Metabolic Brain Disease, Vol. 7, No. 3, 1992
Ascorbic Acid Upregulates Myelin Gene Expression in C6 Glioma Cells I. Laszkiewicz, t R.C. Wiggins, 1 and G. K o n a t 1, 2 Received July 31, 1992; accepted August 12, 1992 The effect of ascorbic acid (AA) on rat glioma C6 cells was studied. At physiological AA concentrations of 0.1 and 1 mM, no morphological and no proliferative alterations in the C6 cultures were detectable. Although the total RNA content per cell was not affected by the AA-treatment, AA upregulated the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes as assessed by northern blot analysis. The steady-state level of the specific mRNAs increased transiently in the AA-treated cells. Three days after AA administration the message levelreached a maximum of 10- and 2-fold over control for the PLP and MAG genes, respectively. The upregulation of the genes was directly related to AA concentration. The present data indicate a possible involvement of AA in the regulation of myelin gene activity in the CNS. KEY WORDS: ascorbic acid; proteolipid protein (PLP); myelin associated glycoprotein (MAG); gene expression; C6 glioma cells.
INTRODUCTION Rat glioma C6 cells represent an attractive model system to study oligodendrocytic differentiation. This cell line expresses genes coding for proteolipid protein (PLP) (Milner et al., 1985; Macklin et al., 1986), and for myelin-associated glycoprotein (MAG) (Bong et al. 1991). PLP is the major protein of myelin membrane and is essential for the formation of the intraperiod line of the myelin sheath (Braun, 1984; Kirschner et al., 1984; Hudson et al., 1989). M A G is thought to play a pivotal role in cell-cell recognition and myelin compaction (Owens & Bunge 1989; Quarles et al., 1990). The expression of both genes in C6 cells is amenable to induction by various factors (Bong et al., 1991; Kanoh et al., 1991;
1 Department of Anatomy, West Virginia University School of Medicine, Morgantown, WV 2 To whom correspondence should be addressed at the Department of Anatomy, West Virginia University, School of Medicine, 4052 HSN, Morgantown, WV 26505. 157 0885-7490/92/0900-0157506.50/0 9 1992 Plenum Publishing Corporation
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Z h u et al., 1992; Ye et al., 1992; Kanoh et al., 1992). Such factors are highly relevant to
modeling myelination, since the induction of the myelin genes is considered an index of oligodendrocyte differentiation into myelin-producing cells. Ascorbic acid (AA) is essential for normal functioning of the mammalian brain, and its concentrations in the brain are higher than in any other organ (Hammarstrom, 1966; Milby et al., 1982). Although brain AA homeostasis is strictly preserved (Schenk et al., 1982), its concentration changes dramatically during development (Kratzing et al., 1985) suggesting a possible involvement of AA in the ontogenetic processes. AA appears to play a role in the PNS myelinogenesis as it is essential for the activation of the P0 protein gene in cultured Schwann cells (Owens & Bunge 1989). This study was undertaken to determine the effect of AA on the expression of myelin-specific genes, namely, the PLP and MAG genes in the CNS-derived C6 ceils.
MATERIALS AND METHODS Cell Culture
C6 rat glioma cells obtained from American Type Culture Collection (Rockville, MD). Stock monolayer cultures were maintained in Ham's F-10/Dulbecco modified Eagle's medium (1:1) (Gibco, Grand Island, NY) with 1% of antibiotic-antimycotic mixture (Gibco) and 10% fetal calf serum (FCS)(Sigma) at 37~ under 95% air/5% CO2. For experimental cultures, 2.0 x 106 cells were plated into 5 cm plastic Petri dish (Coming Co., Coming, NY) and grown in serum-free (defined) medium. Ascorbic acid (Sigma Chemical Co., St Louis, MO) was dissolved in the medium before addition to the culture. Both control and AA-containing media were changed dally. For the measurement of proliferation rate, the cells were trypsinized at different intervals and counted in a hemacytometer. Hybridization Probes
Plasmid p27 containing full-length cDNA (3.2 kb) for rat PLP (Milner et al., 1985) and plasmid plB236 containing partial cDNA (1.5 kb) for rat MAG (Sutcliffe et al., 1983) were obtained from Dr. Milner (Scripps Clinic, La Jolla, CA). High efficiency ssDNA probes were generated on these templates by asymmetric PCR (Bednarczuk et al. 1991) as modified by Konat et a/.(1991) using respective 20-mer antisense primers and 32p-~-dCTP (New England Nuclear, Boston, MA). The probes corresponded to 243-928 bp ofPLP cDNA and to 281-761 bp of MAG cDNA. Northern Blot Analysis
Total RNA was isolated from C6 cells by the method of Chomczynski and Sacchi (1987) and analyzed by Northern blot technique as described by Ye et al. (1992). Briefly, RNA was electrophoresed on 1% agarose-formaldehyde gel, transferred to Nytran nylon membrane (Schleicher and Schuell, Keen, NH) and stained with methyleneblue to determine
Ascorbic Acid and Myelin Gene Expression
159
actual amount of RNA on the membrane. The filters were subsequently hybridized with radiolabeled probes and washed at high stringency (0.2X SSC, 0.1% SDS, 60(2, 60 min). The position of the specific messages was detected by autoradiography. Quantitation of the messages was performed using a LKB soft laser densitometer. The message levels were normalized to the amount of total RNA on the membrane.
RESULTS Two AA concentrations were employed in this study, namely 0.1 mM and 1 mM. AA concentrations higher than 1 mM resulted in the detachment of the cells from substratum, and hence were not used. The addition of AA into the medium at either low (0.1 mM) or high (1 raM) dose had no effect on the morphology or on the proliferation of C6 ceils. As compared to parental (control) cells, no alteration in the amount of total RNA per cell was observed following the exposure of C6 cells to AA, however, the treatment upregulated the expression of myelin-specific genes, i.e, the PLP and MAG genes. As seen from the Northern blot analysis (Fig. 1) the steady-state levels of both PLP-specific mRNAs, namely 3.2 kb and 1.6 kb, as well as 2.5 kb MAG-specific mRNA, were profoundly increased in the AA-treated cells. There was no detectable change in the PLP message ratio (3.2 kb/1.6 kb). The effect of AA on the gene expression was directly related to its concentration (Fig. 2). The message level after 4 days of treatment with 1 mM AA was approximately 5- and 2-fold over the control for the PLP and MAG mRNA, respectively. In cells treated with 0.1
Fig. 1. Expression of PLP and MAG mRNA in control (C) and AA-treated (AA) C6 cells. The cells were cultured for four days without or with 1 mM AA. I0 p.g of total RNA were applied per lane. All the lanes contained equal load as assessed by methylene blue staining (see Materials and Methods). Arrowheads indicate the position of 18S and 28S rRNA bands.
160
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AA C o n c e n t r a t i o n (mM) Fig. 2. Expression of PLP (single-hatched columns) and MAG (double-hatched columns) mRNA in C6 cells exposed to different AA concentrations for four days. The message level was normalized to the amount of total RNA. The results represent averages from three to five experiments and are expressed as the stimulation index; stimulation index for control cells (cultured without AA) equals 1.0. Vertical bars indicate S.D. m M AA, the MAG gene upregulation was approximately 1.8-fold, whereas the PLP gene upregulation was 4-fold. The gene expression in the course of the experiment is presented in Fig. 3. The cells maintained in the presence of 1 mM AA showed a maximal elevation of PLP m R N A to approximately 10-fold over control on day 3, followed by a gradual drop during the next two days to approximately 2-fold over control value. The maximal upregulation of the MAG
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I n c u b a t i o n Time (days) Fig. 3. Time dependence of PLP (circles) and MAG (triangles) mRNA expression in AA-treated C6 cells. The concentration of AA was 1 raM. The results represent averages fi'om three to five experiments and are expressed as the stimulation index; stimulation index for control cells (cultured without AA) equals 1.0. Vertical bars indicate S.D. m R N A was also observed on day 3 but it was only approximately 2-fold over the control value and was only slightly decreased by day 5.
DISCUSSION AA concentrations employed in the present study, i.e., 1 mM and 0.1 mM, brackets the concentration o f 0.28 m M used to promote myelination in Schwann cell-neuronal cocultures
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(vide infra). This is within physiological AA concentration range of 0.9 to 1.2 gmol/g reported for the adult rat brain (Milby et al., 1982). In the fetal and/or newborn brain AA levels are twice as high (Kratzing et al., 1985). Furthermore, a significant portion of brain AA is in the interstitial fluid (Kratzing et al., 1982), and, thus, the extracellular AA concentration in discrete areas may be higher than the average tissue value. AA is an essential supplement to promote axonal ensheathment in Schwann cell-neuronal coculture (Owens & Bunge, 1989). In the absence of AA, axonal interactions upregulate the expression of MAG and galactocerebrosides in Schwann cells, but the expression of the major PNS myelin protein, namely P0, is undetectable. The addition of serum plus AA leads to the deposition of basal lamina, profound upregulation of the Po gene, and the formation of myelin sheath. This, as well as previous study in the coculture system (Moya et al., 1980) indicates that basal lamina and possibly other components of the extracellular matrix (e.g. thin collagenous fibrils) plays a pivotal role in the initiation of myelin assembly. The stimulatory effect of AA on the extracellular matrix formation is in line with the known function of AA in posttranslational processing of collagen where it is a cofactor for proline and lysine hydroxylation (Flier & Underhill, 1986). In addition, in a variety of cell types, AA upregulates the synthesis of collagen by increasing transcription, translation, and stability of procollagen message (Murad et al., 1981; Tajima and Pinnel, 1982; Geesin et al., 1988; McDevitt et al., 1988; Sandell and Daniel, 1988; Owen et al., 1990). In this communication we demonstrate that AA is a potent stimulator of the PLP, and to a lesser extent of the MAG gene expression in the CNS-derived C6 cells. It seems possible that, like in Schwann ceils, the effect of AA on the myelin genes may be mediated by the deposition of extracellular collagenous components. Although the CNS myelinogenesis is not associated with the formation of basal lamina, the extrapolation of the present results would suggest that the deposition of some extracellular matrix elements may be involved in the oligodendrocyte differentiation. On the other hand, AA is known to function in many diverse physiological processes in animal cells acting as a reducing and antioxidant agent (Padh, 1990). Hence, it should also be considered that the observed effect of AA on the myelin genes in C6 cells may not be related to collagen metabolism, but instead, results from other cellular reactions affected by AA. The PLP and MAG gene expression undergoes only a transient upregulation upon AA-treatment. The inability of C6 cells to sustain a high level of the myelin gene expression appears to be an intrinsic feature of these cells as it was observed with other, more potent stimulators (Kanoh et al., 1991; Ye et al., 1992; Zhu et al. 1992; Kanoh et al., 1992).
ACKNOWLEDGMENTS This research was supported by PHS service grant NS13799, DA04072, WVU Medical Corporation and NIH BRG No. 2S07RR05433. REFERENCES Bednarczuk,T.A., Wiggins, R.C and Konat, G. (1991) Generation of high efficiency, single-stranded DNA hybridization probes by PCR. BioTechniques 10: 478.
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Bong, M., Chakrabarti, A., Banik, N., Hogan, E.L., Kanoh, M., Wiggins, R.C. and Konat G. (1991) Differential regulation of myelin gene expression in SV40 T antigen-transferred rat glioma C6 cells. Met. Brain Disease 6:11-21. Braun, P.E. (1984) Molecular Organization of myelin. In Myelin, Morell, P. (Ed.) Plenum, NY, pp. 97-116 Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by a~dic guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. Flier, J.S. and Underhill, L.H. (1986) New concepts in the biology and biochemistry of ascorbic acid. New Engl. J. Med. 314: 892-902. Geesin, J.C., Dart, D., Kanfman, R., Mumd, S. and Pinnel, S.R. (1988) Ascorbic acid specifically increases type I and type TITprocollagen messenger RNA levels in human skin fibroblasts. J. Invest. Dermatol. 90: 420-424. Hammarstrom, L. (1966) Autoradiographic studies on the distribution of C14-1abeled nscorbic acid and dehydroascorbie acid. Acta Physiol. Scand. Suppl. 289: 1-84. Hudson, L.D., Friedrich, V.LJr., Behar, T., Dubois-Dalcq, M. and Lazzarini, R.A. (1989) The initial events in myelin synthesis: Orientation of proteolipid protein in the plasma membrane of cultured oligodendrocytes. J. Cell. Biol. 109: 717-727. Kanoh, M., Ye, P., Zhu, W., Wiggins, R.C. and Konat, G. (1991) Effect of culture conditions on expression of PLP and MAG genes in rat glioma C6 cells. Met. Brain Disease 6: 133-143. Kanoh, M., Wiggins, R.C. and Konat, G. (1991) Differential upregulation of PLP and MAG genes in C6 glioma cells by N2A neuroblastoma conditioned medium. Met. Brain Disease 7:147-156. Kirschner, D.A., Ganser, A.L. and Caspar, D.L.D. (1984) Diffraction studies of molecular organization and membrane interactions in myelin. In Myelin, Morell, P. (Ed.) Plenum, NY, pp. 51-95 Konat, G., Laszkiewicz, I., Bednarczuk, T.A., Kanoh, M., Wiggins, R.C. (1991) Generation of radioactive and nonradioactive ssDNA hybridization probes by polymerase chain reaction. Technique 3: 64-68. Kratzing, C.C., Kelly, J.D. and Oelrichs, B.A. (1982) Ascorbic acid in neural tissues. J. Neurochem. 39: 625-627. Kratzing, C.C., Kelly, J.D. and Kratzing, J.E. (1985) Ascorbic acid in fetal rat brain. J. Neurochem. 44: 1623-1624. Macklin, W.B., Weill, C.L. and Deininger, P.L. (1986) Expression of myelin proteolipid and basic protein mRNA in cultured cells. J. Neurosci. Res. 16: 203-217. McDevitt, C.A., Lipman, J.M., Ruemer, R.J. and Sokoloff, L. (1988) Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and_-aminopropionitrile. J. Orthop. Res. 6: 397-407. Milby, K., Oke, A. and Adams, R.N. (1982) Detailed mapping of ascorbate distribution in rat brain. Nettrosci. Left. 28: 169-174. Milner, R.J., Lai, C., Nave, K-A., Lenoir, D., Ogata, J. and Sutcliffe, J.G. (1985) Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein. Cell 42:931-939. Moya, F., Bunge, M.B. and Bunge, R.P. (1980) Schwann cells proliferate but fail to differentiate in defined medium. Proc. Natl. Acad. Sci. USA 77: 6902-6906. Murad, S., Grove, D., Lindberg, K., Reynolds, G., Sivarajah, A. and Pinnel, S.R. (1981) Regulation of collagen synthesis by ascorbic acid. Proc. Natn. Acad. Sci. USA 78:2879-2882. Owens, G.C. and Bunge, R.P. (1989) Evidence for an early role for myelin-associated glycoprotein in the process of myelination. Glia 2:119-128. Owen, T.A., Aronow, M., Shalhoub, V., Barone, L.M., Wilming, L., Tassinari, M.S., Kennedy, M.B., Pockwinse, S., Lian, J.B. and Stein, G.S. (1990) Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationship in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J. Cell. Physiol. 143: 429-430.
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Padh, H. (1990) Cellular functions of ascorbic acid. Biochem. Cell. Biol. 68:1166-1163. Quarles, R.H., Hammer, J.A. and Trapp, B.D. (1990) The immunoglobulin gene superfamily and myelination. In Dynamic Interactions of Myelin Proteins, Alan R. Liss, Inc., New York, pp. 49-79. Sandell, LJ. and Daniel, J.C. (1988) Effects of ascorbic acid on collagen mRNA levels in short term ehondrocyte cultures. Connect. Tissue Res. 17:11-22. Schenk, J.O., Miller, E., Gaddis, R. and Adams, R.N. (1982) Homeostatic control of ascorbate concentration in CNS extracellular fluid. Brain Res. 253: 353-356. Sutcliffe, J.G., Milncr, RJ., Shinnick, T.M., Bloom, F.E. (1983) Identifying the protein product of brain specific genes with antibodies to chemically synthesized peptides. Cell 33:671-682 Tajima, S. and Pinnel, S.R. (1982) Regulation of collagen synthesis by aseorbic acid. Ascorbie acid increases type I procollagen mRNA. Biochem. Biophys. Res. Commun. 106: 632-637, Ye, P., Kanoh, M., Zhu, W., Laszkiewicz, I., Royland, J.E., Wiggins, R.C. and Konat, G. (1992) Cyclic AMP-induced upregulation of proteolipid protein and myelin associated glycoprotein gene expression in rat glioma C6 ceils. J. Neurosci. Res. 31: 578-583. Zhu, W., Kanoh, M., Ye, P., Laszkiewicz, I., Royland, J., Wiggins, R.C., Konat, G. (1992) Retinoic acid regulation of proteolipid protein and myelin associated glycoprotein gene expression in C6 glioma cells. J. Neurosci. Res. 31: 745-750.
Ascorbic Acid Upregulates Myelin Gene Expression in C6 Glioma Cells I. Laszkiewicz, t R.C. Wiggins, 1 and G. K o n a t 1, 2 Received July 31, 1992; accepted August 12, 1992 The effect of ascorbic acid (AA) on rat glioma C6 cells was studied. At physiological AA concentrations of 0.1 and 1 mM, no morphological and no proliferative alterations in the C6 cultures were detectable. Although the total RNA content per cell was not affected by the AA-treatment, AA upregulated the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) genes as assessed by northern blot analysis. The steady-state level of the specific mRNAs increased transiently in the AA-treated cells. Three days after AA administration the message levelreached a maximum of 10- and 2-fold over control for the PLP and MAG genes, respectively. The upregulation of the genes was directly related to AA concentration. The present data indicate a possible involvement of AA in the regulation of myelin gene activity in the CNS. KEY WORDS: ascorbic acid; proteolipid protein (PLP); myelin associated glycoprotein (MAG); gene expression; C6 glioma cells.
INTRODUCTION Rat glioma C6 cells represent an attractive model system to study oligodendrocytic differentiation. This cell line expresses genes coding for proteolipid protein (PLP) (Milner et al., 1985; Macklin et al., 1986), and for myelin-associated glycoprotein (MAG) (Bong et al. 1991). PLP is the major protein of myelin membrane and is essential for the formation of the intraperiod line of the myelin sheath (Braun, 1984; Kirschner et al., 1984; Hudson et al., 1989). M A G is thought to play a pivotal role in cell-cell recognition and myelin compaction (Owens & Bunge 1989; Quarles et al., 1990). The expression of both genes in C6 cells is amenable to induction by various factors (Bong et al., 1991; Kanoh et al., 1991;
1 Department of Anatomy, West Virginia University School of Medicine, Morgantown, WV 2 To whom correspondence should be addressed at the Department of Anatomy, West Virginia University, School of Medicine, 4052 HSN, Morgantown, WV 26505. 157 0885-7490/92/0900-0157506.50/0 9 1992 Plenum Publishing Corporation
158
Laszkiewicz,Wiggins, and Konat
Z h u et al., 1992; Ye et al., 1992; Kanoh et al., 1992). Such factors are highly relevant to
modeling myelination, since the induction of the myelin genes is considered an index of oligodendrocyte differentiation into myelin-producing cells. Ascorbic acid (AA) is essential for normal functioning of the mammalian brain, and its concentrations in the brain are higher than in any other organ (Hammarstrom, 1966; Milby et al., 1982). Although brain AA homeostasis is strictly preserved (Schenk et al., 1982), its concentration changes dramatically during development (Kratzing et al., 1985) suggesting a possible involvement of AA in the ontogenetic processes. AA appears to play a role in the PNS myelinogenesis as it is essential for the activation of the P0 protein gene in cultured Schwann cells (Owens & Bunge 1989). This study was undertaken to determine the effect of AA on the expression of myelin-specific genes, namely, the PLP and MAG genes in the CNS-derived C6 ceils.
MATERIALS AND METHODS Cell Culture
C6 rat glioma cells obtained from American Type Culture Collection (Rockville, MD). Stock monolayer cultures were maintained in Ham's F-10/Dulbecco modified Eagle's medium (1:1) (Gibco, Grand Island, NY) with 1% of antibiotic-antimycotic mixture (Gibco) and 10% fetal calf serum (FCS)(Sigma) at 37~ under 95% air/5% CO2. For experimental cultures, 2.0 x 106 cells were plated into 5 cm plastic Petri dish (Coming Co., Coming, NY) and grown in serum-free (defined) medium. Ascorbic acid (Sigma Chemical Co., St Louis, MO) was dissolved in the medium before addition to the culture. Both control and AA-containing media were changed dally. For the measurement of proliferation rate, the cells were trypsinized at different intervals and counted in a hemacytometer. Hybridization Probes
Plasmid p27 containing full-length cDNA (3.2 kb) for rat PLP (Milner et al., 1985) and plasmid plB236 containing partial cDNA (1.5 kb) for rat MAG (Sutcliffe et al., 1983) were obtained from Dr. Milner (Scripps Clinic, La Jolla, CA). High efficiency ssDNA probes were generated on these templates by asymmetric PCR (Bednarczuk et al. 1991) as modified by Konat et a/.(1991) using respective 20-mer antisense primers and 32p-~-dCTP (New England Nuclear, Boston, MA). The probes corresponded to 243-928 bp ofPLP cDNA and to 281-761 bp of MAG cDNA. Northern Blot Analysis
Total RNA was isolated from C6 cells by the method of Chomczynski and Sacchi (1987) and analyzed by Northern blot technique as described by Ye et al. (1992). Briefly, RNA was electrophoresed on 1% agarose-formaldehyde gel, transferred to Nytran nylon membrane (Schleicher and Schuell, Keen, NH) and stained with methyleneblue to determine
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actual amount of RNA on the membrane. The filters were subsequently hybridized with radiolabeled probes and washed at high stringency (0.2X SSC, 0.1% SDS, 60(2, 60 min). The position of the specific messages was detected by autoradiography. Quantitation of the messages was performed using a LKB soft laser densitometer. The message levels were normalized to the amount of total RNA on the membrane.
RESULTS Two AA concentrations were employed in this study, namely 0.1 mM and 1 mM. AA concentrations higher than 1 mM resulted in the detachment of the cells from substratum, and hence were not used. The addition of AA into the medium at either low (0.1 mM) or high (1 raM) dose had no effect on the morphology or on the proliferation of C6 ceils. As compared to parental (control) cells, no alteration in the amount of total RNA per cell was observed following the exposure of C6 cells to AA, however, the treatment upregulated the expression of myelin-specific genes, i.e, the PLP and MAG genes. As seen from the Northern blot analysis (Fig. 1) the steady-state levels of both PLP-specific mRNAs, namely 3.2 kb and 1.6 kb, as well as 2.5 kb MAG-specific mRNA, were profoundly increased in the AA-treated cells. There was no detectable change in the PLP message ratio (3.2 kb/1.6 kb). The effect of AA on the gene expression was directly related to its concentration (Fig. 2). The message level after 4 days of treatment with 1 mM AA was approximately 5- and 2-fold over the control for the PLP and MAG mRNA, respectively. In cells treated with 0.1
Fig. 1. Expression of PLP and MAG mRNA in control (C) and AA-treated (AA) C6 cells. The cells were cultured for four days without or with 1 mM AA. I0 p.g of total RNA were applied per lane. All the lanes contained equal load as assessed by methylene blue staining (see Materials and Methods). Arrowheads indicate the position of 18S and 28S rRNA bands.
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AA C o n c e n t r a t i o n (mM) Fig. 2. Expression of PLP (single-hatched columns) and MAG (double-hatched columns) mRNA in C6 cells exposed to different AA concentrations for four days. The message level was normalized to the amount of total RNA. The results represent averages from three to five experiments and are expressed as the stimulation index; stimulation index for control cells (cultured without AA) equals 1.0. Vertical bars indicate S.D. m M AA, the MAG gene upregulation was approximately 1.8-fold, whereas the PLP gene upregulation was 4-fold. The gene expression in the course of the experiment is presented in Fig. 3. The cells maintained in the presence of 1 mM AA showed a maximal elevation of PLP m R N A to approximately 10-fold over control on day 3, followed by a gradual drop during the next two days to approximately 2-fold over control value. The maximal upregulation of the MAG
Ascorbic Acid and Myelin Gene Expression
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I n c u b a t i o n Time (days) Fig. 3. Time dependence of PLP (circles) and MAG (triangles) mRNA expression in AA-treated C6 cells. The concentration of AA was 1 raM. The results represent averages fi'om three to five experiments and are expressed as the stimulation index; stimulation index for control cells (cultured without AA) equals 1.0. Vertical bars indicate S.D. m R N A was also observed on day 3 but it was only approximately 2-fold over the control value and was only slightly decreased by day 5.
DISCUSSION AA concentrations employed in the present study, i.e., 1 mM and 0.1 mM, brackets the concentration o f 0.28 m M used to promote myelination in Schwann cell-neuronal cocultures
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(vide infra). This is within physiological AA concentration range of 0.9 to 1.2 gmol/g reported for the adult rat brain (Milby et al., 1982). In the fetal and/or newborn brain AA levels are twice as high (Kratzing et al., 1985). Furthermore, a significant portion of brain AA is in the interstitial fluid (Kratzing et al., 1982), and, thus, the extracellular AA concentration in discrete areas may be higher than the average tissue value. AA is an essential supplement to promote axonal ensheathment in Schwann cell-neuronal coculture (Owens & Bunge, 1989). In the absence of AA, axonal interactions upregulate the expression of MAG and galactocerebrosides in Schwann cells, but the expression of the major PNS myelin protein, namely P0, is undetectable. The addition of serum plus AA leads to the deposition of basal lamina, profound upregulation of the Po gene, and the formation of myelin sheath. This, as well as previous study in the coculture system (Moya et al., 1980) indicates that basal lamina and possibly other components of the extracellular matrix (e.g. thin collagenous fibrils) plays a pivotal role in the initiation of myelin assembly. The stimulatory effect of AA on the extracellular matrix formation is in line with the known function of AA in posttranslational processing of collagen where it is a cofactor for proline and lysine hydroxylation (Flier & Underhill, 1986). In addition, in a variety of cell types, AA upregulates the synthesis of collagen by increasing transcription, translation, and stability of procollagen message (Murad et al., 1981; Tajima and Pinnel, 1982; Geesin et al., 1988; McDevitt et al., 1988; Sandell and Daniel, 1988; Owen et al., 1990). In this communication we demonstrate that AA is a potent stimulator of the PLP, and to a lesser extent of the MAG gene expression in the CNS-derived C6 cells. It seems possible that, like in Schwann ceils, the effect of AA on the myelin genes may be mediated by the deposition of extracellular collagenous components. Although the CNS myelinogenesis is not associated with the formation of basal lamina, the extrapolation of the present results would suggest that the deposition of some extracellular matrix elements may be involved in the oligodendrocyte differentiation. On the other hand, AA is known to function in many diverse physiological processes in animal cells acting as a reducing and antioxidant agent (Padh, 1990). Hence, it should also be considered that the observed effect of AA on the myelin genes in C6 cells may not be related to collagen metabolism, but instead, results from other cellular reactions affected by AA. The PLP and MAG gene expression undergoes only a transient upregulation upon AA-treatment. The inability of C6 cells to sustain a high level of the myelin gene expression appears to be an intrinsic feature of these cells as it was observed with other, more potent stimulators (Kanoh et al., 1991; Ye et al., 1992; Zhu et al. 1992; Kanoh et al., 1992).
ACKNOWLEDGMENTS This research was supported by PHS service grant NS13799, DA04072, WVU Medical Corporation and NIH BRG No. 2S07RR05433. REFERENCES Bednarczuk,T.A., Wiggins, R.C and Konat, G. (1991) Generation of high efficiency, single-stranded DNA hybridization probes by PCR. BioTechniques 10: 478.
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