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Nerve growth factor-mediated differentiation of a nerve cell line cultured in a hormone-supplemented serum-free medium
Developmental Brain Research, 6 (1983) 243-250 Elsevier Biomedical Press
243
Nerve Growth Factor-Mediated Differentiation of a Nerve Cell Line Cultured in a Hormone-Supplemented Serum-Free Medium HIROSHI HATANAKA
Department of Neurochemistry, Mitsubishi-Kasei Institute of Life Sciences, Machida-shi, Tokyo 194 (Japan) (Accepted June 22nd, 1982)
Key words: nerve growth factor - - tyrosine hydroxylase - - pheochromocytoma cell line - - choline acetyltransferase - serum-free medium - - dexamethasone
In newly-isolated subclone of PC12, designated h, nerve growth factor (NGF) is known to cause an increase of tyrosine hydroxylase (TH) activity concomitantly with that of choline acetyltransferase (CAT) activity16. When the PC12h cells were cultured and maintained for several generations in a hormone-supplemented serum-free medium, only TH activity was selectivelyenhanced by NGF, while CAT and glutamic acid decarboxylaseactivities remained unaffected. PC12h cells cultured in serum-free medium could extend long neurites in response to NGF. The loss of the NGF-mediated increase of CAT activity in PCI2h cells cultured in serumfree medium was fully restored upon re-exposure to serum. The NGF-mediated increase of TH activity in PC12h cells cultured in serum-free medium was additive to that increased by an applications of 10-5-10-1° M dexamethasone. In conclusion, PC12h cells cultured in chemically-definedserum-free medium, having responses to NGF identical to sympathetic neurons, will be a useful model for elucidating the molecular mechanism(s) of NGF-mediated neuronal differentiation. INTRODUCTION Nerve growth factor (NGF) is known to be necessary for the development of sympathetic neurons both in vivo and in vitro14,19, 26. One of the characteristic effects of N G F on sympathetic neurons is a selective induction of tyrosine hydroxylase (TH) activity 25, a key enzyme in catecholamine biosynthesis. The PC12 clonal rat pheochromocytoma cell line is responsive to N G F 1° and shares the highly differentiated properties of the sympathetic neurons a4. PC12 cells are capable of synthesis, storage, release and uptake of catecholamines 10-1~. However, in PC12 cells, N G F is reported to induce choline acetyltransferase (CAT) activity13, 23, but not TH activity 8, in reverse to the adrenergic neurons in vivo. In a previous paper 16, a newly-isolated cell line, PC12h, a subclone of the PCI2 cell, was observed as responding to N G F with the increase of T H activity. CAT activity in PC12h cells also increased by the action of NGF. I report here that by culturing in a hormone-supplemented serum-free medium PC12h cells respond selectively to N G F
0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
with an increase of TH activity, but not of CAT and glutamic acid decarboxylase (GAD) activities. MATERIALS AND METHODS PC12 cells were obtained from Dr. L. A. Greene and subcloned as described previously 16. One subclone, PC12h cell was used throughout the present study unless otherwise noted. Culture medium was the same as described previously 16. Briefly, a basal medium (DF medium) consisted of a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F I 2 media (Gibco) supplemented with 1.9 mg/ml sodium bicarbonate, 15 mM Hepes buffer, 50 U/ml penicillin, 100 #g/ml streptomycin and 30 nM selenium. For the cultivation of PC12h cells in a serum-containing medium, H S / P N C S / D F medium consisted of D F medium supplemented with 5 ~ (v/v) heat-inactivated horse serum (HS, Gibco) and 5 ~ (v/v) precolostrum newborn calf serum (PNCS, MitsubishiKasei Nakashibetsu Serum Center). T I P / D F medium was made for the serum-free cultivation, and consisted of D F medium supplemented with 5 #g/
of PC12h cells cultured in TIP/D!? (A, B) and DF (C)media without (4 land with @a Fig. 1. Phas #e-Contras;t photomicrographs md C) NGF at a cancer Itration of 50 ng/ml. Cells were plated at a density of lo4 cells/sq.cm on collagen-coatt :d F:a1co,n dirrhes, in TIP/DF me odium. Af ier overnight cultivation, the medium was changed to TIP/DF or DF medium with 01 wit1lout NGI F. ClidIS were cultiva rted for 6 days. Bar represents 100 /Lrn. PC1 2h cells cultured in TlP/DF and DF media significantly c:xte!ndedlthel Ieur’ites in response to NGF.
245 ml human transferrin (Sigma), 5/~g/ml bovine insulin (Collab. Res.) and 20 nM progesterone (Sigma) (refs. 3, 16). TIP/DF medium was prepared with minor changes from the serum-free medium originally developed by Bottenstein and Sato a. PC12h cells were usually maintained in a glass culture flask (TD 40, Ikemoto) using HS/PNCS/DF medium. For the serum-free cultivation, cells were plated on 6 cm oi 10 cm collagen-coated Falcon dishes. After overnight cultivation, medium was changed to TIP/DF medium. The cells cultured in TIP/DF medium grew at almost the same rate as those in HS/PNCS/DF medium. PC12h cells cultured in TIP/DF medium can replate on the collagen-coated dishes by using TIP/DF medium. Thus, cells were able to maintain and multiply continually in the TIP/DF medium. NGF (2.5S form) was prepared from male mouse salivary gland by the method of Bocchini and Angeletti 5. TH, CAT and GAD activities were determined as previously reported 15,16. TH activity in an aliquot of cell suspension was measured by the detection of decarboxylated 14CO2 from L-[1-14C]tyrosine (New England Nuclear, final concentration of 0.5 mM) coupled with aromatic amino acid decarboxylase27. CAT activity was measured by the detection of solvent extractable [14C]acetylcholine formed from [1-14C]acetyl CoA (Amersham, final concentration 0.1 mM) 9. GAD activity was measured by the detection of decarboxylated 14CO2 from L-[l-14C]glutamic acid (Amersham, final concentration 1 mM) as previously described 15. Initial reaction rates of all enzyme activities were linear with time and amount of cell suspension. Protein was estimated by the method of Lowry et al. 2° (for TH and CAT), and of Higuchi and Yoshida 17 (for GAD), using bovine serum albumin as the standard. RESULTS Cell culture in serum-free medium PC12h cells can grow in a serum-free medium supplemented with transferrin, insulin and progesterone (TIP). The cells cultured in TIP/DF medium grew at almost the same rate as those in HS/PNCS/ DF medium. The apparent doubling times of PC 12h cells cultured in HS/PNCS/DF and TIP/DF media were about 77 and 89 h, respectively. PC12h cells did
not survive long (over 2 days) in DF medium without hormone-supplementation. PC12h cells cultured even in the TIP/DF medium extended neurites in response to NGF as shown in Fig. lB. Figure 1A shows that the PC12h cells cultured in the TIP/DF medium were indistinguishable from those cultured in serum-containing medium. The rate of the neurite outgrowth was similar to that in serum-containing medium. As previously reported (ref. 16), I have obtained the other subclones, namely PC12a and j, which did not extend the neurite remarkably when cultured in the serum-containing medium (see Fig. 2B, E). When PC12a and j cells were cultured in the TIP/DF medium, both cells extended the neurites as well as PC12h cells (Fig. 2C,
F). NGF-mediated selective increase of T H activity in PC12h cells cultured in serum-free medium As previously described, NGF caused an increase in TH activity in PC12h cells 16, in contrast to the original PC12 cells s in which no increase of TH activity by NGF was observed. The CAT activity also increased by NGF in PC12h cells as in the original PC12 cells. The NGF-responsive TH and CAT activities were still observed in the PC12h cells which were transferred from the serum-containing medium to the TIP/DF medium, as described previously16. In order to eliminate the effect of the residual serum on PC12h cells, PC12h cells were replated and maintained for several generations in TIP/DF medium. Fig. 3 represents the dose-response characteristics for the N G F effects on TH, CAT and glutamic acid decarboxylase (GAD) activities in those cells. Only TH activity was increased by NGF. The EDs0 value of N G F was 10 ng/ml (4 × 10-10 M). Neither CAT nor GAD activity was influenced by the addition of NGF even at the concentration of 300 ng/ml (1 × 10-8 M). These results indicate that the regulation by N G F of the above enzyme activities in PC12h cells cultured in serumfree medium occurred selectively. Then, the selective response of N G F had been masked during the cultivation with serum. The loss of the NGF-mediated increase of CAT activity in PC12h cells by culturing in serum-free medium suggests a factor(s) in serum necessary for the NGF-mediated increase of CAT activity. The lack of the response was
246
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Fig. 2. Phase-contrast photomicrographs of PCI2a c~lls (A, 13 and C) and PcI2j cells (D, E and F) cultured in serum-containing and serum-free media. A and D: cells were plated at the density of 104 cells/sq.cm in HS/PNCS/DF medium and then cultured without N G F for 7 days. B and E : same as A and D except for culture with N G F at the concentration of 50 ng/ml for 7 days. NG Fmediated fiber outgrowth was scarcely observed in both cells. C and F: cells were plated the same as A and D. Culture was carried out in TIP/DF medium with N G F at the concentration of 50 ng/ml for 7 days. Both cells cultured in TIP/DF medium significantly extended the neurites in response to NGF. The morphology of PCI2a and j ceils cultured in TIP/DF mediunl without N G F was: similar to the one in HS/PNCS/DF medium (not shown). Bar represents 100/tn~.
247 ~ l ,
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observed in PC 12h cells cultured for 4 generations in the TIP/DF medium, and was still present after 16 generations cultured in the TIP/DF medium. During the above period, PC12h cells maintained the NGFresponsiveness in the increase of the TH activity. As shown in Fig. 4, the NGF-mediated increase of CAT activity was restored upon re-exposure to serum, indicating that the NGF-responsiveness of the increase of CAT activity in PCI2h cells had not been damaged irreversibly by the cultivation in the TIP/DF medium.
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Fig. 3. Effect of N G F on specific TH (a), CAT (b) and G A D (c) activities in PC12h cells cultured in TIP/DF medium. PC12h cells were maintained in TIP/DF medium. For the determination of TH and CAT activities, the cells (cultured for several generations with T1P/DF medium) were plated at a density of 2.5 × 104 cells/sq.cm on 6 cm collagen-coated Falcon dishes in TIP/DF medium. After overnight culture, the medium was changed to the TIP/DF medium containing the indicated amount of NGF. After further culture for 5 days, the cells were collected. For the determination of G A D activity, PC12h cells were plated at the density of 105 cells/ sq.cm on dishes as above. TH, CAT a n d G A D activities were measured as described in text. Values are means :k S.E.M. on triplicate cultures.
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Fig. 4. Effect of serum on NGF-responsiveness with the increases of specific TH (a) and CAT (b) activities in PC12h cells which had been cultured in TIP/DF medium. PC12h cells were cultivated the same as described in the legend to Fig. 3. Serum (HS or PNCS) was added to TIP/DF medium at the final concentration of 5 ~ (v/v). After 5 days' culture, the cells were served for the determination of enzyme activities. TH and CAT activities were measured as described in the text. Open and stippled columns represent without and with N G F respectively at a concentration of 50 ng/ml.
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Effect of NGF on T H and C A T activities in PC12h cells cultured in serum-free medium with no hormone supplementation PC12h cells could survive in the presence of N G F even in serum-free medium with no hormone supplementation (see Fig. I C). Fig. 6 shows that the high specific activity of T H in P C l 2 h cells was still observed when cultured in D F medium in the presence of N G F and that specific CAT activity gradually decreased during the cultivation in D F medium with N G F . The ratio of specific TH and CAT activities of PCI2h cells cultured in T I P / D F to DF medium in the presence of N G F for 6 days was 0.87 and 1.83, respectively. PC12h cells cultured in I)F medium with N G F could extend the neurites, as shown in Fig. IC.
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Fig. 5. Effect of dexamethasone on NGF-mediated increase of specific TH activity in PC12h cells cultured in the TIP/DF medium. PC12h cells were plated as described in the legend to Fig. 3 and cultured in the TIP/DF medium in the presence of the different amounts of dexamethasone (Sigma). Half number of culture dishes were served with 50 ng/ml of NGF. After culture for 5 days the cells were collected for the determination of TH activity. TH activity was measured as described in the text. Values are mean ± S.E.M. on triplicate cultures. Open and closed circles represent the cultures with and without NGF, respectively.
activity in P C I 2 h cells cultured even in the T I P / D F medium. However, 10 -6 M dexamethasone does not affect cell morphology including neurite outgrowth. C A T activities of the cells cultured in the T I P / D F medium without and with 10 -6 M dexamethasone were 71.6 ± 32.4 and 46.2 ± 7.2 pmol/min/mg protein (n = 3), respectively. These opposite effects of dexamethasone on T H and C A T activities in PC12 cells were reported recently 24. As can be seen in Fig. 5, the N G F - m e d i a t e d increase of T H activity in PC12h cells cultured in the T I P / D F medium was additive to the one elicited by 10-5-10 -~° M dexamethasone. The N G F dose-dependency of T H activity o f PC12h cells cultured with 10 -6 M dexamethasone was almost the same as one shown in Fig. 3 (data not shown). These results indicate t h a t dexamethasone had an independent action on N G F effect to cause an increase of T H activity in PC12h cells.
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PC12h cells cultured in DF meAham,:PCI2h cells were plated as described in the legend to Figi 3 and cultured overnight in TIP/DF medium. At day 0, medium was changed to TIP/DF medium without NGF (open squares): with 50 ng/ml of NGF (closed squares)or DF m~iium with:5O=ng/mtof NGF (closed circles). After the indicated days' culture the cells were used for the determination of enzyme activities. TH and CAT actiVitles were measured as described in text. Values were means S.E.M. on triplicate cultures.
249 DISCUSSION The experiments described here show that when PC12h cells are grown for several generations in a hormone-supplemented serum-free medium, NGF responsiveness on neurotransmitter syntheses becomes selective. In the serum-free culture conditions, TH activity was selectively enhanced by NGF, while CAT activity remained unaffected. Thus, the NGF-enhanced adrenergic property in catecholamine synthesis of PCI2h cells show that those differentiated PC12h cells lesemble the normal sympathetic neuron. The presence of serum in the culture medium may mislead the cell-differentiation mediated by NGF. An ideal culture system should exclude serum, because most cells in vivo are not directly exposed to serum. Since many cell lines have been grown in the defined serum-free media which are supplemented with hormones instead of sera 1,4, these culture s~stems should contribute to the study of the molecular mechanisms of hormone actionsL When HeLa cells were cultured in serum-containing medium, the down-regulation of mitogenesis elicited by epidermal growth factor occurred. However, the receptor down-regulation in HeLa cells was not observed in serum-free culture 28. The character of fl-adrenergic receptor in a glioma cell line changed by the serumfree cultivation7. Recently, Darmon et al. 6 reported that an embryonal carcinoma line C17-$1 (clone 1003) grown in serum-free medium differentiated into limited phenotypes, predominately neuroepithelial and neuronal cells, and a serum addition allowed multiplication of cells and maintenance of undifferentiated phenotype. Neuronal development depends on exogenous environments during the embryonic stages. The developing autonomic neurons in culture are remarkably plastic and are influenced by various chemical environments. LeDouarinlS and her colleagues have shown that the embryonic environment can influence the differentiated fate of neural crest cells. And also, Patterson 22 and his colleagues discovered the similar phenomena using cultures of dissociated rat sympathetic neurons. The type of transmitter (acetylcholine or catecholamine) and the type
of synapses formed by these cells were controlled by the exogenous chemical environments such as the conditioned medium of non-neuronal cells and/ol NGF. However, there has been little detailed information on the mechanisms of the action of the factors which influence the fate-decision of neuronal cells. Cultured neuronal cells, having the factorzesponsiveness, might be expected to be useful in the detailed examination of the fundamental processes. As shown in Fig. 5, dexamethasone had an independent action on NGF effect to cause an increase of TH activity in PC12h cells. This ~esult clearly differs from the effect of dexamethasone in modulating the action of NGF on TH activity in the original PC12 cells21,24. The postulated precursor stem cell of the adrenengic cell derived from the neural crest cell may differentiate into adrenal medullary secretory cells and sympathetic neurons in an early stage of development2L Glucocorticoid and NGF may decide to differentiate into the respective cells. Although both medullary cells and sympathetic ganglionic cells have high catecholamine synthesis activity, the former functions as secretory cell and the latter as a neuron having characteristic neurites. In PC 12h cells cultured in serum-free medium, the exposure to NGF resulted in the acquisition of distinctive sympathetic neuronal properties, including fiber-outgrowth, while dexamethasone caused only an increase in TH activity. The present PC12h cells cultured in the conditions of the chemically-defined medium, having responses to NGF identical to the sympathetic neuron, will be a useful model for elucidating the molecular mechanisms of the NGFmediated effect on neuronal differentiation. Two advantages in using the PC12h cells are the availability of a large number of the pule population, and the priming of NGF response in PC12h cells cultured in the absence of NGF. Also, these cells are able to escape from the effects of unknown factors in serum. ACKNOWLEDGEMENTS I thank Drs. M. Mitsuka, A. Ogura and M. Takahashi for their discussion and critical reading of the manuscript. I also thank Mrs. Junko Fujita-Shimizu for her technical assistance.
250 REFERENCES 1 Barnes, D. and Stao, G., Methods for growth of cultured cells in serum-free medium, ,4nalyt. Bioehem., 102 (1980) 255-270. 2 Barnes, D. and Sato, G., Serum-free cell culture: a unifying approach, Cell, 22 (1980) 649-655. 3 Bottenstein, J. E. and Sato, G. H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. nat. ,4cad. Sci. U.S.A., 76 (1979) 514-517. 4 Bottenstein, J., Hayashi, T., Hutchings, S., Masui, H., Mather, J., McClure, D. B., Ohasa, S., Rizzino, A., Sato, G., Serrero, G., Wolfe, R. and Wu, R., The growth of cells in serum-free hormone-supplemented media. In W. B. Jakoby and I. H. Pastan (Eds.), Methods in Enzymology, Vol. 58, Academic Press, NY, 1979, pp. 94-109. 5 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000-molecular-weight protein, Proc. nat. ,4cad. Sci. U.S.A., 64 (1969) 787--794. 6 Darmon, M., Bottenstein, J. and Sato, G., Neural differentiation following culture of embryonal carcinoma cells in a serum-free defined medium, Develop. Biol., 85 (1981 ) 463-473. 7 Dibner, M. D., Wolfe, R. A. and lnsel, P. A., Replacement of serum with a defined medium increases/3-adrenergic receptor number in cultured glioma cells, Exp. Cell Res., 131 (1981)424~-27. 8 Edgar, D. H. and Thoenen, H., Selective enzyme induction in a nerve growth factor-responsive pheochromocytoma cell line (PCI2), Brain Res., 154 (1978) 186-190. 9 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 10 Greene, L. A. and Tischler, A. S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 2424-2428. 11 Greene, L. A. and Rein, G., Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells, Brain Res., 129 (1977) 247-263. 12 Greene, L.A.and Rein, G., Release of [Z3H]norepinephrine from a clonal line of pheochromocytoma cells (PCI2) by nicotinic cholinergic stimulation, Brain Res., 138 (1977) 521-528. 13 Greene, L. A. and Rein, G., Synthesis, storage and release of acetylcholine by a noradrenergic pheochromocytoma cell line, Nature (Lond.), 268 (1977) 349-351. 14 Greene, L. A. and Shooter, E. M., The nerve growth factor: biochemistry, synthesis, and mechanism of action,
Ann. Rev. Neurosci., 3 (1980) 353-402. 15 Hatanaka, H., Tanaka, M. and Amano, T., A clonal rat pheochromocytoma cell line possesses synthesizing ability of ~,-aminobutyric acid together with catecholamine and acetylcholine, Brain Res., 183 (1980) 490-493. 16 Hatanaka, H., Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line, Brain Res., 222 (1981) 225--233. 17 Higuchi, M. and Yoshida, F., Lowry determination of protein in the presence of sulfhydryl compounds or other reducing agents, Analyt. Bioehem., 77 (1977) 5 4 2 547. 18 LeDouarin, N. M., The ontogeny of the neural crest in avian embryo chimaeras, Nature (Lond.), 286 (1980)663 ..... 669. 19 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 20 Lowry, O. H., Rosebrough, N. J~, Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 21 Otten, U. and Towbin, M., Permissive action of glucocorticoids in induction of tyrosine hydroxylase by nerve growth factor in a pheochromocytoma cells line, Brain Res., 193 (1980) 304-308. 22 Patterson, P. H., Environmental determination of autonomic neurotransmitter functions, Ann, Rev. Neurosci.~ 1 (1978) 1-17. 23 Schubert, D., Heinemann, S. and Kidokoro, Y., Cholinergic metabolism and synapse formation by a rat nerve cell line, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 2579-2583. 24 Schubert, D., LaCorbiere, M., Klier, F. G. and Steinbach, .I, H., The modulation of neurotransmitter synthesis by steroid hormones and insulin, Brain Res., 190 (1980) 67 -79. 25 Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. and Kettler, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglia, Proe. nat. Aead. SoL U.S.A., 68 (1971) 1598-1602. 26 Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, Physiol. Rev., 60 (t980) 1284-1335. 27 Waymire, J. C., Bjur, R. and Weiner, N., Assay of tyrosine hydroxylase by coupled decarboxylation of dopa formed from [1-14C]L-tyrosine, Analyt. Bioehem., 43 (1971) 588-600. 28 Wolfe, R. A., Wu, R. and Sato, G. H., Epidermal growth factor-induced down-regulation of receptor does not occur in HeLa cells grown in defined medium, Proe. nat. Aead. Sci. U.S.A., 77 (1980) 2735-2739.
243
Nerve Growth Factor-Mediated Differentiation of a Nerve Cell Line Cultured in a Hormone-Supplemented Serum-Free Medium HIROSHI HATANAKA
Department of Neurochemistry, Mitsubishi-Kasei Institute of Life Sciences, Machida-shi, Tokyo 194 (Japan) (Accepted June 22nd, 1982)
Key words: nerve growth factor - - tyrosine hydroxylase - - pheochromocytoma cell line - - choline acetyltransferase - serum-free medium - - dexamethasone
In newly-isolated subclone of PC12, designated h, nerve growth factor (NGF) is known to cause an increase of tyrosine hydroxylase (TH) activity concomitantly with that of choline acetyltransferase (CAT) activity16. When the PC12h cells were cultured and maintained for several generations in a hormone-supplemented serum-free medium, only TH activity was selectivelyenhanced by NGF, while CAT and glutamic acid decarboxylaseactivities remained unaffected. PC12h cells cultured in serum-free medium could extend long neurites in response to NGF. The loss of the NGF-mediated increase of CAT activity in PCI2h cells cultured in serumfree medium was fully restored upon re-exposure to serum. The NGF-mediated increase of TH activity in PC12h cells cultured in serum-free medium was additive to that increased by an applications of 10-5-10-1° M dexamethasone. In conclusion, PC12h cells cultured in chemically-definedserum-free medium, having responses to NGF identical to sympathetic neurons, will be a useful model for elucidating the molecular mechanism(s) of NGF-mediated neuronal differentiation. INTRODUCTION Nerve growth factor (NGF) is known to be necessary for the development of sympathetic neurons both in vivo and in vitro14,19, 26. One of the characteristic effects of N G F on sympathetic neurons is a selective induction of tyrosine hydroxylase (TH) activity 25, a key enzyme in catecholamine biosynthesis. The PC12 clonal rat pheochromocytoma cell line is responsive to N G F 1° and shares the highly differentiated properties of the sympathetic neurons a4. PC12 cells are capable of synthesis, storage, release and uptake of catecholamines 10-1~. However, in PC12 cells, N G F is reported to induce choline acetyltransferase (CAT) activity13, 23, but not TH activity 8, in reverse to the adrenergic neurons in vivo. In a previous paper 16, a newly-isolated cell line, PC12h, a subclone of the PCI2 cell, was observed as responding to N G F with the increase of T H activity. CAT activity in PC12h cells also increased by the action of NGF. I report here that by culturing in a hormone-supplemented serum-free medium PC12h cells respond selectively to N G F
0165-3806/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
with an increase of TH activity, but not of CAT and glutamic acid decarboxylase (GAD) activities. MATERIALS AND METHODS PC12 cells were obtained from Dr. L. A. Greene and subcloned as described previously 16. One subclone, PC12h cell was used throughout the present study unless otherwise noted. Culture medium was the same as described previously 16. Briefly, a basal medium (DF medium) consisted of a 1:1 mixture of Dulbecco's modified Eagle's and Ham's F I 2 media (Gibco) supplemented with 1.9 mg/ml sodium bicarbonate, 15 mM Hepes buffer, 50 U/ml penicillin, 100 #g/ml streptomycin and 30 nM selenium. For the cultivation of PC12h cells in a serum-containing medium, H S / P N C S / D F medium consisted of D F medium supplemented with 5 ~ (v/v) heat-inactivated horse serum (HS, Gibco) and 5 ~ (v/v) precolostrum newborn calf serum (PNCS, MitsubishiKasei Nakashibetsu Serum Center). T I P / D F medium was made for the serum-free cultivation, and consisted of D F medium supplemented with 5 #g/
of PC12h cells cultured in TIP/D!? (A, B) and DF (C)media without (4 land with @a Fig. 1. Phas #e-Contras;t photomicrographs md C) NGF at a cancer Itration of 50 ng/ml. Cells were plated at a density of lo4 cells/sq.cm on collagen-coatt :d F:a1co,n dirrhes, in TIP/DF me odium. Af ier overnight cultivation, the medium was changed to TIP/DF or DF medium with 01 wit1lout NGI F. ClidIS were cultiva rted for 6 days. Bar represents 100 /Lrn. PC1 2h cells cultured in TlP/DF and DF media significantly c:xte!ndedlthel Ieur’ites in response to NGF.
245 ml human transferrin (Sigma), 5/~g/ml bovine insulin (Collab. Res.) and 20 nM progesterone (Sigma) (refs. 3, 16). TIP/DF medium was prepared with minor changes from the serum-free medium originally developed by Bottenstein and Sato a. PC12h cells were usually maintained in a glass culture flask (TD 40, Ikemoto) using HS/PNCS/DF medium. For the serum-free cultivation, cells were plated on 6 cm oi 10 cm collagen-coated Falcon dishes. After overnight cultivation, medium was changed to TIP/DF medium. The cells cultured in TIP/DF medium grew at almost the same rate as those in HS/PNCS/DF medium. PC12h cells cultured in TIP/DF medium can replate on the collagen-coated dishes by using TIP/DF medium. Thus, cells were able to maintain and multiply continually in the TIP/DF medium. NGF (2.5S form) was prepared from male mouse salivary gland by the method of Bocchini and Angeletti 5. TH, CAT and GAD activities were determined as previously reported 15,16. TH activity in an aliquot of cell suspension was measured by the detection of decarboxylated 14CO2 from L-[1-14C]tyrosine (New England Nuclear, final concentration of 0.5 mM) coupled with aromatic amino acid decarboxylase27. CAT activity was measured by the detection of solvent extractable [14C]acetylcholine formed from [1-14C]acetyl CoA (Amersham, final concentration 0.1 mM) 9. GAD activity was measured by the detection of decarboxylated 14CO2 from L-[l-14C]glutamic acid (Amersham, final concentration 1 mM) as previously described 15. Initial reaction rates of all enzyme activities were linear with time and amount of cell suspension. Protein was estimated by the method of Lowry et al. 2° (for TH and CAT), and of Higuchi and Yoshida 17 (for GAD), using bovine serum albumin as the standard. RESULTS Cell culture in serum-free medium PC12h cells can grow in a serum-free medium supplemented with transferrin, insulin and progesterone (TIP). The cells cultured in TIP/DF medium grew at almost the same rate as those in HS/PNCS/ DF medium. The apparent doubling times of PC 12h cells cultured in HS/PNCS/DF and TIP/DF media were about 77 and 89 h, respectively. PC12h cells did
not survive long (over 2 days) in DF medium without hormone-supplementation. PC12h cells cultured even in the TIP/DF medium extended neurites in response to NGF as shown in Fig. lB. Figure 1A shows that the PC12h cells cultured in the TIP/DF medium were indistinguishable from those cultured in serum-containing medium. The rate of the neurite outgrowth was similar to that in serum-containing medium. As previously reported (ref. 16), I have obtained the other subclones, namely PC12a and j, which did not extend the neurite remarkably when cultured in the serum-containing medium (see Fig. 2B, E). When PC12a and j cells were cultured in the TIP/DF medium, both cells extended the neurites as well as PC12h cells (Fig. 2C,
F). NGF-mediated selective increase of T H activity in PC12h cells cultured in serum-free medium As previously described, NGF caused an increase in TH activity in PC12h cells 16, in contrast to the original PC12 cells s in which no increase of TH activity by NGF was observed. The CAT activity also increased by NGF in PC12h cells as in the original PC12 cells. The NGF-responsive TH and CAT activities were still observed in the PC12h cells which were transferred from the serum-containing medium to the TIP/DF medium, as described previously16. In order to eliminate the effect of the residual serum on PC12h cells, PC12h cells were replated and maintained for several generations in TIP/DF medium. Fig. 3 represents the dose-response characteristics for the N G F effects on TH, CAT and glutamic acid decarboxylase (GAD) activities in those cells. Only TH activity was increased by NGF. The EDs0 value of N G F was 10 ng/ml (4 × 10-10 M). Neither CAT nor GAD activity was influenced by the addition of NGF even at the concentration of 300 ng/ml (1 × 10-8 M). These results indicate that the regulation by N G F of the above enzyme activities in PC12h cells cultured in serumfree medium occurred selectively. Then, the selective response of N G F had been masked during the cultivation with serum. The loss of the NGF-mediated increase of CAT activity in PC12h cells by culturing in serum-free medium suggests a factor(s) in serum necessary for the NGF-mediated increase of CAT activity. The lack of the response was
246
..........................
. i
...........................................
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Fig. 2. Phase-contrast photomicrographs of PCI2a c~lls (A, 13 and C) and PcI2j cells (D, E and F) cultured in serum-containing and serum-free media. A and D: cells were plated at the density of 104 cells/sq.cm in HS/PNCS/DF medium and then cultured without N G F for 7 days. B and E : same as A and D except for culture with N G F at the concentration of 50 ng/ml for 7 days. NG Fmediated fiber outgrowth was scarcely observed in both cells. C and F: cells were plated the same as A and D. Culture was carried out in TIP/DF medium with N G F at the concentration of 50 ng/ml for 7 days. Both cells cultured in TIP/DF medium significantly extended the neurites in response to NGF. The morphology of PCI2a and j ceils cultured in TIP/DF mediunl without N G F was: similar to the one in HS/PNCS/DF medium (not shown). Bar represents 100/tn~.
247 ~ l ,
600
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,
observed in PC 12h cells cultured for 4 generations in the TIP/DF medium, and was still present after 16 generations cultured in the TIP/DF medium. During the above period, PC12h cells maintained the NGFresponsiveness in the increase of the TH activity. As shown in Fig. 4, the NGF-mediated increase of CAT activity was restored upon re-exposure to serum, indicating that the NGF-responsiveness of the increase of CAT activity in PCI2h cells had not been damaged irreversibly by the cultivation in the TIP/DF medium.
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D e x a m e t h a s o n e has b e e n k n o w n t o i n c r e a s e t h e T H a c t i v i t y in t h e o r i g i n a l P C 1 2 cells21, 24 a n d the
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Fig. 3. Effect of N G F on specific TH (a), CAT (b) and G A D (c) activities in PC12h cells cultured in TIP/DF medium. PC12h cells were maintained in TIP/DF medium. For the determination of TH and CAT activities, the cells (cultured for several generations with T1P/DF medium) were plated at a density of 2.5 × 104 cells/sq.cm on 6 cm collagen-coated Falcon dishes in TIP/DF medium. After overnight culture, the medium was changed to the TIP/DF medium containing the indicated amount of NGF. After further culture for 5 days, the cells were collected. For the determination of G A D activity, PC12h cells were plated at the density of 105 cells/ sq.cm on dishes as above. TH, CAT a n d G A D activities were measured as described in text. Values are means :k S.E.M. on triplicate cultures.
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Fig. 4. Effect of serum on NGF-responsiveness with the increases of specific TH (a) and CAT (b) activities in PC12h cells which had been cultured in TIP/DF medium. PC12h cells were cultivated the same as described in the legend to Fig. 3. Serum (HS or PNCS) was added to TIP/DF medium at the final concentration of 5 ~ (v/v). After 5 days' culture, the cells were served for the determination of enzyme activities. TH and CAT activities were measured as described in the text. Open and stippled columns represent without and with N G F respectively at a concentration of 50 ng/ml.
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Effect of NGF on T H and C A T activities in PC12h cells cultured in serum-free medium with no hormone supplementation PC12h cells could survive in the presence of N G F even in serum-free medium with no hormone supplementation (see Fig. I C). Fig. 6 shows that the high specific activity of T H in P C l 2 h cells was still observed when cultured in D F medium in the presence of N G F and that specific CAT activity gradually decreased during the cultivation in D F medium with N G F . The ratio of specific TH and CAT activities of PCI2h cells cultured in T I P / D F to DF medium in the presence of N G F for 6 days was 0.87 and 1.83, respectively. PC12h cells cultured in I)F medium with N G F could extend the neurites, as shown in Fig. IC.
--~
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dexamethasone (M)
Fig. 5. Effect of dexamethasone on NGF-mediated increase of specific TH activity in PC12h cells cultured in the TIP/DF medium. PC12h cells were plated as described in the legend to Fig. 3 and cultured in the TIP/DF medium in the presence of the different amounts of dexamethasone (Sigma). Half number of culture dishes were served with 50 ng/ml of NGF. After culture for 5 days the cells were collected for the determination of TH activity. TH activity was measured as described in the text. Values are mean ± S.E.M. on triplicate cultures. Open and closed circles represent the cultures with and without NGF, respectively.
activity in P C I 2 h cells cultured even in the T I P / D F medium. However, 10 -6 M dexamethasone does not affect cell morphology including neurite outgrowth. C A T activities of the cells cultured in the T I P / D F medium without and with 10 -6 M dexamethasone were 71.6 ± 32.4 and 46.2 ± 7.2 pmol/min/mg protein (n = 3), respectively. These opposite effects of dexamethasone on T H and C A T activities in PC12 cells were reported recently 24. As can be seen in Fig. 5, the N G F - m e d i a t e d increase of T H activity in PC12h cells cultured in the T I P / D F medium was additive to the one elicited by 10-5-10 -~° M dexamethasone. The N G F dose-dependency of T H activity o f PC12h cells cultured with 10 -6 M dexamethasone was almost the same as one shown in Fig. 3 (data not shown). These results indicate t h a t dexamethasone had an independent action on N G F effect to cause an increase of T H activity in PC12h cells.
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PC12h cells cultured in DF meAham,:PCI2h cells were plated as described in the legend to Figi 3 and cultured overnight in TIP/DF medium. At day 0, medium was changed to TIP/DF medium without NGF (open squares): with 50 ng/ml of NGF (closed squares)or DF m~iium with:5O=ng/mtof NGF (closed circles). After the indicated days' culture the cells were used for the determination of enzyme activities. TH and CAT actiVitles were measured as described in text. Values were means S.E.M. on triplicate cultures.
249 DISCUSSION The experiments described here show that when PC12h cells are grown for several generations in a hormone-supplemented serum-free medium, NGF responsiveness on neurotransmitter syntheses becomes selective. In the serum-free culture conditions, TH activity was selectively enhanced by NGF, while CAT activity remained unaffected. Thus, the NGF-enhanced adrenergic property in catecholamine synthesis of PCI2h cells show that those differentiated PC12h cells lesemble the normal sympathetic neuron. The presence of serum in the culture medium may mislead the cell-differentiation mediated by NGF. An ideal culture system should exclude serum, because most cells in vivo are not directly exposed to serum. Since many cell lines have been grown in the defined serum-free media which are supplemented with hormones instead of sera 1,4, these culture s~stems should contribute to the study of the molecular mechanisms of hormone actionsL When HeLa cells were cultured in serum-containing medium, the down-regulation of mitogenesis elicited by epidermal growth factor occurred. However, the receptor down-regulation in HeLa cells was not observed in serum-free culture 28. The character of fl-adrenergic receptor in a glioma cell line changed by the serumfree cultivation7. Recently, Darmon et al. 6 reported that an embryonal carcinoma line C17-$1 (clone 1003) grown in serum-free medium differentiated into limited phenotypes, predominately neuroepithelial and neuronal cells, and a serum addition allowed multiplication of cells and maintenance of undifferentiated phenotype. Neuronal development depends on exogenous environments during the embryonic stages. The developing autonomic neurons in culture are remarkably plastic and are influenced by various chemical environments. LeDouarinlS and her colleagues have shown that the embryonic environment can influence the differentiated fate of neural crest cells. And also, Patterson 22 and his colleagues discovered the similar phenomena using cultures of dissociated rat sympathetic neurons. The type of transmitter (acetylcholine or catecholamine) and the type
of synapses formed by these cells were controlled by the exogenous chemical environments such as the conditioned medium of non-neuronal cells and/ol NGF. However, there has been little detailed information on the mechanisms of the action of the factors which influence the fate-decision of neuronal cells. Cultured neuronal cells, having the factorzesponsiveness, might be expected to be useful in the detailed examination of the fundamental processes. As shown in Fig. 5, dexamethasone had an independent action on NGF effect to cause an increase of TH activity in PC12h cells. This ~esult clearly differs from the effect of dexamethasone in modulating the action of NGF on TH activity in the original PC12 cells21,24. The postulated precursor stem cell of the adrenengic cell derived from the neural crest cell may differentiate into adrenal medullary secretory cells and sympathetic neurons in an early stage of development2L Glucocorticoid and NGF may decide to differentiate into the respective cells. Although both medullary cells and sympathetic ganglionic cells have high catecholamine synthesis activity, the former functions as secretory cell and the latter as a neuron having characteristic neurites. In PC 12h cells cultured in serum-free medium, the exposure to NGF resulted in the acquisition of distinctive sympathetic neuronal properties, including fiber-outgrowth, while dexamethasone caused only an increase in TH activity. The present PC12h cells cultured in the conditions of the chemically-defined medium, having responses to NGF identical to the sympathetic neuron, will be a useful model for elucidating the molecular mechanisms of the NGFmediated effect on neuronal differentiation. Two advantages in using the PC12h cells are the availability of a large number of the pule population, and the priming of NGF response in PC12h cells cultured in the absence of NGF. Also, these cells are able to escape from the effects of unknown factors in serum. ACKNOWLEDGEMENTS I thank Drs. M. Mitsuka, A. Ogura and M. Takahashi for their discussion and critical reading of the manuscript. I also thank Mrs. Junko Fujita-Shimizu for her technical assistance.
250 REFERENCES 1 Barnes, D. and Stao, G., Methods for growth of cultured cells in serum-free medium, ,4nalyt. Bioehem., 102 (1980) 255-270. 2 Barnes, D. and Sato, G., Serum-free cell culture: a unifying approach, Cell, 22 (1980) 649-655. 3 Bottenstein, J. E. and Sato, G. H., Growth of a rat neuroblastoma cell line in serum-free supplemented medium, Proc. nat. ,4cad. Sci. U.S.A., 76 (1979) 514-517. 4 Bottenstein, J., Hayashi, T., Hutchings, S., Masui, H., Mather, J., McClure, D. B., Ohasa, S., Rizzino, A., Sato, G., Serrero, G., Wolfe, R. and Wu, R., The growth of cells in serum-free hormone-supplemented media. In W. B. Jakoby and I. H. Pastan (Eds.), Methods in Enzymology, Vol. 58, Academic Press, NY, 1979, pp. 94-109. 5 Bocchini, V. and Angeletti, P. U., The nerve growth factor: purification as a 30,000-molecular-weight protein, Proc. nat. ,4cad. Sci. U.S.A., 64 (1969) 787--794. 6 Darmon, M., Bottenstein, J. and Sato, G., Neural differentiation following culture of embryonal carcinoma cells in a serum-free defined medium, Develop. Biol., 85 (1981 ) 463-473. 7 Dibner, M. D., Wolfe, R. A. and lnsel, P. A., Replacement of serum with a defined medium increases/3-adrenergic receptor number in cultured glioma cells, Exp. Cell Res., 131 (1981)424~-27. 8 Edgar, D. H. and Thoenen, H., Selective enzyme induction in a nerve growth factor-responsive pheochromocytoma cell line (PCI2), Brain Res., 154 (1978) 186-190. 9 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 10 Greene, L. A. and Tischler, A. S., Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor, Proc. nat. Acad. Sci. U.S.A., 73 (1976) 2424-2428. 11 Greene, L. A. and Rein, G., Release, storage and uptake of catecholamines by a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells, Brain Res., 129 (1977) 247-263. 12 Greene, L.A.and Rein, G., Release of [Z3H]norepinephrine from a clonal line of pheochromocytoma cells (PCI2) by nicotinic cholinergic stimulation, Brain Res., 138 (1977) 521-528. 13 Greene, L. A. and Rein, G., Synthesis, storage and release of acetylcholine by a noradrenergic pheochromocytoma cell line, Nature (Lond.), 268 (1977) 349-351. 14 Greene, L. A. and Shooter, E. M., The nerve growth factor: biochemistry, synthesis, and mechanism of action,
Ann. Rev. Neurosci., 3 (1980) 353-402. 15 Hatanaka, H., Tanaka, M. and Amano, T., A clonal rat pheochromocytoma cell line possesses synthesizing ability of ~,-aminobutyric acid together with catecholamine and acetylcholine, Brain Res., 183 (1980) 490-493. 16 Hatanaka, H., Nerve growth factor-mediated stimulation of tyrosine hydroxylase activity in a clonal rat pheochromocytoma cell line, Brain Res., 222 (1981) 225--233. 17 Higuchi, M. and Yoshida, F., Lowry determination of protein in the presence of sulfhydryl compounds or other reducing agents, Analyt. Bioehem., 77 (1977) 5 4 2 547. 18 LeDouarin, N. M., The ontogeny of the neural crest in avian embryo chimaeras, Nature (Lond.), 286 (1980)663 ..... 669. 19 Levi-Montalcini, R. and Angeletti, P. U., Nerve growth factor, Physiol. Rev., 48 (1968) 534-569. 20 Lowry, O. H., Rosebrough, N. J~, Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 21 Otten, U. and Towbin, M., Permissive action of glucocorticoids in induction of tyrosine hydroxylase by nerve growth factor in a pheochromocytoma cells line, Brain Res., 193 (1980) 304-308. 22 Patterson, P. H., Environmental determination of autonomic neurotransmitter functions, Ann, Rev. Neurosci.~ 1 (1978) 1-17. 23 Schubert, D., Heinemann, S. and Kidokoro, Y., Cholinergic metabolism and synapse formation by a rat nerve cell line, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 2579-2583. 24 Schubert, D., LaCorbiere, M., Klier, F. G. and Steinbach, .I, H., The modulation of neurotransmitter synthesis by steroid hormones and insulin, Brain Res., 190 (1980) 67 -79. 25 Thoenen, H., Angeletti, P. U., Levi-Montalcini, R. and Kettler, R., Selective induction by nerve growth factor of tyrosine hydroxylase and dopamine-fl-hydroxylase in the rat superior cervical ganglia, Proe. nat. Aead. SoL U.S.A., 68 (1971) 1598-1602. 26 Thoenen, H. and Barde, Y.-A., Physiology of nerve growth factor, Physiol. Rev., 60 (t980) 1284-1335. 27 Waymire, J. C., Bjur, R. and Weiner, N., Assay of tyrosine hydroxylase by coupled decarboxylation of dopa formed from [1-14C]L-tyrosine, Analyt. Bioehem., 43 (1971) 588-600. 28 Wolfe, R. A., Wu, R. and Sato, G. H., Epidermal growth factor-induced down-regulation of receptor does not occur in HeLa cells grown in defined medium, Proe. nat. Aead. Sci. U.S.A., 77 (1980) 2735-2739.