Somatostatin Acts by Inhibiting the Cyclic 3',5'-Adenosine Monophosphate (cAMP)/Protein Kinase A Pathway, cAMP Response Element-Binding Protein (CREB) Phosphorylation, and CREB Transcription Potency

John J. Tentler, John R. Hadcock and Arthur Gutierrez-Hartmann

Departments of Medicine and of Biochemistry, Biophysics,and Genetics, Program in Molecular Biology, Colorado Cancer Center, University of Colorado Health Sciences Center (J.J.T., A.G.-H.), Denver, Colorado 80262,
Wyeth Ayerst Research (J.R.H.), Princeton, New Jersey 08543


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatostatin (SRIF) was discovered as an inhibitor of GH secretion from pituitary somatotroph cells. SRIF analogs are very effective agents used to treat neuroendocrine tumors and are now being used with increasing frequency in clinical trials to treat more aggressive malignancies. However, the cellular components mediating SRIF signal transduction remain largely unknown. We have stably overexpressed the SRIF type 2 receptor (SST2) in GH4 rat somatomammotroph cells, establishing a physiologically relevant model system. In this model, the SRIF analog, BIM23014, inhibited forskolin-induced cAMP accumulation, protein kinase A activation, cAMP response element-binding protein phosphorylation, and Pit-1/GHF-1 promoter activation in an okadaic acid-insensitive manner. Pertussis toxin inhibited the effects of BIM23014, documenting that SST2 signaling was coupled to Gi. Moreover, the inhibitory effects of BIM23014 were reversed by overexpression of protein kinase A catalytic subunit, indicating that SRIF does not act via serine/threonine phosphatases, but, rather, by lowering protein kinase A activity. These data define the components of the SRIF/SST2 receptor signaling pathway and provide important mechanistic insights into how SRIF controls neuroendocrine tumors. As SRIF analogs are effective antitumor agents, and many other related compounds are in development, the knowledge gained here will further our understanding of their mechanism of action in other malignancies as well.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The neuropeptide somatostatin (SRIF) has potent inhibitory effects on cellular proliferation in a wide variety of tissues. Therefore, several long lasting SRIF analogs, such as SMS 201–995 (octreotide), MK 678 (seglitide), and BIM23014 (somatuline), have been developed as anticancer agents (1). Because SRIF was discovered as the key physiological inhibitor of GH secretion in pituitary somatotroph cells, octreotide was first approved for use in treating GH-secreting pituitary somatotroph tumors resulting in acromegaly (2, 3). Octreotide and other stable SRIF analogs cause a significant reduction of both GH secretion and pituitary tumor size in acromegaly. More recently, SRIF analogs have shown significant utility in the treatment of other endocrine and nonendocrine tumors, where it can inhibit tumor progression and in some cases result in tumor shrinkage (4, 5).

Critical to SRIF action is its binding to specific plasma membrane receptors (SSTx, where x is the receptor subtype number). Five distinct SST receptor subtypes (SSTs 1–5) have been characterized and shown to be members of the seven transmembrane-spanning, Gi-protein-coupled superfamily of receptors (6, 7, 8). All five SSTs have been detected in a variety of human tumors. However, the SST2 subtype is the most frequently expressed, providing a potential explanation for the significant effects of SST2-selective agents, such as octreotide, on many different types of malignancies (4, 5, 9). Elucidation of downstream effectors in a native cellular environment has been hampered by the very low expression of SST protein in most cell types. To circumvent this problem, investigators have overexpressed individual SST subtypes in heterologous cells, such as Chinese hamster ovary (CHO) and HEK 293, and reported that SSTs can couple to multiple cellular effector systems, including adenylyl cyclase, Ca2+ and K+ channels, phospholipase A, serine/threonine phosphatases, and tyrosine phosphatases (6, 7, 8, 9, 10). One SST subtype may be linked to more than one effector system, and the actual pattern of SST-effector coupling appears to be cell or tissue specific, most likely based on which Gi proteins and effector systems are present. Although these fibroblast expression studies have provided important insights into the SRIF signaling pathway, these cells are not physiological targets of SRIF and thus may lack critical cellular SRIF signaling component(s). In this report, we describe the use of a novel, physiologically relevant, GH4 pituitary cell line that stably overexpresses the long isoform of the type 2 SRIF receptor (SST2). This model system was characterized and used to begin to identify the signaling components and gene targets of the SRIF pathway mediated by SST2. We find that SRIF inhibits the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway, lowers CREB transcription potency, and decreases GHF-1/Pit-1 promoter activity in a pertussis toxin-sensitive, but okadaic acid (OA)-insensitive, manner. Moreover, we map the effect of SRIF to the PKA step. As the cAMP/PKA pathway is important for pituitary somatotroph ontogeny, proliferation, and cellular function, we conclude that SRIF acts by targeting its inhibitory effects to signaling components critical to somatotroph function.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Development and Characterization of SST2 Expressing Cell Lines
To identify the physiologically relevant signaling components by which SRIF controls cell function, we developed an experimental model system by stably transfecting GH4C1 rat pituitary somatomammotroph tumor cells with a plasmid encoding for a full-length rat SST2A, the long SST2 isoform, and the predominant pituitary SRIF receptor (hereafter termed SST2). Control cells were transfected with an empty vector (CMV). Several clones were isolated and, along with wild-type nontransfected controls, were subjected to saturation binding analysis using [125I]SRIF-14 to determine levels of SST2 protein. The results of the saturation binding analysis are given in Fig. 1Go and expressed as picomoles per mg protein. [125I]SRIF-14 binding to membranes prepared from all GH4C1 cells was saturable and of high affinity at all levels of SST2 expression. The nontransfected wild-type control and six transfected clones expressing different levels of SST2 were then cultured in either the absence or presence of the long acting SRIF analog, BIM23014, and the levels of SST2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) expression were determined by Northern blot analysis (Fig. 1Go). Although endogenous SST2 mRNA levels could not be detected in the wild-type (WT), the CMV control, or the R2/4 cell lines after a 36-h exposure, [125I]SRIF-14 binding analysis revealed that the controls each expressed about 0.4 pmol SST2/mg protein and that the R2/4 clone expressed approximately a 5-fold higher level of SST2 (2.2 pmol/mg protein). Detection of the endogenous SST2 mRNA in the control cells required a 14-day exposure, revealing two bands of 2.3 and 2.6 kb (data not shown), consistent with the size of endogenous SST2 mRNA (11). By contrast, the R2/25, R2/34, R2/20, and R2/21 clones resulted in a detectable signal of about 1.9 kb, consistent with the size of the transfected SST2 complementary DNA (cDNA), and clones R2/20 and R2/21 produced a relatively strong SST2 mRNA signal. Again, the mRNA signals in these clones generally agreed with the levels of SST2 protein detected by saturation binding analysis, with the intermediate clones expressing 15 and 25 pmol/mg protein (40- and 60-fold), and the high expressing clones producing 40 pmol/mg protein (100-fold). The effects of BIM23014 on endogenous SST2 mRNA expression in WT and CMV cells are negligible (data not shown), as are its effects on exogenous SST2 mRNA expression, except for clone R2/34. It is unlikely that BIM23014 affects the CMV promoter activity controlling SST2 expression, because several of the clonal lines are not affected; it is more likely an integration site-specific effect in the R2/34 clone. The GAPDH band (Fig. 1Go, bottom panel) reveals equal loading of RNA and showed that the ratio of exogenous/endogenous SST2 mRNA expression was at least 100-fold, consistent with the data from Scatchard analysis. Together, the Scatchard and Northern data verify the scarcity of endogenous SST2 mRNA and protein and the abundant levels of the transfected SST2. The high expressing R2/20 clone was used in all of the following studies.



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Figure 1. Northern Blot Analysis of SST2 mRNA in Control and SST2-Stable GH4C1 Cell Lines

Clonal cell lines (R2/4, R2/25, R2/34, R2/20, and R2/21) were subjected to saturation binding analysis and found to express varying amounts of SST2 receptor protein (picomoles per mg). WT and CMV represent control nontransfected and pRC/CMV only-transfected wild-type GH4 cells, respectively. Cells were exposed to PBS (-) or 10 nM BIM 23014 (Bachem, Torrance, CA; +) in DMEM plus 10% FBS for 36 h, replenishing the analog at 6-h intervals. Total RNA (20 µg) from each clonal cell line was electrophoresed on a 1.4% agarose-formaldehyde gel and transferred to a nitrocellulose membrane; the membrane probed with a radiolabeled SST2 cDNA hybridization probe at high stringency and exposed to film for 36 h (top panel). The same blot was reprobed with a labeled cDNA for GAPDH (bottom panel) to assure equal loading of RNA. A representative autoradiograph is presented.

 
Effects of BIM23014 on the 3',5'-cAMP/PKA/CREB Pathway in R2/20 Pituitary Cells
The cAMP/PKA/CREB signaling pathway is critical for somatotroph ontogeny, proliferation, and cell-specific gene expression (12, 13, 14). Also, 30–50% of human somatotroph tumors contain activating Gs mutations, resulting in constitutively elevated cAMP levels and constitutively phosphorylated and activated CREB transcription factor (15, 16, 17). As SRIF analogs inhibit somatotroph tumors, we sought to determine whether the SRIF analog BIM23014 inhibited any of the components of the cAMP signaling cascade. The R2/20 clone expressed lower basal levels of cAMP compared with control GH4 cells, and they displayed a 5-fold lower ED50 for SRIF-14-mediated reduction of forskolin (FSK)-stimulated cAMP levels (data not shown). Using a serine 133 phospho-specific CREB antibody (17), we showed that FSK-stimulated CREB phosphorylation (Fig. 2AGo) by about 8.5-fold (Fig. 2BGo). Pretreatment with increasing doses of BIM23014 reversed the FSK-induced CREB phosphorylation, with an IC50 of approximately 7 nM (Fig. 2Go, A and B). BIM23014 (10 nM) alone had no effect (Fig. 2AGo, lane 2, and Fig. 2BGo), and the BIM23014-mediated inhibition of the FSK response was blocked by pretreatment with pertussis toxin (Fig. 2AGo, lanes 12 and 13, and Fig. 2BGo), verifying that the effect of BIM23014 was mediated by Gi, as predicted (6, 7, 8, 10). Also, BIM23014 had minimal effects on FSK-mediated CREB phosphorylation in GH4 WT cells (data not shown), indicating that the effects of BIM23014 were not through an endogenously expressed SST receptor and substantiating the idea that SST2 overexpression amplifies the effects of BIM23014. As CREB phosphorylation at serine 133 directly correlates with its activity as a transcription factor (17), these data suggest that BIM23014 inhibits CREB transcription potency.



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Figure 2. Effect of BIM23014 on FSK-Stimulated Phosphorylation of CREB

A, R2/20 cells were cultured in 1% FBS for 6 h, and then treated with the indicated doses of BIM23014 for 10 min, followed by treatment with FSK (1 µM). After 10 min, the cells were lysed by the addition of Laemmli’s sample buffer. Some cells were pretreated with pertussis toxin (PTX; 100 ng/ml) for 24 h before and during the experimental protocol noted above. Equal amounts (50 µg) of whole cell extracts were resolved by SDS-PAGE, followed by Western blotting, and probed with an antibody specific for [Ser133]phospho-CREB (top panel). The blot was subsequently stripped and reprobed with an antibody to both phospho- and dephospho-CREB (bottom panel). Bands were detected by enhanced chemiluminescence (Amersham). B, Quantification of the Western blot shown in A. Phosphorylation was quantitated by scanning laser densitometry of the autoradiograph (Molecular Dynamics, Sunnyvale, CA), and values were corrected for levels of CREB in each sample.

 
Effect of BIM23014 on Transcription of CREB-Dependent Genes
To determine the functional significance of the BIM23014-mediated attenuation of FSK-induced CREB phosphorylation, we tested the response of the CREB-dependent, pituitary-specific POU homeodomain transcription factor promoter, Pit-1/GHF-1, to FSK with or without BIM23014 (Fig. 3BGo). The Pit-1/GHF-1 promoter is a particularly relevant target, because it has been shown that the Pit-1 protein is one of the factors that controls somatotroph cellular proliferation (18). FSK treatment resulted in a 13-fold activation of a -200Pit-1/CAT reporter construct (set at 100%) transiently transfected into the R2/20 pituitary cells (Fig. 3BGo) (12). Moreover, the dose response of the BIM23014-mediated inhibition of FSK-induced Pit-1 promoter activation (Fig. 3BGo) closely paralleled the inhibition of CREB phosphorylation (Fig. 2Go), with an IC50 of approximately 15 nM. Similarly, the inhibitory effect of BIM23014 on the Pit-1 promoter was reversed by pretreatment with pertussis toxin (Fig. 3BGo). The brief period of BIM23014 treatment (6 h) was insufficient to mediate inhibition of Pit-1 mRNA levels (data not shown). However, as Pit-1 protein autoregulates its own promoter, we used a separate pituitary-specific, CREB-dependent, Pit-1-independent promoter, the -1760 {alpha}-subunit ({alpha}-SU) glycoprotein promoter (19), to insure that the effects were due to inhibition of CREB alone (Fig. 3CGo). FSK stimulated {alpha}-SU promoter activity 22-fold, and 10 nM BIM23014 reduced this activation by 52%, mimicking the effects on the Pit-1 promoter (Fig. 3BGo). Finally, BIM23014 had no effect on the CMV promoter used as an internal control to drive ß-galactosidase, thus showing that the BIM23014 effect was specific for cAMP-responsive promoters. These results corroborate that the BIM23014-mediated effects on CREB phosphorylation status govern its potency as a transcription factor.



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Figure 3. Dose Effects of BIM23014 on -200 Rat GHF-1/Pit-1 Promoter Activity

A, Schematic map of the GHF-1/Pit-1 proximal promoter region. Binding sites for CREB (CRE1 and CRE2) and for Pit-1 (Pit-1B1) are indicated. B, R2/20 cells were cultured in complete DMEM and transfected by electroporation with 9 µg Pit-1-CAT and 3 µg CMV ß-galactosidase (ß-gal), as previously described (40). After transfection, cells were cultured in DMEM plus 1% FBS for 18 h and then treated with FSK (1 µM) and the indicated doses of BIM23014 for 6 h. Some cells were pretreated with pertussis toxin (PTX; 100 ng/ml) for 24 h before and during the transfection protocol. The amounts of extracts used to determine CAT activity were normalized according to the level of ß-galactosidase. The results shown are expressed as a percentage of the value with FSK alone (100% = 13-fold). C, Effects of BIM23014 on FSK-activated human {alpha}-SU. R2/20 cells were cultured in complete medium and transfected by electroporation with 3 µg {alpha}-SU-luciferase and 3 µg CMV-ß-gal. After transfection, cells were cultured in DMEM plus 1% FBS. FSK (1 µM) and the indicated doses of BIM23014 were added 18 h after transfection, and cells were collected 6 h later. Results are corrected for ß-galactosidase activity and expressed as fold promoter activation.

 
Effects of OA on SRIF Action
It has been proposed that activation of serine/threonine phosphatases may mediate some of the effects of SRIF, as the Ser/Thr phosphatase inhibitor OA can reverse the stimulatory effects of SRIF on large conductance Ca2+- and voltage-activated K+ (BK) channel activity (20). Serine/threonine phosphatases 1 (PP1) and 2A (PP2A) have been shown to dephosphorylate CREB in vivo and thus attentuate its transcription activity (21, 22, 23). To investigate the roles of PP1 and PP2A in the BIM23014-mediated reduction in CREB phosphorylation, we treated cells with or without OA, a specific inhibitor of PP1 and PP2A, at a dose (25 nM) that will inhibit both of these phosphatases (22, 23). Western analysis (Fig. 4AGo), using the CREB phospho-specific antibody, and quantification of these data (Fig. 4BGo) show that OA, either alone or in combination with FSK, slightly stimulates CREB phosphorylation, yet OA fails to reverse the BIM23014-mediated inhibition of CREB phosphorylation. To corroborate these results in a functional manner, we tested the ability of 25 nM OA to reverse the inhibitory effects of BIM23014 on the {alpha}-SU promoter (Fig. 4CGo). Again, OA alone activated the {alpha}-SU promoter and enhanced the effects of FSK, verifying that OA is functional. However, OA failed to reverse the inhibitory effects of BIM23014 {alpha}-SU promoter activity. We found that the effects of OA are specific. All transfections were internally controlled with pCMV ß-galactosidase, and this cAMP-unresponsive promoter is unaffected by OA, with or without BIM23014.



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Figure 4. Effects of the Ser/Thr Phosphatase Inhibitor, OA, on Biochemical and Functional Activity of BIM23014

A, R2/20 cells were pretreated with or without 30 nM OA for 6 h (+ or -), then exposed to 10 nM BIM23014 for 10 min, followed by treatment with FSK (1 µM) for 10 min. Phospho-CREB (top panel) was detected by Western analysis as described in Fig. 2Go. The blot was subsequently stripped and reprobed with an antibody to both phospho- and dephospho-CREB (bottom panel). B, Quantification of the Western blot shown in A. Phosphorylation was quantitated by scanning densitometry of the autoradiograph, and values were corrected for levels of CREB in each sample. C, Effects of OA on BIM23014 inhibition of FSK-stimulated {alpha}-SU promoter. R2/20 cells were cotransfected with 3 µg {alpha}-SU-luciferase and 3 µg CMV ß-gal. Cells were treated with or without OA (30 nM) for 6 h (+ or -) before BIM23014 and FSK treatment, which was added for the last 6 h of a 24-h transfection. Results are corrected for ß-galactosidase activity and expressed as fold promoter activation.

 
PKA Catalytic Subunit Expression Reverses Inhibitory Effects of BIM23014 on {alpha}-SU Promoter
The biochemical and functional data presented above indicate that SRIF does not act via PP1 or PP2A to inhibit CREB phosphorylation and activity. Therefore, we postulated that BIM23014 may act by lowering PKA activity. To determine whether BIM23014 is acting upstream or downstream of PKA, an expression vector encoding the ß-catalytic subunit of PKA was cotransfected with the {alpha}-SU reporter gene, and the effects of BIM23014 on PKA promoter activation were assessed (Fig. 5Go). These results show that PKAß, in the absence of BIM23014, activated the {alpha}-SU promoter 18-fold, consistent with the FSK results shown in Fig. 3CGo. However, addition of increasing amounts of BIM23014, from 0.1–100 nM, had no statistically significant effect on the PKA response. These data are in striking contrast to the inhibitory effects of BIM23014 on FSK activation of this same promoter (Fig. 3CGo). More importantly, we have mapped the site of SRIF action between adenylate cyclase and PKA, with the data clearly implicating PKA as a key target. Finally, these data independently corroborate that phosphatases are unlikely to be involved in SRIF action.



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Figure 5. Overexpression of PKA Catalytic Subunit Blocks Effects of BIM23014

R2/20 cells were cotransfected by electroporation with and without (+ or -) 9 µg of an expression vector encoding an RSV-driven PKA catalytic subunit (RSV PKAß) plus 3 µg {alpha}-SU-luciferase and 3 µg CMV ß-galactosidase. The indicated doses of BIM23014 were added 18 h after transfection, and cells were collected 6 h later. Results are corrected for ß-galactosidase activity and expressed as fold promoter activation.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SRIF and its analogs have found clinical utility as potent antisecretory and antiproliferative agents in tumors derived from a wide variety of tissues. Despite this widespread use of SRIF analogs, little is known regarding the functional cellular components that mediate SRIF signal transduction. In this paper, we report the development and characterization of a novel SST2-overexpressing model system in a physiologically relevant cell line, GH4 pituitary cells. Using this system, we demonstrated that the SRIF analog, BIM23014, acts via a Gi-coupled process to inhibit the cAMP pathway, acting at or upstream of PKA and resulting in decreased CREB phosphorylation and transcription potency in an OA-insensitive manner. As cAMP/PKA, CREB, and Pit-1/GHF-1 exert important growth regulatory effects on somatotroph cells (12, 13, 14, 15, 16), we have linked the inhibitory effects of SRIF to specific cellular constituents that are critical for neuroendocrine cellular function and proliferation (24, 25).

Trophic hormone activation of the cAMP/PKA pathway has been shown to be critical for the maintenance of cell-specific function and cell proliferation in many neuroendocrine cell types (24, 25). Underscoring this idea has been the identification of several naturally occurring mutations that result in the constitutive activation of receptors for trophic hormones, such as TSH, LH, and PTH/PTH-related peptide (26, 27, 28, 29), and the demonstration that these receptor mutations cause elevations in cAMP and hyperfunction of the respective endocrine end organ, leading to endocrine tumor formation. Conversely, inactivating mutations of trophic hormone receptors have also been identified, and patients harboring these mutations display a phenotype consistent with the loss of endocrine end organ cell function (30). These trophic hormone receptors typically act by coupling to heterotrimeric Gs{alpha} proteins, and activating mutations of Gs{alpha} have also been identified and denoted the gsp oncogene, because they result in endocrine end-organ hyperfunction and tumor formation (15, 16, 24, 26, 31). Taken together, these findings corroborate the importance of the cAMP/PKA pathway in endocrine cells. Indeed, this pathway has been shown to be critical for normal pituitary somatotroph cell ontogeny, maintenance of cell function, and cellular proliferation (18, 32). For example, an inactivating mutation of the GRF receptor results in a lack of the normal expansion of somatotroph cells during development and leads to the little mouse phenotype (33). Similarly, targeted transgene expression of a cholera toxin to somatotroph cells leads to a persistently activated Gs{alpha}, increased cAMP, and somatotroph hyperplasia and tumorigenesis (13), whereas targeted expression of a dominant negative CREB leads to somatotroph hypoplasia (14). Finally, many GH-secreting somatotroph adenomas from acromegalic patients arise from expression of the constitutively active gsp oncogene, resulting in activated adenylyl cyclase, elevated cAMP levels, persistently phosphorylated CREB, and up-regulated Pit-1 promoter activity (13, 14, 15, 16, 17, 34). Of note, these patients are particularly responsive to the SRIF analog octreotide (35). Thus, our results showing that SRIF acts via SST2 to inhibit the cAMP/PKA/CREB pathway and Pit-1 promoter activity provide important insights into the cellular mechanisms by which SRIF analogs effectively control tumors derived from cAMP-regulated endocrine cells.

Having determined that BIM23014 inhibits FSK-induced CREB phosphorylation, we sought to identify the mechanism of this action. In theory, the inhibitory effects of BIM23014 on CREB phosphorylation could be mediated by either increased phosphatase activity, decreased PKA activity, or a combination of these two possibilities. The results of our studies indicate that in our system, BIM23104 is not activating phosphatases, as it is capable of attenuating CREB phosphorylation and transcription potency in the presence of OA, a specific inhibitor of Ser/Thr phosphatases PP1 and PP2A. The observation that addition of OA alone results in increased basal CREB phosphorylation and {alpha}-SU promoter activity (Fig. 4Go) indicates that OA is entering the cells and exerting its expected effect on CREB phosphorylation, by modulating either PP1 and/or PP2A (21, 22). By contrast, our results showing that overexpression of a PKA catalytic subunit can reverse the inhibitory effects of BIM23014 on {alpha}-SU promoter (Fig. 5Go) indicate that the effect of SRIF maps upstream or at the level of PKA. Furthermore, this finding also supports the idea that phosphatases are not the key regulatory effectors, as it would be expected that SRIF stimulation of phosphatase catalytic activity should diminish the effects of transfected PKA. In this regard, the effects of SRIF on somatotrophs mimic the tonic inhibitory effects of dopamine on lactotrophs, by which dopamine agonists also act via a Gi-coupled D2 receptor to lower cAMP and diminish Pit-1 promoter activity (36, 37). Similarly, these dopamine agonists inhibit pituitary lactotroph cell function and shrink pituitary lactotroph adenomas (38). Together, these data indicate a convergence of mechanisms for the clinical antiproliferative effects of SRIF analogs on somatotrophs and of dopamine analogs on lactotrophs, indicating that the cAMP/PKA pathway is critical to both of these pituitary cell types. Therefore, the mechanism of SRIF action in somatotrophs that we show here may be generalizable to other tumors that use cAMP as a key regulator of cellular growth or to maintain cell function (24, 25).

Although these data clearly implicate the cAMP/PKA pathway as a critical target of SRIF, the possibility remains that SRIF may inhibit other, OA-resistant signaling pathways as well. For example, SRIF might alter calcium-dependent pathways (such as calcineurin/calmodulin kinase, phosphoinositol-3 kinase, or protein kinase C) or tyrosine kinase/MAP kinase pathways. All of these signaling constituents have been shown to play important roles in somatolactotroph cell function (39, 40, 41, 42, 43), and thus, SRIF inhibition of one or more of these signaling cascades would result in a pleiotropic and more effective inhibition of pituitary cell functions. Previous reports have shown that SRIF analogs inhibit the inositol phospholipid/calcium pathway in rat pancreas (44) and clonal hamster ß-cells (45). Also, SRIF analogs have been show to stimulate p66 PTP1C tyrosine phosphatase activity in MCF-7 breast cancer cells (45, 46), AR4–2J rat pancreatic acinar cells (47), and COS, CHO, and NIH3T3 cells stably expressing the SST2 receptor subtype (48), each in a vanadate-sensitive manner. However, the SRIF analog, RC-160, inhibited cellular proliferation of CHO cells overexpressing the SST5 receptor subtype in a vanadate-insensitive manner, indicating differential effector coupling to the distinct SST subtypes (48). These results raise the possibility that SRIF/SST2-activated PTP1C tyrosine phosphatase may dephosphorylate and inactivate membrane receptor and cytoplasmic tyrosine kinases required for cell growth (46, 47, 48, 49). In the studies reported here, we have not examined whether BIM23014 results in activation of tyrosine phosphatase activity, because we have focused on the cAMP/PKA pathway. Therefore, the possibility remains that BIM23014 might induce tyrosine phosphatase activity via the overexpressed SST2 in the GH4 pituitary model system described here.

Finally, and of particular significance for the future discovery of clinically applicable agents, the SSTR2-overexpressing GH4 somatolactotroph model that we have developed can be used to identify and characterize SST2 subtype-selective agonists and antagonists, using the biochemical and functional assays that we have reported here. In this regard, this system provides an important and biologically pertinent model that will allow us to further elucidate the molecular mechanisms of SRIF actions that are selective for the SST2 receptor subtype. These approaches should begin to decipher the structure-function code by which specific SST receptor subtypes target distinct and overlapping downstream effectors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The long isoform of the SST2 SRIF receptor (SST2A) was used for these studies. The SST2 cDNA was isolated by the PCR using specific primers and GH4C1 RNA. Both strands of the SST2 cDNA were sequenced using an Applied Biosystems automated DNA sequencer (Foster City, CA). The full-length rat SST2 SRIF receptor was subcloned into the expression vector, pRC/CMV (Invitrogen, San Diego, CA), and stable transfections of GH4C1 cells were accomplished using a calcium phosphate transfection kit (Stratagene, La Jolla, CA). Six individual clones were selected using 500 µg/ml G-418 and were amplified and maintained in 100 µg/ml G-418. Saturation binding was performed on membranes of WT and transfected GH4C1 cells with 0–2500 pmol [125I]SRIF-14. Nonspecific binding for each point (counts per min bound in the presence of 5 µM cold SRIF-14) ranged from 10–40%. Experiments for each cell line were performed in triplicate. Transient transfection experiments of GH4C1 rat pituitary cell lines were performed as described previously, using pCMV-ß-galactosidase as an internal control for transfection efficiency (40, 50). The -200 Pit-1 promoter and the human glycoprotein {alpha}-SU promoter plasmid constructs have been described previously (12, 19). Cells were harvested, and reporter enzyme activities were assayed as previously described (40, 50). Normalized data are depicted as the mean of several experiments ± SEM.

Cell extracts for Western blotting were prepared from 80% confluent 60-mm dishes. Cells were harvested with Laemmli-SDS sample buffer and cell scraping. Cell extracts were boiled for 5 min and sheared through a 22-gauge needle. Samples were resolved on 12% SDS-polyacrylamide gels and transferred to nitrocellulose as described previously (40). Filters were blocked in 5% nonfat milk and 0.2% Tween-20, probed with antibodies to phospho-CREB (provided by Dr. M. Montminy) and CREB (provided by Dr. D. Klemm), and developed using enhanced chemiluminescence (Amersham Corp., Arlington Heights, IL) according to the manufacturer’s protocols.

Total RNA was prepared from the indicated clones with the RNA Stat 60 Kit (Tel-Test, Inc., Friendswood, TX). Samples (20 µg total RNA) were electrophoresed on a 1.4% agarose-formaldehyde gel and transferred to a nylon-reinforced nitrocellulose membrane. A [{alpha}-32P]CTP-labeled hybridization probe was prepared from the full-length SST2 cDNA template by random primer synthesis. After high stringency hybridization, the membrane was washed three times with 0.1 x saline sodium citrate (SSC) and 0.1% SDS at 65 C for 30 min. The same blot was reprobed with a labeled cDNA for GAPDH to assure equal loading of RNA.


    ACKNOWLEDGMENTS
 
We thank M. C. Eppler for providing cell lines, M. Montminy for providing antibody to phospho-CREB, M. Karin for the -200 GHF-1 promoter construct, and K. N. Farrow, A. P. Bradford, P. Zeitler, S. E. Diamond, and A. James for critical comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Arthur Guitierrez-Hartmann, Departments of Medicine and of Biochemistry, Biophysics, and Genetics, Program in Molecular Biology, Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262.

This work was supported by NIH Grants DK-46868 and DK-37667, and NIH SBIR Subcontract N44-DK-2–2214 (to A.G.-H.). Additionally, partial support was provided by the Lucille P. Markey Charitable Trust and the University of Colorado Cancer Center Core Grant CA-46934.

Received for publication December 11, 1996. Revision received January 31, 1997. Accepted for publication March 17, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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