©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclic AMP-independent Up-regulation of the Human Serotonin Transporter by Staurosporine in Choriocarcinoma Cells (*)

(Received for publication, April 3, 1995; and in revised form, May 15, 1995)

Jayanthi D. Ramamoorthy (1), Sammanda Ramamoorthy (1), Andreas Papapetropoulos (2), John D. Catravas (2), Frederick H. Leibach (1), Vadivel Ganapathy (1)(§)

From the  (1)Departments of Biochemistry and Molecular Biology and (2)Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with staurosporine caused a marked stimulation of serotonin transport activity in these cells. The stimulatory effect was noticeable at nanomolar concentrations of staurosporine, and a treatment time of >4 h was required for staurosporine to elicit the effect. At 40 nM and with a treatment time of 16 h, the stimulation of the transport activity was 3.5-6.0-fold. None of the several other protein kinase inhibitors tested had similar effect except KT 5720, a protein kinase A inhibitor, which showed a small but significant (1.4-fold) stimulatory effect at a concentration of 5 µM. Blockade of RNA synthesis and protein synthesis in the cells prevented completely the stimulation of the transport activity induced by staurosporine. The stimulation was observed not only in intact cells but also in plasma membrane vesicles prepared from staurosporine-treated cells. The stimulation was accompanied by a 5-7-fold increase in the steady state levels of the transporter-specific mRNAs, by a 7-fold increase in the maximal velocity of the transport process, and by a 6-fold increase in the transporter density in the plasma membrane. Even though both staurosporine and cholera toxin had similar effects on the serotonin transport activity in these cells, the effect was not additive when the cells were treated with both reagents together. While treatment of the cells with cholera toxin markedly elevated intracellular levels of cAMP, staurosporine did not have any effect on the cellular levels of this cyclic nucleotide. It is concluded that staurosporine up-regulates the serotonin transport activity in JAR cells by increasing the steady state levels of the serotonin transporter mRNA and by the consequent increase in the transporter density in the plasma membrane and that the process involves a cAMP-independent signaling pathway.


INTRODUCTION

The Na- and Cl-coupled serotonin transporter that is expressed in the serotonergic neurons, platelets, and placenta is a target for cocaine and antidepressants. Molecular cloning studies have demonstrated that a single gene codes for the transporter in neuronal and non-neuronal cells(1, 2, 3) . Human placental choriocarcinoma cells express a serotonin transporter which is identical to the transporter present in normal tissues(4) . These cells have proved to be very useful in studies involving the regulation of the human serotonin transporter(4, 5, 6) . In these cells, the serotonin transporter is up-regulated by cAMP(4) , and the regulation involves transcriptional activation(5) . Calmodulin-dependent processes also participate in the regulation of the transporter, presumably involving phosphorylation/dephosphorylation of the transporter protein(6) . There is evidence in other cell types for regulation of the serotonin transporter by additional cellular signaling pathways such as Ca(7) , protein kinase C(8, 9) , cGMP(10, 11) , and oxidants(12) . However, these signaling pathways modulate the serotonin transporter activity rapidly and the underlying mechanism apparently involves covalent modification of the transporter protein without altering the transporter density. Therefore, cAMP is the only cellular signal thus far known which has been shown to regulate the serotonin transporter by transcriptional activation. The influence of cAMP on the serotonin transporter is not selective for the choriocarcinoma cells since a similar role for cAMP in the regulation of the serotonin transporter has been demonstrated in the brain(13) . The present study identifies a second cellular signaling pathway involved in the transcriptional activation of the serotonin transporter in choriocarcinoma cells, and this pathway is mediated by staurosporine.


EXPERIMENTAL PROCEDURES

Materials

The JAR and BeWo human placental choriocarcinoma cell lines were purchased from the American Type Culture Collection. Culture media (RPMI 1640 and F-12), penicillin, streptomycin, and trypsin were obtained from Life Technologies, Inc. Fetal bovine serum, cholera toxin, iproniazid, imipramine, serotonin, staurosporine, prostaglandin E, apotransferrin, hydrocortisone, thyroxine, phorbol esters, cycloheximide, and actinomycin D were from Sigma. Human recombinant insulin was from Novo Nordisk Pharmaceuticals, Inc. (Princeton, NJ). Protein kinase inhibitors were from Research Biochemicals, Inc. (Natick, MA) and from LC Laboratories (Woburn, MA). Paroxetine was obtained from Beecham Pharmaceuticals (Betchworth, Surrey, United Kingdom). 5-[1,2-H]Hydroxytryptamine (serotonin) binoxalate (specific radioactivity, 25.4 Ci/mmol), L-[3-H]alanine (specific radioactivity, 76.9 Ci/mmol), and [I]RTI-55()(specific radioactivity, 2200 Ci/mmol) were purchased from DuPont NEN. [2-H]Taurine (specific radioactivity, 30.0 Ci/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St Louis, MO).

Culture of JAR and BeWo Cells

The cells were cultured with either RPMI 1640 medium (JAR) or F-12 nutrient mixture (BeWo) as described previously(14) . The media were supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Trypsin-released cells were seeded in 35-mm Petri dishes at a density of 1.5 10 cells/dish and allowed to grow as a monolayer. Twenty-four h after subculturing, the medium was replaced with a hormonally defined medium which did not contain fetal bovine serum. The defined medium consisted of either RPMI 1640 or F-12 nutrient mixture, supplemented with insulin (5 µg/ml), apotransferrin (5 µg/ml), prostaglandin E (2.5 10 mg/ml), hydrocortisone (5 10M), and thyroxine (5 10M). Treatment with different reagents was carried out for indicated time periods in the defined media prior to uptake measurements.

Uptake Measurements in Cells

The dishes containing monolayer cultures of the cells were taken out of the incubator and let stand at room temperature for 2 h. The culture medium was then aspirated, and the cells were washed once with the uptake buffer. One ml of uptake buffer containing radiolabeled substrate (serotonin, alanine, or taurine) was added to the cells and incubated for 3 min. Uptake was terminated by aspirating the buffer and subsequently washing the cells three times with fresh uptake buffer. The cells were lysed with 1 ml of 0.2 N NaOH, 1% SDS, and the lysate was transferred to scintillation vials for quantitation of radioactivity. The composition of the uptake buffer was 25 mM HEPES-Tris, pH 7.5, 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl, 0.8 mM MgSO, 5 mM glucose, and 0.1 mM iproniazid, an inhibitor of monoamine oxidases. Serotonin uptake that occurred independent of the serotonin transporter (i.e. diffusion) was determined by measuring the uptake in the presence of imipramine (0.1 mM), and this component was always <10% of total uptake measured in the absence of imipramine.

Determination of cAMP and cGMP

JAR cells were cultured in 35-mm dishes for 24 h in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. At the end of 24 h, the medium was removed and replaced with a hormonally defined medium. When present, staurosporine and cholera toxin were added to this medium at a concentration of 40 nM and 100 ng/ml, respectively. The incubation was continued for 16 h. In cases where the treatment of the cells with staurosporine or cholera toxin was only for 2 h, the cells were incubated with the defined medium for 14 h, following which staurosporine or cholera toxin was added, and the incubation was continued for 2 h. At the end of the incubation, cell cultures were washed with ice-cold buffer and lysed in 1 ml of 0.1 N HCl. After 30 min, the acid medium was collected and stored frozen until use. Measurement of cAMP and cGMP in these samples was done by radioimmunoassay as described previously (15) .

Preparation of Plasma Membrane Vesicles from Cultured JAR Cells

The cells were cultured in 225-cm flasks for 24 h in RPMI 1640 medium, supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and fetal bovine serum (10%), after which the medium was replaced with the hormonally defined medium containing either dimethyl sulfoxide (control) or staurosporine (40 nM) in dimethyl sulfoxide. The cells were then incubated for 16 h, following which plasma membrane vesicles were prepared from these cells as described previously(6) . Membrane vesicles were suspended in 10 mM HEPES-Tris, pH 7.5, containing 75 mM potassium gluconate, 130 mM mannitol, 2 mM EGTA, and 10 mM KF for serotonin uptake measurements and 10 mM Tris-HCl, pH 7.5, containing 230 mM mannitol, 1 mM MgSO, 2 mM EGTA, and 10 mM KF for RTI-55 binding measurements. Protein concentration of the final membrane suspension was determined and adjusted to 1 mg/ml (RTI-55 binding) or 2.5 mg/ml (serotonin uptake).

Uptake Measurements in Membrane Vesicles

Uptake of serotonin in JAR cell plasma membrane vesicles was measured by the rapid filtration method as described earlier(16, 17) . The uptake medium was 150 mM NaCl buffered with 10 mM HEPES-Tris, pH 7.5. Uptake was measured with a 15-s incubation and at a serotonin concentration of 0.2 µM. Serotonin uptake that occurred by diffusion was determined by measuring the uptake in the presence of 0.1 mM imipramine. This value was subtracted from total uptake to determine the transporter-mediated uptake.

RTI-55 Binding

Equilibrium binding of [I]RTI-55 to the JAR cell plasma membranes was assayed (6) by incubating the membranes (50 µg of membrane protein) with different concentrations of the ligand (0.025-1 nM) at room temperature for 1 h in a total volume of 0.5 ml. The binding buffer was 10 mM Tris-HCl, pH 9.5, containing 300 mM NaCl and 1 mM MgSO. The binding was terminated by addition of 3 ml of ice-cold stop buffer (10 mM Tris-HCl, 200 mM NaCl, pH 7.5) followed by filtration of the mixture on a GF/F glass fiber filter (0.7-µm pore size) which had been presoaked in 0.3% polyethylenimine. The filter was washed with 3 5 ml of the ice-cold stop buffer, and the radioactivity associated with the filter was determined using a gamma counter. Nonspecific binding was determined in the presence of 5 µM paroxetine.

Isolation of Poly(A) RNA and Northern Analysis

Poly(A) RNA was isolated from JAR cells using FastTrack mRNA isolation kit (Invitrogen). Northern hybridization was carried out as described earlier (5, 18) under high stringency conditions using P-labeled human serotonin transporter cDNA as the probe. The same membrane blot was used for probing with P-labeled human -actin cDNA as an internal control for RNA loading and transfer efficiency. This was done by stripping of the blot followed by rehybridization with the -actin cDNA probe.


RESULTS

Influence of Phorbol-12-myristate-13-acetate (PMA) and Staurosporine on Serotonin Uptake in JAR Cells

This investigation was initially started to determine whether the serotonin transporter expressed in JAR cells is regulated by protein kinase C as it has been shown for the serotonin transporter present in platelets (9) and in endothelial cells(8) . Treatment of JAR cells for 2 h with 1 µM PMA, an activator of protein kinase C, was found to cause a significant inhibition of the serotonin transporter activity, measured as the imipramine-sensitive serotonin uptake (Table 1). The inhibition was small (15%), but the effect was consistent in three different experiments. Treatment of the cells with staurosporine, a nonspecific but very potent inhibitor of protein kinases, for 2 h did not have any significant effect on the transporter activity. However, when treatment of the cells with PMA was done in the presence of staurosporine, PMA failed to inhibit the transporter. These experiments indicated that the serotonin transporter expressed in JAR cells is inhibited by protein kinase C, even though the effect is much smaller than found in platelets (40%; (9) ) and in endothelial cells (30%, (8) ).



We then investigated the long term regulation of the serotonin transporter in JAR cells by PMA. Treatment of the cells with 1 µM PMA for 16 h caused a significant increase in the transporter activity (Table 1). This stimulation could be either due to the PMA-induced down-regulation of protein kinase C (which could be expected to relieve the transporter from the inhibition caused by protein kinase C that may be constitutively active to a significant extent even in the absence of PMA) or due to protein kinase C-dependent cellular effects that are unique to the long term treatment with PMA. We studied the influence of staurosporine on the long term stimulatory effect of PMA (Table 1). These experiments led to a surprising and unexpected finding that treatment of the cells with 40 nM staurosporine alone for 16 h caused a 5-fold increase in the transporter activity. The magnitude of increase was not altered by PMA. The stimulatory effect of staurosporine does not appear to be due to the inhibition of protein kinase C. If this were to be the case, one would have expected staurosporine to have either no effect or an inhibitory effect on the transporter activity, based on the long term effect of PMA. Moreover, the stimulation of the transporter activity by staurosporine requires long term treatment because such an effect was not noticed when the cells were treated with staurosporine only for 2 h. This interesting observation prompted us to investigate in more detail the long term regulatory effect of staurosporine on the serotonin transporter.

Stimulation of Serotonin Uptake by Staurosporine in JAR and BeWo Cells

Imipramine-sensitive serotonin uptake in JAR cells was measured after the cells were treated with 40 nM staurosporine for various time periods (1-16 h) at 37 °C (Fig. 1A). There was no noticeable change in the uptake up to 4 h of treatment. With longer periods of treatment, there was marked stimulation of serotonin uptake, 30% at 6 h, 90% at 8 h, and 265% at 16 h. Dose-response studies carried out using a 16-h treatment time revealed that staurosporine is a very potent stimulator of serotonin uptake (Fig. 1B). A significant stimulation was noticeable at a staurosporine concentration as low as 2.5 nM. The magnitude of stimulation increased as the concentration of staurosporine increased. The uptake in cells treated with 40 nM staurosporine was 3.4-fold compared to the uptake in control cells. Similar results were obtained with BeWo cells, another human choriocarcinoma cell line (data not shown). Staurosporine at concentrations greater than 40 nM was found to affect cell viability to a significant extent.


Figure 1: Stimulation of serotonin transporter activity in JAR cells by staurosporine. A, monolayer cultures were treated with or without staurosporine (40 nM) for indicated time periods (1-16 h) at 37 °C, following which imipramine-sensitive uptake of serotonin was measured at a serotonin concentration of 50 nM and with an incubation period of 3 min. Values are means ± S.E. (n = 3). The control uptake values for all treatment periods were similar (1.82 ± 0.06 pmol/mg of protein/3 min) and this value was taken as 100%. B, monolayer cultures were treated for 16 h at 37 °C with varying concentrations of staurosporine (0-40 nM), following which serotonin uptake was measured as described. Values are means ± S.E. (n = 4). The control uptake (100%) measured in cells treated in the absence of staurosporine was 1.41 ± 0.08 pmol/mg of protein/3 min.



Specificity of Staurosporine Effect

This was assessed by measuring the uptake of taurine and alanine in control and staurosporine (40 nM, 16 h)-treated JAR cells and by comparing the results with serotonin uptake (Fig. 2). These experiments showed that under the conditions in which the uptake of serotonin was stimulated 3.6-fold by staurosporine, the uptake of taurine and the uptake of alanine remained unaffected.


Figure 2: Specificity of staurosporine effect. Monolayer cultures were treated with or without staurosporine (40 nM) for 16 h at 37 °C, following which the uptake of serotonin (the imipramine-sensitive component), taurine, and alanine was measured. Final concentration of serotonin, taurine, and alanine was 50, 30, and 5 nM, respectively, and the incubation period for uptake measurement was 3 min. Values are means ± S.E. (n = 4).



Influence of Staurosporine on Kinetic Parameters of the Serotonin Transporter

The serotonin transporter activity in control and in staurosporine (40 nM, 16 h)-treated JAR cells was kinetically analyzed to determine the influence of staurosporine on the kinetic parameters, Michaelis-Menten constant (K) and maximal velocity (V) of the transport system. The results, given in Fig. 3as Eadie-Hofstee plots, show that the staurosporine-induced stimulation of serotonin uptake is accompanied by changes in K as well as in V. Treatment of the cells with staurosporine (40 nM, 16 h) increased the Kvalue from 0.31 ± 0.01 to 0.60 ± 0.03 µM. The V was also markedly affected by staurosporine. The V in control cells was 9.1 ± 0.2 pmol/3 min/mg of protein which increased to 66.6 ± 2.4 pmol/3 min/mg of protein in staurosporine-treated cells.


Figure 3: Influence of staurosporine on the kinetic parameters of the serotonin transporter. Confluent cultures were treated with () or without () staurosporine (40 nM) for 16 h at 37 °C. Imipramine-sensitive serotonin uptake was measured in these cells over a serotonin concentration range of 0.05-1 µM and with an incubation period of 3 min. Results (means ± S.E., n = 4) are given as Eadie-Hofstee plots (i.e.v/sversusv). v, serotonin uptake rate in pmol/mg of protein/3 min; s, serotonin concentration in µM. Correlation coefficient (r) for linear plots was >0.99.



Influence of Cycloheximide and Actinomycin D on the Staurosporine-induced Stimulation of Serotonin Uptake

The lag period observed in the stimulation of serotonin uptake by staurosporine (Fig. 1A) indicated that de novo synthesis of the serotonin transporter protein might be involved in the process. Therefore, we assessed the influence of cycloheximide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of RNA synthesis, on the staurosporine-induced stimulation of serotonin uptake in JAR cells (Table 2). For this purpose, the cells were treated with or without staurosporine (20 nM) in the absence or in the presence of cycloheximide (10 µM) or actinomycin D (0.02 µg/ml) for 16 h, following which the activity of the serotonin transporter was measured by determining the imipramine-sensitive serotonin uptake. Cycloheximide and actinomycin D have been shown under these conditions to block the de novo synthesis of protein and RNA, respectively(5, 19) . The results of the experiments on serotonin uptake revealed that the stimulatory effect of staurosporine was completely abolished by cycloheximide and actinomycin D, strongly suggesting that transcriptional activation might underlie the observed stimulatory effect of staurosporine.



Effects of Protein Kinase Inhibitors on Serotonin Uptake in JAR Cells

Staurosporine is one of the most potent inhibitors of protein kinases, but is rather non-selective. It inhibits protein kinase C as well as cAMP- and cGMP-dependent protein kinases (protein kinase A and protein kinase G). In addition, tyrosine kinases and Ca/calmodulin-dependent kinases are also inhibited by staurosporine. We screened a variety of protein kinase inhibitors for their ability to stimulate serotonin uptake in JAR cells in order to find out whether inhibition of any of these protein kinases underlies the staurosporine-induced stimulation of serotonin uptake (Table 3). The results of the experiment showed that staurosporine is the only protein kinase inhibitor with the ability to enhance serotonin uptake. KT 5720, a selective inhibitor of PKA, showed a small but significant ability to stimulate the uptake. However, H-9, HA-1004, and K-252b which are expected to inhibit PKA at concentrations used in the experiment failed to show any effect. Similarly, chelerythrine which is a selective inhibitor of protein kinase C also had no effect on the uptake. H-89, an inhibitor of PKA and PKG, did not stimulate but instead markedly inhibited the uptake. Genistein and tyrphostin, inhibitors of tyrosine kinases, had no effect. CGS 9343B, a selective calmodulin antagonist, inhibited the uptake. The inability of any of these protein kinase inhibitors to simulate the staurosporine effect strongly suggests that the inhibition of protein kinases by staurosporine may not be related to the stimulation of serotonin uptake induced by staurosporine.



Serotonin Uptake in Plasma Membrane Vesicles Prepared from Control and Staurosporine-treated JAR Cells

The experiments with cycloheximide and actinomycin D indicated that the stimulatory effect of staurosporine might involve de novo synthesis of the serotonin transporter, thus increasing the transporter density in the plasma membrane. An additional supporting evidence for this mechanism would be to demonstrate the staurosporine-induced increase in serotonin uptake in plasma membrane vesicles isolated from these cells. For this purpose, we prepared plasma membrane vesicles from control and staurosporine (40 nM, 16 h)-treated JAR cells and measured in these vesicles imipramine-sensitive serotonin uptake in the presence of appropriate ionic gradients (inwardly directed Na and C1 gradients and outwardly directed K gradient). The results are given in Fig. 4. The value for imipramine-sensitive serotonin uptake at 0.2 µM concentration was 0.97 ± 0.06 pmol/15 s/mg of protein in plasma membrane vesicles prepared from control cells. The corresponding value in vesicles prepared from staurosporine-treated cells was 8.13 ± 0.15 pmol/15 s/mg of protein, a 7-fold increase compared to the control value. Thus, the stimulatory effect of staurosporine on serotonin uptake could be demonstrated at the plasma membrane level.


Figure 4: Serotonin uptake in plasma membrane vesicles prepared from control and staurosporine-treated cells. Monolayer cultures were treated with or without staurosporine (40 nM) for 16 h at 37 °C. Plasma membrane vesicles were isolated from control and treated cells, and serotonin uptake was measured in these membrane vesicles in the presence of an inwardly directed NaCl gradient and an outwardly directed K gradient. Concentrations of serotonin during uptake measurement was 0.2 µM, membrane protein used for each assay was 100 µg, and the incubation period for uptake measurement was 15 s. Uptake was measured in the presence as well as in the absence of imipramine (0.1 mM). Values are means ± S.E. (n = 6).



Binding of [I]RTI-55 to Plasma Membranes Isolated from Control and Staurosporine-treated JAR Cells

We estimated the serotonin transporter density in the plasma membranes derived from control and staurosporine-treated cells by analyzing the binding of [I]RTI-55 to the membranes. RTI-55 is a cocaine analog which is a high affinity ligand to the serotonin transporter and also to the dopamine and norepinephrine transporters(20, 21, 22, 23) . However, since the JAR cells do not express the dopamine and norepinephrine transporters(18, 24) , RTI-55 can be considered as a selective ligand for the serotonin transporter in these cells. We determined the kinetic parameters for RTI-55 binding in JAR cell plasma membranes by measuring equilibrium binding over a concentration range of 0.025-1 nM. The results given in Fig. 5as Scatchard plots revealed that the apparent dissociation constant (K) was 0.30 ± 0.01 nM and the maximal binding capacity (B) was 195 ± 3 fmol/mg of protein in control membranes. The corresponding values for the binding in membranes from staurosporine-treated cells were 0.29 ± 0.01 nM and 1127 ± 21 fmol/mg of protein. These data show that treatment of the cells with staurosporine results in an increase (approximately 6-fold) in the B for RTI-55 binding, without altering the K.


Figure 5: Binding of RTI-55 to plasma membranes isolated from the control and staurosporine-treated cells. Confluent cultures were treated with () or without () staurosporine (40 nM) for 16 h at 37 °C. Plasma membranes were isolated from control and treated cells and used for measurement of [I]RTI-55 binding as described under ``Experimental Procedures.'' Concentration range for RTI-55 was 0.025-1.0 nM. Nonspecific binding was measured in the presence of 5 µM paroxetine, and this component constituted <5% of total binding. Results (means ± S.E., n = 3) are given as Scatchard plots (i.e.BversusB/F). B, RTI-55 bound in fmol/mg of protein; F, concentration of free RTI-55 in nM. Correlation coefficient (r) for the linear plots was >0.99.



Influence of Staurosporine on Steady State Levels of the Serotonin Transporter mRNAs

Since co-treatment with actinomycin D completely blocks the stimulatory effect of staurosporine on serotonin uptake, it was inferred that staurosporine most likely causes transcriptional activation of the serotonin transporter gene. Therefore, poly(A) RNA was isolated from control and staurosporine (40 nM, 16 h)-treated JAR cells and analyzed by Northern hybridization using the human serotonin transporter cDNA as the probe(1) . The same membrane blot was reprobed with the -actin cDNA probe as an internal control for RNA loading and transfer efficiency. JAR cells as well as normal placenta express three different serotonin transporter mRNA transcripts, 6.8, 4.9, and 3.0 kilobases in size. Treatment of the JAR cells with staurosporine increased the steady state levels of all three transcripts (Fig. 6) Taking into consideration the small difference in the levels of the -actin mRNA between the two lanes containing the RNA samples from control and staurosporine-treated cells, the increase in the 6.8-, 4.9-, and 3.0-kilobase serotonin transporter mRNA transcripts was 4.5-, 7.1-, and 6.5-fold, respectively.


Figure 6: Influence of staurosporine on steady state levels of serotonin transporter mRNAs and -actin mRNA. Confluent cultures were treated with (lane B) or without (lane A) staurosporine (40 nM) for 16 h at 37 °C. Poly(A) RNA was isolated from control and treated cells. The size-fractionated mRNA was probed with P-labeled human placental serotonin transporter cDNA and with P-labeled human -actin cDNA by sequential hybridization. Results are from a representative experiment. Comparable results were obtained in three different experiments.



Relationship between Staurosporine and Cholera Toxin in Inducing the Serotonin Transporter Activity in JAR Cells

We have shown previously that treatment of JAR cells with cholera toxin enhances the serotonin transporter activity (4) and that this effect is accompanied by increased serotonin transporter mRNA levels and also by increased binding of RTI-55 to the membranes(5) . Since the increase in serotonin transporter activity induced by staurosporine also showed a similar phenomenon by being accompanied by increases in mRNA levels and RTI-55 binding, it was of interest to determine whether or not cholera toxin and staurosporine can act synergistically. The results given in Table 4show that when the cells were treated independently with either cholera toxin (100 ng/ml) or staurosporine (40 nM) for 16 h, the increase in imipramine-sensitive serotonin uptake was 3.3- and 6.0-fold, respectively. However, when the cells were treated with cholera toxin and staurosporine together, the increase in the uptake was 6.6-fold. This value was almost the same as the value obtained with staurosporine alone, indicating that the effect of cholera toxin and staurosporine were not additive.



Influence of Staurosporine on Intracellular Levels of cAMP and cGMP

Cholera toxin treatment leads to elevation of intracellular levels of cAMP in JAR cells, and this effect is accompanied by an increase in the activity of the serotonin transporter(4) . Since the influence of cholera toxin and the influence of staurosporine on serotonin uptake are not additive, it was important to determine whether treatment of the cells with staurosporine causes elevation of intracellular cAMP levels as treatment with cholera toxin does. Therefore, we measured cAMP levels in control cells and in cells treated with either staurosporine or cholera toxin (Table 5). It was found that staurosporine did not alter the cellular content of cAMP, whether the treatment time was 2 or 16 h. In contrast, treatment with cholera toxin for 2 h increased the cAMP levels 22-fold. Even after treatment with cholera toxin for 16 h, the cAMP levels remained elevated (8-fold) compared with control cells. The cellular content of cGMP was not affected by staurosporine and by cholera toxin.




DISCUSSION

This paper reports for the first time that staurosporine is a potent inducer of the expression of serotonin transporter in the human placental choriocarcinoma cells. The underlying mechanism appears to be transcriptional activation of the serotonin transporter gene. Supporting evidence for this mechanism includes (i) a substantial lag period before the effect of staurosporine on the serotonin transporter activity becomes noticeable, (ii) a marked increase in the steady state levels of the serotonin transporter-specific mRNAs, and (iii) effective blockade of the staurosporine effect by actinomycin D, and inhibitor of transcription. The increase in the steady state levels of the transporter mRNAs resulting from the transcriptional activation is accompanied by a parallel increase in the de novo synthesis of the transporter protein. This is evidenced from (i) the blockade of the staurosporine effect on the serotonin transport activity by cycloheximide, an inhibitor of protein synthesis, (ii) the staurosporine-mediated increase in the maximal velocity of the transport system, (iii) the staurosporine-mediated increase in the transporter density in the plasma membrane as seen from the increase in the binding of RTI-55, a ligand specific for the serotonin transporter in JAR cells, and (iv) the increased serotonin transport in plasma membrane vesicles isolated from staurosporine-treated cells versus control cells. These effects of staurosporine are very similar to those of cholera toxin. However, in contrast to cholera toxin treatment, staurosporine treatment does not increase cAMP levels in JAR cells. Therefore, the transcriptional activation of the serotonin transporter gene by staurosporine is mediated by a cAMP-independent signaling pathway. Interestingly, the effects of staurosporine and cholera toxin are not additive, indicating that the signaling pathways, though one is cAMP-dependent and the other is cAMP-independent, converge at some point prior to the transcriptional activation of the transporter gene. The physiological significance of such a dual pathway is not known. One possibility is that it enables the cell to integrate the biological effects of different external signals efficiently by providing a means for cross-talk between the two pathways. The organization of the human serotonin transporter gene has recently been reported(25) . The gene is composed of 14 exons spanning 31 kilobases, and the promotor region contains cAMP response element which is the binding site for the cAMP-dependent transcription factor, CREB(26, 27) .

The precise nature of the signaling pathway involved in the transcriptional activation of the serotonin transporter gene in JAR cells by staurosporine remains to be elucidated. The effect of staurosporine does not appear to be related to its ability to block the protein kinases. There is precedence for stimulation of gene expression by staurosporine which is not mediated through inhibition of protein kinases. Staurosporine induces differentiation and neurite outgrowth in PC12 cells(28, 29, 30) . In addition, it potentiates markedly the ability of epidermal growth factor to cause differentiation in these cells (31) . The underlying mechanism for this potentiation has been shown to be the up-regulation of the receptor for epidermal growth factor, resulting from induction of the receptor gene expression(31) . Similarly, the receptor for tumor necrosis factor in myeloid and epithelial cells is also up-regulated by staurosporine(32) . However, the cellular signaling pathway involved in staurosporine-mediated stimulation of gene expression remains unknown.

The physiological functions of the serotonin transporter expressed in the normal placental syncytiotrophoblast have not been identified. It has been speculated that the transporter is involved in the clearance of the vasoactive monoamine serotonin from the intervillous space and in the transplacental transfer of serotonin to the developing fetus (33, 34, 35) . These processes may be crucial to the normal placental function and for optimal development of the fetus. Therefore, a thorough understanding of the cellular mechanisms underlying the regulation of the serotonin transporter in human placenta is important. The choriocarcinoma cells offer an excellent model system to study this phenomenon. Moreover, it is very likely that the studies on the regulation of the serotonin transporter in these cells may become relevant to understand the regulation of the serotonin transporter in tissues other than the placenta (e.g. serotonergic neurons, endothelial cells, and astrocytes). To our knowledge, choriocarcinoma cells are the only cultured cell lines of human origin which possess endogenous serotonin transporter activity and thus are valuable to studies involving the regulation of the human serotonin transporter. The role of this transporter in the function of the serotonergic neuronal pathways is well known. These neuronal pathways are involved in a variety of physiological functions. Alterations in the function of these neurons are believed to underlie several pathological conditions. Therefore, unraveling the various cell signaling mechanisms which participate in the regulation of the serotonin transporter has immense clinical relevance.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HD 27487. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100. Tel.: 706-721-2100; Fax: 706-721-6608.

The abbreviations used are: RTI-55, 2-carbomethoxy-3-(4-iodophenyl)tropane; PMA, 4-phorbol-12-myristate-13-acetate.


ACKNOWLEDGEMENTS

We thank Sarah Taylor for expert secretarial assistance.


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