Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, New York 10987
Received August 23, 2000; accepted July 27, 2001
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ABSTRACT |
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Key Words: lead toxicity; glial cells; thrombospondin-1; heparin sulfate 6-sulfotransferase; neuropilin-1; PbR11 cells; C6 cells.
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INTRODUCTION |
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In the developing brain, higher levels of lead affect many sites, including neurons, astroglia, and the microvasculature (reviewed in Chang, 1996). Young rodents exposed to lead exhibit abnormal development of the hippocampus and cerebral cortex, reduction in the number and diameter of axons in the optic nerve, and demyelinization of peripheral nerves (Rodier, 1995
). In rats exposed to lead during development, a number of changes are also seen in the cerebellum (Lorton and Anderson, 1986
). Lead damages the blood-brain barrier (BBB) in rats when given during the period of active microvessel growth. Rat pups become progressively resistant to lead-induced neurotoxic effects as they mature (Holtzman et al., 1982
).
Although neurons retain the ability to form new synapses throughout life, it is critical for the organism to develop proper circuitry during development. During this period, the radial glia provide a scaffold for the migrating neurons. The development of glial pathways in the cerebellum is critical for neuronal migration and synapse formation as well as microvessel formation (Laterra and Goldstein, 1991). A critical sequence of gene expressions is necessary for these events, and disruption of this sequence could result in permanent damage.
Once development is complete, the radial glia transform into various specialized cells, including astrocytes (astroglia), which make up a substantial proportion of the nervous system. The functions of astrocytes and their role as modulators in metal-induced neurotoxicity have been reviewed (Aschner and Kimelberg, 1996). Astroglia serve important functions in the nervous system, including control of neurotransmitter concentrations, signaling of neurons by release of neuroactive substances such as growth factors and cytokines, secreting components of the extracellular matrix, and interaction with non-neuronal cells, especially capillary epithelial cells. The coating of axons by sheets of glial tissue provides insulation and speeds up neurotransmission.
Astroglia in the brain remain intimately associated with both neurons and the capillary endothelial cells that make up the BBB. Development of the BBB begins in utero and is complete about 6 months after birth. Highly impermeable tight junctions between endothelial cells are responsible for the BBB, and evidence suggests that interactions between astrocytes and endothelial cells are responsible for specific differentiation of the endothelial cells in formation of the BBB (Janzer and Raff, 1987). Neural endothelial cells develop into tubelike capillary structures when cocultured with rat C6 glioma cells, a model for astrocytes. This process is inhibited by lead (Laterra et al., 1992
). Epidemiological studies also implicate lead in the pathogenesis of hypertension (Schwartz, 1995
).
Astroglia develop the ability to sequester lead into nonmitochondrial sites so as to protect not only themselves from toxic effects, but also the more sensitive neurons. This is the "lead sink" hypothesis (Tiffany-Castiglioni et al., 1989). Astroglia accumulate 24 times more lead than mature neural cells (Lindahl et al., 1999
). Both nuclear and cytoplasmic inclusions containing lead are seen in astroglia (Holtzman et al., 1982
). The ability of glial cells to sequester lead develops in response to their interaction with neural cells (Lindahl et al., 1999
).
It has been suggested that astrocytes possess the ability to adapt to lead by altering gene expression patterns (Opanashuk and Finkelstein, 1995). Here we report the identification of seven genes whose expressions are upregulated after long-term treatment with lead, a treatment that rendered these cells lead-resistant. It is clear that the changes in gene regulation occur not only in genes likely to confer resistance, but perhaps more importantly, also in genes whose expressions control important glial-induced processes during neural development.
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MATERIALS AND METHODS |
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The cells are grown as a monolayer in Ham's F10 medium containing 15% horse serum, 2.5% fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cytotoxicity is determined by colony formation. Exponentially growing cells are trypsinized, counted, and replated at 300 cells per 60-mm dish. Lead (or other test agent) is added to the medium immediately after attachment (three plates/dose), and remains in the medium throughout the incubation. Cells are incubated for 10 days without changing medium, fixed with methanol, and stained with Giemsa. The number of colonies is scored, and survival is defined as the fraction of the colony number in the treated group compared to the control group. Three independent experiments are performed, and the mean value and standard errors of survival percentages for each treated group are calculated.
Cloning of genes upregulated in PbR11 cells.
A subtracted library was generated using suppression subtractive hybridization (SSH). PolyA RNA (1.5 µg) from both wild-type C6 and PbR11 cells were used to make cDNA, using a FastTrack kit (Invitrogen, Carlsbad, CA). SSH was performed using a PCR-select cDNA subtraction kit (Clontech, Palo Alto, CA) according to the manufacturer's protocol. Forward (wild-type C6 cDNA as the driver and PbR11 cDNA as the tester) and reverse (PbR11 cDNA as the driver and wild-type C6 cDNA as the tester) SSHs were performed independently. Briefly, the tester and the driver cDNAs were digested with RsaI, and the tester cDNA was ligated to the adapter DNA. After first and second hybridizations with the tester and the driver cDNA, the resulting mixture was amplified by PCR using flanking and nested primers that anneal the adapter DNA to produce subtracted PCR fragments. PCR reactions were performed with the Advantage PCR system (Clontech, Palo Alto, CA). The cDNA fragments generated by PCR were subcloned into pT-Adv vector (Clontech, Palo Alto, CA) and transformed into TOP10F` super competent cells (Clontech) to establish a library of potentially subtracted cDNAs.
After removal of the adapter sequences from SSH-PCR products by RsaI digestion, the resulting PCR-amplified cDNA fragments were used to make the probes from forward and reverse subtraction, respectively. Randomly picked colonies from the SSH library were inoculated on duplicated nylon membranes and incubated on LB-agar supplemented with ampicillin (50 µg/ml) overnight at 37°C. Cells were lysed and DNA was denatured using 0.5 M NaOH, 1.5 M NaCl, then neutralized using 0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl. Hybridizations of this colony array were performed using digoxegenin (DIG)-labeled forward and reverse subtraction probes, respectively. Colonies that were positive with forward probe but negative with reverse probe were selected.
Plasmids were isolated using a High Pure Plasmid Isolation Kit (Boehringer Mannheim, Indianapolis, IN) from colonies identified by differential screening of the SSH library. DNA samples were denatured by heating in a boiling water bath for 5 min and chilling rapidly on ice and applied to nitrocellulose membrane using the S&S Minifold II slot blotter. Reverse Northern blot membranes were prehybridized in hybridization solution at 68°C for 2 h. Total cDNA from wild-type C6 and lead-resistant PbR11 cells were used to make probes for reverse Northern blot. Probes were labeled by random priming with DIG using the DIG Nucleic Acid Labeling kit (Boehringer Mannheim, Indianapolis, IN), heat denatured, added to the membranes, and allowed to hybridize at 68°C overnight. Membranes were washed at room temperature twice (5 min each) with low stringency (2 x SSC, 0.1% SDS) washing solution, followed by twice (15 min each) at 68°C with high-stringency (0.1 x SSC, 0.1% SDS) washing. Color reactions were performed using DIG nucleic acid detection kit (Boehringer Mannheim, Indianapolis, IN).
Virtual Northern blot.
Total RNA was isolated from wild-type and lead-resistant C6 cells using the TRI Reagent kit (Molecular Research Center, Inc., Cincinnati, Ohio). PCR-amplified cDNA was prepared using SMARTTM PCR cDNA synthesis kit (Clontech, Palo Alto, CA). Briefly, first-strand cDNA was synthesized from 1 µg total RNA using cDNA synthesis primer and SMART II oligonucleotide by SuperScriptTM II reverse transcriptase (Boehringer Mannheim, Indianapolis, IN). Double-strand cDNA was obtained by PCR using the first-strand cDNA as the template. Preliminary experiments were done to determine the optimal number of cycles for each sample so that the double-strand cDNA produced by PCR will remain in the exponential phase of amplification. Amplification was carried out using the parameters recommended by the kit in a GeneAmp PCR System 2400 DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT). Double-stranded cDNA (4 µg) was separated on a 1% agarose gel, then transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) in 10 x SSC buffer. The filter was hybridized with DIG-labeled probes made from cDNA fragments of genes of interest (data not shown). At this point, all cDNA fragments overexpressed in PbR11 cells were sequenced at New York University's DNA sequencing Center.
Northern blot.
Total RNA was isolated as above. mRNA was isolated from the total RNA samples using oligo(dT)-cellulose columns (Molecular Research Center, Inc., Cincinnati, Ohio). mRNA (5 µg) was separated on a 1% formaldehyde agarose gel, then transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) in 20 x SSC buffer. The membrane was hybridized in a High Efficiency Hybridization System (Molecular Research Center, Inc., Cincinnati, Ohio) with 32P-labeled probe generated by random priming using the High Prime Kit (Boehringer Mannheim, Indianapolis, IN). Gene-specific mRNA was detected using the STORM 860 PhosphorImager. Gene expression level is determined by calculating the ratio of band density of gene of interest divided by that of ß-actin. Results are expressed in arbitrary units by setting the value for the C6 cell control at 1.0. Northern blot data show mean ± SD of arbitrary units from three independent experiments.
Statistical analysis.
Statistical significance of differences in the toxicities of compounds toward different cell lines was determined by Student t-test. Statistical significance of differences in Northern blots between C6 cells and lead-treated C6 cells or lead-resistant PbR11 cells was also determined by Student t-test. Linear dose-response relationships were determined by a t-test that tests the null hypothesis that the slope of the regression line (of response vs. dose) is zero. Results were considered significant at the level p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
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RESULTS |
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NRP-1 is a cell-surface receptor which mediates cellcell adhesion in a Ca-independent manner. Ligands for NRP-1 belong to two families: members of the vascular epithelial growth factor (VEGF) family (Makinen et al., 1999; Migdal et al., 1998
; Tordjman et al., 1999
), and members of the semaphorin/collapsin family (Chedotal et al., 1998
; Kolodkin et al., 1997
; Takahashi et al., 1999
). VEGF proteins regulate angiogenesis, vasculogenesis, and vascular permeability (Soker et al., 1998
). In addition, binding of VEGF165 to NRP-1 on bone marrow stromal cells increases expression of two cytokines known to regulate early hematopoiesis (Tordjman et al., 1999
). The semaphorins are the largest family of repulsive axon guidance proteins. Those binding to NRP-1 repel sensory, sympathetic, and motor axons. Axon guidance in the hippocampus (a target of lead toxicity) is controlled by this type of chemorepulsion (Chedotal et al., 1998
).
In line with the role of NRP-1 as a receptor for both VEGF and semaphorin family members, overexpression of NRP-1 ectopically in transgenic mice is embryo lethal and results in severe abnormalities of the nervous system (defasciculation, disorganization, abnormal sprouting) as well as abnormalities of the cardiovascular system such as excess capillaries and blood vessels (Kitsukawa et al., 1995). Targeted disruption of the NRP-1 gene is also embryo lethal in mice with severe abnormalities of both nervous and cardiovascular system (Kitsukara et al., 1997
). Long-term exposure to lead during development results in abnormalities of the nervous and vascular systems. Misregulation of NRP-1 might be a mediator of these effects. We suggest that lead interferes with the ability of NRP-1 to bind one or more of its ligands. Such binding may be necessary for the growth or survival of C6 cells, which then become resistant to lead by upregulating NRP-1 expression. If so, then antisense expression of NRP-1 should limit C6 cell growth or survival. This is currently under investigation.
Heparin Sulfate 6-Sulfotransferase
Heparins are polysaccharides composed of glucuronic acid and N-acetylglucosamine residues, some of which become sulfated by either N-sulfotransferases or O-sulfotransferases. The heparin sulfate 6-sulfotransferase (HS6ST) enzyme adds a sulfate to oxygen 6 in N-acetylglucosamine. These sulfation modifications and others create many distinct fine structures in heparin chains. Heparin sulfate chains are covalently linked to a number of core proteins (syndean, glypican, or perlecan) that determine their localization at the cell surface or extracellular matrix. Vascular endothelial cells make perlecan, which localizes to the extracellular matrix (Aviezer et al., 1994).
Our sequence is 90% identical to the 3` untranslated region of the mouse HS6ST gene (accession AB024566) and gives a > 3-kb transcript on a Northern blot, consistent with the full-length mouse transcript size. The rat sequence has not been determined.
A number of growth factors have been found to bind to heparin sulfate, and the sulfations at various residues are essential for these functions (Kornblum et al., 1999). The binding of vascular epithelial growth factor-B(167) [VEGF-B(167)], to NRP-1 is mediated by its heparin-binding domain (Makinen et al., 1999
). The heparin sulfatebinding basic fibroblast growth factor (bFGF, also called FGF-2) has recently been found to play a role in neuronal differentiation (Okada-Ban et al., 2000
). The binding of bFGF to its receptor on endothelial cells is promoted by heparin sulfate. bFGF bound to heparin sulfate on the cell surface is protected from proteolytic degradation and maintains its ability to stimulate proliferation and plasminogen activator activity. Low concentrations of lead inhibit bFGF stimulation of endothelial cells as well as inhibiting the sulfation of heparin (Fujiwara and Kaji, 1999
; Kaji et al., 1995
). This suggests that the inhibition of endothelial cell proliferation by lead may be due to decreased ability of the cells to respond to bFGF due to loss of sulfated heparin. We suggest that HS6ST is a lead-sensitive enzyme, and that upregulation in lead-resistant cells may be a mechanism to overcome this inhibition. This assumes that glial cells also require heparin sulfate for effective growth factor responsiveness. This is now under investigation.
Thrombospondin 1
Thrombospondin 1 (TSP-1) is a 420-kDa extracellular matrix glycoprotein. The sequence we found is 94% identical to the mouse sequence (accession MN011580). The rat sequence has not been determined. Although it was first discovered in platelets, it is best known now as a tumor-suppressor gene and inhibitor of angiogenesis (Good et al., 1990). However, it is found in a variety of cells and has important roles in embryogenesis, wound repair, inflammation, and tumorigenesis (Enenstein et al., 1998
; Guo et al., 2000
; Scott-Drew and ffrench-Constant, 1997). The ability of TSP-1 to control various biological processes resides in its interactions with cell surface and extracellular matrix proteins. TSP-1 binds to integrins, nonintegrin receptors, and heparin sulfate proteoglycans with strong affinity and influences cell-to-cell and cell-to-matrix interactions, thus modulating cellular adhesion, migration, proliferation, and differentiation.
TSP-1 is widely expressed in the developing central nervous system, where it promotes neurite outgrowth (Scott-Drew and ffrench-Constant, 1997). It is present during postnatal development in tracts of myelinated axons. At this stage, the axons have generally reached their targets, but have not yet completed their myelination, which occurs by specialized oligodendrocyte glial cells. Glial cells make TSP-1, which enhances the migration and maturation of oligodendrocytes necessary for myelination.
TSP-1 acts as an immediate early-response gene. Its promoter region contains AP-1, AP-2, NFB, and ATF-1 and SRE sites (Salnikow et al., 1997
). It is upregulated by TGFß1 and platelet-derived growth factor (PDGF) and downregulated by bFGF and ATF-1. TSP-1 also activates TGFß (Murphy-Ullrich and Poczatek, 2000
). Nickel-transformed cells show a downregulation of TSP-1, which has been attributed to negative regulation by ATF-1 transcription factor (Salnikow et al., 1997
).
The binding of TSP-1 to heparin sulfate proteoglycans is of particular interest because loss of heparin sulfate at the cell surface or removal of the sulfate from heparin inhibits the binding of TSP-1 (Feitsma et al., 2000). The upregulation of HS6ST in lead-resistant cells concurrent with upregulation of TSP-1 might be a response to the inhibition by lead of HS6ST activity (see above) and its effect in downregulating TSP-1 transcription (Fig. 4
).
Heat Shock Protein 90
Our sequence is 95% identical with a heat shock protein 90 (HSP90) sequence found in rat brain (accession S45392). HSP90 is one of the most abundant proteins in the eukaryotic cell cytosol. It is a molecular chaperone, a protein needed to ensure proper protein folding of other proteins. However, unlike some other chaperones, HSP90 appears to be necessary for the proper folding of only a select subset of proteins, including some protein kinases, steroid hormone receptors, and telomerase (Buchner, 1999; Caplan, 1999
). HSP90 binds to the inactive conformation of these proteins prior to the final processing step (Buchner, 1999
). HSP90 does not act alone, but acts in concert with other cochaperones. It is essential in yeast and Drosophila, where homozygous mutants are lethal.
Agents that cause protein damage can divert HSP90 away from its usual targets toward other partially denatured proteins. In response to such stress, HSP90 is induced up to 10-fold (Buchner, 1999). In yeast, high concentrations of HSP90 are required for growth at elevated temperatures. High HSP90 is thought to prevent the aggregation or collapse of thermally or chemically damaged proteins by promoting their refolding. The increased expression of HSP90 in lead-resistant cells is almost certainly a major mechanism for their lead resistance. Lead is known to inhibit a number of enzymes via its affinity for sulfhydryl groups (Fishbein, 1998
) and can also inhibit binding of zinc finger transcription factors to DNA (Hanas et al., 1999
). HSP90 may be able to prevent further deleterious effects of proteins with bound lead.
Ubiquitin-like Activating Enzyme E1C
Ubiquitins are highly conserved small proteins that become covalently attached to other proteins in order to mark them for degradation by the 26S proteosome or to alter their activities. Ubiquitin-mediated proteolysis is an important pathway for the nonlysosomal degradation of proteins. The first step in the ubiquitination of proteins is the ATP-dependent activation of ubiquitin by a ubiquitin-activating enzyme (E1), leading to the formation of a thiol ester linkage between a glycine of ubiquitin and a cysteine residue on E1. This is followed by activating (E2) and ligating (E3) enzymes (reviewed in Hochstrasser, 1996).
Ubiquitin-like activating enzyme (UBA3) encodes an enzyme homologous to the C-terminal half of E1, but the enzyme forms a thiol ester linkage with the ubiquitin-like protein NEDD8 rather than with ubiquitin (Gong and Yeh, 1999). Our sequence is 91% identical with the mouse NEDD8 conjugating enzyme sequence (accession NM 011666). The rat sequence has not been determined. NEDD8 conjugates proteins with a specificity different from ubiquitin, being found on a limited number of mostly nuclear proteins. NEDD8-conjugates do not appear to be tagged for degradation. All members of the cullin family are substrates for NEDD8 conjugation (Hori et al., 1999
). Cullins take part in a multiprotein complex, SCF(Skp2), that acts as a ubiquitin ligase (E3) to ubiquinate the cell cycle inhibitor p27(Kip1) (Podust et al., 2000
), thus marking it for destruction. SCF(ß(TrCP)), which also contains an NEDD8-modified cullin, ubiquinates I
B
, the inhibitor of the transcription factor NF
B, resulting in NF
B activation (Read et al., 2000
). Another SCF ubiquitin ligase containing NEDD8-modified cullin is found in the centrosome and appears to control centrosome replication and separation (Freed et al., 1999
).
The upregulation of UBA3 in lead-resistant PbR11 cells might be in response to the ability of lead to block UBA3 activity. A thiol ester is formed between a cysteine residue on UBA3 and NEDD8, and it might be expected that lead can react with this cysteine, thereby blocking the reaction.
Rat Endogenous Retrovirus
We have found an overexpressed sequence in PbR11 that is 91% identical to a rat endogenous retrovirus (RERV; accession D90005). This sequence is expressed only in PbR11 cells, and not in C6 cells either constitutively or after lead treatment (Fig. 7). RERV expression was found to be upregulated in advanced rat prostatic tumors (Bussemakers et al., 1992
). Endogenous retroviruses are most likely the descendants of retroviruses that have been reverse-transcribed into DNA and then integrated into the host genome as a provirus. The 3` and 5` ends contain long terminal repeats (LTRs) containing promoter, enhancer, and repressor sequences. These sequences are potentially dangerous in that they can undergo recombination. Oncogenes from the host sometimes come under control of these elements, leading to their overexpression (cis-activation), with oncogenic consequences. For example, during carcinogen-induced rat mammary carcinogenesis, the 3` LTR of a retrovirus inserted into the c-Ha-ras gene and caused its overexpression (Bera et al., 1998
). In addition, insertional mutagenesis causing loss of function of tumor suppressor genes could occur during retrotransposition. There is still controversy over whether human endogenous retroviruses contribute to carcinogenesis or other pathology (see reviews by Fan, 1994
, and Lower, 1999
). It is of interest that some epidemiological evidence links lead exposure to increased risk of overall cancers as well as cancers of the stomach, lung, and bladder (Fu and Boffetta, 1995
).
2C9 (and c-fos)
One sequence overexpressed in PbR11 was 99% identical to a sequence called 2C9 (accession S74257), seen once before in a cDNA library constructed from rat cells transfected with v-fos (Hennigan et al., 1994). 2C9 has no known translation products, although a few small open reading frames can be found. It is expressed only in PbR11 cells and not in uninduced or lead-induced C6 cells (Fig. 7
). As it seemed that 2C9 is regulated by Fos, we performed a Northern blot on C6 and PbR11 cells, using a human c-fos probe, and found a 3-fold increase in fos mRNA abundance in PbR11 cells (data not shown). Low concentrations of lead increased c-fos expression in PC12 cells, a response most likely mediated by the ability of lead to activate protein kinase C (Kim et al., 2000
). However, higher concentrations of lead inhibit protein kinase C (Tomsig and Suszkiw, 1995
). It is possible that PbR11 cells have upregulated fos in order to overcome lead's inhibitory effect.
Fos is an immediate early gene that responds rapidly to stimuli such as growth factors and various stressors. There are four major members of the Fos family of proteins, each possessing a leucine zipper motif to enable dimerization with Jun family members to form transcription factor AP-1. The involvement of Fos in activated neurons is reviewed by Kovacs (1998). Long-term adaptation in the brain is thought to be mediated by long-term changes in transcription factors, especially of Fos family members (Nestler et al., 1999).
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DISCUSSION |
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NOTES |
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