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Endocrinology Vol. 143, No. 1 13-22
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Regulation of the PRL Promoter by Akt through cAMP Response Element Binding Protein

Jun Hayakawa, Masahide Ohmichi, Keiichi Tasaka, Yuki Kanda, Kazushige Adachi, Yukihiro Nishio, Koji Hisamoto, Seiji Mabuchi, Shuji Hinuma and Yuji Murata

Department of Obstetrics and Gynecology (J.H., M.O., K.T., Y.K., K.A., Y.N., K.H., S.M., Y.M.), Osaka University Medical School, Osaka 565-0871, Japan; and Discovery Research Laboratories I (S.H.), Pharmaceutical Discovery Research Division, Takeda Chemical Industries Co., Ltd., Ibaraki 300-4293, Japan

Address all correspondence and requests for reprints to: Dr. Masahide Ohmichi, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: masa{at}gyne.med.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Regulation of the PI3K-protein kinase B/Akt (serine/threonine kinase) cascade by PRL-releasing peptide (PrRP) and insulin in GH3 rat pituitary tumor cells was investigated. PrRP and insulin rapidly and transiently stimulated the activation of Akt, and the PI3K inhibitor wortmannin blocked the PrRP- or insulin-induced activation of Akt. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the ß-adrenergic receptor kinase I, which specifically blocks signaling mediated by the ß{gamma} subunits of G proteins, completely blocked the PrRP-induced Akt activation, suggesting that Gi/Go proteins are involved in PrRP-induced Akt activation, as they are in the activation of ERK by PrRP. Moreover, to determine whether a PI3K-Akt cascade regulates rat PRL (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP and insulin activated the rPRL promoter activity. Pretreatment with wortmannin or cotransfection with a dominant-negative Akt partially but significantly inhibited the induction of the rPRL promoter by PrRP or insulin. Cotransfection with a constitutively active Akt induced the rPRL promoter activity and cotransfection with a dominant-negative cAMP response element-binding protein (CREB) completely abolished the response of the rPRL promoter to the constitutively active Akt. Furthermore, either treatment with PrRP and insulin or transfection with the constitutively active Akt induced the phosphorylation of CREB. These results suggest that PrRP and insulin activate a PI3K-Akt cascade that is necessary to elicit rPRL promoter activity via a CREB-dependent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE PREVIOUSLY REPORTED that PRL-releasing peptide (PrRP) differentially activates ERK and c-Jun N-terminal protein kinase (JNK) and that both cascades are necessary to elicit rat PRL (rPRL) promoter activity via an Ets-dependent mechanism (1). It has been shown that Raf, ERK, and Ets are crucial components of the downstream transmission of the Ras signal in the regulation of the PRL promoter activity (2, 3). Transcription of the PRL gene is also modulated by a number of ligands that bind to plasma membrane receptors, including dopamine, TRH, epidermal growth factor (EGF), insulin, and IGF-1 (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Furthermore, PRL gene expression is stimulated by activation of PKA and PKC cascades (14, 15, 16, 17).

It was reported that IGF-1 stimulates rPRL gene expression via a PI3K-dependent mechanism (13). The serine/threonine kinase termed Akt or protein kinase B has been identified as a downstream component of survival signaling through PI3K (18, 19, 20, 21, 22). Akt may be regulated by both phosphorylation and the direct binding of PI3K lipid products to the Akt pleckstrin homology domain. Activation of receptor tyrosine kinases and G protein-coupled receptors, and stimulation of cells by mechanical force, can lead to activation of the PI3K-Akt cascade (23, 24, 25). Akt can then phosphorylate substrates such as glycogen synthase kinase-3, 6-phosphofructo-2- kinase, and Bad and thereby regulate various cellular processes including glucose metabolism and cell survival (26, 27).

Previous studies demonstrated that the -100 to -85 region of the rPRL gene might contribute to basal and hormonally regulated expression of the PRL gene (28, 29). This region contains the sequence [(-99)TGACGGAA(-92)], which is an asymmetrical form of the canonical symmetric cAMP response element (CRE) [TGACGTCA]. It was reported that activation of the rPRL promoter was induced by a constitutively active form of CRE-binding protein (CREB) (30). Therefore, a possible role for CREB in the regulation of rPRL gene expression has been explored. More recently, it was found that CREB is a regulatory target for Akt (31).

Taken together, these facts led us to examine whether PrRP, whose receptor belongs to the G protein-coupled receptor family, and insulin, whose receptor belongs to the receptor tyrosine kinase family, stimulate the activity of PI3K-Akt and whether this cascade plays a role in the transcriptional activation of the rPRL gene in GH3 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Wortmannin and phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma (St. Louis, MO). PrRP was a gift from Takeda Chemical Industries Co., Ltd. (Tsukuba, Japan). Insulin was from Eli Lilly & Co. (Indianapolis, IN). ECL Western blotting detection reagents were obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). An Akt kinase assay kit, including GSK-3 fusion protein and a phospho-specific GSK-3{alpha}/ß antibody, was obtained from New England Biolabs, Inc. (Beverly, MA). A phospho-specific CREB antibody was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY).

Cell cultures
GH3 cells were cultured at 37 C in DMEM containing 10% FBS in a water-saturated atmosphere of 95% O2 and 5% CO2.

Construction of expression plasmids
The vectors encoding the various hemagglutinin (HA)-tagged forms of Akt, wild-type, kinase-dead (K179M mutant), and a constitutively active mutant form of Akt, used in this study have been described previously (32). The vectors encoding the various forms CREB, dominant interfering forms of CREB (K-CREB and M1-CREB) and a constitutively active mutant form of CREB (VP-16 CREB), used in this study have been described previously (33). The ßARKct peptide-encoding minigene, containing cDNA encoding the carboxyl-terminal 195 amino acids of ßARK1, was prepared as described previously (34, 35). The reporter construct pA3-425PRLluc (36, 37, 38) contains a 498-bp fragment encompassing positions -425 to +73 of the rPRL gene ligated upstream of the luciferase reporter gene in pA3luc (39) and contains three polyadenylation sites. The reporter construct pA3-425PRLluc was a kind gift from Dr. A. Gutierrez-Hartmann (University of Colorado Health Sciences Center, Denver, CO). The pAPr-etsZ, encoding the consensus DNA-binding domain of Ets-2, was a kind gift from Dr. M. C. Ostrowski (Ohio State University, Columbus, OH) (40).

Assay of Akt kinase activity
Cells were incubated in the absence of serum for 16 h and then treated with various materials. They were then washed twice with PBS and lysed in ice-cold lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol-phosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) (41, 42). The extracts were centrifuged to remove cellular debris, and the protein content of the supernatants was determined using the Bio-Rad Laboratories, Inc. protein assay reagent. Two hundred fifty micrograms protein from the lysate samples was incubated with gentle rocking at 4 C overnight with immobilized Akt antibody cross-linked to agarose hydrazide beads. After Akt was selectively immunoprecipitated from the cell lysates, the immunoprecipitated products were washed twice in lysis buffer and twice in kinase assay buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM ß-glycerol-phosphate, 0.1 mM sodium orthovanadate and 2 mM dithiothreitol) (41, 42) and resuspended in 40 µl kinase assay buffer containing 200 µM ATP and 1 µg GSK-3{alpha} fusion protein. The kinase reaction was allowed to proceed at 30 C for 30 min and stopped by the addition of Laemmli SDS sample buffer (43). Reaction products were resolved by 15% SDS-PAGE and subjected to Western blotting with a phospho-GSK-3{alpha} antibody (41, 42). Occasionally two bands were detected by Western blotting, the smaller of which might represent an N-terminally deleted product.

Assay of Akt activity using a transient expression system
GH3 cells cultured in 100-mm dishes were transfected with 1 µg HA-tagged wild-type Akt in combination with 9 µg pRK or pRK-ßARK1 using LipofectAMINE as described previously (1, 44, 45). At 72 h after transfection, serum-deprived cells were incubated with 1 µM PrRP for 5 min, and expressed HA-tagged Akt was immunoprecipitated with anti-HA antibody. The Akt activity in the immunoprecipitate was measured as described above. The transfection efficiency of each experiment was 8–10% as assessed by ß-galactosidase staining after transfection of a ß-galactosidase expression plasmid.

rPRL promoter assay
GH3 cells cultured in 24-well plates were transfected with pA3-425PRLluc and CMV-ß-galactosidase plasmid (to normalize for cell viability and transfection efficiency) in combination with the indicated plasmids using LipofectAMINE. At 48 h after transfection, serum- deprived cells were incubated with 1 µM PrRP or 1 µM insulin for 12 h. In some of the experiments, cells were treated with 2 x 10-7 M wortmannin for 15 min before the addition of 1 µM PrRP or 1 µM insulin. Cell extracts were prepared by lysing the cells with three sequential freeze-thaw cycles in 100 mM potassium phosphate, pH 7.8, and 10 mM dithiothreitol. The frozen/thawed cells were vigorously vortexed to enhance cell lysis. Unlysed cells and insoluble material were pelleted at 10,000 rpm for 10 min at 4 C. The aliquots of the supernatant were used in the subsequent luciferase and ß-galactosidase assays.

Luciferase was assayed as previously described (14). Briefly, the luciferase assay mixture contained 100 mM KPO4, pH 7.8, 1 mM dithiothreitol, 3.7 mM MgSO4, 530 µM ATP, and 470 µM luciferin plus 20 µl cell extract in a final volume of 100 µl. Luciferin was added just before measuring light units, which were measured in duplicate during the first 40 sec of the reaction at 25 C in a luminometer (15).

ß-Galactosidase was assayed as previously described (14). The ß- galactosidase buffer contained 60 mM sodium phosphate, pH 7.5, 1 mM MgCl2, 0.80 mg/ml O-nitrophenyl-ß-{delta}-galactopyranoside, and 40 mM ß-mercaptoethanol. A standard curve for reactions containing 100 µU to 2 mU ß-galactosidase was made with each assay. A 30-µl aliquot of cell extract was incubated with assay buffer until color developed (30–120 min), and the reaction was then stopped by adding Na2CO3 to a final concentration of 625 mM. Absorbance was then read at 405 nm.

Luciferase light units were normalized relative to the activity of ß-galactosidase. The control value was then set at 1, and the data were expressed as fold-stimulation relative to control. Data are expressed as the mean ± SE.

Phosphorylation of CREB
Nuclear extracts were prepared as previously described (46). One hundred fifty micrograms protein were resolved by SDS-PAGE and subjected to Western blotting with a phospho-CREB antibody (46).

Statistics
Statistical analysis was performed using t test, and P < 0.01 was considered significant. Data are expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of Akt
To evaluate whether Akt is activated by PrRP or insulin in GH3 cells, cell lysates were prepared from cells exposed to 1 µM PrRP or 1 µM insulin for various times (Fig. 1AGo). The lysates were subjected to immunoprecipitation with immobilized anti-Akt antibody, the immunoprecipitates were incubated with ATP and GSK-3{alpha} fusion protein, and the kinase reaction products were analyzed by Western blotting with anti-phospho-GSK-3{alpha}/ß antibody. Activation of Akt by PrRP in GH3 cells reached a plateau at 5 min and declined thereafter (Fig. 1AGo, i, upper panel). We confirmed that the total amount of Akt in each lane was the same (Fig. 1AGo, i, lower panel). Activation of Akt by insulin reached a plateau from 5 to 10 min and declined thereafter (Fig. 1AGo, ii). Because Akt is an effector of the survival signaling downstream from PI3K, we next determined whether stimulation of cells with PrRP or insulin increased the activity of Akt through a PI3K-dependent mechanism. Cells were stimulated with PrRP (Fig. 1BGo, lane 2) or insulin (Fig. 1BGo, lane 3) in the presence or absence of wortmannin, a PI3K inhibitor, and the kinase activity of Akt was assayed. The induction of Akt activity by PrRP or insulin was inhibited by wortmannin (Fig. 1BGo, lanes 4 and 5). These results indicate that PrRP and insulin activate Akt activity in GH3 cells through a PI3K-dependent mechanism.



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  		Figure 1. The effect of PrRP on the activity of Akt. GH3 cells were grown in 100-mm dishes. A, Cells were treated with 1 µM PrRP (i) or 1 µM insulin (ii) for the indicated times (lanes 2–5). B, Cells were pretreated (lanes 4 and 5) or not pretreated (lanes 1, 2, and 3) with 2 x 10-7 M wortmannin for 15 min and then were treated with 1 µM PrRP or 1 µM insulin for 5 min. Lysates were subsequently subjected to immunoprecipitation with immobilized anti-Akt antibody, and the kinase reaction was carried out in the presence of ATP and GSK-3{alpha} fusion protein, as described in Materials and Methods. After the reactions were stopped with Laemmli sample buffer, samples were resolved by 12% SDS-PAGE and then analyzed by Western blotting with an anti-phospho-GSK-3{alpha}/ß antibody. For analysis of the total amount of Akt (i, lower panel), 250 µg protein from the lysate samples were resolved by 8% SDS-PAGE and then subjected to Western blotting with anti-Akt antibody. Experiments were repeated three times with essentially identical results.

 
Gß{gamma}-mediated PrRP-induced Akt activation
It has been shown that the receptor for PrRP (47, 48) is a member of the superfamily of G protein-coupled receptors. To determine what type of G protein is coupled to this receptor, we pretreated GH3 cells (Fig. 2AGo, left panel) with 100 ng/ml pertussis toxin (PTX) for 4 h to inactivate Gi and Go proteins, and then treated the cells with 1 µM PrRP for 5 min. PTX at 100 ng/ml almost completely blocked the PrRP-induced Akt activation (Fig. 2AGo, lane 4). Thus, the effect of PrRP on Akt activity involves a PTX-sensitive G protein such as Gi or Go.



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  		Figure 2. Gß{gamma}-mediated PrRP-induced Akt activation. A, Cells were pretreated (lanes 2 and 4) or not pretreated (lanes 1 and 3) with 100 ng/ml PTX for 4 h and then were treated with (lanes 3 and 4) or without (lanes 1 and 2) 1 µM PrRP for 5 min. Akt activity was measured as described in the legend for Fig. 1Go. Experiments were repeated three times with essentially identical results. B, Cells were transfected with pRK (lanes 1 and 2) or pRK-ßARK1 (lanes 3 and 4) together with HA-tagged wild-type Akt expression plasmid and after 72 h were stimulated with 1 µM PrRP (lanes 2 and 4) for 5 min. For analysis of the effects of ectopically expressed pRK or pRK-ßARK1 on Akt activity, immune complexes were precipitated with protein A Sepharose, and the kinase reaction was carried out in the presence of cold ATP and GSK-3{alpha} fusion protein, as described in the legend to Fig. 1Go. Experiments were repeated three times with essentially identical results.

 
It has been reported that the carboxyl terminus of the ß-adrenergic receptor kinase, containing its Gß{gamma}-binding domain, is a cellular Gß{gamma} antagonist capable of specifically distinguishing between G{alpha}- and Gß{gamma}-mediated processes (35). To examine the effect of the Gß{gamma} subunit-sequestrant ßARKct peptide on PrRP-induced exogenous Akt activity, an HA-tagged wild-type Akt expression plasmid was used to distinguish exogenous Akt from endogenous Akt. We transfected cells with pRK or pRK-ßARK1 together with an HA-tagged wild-type Akt expression plasmid and after 72 h stimulated them with 1 µM PrRP for 5 min (Fig. 2BGo). Cell lysates were immunoprecipitated with anti-HA antibody and examined for the exogenous Akt activity. Transfection with pRK-ßARK1 completely abolished the PrRP-induced Akt activation in GH3 cells (Fig. 2BGo, lane 4). These results suggest that Akt activation by PrRP is mediated by Gß{gamma} in GH3 cells.

Role of PKC and Ca2+ in activation of Akt by PrRP
Many G protein-linked receptors mediate stimulation of ERK activity via the PLC-dependent activation of PKC (49, 50). Activation of ERK by PrRP is partly mediated by PKC (1). Therefore, the role of PKC in PrRP-induced Akt activation was examined (Fig. 3Go, lane 3). Pretreatment with 1 µM PMA for 16 h to deplete most PKC isoforms partially attenuated the PrRP-induced Akt activation (Fig. 3Go, lane 3), as in the case of PrRP-induced ERK activation (1).



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  		Figure 3. The effect of the downregulation of PKC or chelation of extracellular Ca2+ on PrRP-induced Akt. Cells were pretreated with 1 µM PMA for 16 h (lane 3) or with 3 mM EGTA for 1 min (lane 4) and then were treated with 1 µM PrRP for 5 min (lanes 2–4). Akt activity was measured as described in the legend to Fig. 1Go. Experiments were repeated three times with essentially identical results.

 
It has been reported that Ca2+ influx is important as a signal-transduction pathway in PRL secretion by pituitary cells (51) and that PrRP can induce Ca2+ influx (52). However, PrRP-induced ERK activation is not dependent on extracellular Ca2+ (1). We therefore evaluated the effect of Ca2+ on the PrRP-induced Akt activation (Fig. 3Go, lane 4). Interestingly, elimination of extracellular Ca2+ by treatment with 3 mM EGTA for 1 min (53) attenuated the PrRP-induced Akt activation (Fig. 3Go, lane 4). These results suggest that PrRP-induced Akt activation is dependent on extracellular Ca2+.

Stimulation of PRL promoter activity by PrRP or insulin
We next sought to determine whether the PI3K-Akt cascade is involved in the regulation of PRL synthesis by PrRP or insulin. An rPRL promoter (-425 bp)-luciferase reporter construct was transiently transfected into GH3 cells. As shown in Fig. 4AGo, addition of 1 µM PrRP or insulin for 12 h enhanced the luciferase activity. To examine whether the stimulation of the rPRL promoter by PrRP or insulin is the result of activation of the PI3K-Akt cascade, either wortmannin, a PI3K inhibitor, or an expression vector, HA-AktK179M, encoding an Akt derivative rendered kinase-inactive by a point mutation within the catalytic domain (32, 41, 42) was used. Pretreatment with 2 x 10-7 M wortmannin for 15 min partially but significantly attenuated the PrRP- or insulin-induced rPRL promoter activation (Fig. 4AGo). In addition, cotransfection with HA-AktK179M also partially but significantly attenuated the PrRP (Fig. 4BGo)- or insulin (Fig. 4CGo)-induced rPRL promoter activation, whereas cotransfection with control vector had no effect on the response to PrRP or insulin. Moreover, cotransfection with an expression vector for an Akt derivative rendered constitutively active by targeting it to the plasma membrane with a myristoyl tag (HA-m{Delta}4-129Akt) (32, 41, 42) significantly enhanced the rPRL promoter activity, compared with that in cells transfected with the control vector (Fig. 4DGo). These results suggest that the PI3K-Akt cascade is involved in both PrRP- and insulin- induced rPRL promoter activation.



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  		Figure 4. The role of the PI3K-Akt cascade in the PrRP- and insulin-dependent stimulation of the rPRL promoter. A, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal. After transfection, cells were incubated with or without 2 x 10-7 M wortmannin for 15 min as indicated and then treated with 1 µM PrRP or 1 µM insulin for 12 h before harvesting. B, C, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal, with 0.4 µg of empty vector (CMV-6) or 0.4 µg of expression vector for kinase-inactive HA-AktK179M (inactive Akt), as indicated. After transfection, cells were treated with 1 µM PrRP (B) or 1 µM insulin (C) for 12 h before harvesting. D, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal, with 0.4 µg of empty vector (CMV-6) or 0.4 µg of expression vector for constitutively active HA-m{Delta}4-129Akt (active Akt), as indicated. Luciferase activity was normalized relative to ß-galactosidase activity, and the basal activity of pA3-425PRLluc was set at 1.0. Data are expressed as the mean fold activation ± SE of six transfections. **, P < 0.01, compared with control.

 
CREB is a nuclear acceptor of the PI3K-Akt signaling cascade
It was reported that CREB is a regulatory target for the PI3K-Akt cascade (31). The rPRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element. It has also been reported that a cAMP analog increased the rPRL promoter activity (54). Therefore, to determine the functional role of CREB in the stimulation of rPRL promoter activity induced by the Akt cascade, we tested the effects of two distinct dominant interfering forms of CREB on the response of cells either to treatment with PrRP or insulin (Fig. 5AGo) or to cotransfection with HA-m{Delta}4-129Akt (Fig. 5BGo). K-CREB (33), in which Arg287 is converted to Leu, forms dimers with endogenous CREB proteins via its leucine zipper domain, and thereby inhibits the binding of endogenous CREB to the promoters of CREB-responsive genes. M1-CREB (33), in which Ser133 is converted to Ala, competes with endogenous CREB proteins for binding to the promoters of CREB- responsive genes. However, although it binds to DNA, M1-CREB does not activate transcription. When expressed from vectors cotransfected into GH3 cells, both K-CREB and M1-CREB partially but significantly inhibited the PrRP- or insulin-induced rPRL promoter activation (Fig. 5AGo) and abolished the HA-m{Delta}4-129Akt-induced rPRL promoter activation (Fig. 5BGo). VP-16 CREB (33) is a constitutively active mutant form of CREB in which the full-length CREB protein is fused at its NH2-terminus to the transactivation domain of the viral transcriptional coactivator VP16. Cotransfection with a vector expressing VP-16 CREB significantly enhanced the rPRL promoter activity, compared with that in cells cotransfected with control vector (Fig. 5BGo). These results suggest that CREB is a nuclear acceptor for the stimulation of rPRL promoter activity induced by the Akt cascade.



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  		Figure 5. The role of CREB in the Akt-dependent stimulation of the rPRL promoter. A, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal, with 0.4 µg of empty vector (pRK-5) or 0.4 µg of expression vector for two distinct dominant-interfering forms of CREB (M1-CREB or K-CREB), as indicated. After transfection, cells were treated with 1 µM PrRP or 1 µM insulin for 12 h before harvesting. B, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal, with 0.4 µg of empty vector (CMV-6) or 0.4 µg of expression vector for constitutively active HA-m{Delta}4-129Akt (active Akt), in addition to 0.4 µg of empty vector (pRK-5), 0.4 µg of expression vector for constitutively active CREB (VP-16 CREB), or 0.4 µg of expression vector for two distinct dominant-interfering forms of CREB (M1-CREB or K-CREB), as indicated. Luciferase activity was normalized relative to ß-galactosidase activity, and the basal activity of pA3-425PRLluc was set at 1.0. Data are expressed as the mean fold activation ± SE of six transfections. **, P < 0.01, compared with the respective control.

 
Stimulation of phosphorylation of CREB by PrRP or insulin
It is known that CREB is mainly phosphorylated at Ser133 and that the phosphorylation is essential for gene activation by CREB (55). Therefore, we examined the effects of PrRP and insulin on the phosphorylation of CREB. Cultured cells were exposed to 1 µM PrRP for 5 min, 1 µM insulin for 5 min, or 10 µM forskolin for 5 min. Nuclear extracts were prepared and analyzed by Western blotting using antiphospho CREB antibody. PrRP, insulin, and forskolin all induced the phosphorylation of CREB (Fig. 6AGo). In addition, we examined the effect of activated Akt on the phosphorylation of CREB. Transfection with an expression vector for constitutively kinase-active Akt (HA-m{Delta}4-129Akt) significantly enhanced the phosphorylation of CREB, compared with transfection with control vector (Fig. 6BGo).



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  		Figure 6. Phosphorylation of CREB by PrRP, insulin, forskolin, or active Akt. A, Cells were treated with 1 µM PrRP for 5 min, 1 µM insulin for 5 min, or 10 µM forskolin for 5 min. B, Cells were transiently cotransfected with 0.4 µg of empty vector (CMV-6) or 0.4 µg expression vector for constitutively active HA-m{Delta}4-129Akt (active Akt). Nuclear extracts were prepared and analyzed by Western blotting using anti-phospho CREB antibody, as described in Materials and Methods.

 
Involvement of Ets sites in the CREB-binding element on the rPRL promoter
The promoter region of rPRL that we used contains the CRE-like element. Previous studies showed that CREB does not exhibit high-affinity binding to the CRE-like element (28, 29). It has been reported that the CRE-like element overlaps an Ets factor-binding element (56, 57) and that CREB-binding protein interacts with Ets-1 and Ets-2 (58). Several putative Ets sites [(A/C)GGAA], located at positions -295, -185, -165, and -96, are found in the rPRL promoter. Therefore, we examined whether an Ets site is involved in the CREB-binding element on the rPRL promoter. The activation of the rPRL promoter induced by treatment with either insulin or forskolin (Fig. 7AGo) or by cotransfection with vectors expressing a constitutively active form of either Akt or CREB (Fig. 7BGo) was significantly inhibited by cotransfection with a vector (pAPr-etsZ) encoding a dominant-negative Ets (40) that competes with endogenous Ets proteins for binding to the promoters of Ets-responsive genes. We previously reported that the activation of the rPRL promoter induced by treatment with PrRP was completely inhibited by cotransfection with pAPr-etsZ (1). These results suggest that an Ets site is also involved in the CREB-binding element on the rPRL promoter.



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  		Figure 7. Dominant-negative Ets inhibits the Akt- dependent stimulation of the rPRL promoter. A, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal, with 0.4 µg of empty vector (pAPr) or 0.4 µg of expression vector for dominant-negative Ets (pAPr-etsZ), as indicated. After transfection, cells were treated with 1 µM insulin or 10 µM forskolin for 12 h before harvesting. B, Cells were transiently cotransfected with 0.4 µg of the reporter construct pA3-425PRLluc and 0.04 µg of an internal control, pCMVßgal, with 0.4 µg of empty vector (CMV-6) or 0.4 µg of expression vector for constitutively active HA-m{Delta}4-129Akt (active Akt), or 0.4 µg of empty vector (pRK-5) or 0.4 µg of expression vector for constitutively active CREB (VP-16 CREB), in addition to 0.4 µg of empty vector (pAPr) or 0.4 µg of expression vector for dominant-negative Ets (pAPr-etsZ), as indicated. Luciferase activity was normalized relative to ß-galactosidase activity, and the basal activity of pA3-425PRLluc was set at 1.0. Data are expressed as the mean fold activation ± SE of six transfections. **, P < 0.01, compared with the respective control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results presented here show that PrRP and insulin caused Akt activation in a PI3K-dependent manner. PrRP-induced Akt activation was mediated by Gi/Go proteins in a manner that was partly dependent on PKC and was also dependent on extracellular Ca2+ in GH3 cells. Either treatment with PI3K inhibitor wortmannin or cotransfection with a dominant-negative Akt construct partially but significantly inhibited the PrRP- and insulin-induced rPRL promoter activation. Cotransfection with a constitutively active form of Akt also enhanced the rPRL promoter activity. Moreover, either treatment with PrRP or insulin or transfection with a constitutively active form of Akt induced the phosphorylation of CREB. Cotransfection with a constitutively active form of CREB enhanced the rPRL promoter activity. In addition, cotransfection with a dominant-negative CREB construct significantly inhibited activation of the rPRL promoter by a constitutively active form of Akt. Thus, because it has been reported that CREB is a substrate for Akt (31), there is a possibility that PrRP and insulin activate the PI3K-Akt cascade and this cascade is necessary to elicit rPRL promoter activity via a CREB-dependent mechanism.

Although PrRP was the first hypothalamic peptide hormone shown to specifically stimulate PRL production in the pituitary gland, its physiological relevance has yet to be established. We reported previously that PrRP induces rPRL promoter activity (1). As reported recently (59, 60), the ability of PrRP, identified originally as a ligand for a receptor found in abundance in the pituitary gland, to stimulate PRL release may not represent its primary biological function. Thus, PrRP might be a potent factor capable of promoting PRL synthesis, like insulin and IGF-1, rather than a factor that promotes PRL release.

Akt plays a central role in regulating a number of key biological functions, including protein synthesis, glucose homeostasis, and cell survival and death. The mechanism by which tyrosine kinase growth factor receptors stimulate Akt has recently been defined. In contrast, the existence of a biochemical route connecting this kinase to the large family of receptors that signal through heterotrimeric G proteins has yet to be explored. TRH binds to a G protein-coupled receptor, presumably of the PTX-insensitive Gq family, and activates multiple signaling pathways in pituitary cells (61). We previously showed the involvement of a PTX-insensitive G protein-coupled (Gq) in TRH-induced ERK activation (61) and a PTX-sensitive G protein-coupled (Gi or Go) in PrRP-induced ERK activation (1). It was reported that Gi-mediated ERK activation is initiated by stimulation of PI3K activity, followed by a pathway common to tyrosine-kinase receptors (62). In this study, PrRP induced Akt activation, and both pretreatment with PTX and expression of ßARK1 blocked the PrRP-induced Akt activation, suggesting that PrRP coupling to a PTX-sensitive G protein-coupled (Gi or Go) may be responsible for the activation of Akt. Our data support the recent findings that Gß{gamma} heterodimers induce the activation of Akt in a PI3K-dependent fashion (63).

Many G protein-linked receptors can mediate stimulation of ERK activity via the PLC-dependent activation of PKC (49, 50). PKC{alpha} activates Raf-1 by direct phosphorylation (64). Apparent downregulation of PKC by prolonged incubation with PMA attenuated the stimulation of ERK activity by TRH (1, 61) and TRH-induced Raf-1 phosphorylation (61), suggesting that TRH stimulation of ERK activity is likely to be mediated by Gq-PKC. On the other hand, downregulation of PKC by prolonged incubation with PMA did not completely attenuate the stimulation of ERK activity by PrRP (1). In this study, pretreatment with 1 µM PMA for 16 h also did not completely attenuate the PrRP-induced Akt activation, suggesting that PrRP stimulation of ERK and Akt activity is not likely to be mainly mediated by Gq-PKC. Although it was also reported that downregulation of PKC by PMA did not significantly influence the effects of IGF-1 on PI3K activation or Akt phosphorylation (65), pretreatment with 1 µM PMA for 16 h does not deplete all PKC isoforms. Thus, there is still a possibility that either novel or atypical PKC isozymes are involved, as shown in some cases previously reported (66, 67).

Ca2+ is a critical mediator of the induction of PRL secretion by TRH in both primary cultures of rat anterior pituitary cells (51) and GH3 cells (68). In addition, the regulation of the PRL promoter by TRH is dependent on Ca2+ influx (69). Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. It was reported that Akt activation was regulated not only through the PI3K cascade but also the Ca2+/calmodulin protein kinase cascade (70). Elimination of extracellular Ca2+ by treatment with 3 mM EGTA for 1 min attenuated PrRP-induced Akt activation (Fig. 3Go) but not ERK activation (1). These data suggest that the cascade of PrRP-induced Akt activation might be different from that of PrRP-induced ERK activation, as was shown in the case of cisplatin-induced Akt and ERK activation (41).

Although the promoter of rPRL that we used contains a CRE-like element, DNase footprinting experiments have indicated that CREB does not exhibit high-affinity binding to the rPRL CRE-like element (28, 29). We also obtained similar results in mobility shift experiments (data not shown). In addition, it was reported that the activation of the rPRL promoter induced by a constitutively active form of CREB (VP-16 CREB) did not involve a direct interaction with the rPRL CRE-like element (30). Thus, the CREB binding element in the rPRL promoter has not yet been identified. Insulin, EGF, and cAMP act at an overlapping element at -100/-66 that is also critical for high-level basal transcription of the PRL gene (56, 57). This control region contains a cAMP responsive sequence, TGACGGA. Overlapping this element is an Ets-factor binding sequence, CGGAAA. This element has been shown to mediate the effects of insulin, IGF-I, and EGF (56, 57).

We previously reported that PrRP uses both ERK and JNK cascades to elicit rPRL promoter activity, with an Ets site as the responsible region. The ability of wortmannin and a dominant-negative Akt construct to block activation of the rPRL promoter by PrRP or insulin is partial rather than complete. In addition, whereas dominant-negative CREB constructs abolished the constitutively active Akt-induced rPRL promoter activation, they partially but significantly inhibited the rPRL promoter activation by PrRP or insulin. These results suggest that ERK and/or JNK pathways activated by PrRP or insulin would still be functional even in the presence of wortmannin, a dominant-negative Akt construct, or a dominant-negative CREB construct. Although it was reported that Ets-2 is also a target for Akt activation (71), a constitutively active Akt did not induce the phosphorylation of Ets-domain transcription factor Elk-1 (data not shown), suggesting that there is no cross-talk between the Akt and ERK/JNK cascades at the level of transcription factors in the regulation of the rPRL promoter. Several putative Ets sites [(A/C)GGAA], located at positions -295, -185, -165, and -96, are found in the rPRL promoter. The activation of the rPRL promoter induced by treatment with insulin or PrRP (1) or by cotransfection with an expression vector for a constitutively active form of either Akt or CREB was completely inhibited by cotransfection with an expression vector (pAPr-etsZ) for a dominant-negative Ets construct that competes with endogenous Ets proteins for binding to the promoters of Ets-responsive genes. It was also reported that CREB-binding protein interacts with Ets-1 and Ets-2 (58). Thus, Ets sites appear to be involved in the CREB binding element on the rPRL promoter, suggesting that ERK, JNK, and Akt signaling cascades may converge on Ets sites on the rPRL promoter. Because there are a number of sites for Ets binding in the promoter of rPRL, further studies to examine which Ets sites might be the elements responsible for PrRP and Akt activation are necessary.

Akt mediates a variety of biological activities. It was reported that Akt is involved in regulation of insulin-induced vascular endothelial growth factor mRNA expression (72) and protein synthesis but not glucose transport (73). Although there have been several reports that MAPK is involved in regulating PRL synthesis (2, 13, 74), this is the first report that an Akt cascade is involved in PRL synthesis. However, the complete role of the Akt cascade in the action of PrRP or insulin in lactotrophs remains to be elucidated. Apart from contributing to mediating transcriptional responses to PrRP or insulin, Akt activation may be associated with the survival function (32, 41) of the lactotroph for long-term maintenance of the phenotype.


   Acknowledgments
 
We thank Dr. A. Gutierrez-Hartmann for the gift of the reporter construct pA3-425PRLluc, Dr. M. E. Greenberg for the gift of the vectors encoding the various HA-tagged forms of Akt and the vectors encoding the various forms CREB, Dr. M. C. Ostrowski for the gift of pAPr-etsZ, and Dr. K. Touhara for the gift of pRK and pRK-ßARK1.


   Footnotes
 
Abbreviations: Akt, Serine/threonine kinase; CRE, cAMP-response element; CREB, CRE-binding protein; EGF, epidermal growth factor; HA, hemagglutinin; JNK, c-Jun N-terminal protein kinase; PMA, phorbol-12-myristate-13-acetate; PrRP, PRL-releasing peptide; PTX, pertussis toxin; rPRL, rat PRL.

Received July 9, 2001.

Accepted for publication September 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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