Prolactin-Regulated Tyrosine Hydroxylase Activity and Messenger Ribonucleic Acid Expression in...

Prolactin-Regulated Tyrosine Hydroxylase Activity andMessenger Ribonucleic Acid Expression in MediobasalHypothalamic Cultures: The Differential Role of SpecificProtein Kinases

Frank Y. Ma, David R. Grattan, Vincent Goffin, and Stephen J. Bunn

The Centre for Neuroendocrinology (Y.F.M., D.R.G., S.J.B), Department of Anatomy and Structural Biology, The Universityof Otago, Dunedin 9001, New Zealand; and Institut National de la Sante et de la Recherche Medicale (V.G.), U-584,Molecular Endocrinology, Faculte de Medecine Necker, 75730 Paris, France

Prolactin secretion from the anterior pituitary is tightly reg-ulated by feedback onto the hypothalamic neuroendocrinedopaminergic (NEDA) neurons. Prolactin stimulates theseneurons to synthesize and secrete dopamine, which acts viathe pituitary portal vasculature to inhibit prolactin secretionfrom the pituitary lactotrophs. Despite the physiological im-portance of this feedback, relatively little is known about thesignaling mechanisms responsible for prolactin activation ofNEDA neurons. This issue has been examined here using a cellculture preparation of the fetal rat mediobasal hypothalamus.Prolactin stimulated a time- and concentration-dependent in-crease in catecholamine synthesis, which was maximal after60–120 min (1 �g/ml prolactin) and inhibited by the prolactinantagonist �1–9-G129R-hPRL. This prolactin response was ac-companied by a rise in the site-specific (ser-19, -31, and -40)phosphorylation of tyrosine hydroxylase (TH), the rate-limit-

ing enzyme in catecholamine synthesis. Consistent with thisobservation, the prolactin-induced increase in catecholaminesynthesis was abolished by inhibitors of protein kinase A andprotein kinase C (PKC). Prolactin incubation also resulted ina PKC-dependent activation of the MAPK pathway, althoughthis was not required for the stimulation of catecholaminesynthesis. In addition to increasing TH phosphorylation andcatecholamine synthesis, prolactin also increased TH mRNAexpression. In contrast to catecholamine synthesis, this latterresponse was not suppressed by inhibition of protein kinaseA or PKC. These results indicate that although prolactin con-trols catecholamine synthesis in NEDA neurons by regulatingboth TH activity and TH mRNA expression, it employs dis-tinct, nonoverlapping, signaling pathways to achieve theseends. (Endocrinology 146: 93–102, 2005)

PROLACTIN IS A HORMONE of major physiologicalimportance with many actions in addition to its well-

described role in lactation (1, 2). The secretion of prolactinfrom the anterior pituitary lactotrophs is subject to a numberof regulatory factors, the most important being an inhibitoryinput from dopamine carried in the hypophysial portal sys-tem (1). Dopamine found within these vessels is synthesizedand secreted by the neuroendocrine dopaminergic (NEDA)neurons located in the arcuate and periventricular nuclei ofthe mediobasal hypothalamus. The NEDA neurons arepresent as three distinct groups, referred to as the tuberoin-fundibular, tuberohypophyseal, and periventricular-hypo-physeal dopaminergic neurons. The relative contribution ofeach of these NEDA populations to the control of prolactinsecretion is not fully resolved, although the tuberoinfun-dibular dopaminergic (TIDA) neurons, which deliver dopa-mine directly into the median eminence, appear to be par-

ticularly important (1). Circulating prolactin stimulates theactivity of all three populations of NEDA neurons, thus in-creasing dopamine release and ultimately suppressing itsown secretion from the anterior pituitary (3). Whereas thisshort-loop feedback pathway is well established, the cellularmechanisms by which prolactin stimulates NEDA neuronactivity remains to be resolved.

Prolactin receptors have been localized within the hypo-thalamus including the arcuate nucleus and median emi-nence and have been identified colocalized on TIDA neurons(4, 5). Isolated dopaminergic neurons from the fetal rat hy-pothalamus have also been shown to express prolactin re-ceptors (6). When maintained in culture, these neurons re-spond to prolactin receptor activation by increasing their rateof dopamine synthesis (6). Thus, it would appear likely thatTIDA, and perhaps other NEDA neurons, are directly re-sponsive to prolactin. As noted above, the cellular mecha-nisms underlying such a response are not fully resolved.Whereas data from signal transducer and activator of tran-scription-5b-deficient mice suggest that this transcriptionfactor is required for prolactin-induced activation of theseneurons (7), other studies indicate that prolactin may alsoexert nontranscription regulation of dopamine synthesis andrelease. In vivo studies suggest that the TIDA neuron re-sponse to prolactin is relatively rapid (8) with the initialincrease in dopamine synthesis being independent of tran-scription and translation, followed by a delayed (12 h) further

First Published Online September 23, 2004Abbreviations: CaMKII, Ca2�/calmodulin-dependent protein kinase

II; HBS, HEPES-buffered saline; MAPKAPK, MAPK-activated proteinkinase; MEK, MAPK/ERK kinase; NEDA, neuroendocrine dopaminer-gic; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; TH, tyrosine hydroxylase; TIDA, tuberoinfundibu-lar dopaminergic.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/05/$15.00/0 Endocrinology 146(1):93–102Printed in U.S.A. Copyright © 2005 by The Endocrine Society

doi: 10.1210/en.2004-0800

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increase, which was sensitive to cycloheximide inhibition (9).In vitro studies using rat hypothalamic slices have also re-ported a relatively rapid (2 h) prolactin-induced increase indopamine synthesis (10), whereas studies using hypotha-lamic fetal cells in culture reported the need for long-termprolactin exposure (1–10 d) to significantly increase dopa-mine levels (6).

The rate of dopamine synthesis in TIDA and other dopa-minergic neurons is controlled by the activity of tyrosinehydroxylase (TH), the rate-limiting enzyme in catecholaminebiosynthesis (11, 12). TH is subject to many regulatory inputsboth chronically at the level of gene transcription and trans-lation and acutely at the level of the enzyme activity. THactivity is tightly regulated by end-product inhibition, in thatdopamine suppresses its activity by competing with thebinding of the tetrahydrobiopterin BH4 cofactor (12). Phos-phorylation of TH, within its N-terminal regulatory domain,reduces the affinity of dopamine binding, thus relieving theinhibition and elevating TH activity. Physiologically relevantregulation of TH through its phosphorylation has been welldescribed in a number of tissues and appears to involve threespecific serine residues, Ser-19, -31, and -40 (13). The proteinkinases responsible for the phosphorylation of each of theseresidues and their precise roles in the activation of the en-zyme are still relatively unclear. At present, however, it isprobable that Ser-19 is phosphorylated by Ca2�/calmodulin-dependent protein kinase II (CaMKII), MAPK-activated proteinkinase (MAPKAPK) 2, and p38-regulated/activated kinase(14–16). Ser-31 appears to be exclusively phosphorylated by theMAPKSs ERK1/2 (17). In contrast, Ser-40 may be phosphory-lated by a number of protein kinases including protein kinaseA (PKA), protein kinase C (PKC), protein kinase G, CaMKII,MAPKAPK1/2, and the mitogen- and stress-activated proteinkinase 1 (11, 16).

Despite its physiological importance, our understandingof the intracellular pathways by which prolactin receptoractivation couples to either TH expression or TH phosphor-ylation is still incomplete. The aim of this current study wastherefore to conduct a detailed investigation into the kinasepathways activated in TIDA neurons by prolactin and de-termine their influence on the level of dopamine synthesis.To achieve this aim, we employed cell cultures preparedfrom the mediobasal hypothalamus of the embryonic rat pupand determined the effect of protein kinase inhibitors on theability of prolactin to increase both TH activity (as reflectedin the level of catecholamine synthesis) and TH mRNAexpression.

Materials and MethodsHypothalamic cell cultures

Cultures of the rat mediobasal hypothalamus were prepared usingthe method developed by Arbogast and Voogt (6) modified as describedpreviously (18). All animal procedures were approved by the Universityof Otago Animal Ethics Committee. Briefly, mediobasal hypothalamifrom 28–56 embryonic d 18–21 fetal rats were excised to a depth ofapproximately 1 mm, limited anteriorly by optic chiasm, posteriorly bythe border of the mamillary bodies, and laterally by the hypothalamicfissures. Tissue blocks were pooled, rinsed with buffer (137 mm NaCl,5.4 mm KCl, 59 mm sucrose, 0.2 mm Na2HPO4, 0.2 mm KH2PO4, 5.5 mmglucose, 100,000 U/liter penicillin, 100 mg/liter streptomycin, and 250�g/liter Fungizone adjusted to pH 7.4), and roughly chopped. Tissue

fragments were then incubated with 10 ml trypsin (2.5 mg/ml in thesame buffer) at 37 C for 5 min at which time 1 ml DNase solution (1mg/ml) was added and the incubation continued for a further 5 min. Thedigestion was terminated by the addition of 12 ml of soybean trypsininhibitor solution (0.3 mg/ml) followed by centrifugation at 800 � g for5 min. The resultant pellet was resuspended, washed with buffer, andagain centrifuged at 800 � g for 5 min before being dispersed at a densityof approximately 106 cells/ml in DMEM (Life Technologies, Inc., Rock-ville, MD) containing 2.5% fetal bovine serum and 5% horse serum. Cellswere plated out at 1 ml/well (106 cells/well) onto poly-d-lysine-coated24-well culture plates or for immunocytochemical investigations poly-d-lysine-coated glass coverslips in 24-well culture plates. Plated cellswere then transferred to 5% CO2 humidified incubator at 37 C. After thefirst 24 h in culture, the medium was removed and replaced with aserum- and phenol red-free DMEM containing high glucose and KCl(each at 25 mm) and the following reagents (each obtained from Sigma,St. Louis, MO): linolenic acid (1 �g/ml), linoleic acid (1.5 �g/ml), pu-trescine (4.4 �g/ml), and ITS� (insulin-transferrin-sodium selenite sup-plement) (1:100 dilution). The medium was replaced every second dayand the cells routinely used for experimentation after 14–21 d in culture.

Immunocytochemistry

Cells cultured on glass coverslips were removed from the incubator,washed twice (5 min each) with 1 ml of 10 mm PBS, fixed with 2%formaldehyde in PBS for 20 min at room temperature, and then per-meabilized with 90% ethanol/10% acetic acid (5 min at �20 C). Cellswere then washed three times for 5 min each in 1 ml PBS followed by1 h in PBS containing 5% goat serum and then 48 h at 4 C with acombination of both primary antibodies, polyclonal anti-TH (AB151diluted 1:200, from Chemicon, Temecula, CA) and monoclonal antip-rolactin receptor (rat) (MA1–610 diluted 1:100, from Affinity Biore-agents, Golden, CO) made up in PBS containing 5% goat serum. The cellswere then washed (6 � 5 min in PBS) and incubated with a combinationof secondary antibodies antimouse Alexa488 and antirabbit Alexa568both diluted 1:1000 in PBS (Molecular Probes, Eugene, OR) for 30 minat room temperature. Coverslips were mounted in Vectashield (VectorLaboratories, Burlingame, CA) and examined under fluorescence mi-croscopy using an AX70 microscope (Olympus, Tokyo, Japan) fittedwith a Spot-RT color digital camera (Diagnostic Instruments Inc., Ster-ling Heights, MI).

Catecholamine synthesis

Catecholamine synthesis was measured using the method originallydeveloped for adrenal chromaffin cells (19) modified as described pre-viously (18). Cells were removed from the incubator and washed briefly(twice for 5 min) with 400 �l carbogen-gassed HEPES-buffered saline(HBS) of the following composition: 150 mm NaCl, 15 mm HEPES, 5.5mm glucose, 3.8 mm K2HPO4, 1 mm MgSO4, 1.5 mm CaCl2, and 0.5 mmsodium ascorbate, adjusted to pH 7.4 at 37 C. The last wash was replacedwith 200 �l HBS containing 20 �m l-[carboxyl-14C] tyrosine (0.22 �Ci perwell, purchased from Amersham Biosciences NZ Ltd. Auckland, NewZealand) in the presence or absence of a stimulating agent, routinely1 �g/ml prolactin (ovine prolactin obtained from Sigma). Each well wasimmediately fitted with a tube sealed with a rubber stopper from whichwas suspended a small plastic cap containing 200 �l of 1 m NaOH toabsorb the 14CO2 produced by the cells during the decarboxylation stepin catecholamine synthesis. The incubation was terminated by the in-jection of 400 �l of ice-cold 10% trichloroacetic acid through the rubberstopper. The plates, with their sealed wells in place, were then kept at4 C for 2 h before an aliquot of the NaOH solution was collected and the14C radioactivity measured by liquid scintillation spectrometry. Whenexamining the effect of prolactin antagonist or protein kinase inhibitors,these agents were included during both a 15-min preincubation periodand the subsequent stimulation. PD-98059, KN-92, KN-93, and H89 werepurchased from Sigma; H85 from Seikagaku (Tokyo, Japan); and bisin-domaleimide I and V, actinomycin D, and cycloheximide from Calbio-chem (San Diego, CA). Recombinant prolactin receptor antagonist �1–9-G129R-hPRL was prepared and purified as described previously (20).Catecholamine synthesis was expressed relative to basal levels (cellsincubated with buffer alone for the same time period) or, where ap-propriate, a control value obtained from cells incubated in the absence

94 Endocrinology, January 2005, 146(1):93–102 Ma et al. • Prolactin Regulation of Tyrosine Hydroxylase

of an antagonist and statistical comparisons performed using a Mann-Whitney U test (unless otherwise stated).

TH mRNA expression

Cells were removed from the incubator and briefly washed with 500�l serum-free DMEM and then returned to the incubator in a further 500�l serum-free DMEM with or without prolactin (1 �g/ml) in the pres-ence or absence of an appropriate protein kinase inhibitor. After 4 hincubation, each well was extracted into 200 �l TRIzol reagent and totalRNA isolated as per manufacturer’s instructions. The resultant RNA wasreverse transcribed using GeneAmp Gold RNA PCR kit (PE AppliedBiosystems, Foster City, CA) and quantitative real-time RT-PCR for THand �-actin performed using the Taqman system (PE Applied Biosys-tems). Primer and probe details were exactly as described previously(18). The reaction mix was also prepared as previously described and anABI PRISM 7700 sequence detection system (Centre for Gene Research,University of Otago) used to detect fluorescence during each PCR underconditions exactly as described previously (18). Data were captured andanalyzed using sequence detector software (SDS, version 3.0, PE Ap-plied Biosystems). TH mRNA levels in each sample were normalizedwith reference to its �-actin mRNA levels and then expressed as relativeto TH mRNA in control cells (i.e. those incubated with prolactin in theabsence of kinase inhibitors) determined in the same experiment. Sta-tistical analysis was performed using a Mann-Whitney U test.

Reverse-transcribed RNA extracted from cells was also probed for thelong and short forms of the prolactin receptor. The forward primer forthe prolactin receptor short and long form was 5�-ATACTG GAG TAGATG GAG CCA GGA GAG TTC-3� corresponding to nucleotides 624–653 of the prolactin receptor cDNA sequence. The reverse primer for theshort form was 5�-TCC TAT TTG AGT CTG CAG CTT CAG TAG TCA-3�corresponding to nucleotides 924–953 of the cDNA sequence, and thereverse primer for the long form was 5�-CTT CCG TGA CCA GAG TCACTG TCG GGA TCT-3� corresponding to nucleotides 1014–1043 of thecDNA sequence (21). The cDNA was amplified through 30 PCR cycles,the products resolved on 1.5% agarose gels, and visualized withethidium bromide staining.

Activation of ERK1/2

The phosphorylation of ERK1/2 was measured by immunoblottingusing activation state-specific antibodies. Cells were washed twice for 5min with 400 �l HBS and then preincubated for 15 min with PD98059(50 �m) or bisindolylmaleimide I (3 �m) before being stimulated for 15min with or without prolactin (1 �g/ml) in the continued presence orabsence of the appropriate inhibitor. The cells were then lysed in ice-cold10 mm Tris buffer (three wells collected in a total of 200 �l) containing5 mm EDTA, 50 mm NaF, 50 mm NaCl, 1% Triton-X100, 1 mm sodiumorthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 10 �l /ml ofprotease inhibitor cocktail (Sigma). The lysate was sonicated, centri-fuged at 12,000 � g for 10 min and the resultant supernatant fractionatedby SDS-PAGE and transferred onto nitrocellulose membranes. Thesemembranes were probed overnight at 4 C using a phospho-p44/42MAPK (Thr202/Tyr204)-specific monoclonal antibody (at 1:1000 dilu-tion, Cell Signaling Technology, Beverly, MA). The membranes werethen washed with 10 mm Tris-buffered saline containing 0.05% Tween20 and reincubated with horseradish peroxidase-coupled anti-IgG an-tibody (1:5000 dilution for 60 min at room temperature), washed again,and the image developed using enhanced chemiluminescence. Relativelevels of phosphorylation of ERK1/2 were determined using densito-metric image analysis (National Institutes of Health Image) and ex-pressed as a percentage of that obtained from cells incubated with bufferalone and examined on the same immunoblot. Statistical analysis wasperformed using a Mann-Whitney U test.

TH phosphorylation

Cells were washed and stimulated with or without prolactin (1 �g/mlfor 120 min), extracted in lysis buffer, and processed for immunoblottingas described above. Blots were probed overnight at 4 C using antibodiesraised against TH (AB151 diluted 1:200, Chemicon) or specific TH phos-phorylation sites (ser-19, ser-31, or ser-40; 1:10,000, kindly provided byProf. Peter Dunkley, University of Newcastle, New South Wales, Aus-

tralia) and then washed and labeled with secondary antibodies as de-scribed above. The density of immunoreactive bands was visualized andquantitated on the Typhoon system (Molecular Dynamics, Sunnyvale,CA) and expressed as a percentage of those detected in cells incubatedwith buffer alone and examined on the same immunoblot. Details re-garding the generation and characterization of these antibodies weredescribed by Cammarota et al. (22).

Results

Cultures prepared from the fetal rat mediobasal hypothal-amus contained a population of TH immunoreactive cells.Cell counts indicated that these TH-positive cells constitutedapproximately 7 � 1% of the total cell population (deter-mined from six separate cell culture preparations). Some,although not all, of these TH-positive cells also expressedprolactin receptors, as shown by immunocytochemistry (Fig.1A). It should be noted that prolactin receptor expression wasnot limited to TH-positive cells, in that other unidentified celltypes were also immunoreactive for this antibody (Fig. 1A).RT-PCR performed on extracts of these cultures indicatedthat the long, rather than short, form of prolactin receptorwas the predominant form expressed in these cultures (Fig. 1B).

Incubation with prolactin stimulated a time- and concen-tration-dependent increase in catecholamine synthesis, pre-sumably reflecting an increase in TH activity (Fig. 2, A andB). This response was slow and fairly modest but highlyconsistent with a statistically significant increase in catechol-

FIG. 1. The expression of prolactin receptors on TH immunoreactiveneurons in mediobasal hypothalamic cultures. A, Cell cultures wereprepared as described in Materials and Methods and then dual la-beled with antibodies directed against TH (left panel) and prolactinreceptors (right panel). Note that prolactin receptor immunoreactiv-ity was present on, but not limited to, cells that were also immuno-reactive for TH (a non-TH-immunoreactive cell indicated by arrow,immediately adjacent to a TH-positive cell). B, Mediobasal hypotha-lamic cell cultures were prepared and maintained as described inMaterials and Methods. After 14 d in culture, total mRNA was ex-tracted and mRNA for �-actin (lane 1), short-form prolactin receptor(lane 2), long-form prolactin receptor (lane 3), TH (lane 4), and shortand long forms of the prolactin in choroid plexus (lane 5 and 6, respec-tively) detected by RT-PCR as described in Materials and Methods.

Ma et al. • Prolactin Regulation of Tyrosine Hydroxylase Endocrinology, January 2005, 146(1):93–102 95

amine production (�120% of the basal response) seen aftera 60-min incubation with prolactin (1 �g/ml). Extendingthe incubation period to 120 min did not further increasecatecholamine synthesis (Fig. 2A). A prolactin-stimulatedincrease in catecholamine synthesis was evident with con-centrations of prolactin as low as 10 ng/ml and was con-centration dependent, although no apparent maximum wasreached with concentrations up to 1 �g/ml (Fig. 2B). Theresponse to this highest concentration of prolactin was in-hibited by the prolactin antagonist, �1–9-G129R-hPRL (10�g/ml), although it should be noted that catecholamine pro-

duction in these cells was still significantly higher than thatin cells incubated with the antagonist alone (Fig. 2B). Theinclusion of serum (2.5% fetal calf serum and 5% horse se-rum) in the culture medium did not alter responsiveness toprolactin, with a 120-min incubation with prolactin (1 �g/ml), resulting in a 119 � 5% basal response in the presenceof serum, compared with 119 � 3% basal in its absence (n �3 from separate cell culture preparations).

It is well recognized that TH activity, and thus the rate ofcatecholamine synthesis, can be regulated by the phosphor-ylation of specific serine residues located at the N terminalof the enzyme. To determine whether this was the mecha-nism underlying prolactin’s action on these hypothalamicneurons, the effect of selected protein kinase inhibitors wasdetermined. Incubation with H89 (3 �m), an inhibitor ofPKA, resulted in a small but significant decrease in the basalcatecholamine synthesis measured over 120 min (Fig. 3A).This inhibitor also markedly reduced the prolactin-inducedincrease in this response activity, such that it was no longersignificantly greater than the basal activity over this timeperiod (Fig. 3A). In contrast, the same concentration of H85,an inactive analog of H89, was without effect on either thebasal or prolactin-induced response (Fig. 3A). Bisindolyma-leimide I (3 �m), a PKC antagonist, also fully inhibited theprolactin-induced increase in catecholamine synthesis butunlike H89 had no significant effect on the basal response(Fig. 3B). Bisindolymaleimide V, an inactive analog of bisin-dolymaleimide I, was without effect on either basal orprolactin-stimulated activity (Fig. 3B). KN-93 (3 �m), whichis known to inhibit Ca2�-dependent protein kinases includ-ing CaMKII, had no effect on basal catecholamine synthesisbut caused a partial inhibition (about 50%) of the prolactin-induced response (Fig. 3C). It is important to note, however,that KN-92, an inactive analog of KN-93, also significantlysuppressed the prolactin-stimulated response, albeit to alesser extent than KN-93 (Fig. 3C).

The possible involvement of MAPKs in the prolactin-induced increase in catecholamine synthesis in these cellswas first investigated by determining whether the MAPKsERK1/2 were activated in response to prolactin. As depictedin Fig. 4A, incubation with prolactin (1 �g/ml for 15 min)resulted in an increased phosphorylation and thus activationof ERK1 (Fig. 4A). Phosphorylation of ERK1 was abolishedby both the MAPK/ERK kinase (MEK) inhibitor PD98059 (50�m) and the PKC inhibitor bisindolymaleimide I (3 �m) (Fig.4A). Interestingly, inhibition of MEK, but not PKC, resultedin a significant decrease in ERK1 phosphorylation seen underbasal conditions. The effect of prolactin and the above in-hibitors on ERK2 phosphorylation was essentially identicalwith that on ERK1 (data not shown). The level of TH ex-pression detected on these Western blots was not signifi-cantly altered after stimulation with prolactin at 115.7 � 16%basal levels (P 0.05 using a Wilcoxon sign-ranks test). Inagreement with its action on ERK phosphorylation, incuba-tion with PD98059 (50 �m) also caused a marked reductionin the basal rate of catecholamine synthesis (Fig. 4B). Incontrast, whereas the overall rate of catecholamine synthesisin the presence of prolactin was reduced in the presence ofPD98059, this apparent inhibition was entirely due to thedecreased basal activity, with the prolactin response being

FIG. 2. Prolactin stimulates a time- and concentration-dependent in-crease in catecholamine synthesis. Cells were incubated with or with-out prolactin (1 �g/ml) for 15–120 min (A) or increasing concentra-tions of prolactin for 120 min (B). The presence of the prolactinantagonist �1–9-G129R-hPRL (10 �g/ml) is indicated by the darkshaded columns and resulted in a significant suppression of prolactin-induced catecholamine synthesis. Catecholamine synthesis was de-termined as described in Materials and Methods and expressed as apercentage of the response obtained under basal conditions for thesame time period. Data are presented as the mean � SEM. *, P 0.05;**, P 0.01, compared with the response in cells incubated for thesame time period with buffer alone; #, P 0.01, compared with thematched prolactin response in the absence of antagonist; �, P 0.05,compared with the basal response in the presence of the prolactinantagonist (Mann-Whitney U test, n � 6–12 from three to four sep-arate cell culture preparations).


122% of basal in the absence of the inhibitor ,compared with123% of basal in its presence (Fig. 4B). Thus, whereas PKCappears to be involved in both prolactin-induced ERK1/2activation and prolactin-induced TH activity, the ERK1/2pathway is not itself required for the stimulation of cate-cholamine synthesis. To examine this proposal further, theeffect of PD98059 on the ability of phorbol-12-myristate-13-acetate (PMA), a PKC activator, to stimulate catecholaminesynthesis was determined. As described previously (18),PMA (100 nm for 60 min) significantly increased the levelof catecholamine synthesis to about 115% of basal levels(Fig. 4C). The inclusion of PD98059 caused a concentration-dependent decrease in the net response to PMA, but, aswith prolactin, this resulted from a parallel concentration-dependent decrease in basal activity (Fig. 4C).

The involvement of multiple protein kinases in thisprolactin-stimulated response was supported by the findingthat TH protein extracted from these cells was phosphory-lated on multiple serine residues. As presented in Fig. 5, theuse of phosphorylation site-specific antibodies revealed thatprolactin (1 �g/ml for 60 min) stimulated a significant in-crease in phosphorylation of ser-19, -31, and -40 residueswhen compared with cells incubated under basal condition.Whereas the increase in ser-40 phosphorylation appears to belarger than the other two sites, this apparent difference wasnot statistically significant. The level of total TH expressiondetected on these Western blots was not significantly alteredafter stimulation with prolactin at 110.7 � 9.5% basal levels(P 0.05 using Wilcoxon sign-ranks test on the originaldensitometric data).

In addition to increasing TH activity (as reflected by a risein catecholamine synthesis), incubation with prolactin sig-nificantly increased the level of TH mRNA expression after240 min incubation (Fig. 6A). In contrast to the prolactin-induced increase in catecholamine synthesis, this rise in THmRNA was not inhibited by H89, bisindolymaleimide I, KN-93, or PD98059 (Fig. 6A). Interestingly, inhibition of tran-scription or translation with actinomycin D (1 �m) or cyclo-hexamide (1 �m) decreased basal catecholamine synthesisbut failed to suppress the stimulated response to prolactin(Fig. 6B).

Discussion

The primary aim of this project was to investigate theintracellular mechanisms by which prolactin activity regu-lates dopamine synthesis in NEDA neurons. As outlined inthe introductory text, the key regulatory step in this pathwayis the activity of TH. The experimental approach used hereto examine TH activity in cultured mediobasal hypothalamicneurons was based on the liberation of 14CO2 from cellsloaded with 14C-tyrosine. TH converts the 14C-tyrosine to14C-DOPA with is then decarboxylated by aromatic aciddecarboxylase to dopamine, releasing 14CO2. Thus, whereasthe assay is dependent on the activity of both enzymes, thelatter is not rate limiting and has been shown to be relativelyinsensitive to acute regulation (23). Thus, the rate of 14CO2accumulation provides a sensitive and reliable index of in situTH activity (19). Whereas the ability to measure cellulardopamine synthesis in the intact living cell has clear advan-

FIG. 3. The effect of selected protein kinase inhibitors on prolactin-stimulated catecholamine synthesis in mediobasal hypothalamic cul-tures. Cell cultures were preincubated with or without (open columns)selected protein kinase inhibitors (all at 3 �M), H89 (dark columns) orH85 (light columns) (A), bisindolymaleimide I (dark columns) or bisin-dolymaleimide VI (light columns) (B), or KN-93 (dark columns) or KN-92(light columns) (C), for 15 min and then incubated for a further 120 minwith or without prolactin (1 �g/ml) in the continued presence or absenceof the appropriate protein kinase inhibitor. Catecholamine synthesiswas measured as described in Materials and Methods and expressed asa percentage of the basal response in the absence of inhibitors. Data arepresented as the mean � SEM (Mann-Whitney U test, n � 9–18 fromthree to six separate cell preparations). *, P 0.001, compared withmatched basal response; #, P 0.05, compared with control basal; ##,P 0.01, compared with control prolactin response.


tages, there are also limitations in terms of identifying thelocus of an action. TH activity may, for example, be sensitiveto multiple factors in addition to its phosphorylation status,including the rate of tyrosine uptake and cofactor availabil-ity, which may themselves be influenced by receptor stim-ulation or protein kinase activity.

Preliminary immunocyotochemical studies confirmedearlier reports that prolactin receptors are expressed on THimmunoreactive cells in these cultures (6). Whereas not fullyquantitated, our observations suggest that prolactin recep-

FIG. 4. Prolactin activates the ERK1/2 pathway in mediobasal hy-pothalamic cell cultures. A, Cells were preincubated with or without(open columns) PD98059 (50 �M, filled columns) or bisindolymaleim-ide I (3 �M, light columns) for 15 min and then for a further 15 minwith or without prolactin (1 �g/ml) in the continued presence orabsence of the appropriate inhibitor. Cells were lysed and phosphor-ylated ERK1/2 levels determined as described in Materials and Meth-ods. Data are presented as a percentage of phospho-ERK1 levelsmeasured under control basal conditions, determined on the same

FIG. 5. Prolactin increases the phosphorylation of specific serine res-idues on TH from mediobasal hypothalamic cultures. Cells were in-cubated with (shaded columns) or without (open columns) prolactin (1�g/ml) for 60 min. After incubation cells were lysed and the phos-phorylation of ser-19, -31, and -40 residues of TH determined usingphosphorylation site-specific antibodies as described in Materials andMethods. Data are expressed as a percentage of the site-specific phos-phorylation measured in cells incubated with buffer alone and rep-resent the mean � SEM (*, P 0.01, compared with the relevant basalresponse; Mann-Whitney U test, n � 4 independent experimentsperformed on separate cell culture preparations). Inserts present rep-resentative phospho-TH immunoblot positions above the appropriatecolumns.

immunoblot. *, P 0.001 and #, P 0.05, compared with control basallevels (Mann-Whitney U test, n � 3–6 from independent experimentsperformed on separate cell culture preparation). Inserts present rep-resentative phospho-ERK immunoblots positioned above the appro-priate columns. B, Cells were preincubated with or without (opencolumns) PD98059 (50 �M, filled columns) for 15 min followed by afurther 120 min with or without prolactin (1 �g/ml) in the continuingpresence or absence of the inhibitor. Catecholamine synthesis wasmeasured as described in Materials and Methods and expressed as apercentage of the basal response in the absence of inhibitor. Data arepresented as the mean � SEM. *, P 0.001, compared with the controlbasal response; #, P 0.001, compared with the basal response in thepresence of PD98059 (Mann-Whitney U test, n � 9 from three sep-arate cell preparations). C, Cells were preincubated with increasingconcentrations of PD98059 for 15 min and then for a further 60 minwith (shaded columns) or without (open columns) PMA (100 nM) in thecontinued presence or absence of the inhibitor. Catecholamine syn-thesis was measured as described in Materials and Methods andexpressed as a percentage of the basal response in the absence ofinhibitor. Data are presented as the mean � SEM. *, P 0.01, com-pared with the matched basal response; #, P 0.05, compared withthe appropriate response in the absence of PD98059 (ANOVA, n � 3).


tors are present on most of the TH-expressing cells in thesecultures. Prolactin receptor immunoreactivity was also evi-dent on a population of non-TH expressing cells. The identityof these cells was not investigated here, but an earlier study

suggested that they were neuronal rather than glial in origin(6). Whereas most TH-immunoreactive cells express prolac-tin receptors and are thus are presumably capable of re-sponding directly to prolactin, other non-TH immunoreac-tive cells could also respond to prolactin and therebypotentially mediate a secondary, indirect, action on the TH-positive neurons. RT-PCR examination suggested that re-gardless of specific cellular location, the vast majority ofprolactin receptor expression in these hypothalamic cultureswas of the long, rather than short form of the receptor, anobservation in agreement with in vivo studies (24, 25). Itshould be noted, however, that the short-form primers wereable to detect the short-form prolactin receptor in choroidplexus (Fig. 1B) and a number of other rat brain regions (21).

The concentration of prolactin required to stimulate a sig-nificant increase in catecholamine synthesis in these culturesis in excellent agreement with those reported previously formore chronic prolactin actions on similar cultures (6, 10, 26).The majority of experiments reported here have used a pro-lactin concentration of 1 �g/ml, which gave a reliable andrelatively robust response. This concentration is a littlehigher than the high end of the physiological range, such asduring the preovulatory surge but less than concentrationsreported in the portal blood (27). The specificity of prolactinaction in these experiments is supported by the inhibitionobserved using the prolactin antagonist �1–9-G129R-hPRL.This prolactin analog acts as a pure competitive antagonistat the prolactin receptor, and similar concentrations of this orrelated analogs (20, 28, 29) have been shown to inhibit avariety of prolactin-induced responses in other cells andtissues. The partial inhibition of prolactin response observedin the current studies reflects the fact that the antagonist hasa 10-fold lower affinity for the prolactin receptor than wild-type human prolactin (22).

It should be noted that the culture preparations used in thecurrent study have been maintained in a serum-free envi-ronment for at least 14 d before stimulation with prolactin.In the earlier studies mentioned above (6), cells were foundto be unresponsive to prolactin unless maintained in thepresence of serum. The reason for this difference, or indeedthe explanation underlying the need for serum in the earlierstudies, is unknown, but the ability to maintain prolactin-responsive cells in a defined culture environment is clearlyan experimental advantage. Thus, in the absence of postnatalexposure to prolactin, circulating steroids, or other serum-derived factors, TH-containing cells from the mediobasalhypothalamus of the rat fetus appear capable of expressingfunctional prolactin receptors.

The ability of prolactin to stimulate a relatively rapid in-crease in catecholamine synthesis in these cultures stronglysuggests that it is promoting the phosphorylation and thusactivation of TH. As outlined in the introductory text, TH inother cell types can be phosphorylated on three specificserine residues, ser-19, -31, and -40, through a variety ofprotein kinases (11, 12). In many cases this phosphorylationleads to a rise in catalytic activity of TH. Previous in vivostudies strongly suggested that protein kinases play a keyrole in regulating TH activity in the arcuate nucleus andmedian eminence (10, 30, 31), although the pathway bywhich prolactin mediates these actions is not well docu-

FIG. 6. Prolactin increases TH mRNA levels in mediobasal hypotha-lamic cultures. A, Cells were incubated for 4 h with or without pro-lactin (1 �g/ml) in the presence (shaded columns) or absence (opencolumns) of the indicated protein kinase inhibitor, H89 (10 �M),KN-93 (3 �M), bisindolymaleimide I (BisM, 3 �M), or PD98059 (3 �M).Cells were then extracted and TH mRNA levels measured usingreal-time RT-PCR as described in Materials and Methods. Data areexpressed as a percentage of the level detected in cells stimulated withprolactin in the absence of kinase inhibitors and are presented asmeans � SEM. *, P 0.05, compared with cells incubated under basalcondition in the presence of appropriate kinase inhibitor (Mann-Whit-ney U test, n � 3–6 from separate cell culture preparations). B, Cellswere preincubated for 15 min with or without actinomycin D (1 �M,dark columns) or cycloheximide (1 �M, light columns) and then for afurther 120 min with or without prolactin (1 �g/ml) in the continuedpresence of the appropriate inhibitor. Catecholamine synthesis wasdetermined as described in Materials and Methods and is expressedas a percentage of that detected in cells incubated under basal con-dition in the absence of inhibitor. *, P 0.01, compared with activityin cells incubated under matched basal conditions; #, P 0.05, com-pared with cells incubated with buffer alone (Mann-Whitney U test,n � 4 from separate cell culture preparations).


mented. The data presented in Fig. 3a suggests that PKAactivity may be required for prolactin stimulation of TH. Theincrease in catecholamine synthesis induced by prolactin wasabolished by H89 and unaffected by its inactive analog H85.Previous studies demonstrated that increased cAMP levelsstimulate the rate of catecholamine synthesis in both hypo-thalamic slices and cultures (30, 32).

Given that ser-40 phosphorylation of TH leads to its ac-tivation (11, 13) and that prolactin increases the phosphor-ylation of this residue (Fig. 5), it is tempting to suggest thatprolactin achieves this activation via a PKA-mediated path-way. Caution should be taken, however, before reaching thisconclusion. There is little evidence that prolactin receptorsare coupled to adenylyl cyclase, although it is possible thatthis enzyme could be activated secondarily to a prolactin-induced increase in intracellular Ca2� (33, 34). More impor-tantly, however, we and others have shown that H89 inhibitsTH activation by other agents that are unlikely to act viaPKA. The PMA-induced increase in catecholamine synthesisin these hypothalamic cultures, for example, is abolished byH89 (18). Similarly, TH activity in adrenal medullary chro-maffin cells is inhibited by H89, even when this appears toinvolve little or no stimulation of PKA (35). Thus, whereas itcan be reasonably concluded that PKA activity, or at least anH89-sensitive event, is required for the activation of TH inNEDA neurons, its precise role in the prolactin-driven re-sponse remains somewhat equivocal. As outlined in the in-troductory text, TH ser-40 is the substrate for a number ofprotein kinases in addition to PKA including CaMKII, PKC,and MAPKAPKs downstream of ERK1/2. It is possible thatone or more of these kinases is responsible for prolactinstimulation of catecholamine biosynthesis.

In addition to increasing TH ser-40 phosphorylation, pro-lactin incubation stimulated the phosphorylation of ser-19(Fig. 5). Whereas this residue is believed to be a substrate forCaMKII, the data in Fig. 2B provide only weak evidence tosupport involvement of CaMKII in this prolactin response.Preincubation with KN-93, an inhibitor of CaMKII, resultedin only a partial reduction in the prolactin response. A sig-nificant inhibition was also seen, however, with its inactiveanalog KN-92. In contrast, we previously demonstrated thatactivation of catecholamine synthesis in these cultures byangiotensin II or K�-depolarization was completely inhib-ited by KN-93 and unaffected by the inactive analog (18). Theability of KN-93 and KN-92 to partially inhibit the prolactinresponse may result from suppression of Ca2� influx (36), arecognized action of these compounds. Whereas not char-acterized in NEDA neurons, prolactin has been reported toincrease Ca2� influx in some transfected cells (33). If this isthe case here, then it suggests that Ca2� influx may play a rolein the prolactin stimulation of catecholamine synthesis, al-though the significance of that role cannot be assessed with-out knowing the extent of KN-93/92 action on the Ca2�

influx.The data provide good evidence for an involvement of

PKC in prolactin-induced activation of catecholamine syn-thesis in these hypothalamic cultures. The response to pro-lactin was abolished by bisindolymaleimide I but unaffectedby its inactive analog (Fig. 3B). PKC has been shown to beactivated in response to prolactin in a number of tissues

including the hypothalamus (2, 10, 37). A previous studymeasuring prolactin-stimulated TH activity in rat hypotha-lamic slices employed selected protein kinase inhibitors toconclude that PKC but not CaMKII or PKA was involved inprolactin’s action (10). The importance of PKC in regulatingTH activity in these cells is further supported by data fromourselves and others showing that responses to angiotensinII and neurotensin were also inhibited by PKC antagonists(18, 38). In contrast to the actions of the PKA antagonist H89(discussed above), the PKC antagonist appeared to be selec-tive, in that it only reduced responses to PMA and not thoseto dibutyl cAMP or K� depolarization (18). The possiblemechanism of PKC action is discussed below in the contextof the MAPK pathway.

Stimulation of prolactin receptors on many cell types leads toactivation of the MAPK pathway (2). As can be seen in Fig. 4A,in agreement with this general finding, prolactin caused anincrease in ERK1/2 phosphorylation in these hypothalamiccultures. It should be noted that, whereas these experiments donot preclude a prolactin-induced increase in ERK expression,such a response is unlikely after this relatively short, 15-minincubation period. ERK1/2 activation is well documented toinvolve both PKC-dependent and PKC-independent pathways(39). The prolactin stimulation of ERK1/2 phosphorylation ap-peared to involve the former pathway in that it was abolishedby the PKC inhibitor bisindolymaleimide I. Interestingly, theobservation that the MEK inhibitor PD98059 reduced basal THactivity suggests an additional PKC-independent phosphory-lation of ERK1/2, which is evident under basal conditions.Despite apparently complete inhibition of prolactin-mediatedERK1/2 phosphorylation, PD98059 did not inhibit prolactinactivation of catecholamine synthesis, although it did cause aconcentration-dependent decrease in the level of catecholaminesynthesis occurring under basal conditions. The role of theERKs in TH regulation is particularly interesting because theyare the only kinases currently identified to phosphorylate ser-31(13). Whereas data presented here clearly show that prolactinincreases ser-31 phosphorylation (Fig. 5) and that this is prob-ably mediated by a PKC-dependent activation of ERK1/2, thisresponse does not appear to be necessary for the prolactin-induced increase in catecholamine synthesis. As noted in theintroductory text, an increase in the TH ser-31 phosphorylationhas been reported in other cells in response to many differentstimuli, but the precise role of this phosphorylation in regulat-ing TH activity is not fully resolved. In contrast to its apparentlack of involvement in the prolactin-stimulated response, MEKactivity does appear to be important for maintaining basal lev-els of catecholamine synthesis. Although it is clear that MEK-inhibition decreases the level of basal ERK1/2 phosphorylation,it remains to be determined whether this is associated with adecrease in TH ser-31 phosphorylation.

Thus, the role of PKC in the prolactin-induced increase incatecholamine synthesis in these NEDA neuronal cultures iscomplex. PKC activity appears to be essential for prolactinstimulation both of catecholamine synthesis and MAPK ac-tivation in these cells, but this latter pathway is not requiredfor prolactin activation of catecholamine synthesis. PKC maytherefore be stimulating catecholamine synthesis through analternative as-yet-unidentified mechanism. One interestingpossibility is that PKC is mediating the phosphorylation of


the ser-40 residue and thus having a direct effect on THactivity. Alternatively, PKC may be having its stimulatoryaction by acting on another component of the catecholaminesynthesis machinery.

In addition to increasing the rate of catecholamine syn-thesis, prolactin induced TH mRNA expression in these cul-tures. As would be expected, this transcriptional responseappeared to be slower in onset than the increase in activity,requiring 4 h incubation to reach significance. This observa-tion is in agreement with previous in vivo evidence showingthat prolactin induces both an acute and chronic activationof TH (9). Whereas earlier reports indicated that the genomicresponse may require up to 12 h to be induced, more recentevidence suggests that as little as 3 h exposure to high pro-lactin is sufficient (40). It should be noted, however, that thedata obtained using cycloheximide and actinomycin D in-dicate that the increased TH mRNA expression in responseto prolactin does not make a significant contribution to therate of catecholamine synthesis during the first 2 h. In aprevious study, we reported that angiotensin II also stimu-lates both TH expression and TH activity in these cultures(18). The former response required PKC and the MAPK ac-tivity, in that it was inhibited by their respective antagonists.In contrast, prolactin-induced TH mRNA expression wasunaffected by either of these inhibitors. Thus, as discussedearlier, prolactin appears to stimulate the ERK1/2 MAPKpathway in these cells in a PKC-dependent manner, but theobserved increase in TH mRNA is not dependent on thispathway. The reasons for these differences are unclear butsuggest that, whereas both prolactin and angiotensin II usesomewhat similar pathways to acutely activate TH, the path-ways diverge to regulate its expression. Such a conclusion isconsistent with our previous report that the prolactin acti-vation of TIDA neurons in vivo appears to require the signaltransducer and activator of transcription-5b signaling path-way (7). Because both angiotensin II and prolactin are likelyto regulate the activity of NEDA neurons in vivo, such con-vergent and divergent signaling pathways may have impor-tant consequences for the integrated regulation of NEDAneuron activity and thus prolactin secretion.

Acknowledgments

We acknowledge the valuable technical and intellectual assistancefrom Professor Peter Dunkley and Dr. Larisa Bobrovskaya in preparingthis manuscript.

Received June 25, 2004. Accepted September 17, 2004.Address all correspondence and requests for reprints to: Stephen

Bunn, The Centre for Neuroendocrinology, Department of Anatomy andStructural Biology, The University of Otago, P.O. Box 913, Dunedin 9001,New Zealand. E-mail: [email protected].

This work was supported by a grant from the Marsden Fund, NewZealand. F.Y.M. was the recipient of a University of Otago scholarship.

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