©Copyright 1992 by The Humana Press Inc. All rights of any nature whatsoever reserved. 1044-739319211603-0303 $03.00
Characterization• GINELL RISTIC POST' AND GLYN DAWSON* 2,3 i Departments of Biochemistry and Molecular Biology, 2 Department of Pediatrics, and 3 Committee on Neurobiology, Joseph P. Kennedy Jr. Mental Retardation Research Center, University of Chicago, Chicago, IL 60637 Mailing address: Dr. Glyn Dawson, Department of Pediatrics, HM4068, University of Chicago, 5841 South Maryland Avenue, Chicago IL 60637 ,
Received September 20, 1991; Accepted November 8, 1991
ABSTRACT A novel clonal cell line derived from a human glioma (HOG) was found to express some oligodendrocyte-specific proteins including a 15-kDa form of myelin basic protein (MBP) and high 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Expression of the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide) was low. HOG cells did not express the characteristic astrocyte markers glial fibrillary acidic protein (GFAP) or significant glutamine synthetase (GS) activity. After initial plating, HOG cells were flat and epitheloid and thus showed a limited oligodendrocyte-like morphology. However, after cells became more confluent, some cells were phasebright and elaborated short processes. Receptor types expressed by HOG cells included A,-adenosine, prostaglandin E t (PGE,), and i3,adrenergic receptors (3-ARs) linked to stimulation of adenylate cyclase, and muscarinic cholinergic and H l -histamine coupled to phosphatidyinositol turnover (Post and Dawson, 1991). HOG cells should therefore provide a useful model for studying the extracellular regulation and phosphorylation of oligodendrocyte-specific proteins.
Index Entries: Muscarinic cholinergic receptor; histaminergic receptor; oligodendrocyte; glutamine synthetase; 2',3'-cyclic nucleotide 3'-phosphodiesterase; myelin basic protein.
*Author to whom all correspondence and reprint requests should be addressed. Molecular and Chemical Neuropathology
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Abbreviations: ,6-AR, ,(3-adrenergic receptor; CCH, chick cerebral hemispheres; CNPase, 2',3'-cyclic nucleotide 3'-phosphodiesterase; GFAP, glial fibrillary acidic protein, GS, glutamine synthetase; HOG, human oligodendroglioma; IBMX, 3-isobutyl-l-methylxanthine; InsP, inositol monophosphate, MBP, myelin basic protein, PGE I prostaglandin E l , PKC, protein kinase C, PLP proteolipid protein; TPA, 12-O-tetradecanoylphorbol-13-acetate. ,
INTRODUCTION Despite intense interest in the signals that initiate and control the assembly and maintenance of myelin, very little is known about the ability of oligodendrocytes to respond to myelinogenic signals (Vartanian et al., 1986, 1988). The responsiveness of these cells has been difficult to study owing to problems in isolating sufficient quantities of pure (i.e., astrocyte-free), metabolically active oligodendrocytes for receptor and second messenger studies. Previous studies of isolated cultures of oligodendrocytes (McCarthy and deVellis, 1980; Vartanian et al., 1986, 1988) and a mouse oligodendroglioma cell line (Vartanian et al., 1985) have demonstrated that oligodendrocytes express ,6-adrenergic and PGE 1 receptors coupled to activation of adenylate cyclase. Ritchie et al. (1987) reported that carbachol (a muscarinic cholinerigic agonist), bradykinin, histamine, and norepinephrine all increased 3 H]inositol phosphate accumulation in primary cultures of rat oligodendrocytes prelabeled with 3 H] inositol, but the increases were very modest. Using a video imaging system and the calcium indicator dye Fura-2AM, Castros et al. (1990) observed that histamine, norepinephrine, and ATP increased Caz+ influx in different populations of oligodendrocytes, indicating heterogeneous receptor expression in neonatal rat oligodendrocytes. Clonal cell lines in tissue culture provide a system in which a single cell can be subcultured through many generations to generate large quantities of homogeneous cells for detailed study and this has been successful for astrocytic cells lines (Pfieffer et al., 1977). Thus far it has not been possible to generate clonal cell lines derived from human oligodendrocytes and the only established clonal cell lines described that express any of the biochemical functions of oligodendrocytes are the mouse G26 oligodendroglioma cells (Dawson et al., 1977; Sundarraj et al., 1975) and rat C6 glioma cells (Benda et al., 1968). Subclones derived from the G26 glioma (Zimmerman, 1955) express astrocyte specific glial fibrillary protein (GFAP) (Bignami and Stoolmiller, 1979), the myelin lipids, galactosylceramide and sulfatide (Dawson et al., 1977), and CNPase activity (Seeds and Marks, 1979), but do not express MBP (Sundarraj et al., 1975). C6 Glioma cells are even less oligodendrocyte-like and exhibit differential glial specific enzymes with cell passage (Parker et al., 1980). Thus the activity of CNPase (an enzyme marker for oligodendro[
[
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cytes [Poduslo, 1975] ), is elevated in early cell passages, whereas late passage cells express high glutamine synthetase activity, an enzyme marker for astrocytes (Martinez-Hernandez et al., 1977). Rat C6 glioma cells also express the astrocytic marker GFAP (Bissell et al., 1975), and do not synthesize the myelin lipids galactosylceramide and sulfatide, or any forms of the myelin-associated proteins, MBP or proteolipid protein (PLP), making them of little value for studying the regulation of oligodendrocytespecific properties. We therefore biochemically characterized an oligodendroglioma-derived clonal cell line using glial specific markers and we identified cell surface membrane receptors, which activate the adenylate cyclase and inositol phosphate signaling pathways.
MATERIALS AND METHODS Matalon's modified Eagle's medium (EMS), Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were from Gibco (Grand Island, NY). Gentamicin solution, 3-isobutyl-l-methyl-xanthine (IBMX), forskolin, isoproterenol, antiserum to 3',5'-cyclic adenosine monophosphate (cAMP), adenosine 5' triphosphate (ATP), adenosine 2',3' cyclic monophosphate, alkaline phosphatase, phospho(enol) pyruvate (PEP), pyruvate kinase and rabbit antiglial fibrillary protein were from Sigma Chemical (St. Louis, MO). Goat antirabbit conjugated alkaline-phosphatase (AP) and AP color development reagents BCIP (5-bromo 4-chloro 3-indolyl phosphate p-toluidine salt) and NBT (p-nitro blue tetrazolium chloride) were purchased from BioRad (Richmond, CA). Dowex 1x8 (chloride form) was obtained from BioRad and converted to the formate form. ICI 118,551 and metroprolol were generous gifts from Dr. L. Seiden (University of Chicago). Antiserum to myelin basic protein was a gift from Dr. Anthony Campagnoni (University of California at Los Angeles). 3 H]myo-Inositol, and 125 1]2-0-succinyl cyclic AMP tyrosine methyl ester were purchased from Amersham (Arlington Heights, IL). [
[
Cell Culture The human oligodendroglioma cell line (HOG) was established from a surgically removed oligodendroglioma, subcloned and maintained by continuous cell culture, as described previously (Post and Dawson, 1991). HOG cells with passages between 4 and 20 were initially cultured on 100-mm Falcon plastic tissue culture dishes in Matalon's modified Eagle's medium (EMS) supplemented with 5% fetal calf serum (FCS) and gentamicin (6 mg/L), and maintained at 37°C in an atmosphere of 10% CO 2 /90% air. Cells were routinely passaged at d 5 in culture at an approximate density of 0.5 million cells per 100-mm dish. For CNPase and GS assays, experiments were performed in 100-mm plates on d 4 in culture. Karyo-
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type analysis confirmed the human origin of the cells although some chromosomes were deleted and several translocations were present. Mouse neuroblastomax 18-d Chinese hamster embryo hybrid cell line (NCB-20) was obtained from Dr. J. Minna (Veterans Administration Hospital, Washington DC) and grown as described previously (BerryKravis and Dawson, 1983). Primary rat oligodendrocyte- and astrocyte-enriched cultures were prepared from 4-d-old rat cerebral hemispheres by a modification of the method of McCarthy and deVellis (1980) as described previously (Kendler and Dawson, 1990).
Western Blot Analysis HOG cells, rat oligodendrocyte- and astrocyte-enriched cultures were mechanically harvested with a rubber policeman, pelleted at 600g for 10 min, then resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel buffer (Laemmli, 1970). Oligodendrocyte (80 µg), astrocyte (200 µg) and HOG cell proteins (200 µg) were separated on 9% acrylamide slab gels as described by Laemmli (1970). Following electrophoretic transfer of SDS-PAGE separated cellular proteins to nitrocellulose (Burnette, 1981), antigen was identified by an overnight incubation at room temperature with either rabbit anti-GFAP (1:25), rabbit antiMBP (1:200) or normal rabbit serum (1:500) in Tris buffered saline (TBS) containing 1% gelatin and 0.05% Tween-20, pH 7.5 (T-TBS). The nitrocellulose was washed in T-TBS then incubated for 2 h in goat antirabbit conjugated alkaline-phosphatase at a dilution of 1:3000. The alkaline phosphatase color development reagents BCIP and NBT were used to detect antigens bound to nitrocellulose (according to Biorad immunoblot alkaline phosphatase assay kit).
In Vitro Enzyme Assays CNPase activity was determined using the spectrophotometric method of Prohaska et al. (1973). A standard curve was prepared using known concentrations of 2'-AMP and used to calculate CNPase activity, which is expressed in µmol of 2'-AMP made from 2',3'-cAMP per min. Glutamine synthetase was determined using the method of Berl (1966). GS activity is expressed as µmol of -y-glutamylhydroxamate formed per 15 min. Synthetic y-glutamylhydroxamate was used for the standard curve.
Ligand Binding Assays HOG cells were harvested mechanically with a rubber policeman, pelleted at 600g for 10 min, then resuspended in ice-cold PBS, pH 7.4, containing 1 mM ascorbate and 1 mg/mL BSA to give a cell pellet protein content of 1.0 mg/mL as determined by the method of Lowry et al. (1951).
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Aliquots (700 1 L) of this whole cell preparation were then added to triplicate test tubes containing 100 NtL of 125 1]iodocyanopindolol (at final concentrations of 6-180 pM) and either 100 µL of phosphate buffered saline (PBS) or PBS with 5.5 nM ICI 118,551 or 50 mM metroprolol, (3 2 - and ,6,-AR specific antagonists, respectively. After incubation for 1 h at 26°C, 1.0 mL of ice-cold PBS was added to each test tube and the contents were filtered through a Brandel cell harvester (Gaithersburg, MD) onto Whatman GF/C filter papers. Filters were washed three times with 5.0 mL of ice-cold PBS and the bound radioactivity was quantitated on a gamma-counter. Specific binding was defined as the amount of radioligand retained by the filters in the absence of competing drug minus the amount of radioligand bound in the presence of ICI 118,551 or metroprolol. Data was analyzed using a nonlinear least squares fit program (Yamaoka et al., 1981). [
Measurement of Cyclic AMP Levels All drugs used were prepared in water or 50% ethanol and added in 1:100 dilution to cell cultures growing in Costar 35-mm tissue culture wells. 3-Isobutyl-l-methylxanthine (IBMX; 0.5 mM) was added to all samples. Following 30-min incubations at 37°C, the medium was aspirated, the reactions terminated by addition of 1 mL of ice-cold 5% TCA, and assayed for cAMP content by radioimmunoassay via competition with a 125 I-labeled cAMP derivative for binding to anti-.cAMP antisera with modifications to the method of Harper and Brooker (1975) as described previouisly (Berry-Kravis and Dawson, 1983).
RESULTS Shortly after human oligodendroglioma (HOG) cells adhered to plastic tissue culture dishes, they appeared flat and epithelioid. After a few days in culture, some cells became phase-bright and exhibited thin, short processes (Fig. 1). As shown in the inset of Fig. 2, HOG cells typically have a doubling time of approximately 2 d.
Western Blot Analysis Immunoblots of rat oligodendrocyte proteins (80 g!lane) revealed the 21.5 kDa, 18.5 kDa, 17 kDa, and 14 kDa forms of MBP (Barbarese et al., 1977), when probed with an antibody to the bovine 18.5 kDa form of MBP (Fig. 3, lane 5); but under identical conditions, blots of HOG cell lysates (200 tg protein), showed only a single band at 15 kDa (Fig. 3, lane 4). The band at 50 kDa is not believed to be MBP, but may represent actin (Fig. 3, lane 5). Immunoblots of neonatal rat astrocyte cell lysates revealed two mol-wt forms of GFAP bands at approximately 48 kDa and 55 kDa,
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Fig. 1. Human oligodendroglioma cells in culture. HOG cells were maintained by continuous cell culture as described in Materials and Methods. The photo shows cultured cells 4 d after passaging cells onto plastic tissue culture dishes. Magnification x200. which is typical for mammalian forms of GFAP (Shelanski and Liem, 1979) (Fig. 3, lane 3), but HOG cell lysates did not react with antibody to GFAP (Fig. 3, lane 2).
Biochemical Assays of Glial Specific Enzymes In HOG cells CNPase specific activity peaked at 4 d in culture and then actually declined for a few more days until it reached 42% of maximum (Fig. 2 and inset). However, CNPase specific activity in HOG cells was higher than the activity measured in neuronal cell hybrids (NCB-20 cells) at 4 d in culture (0.17 µmol/20 min%mg protein; data not shown). The amount of glutamine synthetase (GS) activity was determined in three different types of CNS-derived cells (Table 1). Chick cerebral hemispheres (CCH; from 18-d-old embryos) provided a source of astrocytes (GS positive cells) in which GS activity has been extensively characterized (Martinez-Hernandez et al., 1977; Sakellaridis et al., 1983) and these showed the highest amount of GS activity (1.16 µmol y-glutamylhydroxamic acid formed/15 min/mg protein) and HOG cells were even lower (0.08 to 0.14 µmol -y-glutamylhydroxamic acid formed/15 min/mg protein). Furthermore, there was not increase or induction of GS activity in HOG cells with days in culture, as shown previously for CNPase activity.
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0.6
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Fig. 2. CNPase activity and total protein present in HOG cells at various times in cultures. This figure shows CNPase activity and total protein present in HOG cells after various days in culture. CNPase activity was assayed in HOG cells after 2, 4, 6, and 8 d in culture and in NCB-20 cell homogenates (after 4 d of culture) as described in experimental procedures. A standard curve was prepared using known concentrations of 2' AMP and used to calculate enzyme activity, which is expressed as µmol 2' AMP formed; 20 min /dish (—O —) and as i mol 2' AMP formed/20 min/mg protein (--O --). The protein content was determined by the method of Lowry et al. (1951). The inset indicates the total protein content per 100-mm plate; the vertical axis is protein content in mg and the horizontal axis shows the corresponding culture day. The data shown are the means ± SEM of quadruplicate determinations performed in a single experiment. CNPase specific activity (expressed as µmol 2' AMP formed/20 min/dish) and total protein content in HOG cells increased with days in culture. When expressed as µmol 2' AMP formed/20 min/mg protein, CNPase specific activity decreased after 6 d in culture owing to an increase in total protein. Neuronal cell hybrids, NCB-20 cells, had little CNPase specific activity (0.17 mmol 2' AMP formed!20 min/mg protein) and served as a negative control (data not shown).
Receptor- Mediated Second Messenger Production in HOG Cells Isoproterenol, adenosine, and PGE 1 increased the production of cAIMP from ATP in the presence of IBMX (Table 2, Panel A). Thus the major functional receptors coupled through G s to adenylate cyclase expressed on HOG cells are r3-adrenergic, A 2 -adenosine and PGE I receptors, respectively. Direct stimulation of adenylate cyclase by forskolin in the
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a
Fig. 3. Western blot analysis of CNS .astrocytes and oligodendrocytes and HOG cells using antibodies to myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Following electrophoretic transfer of SDS-PAGE separated cellular proteins to nitrocellulose, antigen was identified after an overnight incubation with either rabbit anti-MBP (1:200 dilution), rabbit anti-GFAP (1:25 dilution), or normal rabbit serum, followed by AP-conjugated secondary antibody and color development. Nonspecific antibody binding was defined with normal rabbit serum, which reacted with HOG cell proteins; however, these bands do not coincide with the mol wt of MBP or GFAP proteins (lane 1) Prestained mol-wt markers, electrophoretically separated and transferred to nitrocellulose, indicate the approximate mol wt (lane 6). Eighty micrograms of total cell homogenate from cultured rat oligodendrocytes showed the typical pattern of four MBP bands at 14 kDa, 17.5 kDa, 18.5 kDa, and 21 kDa (lane 5), whereas HOG cells (200 µg total protein) showed only a single band at 15 kDa when incubated under identical conditions (lane 4). Cultured rat astrocytes expressed the typical 57, 66, and 78 KDa forms of GFAP, as detected with anti-GFAP antibody (lane 3), whereas identical treatment of HOG cell lysates did not result in any GFAP-positive bands (lane 2). presence of IBMX led to a 25-fold increase in intracellular cAMP (Table 2, panel B), but there was no evidence for inhibition of forskolin stimulatedcAMP levels by a 2 -adrenergic, µ or 8-opioid, A l -adenosine, D 2 dopamine, M 2 -cholinergic, or serotonin agonists. As shown in Fig. 4, cAMP production in HOG cells was stimulated by 6 2 -AR specific agonists (clenbuterol and albuterol), but not by the /3 1 specific agonist, prenaterol. These results indicate that the 3 2 -AR subtype is the major functional ,3-AR expressed on HOG cells. -
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Characterization of Human Oligodendroglioma Table 1 Glutamine Synthetase Activity in HOG and NCB-20 Cell Lines and in Chick Embryo Cerebral Hemispheres Cell type
Days in culture embryonic age
µmol y-glutamylhydroxamic acid formed 15 mm/mg protein
CCH HOG HOG HOG NCB-20
E18 C4 C6 C8 C4
1.16±0.02 0.08±0.05 0.14±0.02 0.14±0.03 0.20±0.01
HOG cells (plated at 0.5 million cellsi100-mm plate and harvested after 4, 6, and 8 d in culture). NCB -20 cells (harvested after 4 d in culture), and cerebral hemispheres of 18 -d-old chick embryos (CCH) are shown for comparison. Aliquots of cell homogenates were tested for GS activity as described in the text. The data shown are the means +_ SEM of quadruplicate determinations performed in a single experiment. CCHs contain neurons, astrocytes, and oligodendrocytes, and thus provided a source of astrocytes (GS positive cells). NCB -20 cells are neuronal cell hybrids, and as expected, they exhibited very low GS specific activity at 4 d in culture. HOG cells were found to have minimal GS activity when compared to both NCB -20s and astrocyte-containing CCHs. Furthermore, GS activity was not induced with time in culture, as seen with CNPase (Fig. 2).
We have previously shown that HOG cells express muscarinic cholinergic and histamine receptors linked to phosphoinositide turnover (Post and Dawson, 1991).
6 -Adrenergic Receptor Binding Studies Binding of 125 I]ICYP to f3,-ARs was determined in the presence of 5.5 nM ICI 118,551, a f3-AR antagonist that is 125 times more selective for f 2 ARs (Bilski et al., 1983) (Fig. 5: open circles). Similarly, binding to f 2 -ARs was assessed in the presence of 50 nM metroprolol, a 10-fold selective 13 1 -AR antagonist (Fig. 5; closed circles). The data was analyzed using a nonlinear least squares fit program (Yamaoka et al., 1981) to determine the following binding parameters ,3 2 -ARs had a Kai of 51 pM and B,n was 70 fmol/mg, whereas (3,-ARs had a K of 32 pM and Bm of only 4 fmol/mg. Results from binding analysis confirmed the previous observation that f3 2 -ARs are the major subtype of N -ARs expressed on HOG cells. [
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DISCUSSION Isolated oligodendrocytes can express many of the proteins and lipids associated with myelin membranes (Szuchet et al., 1986), yet it has been difficult to isolate large quantities of pure oligodendrocytes cultures for detailed biochemical studies. It is especially difficult to study cultured human oligodendrocytes because of the limited availability of viable human brain tissue. Single cell imaging of cultured oligodendrocytes offers some promise of resolving this problem, but these oligodendrocyte Molecular and Chemical Neuropathology
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Post and Dawson Table 2 Effect of Various Receptor Agonists on the Production of Intracellular Cyclic AMP in HOG Cells
Agonist
Conc (µ[M)
Basal (0.5 mM IBMX) Isoproterenol PGE I Chloroadenosine Dopamine Fenoldopam Serotonin NPY Neurotensin VIP DADLE U69593 Morphiceptin Histamine Carbachol
1 1 100 10 10 100 100 100 100 10 10 10 100 1000
FORSKOLIN and Clonidine LY171555 Chloroadenosine DADLE Morphiceptin Carbachol Histamine
1 10 10 10 10 10 10 10
A Receptor type ,61 /02- Adrenergic
PGE1 A2-Adenosine D1/D2-dopamine D1-Dopamine 5HT1-Serotonin NPY Neurotensin VIP S-opiate x-opiate µ-opiate H2 Histaminergic M2-Cholinergic
Gr
pmol cAMP/mg
G G5 Gs G,G; G, Gi G. G? G, G; G? G; GS G;
41± 2 1332±31 862±97 120± 7 40± 2 27± 1 48± 4 22 ± 11 33+ 4 49±12 33 + 2 32± 4 34± 1 53± 2 56± 1
Gf G; G; Gi
1037± 9 1914±36 1002±51 1170±50 1040± 9
;
B c 2 -Adrenergic D2-Dopamine Al-Adenosine 6-opiate µ-opiate M2-Cholinergic H2-Histaminergic
G;
974±73
G; G5
1115±50 1219±26
Some of the receptor types copuled to G s and/or G; that alter intracellular cAMP levels are shown in A. HOG cells were incubated for 30 min with specific adenylate cyclase-linked receptor agonists at the concentrations (in µNI) shown. Some of the receptor types that are coupled to Gi and thus inhibit the formation of cAMP are listed in B. HOG cells were preincubated for 10 min with and without the agonists (at 10 .M final concentration) listed, which are specific for those receptors coupled to G. Forskolin (1 µM) was added and the incubation continued for 30 min. The amount of cAMP generated was measured by RIA as described in the text. Data shown are the mean +SEM for each agonist. Each experiment was performed in triplicate and repeated twice_
cultures often comprise a heterogeneous population of cells (Castros et al., 1990). Clonal cell lines are an attractive alternative because large quantities of homogeneous cells can be generated and they offer the opportunity to study a metabolically active cell population. Two disadvantages of clonal cell lines are that they may represent immature "blast" cells that have not yet differentiated or tumor cells often have alterations in their
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0 400
200
-8
-7
-6
log [Agonist] (M)
Fig. 4. Effect of specific Pi and Pz adrenergic receptor agonists on the production of intracellular cyclic AMP in HOG cells. HOG cells were incubated for 30 min with one p 1 -AR specific agonist (prenaterol, *) and two different p 2-AR specific agonists (clenbuterol [0] and albuterol [ J) at the concentrations shown. Intracellular cAMP was measured by RIA as described in the text. Data shown are the means of duplicate assays performed on one experiment done in triplicate. The error bars indicate the SEM. Basal cAMP was 31.64 ± 6.89 pmol/mg protein. genome that correlate with a loss or diminution of at least some of the differentiated characteristics of oligodendrocytes. For example, rat C6 glioma cells have been used to study the differential expression of astrocyte-specific GS and oligodendrocyte-specific CNPase (Parker et al., 1980). In contrast, HOG cells express some of the proteins of oligodendrocytes, including a 15-kDA form of myelin basic protein and together with CNPase, but not astrocyte markers such as GFAP and GS. The finding of a low-mol-wt form of MBP was unexpected because, based on in vitro translation of human fetal spinal cord poly A mRNA (Roth et al., 1986), we would expect human oligodendrocytes to express three major MBP forms with approximate mol wts of 17 kDa, 18.5 kDa, and 21.5 kDa. However, human fetal spinal cord has recently been shown to express also a 15-kDa form of MBP, and in HOG cells a 2.6 kb mRNA can be transcribed into this 15-kDa MBP (Kashima et al., 1991). HOG cells provide a useful model system in which to study these myelin specific proteins and how their expression relates to activation of signal transduction pathways by specific neurotransmitters.
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50
40
e
30
20
to 0
0 0
30
90
60
120
150
180
125I-CYP (pM)
Fig. 5. Saturation binding of 125 I]cyanopindolol to HOG cells in the presence of f3-AR selective antagonists. Whole cells were incubated with 125 I]cyanopindolol 125 1-C1P) in the concentration range of 6-180 pM. The incubation mixture also contained either 5.5 nM ICI 118,551 (0) (125-fold more specific for ,6 2 - ARs and thus preferentially blocks binding to 0 2 -ARs or 50 nM metroprolol (e) (10-40-fold more specific for f3 1 -ARs and thus preferentially blocks binding to (3 1 -ARs. Specific binding was measured as described in the text. Data shown are the means of triplicate determinations in a single experiment, which was repeated twice. Binding parameters, Kd and B m , were determined from the aforementioned data by a nonlinear least squares fit program (Yamaoka et al., 1981): [
[
(
fie AR -
Kd = 50.9±7.5 pM B,,, = 69.6 ±3.8 fmol/mg (3 1 -AR Kd = 32.0±11.8 pM
HOG cells were found to express neurotransmitter receptors coupled to the activation of adenylate cyclase. For example, HOG cells express both Q-adrenergic and PGE1 receptors that have been previously identified in sheep oligodendrocytes prior to remyelination (Vartanian et al., 1988), in primary cultures of rat oligodendrocytes (McCarthy and deVellis, 1980), in a mouse oligodendroglioma cell line (Vartanian et al., 1985), and in primary cultures of astrocytes (Trimmer and McCarthy, 1986). However mouse oligodendroglioma cells express primarily the X3 1 -AR subtype, whereas HOG and 1321N1 human astrocytoma cells (Harden and McCarthy, 1982) express only (3 2 -ARs. HOG cells also express adenosine receptors linked to the stimulation of adenylate cyclase. These receptors have been identified in astrocytes (Van Calker and Hamprecht, 1980), but not in 1321N1 human astrocytoma cells. Neither HOG nor 1321N1 astrocytoma cells responded to the a-adrenergic and muscarinic cholinergic receptor Molecular and Chemical Neuropathology
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agonists or the peptide somatostatin, which have been shown to reduce cAMP levels in primary cultures of astrocytes (Evans et al., 1984; Van Calker and Hamprecht, 1980). HOG cells were found to express muscarinic and H1-histamine receptors coupled to the activation of PLC. These receptors may in turn activate PKC the major kinase involved in phosphorylation of two proteins thought to be essential to myelination, MBP and CNPase (Vartanian et al., 1986). Ritchie et al. (1987) also found evidence for muscarinic and histamine receptors in primary cultures of rat oligodendrocytes. We did not find evidence for functional bradykinin or a l -adrenergic receptors previously reported in rat oligodendrocytes (Ritchie et al., 1987) and primary glial cultures (Wilson et al., 1990). Muscarinic and histamine receptors have been identified in 1321N1 human astrocytoma cells (Nakahata and Harden, 1987), but their activation and desensitization profiles were markedly different (Post and Dawson, 1991), suggesting differences between oligodendrocytes and astrocytes in the regulation of signal transduction. In summary, we have presented evidence for a human cell line (HOG) that expresses at least two oligodendrocyte-specific proteins, CNPase and a 15 kDa form of MBP, as well as receptors linked to the activation of second messenger systems presumed to be important for the regulation of oligodendrocyte metabolism.
ACKNOWLEDGMENTS Supported by USPHS Grand HD-06426 and a Medical Science Training Grant Award (GNI-07281) to G. R. Post.
REFERENCES Barbarese E., Braun P. E., and Carson J. H. (1977) Identification of prelarge and presmall basic proteins in mouse myelin and their structural relationship to large and small basic proteins. Proc. Nat. Acad. Sci. USA 74, 3360-3364. Benda P., Lightbody J., Sato G., Levine L., and Sweet W. (1968) Differentiated rat glial cell strain in tissue culture. Science 161, 370-371. Berl S. (1966) Glutamine synthetase-determination of its distribution in brain during development. Biochemistry 5, 916-922. Berry-Kravis E. and Dawson G. (1983) Characterization of an adenylate cyclaselinked serotonin (5HT1) receptor in a neuroblastoma x brain explant hybrid cell line (NCB-20), J. Neurochem. 40, 977-985. Bignami A. and Stoolmiller A. C. (1979) Astroglia-specific protein (GFA) in clonal cell lines derived from the G26 mouse glioma. Brain Res. 163, 353-357. Bilski A. J., Halliday S. E., Fitzgerald J. D., and Wale J. L. (1983) The pharmacology of a,(32-selective adrenoceptor antagonist (ICI 118,551). J. Cardiovascular Pharm. 5, 430-437. Molecutarand Chemical Neuropathology
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Bissell M. G., Eng L. F., Herman M. M., Bensch K. G., and Miles L. E. M. (1975) Quantitative increase of neuroglia-specific GFA protein in rat C-6 glioma cells in vitro. Nature (London) 255, 633-634. Burnette W. N. (1981) "Western blotting": Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein. Anal. Biochem. 112, 195-203. Castros N. E., Levison S. W., and McCarthy K. D. (1990) Cultured oligodendroglia express receptors linked to calcium mobilization. Trans. Am. Soc. Neurochem. 21, 287 (abstr). Dawson G., Sundarraj N., and Pfeiffer S. E. (1977) Synthesis of myelin glycosphingolipids (galactosylceramide and galactosyl (3-0-sulfate) ceramide (sulfatide) by cloned cell lines derived from mouse and rat neurotumors. J. Biol. Chem. 252, 2777-2779. Evans T., McCarthy K. D., and Harden T. K. (1984) Regulation of cyclic AMP accumulation by peptide hormone receptors in immunocytochemically defined astroglial cells. J. Neurochem. 43, 131-138. Harper J. and Brooker G. (1975) Femtomole-sensitive RIA for cAMP and cAMP after 2'O-acetylation by acetic anhydride in aqueous solution. Cyclic. Nucl. Res. 1, 207-218. Harden T. K. and McCarthy K. D. (1982) Identification of the beta adrenergic receptor subtype on astroglia purified from rat brain. J. Pharmacol. Exp. Ther. 226, 600-605. Kashima T., Merrill J. E., Dawson G., and Campagnoni A. T. (1991) Molecular characterization of two incompletely differentiated human oligodendroglioma cell lines. Trans. Am. Soc. Neurochem. 22, 135 (abstract). Kendler, A. and Dawson G. (1990) Progressive hypoxia inhibits the de novo synthesis of galactosylceramide in cultured oligodendrocytes. J. Biol. Chem. 21, 12259-12266. Lowry D. H., Rosebrough J. G., Farr A. L., and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 93, 265-275. Martinez-Hernandez A, Bell K. P., and Norenberg M. D. (1977) Glutamine synthetase-glial localization in the brain. Science 195, 1365-1358. McCarthy K. D. and deVellis J. (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890-902. Nakahata N. and Harden T. K. (1987) Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H1-histamine receptors on human astrocytoma cells. Biochem. J. 241, 337-344. Parker K. K., Norenberg M. D., and Vernadakis A. (1980) "Transdifferentiation" of C6 glial cells in culture. Science 208, 179-181. Pfeiffer S. E., Betchart B., Cook J., Mancini P., and Morris R. (1977) Glial cell lines, in Cell, Tissue and Organ Cultures in Neurobiology (Federoff S. and Hertz L. eds.), pp. 287-346, Academic, New York. Poduslo S. E. (1975) The isolation and characterization of a plasma membrane and myelin fraction derived from oligodendroglia of calf brain. J. Neurochem. 24, 647-664.
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Post G. R. and Dawson G. (1991) Regulation of carbachol- and histamine-induced inositol phospholipid hydrolysis in a human oligodendroglioma. Glia, in press. Prohaska J. R., Clar D. A., and Wells W. W. (1973) Improved rapidity and precision in determination of brain 2', 3'-cyclic nucleotide 3'-phosphohydrolase. Anal. Biochem. 56, 275-282. Ritchie T., Cole R., Kim H.-S., deVellis J., and Noble E. P. (1987) Inositol phospholipid hydrolysis in cultured astrocytes and oligodendrocytes. Life. Sci. 41, 31-39. Roth H. J., Kronquist K., Prerorius P. J., Crandell B. F., and Campagnoni A. T. (1986) Isolation and characterization of a cDNA coding for a novel 17.3K myelin basic protein (MEP) variant. J. Neurosci. Res. 16, 227-238. Sakellaridis N., Bau D., Mangoura D., and Vernadakis A. (1983) Developmental profiles of glial enzymes in chick embryo: In vivo and in culture. Neurochem. Int. 5, 685-689. Seeds N. W. and Marks M. J. (1979) Sulfatide synthesis by neuronal cell lines. Dev. Neurosci. 2, 249-253. Shelanski M. L. and Liem R. K. H. (1979) Neurofilaments. J. Neurochem. 33, 5-13. Sundarraj N., Schachner M., and Pfeiffer S. E. (1975) Biochemically differentiated mouse glial lines carrying a nervous system specific surface antigen (NS-1). Proc. Nat. Acad. Sci. USA 72, 1927-1931. Szuchet S., Polak P. E., and Yim S. H. (1986) Mature oligodendrocytes cultured in the absence of neurons recapitulate the ontogenic development of myelin membranes. Dev. Neurosci. 8, 208-221. Trimmer P. A. and McCarthy K. D. (1986) Immunocytochemically defined astroglia from fetal, newborn, and young rats express ,3-adrenergic receptors in vitro. Dev. Brain Res. 27, 151 -165. Van Calker D. and Hamprecht B. (1980) Effects on neurohormones on glial cells, in Advances in Cellular Neurobiology, vol. 1 (Fedoroff S. and Hertz L. eds.), pp. 31-67, Academic, New York. Vartanian T., Szuchet S., Dawson G., and Campagnoni A. T. (1986) Oligodendrocyte adhesion activates protein kinase C- mediated phosphorylation of myelin basic protein. Science 234, 1395-1398. Vartanian T., Sprinkle T. J., Dawson G., and Szuchet S. (1988). Oligodendrocyte substratum adhesion modulates expression of adenylate cyclase-linked receptors. Proc. Natl. Acad. Sci. USA 85, 939-943. Vartanian T. K., Szuchet S., and Dawson G. (1985) Metabolic regulation in oligodendrocytes and oligodendrogliomas. Trans. Am. Soc. Neurochem. 16, 259 (abstr). Wilson K. M., Gilchrist S., and Minneman K. P. (1990) Comparison of a1-adrenergic receptor-stimulated inositol phosphate formation in primary neuronal and glial cultures. J. Neurochem. 55, 691-697. Yamaoka K., Tanigawara Y., Nakagawa T., and Uno T. (1981) A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmc. Dyn. 4, 879-885. Zimmerman H. M. (1955) The nature of gliomas as revealed by animal experimentation. Am. J. Pathol. 31, 1 -29.
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Characterization• GINELL RISTIC POST' AND GLYN DAWSON* 2,3 i Departments of Biochemistry and Molecular Biology, 2 Department of Pediatrics, and 3 Committee on Neurobiology, Joseph P. Kennedy Jr. Mental Retardation Research Center, University of Chicago, Chicago, IL 60637 Mailing address: Dr. Glyn Dawson, Department of Pediatrics, HM4068, University of Chicago, 5841 South Maryland Avenue, Chicago IL 60637 ,
Received September 20, 1991; Accepted November 8, 1991
ABSTRACT A novel clonal cell line derived from a human glioma (HOG) was found to express some oligodendrocyte-specific proteins including a 15-kDa form of myelin basic protein (MBP) and high 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity. Expression of the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide) was low. HOG cells did not express the characteristic astrocyte markers glial fibrillary acidic protein (GFAP) or significant glutamine synthetase (GS) activity. After initial plating, HOG cells were flat and epitheloid and thus showed a limited oligodendrocyte-like morphology. However, after cells became more confluent, some cells were phasebright and elaborated short processes. Receptor types expressed by HOG cells included A,-adenosine, prostaglandin E t (PGE,), and i3,adrenergic receptors (3-ARs) linked to stimulation of adenylate cyclase, and muscarinic cholinergic and H l -histamine coupled to phosphatidyinositol turnover (Post and Dawson, 1991). HOG cells should therefore provide a useful model for studying the extracellular regulation and phosphorylation of oligodendrocyte-specific proteins.
Index Entries: Muscarinic cholinergic receptor; histaminergic receptor; oligodendrocyte; glutamine synthetase; 2',3'-cyclic nucleotide 3'-phosphodiesterase; myelin basic protein.
*Author to whom all correspondence and reprint requests should be addressed. Molecular and Chemical Neuropathology
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Abbreviations: ,6-AR, ,(3-adrenergic receptor; CCH, chick cerebral hemispheres; CNPase, 2',3'-cyclic nucleotide 3'-phosphodiesterase; GFAP, glial fibrillary acidic protein, GS, glutamine synthetase; HOG, human oligodendroglioma; IBMX, 3-isobutyl-l-methylxanthine; InsP, inositol monophosphate, MBP, myelin basic protein, PGE I prostaglandin E l , PKC, protein kinase C, PLP proteolipid protein; TPA, 12-O-tetradecanoylphorbol-13-acetate. ,
INTRODUCTION Despite intense interest in the signals that initiate and control the assembly and maintenance of myelin, very little is known about the ability of oligodendrocytes to respond to myelinogenic signals (Vartanian et al., 1986, 1988). The responsiveness of these cells has been difficult to study owing to problems in isolating sufficient quantities of pure (i.e., astrocyte-free), metabolically active oligodendrocytes for receptor and second messenger studies. Previous studies of isolated cultures of oligodendrocytes (McCarthy and deVellis, 1980; Vartanian et al., 1986, 1988) and a mouse oligodendroglioma cell line (Vartanian et al., 1985) have demonstrated that oligodendrocytes express ,6-adrenergic and PGE 1 receptors coupled to activation of adenylate cyclase. Ritchie et al. (1987) reported that carbachol (a muscarinic cholinerigic agonist), bradykinin, histamine, and norepinephrine all increased 3 H]inositol phosphate accumulation in primary cultures of rat oligodendrocytes prelabeled with 3 H] inositol, but the increases were very modest. Using a video imaging system and the calcium indicator dye Fura-2AM, Castros et al. (1990) observed that histamine, norepinephrine, and ATP increased Caz+ influx in different populations of oligodendrocytes, indicating heterogeneous receptor expression in neonatal rat oligodendrocytes. Clonal cell lines in tissue culture provide a system in which a single cell can be subcultured through many generations to generate large quantities of homogeneous cells for detailed study and this has been successful for astrocytic cells lines (Pfieffer et al., 1977). Thus far it has not been possible to generate clonal cell lines derived from human oligodendrocytes and the only established clonal cell lines described that express any of the biochemical functions of oligodendrocytes are the mouse G26 oligodendroglioma cells (Dawson et al., 1977; Sundarraj et al., 1975) and rat C6 glioma cells (Benda et al., 1968). Subclones derived from the G26 glioma (Zimmerman, 1955) express astrocyte specific glial fibrillary protein (GFAP) (Bignami and Stoolmiller, 1979), the myelin lipids, galactosylceramide and sulfatide (Dawson et al., 1977), and CNPase activity (Seeds and Marks, 1979), but do not express MBP (Sundarraj et al., 1975). C6 Glioma cells are even less oligodendrocyte-like and exhibit differential glial specific enzymes with cell passage (Parker et al., 1980). Thus the activity of CNPase (an enzyme marker for oligodendro[
[
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cytes [Poduslo, 1975] ), is elevated in early cell passages, whereas late passage cells express high glutamine synthetase activity, an enzyme marker for astrocytes (Martinez-Hernandez et al., 1977). Rat C6 glioma cells also express the astrocytic marker GFAP (Bissell et al., 1975), and do not synthesize the myelin lipids galactosylceramide and sulfatide, or any forms of the myelin-associated proteins, MBP or proteolipid protein (PLP), making them of little value for studying the regulation of oligodendrocytespecific properties. We therefore biochemically characterized an oligodendroglioma-derived clonal cell line using glial specific markers and we identified cell surface membrane receptors, which activate the adenylate cyclase and inositol phosphate signaling pathways.
MATERIALS AND METHODS Matalon's modified Eagle's medium (EMS), Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum were from Gibco (Grand Island, NY). Gentamicin solution, 3-isobutyl-l-methyl-xanthine (IBMX), forskolin, isoproterenol, antiserum to 3',5'-cyclic adenosine monophosphate (cAMP), adenosine 5' triphosphate (ATP), adenosine 2',3' cyclic monophosphate, alkaline phosphatase, phospho(enol) pyruvate (PEP), pyruvate kinase and rabbit antiglial fibrillary protein were from Sigma Chemical (St. Louis, MO). Goat antirabbit conjugated alkaline-phosphatase (AP) and AP color development reagents BCIP (5-bromo 4-chloro 3-indolyl phosphate p-toluidine salt) and NBT (p-nitro blue tetrazolium chloride) were purchased from BioRad (Richmond, CA). Dowex 1x8 (chloride form) was obtained from BioRad and converted to the formate form. ICI 118,551 and metroprolol were generous gifts from Dr. L. Seiden (University of Chicago). Antiserum to myelin basic protein was a gift from Dr. Anthony Campagnoni (University of California at Los Angeles). 3 H]myo-Inositol, and 125 1]2-0-succinyl cyclic AMP tyrosine methyl ester were purchased from Amersham (Arlington Heights, IL). [
[
Cell Culture The human oligodendroglioma cell line (HOG) was established from a surgically removed oligodendroglioma, subcloned and maintained by continuous cell culture, as described previously (Post and Dawson, 1991). HOG cells with passages between 4 and 20 were initially cultured on 100-mm Falcon plastic tissue culture dishes in Matalon's modified Eagle's medium (EMS) supplemented with 5% fetal calf serum (FCS) and gentamicin (6 mg/L), and maintained at 37°C in an atmosphere of 10% CO 2 /90% air. Cells were routinely passaged at d 5 in culture at an approximate density of 0.5 million cells per 100-mm dish. For CNPase and GS assays, experiments were performed in 100-mm plates on d 4 in culture. Karyo-
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type analysis confirmed the human origin of the cells although some chromosomes were deleted and several translocations were present. Mouse neuroblastomax 18-d Chinese hamster embryo hybrid cell line (NCB-20) was obtained from Dr. J. Minna (Veterans Administration Hospital, Washington DC) and grown as described previously (BerryKravis and Dawson, 1983). Primary rat oligodendrocyte- and astrocyte-enriched cultures were prepared from 4-d-old rat cerebral hemispheres by a modification of the method of McCarthy and deVellis (1980) as described previously (Kendler and Dawson, 1990).
Western Blot Analysis HOG cells, rat oligodendrocyte- and astrocyte-enriched cultures were mechanically harvested with a rubber policeman, pelleted at 600g for 10 min, then resuspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel buffer (Laemmli, 1970). Oligodendrocyte (80 µg), astrocyte (200 µg) and HOG cell proteins (200 µg) were separated on 9% acrylamide slab gels as described by Laemmli (1970). Following electrophoretic transfer of SDS-PAGE separated cellular proteins to nitrocellulose (Burnette, 1981), antigen was identified by an overnight incubation at room temperature with either rabbit anti-GFAP (1:25), rabbit antiMBP (1:200) or normal rabbit serum (1:500) in Tris buffered saline (TBS) containing 1% gelatin and 0.05% Tween-20, pH 7.5 (T-TBS). The nitrocellulose was washed in T-TBS then incubated for 2 h in goat antirabbit conjugated alkaline-phosphatase at a dilution of 1:3000. The alkaline phosphatase color development reagents BCIP and NBT were used to detect antigens bound to nitrocellulose (according to Biorad immunoblot alkaline phosphatase assay kit).
In Vitro Enzyme Assays CNPase activity was determined using the spectrophotometric method of Prohaska et al. (1973). A standard curve was prepared using known concentrations of 2'-AMP and used to calculate CNPase activity, which is expressed in µmol of 2'-AMP made from 2',3'-cAMP per min. Glutamine synthetase was determined using the method of Berl (1966). GS activity is expressed as µmol of -y-glutamylhydroxamate formed per 15 min. Synthetic y-glutamylhydroxamate was used for the standard curve.
Ligand Binding Assays HOG cells were harvested mechanically with a rubber policeman, pelleted at 600g for 10 min, then resuspended in ice-cold PBS, pH 7.4, containing 1 mM ascorbate and 1 mg/mL BSA to give a cell pellet protein content of 1.0 mg/mL as determined by the method of Lowry et al. (1951).
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Aliquots (700 1 L) of this whole cell preparation were then added to triplicate test tubes containing 100 NtL of 125 1]iodocyanopindolol (at final concentrations of 6-180 pM) and either 100 µL of phosphate buffered saline (PBS) or PBS with 5.5 nM ICI 118,551 or 50 mM metroprolol, (3 2 - and ,6,-AR specific antagonists, respectively. After incubation for 1 h at 26°C, 1.0 mL of ice-cold PBS was added to each test tube and the contents were filtered through a Brandel cell harvester (Gaithersburg, MD) onto Whatman GF/C filter papers. Filters were washed three times with 5.0 mL of ice-cold PBS and the bound radioactivity was quantitated on a gamma-counter. Specific binding was defined as the amount of radioligand retained by the filters in the absence of competing drug minus the amount of radioligand bound in the presence of ICI 118,551 or metroprolol. Data was analyzed using a nonlinear least squares fit program (Yamaoka et al., 1981). [
Measurement of Cyclic AMP Levels All drugs used were prepared in water or 50% ethanol and added in 1:100 dilution to cell cultures growing in Costar 35-mm tissue culture wells. 3-Isobutyl-l-methylxanthine (IBMX; 0.5 mM) was added to all samples. Following 30-min incubations at 37°C, the medium was aspirated, the reactions terminated by addition of 1 mL of ice-cold 5% TCA, and assayed for cAMP content by radioimmunoassay via competition with a 125 I-labeled cAMP derivative for binding to anti-.cAMP antisera with modifications to the method of Harper and Brooker (1975) as described previouisly (Berry-Kravis and Dawson, 1983).
RESULTS Shortly after human oligodendroglioma (HOG) cells adhered to plastic tissue culture dishes, they appeared flat and epithelioid. After a few days in culture, some cells became phase-bright and exhibited thin, short processes (Fig. 1). As shown in the inset of Fig. 2, HOG cells typically have a doubling time of approximately 2 d.
Western Blot Analysis Immunoblots of rat oligodendrocyte proteins (80 g!lane) revealed the 21.5 kDa, 18.5 kDa, 17 kDa, and 14 kDa forms of MBP (Barbarese et al., 1977), when probed with an antibody to the bovine 18.5 kDa form of MBP (Fig. 3, lane 5); but under identical conditions, blots of HOG cell lysates (200 tg protein), showed only a single band at 15 kDa (Fig. 3, lane 4). The band at 50 kDa is not believed to be MBP, but may represent actin (Fig. 3, lane 5). Immunoblots of neonatal rat astrocyte cell lysates revealed two mol-wt forms of GFAP bands at approximately 48 kDa and 55 kDa,
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Fig. 1. Human oligodendroglioma cells in culture. HOG cells were maintained by continuous cell culture as described in Materials and Methods. The photo shows cultured cells 4 d after passaging cells onto plastic tissue culture dishes. Magnification x200. which is typical for mammalian forms of GFAP (Shelanski and Liem, 1979) (Fig. 3, lane 3), but HOG cell lysates did not react with antibody to GFAP (Fig. 3, lane 2).
Biochemical Assays of Glial Specific Enzymes In HOG cells CNPase specific activity peaked at 4 d in culture and then actually declined for a few more days until it reached 42% of maximum (Fig. 2 and inset). However, CNPase specific activity in HOG cells was higher than the activity measured in neuronal cell hybrids (NCB-20 cells) at 4 d in culture (0.17 µmol/20 min%mg protein; data not shown). The amount of glutamine synthetase (GS) activity was determined in three different types of CNS-derived cells (Table 1). Chick cerebral hemispheres (CCH; from 18-d-old embryos) provided a source of astrocytes (GS positive cells) in which GS activity has been extensively characterized (Martinez-Hernandez et al., 1977; Sakellaridis et al., 1983) and these showed the highest amount of GS activity (1.16 µmol y-glutamylhydroxamic acid formed/15 min/mg protein) and HOG cells were even lower (0.08 to 0.14 µmol -y-glutamylhydroxamic acid formed/15 min/mg protein). Furthermore, there was not increase or induction of GS activity in HOG cells with days in culture, as shown previously for CNPase activity.
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Fig. 2. CNPase activity and total protein present in HOG cells at various times in cultures. This figure shows CNPase activity and total protein present in HOG cells after various days in culture. CNPase activity was assayed in HOG cells after 2, 4, 6, and 8 d in culture and in NCB-20 cell homogenates (after 4 d of culture) as described in experimental procedures. A standard curve was prepared using known concentrations of 2' AMP and used to calculate enzyme activity, which is expressed as µmol 2' AMP formed; 20 min /dish (—O —) and as i mol 2' AMP formed/20 min/mg protein (--O --). The protein content was determined by the method of Lowry et al. (1951). The inset indicates the total protein content per 100-mm plate; the vertical axis is protein content in mg and the horizontal axis shows the corresponding culture day. The data shown are the means ± SEM of quadruplicate determinations performed in a single experiment. CNPase specific activity (expressed as µmol 2' AMP formed/20 min/dish) and total protein content in HOG cells increased with days in culture. When expressed as µmol 2' AMP formed/20 min/mg protein, CNPase specific activity decreased after 6 d in culture owing to an increase in total protein. Neuronal cell hybrids, NCB-20 cells, had little CNPase specific activity (0.17 mmol 2' AMP formed!20 min/mg protein) and served as a negative control (data not shown).
Receptor- Mediated Second Messenger Production in HOG Cells Isoproterenol, adenosine, and PGE 1 increased the production of cAIMP from ATP in the presence of IBMX (Table 2, Panel A). Thus the major functional receptors coupled through G s to adenylate cyclase expressed on HOG cells are r3-adrenergic, A 2 -adenosine and PGE I receptors, respectively. Direct stimulation of adenylate cyclase by forskolin in the
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a
Fig. 3. Western blot analysis of CNS .astrocytes and oligodendrocytes and HOG cells using antibodies to myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). Following electrophoretic transfer of SDS-PAGE separated cellular proteins to nitrocellulose, antigen was identified after an overnight incubation with either rabbit anti-MBP (1:200 dilution), rabbit anti-GFAP (1:25 dilution), or normal rabbit serum, followed by AP-conjugated secondary antibody and color development. Nonspecific antibody binding was defined with normal rabbit serum, which reacted with HOG cell proteins; however, these bands do not coincide with the mol wt of MBP or GFAP proteins (lane 1) Prestained mol-wt markers, electrophoretically separated and transferred to nitrocellulose, indicate the approximate mol wt (lane 6). Eighty micrograms of total cell homogenate from cultured rat oligodendrocytes showed the typical pattern of four MBP bands at 14 kDa, 17.5 kDa, 18.5 kDa, and 21 kDa (lane 5), whereas HOG cells (200 µg total protein) showed only a single band at 15 kDa when incubated under identical conditions (lane 4). Cultured rat astrocytes expressed the typical 57, 66, and 78 KDa forms of GFAP, as detected with anti-GFAP antibody (lane 3), whereas identical treatment of HOG cell lysates did not result in any GFAP-positive bands (lane 2). presence of IBMX led to a 25-fold increase in intracellular cAMP (Table 2, panel B), but there was no evidence for inhibition of forskolin stimulatedcAMP levels by a 2 -adrenergic, µ or 8-opioid, A l -adenosine, D 2 dopamine, M 2 -cholinergic, or serotonin agonists. As shown in Fig. 4, cAMP production in HOG cells was stimulated by 6 2 -AR specific agonists (clenbuterol and albuterol), but not by the /3 1 specific agonist, prenaterol. These results indicate that the 3 2 -AR subtype is the major functional ,3-AR expressed on HOG cells. -
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Characterization of Human Oligodendroglioma Table 1 Glutamine Synthetase Activity in HOG and NCB-20 Cell Lines and in Chick Embryo Cerebral Hemispheres Cell type
Days in culture embryonic age
µmol y-glutamylhydroxamic acid formed 15 mm/mg protein
CCH HOG HOG HOG NCB-20
E18 C4 C6 C8 C4
1.16±0.02 0.08±0.05 0.14±0.02 0.14±0.03 0.20±0.01
HOG cells (plated at 0.5 million cellsi100-mm plate and harvested after 4, 6, and 8 d in culture). NCB -20 cells (harvested after 4 d in culture), and cerebral hemispheres of 18 -d-old chick embryos (CCH) are shown for comparison. Aliquots of cell homogenates were tested for GS activity as described in the text. The data shown are the means +_ SEM of quadruplicate determinations performed in a single experiment. CCHs contain neurons, astrocytes, and oligodendrocytes, and thus provided a source of astrocytes (GS positive cells). NCB -20 cells are neuronal cell hybrids, and as expected, they exhibited very low GS specific activity at 4 d in culture. HOG cells were found to have minimal GS activity when compared to both NCB -20s and astrocyte-containing CCHs. Furthermore, GS activity was not induced with time in culture, as seen with CNPase (Fig. 2).
We have previously shown that HOG cells express muscarinic cholinergic and histamine receptors linked to phosphoinositide turnover (Post and Dawson, 1991).
6 -Adrenergic Receptor Binding Studies Binding of 125 I]ICYP to f3,-ARs was determined in the presence of 5.5 nM ICI 118,551, a f3-AR antagonist that is 125 times more selective for f 2 ARs (Bilski et al., 1983) (Fig. 5: open circles). Similarly, binding to f 2 -ARs was assessed in the presence of 50 nM metroprolol, a 10-fold selective 13 1 -AR antagonist (Fig. 5; closed circles). The data was analyzed using a nonlinear least squares fit program (Yamaoka et al., 1981) to determine the following binding parameters ,3 2 -ARs had a Kai of 51 pM and B,n was 70 fmol/mg, whereas (3,-ARs had a K of 32 pM and Bm of only 4 fmol/mg. Results from binding analysis confirmed the previous observation that f3 2 -ARs are the major subtype of N -ARs expressed on HOG cells. [
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DISCUSSION Isolated oligodendrocytes can express many of the proteins and lipids associated with myelin membranes (Szuchet et al., 1986), yet it has been difficult to isolate large quantities of pure oligodendrocytes cultures for detailed biochemical studies. It is especially difficult to study cultured human oligodendrocytes because of the limited availability of viable human brain tissue. Single cell imaging of cultured oligodendrocytes offers some promise of resolving this problem, but these oligodendrocyte Molecular and Chemical Neuropathology
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Post and Dawson Table 2 Effect of Various Receptor Agonists on the Production of Intracellular Cyclic AMP in HOG Cells
Agonist
Conc (µ[M)
Basal (0.5 mM IBMX) Isoproterenol PGE I Chloroadenosine Dopamine Fenoldopam Serotonin NPY Neurotensin VIP DADLE U69593 Morphiceptin Histamine Carbachol
1 1 100 10 10 100 100 100 100 10 10 10 100 1000
FORSKOLIN and Clonidine LY171555 Chloroadenosine DADLE Morphiceptin Carbachol Histamine
1 10 10 10 10 10 10 10
A Receptor type ,61 /02- Adrenergic
PGE1 A2-Adenosine D1/D2-dopamine D1-Dopamine 5HT1-Serotonin NPY Neurotensin VIP S-opiate x-opiate µ-opiate H2 Histaminergic M2-Cholinergic
Gr
pmol cAMP/mg
G G5 Gs G,G; G, Gi G. G? G, G; G? G; GS G;
41± 2 1332±31 862±97 120± 7 40± 2 27± 1 48± 4 22 ± 11 33+ 4 49±12 33 + 2 32± 4 34± 1 53± 2 56± 1
Gf G; G; Gi
1037± 9 1914±36 1002±51 1170±50 1040± 9
;
B c 2 -Adrenergic D2-Dopamine Al-Adenosine 6-opiate µ-opiate M2-Cholinergic H2-Histaminergic
G;
974±73
G; G5
1115±50 1219±26
Some of the receptor types copuled to G s and/or G; that alter intracellular cAMP levels are shown in A. HOG cells were incubated for 30 min with specific adenylate cyclase-linked receptor agonists at the concentrations (in µNI) shown. Some of the receptor types that are coupled to Gi and thus inhibit the formation of cAMP are listed in B. HOG cells were preincubated for 10 min with and without the agonists (at 10 .M final concentration) listed, which are specific for those receptors coupled to G. Forskolin (1 µM) was added and the incubation continued for 30 min. The amount of cAMP generated was measured by RIA as described in the text. Data shown are the mean +SEM for each agonist. Each experiment was performed in triplicate and repeated twice_
cultures often comprise a heterogeneous population of cells (Castros et al., 1990). Clonal cell lines are an attractive alternative because large quantities of homogeneous cells can be generated and they offer the opportunity to study a metabolically active cell population. Two disadvantages of clonal cell lines are that they may represent immature "blast" cells that have not yet differentiated or tumor cells often have alterations in their
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0 400
200
-8
-7
-6
log [Agonist] (M)
Fig. 4. Effect of specific Pi and Pz adrenergic receptor agonists on the production of intracellular cyclic AMP in HOG cells. HOG cells were incubated for 30 min with one p 1 -AR specific agonist (prenaterol, *) and two different p 2-AR specific agonists (clenbuterol [0] and albuterol [ J) at the concentrations shown. Intracellular cAMP was measured by RIA as described in the text. Data shown are the means of duplicate assays performed on one experiment done in triplicate. The error bars indicate the SEM. Basal cAMP was 31.64 ± 6.89 pmol/mg protein. genome that correlate with a loss or diminution of at least some of the differentiated characteristics of oligodendrocytes. For example, rat C6 glioma cells have been used to study the differential expression of astrocyte-specific GS and oligodendrocyte-specific CNPase (Parker et al., 1980). In contrast, HOG cells express some of the proteins of oligodendrocytes, including a 15-kDA form of myelin basic protein and together with CNPase, but not astrocyte markers such as GFAP and GS. The finding of a low-mol-wt form of MBP was unexpected because, based on in vitro translation of human fetal spinal cord poly A mRNA (Roth et al., 1986), we would expect human oligodendrocytes to express three major MBP forms with approximate mol wts of 17 kDa, 18.5 kDa, and 21.5 kDa. However, human fetal spinal cord has recently been shown to express also a 15-kDa form of MBP, and in HOG cells a 2.6 kb mRNA can be transcribed into this 15-kDa MBP (Kashima et al., 1991). HOG cells provide a useful model system in which to study these myelin specific proteins and how their expression relates to activation of signal transduction pathways by specific neurotransmitters.
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50
40
e
30
20
to 0
0 0
30
90
60
120
150
180
125I-CYP (pM)
Fig. 5. Saturation binding of 125 I]cyanopindolol to HOG cells in the presence of f3-AR selective antagonists. Whole cells were incubated with 125 I]cyanopindolol 125 1-C1P) in the concentration range of 6-180 pM. The incubation mixture also contained either 5.5 nM ICI 118,551 (0) (125-fold more specific for ,6 2 - ARs and thus preferentially blocks binding to 0 2 -ARs or 50 nM metroprolol (e) (10-40-fold more specific for f3 1 -ARs and thus preferentially blocks binding to (3 1 -ARs. Specific binding was measured as described in the text. Data shown are the means of triplicate determinations in a single experiment, which was repeated twice. Binding parameters, Kd and B m , were determined from the aforementioned data by a nonlinear least squares fit program (Yamaoka et al., 1981): [
[
(
fie AR -
Kd = 50.9±7.5 pM B,,, = 69.6 ±3.8 fmol/mg (3 1 -AR Kd = 32.0±11.8 pM
HOG cells were found to express neurotransmitter receptors coupled to the activation of adenylate cyclase. For example, HOG cells express both Q-adrenergic and PGE1 receptors that have been previously identified in sheep oligodendrocytes prior to remyelination (Vartanian et al., 1988), in primary cultures of rat oligodendrocytes (McCarthy and deVellis, 1980), in a mouse oligodendroglioma cell line (Vartanian et al., 1985), and in primary cultures of astrocytes (Trimmer and McCarthy, 1986). However mouse oligodendroglioma cells express primarily the X3 1 -AR subtype, whereas HOG and 1321N1 human astrocytoma cells (Harden and McCarthy, 1982) express only (3 2 -ARs. HOG cells also express adenosine receptors linked to the stimulation of adenylate cyclase. These receptors have been identified in astrocytes (Van Calker and Hamprecht, 1980), but not in 1321N1 human astrocytoma cells. Neither HOG nor 1321N1 astrocytoma cells responded to the a-adrenergic and muscarinic cholinergic receptor Molecular and Chemical Neuropathology
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agonists or the peptide somatostatin, which have been shown to reduce cAMP levels in primary cultures of astrocytes (Evans et al., 1984; Van Calker and Hamprecht, 1980). HOG cells were found to express muscarinic and H1-histamine receptors coupled to the activation of PLC. These receptors may in turn activate PKC the major kinase involved in phosphorylation of two proteins thought to be essential to myelination, MBP and CNPase (Vartanian et al., 1986). Ritchie et al. (1987) also found evidence for muscarinic and histamine receptors in primary cultures of rat oligodendrocytes. We did not find evidence for functional bradykinin or a l -adrenergic receptors previously reported in rat oligodendrocytes (Ritchie et al., 1987) and primary glial cultures (Wilson et al., 1990). Muscarinic and histamine receptors have been identified in 1321N1 human astrocytoma cells (Nakahata and Harden, 1987), but their activation and desensitization profiles were markedly different (Post and Dawson, 1991), suggesting differences between oligodendrocytes and astrocytes in the regulation of signal transduction. In summary, we have presented evidence for a human cell line (HOG) that expresses at least two oligodendrocyte-specific proteins, CNPase and a 15 kDa form of MBP, as well as receptors linked to the activation of second messenger systems presumed to be important for the regulation of oligodendrocyte metabolism.
ACKNOWLEDGMENTS Supported by USPHS Grand HD-06426 and a Medical Science Training Grant Award (GNI-07281) to G. R. Post.
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