitumorigenic and antimetastatic properties. Although the diversity of PEDF activities seems to be complex, they are consistent with the varied mechanisms that regulate this multimodal factor. If PEDF is to be used for cancer management, a deeper appreciation of its many functions and mechanisms of action is needed.

Structure-Function Studies on PEDF

Pigment epithelium-derived factor (PEDF) is a neurotrophic factor. In vitro it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule cell neurons. By virtue of amino acid sequence homology PEDF is a unique serpin member.

PIGMENT EPITHELIUM-DERIVED FACTOR (PEDF) HAS DIRECT EFFECTS ON THE METABOLISM AND PROLIFERATION OF MICROGLIA AND INDIRECT EFFECTS ON ASTROCYTES

Pigment epithelium‐derived factor (PEDF), a neurotrophic agent first identified in conditioned medium from cultured human retinal pigment epithelial cells, induces neuronal differentiation with neurite outgrowth in Y‐79 retinoblastoma cells and has a neurotrophic survival effect on cerebellar granule cells in culture. In the present study, we investigated the effects of human recombinant PEDF (rPEDF) on proliferation and activation of microglia and astrocytes isolated from newborn rat brain. rPEDF treatment caused microglia to round up morphologically, increased their metabolic activity (measured by both MTS conversion and acid phosphatase activity), but blocked proliferation (mitosis). This blocking effect could be demonstrated in cultures stimulated to proliferate by addition of granulocyte‐macrophage colony stimulating factor. The effect of rPEDF on microglial metabolic activity showed a dose–response relationship both in serum‐containing medium and in chemically defined medium and was blocked with anti‐PEDF antibody. rPEDF had no direct effect on the metabolic activity or proliferation of cultured astrocytes but blocked their proliferation in astrocyte‐microglia co‐cultures. Proliferation of isolated astrocytes was also blocked by conditioned medium from microglia treated with PEDF (PMCM). The effect of PMCM on astrocytes was not blocked by an antibody to transforming growth factor‐β. These results demonstrate that PEDF activates microglial metabolism while blocking proliferation and suggest that a soluble factor(s) released by rPEDF‐stimulated microglia blocks the proliferation of astrocytes. Thus, PEDF could play an important role in regulation of glial function and proliferation in the central nervous system. J. Neurosci. Res. 49:710–718, 1997. Published 1997 Wiley‐Liss, Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

Differentiation of Y79 retinoblastoma cells with pigment epithelial-derived factor and interphotoreceptor matrix wash: effects on tumorigenicity.

We investigated the in vivo differentiation potential of Y79 human retinoblastoma cells following pre-treatment with two novel neurotrophic agents: PEDF (human recombinant pigmented-epithelial derived factor) or IPM (interphotoreceptor matrix) wash. These agents were able to induce a significant degree of morphological differentiation in vitro. However, 48 days after subretinal transplantation of pre-treated cells, massive tumor formation was apparent. In contrast, Y79 cells pre-treated with retinoic acid/sodium butyrate, which attain a lesser degree of morphological differentiation, did not produce tumors over a 30 to 60 day-survival time (del Cerro et al., Brain Research, 12-22, 1992). We conclude that for PEDF and IPM, the degree of in vitro differentiation and the degree of mitotic arrest are independent features.

Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli

The ribonuclease H (RNase H) domain of human immune‐deficiency virus (HIV‐1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the λ pl promoter. The domain corresponds to residues 427‐560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion‐exchange and size exclusion chromatography. The purified protein appears to be in a native‐like homogeneous conformational state as determined by 1H‐NMR spectroscopy and circular dichroism measurements. HIV‐protease treatment of the RNase H domain resulted in cleavage between Phe‐440 and Tyr‐441.