We have analyzed the effects of cAMP-elevating drugs (dibutyryl cAMP, forskolin, and isobutyl methylxan-thine) on growth properties and myelin-specific gene expression in the peripheral neurinoma cell line D6P2T. The steady-state levels of RNA and Polypeptide for the two major PNS myelin proteins, P0 glycoprotein (P0) and myelin basic protein (MBP), were measured by Northern blotting and immunoblotting, respectively. The levels of the two RNAs in individual cells were examined by in situ hybridization. The transcriptional activities of the P0 and MBP genes were analyzed by nuclear run-off experiments. Treatment with cAMP-elevating agents caused cell aggregation and dose-dependent increase in growth control. Expression of P0 RNA was constitutive in untreated cells and was repressed at high doses. Expression of MBP RNA was induced at low doses and repressed at higher doses. For both MBP and P0 the effects on gene expression were first detected after a lag of approximately 6h, were manifested in all cells and were mediated, at least in part, at the transcriptional level. The level of P0 Polypeptide was proportional to the level of P0 RNA, but MBP Polypeptide was not detectable even under conditions where MBP RNA was induced. The results with this clonal model suggest that cAMP plays a pivotal role in regulation of growth and gene expression during Schwann cell differentiation.

Myelin is a multilamellar membrane sheath that wraps around axons, providing electrical insulation and facilitating saltatory nerve conduction. It is formed as a plasma membrane extension of Schwann cells in the peripheral nervous system and of oligodendrocytes in the central nervous system. Both Schwann cells and oligodendrocytes undergo dramatic metamorphoses in cellular phenotype during myelination. Prior to myeli-nogenesis they are migratory, proliferative cells which exhibit relatively few differentiated features. During myelinogenesis they cease proliferation and migration, form intimate associations with axons and express a variety of myelin-specific genes. When myelin formation is complete, the expression of myelin-specific genes is repressed. The work described here is concerned with the general question of how these changes in phenotype are regulated.

Previous studies on differentiation of glial cells have used primary culture systems (Pfeiffer, 1984). Oligo-dendrocytes maintained in primary culture express a variety of myelin-related components (Mirsky et al. 1980). In contrast, Schwann cells in primary culture repress the expression of myelin components unless suitable axonal contact and collagen containing subtrata are provided (Bunge et al. 1983). The reason for this difference in the regulation of myelin gene expression is not clear. cAMP-elevating drugs have been shown to stimulate expression of some myelin components in both oligodendrocytes and Schwann cells in culture (McMorris, 1983; Sobue & Pleasure, 1984; Sobue et al. 1986; Pleasure et al. 1985; Shuman et al. 1988; Lemke and Chao, 1988).

P0 glycoprotein (P0) and myelin basic protein (MBP) are the major protein components of peripheral nervous system myelin. P0 is expressed exclusively in Schwann cells (Trapp et al. 1981) as a single protein species encoded by a single species of mRNA (Lemke & Axel, 1985). MBP is expressed in both Schwann cells and oligodendrocytes (Lees & Brostoff, 1984) as a group of structurally related Polypeptides encoded by a family of mRNAs generated from a single gene by differential combinatorial splicing (Takahashi et al. 1985; de Ferra et al. 1985). Developmental studies on P0 and MBP gene expression indicate that both are induced at the beginning of myelination, reach a maximum level of expression during the period of most rapid myelin formation, and are repressed after myelination is complete (Carson et al. 1983; Zeller et al. 1984; Lemke & Axel, 1985). Quantitative immunocytochemical studies in developing rat PNS indicate that P0 expression precedes MBP expression by several days (Hahn et al. 1987). For both P0 and MBP, the RNA and Polypeptide developmental profiles are parallel (Lemke & Axel, 1985; Carson et al. 1983), suggesting that protein expression is largely determined by the steadystate level of the corresP0nding mRNA. The work described in this paper was undertaken to determine how P0 and MBP gene expression is regulated in the peripheral neurinoma cell line D6P2T as a model for understanding the regulation of gene expression during normal Schwann cell development. A preliminary report of this work has appeared (Yang et al. 1987).

Cell culture

Tumor cell line RT4-D6 was obtained from Dr Sueoka (University of Colorado) (Imada & Sueoka, 1978; Imada et al. 1978; Tomozawa & Sueoka, 1978; Tomozawa et al. 1985). The original tumor, RT4, gave rise to a pluripotent stem cell line RT4-AC, which spontaneously converts in culture to a variety of different cell types. Two neuronal lines, RT4-B and RT4-E, and one glial line RT4-D were established (Imada & Sueoka, 1978; Imada et al. 1978; Tomozawa & Sueoka, 1978; Tomozawa et al. 1985; Droms & Sueoka, 1987). RT4-D6 was subcloned from the glial line to generate fine D6P2T that exhibits a variety of Schwann cell characteristics (Bansal & Pfeiffer, 1987). Unless otherwise indicated, cells were cultured in Dulbecco’s modified Eagle’s medium (DME) supplemented with 5 % fetal calf serum in a humidified atmosphere of CO2: air (5 % : 95 %) at 37 °C. Medium was changed every three days. For analysis of RNA or protein, cells were seeded into 100 mm culture dishes (2 × 106 cells/dish) and grown for three days. Treatment with drugs affecting intracellular cAMP levels were initiated by replacing the growth medium with fresh medium containing the desired concentration of the appropriate agent. After culture the cells were collected, frozen in liquid nitrogen, and stored at −70°C. For analysis of cell proliferation, cells were grown in twenty-four multiwell culture plates (3 × 104cells/well), collected and resuspended in 0·04 % Nonidet-P40 to dissolve the plasma membrane and release the nuclei which were counted by hemocytometry.

Cyclic AMP elevating drugs

D6P2T cells were treated with the following pharmacological agents that increase intracellular cAMP: (1) dibutyryl cAMP (DBC) which is a cAMP analogue that enters cells by passive diffusion (Miller, 1977); (2) forskolin (FK) which stimulates adenylate cyclase (de Souza et al. 1983) thereby increasing cAMP synthesis; and (3) isobutyl methylxanthine (IBMX) which inhibits phosphodiesterase (Russell, 1979) thereby decreasing cAMP breakdown.

Isolation of total RNA

Total RNA was isolated by the guanidinium isothiocyanate cesium chloride method (Glisin et al. 1974) and stored at −70°C. Yields were approximately 100 Mg of RNA per confluent 100 mm culture, and 2·7, 3·8, and 3 mg g−1 wet weight from 14 day old rat sciatic nerve, brain and liver, respectively.

Northern analysis of specific RNA

Total RNA (10 μg) was denatured with dimethylsulfoxide and glyoxal and analyzed by gel electrophoresis (McMaster & Carmichael, 1977). Ribosomal RNA was quantified by ethidium bromide staining in order to normalize for small variations in RNA concentration among the samples. MBP and P0 RNA were analyzed by Northern blotting. The separated RNA was transferred to charge-modified nylon membrane (Gene Screen Plus, New England Nuclear Corp.) by capillary transfer as described by the manufacturer. Specific RNAs were identified by overnight hybridization at 42 °C with denatured 32P-labeled cDNA probes prepared by random oligonucleotide priming (Feinberg & Vogelstein, 1983). The cDNA probe for MBP mRNA was prepared using the 1·5 kb EcoRI insert from pMBP-1 (Roach et al. 1983) containing a partial cDNA for rat 14K MBP. The cDNA probe for P0 mRNA was prepared using the 1·85 kb EcoRI insert from pSN63 (Lemke & Axel, 1985) containing a fulllength cDNA for rat P0. The blots were washed at 65°C and exposed to film with intensifying screens at −70 °C. Exposure times were adjusted (30 min to 6 h) to insure that the band intensities were within the linear response range of film. Autoradiogram bands were quantified by densitometry. The integrated area of the band on the autoradiogram was normalized to the integrated area of the ethidium bromide stained ribosomal RNA bands in the same sample.

Protein determination

Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.

Immunoblotting

The cell pellet was sonicated in PBS (1 mg protein ml−1) and incubated overnight in 10 % SDS. Samples were incubated in sample buffer containing 1 % mercaptoethanol and subjected to 13 % Polyacrylamide gel electrophoresis (PAGE) (Maizel, 1971). The separated proteins were electrophoretically transferred to nitrocellulose membrane (Towbin et al. 1979). The membrane was incubated first with either affinity-purified rabbit anti-14K MBP (1:200) (Barbarese et al. 1977) or rabbit anti-P0 (1:1000) (obtained from B. Trapp), then with affinitypurified goat anti-rabbit IgG (1:1000) conjugated to alkaline phosphatase (obtained from Cappel laboraories). The bands were visualized by incubating with alkaline phosphatase substrate (Blake et al. 1984) and quantified by reflectance densitometry (Kerner & Carson, 1984).

Immunodot blotting

The cell pellet was sonicated in PBS, heated at 95 °C for 15 min in 1 % SDS, and then incubated 30 min at 37 °C. Appropriate dilutions were prepared in 96-multiwell plates, filtered (maximum of 500 μg per well) onto a prewet nitrocellulose paper in a Schleicher & Schuell microfiltration apparatus at room temperature under vacuum for 30 min. The paper was stained, as described above for immunoblotting, with affinity-purified rabbit anti-14K MBP (1:200), and phosphatase-conjugated affinity-purified goat anti-rabbit IgG (1:1000). The level of MBP was determined by comparison to serial dilutions of purified rabbit MBP. The lower limit of sensitivity was 100 pg per spot.

In situ hybridization

The in situ hybridization procedure of Singer et al. (1986) was used. 35S-labeled cDNA probes for P0 and MBP RNA were prepared by random oligonucleotide priming (Feinberg & Vogelstein, 1983) using the 1·85 kb EcoRI insert from pSN63 (Lemke & Axel, 1985) or the 1·5 kb EcoRl insert from pMBP-1 (Roach et al, 1983). The autoradiography procedure of Oliver (1984) was used. The developed slides were counter stained for 5 min with 0·2 % Giemsa stain in PBS. To quantify grain densities over individual cells dark-field images of random fields were digitized using the Argus 100 image-processing sytem (Hamamatsu). The grain density over each cell was determined by integrating the total intensity over the cell and subtracting the total intensity over an adjacent background region of equivalent area. Grain densities obtained by this technique were proP0rtional to actual grain counts over a wide range.

Nuclear run off

This assay measures the transcriptional activity of specific genes in isolated nuclei (McKnight & Palmiter, 1979; Groudine et al. 1981; Greenberg & Ziff, 1984). The procedure of Greenberg & Ziff (1984) was used with minor modifications. Medium was removed from two to three 100 mm culture plates. The cells (approximately 3 × 107) were washed two times with ice-cold phosphate-buffered saline (PBS), scraped in 2ml of PBS, and centrifuged at 500g for 5min. The cell pellet was resuspended in 1ml lysis buffer (10 mm-Tris-HCl pH7·5, 10 mm-NaCl, 3 mm-MgC12, 0·5% (v/v) NP40), incubated 10 min on ice, centrifuged at 500g for 5 min. The nuclear pellet was washed once with 2 ml of lysis buffer and centrifuged at 500g for 5 min. The final nuclear pellet was resuspended in 100 μl of freezing buffer (50 mm-Tris-HCl pH 8·3, 40 % (v/v) glycerol, 5 m.m-MgCl2, 0·1 mm-EDTA) and frozen in liquid N2. For transcription assays, the nuclei were thawed and mixed with 100 μl of reaction buffer (10 mm-Tris-HCl pH 8·0, 5 mm-MgC12, 300 mm-KCl), 0·5 HIM each of ATP, CTP, GTP and 150 μCi a32PUTP(800 Ci mmol−1, NÈN), and incubated at 30 °C for 30min. The reaction was terminated by the addition of 1 μl of 10 mg ml−1 DNase I (BRL, RNase-free DNase I) and incubation at 37°C for 5 min. After that, 23 μl of 10 % SDS and 2 μl of 10 mg ml−1 proteinase K (BRL) were added and incubated at 42 °C for 30 min. RNA was isolated by phenol–chloroform extraction, ethanol precipitation, and reprecipitation at 4 °C for 24h with 50 μl 10 mm-Hepes pH 7·5, 1 mm-EDTA) and 50 μl of lithium chloride. The RNA pellet was resuspended in 50 μl 10 mm-Hepes pH 7·5, 10 mm-EDTA, 0·2% SDS, 0·l m-NaCl, partially hydrolysed with 10 μl of 1 N-NaOH on ice for 10 min to fragment the RNA, and neutralized by adding 20 μl of 2 m-Hepes pH 5·6. The 32P-labelled RNA was then denatured at 70°C for 10min, cooled on ice, and added to hybridization solution (1% SDS, 1 m-NaCl, and 10 % dextran sulfate) at 5 × 106 cts min−1 ml−1.

To prepare immobilized DNA on filters, 10 μg of plasmid DNA was denatured with 0·2 N-NaOH at room temperature for 30 min, heated to 100 °C for 1 min, neutralized with 10 volumes of ice cold 6 × SSC, and blotted onto Gene Screen Plus filter (NEN) with slow flow rate. The recombinant plasmid DNAs were: pSVLmbp (Barbarese et al. 1988b) containing a partial cDNA of 1·5 kb for rat 14K MBP in the vector pJC119; Psvlp0 (Barbarese et al. 1988b) containing a full-length cDNA of 1·85 kb for rat P0 in the vector pJC119; pTl containing a partial cDNA of 1·36 kb for chicken α-tubulin (Cleveland et al. 1980) in the vector pBR322.

[32P]labeled RNA solution was hybridized to the filter at 65 °C for 72 h. The filter was then washed two times with wash solution (1 × SSC and 1 % SDS) at room temperature, 5 min each, and two times at 65 °C, 30min each. The filter was further incubated at 37 °C in 2 × SSC with 10 μg ml−1 RNase A (Sigma) for 30 min, and washed again in wash solution with 10 μl of 10 mg ml proteinase K (BRL) at 65 °C for 1h, air dried, and exposed to Kodak XAR-5 X-ray film with intensifying screen for 24 h.

Growth properties

Untreated D6P2T cells grown at low cell density have spindle-shaped cell bodies and extend long bipolar processes (Fig. 1A). In this respect they resemble Schwann cells grown in vitro (Wood, 1976). At confluency they remain spindle-shaped but extend shorter processes. At very high density the cells overlap and become spherical. Untreated cells have a doubling time of ∼20h and a saturation density of ∼2×106cells/well (Fig. 2). D6P2T cells grown in the presence of cAMP-elevating drugs exhibit decreased substrate adherence and clump together (Fig. 1B). Aggregation can be prevented by growing the cells on Polyornithine-coated dishes (data not shown). Treatment with cAMP-elevating drugs does not significantly affect the doubling time of the cells, but does increase density-dependent growth control in a dose-dependent manner, resulting in a reduction of the saturation density from ∼2 × 106 (untreated) to ∼5 × 105 cells/well (Fig. 2). We have not seen any evidence for extensive cell death in treated cultures. These results indicate that elevated intracellular cAMP has three specific effects on the growth properties of D6P2T cells: (1) decreased cell-substratum adhesion, (2) increased cell-cell adhesion, and (3) increased density-dependent growth control. It is possible that some or all of these phenomena are interrelated and/or may reflect changes in the surface properties of D6P2T cells.

Fig. 1.

Effect of DBC on D6P2T cell morphology. Phase contrast photomicrograph of D6P2T cells. (Panel A) Cells grown in DME plus 5 % fetal calf serum. (Panel B) Cells grown in DME plus 1 mm-DBC for two days. Bar: 37 μm.

Fig. 1.

Effect of DBC on D6P2T cell morphology. Phase contrast photomicrograph of D6P2T cells. (Panel A) Cells grown in DME plus 5 % fetal calf serum. (Panel B) Cells grown in DME plus 1 mm-DBC for two days. Bar: 37 μm.

Fig. 2.

Effect of DBC on proliferation of D6P2T cells Cells were grown in different concentrations of DBC and counted at intervals. The number of cells is plotted on a log scale. Each P0int represents the mean of three samples. At day 5, the cell number in cultures grown in 200, 500, and 1000 μm-DBC are significantly different from controls (P<0·05; two-tailed Student’s t-test).

Fig. 2.

Effect of DBC on proliferation of D6P2T cells Cells were grown in different concentrations of DBC and counted at intervals. The number of cells is plotted on a log scale. Each P0int represents the mean of three samples. At day 5, the cell number in cultures grown in 200, 500, and 1000 μm-DBC are significantly different from controls (P<0·05; two-tailed Student’s t-test).

P0- and MBP-RNA expression

We examined the steady-state levels of P0 and MBP RNA in D6P2T cells and in various control tissues by Northern blotting (Fig. 3). P0 RNA is undetectable in brain or liver while in sciatic nerve there is a single band of ∼1·9 kb as reported previously by Lemke & Axel (1985). Untreated D6P2T cells express a single band of P0 RNA which is approximately the same size as in rat sciatic nerve. This indicates that P0 gene expression is constitutive in untreated D6P2T cells, and suggests that P0 gene transcription and RNA processing are qualitatively similar in D6P2T cells and in sciatic nerve since the size of the P0 RNA is comparable. The steady-state level of P0 RNA (expressed as units of P0 RNAμg total RNA) in untreated D6P2T is approximately 3 % of the level in 14 day old rat sciatic nerve Treatment of D6P2T cells for 48 h with 1 mm-DBC reduced the steady-state level of P0 RNA to ∼0·6% of the level in rat sciatic nerve. Butyrate (1mm), which should not affect intracellular cAMP levels, did not affect P0 RNA levels, indicating that the effects of DBC are attributable to its action as a cAMP analogue rather than to the butyrate moiety.

Fig. 3.

Northern analysis of P0 and MBP RNA. Total RNA was denatured with dimethylsulfoxide and glyoxal, subjected to agarose gel electrophoresis, blotted to Gene Screen Plus membranes and hybridized with 32P-labelled probes. ExP0sure time was 1h with intensifying screen. (Panel A) Hybridized with P0 cDNA insert from plasmid pSN63. (Panel B) Hybridized with MBP cDNA insert from pMBP-1. RNA from 14 day rat: lane 1, brain; lane 2, liver; lane 3, sciatic nerve. Lane 4, RNA from untreated D6P2T cells. RNA from D6P2T cells grown for 48 h in: lane 5, 1 mm-DBC; lane 6, 1 mm butyrate. RNA sizes were determined by comparison to molecular weight markers including BMV RNA (3·3, 3, 2·27, 0·85 kb) and ribosomal RNA (4·9, 1·9kb).

Fig. 3.

Northern analysis of P0 and MBP RNA. Total RNA was denatured with dimethylsulfoxide and glyoxal, subjected to agarose gel electrophoresis, blotted to Gene Screen Plus membranes and hybridized with 32P-labelled probes. ExP0sure time was 1h with intensifying screen. (Panel A) Hybridized with P0 cDNA insert from plasmid pSN63. (Panel B) Hybridized with MBP cDNA insert from pMBP-1. RNA from 14 day rat: lane 1, brain; lane 2, liver; lane 3, sciatic nerve. Lane 4, RNA from untreated D6P2T cells. RNA from D6P2T cells grown for 48 h in: lane 5, 1 mm-DBC; lane 6, 1 mm butyrate. RNA sizes were determined by comparison to molecular weight markers including BMV RNA (3·3, 3, 2·27, 0·85 kb) and ribosomal RNA (4·9, 1·9kb).

MBP RNA is undetectable in liver but is expressed as a heterodisperse band of ∼2kb in brain and sciatic nerve. Untreated D6P2T cells do not express detectable MBP RNA, but DBC-treated cells express a single MBP RNA band which is approximately the same size as the 2 kb MBP RNA expressed in brain and sciatic nerve. This indicates that MBP gene expression is induced by elevated cAMP levels and suggests that MBP transcription and RNA processing are qualitatively similar in D6P2T cells and in rat brain and sciatic nerve since the size of the MBP RNA is comparable. The steady-state level of MBP RNA in DBC-treated D6P2T cells is ∼0·2 % of the level in sciatic nerve from 14 day old rat. Butyrate did not induce expression of MBP RNA.

The effects of DBC on P0 and MBP gene expression are not due to cell aggregation because similar effects are seen when the cells are grown on Polyomithine which prevents aggregation (data not shown).

Dose-response characteristics for cAMP-elevating drugs

To examine the dose–response characteristics for the effects of cAMP-elevating drugs on P0 and MBP gene expression, cells were treated for 48 h with various concentrations of FK, IBMX or DBC before isolation and analysis of the steady-state levels of P0 and MBP RNA (Fig. 4 and 5). In the case of FK, a dose of 1 μM induces MBP RN A. Doses of FK above 100 μm repress both P0 and MBP RNA. Under some growth conditions repression is seen at lower doses of FK (data not shown), indicating that the effects of FK are dependent on the physiological state of the cell. In the case of IBMX, a dose of 50 μM induces MBP RNA. There also appears to be some stimulation of P0 RNA expression at this dose. Doses of IBMX above 500 μM repress both P0 and MBP RNA. In the case of DBC, a dose of 50 μM induces MBP RNA while doses above 100 μM repress P0 RNA. Repression of MBP RNA at high doses was not observed with DBC. If one assumes that the level of intracellular cAMP is proportional to the dose of cAMP-elevating drug, the results indicate that a small increase in intracellular cAMP levels induces both P0 and MBP RNAs (induction is not as pronounced for P0 because of the constitutive basal level of P0 RNA expression in untreated D6P2T cells) while a larger increase in intracellular cAMP represses both RNAs (except in the case of DBC which did not repress MBP RNA at high doses).

Fig. 4.

Dose–response curves for effects of FK, IBMX and DBC on P0 and MBP RNA. D6P2T cells were treated for 48 h with various concentrations of FK (Panels A and D), IBMX (Panels B and E) or DBC (Panels C and F). Total RNA was isolated and subjected to Northern analysis with either P0 cDNA (Panels A, B and C) or MBP cDNA (Panels D, E and F). Ten μg of total RNA was applied to each lane.

Fig. 4.

Dose–response curves for effects of FK, IBMX and DBC on P0 and MBP RNA. D6P2T cells were treated for 48 h with various concentrations of FK (Panels A and D), IBMX (Panels B and E) or DBC (Panels C and F). Total RNA was isolated and subjected to Northern analysis with either P0 cDNA (Panels A, B and C) or MBP cDNA (Panels D, E and F). Ten μg of total RNA was applied to each lane.

Fig. 5.

Quantification of dose–response curves for FK, IBMX and DBC on P0 and MBP RNA. The levels of P0 and MBP RNA in each lane in Fig. 4 were quantified by densitometry. Variations in the amount of RNA applied to the gel were corrected by normalizing to the amount of 18S ribosomal RNA determined by ethidium bromide staining of the gel. Open squares: levels of P0 RNA; Closed squares: levels of MBP RNA.

Fig. 5.

Quantification of dose–response curves for FK, IBMX and DBC on P0 and MBP RNA. The levels of P0 and MBP RNA in each lane in Fig. 4 were quantified by densitometry. Variations in the amount of RNA applied to the gel were corrected by normalizing to the amount of 18S ribosomal RNA determined by ethidium bromide staining of the gel. Open squares: levels of P0 RNA; Closed squares: levels of MBP RNA.

Kinetics of cAMP regulation of P0 and MBP RNA

The dose–response curves for the effects of FK and IBMX on P0 and MBP RNA are biphasic, with induction at low doses and repression at high doses. This complicates kinetic analysis of the response with these two drugs since at a particular dose one might expect an initial induction phase as cAMP levels begin to rise followed by a repression phase as cAMP levels rise further. To avoid these potential complications we examined the kinetics of the response with DBC because with this drug induction of P0 RNA at low doses and repression of MBP at high doses are not observed. Thus, at a single dose one can examine repression of P0 RNA and induction of MBP RNA without the confounding variables of the antagonistic responses. D6P2T cells were grown in Imm-DBC for various lengths of time before isolation and analysis of specific RNAs. The results (Fig, 6) show reciprocal kinetics for cAMP regulation of the two genes. The level of P0 RNA is unchanged for the first 6 h of DBC treatment, decreases rapidly during the next 36h, and continues to decrease at a slower rate up to 9 days. MBP RNA is first detected after 6 h of DBC treatment, increases rapidly up to 72 h, and continues to increase slowly up to 9 days. These results indicate that repression of P0 RNA and induction of MBP RNA are both ‘delayed’ cAMP responses.

Fig. 6.

Time course for DBC repression of P0 RNA and induction of MBP RNA. D6P2T cells were treated with 1 mm-DBC for various lengths of time. The levels of P0 RNA and MBP RNA were determined by Northern blot analysis and are expressed as arbitrary units with the highest value defined as 1. The values shown represent the mean of two experiments. Open squares: P0 RNA levels; Closed squares: MBP RNA levels.

Fig. 6.

Time course for DBC repression of P0 RNA and induction of MBP RNA. D6P2T cells were treated with 1 mm-DBC for various lengths of time. The levels of P0 RNA and MBP RNA were determined by Northern blot analysis and are expressed as arbitrary units with the highest value defined as 1. The values shown represent the mean of two experiments. Open squares: P0 RNA levels; Closed squares: MBP RNA levels.

cAMP regulation of P0 and MBP gene transcription

The effect of DBC treatment on transcription of P0 and MBP RNA in D6P2T cells was examined using a nuclear run-off technique. Nuclei were isolated from D6P2T cells after 24 to 60 h of DBC treatment and the amount of P0 or MBP transcription was determined. Tubulin transcription was also assayed as a control. The results (Fig. 7) indicate that during DBC treatment P0 transcription is repressed, MBP transcription is induced, and tubulin transcription is stimulated. The kinetics and the extent of repression of P0 transcription and induction of MBP transcription during DBC treatment are generally consistent with the changes in steady-state levels P0 and MBP RNA during DBC treatment shown in Fig. 4, indicating that cAMP repression of P0 gene expression and induction of MBP gene expression is manifested, at least partially, at the transcriptional level. Experiments to measure P0 and MBP RNA turnover after inhibiting transcription with actinomycin D did not reveal any effects of elevated cAMP on RNA stability (data not shown).

Fig. 7.

Run-off analysis of P0 and MBP gene transcription in DBC-treated cells. Nuclei were isolated from D6P2T cells treated with 1 mm-DBC for various lengths of time and were assayed for P0, MBP and tubulin transcriptional activity by transcription run-off as described in Materials and methods. The results shown are from a representative experiment. In the case of P0 and tubulin, similar results were obtained in three separate experiments. In the case of MBP, transcription was below the level of detection in some experiments, however, significant induction of transcription was measured in two separate experiments. Plasmid pJC119 which is the cloning vector for the P0 and MBP cDNAs was used as a control for nonspecific hybridization. Specific transcriptional activity was determined by subtracting the nonspecific hybridization to pJC119 and is expressed in arbitrary units with the highest value defined as 1. Open squares: P0 RNA synthesis; Closed squares: MBP RNA synthesis; Open circles: tubulin RNA synthesis.

Fig. 7.

Run-off analysis of P0 and MBP gene transcription in DBC-treated cells. Nuclei were isolated from D6P2T cells treated with 1 mm-DBC for various lengths of time and were assayed for P0, MBP and tubulin transcriptional activity by transcription run-off as described in Materials and methods. The results shown are from a representative experiment. In the case of P0 and tubulin, similar results were obtained in three separate experiments. In the case of MBP, transcription was below the level of detection in some experiments, however, significant induction of transcription was measured in two separate experiments. Plasmid pJC119 which is the cloning vector for the P0 and MBP cDNAs was used as a control for nonspecific hybridization. Specific transcriptional activity was determined by subtracting the nonspecific hybridization to pJC119 and is expressed in arbitrary units with the highest value defined as 1. Open squares: P0 RNA synthesis; Closed squares: MBP RNA synthesis; Open circles: tubulin RNA synthesis.

In situ hybridization of P0 and MBP RNA

In order to determine whether the effects of elevated cAMP on P0 and MBP RNAs were exhibited by the entire D6P2T cell Population or by a subpopulation of cAMP-responsive cells the cultures were examined by in situ hybridization with cDNA probes for P0 and MBP RNAs. Representative fields are shown in Fig. 8. In untreated cultures all cells show grain densities greater than background when probed for P0 RNA (grain density/cell = 1021 ± 479) but no cells show grain densities greater than background when probed for MBP RNA (grain density/cell<10). In DBC-treated cultures all the cells show grain densities greater than background when probed for both P0 (grain density/cell = 546 ± 396) and MBP (grain density/cell = 604 ± 437), but for P0 RNA the grain density per cell is decreased compared to untreated cells. These results indicate that the entire D6P2T Population exhibits cAMP regulation of P0 and MBP RNA expression.

Fig. 8.

In situ hybridization of P0 and MBP RNA in untreated and DBC-treated D6P2T cells. Cultures of P2T cells were grown for 48 h either untreated (Panels A and C) or in the presence of 1 mm-DBC (Panels B and D). The cells were subjected to in situ hybridization with 35S-labeled probe for P0 RNA (Panels A and B) or MBP RNA (Panels C and D) as described in Materials and methods. Shown are Giemsa counter-stained cells with brightfield illumination. Bar: 10 μm.

Fig. 8.

In situ hybridization of P0 and MBP RNA in untreated and DBC-treated D6P2T cells. Cultures of P2T cells were grown for 48 h either untreated (Panels A and C) or in the presence of 1 mm-DBC (Panels B and D). The cells were subjected to in situ hybridization with 35S-labeled probe for P0 RNA (Panels A and B) or MBP RNA (Panels C and D) as described in Materials and methods. Shown are Giemsa counter-stained cells with brightfield illumination. Bar: 10 μm.

P0 Polypeptide expression

The results described above indicate that under appropriate conditions D6P2T cells express detectable levels of P0 and MBP RNA. The expression of the corresponding Polypeptides in untreated and DBC-treated D6P2T cells was analyzed by immunoblotting (Fig. 9). In untreated cells, P0 polypeptide is detected as two bands of 25 and 30K, which is similar to the pattern observed in P0 in rat sciatic nerve (Fig. 4). According to Cammer et al. (1980), the 30K band represents the reduced form of 25K P0. The level of P0 in untreated D6P2T cells is 4% of the level in sciatic nerve. After 48 h of treatment with 1 mm-DBC the level of P0 is reduced to <2 % of the untreated control. These results indicate that the P0 polypeptide expressed in D6P2T cells is similar, if not identical, to the P0 glycoprotein in PNS myelin, and that the level of P0 polypeptide expression reflects the level of P0 RNA in these cells. In contrast to P0, MBP was undetectable by either immunoblotting or immunodot blotting (<0-01 % the level in sciatic nerve) in either untreated or DBC treated cells (data not shown).

Fig. 9.

Immunoblot analysis of P0 Polypeptide. Proteins were analyzed by SDS–PAGE and immunoblotting. Homogenates of: lane 1, 14 day rat brain (10 μg protein); lane 2, 14 day rat sciatic nerve (5 μg protein); lane 3, untreated D6P2T cells (100 μg protein); lane 4, D6P2T cells grown in 1 mm-DBC for 48 h (100pg protein). Molecular weights were estimated by comparison to standards of 92·5, 66·2, 31, 21·5, and 14·4K.

Fig. 9.

Immunoblot analysis of P0 Polypeptide. Proteins were analyzed by SDS–PAGE and immunoblotting. Homogenates of: lane 1, 14 day rat brain (10 μg protein); lane 2, 14 day rat sciatic nerve (5 μg protein); lane 3, untreated D6P2T cells (100 μg protein); lane 4, D6P2T cells grown in 1 mm-DBC for 48 h (100pg protein). Molecular weights were estimated by comparison to standards of 92·5, 66·2, 31, 21·5, and 14·4K.

The results presented in this paper indicate that intracellular cAMP functions as a biphasic regulator of P0 and MBP gene expression in D6P2T cells, inducing expression at low levels and repressing expression at high levels. Repression of P0 RNA and induction of MBP RNA are mediated at least in part by effects on gene transcription. It remains to be determined if induction of P0 and repression of MBP RNA are also transcriptionally mediated. At comparable doses of cAMP-elevating drug the effects on steady-state RNA levels are more pronounced than the effects on the corresponding gene transcription. For example, 1 mm-DBC results in a fivefold decrease in the level of P0 RNA while P0 transcriptional activity is decreased only two-fold. This may mean that elevated cAMP also affects gene expression post transcriptionally.

Repression of P0 (see also Kreider et al. 1988) and MBP gene expression at high doses of cAMP-elevating drugs could in principal represent a nonphysiological response reflecting inhibition of all macromolecular biosynthesis due to toxic effects of the drugs at these levels. This is unlikely for several reasons. First, at levels of DBC which repress P0 RNA expression, MBP expression remains high, and tubulin RNA actually increases (data not shown). Second, other workers have shown increased expression of a variety of myelin comP0nents at comparable doses of cAMP-elevating drugs (McMorris, 1983; Sobue et al. 1986; Droms & Sueoka, 1987; Bansal & Pfeiffer, 1987). Third, other aspects of Schwann cell behaviour, such as mitosis, also exhibit biphasic regulation by cAMP-elevating drugs (Raff et al. 1978). For these reasons we feel that repression of P0 and MBP gene expression at high doses of cAMP-elevating drugs reflects a physiological effect of high intracellular levels of cAMP.

In addition, there is evidence that repression of P0 and MBP gene expression is an important aspect of regulation in Schwann cells in vivo and in culture. In the absence of axonal contact, rat Schwann cells in culture undergo a marked down-regulation in the P0 and MBP gene expression (Mirsky et al. 1980; Poduslo et al. 1984). Recently Shuman et al. (1988) and Lemke & Chao (1988) have investigated the response of primary cultures of Schwann cells to agents such as forskolin that raise the intracellular concentration of cAMP. Shuman et al. (1988) were unable to detect either P0 or MBP Polypeptide in cultures treated with 100–200 μM- forskolin. The present data with D6P2T cells suggest that these relatively high doses of forskolin may have repressed P0 expression. The Possible presence of MBP RNA without MBP Polypeptide was not investigated. Lemke & Chao (1988), in contrast, observed that treatment of cultured rat neonatal Schwann cells with forskolin at low concentrations between 0 and 100 μM resulted in marked increases in both P0 and MBP RNA in a dose-dependent manner. The threshold for the MBP response was at least ten-fold higher than for P0. The present results with D6P2T cells are consistent with these latter studies. It is Possible that higher concentrations of forskolin would similarly down-regulate P0 expression in primary Schwann cell cultures. The constitutive P0 expression in untreated D6P2T cells could be explained if these cells have an elevated basal level of cAMP.

In other systems the phenotypic effects of elevated cAMP levels are mediated primarily through the activation of cAMP-dependent protein kinase which phos-phorylates many different substrate proteins, affecting their activities in various ways. This provides a rapid method for altering the cellular phenotype in response to extracellular signals. In D6P2T cells there is a lag of several hours before the effects of elevated cAMP on P0 and MBP gene expression are apparent. This suggests that the observed effects are not directly mediated by phosphorylation of transcription factors, but may involve intermediate steps between the phosphorylation event and the effects on transcription. Similar ‘delayed’ cAMP responses have been observed with a variety of other genes (Roesler et al. 1988) including myelinspecific functions (McMorris et al. 1983; Sobue et al. 1986; Droms & Sueoka, 1987).

P0 gene expression is qualitatively similar in D6P2T cells and in Schwann cells. The size of P0 RNA is the same, suggesting that transcription and post-transcriptional RNA processing are similar. The ratio of P0 RNA to P0 polypeptide is approximately the same, suggesting that the translational efficiency of P0 RNA, and post-translational stability of P0 polypeptide are similar. The P0 polypeptides have the same apparent molecular weight, behavior on SDS–PAGE, and antigenic properties, suggesting that the pattern of posttranslational processing of P0 is similar. The level of constitutive P0 gene expression in D6P2T cells is comparable to the levels in neonatal sciatic nerve (Brockes et al. 1981). However, the two preparations cannot be directly compared on a per cell basis because of cellular heterogeneity. Sciatic nerve contains cells other than Schwann cells, and it is difficult to estimate the contribution of non-Schwann cell RNA to the total RNA. D6P2T cells, on the other hand, represent a homogeneous cell population. Despite these uncertainties, it is likely that the level of P0 gene expression in D6P2T cells is comparable, on a per cell basis, to the levels in Schwann cells during the initial stages of myelination.

MBP gene expression in D6P2T cells is also similar in some respects to MBP gene expression in Schwann cells, but exhibits important differences. The size of MBP RNA is similar in the two systems. MBP RNA in sciatic nerve is comprised of several different RNA species produced from a single gene by differential splicing, and these species encode several different forms of MBP. It is not clear from the data presented here which, if any, of these RNA species are expressed in D6P2T cells since expression of MBP RNA in these cells does not result in the expression of detectable MBP polypeptide. In work reported elswhere (Gandelman et al. 1989) we have examined the nature of the MBP gene expression in DBC-treated D6P2T cells in more detail.

The phenotype of D6P2T cells described in this paper indicates that this cell line represents a useful model for Schwann cell differentiation The similarities between Schwann cells and D6P2T cells can be summarized as follows. Schwann cells are the predominant cell type in peripheral nerves; the D6P2T cell line is derived from a peripheral neurinoma. Schwann cells exhibit a bipolar spindle-shaped morphology prior to their association with the axon during myelination (Bunge et al. 1983); untreated D6P2T cells exhibit a similar morphology. Schwann cells express a variety of myelin-specific markers including galactocerebroside, sulfatide, and 2’,3’-cyclic 3’-nucleotide phosphohydrolase (Mirsky et al. 1983); D6P2T cells also express these markers (Bansal & Pfeiffer, 1987). Schwann cells proliferate rapidly prior to myelination and cease proliferation at the beginning of myelination; untreated D6P2T cells proliferate rapidly and exhibit increased growth control when cAMP levels increase. During the initial stages of myelin formation Schwann cells express P0 glycoprotein but not MBP (Hahn et al. 1987); this is the pattern of gene expression exhibited by untreated D6P2T cells. As myelination proceeds, MBP gene expression in Schwann cells is induced, and when myelination is complete both P0 and MBP gene expression are repressed; this is the pattern of gene expression exhibited by D6P2T cells when cAMP levels increase. During myelination, Schwann cells exhibit increased cell-cell adhesion, reflected by adhesion of the Schwann cell plasma membrane to the axolemma, and adhesion of adjacent myelin lamella to each other; increased cAMP causes increased cell-cell adhesion in D6P2T cells. These parallels suggest that D6P2T cells are similar to developmentally immature Schwann cells and that the effects of cAMP on D6P2T cells provides an informative model for Schwann cell differentiation in vivo. It should be noted that Schwann cells which are maintained in culture in the presence of various growth factors and other stimulatory agents exhibit a phenotypic response to elevated cAMP which is similar although not identical to that of D6P2T cells (Sobue & Pleasure,. 1984; Sobue et al. 1986; Pleasure et al. 1985; Shuman et al. 1988; Lemke & Chao, 1988).

In light of the arguments outlined above it appears that the changes in morphology, proliferation and gene expression that take place in Schwann cells during myelin formation are regulated, at least in part, by changes in intracellular cAMP levels. We propose that cAMP regulation in Schwann cells is mediated as follows: the cAMP level in immature Schwann cells is low prior to initiation of myelination, and the cells proliferate rapidly but do not express myelin-specific markers. Interaction of Schwann cells with axolemmal membranes increases intracellular cAMP levels slightly which blocks proliferation and induces the expression of some myelin-specific components, including galactocerebroside, sulfatide, 2’,3’-cyclic nucleotide 3’-phos-phohydrolase and P0 (Sobue et al. 1986; Lemke and Chao, 1988). We believe that untreated D6P2T cells reflect this stage in Schwann cell differentiation. As myelination proceeds, the cAMP level in the Schwann cell rises further which initially induces MBP gene expression (Lemke & Chao, 1988) and ultimately represses both P0 and MBP gene expression. Simul-taneous expression of both P0 and MBP is possible if cAMP levels are maintained at intermediate levels or if Schwann cells contain separate pools of cAMP which are regulated independently. The biphasic effects of cAMP on P0 and MBP gene expression could provide a sensitive mechanism for modulating the relative levels of expression of P0 and MBP during myelination.

Developmental changes in the level of cAMP in Schwann cells could either be intrinsically programmed in the Schwann cell or modulated by external signals through receptor-mediated activation of adenylate cyclase. This general model provides a common basis for the metamorphoses in growth properties, morphology and myelin gene expression that take place in Schwann cells during myelination and stipulates a pivotal role for cAMP in regulating the process of differentiation in these cells.

     
  • DBC

    dibutyryl cAMP

  •  
  • MBP

    myelin basic protein

  •  
  • P0

    myelin P0 glycoprotein

  •  
  • K

    × 103Mr.

This work was supported by NINCDS grants NS15190 (JHC) and NS10861 (SEP). We would like to acknowledge the assistance of Dr E. Barbarese (UCHC, Farmington, CT) in performing the in situ hybridization experiments.

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