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Secretion of S-100 from rat C6 glioma cells
Brain Research. 436 (1987) 367-370
367
Elsevier BRE 22634
Secretion of S-IO0 from rat C6 glioma ceils Linda J. Van Eldik 1'2 and Danna B. Zimmer l Departments of t Cell Biology and 2Pharmacology and Howard Hughes Medical Institute. Van derbilt University, Nashville. TN 37232 ( U. S. A. )
(Acceptcd 1 September 1987) Key words: Radioimmunoassay; Rat glioma celi; Secretion; S-100
Conditioned media from rat C6 glioma cells contain significant levels of S-100 immunoreactivity as determined by radioimmunoassay. The levels of extracellular S-100 detected could not be accounted for by the release of intracellular S-100 into the media from lysed cells. The extracellular form of S-100 exhibits fractionation properties and immunological characteristics that are different from those of the intracellular form of S-100 in C6 cells. While the intracellular S-100 levels increase as a function of days in culture, the extracellular S-100 levels are high until the cells reach confluency and are lower in postconfluent cultures. Altogether, our data suggest that C6 gliema cells secrete S..IO0, and that the quantitative levels of the intracellular and secreted forms of S-100 are differentially regulated.
S-100 is an acidic calcium binding protein which was first isolated 12 as a brain-specific protein fraction. The bovine brain S-100 fraction is composed of primarily two polypeptides, S-100a and S-100fl, which share approximately 50% identity in amino acid sequence 7"8. Although S-100 proteins are found in high levels in brain tissue, they are also present in numerous other tissues 5"16"2°.In the brain, S-100 is localized primarily in glial cells 2"3 and its appearance in brain tissue correlates with maturation of the nervous system 4a5'21. Cultured rat C6 glioma cells also contain S-1001 and are a useful modei system for studying the regulation of these proteins. Previous studies 9"19have demonstrated that conditioned media from rat C6 glioma cell cultures contain a neurite extension factor which appears to be different from nerve growth factor. The neurite extension factor from C6 conditioned media was found to be a heat stable, acidic, low molecular weight protein. More recent studies l° have shown that a neurite extension factor activity from bovine brain is associated with a disulfide form of S-100fl. Taken together, these data suggest that the neurite extension factor activity found in rat C6 glioma conditioned media
may also be S-100fl. As a first step towards addressing this hypothesis, we examined the conditioned media from C6 glioma cells for the presence of S-100 using a radioimmunoassay. Since previous studies ~1a3 have shown that intracellular S-100 levels vary with cell density, we also analyzed the conditioned media from C6 cultures at different times after plating the cells in order to examine whether the levels of extracellular S-100 might vary. The rat C6 glioma cell line was obtained from the AmeriL:an Type Culture Collection (Rockville, MD) and grown in F10 media (Gibco, Grand Island, NJ) supplemented with 15% (v/v) horse serum (Hycione Laboratories, Logan, UT), 2.5% (v/v) fetal calf serum (Hyclone Laboratories, Logan, UT), 2 !~g/ml fungizone (Squibb, Princeton, N J), 50 U/ml penicillin and 50/tg/ml streptomycin (Gibco, Grand Island, N J). Stock cultures were subcultured by trypsinization and plated in T-flasks of 175 cm 2 surface area (Falcon, Oxnard, C A ) containing 50 ml of media at a plating density of approximately 8 x 106 cells/flask. At various times after plating, the coqditioned media and cells were harvested for analysis. All cultures were fed 48 h before harvesting. For each time point,
Correspondence: D.B. Zimmer, 717 Light Hail. Howard Hughes Medical Institute. Vanderbiit University Medical Center. Nashville, TN 37232, U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishv 's B.V. (Biomedical Division)
368 1 flask was trypsinized and the cell number/flask was determined using a hemocytometer. The conditioned media was harvested from the remaining flasks, centrifuged at 1250 g in a Beckman TJ-6 centrifuge to remove cell debris, and stored at -20 °C until use. S-100 radioimmunoassays were done using rabbit polyclonal antisera specific for the individual S-100 polypeptides, S-100a and S-100fl 2°. Since the concentration of S-100 in the unfractionated conditioned media was too low to be detected by radioimmunoassay, 3 concentrated fractions af conditioned media (a 0-60% pellet, 60-80% pH 4 pellet, and 60-80% pH 4 supernatant) were prepared by ammonium sulfate precipitation as previously described 2°. Before radioimmunoassay, all fractions were extensively dialyzed to remove the ammonium sulfate. When C6 cell extracts were fractionated in a similar fashion and analyzed by radioimmunoassay, >95% of the S-100 immunoreactivity was found in the 60-80% pH 4 pellet. These data are in agreement with our previous resuits 2° with rat brain extracts. However, analysis of the S-100 immunoreactivity in ammonium sulfate fractions of the C6 conditioned media showed that S100 immunoreactivity was present in all 3 fractions. When conditioned media which contained exogenously added purified bovine brain S-100a and S100/3 was fractionated, 72% of the S-100a and 95% of the S-100fl immunoreactivity could be recovered in the 0-60% pellet and 60-80% pH 4 pellet fractions. No exogenously added S-100a and S-100fl was recovered in the 60-80% pH 4 supernatant. Altogether, these data suggest that the conditioned media alters the fractionation properties of S-100 and/or that the extrace!lular form of S-100 may have different fractionation properties from the intracellular form. Therefore, the immunoreactivity in the 0-60% pellet, 60-80% pH 4 pellet, and 60-80% pH 4 supernatant fractions was used in calculating the levels of extracellular S-100. In addition, we also observed significant levels of S-100a and S-100fl immunoreactivity in the 0-60% pellet fraction of media which had not been incubated with cells. These values accounted for approximately 53% of the total S-100a and 35% of the total S-100fl immunoreactivity detect~.d Jn the conditioned media and were subtracted from the immunoreactivity in the conditioned media before the extracellular levels ef S-100 were calcu-
lated. The inhibition of binding curves for the conditioned media and cell pellet fractions were parallel to those of purified S-100a and S-100fl. The amount of immunoreactive S-100 in the conditioned media from C6 cells at various stages of cell density is s~own in Fig. 1. For comparison, the immunoreactive levels of intracellular S-100 from the same experiments are also shown. The level of S-100 in conditioned media from subconfluent C6 cells (1-3 days after plating) was approximately 25-fold higher than the intracellular levels of S-100. The amount of extracellular S-100 increased significantly (from 38 fg/cell to 80 fg/cell) when the cells reached confluency (4-5 days after plating), and then decreased in postconfluent cells to about 8 fg/cell by day 16. In addition, there was a significant drop in the ratio of extracellular S-IO0 to intracellular S-IO0 in postconfluent cell populations. These data suggest that C6 cells secrete S-IO0 into the media, possibly in a regulated fashion. While secreted S-IO0 levels are high in subconfluent and confluent cultures and lower in postconfluer.t cultures, intracellular S-I~O levels are low in subconfluent and confluent populations and
m
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7
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/
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40
. . j 20 O O ~¢/)
0
5
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15
Time After Plating (days) Fig. 1. Immunoreactive levels of extracellular S-100 from conditioned media of C6 cells. At various times after plating the cells, the immunoreactive S-100 levels were determined by radioimmunoassay of 3 ammonium sulfate fractions of conditioned media: 0-60% ammonium sulfate pellet, 60-80% ammonium sulfate pH 4 pellet, and 60-80% ammonium sulfate pH 4 supernatant. The mean fg S-100 per cell +_ the S.E.M. was then calculated as described in the text. The total number of determinations using 3 different preparations for each growth condition was three. The extracellular (O) levels of S-100 are shown as a function of days after plating the cells. For comparison, the intracellular (©) levels of S-100 as determined by radioimmunoassay of a 60-80% ammonium sulfate pH 4 pellet are also shown. In these experiments, the cells reached confluency at days 4-5.
369 increase in postconfluent populations. These data suggest that the intracellular and extraceUular forms of S-100 are differentially regulated in C6 cells. It is interesting to note that while imracellular S100 immunoreactivity was exclusively S-100fl, a significant portion of the total extracellular S-100 immunoreactivity was due to reactivity with the anti-S100a antibody: 73% at 3 days, 11% at 5 days, 44% at 9 days, and 47% at 16 days. This may reflect secretion of both S-100a- and S-100fl-like polypeptides. Alternatively, the S-100a immunoreactivity may result from the secreted form of S-100 having immunological properties which are different from those of the intracellular form of S-100. Purification and characterization of the secreted form of S-100 will be necessary before its relationship to the well-characterized S-100a and S-100fl polypeptides can be ascertained. In order to determine what percentage of the extracellular S-100 could be due to the release of intracellular S-100 into the media by iysed cells, we examined the conditioned media for the presence of a soluble cytoplasmic marker enzyme, lactate dehydrogenase (LDH)~ L D H activity was measured at 340_ nm in a 1.0 ml reaction mixture containing 0.2 mM N A D H and 7 mM sodium pyruvate in 94 mM phosphate buffer, pH 7.0. Control experiments showed that when purified L D H (Boehringer Mannheim, Indianapolis, IN) was added to conditioned media, 94% of the exogenously added L D H activity could be recovered. Therefore, the low levels of L D H seen in the C6 conditioned media were not due to inactivation of the enzyme by our experimental procedures. Analysis of the L D H activity in conditioned media is shown in Table I. For comparison, Table I also shows the L D H activity in C6 cell homogenates from the
same experiments in which the conditioned media was obtained. Based on these results, it is possible that as much as 24% of the S-100 detected in the conditioned media from subconfluent cultures could be due to the breakage of cells and the release of intracellular S-100 into the media rather than active secretion of S-100. However, reduction of the extracellular S-100 levels measured from cultures at subconflaenc~ or confluency by 24% and 10% respectively would still result in a 20:1 ratio of extraceilular to intracellular S-100. Thus, the total amount of extracellular S-100 detected cannot be accounted for by a non-specific effect of cell lysis. Consistent with this is the observation that the percent of viable cells as measured by Trypan blue exclusion was > 9 8 % . Our data suggest that the majority of S-100 immunoreactivity detected in the conditioned media of C6 cells at various growth stages is probably due to the active secretion of S-100 from C6 cells. The data presented here suggesting that S-100 is secreted from glial cells are in agreement with previous observations that S-100 immunoreactivity can be released into the extracellular space of the brain 14 or into the media from cultures of adipocytes ~7'18and rat anterior pituitary ceUs6. The function of the secreted form of S-100 is not known. However, the recent finding l° that S-100fl has neurite extension factor activity raise~ the possibility that a form of S-100 may be the neurite extension factor present in the conditioned media of rat C6 cells. Potential neurite extension factor activity of S-100fl is consistent with previous studies that have suggested a role for S-100 in nervous system development. For example, studies on the levels of S-100 m R N A and protein during brain development have shown that S-100 levels increase during the time that neuronal processes are
TABLE I Lactate dehydrogenase activit), h~ C6 conditioned media and cells
"' LDH activity is expressed as units of activity/107cells + S.E.M. where 1 unit = 1!~molpyruvate hydtolyzed/min at 25 °C. ~' % Lvsed cells = LDHcm/(LDHcp + LDHcm) x 100, where LDHcm = LDH activity in the conditioned media, and LDHcp = LDH activity in the cell pellet. Time after plating cells (days)
LDH activity in conditioned media:. LDH activity in cells:' % Lysed cellsb
3
5
9
J~
1.32 +__0.23 4.20 + 1.5 24%
0.37 +__0.03 3.34 +__1.2 10%
0.21 __+0.06 i0.87 +- 1.4 2%
0.27 + 0.01 14.84 +- 2. ! 2%
370 elongating (for review, see ref. 3). It is possible that
S-100 will be i m p o r t a n t areas for future research.
during d e v e l o p m e n t of the central nervous system, S100 is secreted from glial cells and then acts in a paracrine fashion 1o stimulate neurite outgrowth. The m e c h a n i s m s by which S-100 is targeted for secretion and by which the neuron r e s p o n d s to extracellular
W e thank Jay H o d g e and R e b e c c a M i l l e r for careful and c o m p e t e n t assistance with these studies. These studies were s u p p o r t e d in part by N a t i o n a l Institutes of H e a l t h G r a n t GM-33481.
1 Benda, P., Lightbody, J., Sato, G., Levine, L. and Sweet, W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 2 Cocchia, D., Immunocytochemical localization of S-100 protein in the brain of adult rat. An ultrastructural study, Cell Tissue Res., 214 (1981) 529-540. 3 Donato, R., S-100 proteins, Cell Calcium, 7 (1986) 123-145. 4 Haglid, K., Hansson, H. and Ronnback, L., S-100 in the central ner:cas system of rat, rabbit and guinea pig during postnatal development, Brain Research, 123 (1977) 331-345. 5 Hidaka, H., Endo, T., Kawamoto, S., Yamada, E , Umekawa, H., Tanabe, K. and Hara, K., Purification and characterization of adipose tissue S-100fl protein, J. Biol. Chem., 258 (1983) 2705-2709. 6 Ishikawa, H., Nogami, H. and Shirasawa, H., Novel clonal strains from adult rat anterior pituitary producing S-100 protein, Nature (Lond.), 303 (1983) 711-713. 7 Isobe, T. and Okuyama, T., The amino acid sequence of S100 protein (PAP 1-b protein) and its relation to the calcium-binding proteins, Eur. J. Biochem., 89 (1978) 379-388. 8 Isobe, T. and Okuyama, T., The amino acid sequence of the a-subunit in bovine brain S-100a protein, Eur. J. Biochem., 116 (1981) 79-86. 9 Kligman, D., Neurite outgrowth from cerebral cortical neurons is promoted by medium conditioned over heart cells, J. Neurosci. Res., 8 (1982)281-287. 10 K]igman, D. and Marshak, D.R., Purification and characterization of a neurite extension factor from bovine brain, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 7136-7139. ll Labourdette, G. and Marks, A., Synthesis of S-100 protein in monolayer cultures of rat glial cells, Eur. J. Biochem., 58 (1975) 73-79. 12 Moore, B.W., A soluble protein characteristic of the net-
vous system, Piochem. Biophys. Res. Commun., 19 (1965) 739-744. 13 Pfeiffer, S.E., Herschman, H.R., Lightbody, J.E. and Sato, G., Synthesis by a cional line of rat glial cells of a protein unique to the nervous system, J. Cell Physiol., 75 (1971) 329-339. 14 Shashoua, V.E., Hesse, G.W. and Moore, B.W., Proteins of the brain extraceUular fluid: evidence for release of S100 protein, J. Neurochem., 42 (1984) 1536-1541. 15 Stewart, J. and Urban, I., The postnatal accumulation of S100 protein in mouse central nervous system, Dev. Biol., 29 (1972) 372-384. 16 Suzt~ki, F., Nakajima, T. and Kato, K., Peripheral distribution of nervous system-specific S-100 protein in rat, J. Biochem., 92 (1982) 835-838. 17 Suzuki, F., Kato, K. and Nakajima, T., Enhancement of adipose S-100 protein release by catecholamines, J. Biochem., 94 (1983) 1707-1710. 18 Suzuki, F., Kato, K. and Nakajima, T., Regulation of nervous system specific S-100 protein and enolase levels in adipose tissues by catecholamines, J. Neurochem., 42 (1984) 130-134. 19 Unsicker, K., Vey, J., Hofmann, H.-D., Muller, T.H. and Wilson, A.J., C6 glioma-cell conditioned medium induces neurite outgrowth and survival of rat chromaffin cells in vitro: comparison with the effects of nerve growth factor, Proc. Natl. Acad. Sci. U.S.A., 81 (1984)2242-2246. 20 Zimmer, D.B. and Van Eldik, L.J., Tissue distribution of rat S-100a and S-100fl and S-100-binding proteins, Am. J. Physiol: Cell Physiol., 252 (1987) C285-C289. 21 Zuckerman, D., Herschman, H.R. and Levine, L., Appearance of a brain specific antigen (the S-100 protein) during human foetal development, J. Neurochem., 17 (1970) 247-251.
367
Elsevier BRE 22634
Secretion of S-IO0 from rat C6 glioma ceils Linda J. Van Eldik 1'2 and Danna B. Zimmer l Departments of t Cell Biology and 2Pharmacology and Howard Hughes Medical Institute. Van derbilt University, Nashville. TN 37232 ( U. S. A. )
(Acceptcd 1 September 1987) Key words: Radioimmunoassay; Rat glioma celi; Secretion; S-100
Conditioned media from rat C6 glioma cells contain significant levels of S-100 immunoreactivity as determined by radioimmunoassay. The levels of extracellular S-100 detected could not be accounted for by the release of intracellular S-100 into the media from lysed cells. The extracellular form of S-100 exhibits fractionation properties and immunological characteristics that are different from those of the intracellular form of S-100 in C6 cells. While the intracellular S-100 levels increase as a function of days in culture, the extracellular S-100 levels are high until the cells reach confluency and are lower in postconfluent cultures. Altogether, our data suggest that C6 gliema cells secrete S..IO0, and that the quantitative levels of the intracellular and secreted forms of S-100 are differentially regulated.
S-100 is an acidic calcium binding protein which was first isolated 12 as a brain-specific protein fraction. The bovine brain S-100 fraction is composed of primarily two polypeptides, S-100a and S-100fl, which share approximately 50% identity in amino acid sequence 7"8. Although S-100 proteins are found in high levels in brain tissue, they are also present in numerous other tissues 5"16"2°.In the brain, S-100 is localized primarily in glial cells 2"3 and its appearance in brain tissue correlates with maturation of the nervous system 4a5'21. Cultured rat C6 glioma cells also contain S-1001 and are a useful modei system for studying the regulation of these proteins. Previous studies 9"19have demonstrated that conditioned media from rat C6 glioma cell cultures contain a neurite extension factor which appears to be different from nerve growth factor. The neurite extension factor from C6 conditioned media was found to be a heat stable, acidic, low molecular weight protein. More recent studies l° have shown that a neurite extension factor activity from bovine brain is associated with a disulfide form of S-100fl. Taken together, these data suggest that the neurite extension factor activity found in rat C6 glioma conditioned media
may also be S-100fl. As a first step towards addressing this hypothesis, we examined the conditioned media from C6 glioma cells for the presence of S-100 using a radioimmunoassay. Since previous studies ~1a3 have shown that intracellular S-100 levels vary with cell density, we also analyzed the conditioned media from C6 cultures at different times after plating the cells in order to examine whether the levels of extracellular S-100 might vary. The rat C6 glioma cell line was obtained from the AmeriL:an Type Culture Collection (Rockville, MD) and grown in F10 media (Gibco, Grand Island, NJ) supplemented with 15% (v/v) horse serum (Hycione Laboratories, Logan, UT), 2.5% (v/v) fetal calf serum (Hyclone Laboratories, Logan, UT), 2 !~g/ml fungizone (Squibb, Princeton, N J), 50 U/ml penicillin and 50/tg/ml streptomycin (Gibco, Grand Island, N J). Stock cultures were subcultured by trypsinization and plated in T-flasks of 175 cm 2 surface area (Falcon, Oxnard, C A ) containing 50 ml of media at a plating density of approximately 8 x 106 cells/flask. At various times after plating, the coqditioned media and cells were harvested for analysis. All cultures were fed 48 h before harvesting. For each time point,
Correspondence: D.B. Zimmer, 717 Light Hail. Howard Hughes Medical Institute. Vanderbiit University Medical Center. Nashville, TN 37232, U.S.A.
0006-8993/87/$03.50 © 1987 Elsevier Science Publishv 's B.V. (Biomedical Division)
368 1 flask was trypsinized and the cell number/flask was determined using a hemocytometer. The conditioned media was harvested from the remaining flasks, centrifuged at 1250 g in a Beckman TJ-6 centrifuge to remove cell debris, and stored at -20 °C until use. S-100 radioimmunoassays were done using rabbit polyclonal antisera specific for the individual S-100 polypeptides, S-100a and S-100fl 2°. Since the concentration of S-100 in the unfractionated conditioned media was too low to be detected by radioimmunoassay, 3 concentrated fractions af conditioned media (a 0-60% pellet, 60-80% pH 4 pellet, and 60-80% pH 4 supernatant) were prepared by ammonium sulfate precipitation as previously described 2°. Before radioimmunoassay, all fractions were extensively dialyzed to remove the ammonium sulfate. When C6 cell extracts were fractionated in a similar fashion and analyzed by radioimmunoassay, >95% of the S-100 immunoreactivity was found in the 60-80% pH 4 pellet. These data are in agreement with our previous resuits 2° with rat brain extracts. However, analysis of the S-100 immunoreactivity in ammonium sulfate fractions of the C6 conditioned media showed that S100 immunoreactivity was present in all 3 fractions. When conditioned media which contained exogenously added purified bovine brain S-100a and S100/3 was fractionated, 72% of the S-100a and 95% of the S-100fl immunoreactivity could be recovered in the 0-60% pellet and 60-80% pH 4 pellet fractions. No exogenously added S-100a and S-100fl was recovered in the 60-80% pH 4 supernatant. Altogether, these data suggest that the conditioned media alters the fractionation properties of S-100 and/or that the extrace!lular form of S-100 may have different fractionation properties from the intracellular form. Therefore, the immunoreactivity in the 0-60% pellet, 60-80% pH 4 pellet, and 60-80% pH 4 supernatant fractions was used in calculating the levels of extracellular S-100. In addition, we also observed significant levels of S-100a and S-100fl immunoreactivity in the 0-60% pellet fraction of media which had not been incubated with cells. These values accounted for approximately 53% of the total S-100a and 35% of the total S-100fl immunoreactivity detect~.d Jn the conditioned media and were subtracted from the immunoreactivity in the conditioned media before the extracellular levels ef S-100 were calcu-
lated. The inhibition of binding curves for the conditioned media and cell pellet fractions were parallel to those of purified S-100a and S-100fl. The amount of immunoreactive S-100 in the conditioned media from C6 cells at various stages of cell density is s~own in Fig. 1. For comparison, the immunoreactive levels of intracellular S-100 from the same experiments are also shown. The level of S-100 in conditioned media from subconfluent C6 cells (1-3 days after plating) was approximately 25-fold higher than the intracellular levels of S-100. The amount of extracellular S-100 increased significantly (from 38 fg/cell to 80 fg/cell) when the cells reached confluency (4-5 days after plating), and then decreased in postconfluent cells to about 8 fg/cell by day 16. In addition, there was a significant drop in the ratio of extracellular S-IO0 to intracellular S-IO0 in postconfluent cell populations. These data suggest that C6 cells secrete S-IO0 into the media, possibly in a regulated fashion. While secreted S-IO0 levels are high in subconfluent and confluent cultures and lower in postconfluer.t cultures, intracellular S-I~O levels are low in subconfluent and confluent populations and
m
(.I
¢n 80
E ¢J
,;_L'k,
"- 60
7
",
/
V
40
. . j 20 O O ~¢/)
0
5
10
15
Time After Plating (days) Fig. 1. Immunoreactive levels of extracellular S-100 from conditioned media of C6 cells. At various times after plating the cells, the immunoreactive S-100 levels were determined by radioimmunoassay of 3 ammonium sulfate fractions of conditioned media: 0-60% ammonium sulfate pellet, 60-80% ammonium sulfate pH 4 pellet, and 60-80% ammonium sulfate pH 4 supernatant. The mean fg S-100 per cell +_ the S.E.M. was then calculated as described in the text. The total number of determinations using 3 different preparations for each growth condition was three. The extracellular (O) levels of S-100 are shown as a function of days after plating the cells. For comparison, the intracellular (©) levels of S-100 as determined by radioimmunoassay of a 60-80% ammonium sulfate pH 4 pellet are also shown. In these experiments, the cells reached confluency at days 4-5.
369 increase in postconfluent populations. These data suggest that the intracellular and extraceUular forms of S-100 are differentially regulated in C6 cells. It is interesting to note that while imracellular S100 immunoreactivity was exclusively S-100fl, a significant portion of the total extracellular S-100 immunoreactivity was due to reactivity with the anti-S100a antibody: 73% at 3 days, 11% at 5 days, 44% at 9 days, and 47% at 16 days. This may reflect secretion of both S-100a- and S-100fl-like polypeptides. Alternatively, the S-100a immunoreactivity may result from the secreted form of S-100 having immunological properties which are different from those of the intracellular form of S-100. Purification and characterization of the secreted form of S-100 will be necessary before its relationship to the well-characterized S-100a and S-100fl polypeptides can be ascertained. In order to determine what percentage of the extracellular S-100 could be due to the release of intracellular S-100 into the media by iysed cells, we examined the conditioned media for the presence of a soluble cytoplasmic marker enzyme, lactate dehydrogenase (LDH)~ L D H activity was measured at 340_ nm in a 1.0 ml reaction mixture containing 0.2 mM N A D H and 7 mM sodium pyruvate in 94 mM phosphate buffer, pH 7.0. Control experiments showed that when purified L D H (Boehringer Mannheim, Indianapolis, IN) was added to conditioned media, 94% of the exogenously added L D H activity could be recovered. Therefore, the low levels of L D H seen in the C6 conditioned media were not due to inactivation of the enzyme by our experimental procedures. Analysis of the L D H activity in conditioned media is shown in Table I. For comparison, Table I also shows the L D H activity in C6 cell homogenates from the
same experiments in which the conditioned media was obtained. Based on these results, it is possible that as much as 24% of the S-100 detected in the conditioned media from subconfluent cultures could be due to the breakage of cells and the release of intracellular S-100 into the media rather than active secretion of S-100. However, reduction of the extracellular S-100 levels measured from cultures at subconflaenc~ or confluency by 24% and 10% respectively would still result in a 20:1 ratio of extraceilular to intracellular S-100. Thus, the total amount of extracellular S-100 detected cannot be accounted for by a non-specific effect of cell lysis. Consistent with this is the observation that the percent of viable cells as measured by Trypan blue exclusion was > 9 8 % . Our data suggest that the majority of S-100 immunoreactivity detected in the conditioned media of C6 cells at various growth stages is probably due to the active secretion of S-100 from C6 cells. The data presented here suggesting that S-100 is secreted from glial cells are in agreement with previous observations that S-100 immunoreactivity can be released into the extracellular space of the brain 14 or into the media from cultures of adipocytes ~7'18and rat anterior pituitary ceUs6. The function of the secreted form of S-100 is not known. However, the recent finding l° that S-100fl has neurite extension factor activity raise~ the possibility that a form of S-100 may be the neurite extension factor present in the conditioned media of rat C6 cells. Potential neurite extension factor activity of S-100fl is consistent with previous studies that have suggested a role for S-100 in nervous system development. For example, studies on the levels of S-100 m R N A and protein during brain development have shown that S-100 levels increase during the time that neuronal processes are
TABLE I Lactate dehydrogenase activit), h~ C6 conditioned media and cells
"' LDH activity is expressed as units of activity/107cells + S.E.M. where 1 unit = 1!~molpyruvate hydtolyzed/min at 25 °C. ~' % Lvsed cells = LDHcm/(LDHcp + LDHcm) x 100, where LDHcm = LDH activity in the conditioned media, and LDHcp = LDH activity in the cell pellet. Time after plating cells (days)
LDH activity in conditioned media:. LDH activity in cells:' % Lysed cellsb
3
5
9
J~
1.32 +__0.23 4.20 + 1.5 24%
0.37 +__0.03 3.34 +__1.2 10%
0.21 __+0.06 i0.87 +- 1.4 2%
0.27 + 0.01 14.84 +- 2. ! 2%
370 elongating (for review, see ref. 3). It is possible that
S-100 will be i m p o r t a n t areas for future research.
during d e v e l o p m e n t of the central nervous system, S100 is secreted from glial cells and then acts in a paracrine fashion 1o stimulate neurite outgrowth. The m e c h a n i s m s by which S-100 is targeted for secretion and by which the neuron r e s p o n d s to extracellular
W e thank Jay H o d g e and R e b e c c a M i l l e r for careful and c o m p e t e n t assistance with these studies. These studies were s u p p o r t e d in part by N a t i o n a l Institutes of H e a l t h G r a n t GM-33481.
1 Benda, P., Lightbody, J., Sato, G., Levine, L. and Sweet, W., Differentiated rat glial cell strain in tissue culture, Science, 161 (1968) 370-371. 2 Cocchia, D., Immunocytochemical localization of S-100 protein in the brain of adult rat. An ultrastructural study, Cell Tissue Res., 214 (1981) 529-540. 3 Donato, R., S-100 proteins, Cell Calcium, 7 (1986) 123-145. 4 Haglid, K., Hansson, H. and Ronnback, L., S-100 in the central ner:cas system of rat, rabbit and guinea pig during postnatal development, Brain Research, 123 (1977) 331-345. 5 Hidaka, H., Endo, T., Kawamoto, S., Yamada, E , Umekawa, H., Tanabe, K. and Hara, K., Purification and characterization of adipose tissue S-100fl protein, J. Biol. Chem., 258 (1983) 2705-2709. 6 Ishikawa, H., Nogami, H. and Shirasawa, H., Novel clonal strains from adult rat anterior pituitary producing S-100 protein, Nature (Lond.), 303 (1983) 711-713. 7 Isobe, T. and Okuyama, T., The amino acid sequence of S100 protein (PAP 1-b protein) and its relation to the calcium-binding proteins, Eur. J. Biochem., 89 (1978) 379-388. 8 Isobe, T. and Okuyama, T., The amino acid sequence of the a-subunit in bovine brain S-100a protein, Eur. J. Biochem., 116 (1981) 79-86. 9 Kligman, D., Neurite outgrowth from cerebral cortical neurons is promoted by medium conditioned over heart cells, J. Neurosci. Res., 8 (1982)281-287. 10 K]igman, D. and Marshak, D.R., Purification and characterization of a neurite extension factor from bovine brain, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 7136-7139. ll Labourdette, G. and Marks, A., Synthesis of S-100 protein in monolayer cultures of rat glial cells, Eur. J. Biochem., 58 (1975) 73-79. 12 Moore, B.W., A soluble protein characteristic of the net-
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