1 Department of Biomedical Sciences, University of Maryland, Baltimore, MD 21201, USA, 2 Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD 21201, USA and 3 Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA
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Abstract |
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Key Words: adenylyl cyclasecAMP pathway calcyon cell cycle corticogenesis D1 dopamine receptor phospholipase Cß/inositol triphosphate pathway receptor tyrosine kinaseRasRaf-1mitogen-activated protein kinase pathway
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Introduction |
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The present work begins to address the molecular mechanism by which D1Rs prevent cerebral cortical precursor cells from entering the S phase of the cell cycle. EGF-supported cultures of cerebral cortical precursor cells were selected as the in vitro model for this study because we found them to be more proliferatively active than those supported by FGF (Zhang and Lidow, 2002). It has been postulated that such cultures represent the subventricular proliferative zone of the fetal cerebral wall (Burrows et al., 1997
; Raballo et al., 2000
). We have reported (Zhang and Lidow, 2002
) that these cultures contain a mixture of multipotential, neuronal, and glial precursor cells, with a slight predominance of the latter cell type. More than 50% of the cells are proliferatively active; this includes all three aforementioned cell phenotypes, although multipotential and glial precursors display slightly higher proliferative activity as compared to neuronal precursors. Virtually all cells express both a and b subtypes of the D1R receptor, and stimulation of D1Rs results in a reversible supression of cell cycle progression into the S phase for all three cell phenotypes identified in these cultures. This supression is not accompanied by increases in the rates for either apoptosis or cell differentiation (Zhang and Lidow, 2002
).
We focused on examining the ability of D1R to regulate three specific proteins. The first two proteins, cyclin D and P27 (Kip1), are major translators of extracellular influences on the cell progression through the G1-to-S restriction point of the cell cycle, with the promoting and inhibiting activities respectively (Sheaff and Roberts, 1998; Puri et al., 1999
). Alterations in the levels of P27 have been described by some researchers as a part of cell differentiation (Tikoo et al., 1997
; Perez-Juste and Aranda, 1999
). However, detailed analysis showed that this does not reflect some additional activity of this protein, but rather is due its involvement in the supression of the cell cycle, which is a prerequisite for differentiation of many cell types (Zezula et al., 2001
; Munoz et al., 2003
). The third protein, Raf-1 (c-Raf), is a component of the receptor tyrosine kinase (Trk)RasRaf-1MAP kinase (MEKERK) pathway involved in EGF-induced elevation of the levels of cyclin D (Denhardt, 1999
), a primary cell cycle regulatory target of the mitogenic activity of this growth factor (Bogdan and Klambt, 2001
). Our interest in Raf-1 further derives from its role as a factor integrating inputs of a wide range of extracellular signals (Denhardt, 1999
; Gomperts et al., 2002
). Consequently, Raf-1 is likely to be a part of the D1R-initiated cascade counteracting the EGF-induced up-regulation of cyclin D. In the case of Raf-1, not only its levels but also phosphorylation on the serine 338 (S338) residue were evaluated, since it has been demonstrated that phosphorylation on this residue reflects Raf-1 activation (Diaz et al., 1997
; Mason et al., 1999
).
First, we confirmed our earlier finding (Zhang and Lidow, 2002) that the entry of cortical precursor cells in the EGF-supported primary cultures into the S phase of the cell cycle could be reversibly suppressed by agonist-induced activation of D1Rs. Then, we ascertained that this was accompanied by changes in the levels of the three proteins selected for examination in this study. Second, since activation of adenylyl cyclase (AC) and elevation of the intracellular levels of cAMP are known to constitute major consequences of the D1R stimulation (Sibley et al., 1993
; Missale et al., 1998), we determined whether the ACcAMP second messenger cascade was involved in the regulation of the above-mentioned proteins. This also included an assessment of the possible role of protein kinase A (PKA), a major cAMP-dependent protein kinase that has the potential to influence Raf-1 activation (Stork and Schmitt, 2002
). In addition, we tested the ability of cAMP cascade antagonists to block the D1R agonist-induced supression of the cell cycle. Third, we evaluated the possible involvement of D1R-induced stimulation of phospholipase Cß (PLCß)/inositol triphosphate (DAG/IP3) second messenger pathways (Undie, 1999
). As a part of the last objective, we examined whether activation of D1Rs results in altered levels of the recently-discovered protein, calcyon, which links D1R to PLCß-associated second messenger cascades, (Bergson et al., 2003
), and whether the pattern of these alterations resembles those seen in the levels of cyclin D, P27, or Raf-1. In this regard, it is notable that a calcyon-like protein isolated from the broad bean Vicia faba is reported to be capable of affecting cell cycle in mammalian cells (Ng and Ye, 2003
).
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Materials and Methods |
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SKF38393 [1-phenyl-2,3,4,5-tetrahydro-(H1)-3-benzazepine-7,8-diol], a conventional D1R agonist, which stimulates both AC and PLCß (Roberts-Lewis, 1986; Johansen et al., 1991
; Undie and Friedman, 1994
; Jin et al., 1998
), and the D1R antagonist, SCH23390[R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5,-tetrahydro-1H-benzazepine; Lidow et al., 2001
], were purchased from Sigma (St Louis, MO). SKF83959[3-methyl-6-chloro-7,8-dihydroxy-1-[3methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine], a D1R agonist, which activates PLCß without a concomitant AC stimulation (Panchalingam and Undie, 2001
; Jin et al., 2003
), was also obtained from Sigma under the auspices of the National Institutes of Mental Health chemical synthesis program. The AC activator, forskolin (7-b-acetoxi-8,13-epoxi-1a,6b,9a-trihydroxylabd-14-en-11-one; Seamon and Daly, 1998
), the cell permeable cAMP analogue, Sp-8-Br-cAMP (Sp-8-bromo-adenosine-3,5-cyclic phosphorothioate; Kawasaki et al., 1998
), and the PKA inhibitor, H-89 {N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; Chijiwa et al., 1990
}, were purchased from Calbiochem (San Diego, CA). The AC inhibitor, SQ 22,536 [9-{tetrahydro-2-furanyl}-9H-purin-6-amine; SQ 22,536; Fabbri et al., 1991
] was purchased from A.G. Scientific (San Diego, CA).
Cell Cultures
EGF-supported cultures were prepared and maintained as described in Zhang and Lidow (2002). Briefly, pregnant CD1 mice (Charles River, Wilmington, MA) were anesthetized at E14 via peritoneal injection of 50 mg/kg sodium pentobarbital (Abbott Pharmaceuticals, Abbott Park, IL), and the uteri were aseptically removed. The cerebral walls of the embryos were dissected and transferred into ice-cold defined serum-free medium containing 7.15% DMEM, 5.3% F12, 2.0 mM glutamine (all from GIBCO, Grand Island, NY), 5.5 µM HEPES (ICN, Cleveland, OH), 0.6% glucose, 3 mM NaHCO3, 3 nM sodium selenite, 20 µg/ml insulin, 8.9 µg/ml putrescine, 2 nM progesterone and 100 µg/ml apo-transferrin (all from Sigma, St Louis, MO). The collected tissue was dissociated with a sterile fire-polished Pasteur pipette, and the dissociated cells were plated at a density of 105 cells/cm2 in 60 mm diameter petri dishes (Nunc, Rochester, NY) using the same growth medium supplemented with 20 ng/ml EGF (Sigma, St Louis, MO). The Petri dishes were placed into a water-jacketed incubator (Forma Scientific, Marietta, OH) providing 5% CO2 and 95% air at 37°C. In all experiments, drug treatments were initiated 48 h after plating. In most cases, cells were collected for analyses following 48 h of drug exposure (96 h after plating). For evaluation of the effects of D1R stimulation on the progression through the cell cycle of bromodeoxyuridine (BrdU) pulse-labeled cells, 82 mM BrdU was added to the cultures for 1 h. After that, the BrdU containing media was replaced by either dopamine drug-free or D1R agonist-containing media. The cell samples were collected for analysis every 3 h for 27 h. To verify the ability of cells to recover their proliferative activity after exposure to D1R agonist, cell cultures exposed to the agonist for 48 h (between 48 and 96 h after plating) were allowed to grow for additional 4 h in its absence. In the latter case, the control was represented by cultures grown in the absence of D1R drugs for 100 h after plating. To examine the time-course of changes in the levels of specific proteins, cells were collected after 12, 24, or 48 h of drug exposure (60, 72, or 96 h after plating, respectively).
Flow Cytometric Analysis of the Cell Cycle
Cultured cerebral cortical precursor cells were collected, pelleted by 10 min centrifugation at 300 x g on a Clinical Centrifuge (IEC, Austin, TX), and fixed in 70% ethanol for 30 min. The fixed cells were stained for 30 min with 5 µg/ml propidium iodine (PI; Molecular Probes, Eugene, OR) in the presence of 1 mg/ml RNase (Sigma, St Louis, MO). When BrdU labeling also needed to be detected, the PI-labeled cells were re-suspended for 20 min in 0.1 N HCl containing 0.25% triton X-100. They were then washed in 0.1 M Na2B4O7, incubated overnight at 4°C with FITC-conjugated anti-BrdU antibodies (Sigma, St louis, MO) diluted 1:200 in a blocking solution containing 0.5% Tween 20 and 1% BSA, and washed again in phosphate buffered saline (pH 7.4). Flow cytometric analysis was conducted on a FACS Vantage Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). The data acquisition and analyses were performed using CellQuest Pro (Becton Dickinson, Franklin Lakes, NJ) and ModFit LT (Versity, Topsham, ME) software. The percentages of cells in different stages of the cell cycle as well as the percentage of apoptotic cells were calculated based on PI staining. The cell cycle stage of BrdU-labeled cells was also assessed using PI staining of these cells.
PLCß Activity
Activity of PLCß in cultured cells was assessed be measuring phosphatidylinositol hydrolysis as described in Undie (1999) and Panchalalingham and Undie (2001)
. Briefly, 6 µCi/ml 2-[3H]inositol (American Radiolabeled Chemicals, St Louis, MO) was added into the culture media followed by the addition of LiCl (5 mM) 30 min later. At that time, D1R ligands may also be introduced into the media. Incubation continued for 90 min. The cells were collected and pelleted as described above. The concentration of the total protein in the pellet was determined using a modified Lowry Protein Assay kit (Pierce, Upland, IN). The pellet was resuspended in 1.0 ml chloroform/methanol/1 M HCl (100/200/1) mixture, and shaken for 15 min. Aqueous and organic phases were separated by further additions of 0.5 ml chloroform and 0.75 ml deionized water and centrifugation at 1000 x g for 5 min. Aliquots of the upper phase were taken and the content of inositol phosphates analyzed by Dowex anion exchange chromatography.
Western Blots
Cells were collected and pelleted as described above. They were then homogenized by sonication with a Sonic Dismembrator (Fisher, Suwanee, GA) in 0.5 M TrisHCl (pH 7.6) containing 0.1 M NaCl, 10 mM phenylmethylsulfonyl fluoride, 2mg/ml aprotinin, 2 mg/ml leupeptin, and 2 mg/ml pepstatin (all from Sigma, St Louis, MO), and the concentration of the total protein in each sample was determined using a modified Lowry Protein Assay kit (Pierce, Upland, IN). Sample aliquots containing 40 µg total protein were mixed 1:1 with a sample buffer containing 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromophenol blue, and 0.125 M TrisHCl, (pH 6.8; all from Sigma, St Louis, MO) and incubated at 70°C for 10 min. The resultant mixtures were loaded onto precast 415% gradient gels (Bio-Rad, Hercules, CA) and run at 100 mV for 1.5 h. Every gel also included a standard homogenate made from the same drug-naive culture. This standard homogenate was used to normalize the data between different experimental runs by always designating the densitometric values obtained from blots of this homogenate as equal to 1. Proteins resolved by electrophoresis were transferred onto Hybond ECL nitrocellulose membranes (Amersham, Piscataway, NJ) at 100 mV for 1.5 h. After blocking in 5% dry milk and 0.1% Tween 20 in phosphate buffered saline (pH 7.6), the membranes were processed for immunolabeling overnight at 4°C with one of the following antibodies: mouse anti-cyclin D1 (dilution 1:500; Zymed, San Francisco, CA), mouse anti-P27 (dilution 1:500; Zymed, San Francisco, CA), rabbit anti-Raf-1 (dilution 1:1000; Sigma, St Louis, MO), rabbit anti-Raf-1 phosphorylated on S338 residue (dilution 1:500; Upstate, Waltham, MA), or rabbit anti-calcyon (1:50; generated in house and described in Lezcano et al., 2000; Koh et al., 2003
). Incubation with secondary peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies (dilution 1:2000; Sigma, St Louis, MO) was conducted for 1 h at room temperature. The labeled bands were visualized on X-Omat AR Films (Kodak, Rochester, NY) using a SuperSignal Chemiluminescence kit (Pierce, Upland, IN). The uniformity of gel loading and protein transfer was verified by stripping the membranes of the antibodies, used in the initial immunolabeling, and processing them again for immunodetection of ß-actin. The latter protein is well suited for these purposes because our previous studies have demonstrated that its levels in the primary cerebral cortical precursor cells remains virtually unchanged for up to 6 days of culturing (Zhang and Lidow, 2002
). Mouse monoclonal antibodies for ß-actin (1:1000 dilution) and goat anti-mouse peroxidase-conjugated secondary antibodies (1:10000 dilution) were purchased from Sigma (St Louis, MO). Film images of blots were digitized using a UC 1260 flat bed scanner (Umax, Dallas, TX) and subjected to densitometric analysis using IPLab software (Scanalytics, Fairfax, VA). This analysis was performed only on films generated by blots with variability in gel loading/protein transfer of <5% and in which optical densities of images were within the linear range of the relationship between the intensities of chemiluminescent immunosignal and the resultant optical densities of the film brand employed in the present study.
Statistical Analysis
All experiments were repeated five times. Within every repeat, each culture condition was reproduced in triplicate. The statistical analysis of the concentration-dependence of the effects of agonists (which also included the effects of D1R antagonist and ACcAMPPKA pathway agonists and antagonists) was performed by a one-way ANOVA followed by a Dunnett's post hoc test. The ability of cultured cells to recover their proliferative activity after discontinuation of agonist exposure was evaluated using two-tailed Student's t-test. The temporal effects of drug exposure were examined using a two-way ANOVA, with the presence of drugs and duration of exposure as variables. This was followed by a Tukey's post hoc test.
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Results |
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Prior to examining the capacity of the conventional (AC/PLCß-stimulating) D1R agonist, SKF38393 to regulate specific cell-cycle-associated proteins, we confirmed the ability of this ligand to prevent EGF-supported fetal cerebral cortical precursor cells from entering the S phase of the cell cycle. For this purpose, we compared the cell cycle progression of BrdU pulse-labeled cells in the presence and absence of 75 µM SKF38393(the cells were exposed to BrdU for 1 h immediately prior addition of the ligand). We found that in both SKF38393exposed and un-exposed cultures, the highest percentages of BrdU-labeled cells in S phase were detected shortly after BrdU exposure and 15 h later (Fig. 1A). In addition, in both culture types maximal percentages of BrdU-labeled cells in G2-M phases were observed between 3 and 6 h and between 18 and 21 h after BrdU exposure (Fig. 1A). Finally, the highest percentages of BrdU-labeled cells in G0G1 phases were observed
9 and
24 h after BrdU exposure independently of the presence or absence of the D1R agonist (Fig. 1A). While the presence of SKF38393failed to produce notable changes in the maximal percentage of BrdU-labeled cells seen in the G0G1 phases of the cell cycle, it decreased the percentage of these cells re-entering the S phase, which also resulted in a decreased percentage of the cells passing through the G2-M phases (Fig. 1A). These findings indicate that exposure to SKF38393did not interfere either with the total length of the cell cycle or with the length of its phases. However, in agreement with our previous studies (Zhang and Lidow, 2002
), this D1R agonist suppressed cell entry into the S phase. To further assess this aspect of SKF38393actions, we also determined the percentages of cells in G0G1, S, and G2-M phases in cultures exposed for 48 h to 0100 µM SKF38393 There was a concentration-dependent rise in the percentage of G0G1-phase cells accompanied by a decline in the percentage of S-phase and G2M-phase cells. This produced an increase in G0G1/SG2M ratio, which achieved the levels of statistical significance at 50 µM SKF38393and above (Fig. 1B). Such elevation in G0G1/SG2M provides further support of SKF39393induced decrease in the percentage of cells entering the S phase of the cell cycle (Zhang and Lidow, 2002
). The D1R specificity of SKF38393induced alterations in G0G1/SG2M was confirmed by the observation that they were absent when the cell cultures were exposed to this agonist (75 µM) in the presence of 10 µM of the D1R antagonist, SCH23390(Fig. 1B). SCH23390alone had no effect on the cell cycle (not shown). We also found that, 4 h after discontinuation of 48 h long exposure to 75 µM SKF38393 the G0G1/SG2M ratio in the recovering cell cultures was significantly higher than G0G1/SG2M ratio obtained from cells cultured for a comparable period of time without this drug ever being present in the media (for recovering and control cultures, these ratios were 2.4 ± 0.5 and 6.3 ± 0.7 respectively, P of t-test > 0.05). This is in agreement with our earlier finding that D1R stimulation largely induces accumulation of proliferating cells in the G1 phase of the cell cycle, rather than forces these cells to exit the cycle and undergo terminal differentiation (Zhang and Lidow, 2002
).
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Visualization of the Proteins of Interest
Immunolabeling of the Western blots generated from our cultures with antibodies to cyclin D and P27 visualized single bands with molecular weights of 36 kD and 27 kD respectively (Figs 2A and 3), which corresponded to the mol. wts of these proteins (Matsushime et al., 1992; Slingerland and Pagano, 2000
). Both antibodies to total Raf-1 and to Raf-1 phosphorylated on S338 residue also produced a single band of the appropriate mol. wt of 74 kD (Figs 2A and 3; Stanton et al., 1989
). Anti-calcyon antisera showed a 34 kD band (Fig. 7A,B) corresponding to that of calcyon modified by N-linked oligosaccharides (Lezcano et al., 2000
; Koh et al., 2003
). Antibodies to ß-actin, used for verification of the uniformity of gel loading and protein transfer, labeled a protein with the mol. wt of 43 kD (Fig. 3), the weight of ß-actin (Otey et al., 1987
).
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The aim of this part of the study was to determine whether SKF38393induced supression of S phase entry in the EGF-supported primary cultures of cerebral cortical precursor cells is accompanied by changes in the levels of cyclin D, P27, and Raf-1 proteins, as well as the level of Raf-1 phosphorylation at S338 residue.
The study began with the examination of the effects induced by 12, 24, and 48 h of exposure to 75 µM SKF38393 Agonist-exposed cultures showed a time-dependent decrease in the levels of cyclin D and Raf-1 and an increase in the levels of P27 as compared to those in the age-matched drug-naive cultures (Fig. 2). This increase reached statistical significance at 24 h of SKF38393exposure. SKF38393exposed cultures also displayed elevated phosphorylation of Raf-1 on S338 residue, with this effect already achieving statistical significance by 12 h of agonist exposure (Fig. 2). These observations suggest that the D1R-induced alterations in the proteins examined in this study are more robust after 48 h of exposure. Therefore, all future analyses were performed at that time point.
The concentration dependence of the effects produced by 48 h long exposure to SKF38393was examined at the concentration ranging from 0 to 100 µM. The levels of cyclin D and Raf-1 decreased in a concentration-dependent manner (Figs 3 and 4), with statistical significance being detected at 25 µM of the agonist and above (Fig. 4). In contrast, examination of the changes in the levels of P27 revealed a bell-shaped curve. P27 levels increased steadily when the cultures were exposed to 050 µM of SKF38390 but began to decline at higher (75100 µM) concentrations of this agonist (Figs 3 and 4). The alterations in P27 levels were statistically significant between 25 and 75 µM of SKF38393(Fig. 4). The extent of Raf-1 phosphorylation on S338 residue underwent yet another pattern of changes. It showed a concentration-dependent increase (Figs 3 and 4), with the statistical significance being achieved at the agonist concentrations of 50 µM and above (Fig. 4). All these SKF38393induced changes were preventable by co-incubation with 10 µM SCH23390(tested against 75 µM of SKF38393 Figs 3 and 5). SCH23390alone produced no detectable effects (not shown).
Examination of a Possible Involvement of ACcAMP Second Messenger Pathway
Activation of AC has been long identified as the most common consequence of D1R stimulation (Missale et al., 1989; Sibley et al., 1993
), and the ACcAMPPKA cascade has been implicated in inhibiting Raf-1 and suppressing cell cycle progression in several reports (Cho-Chung et al., 1995
; Denhardt, 1999
; Stork and Schmitt, 2002
). Therefore, it was reasonable for us to examine whether this cascade was also involved in the molecular and cell cycle suppressing effects observed in the present study.
We found that neither 10 µM of the AC inhibitor SQ 22,536 nor 10 µM of the PKA inhibitor, H-89 were able to counteract the changes in the levels of cyclin D, P27, and Raf-1 produced by 75 µM SKF38393(Figs 3 and 5A). Furthermore, exposure to 10 µM of the AC activator, forskolin, and 100 µM of the cell-permeable cAMP analog, Sp-8-Br-cAMP also failed to elicit detectable changes in the levels of these proteins (Figs 3 and 5A). In contrast, the SKF38393induced up-regulation of the S338 residue phosphorylation of Raf-1 was both blockable by SQ 22,536 and H-89 and reproducible by forskolin and Sp-8-Br-cAMP (Figs 3 and 5B).
Co-incubation for 48 h of tissue cultures with 75 µM SKF38393and either 10 µM SQ 22 536 or 10 µM H-89 produced a small statistically insignificant reduction in the G0G1/SG2M ratio as compared to that in cultures exposed to the agonist alone (Fig. 5C).
Effects of SKF83959
Since the above-described observations have suggested that the ACcAMP second messenger pathway is unlikely to be responsible for the D1R stimulation-induced alterations in the levels of the three cell-cycle-controlling proteins examined in this study, we decided to assess possible involvement of D1R-driven PLCß-associated pathways (Undie, 1999; Bergson et al., 2003
). Unfortunately, direct agonists and antagonists of these pathways cannot be used under the present circumstances because mitogenic effects of EGF involve PLC
, the activity of which overlaps with that of PLCß (Fedi et al., 2000), and all presently available drugs are incapable of distinguishing between members of the PLCß- and PLC
-initiated intracellular cascades. Therefore, it would be impossible to discern which of the cell-cycle-associated effects of these drugs specifically relate to their interference with the neurotransmitter receptor-PLCß cascade rather than the EGF-PLC
cascade. We took a different approach by employing SKF83959 a novel D1R agonist that induces receptor-coupled stimulation of PLCß without concomitant activation of AC (Panchalingam and Undie, 2001
; Jin et al., 2003
).
We began by examining whether activation of D1R in our primary cultures of EGF-supported cerebral cortical precursor cells could lead to stimulation of PLCß and, thus, increase in phosphatidylinositide hydrolysis. We found that application of SKF83959(10 µM) nearly doubled the levels of [3H]inositol phosphate accumulation (Fig. 6A). This accumulation was absent when SKF83959was added to the cultures in combination with the D1R antagonist, SCH23390(0.1 µM; Fig. 6A).
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SKF38393- and SKF83959-induced Changes in Calcyon Levels
Recently, it has been demonstrated that one of the mechanisms by which D1R may stimulate PLCß involves the D1R-interacting protein, calcyon, which acts as a molecular linker between these receptors and Gq proteins coupled to heterologous non-dopamine receptors (Lezcano et al., 2000; Bergson et al., 2003
; Dai and Bergson, 2003
). In order for calcyon to work it must be primed, which can be achieved in several ways (Bergson et al., 2003
). It would not be unreasonable to expect that at least some active calcyon molecules could be present in our cultured cells. We felt that it might be informative to learn whether the levels of this protein in our cultures were regulated by SKF38393and SKF83959and whether the observed changes resembled those in the levels of the three cell-cycle-regulatory proteins examined in this study. We found that, indeed, calcyon levels in our cultures were affected by both agonists in a concentration-dependent manner. Furthermore, alterations in calcyon levels produced by 48 h long exposure to 0100 µM SKF38393showed a bell-shaped curve. They increased steadily when the cultures were exposed to 050 µM of SKF38393 but declined at higher concentrations of this agonist (Fig. 7A,C). The alterations in calcyon levels were statistically significant between 25 and 75 µM of SKF38393(Fig. 7C). In contrast, 48 h long exposure to 010 µM of SKF83959induced a concentration-dependent decrease in calcyon levels; at 10 µM SKF83959 this decrease was statistically significant (Fig. 7B,D). These effects of SKF38393(75 µM) and SKF83959(10 µM) were blocked by 10 and 0.1 µM SCH23390respectively (Fig. 7). The changes in the calcyon levels produced by both drugs were very similar to the earlier-described changes in the levels of P27.
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Discussion |
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A novel finding of this study is that D1R stimulation-induced supression of the proliferative activity in EGF-supported primary cultures of cerebral cortical precursor cells is accompanied by changes in the levels of cyclin D and P27 and levels and activation of Raf-1. These three proteins are known to be closely associated with regulation of the G1 to S transition of the cell cycle (Sheaff and Roberts, 1998; Denhardt, 1999
; Puri et al., 1999
). Therefore, it is reasonable to propose that the mechanism of the observed D1R influence over the cerebral cortical precursor cell progression into the S phase of the cell cycle, at least in part, involves these proteins.
It is also interesting that, based on our observations, the application of D1 agonists to the EGF-supported cerebral cortical precursor cells induces both cell-cycle-suppressing and cell-cycle-promoting molecular changes. Thus, in the case of Raf-1 we observed a combination of a decrease in its levels (which should lead to an inhibition of TrkRasRaf-1MEKERK cascade and consequently a supression of the EFG-promoted cell cycle; Denhardt, 1999), and an increase in its phosphorylation on S338 residue, which indicates a heightening of its TrkRas-dependent activation and consequently a potentiation of the above-mentioned cascade that is associated with promotion of the cell cycle (Diaz et al., 1997
; Mason et al., 1999
). Also, between the two direct cell-cycle-regulatory factors examined in this study, the cell-cycle-promoting protein, cyclin D, alone showed a persistent dose-dependent D1R agonist-induced down-regulation, which is consistent with the observed cell-cycle-inhibiting action of D1Rs. In contrast, the dose dependent up-regulation of the cell cycle progression-inhibiting protein, P27, was seen only up to 50 µM SKF38393 With further increases in the SKF38393concentration, the magnitude of the P27 up-regulation declined until this up-regulation completely disappeared at 100 µM of the agonist. Furthermore, SKF83959concentrations examined in this study produced a decrease in P27 levels suggesting a cell-cycle-promoting activity. This indicates that the D1R-induced supression of the cell cycle progression in our EGF-supported primary cultures of cerebral cortical precursor cells represents a net effect of competing cell-cycle-promoting and inhibiting molecular changes, with the latter changes being more predominant in this case.
It should be noted that, as was mentioned earlier, our cultures contain a mixture of multipotential, glial, and neuronal precursor cells. The methodology used in this study does not allow us to determine whether the observed protein changes occur in all or only some subpopulations of these cells. We hope that future studies will be able to address this issue.
cAMP Second Messenger Cascade is not Responsible for D1R-induced Alterations in the Levels of Raf-1, Cyclin D and P27
The central finding of this study is that the D1R-induced increase in the intracellular levels of cAMP, as well as the resultant activation of the major cAMP-dependent protein kinase, PKA, may not be involved in alterations in any of the three proteins examined in our cultured cells. This was demonstrated by the inability of the AC inhibitor, SQ 22,536, and the PKA inhibitor, H-89, to prevent SKF38393from inducing such alterations, as well as the failure of the AC stimulator, forskolin, and the cell permeable cAMP analog, Sp-8-Br-cAMP, to reproduce the SKF38393induced changes. Such lack of regulatory activity by the cAMP cascade agonists and antagonists is unlikely to be due to their application at ineffective concentrations since the same drug applications were fully capable of respectively blocking or replicating the SKF38393induced increase in the cycle-promoting activation of Raf-1 (seen as up-regulation of its phosphorylation on S338 residue). The observed lack of cAMP and/or PKA participation in cell cycle suppression through Raf-1, cyclin D, and P27 is very surprising since inhibition of Raf-1 activity, down-regulation of cyclin D, and up-regulation of P27 have long been considered among the major cell cycle regulatory actions of the cAMPPKA intracellular cascade (Hafner et al., 1994; Kato et al., 1994
; Cho-Chung et al., 1995
; Ward et al., 1996
; Cospedal et al., 1999
; van Oirschot et al., 2001
; Stork and Schmitt, 2002
; Shibata et al., 2003
). Furthermore, our data suggest that EGF-supported cerebral cortical precursor cells represent one of the relatively rare biological systems in which cAMP second messenger pathway is capable of promoting the activation of Raf-1 (Stork and Schmitt, 2002
). As mentioned above, this represents a stimulatory influence on cell proliferation (Diaz et al., 1997
; Mason et al., 1999
). It is important to note, however, that cAMP-induced inhibition of Raf-1 might be present in other groups of proliferating cerebral cortical neural cells. This is indicated by the reported involvement of this second messenger in deactivation of Raf-1 in cultures of bFGF-supported cortical astrocytes (Kurino et al., 1996
).
Possible Involvement of PLCß-associated Second Messenger Cascades
The apparent lack of involvement of the cAMP second messenger cascade in the changes of the levels of Raf-1, cyclin D and P27 seen in our cell cultures does not necessarily exclude this cascade from participating in the cell cycle supression in these cultures through some other intracellular venues. Indeed, we observed that the addition of antagonists of the cAMP pathway to our SKF38393treated cultures produced some reduction in the cell cycle suppressing activity of this D1 receptor agonist. However, since exposures of similar cultures to the D1R agonist, SFK83959 which stimulates only PLCß, resulted in supression the cell cycle progression, it is reasonable to propose that activation of the cAMP cascade is not necessary for the cell cycle regulatory actions of D1Rs in EGF-supported cerebral cortical precursor cells. These actions may be conducted via the PLCß-associated second messenger cascades. Moreover, the ability of SKF83959to mimic SKF38393induced concentration-dependent down-regulation of Raf-1 and cyclin D levels suggests that these D1R-induced effects may also be driven by PLCß stimulation. While we are unaware of any studies that have examined PLCß cascade-induced regulation of Raf-1 levels, the ability of this cascade to affect the levels of cyclin D has been described in several papers (Bianchi et al., 1994; Fukumoto et al., 1997
; Frey et al., 2000
). The concentration-dependent patterns of P27 regulation by SKF83959did not match those produced by SKF38393 Nevertheless, D1R may still affect the levels of this protein via PLCß-associated cascades. Such a possibility comes out of our observation of the SKF38393 and SKF83959induced changes in the levels of calcyon, a protein linking D1Rs to PLCß stimulation. This protein also was differentially regulated by SKF38393and SKF83959 with its concentration-dependent changes in response to both agonists resembling closely the concentration-dependent changes induced by these agonists in the levels of P27. It is possible that the observed alterations in the levels of calcyon and P27 represent two independent parallel intracellular processes, but it is tempting to speculate that there is a causal relationship between them, and, since calcyon is a part of the D1RGq proteinPLCß signalling system, this relationship would indicate the PLCß involvement in P27 regulation by D1Rs. The latter would not be unexpected because the ability of PLCß-associated pathways to regulate the levels of P27 is well documented (Ashton et al., 1999
; Chen et al., 1999
; Frey et al., 2000
). The fact that SKF38393induced changes in the levels of calcyon do not match the changes in the levels of Raf-1 and cyclin D may indicate that the D1R-induced regulation of the latter two proteins involves a calcyon-independent D1R stimulation of Gq proteinPLCß signalling. The existence of this calcyon-independent pathway is suggested by the ability of D1Rs to induce IP3 formation in striatal neurons (Undie and Friedman, 1992
) lacking synaptic calcyon (Lezcano and Bergson, 2002
). It should also be noted that the present study employed only an indirect examination of the involvement of PLCß cascades in D1R regulation of the cell cycle and levels of cell cycle proteins. Therefore, it is conceivable that at least some of the observed effects may be related to SKF83959SKF83959-induced stimulation of some other, currently un-identified, non-AC- and non-PLCß-associated second messenger pathways. Presently, we are conducting studies aimed at fully resolving the question of the involvement of PLCß and its second messenger cascades in the D1R regulation of the cell cycle in cerebral cortical precursor cells.
Address correspondence to Michael S. Lidow, Department of Biomedical Sciences, University of Maryland, Baltimore, 5-A-12, HHH, 666 W. Baltimore St, Baltimore, MD 21201, USA. Email: mlidow{at}umaryland.edu.
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Bergson C, Levenson R, Goldman-Rakic PS, Lidow MS (2003) Dopamine receptor-interacting proteins: the Ca2+ connection in dopamine signaling. Trends Pharmacol Sci 24:486492.[CrossRef][ISI][Medline]
Bianchi S, Fabiani S, Muratori M, Arnold A, Sakaguchi K, Miki T, Brandi ML (1994) Calcium modulates the cyclin D1 expression in a rat parathyroid cell line. Biochem Biophys Res Commun 204:691700.[CrossRef][ISI][Medline]
Bogdan S, Klambt C (2001) Epidermal growth factor receptor signaling. Curr Biol 11:R292R295.[CrossRef][ISI][Medline]
Burrows RC, Wancio D, Levitt P, Lillien L (1997) Response diversity and the timing of progenitor cell maturation are regulated by developmental changes in EGFR expression in the cortex. Neuron 19:251267.[ISI][Medline]
Chen YJ, Lin JK, Lin-Shiau SY (1999) Proliferation arrest and induction of CDK inhibitors p21 and p27 by depleting the calcium store in cultured C6 glioma cells. Eur J Cell Biol 78:824831.[ISI][Medline]
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:52675272.
Cho-Chung YS, Pepe S, Clair T, Budillon A, Nesterova M (1995) cAMP-dependent protein kinase: role in normal and malignant growth. Crit Rev Oncol Hematol 21:3361.[CrossRef][ISI][Medline]
Cospedal R, Lobo M, Zachary I (1999) Differential regulation of extracellular signal-regulated protein kinases (ERKs) 1 and 2 by cAMP and dissociation of ERK inhibition from anti-mitogenic effects in rabbit vascular smooth muscle cells. Biochem J 342:407414.[CrossRef][ISI][Medline]
Dai R, Bergson C (2003) Structure and expression of the murine calcyon gene. Gene 311:111117.[CrossRef][ISI][Medline]
Denhardt DT (1999) Signal transduction pathways and regulation of the mammalian cell cycle: cell type-dependent integration of external signals. In: The molecular basis of cell cycle and growth control (Stein GS, Baserga R, Giordano A, Denhardt DT, eds), pp. 225304. New York: John Wiley.
Diaz B, Barnard D, Filson A, MacDonald S, King A, Marshall M (1997) Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling. Mol Cell Biol 17:45094516.[Abstract]
Fabbri E, Brighenti L, Ottolenghi C (1991) Inhibition of adenylate cyclase of catfish and rat hepatocyte membranes by 9-(tetrahydro-2-furyl)adenine (SQ 22536). J Enzyme Inhib 5:8798.[Medline]
Frey MR, Clark JA, Leontieva O, Uronis JM, Black AR, Black JD (2000) Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium. J Cell Biol 151:763778.
Fukumoto S, Nishizawa Y, Hosoi M, Koyama H, Yamakawa K, Ohno S, Morii H (1997) Protein kinase C delta inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression. J Biol Chem 272:1381613822.
Gomperts BD, Kramer IM, Tatham PER (2002) Signal transduction. San Diego, CA: Academic Press.
Hafner S, Adler HS, Mischak H, Janosch P, Heidecker G, Wolfman A, Pippig S, Lohse M, Ueffing M, Kolch W (1994) Mechanism of inhibition of Raf-1 by protein kinase A. Mol Cell Biol 14:66966703.[Abstract]
Jin LQ, Cai G, Wang HY, Smith C, Friedman E (1998) Characterization of the phosphoinositide-linked dopamine receptor in a mouse hippocampalneuroblastoma hybrid cell line. J Neurochem 71:19351943.[ISI][Medline]
Jin LQ, Goswami S, Cai G, Zhen X, Friedman E (2003) SKF83959selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain. J Neurochem 85:378386.[CrossRef][ISI][Medline]
Johansen PA, Hu XT, White FJ (1991) Relationship between D1 dopamine receptors, adenylate cyclase, and the electrophysiological responses of rat nucleus accumbens neurons. J Neural Transm 86:97113.[Medline]
Kato JY, Matsuoka M, Polyak K, Massague J, Sherr CJ (1994) Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell 79:487496.[ISI][Medline]
Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM (1998) A family of cAMP-binding proteins that directly activate Rap1. Science 282:22752279.
Koh PO, Bergson C, Undie AS, Goldman-Rakic PS, Lidow MS (2003) Up-regulation of the D1 dopamine receptor-interacting protein, calcyon, in patients with schizophrenia. Arch Gen Psychiatry 60:311319.
Kurino M, Fukunaga K, Ushio Y, Miyamoto E (1996) Cyclic AMP inhibits activation of mitogen-activated protein kinase and cell proliferation in response to growth factors in cultured rat cortical astrocytes. J Neurochem 67:22462255.[ISI][Medline]
Lezcano N, Bergson C (2002) D1/D5 dopamine receptors stimulate intracellular calcium release in primary cultures of neocortical and hippocampal neurons. J Neurophysiol 87:21672175.
Lezcano N, Mrzljak L, Eubanks S, Levenson R, Goldman-Rakic P, Bergson C (2000) Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein. Science 287:16601664.
Lidow MS, Roberts A, Zhang L, Koh PO, Lezcano N, Bergson C (2001) Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors. Eur J Pharmacol 427:187193.[CrossRef][ISI][Medline]
Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ, Marais R (1999) Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 18:21372148.
Matsushime H, Ewen ME, Strom DK, Kato JY, Hanks SK, Roussel MF, Sherr CJ (1992) Identification and properties of an atypical catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1 cyclins. Cell 71:323334.[ISI][Medline]
Missale C, Nash SR, Robinson SW, Jaber M, Caron MG (1989) Dopamine receptors: from structure to function. Physiol Rev 78:189225.
Munoz JP, Sanchez JR, Maccioni RB (2003) Regulation of p27 in the process of neuroblastoma N2A differentiation. J Cell Biochem 89:539549.[CrossRef][ISI][Medline]
Ng TB, Ye XY (2003) Fabin, a novel calcyon-like and glucanase-like protein with mitogenic, antifungal and translation-inhibitory activities from broad beans. Biol Chem 384:811815.[CrossRef][ISI][Medline]
Otey CA, Kalnoski MH, Bulinski JC (1987) Identification and quantification of actin isoforms in vertebrate cells and tissues. J Cell Biochem 34:113124.[ISI][Medline]
Panchalingam S, Undie AS (2001) SKF83959exhibits biochemical agonism by stimulating [35S]GTP gamma S binding and phosphoinositide hydrolysis in rat and monkey brain. Neuropharmacology 40:826837.[CrossRef][ISI][Medline]
Perez-Juste G, Aranda A (1999) The cyclin-dependent kinase inhibitor p27(Kip1) is involved in thyroid hormone-mediated neuronal differentiation. J Biol Chem 274:50265031.
Puri PL, MacLachlan TK, Levrero M, Giordano A (1999) the intrinsic cell cycle: from east to mammals. In: The molecular basis of cell cycle and growth control (Stein GS, Baserga R, Giordano A, Denhardt DT, eds), pp. 1579. New York: John Wiley.
Raballo R, Rhee J, Lyn-Cook R, Leckman JF, Schwartz ML, Vaccarino FM (2000) Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex. J Neurosci 20:50125023.
Roberts-Lewis JM, Roseboom PH, Iwaniec LM, Gnegy ME (1986) Differential down-regulation of D1-stimulated adenylate cyclase activity in rat forebrain after in vivo amphetamine treatments. J Neurosci 6:22452251.[Abstract]
Seamon KB, Daly JW (1998) Forskolin: its biological and chemical properties. Adv Cyclic Nucleotide Protein Phosphorylation Res 20:1150.
Sheaff RJ, Roberts JM (1998) Regulation of G1 phase. In Cell cycle control (Pagano M, ed), pp. 134. New York: Springer.
Shibata K, Katsuma S, Koshimizu T, Shinoura H, Hirasawa A, Tanoue A, Tsujimoto G (2003) Alpha 1-adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1. J Biol Chem 278:672678.
Sibley DR, Monsma FJ Jr, Shen Y (1993) Molecular neurobiology of dopaminergic receptors. Int Rev Neurobiol 35:391415.[ISI][Medline]
Slingerland J, Pagano M (2000) Regulation of the cdk inhibitor p27 and its deregulation in cancer. J Cell Physiol 183:1017.[CrossRef][ISI][Medline]
Stanton VP Jr, Nichols DW, Laudano AP, Cooper GM (1989) Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol Cell Biol 9:639647.[ISI][Medline]
Stein GS, Stein JL, Lian JB, Last TJ, Owen TA, McCable L (1998) Synchronisation of normal diploid and transformed mammalian cells. In: Cell biology: a laboratory handbook (Celis JE, ed.), Vol, I, pp. 253260. San Diego, CA: Academic Press.
Stork PJ, Schmitt JM (2002) Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12:258266.[CrossRef][ISI][Medline]
Tikoo R, Casaccia-Bonnefil P, Chao MV, Koff A (1997) Changes in cyclin-dependent kinase 2 and p27kip1 accompany glial cell differentiation of central glia-4 cells. J Biol Chem 272:442447.
Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van der Kooy D (1999) Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 208:166188.[CrossRef][ISI][Medline]
Undie AS (1999) Relationship between dopamine agonist stimulation of inositol phosphate formation and cytidine diphosphate-diacylglycerol accumulation in brain slices. Brain Res 816:286294.[CrossRef][ISI][Medline]
Undie AS, Friedman E (1992) Selective dopaminergic mechanism of dopamine and SKF38393stimulation of inositol phosphate formation in rat brain. Eur J Pharmacol 226:297302.[CrossRef][Medline]
Undie AS, Friedman E (1994) Inhibition of dopamine agonist-induced phosphoinositide hydrolysis by concomitant stimulation of cyclic AMP formation in brain slices. J Neurochem 63:222230.[ISI][Medline]
van Oirschot BA, Stahl M, Lens SM, Medema RH (2001) Protein kinase A regulates expression of p27kip1 and cyclin D3 to suppress proliferation of leukemic T cell lines. J Biol Chem 276:3385433860.
Wang F, Bergson C, Howard RL, Lidow MS (1997) Differential expression of D1 and D5 dopamine receptors in the fetal primate cerebral wall. Cereb Cortex 7:711721.[Abstract]
Ward AC, Csar XF, Hoffmann BW, Hamilton JA (1996) Cyclic AMP inhibits expression of D-type cyclins and cdk4 and induces p27Kip1 in G-CSF-treated NFS-60 cells. Biochem Biophys Res Commun 224:1016.[CrossRef][ISI][Medline]
Yamada M, Ikeuchi T, Hatanaka H (1997) The neurotrophic action and signalling of epidermal growth factor. Prog Neurobiol 51:1937.[CrossRef][ISI][Medline]
Zezula J, Casaccia-Bonnefil P, Ezhevsky SA, Osterhout DJ, Levine JM, Dowdy SF, Chao MV, Koff A (2001) p21cip1 Is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal. EMBO Rep 2:2734.
Zhang L, Lidow MS (2002) D1 dopamine receptor regulation of cell cycle in FGF- and EGF-supported primary cultures of embryonic cerebral cortical precursor cells. Int J Dev Neurosci 20:593606.[CrossRef][ISI][Medline]