help button home button Am J Pathol R & D Systems
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oka, H.
Right arrow Articles by Lloyd, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oka, H.
Right arrow Articles by Lloyd, R. V.
(American Journal of Pathology. 1999;155:1893-1900.)
© 1999 American Society for Investigative Pathology

Regular Articles

Pituitary Adenylate Cyclase-Activating Polypeptide Inhibits Transforming Growth Factor-ß1-Induced Apoptosis in a Human Pituitary Adenoma Cell Line

Hidehiro Oka*{dagger}, Long Jin*, Elzbieta Kulig*, Bernd W. Scheithauer* and Ricardo V. Lloyd*

From the Department of Laboratory Medicine and Pathology,*
Mayo Clinic and Mayo Foundation, Rochester, Minnesota; and the Department of Neurosurgery,{dagger}
Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from hypothalamic tissues based on its ability to stimulate cAMP production in cultured anterior pituitary cells. Recent studies have suggested a functional role for PACAP in the apoptosis of brain cells. However, the role of PACAP in regulating apoptosis in human pituitary adenomas has not previously been examined. Analysis of the cultured human pituitary adenoma cell line HP75, which expresses all three major PACAP receptors, showed that both PACAP-38 and PACAP-27 inhibited TGF-ß1-induced apoptosis. Treatment with the PACAP receptor antagonists PACAP 6–38 (PACAP type I receptor antagonist) and (p-chloro-D-Phe6, Leu17)-VIP (PACAP type II receptor antagonist) blocked the effects of PACAP-38 on the inhibition of transforming growth factor-ß1 (TGF-ß1)-induced apoptosis, confirming the specificity of the role of PACAP. Treatment with forskolin but not phorbol 12-myristate 13-actetate (PMA) also inhibited TGF-ß1-induced apoptosis. TGF-ß1 treatment was associated with an increase in mitogen-activated protein kinase (MAP kinase) when analyzed by Western blotting, but PACAP inhibition of TGF-ß1-induced apoptosis was not associated with activation of MAP kinase. Immunocytochemical analysis of the cell cycle cyclin-dependent kinase inhibitor p27 showed that treatment with TGF-ß1, forskolin, PMA, and PACAP increased p27 expression in cultured HP75 cells. These results indicate that PACAP is a highly specific inhibitor of TGF-ß1-induced apoptosis in the HP75 human pituitary adenoma cell line and that PACAP, TGF-ß1, forskolin, and PMA all stimulate expression of the TGF-ß-regulated cell cycle protein p27 in the HP75 human pituitary adenoma cell line. The HP75 cell line can be used as a model to study the regulation of apoptosis in human pituitary cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from hypothalamic tissues by its ability to stimulate cAMP production in cultures of anterior pituitary cells at a potency 1000 times greater than that of vasoactive intestinal polypeptide (VIP).1 PACAP is present in tissues as a 38-amino acid (PACAP-38) and as a shorter 27-amino acid peptide (PACAP-27) and shares approximately 68% sequence homology with VIP at its N-terminal portion.1,2 It stimulates the release of several pituitary hormones, including luteinizing hormone (LH),3 growth hormone (GH),4 and interleukin-6 (IL-6) from folliculo-stellate cells.5 PACAP also stimulates ACTH release from the clonal corticotroph AtT20 cell line and GH and/or prolactin release from the clonal somatotroph GH3 cell line.6 Thus PACAP fulfills various criteria of a hypophysiotropic factor.

Recent studies have demonstrated a relationship between PACAP and apoptosis. Arimura et al described a neurotrophic biphasic effect of low PACAP-38 concentrations on gp120-induced apoptosis in hippocampal cultures.7 Other studies have suggested that PACAP plays a role in preventing apoptosis in cerebellar granule neurons.8,9 Recently, all three major PACAP receptors were demonstrated in normal and neoplastic human pituitaries; however, the role of PACAP in regulating apoptosis in human pituitary adenoma has not previously been examined.10,11

Programmed cell death or apoptosis has been recognized as contributing to a range of developmental and remodeling events in tissues and to tumor regression in response to treatment with chemotherapeutic agents. In lymphoid cells, cellular signal transducers such as cAMP may stimulate apoptosis, and this molecule can substitute for glucocorticoids in triggering apoptosis.12 cAMP acts as a second messenger molecule that regulates the activity of protein kinase A (PKA) or PKA-related enzymes and a wide range of cellular processes.13,14 Protein kinase C (PKC), which is activated by diacylglycerol located on the cell surface membrane, is involved in the regulation of a number of cellular events, including growth and differentiation in a wide range of tissues. The levels of PKC activity and PKC mRNA expression were higher in pituitary adenoma cells than in normal human and rat pituitary cells,15,16 suggesting a possible role of PKC in pituitary tumor development.

Recent in vitro studies suggest that transforming growth factor-ß (TGF-ß) and related peptides may have important roles in pituitary cell proliferation and in hormone expression.17-19 Various isoforms of TGF-ß are expressed in rat18-21 and human22,23 pituitary cells. TGF-ß has an inhibitory effect on the cell cycle directed at the G1-to-S phase transition, and this inhibition is reversible after removal of this cytokine.24,25 Some of the actions of TGF-ß are mediated by cell cycle inhibitory proteins such as p27kip1 (p27) and p15.26-28 p27 in turn may function as a negative regulator of G1 cell cycle progression and may mediate TGF-ß-induced G1 arrest. p27 protein, which interacts with cyclin-cdk complexes, including cyclin E-cdk2,26-28 is expressed at higher levels in quiescent cells than in proliferating cells, which may implicate this cell cycle protein in cell death.

We examined the role of PACAP in modulating apoptosis in a human pituitary adenoma cell line. Our results show that PACAP is a highly specific inhibitor of TGF-ß1-induced apoptosis in this human pituitary adenoma cell line in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture

An immortalized human pituitary HP75 cell line, developed originally from a nonfunctioning pituitary adenoma with a replication-defective recombinant human adenovirus with a SV40 early large T-antigen,29 was used in this study. The HP75 cells were maintained in Dulbecco’s minimum essential medium (DMEM) with 15% horse serum and 2.5% fetal bovine serum with 1 µg/ml insulin and 1% antiboitics (all from Life Technologies, Grand Island, NY).

For the apoptosis experiments, the HP75 cells were plated onto 35-mm plastic dishes at approximately 0.5 x 106 cells/dish for 4 hours. After the complete medium was removed and the cells were washed twice with DMEM, the HP75 cells were incubated in a serum-free DMEM without insulin or dexamethasone for about 24 hours. The following reagents were used in cell culture studies, and the final concentration was obtained after preliminary titration experiments: porcine TGF-ß1 (1 x 10-9 mol/L; R&D Systems, Minneapolis, MN); PACAP38 and PACAP27-NH2 (2.5 x 16-7 mol/L; Peninsula Lab, Belmont, CA); PACAP 6–38 (PACAP type I receptor antagonist) and (p-chloro-D-Phe,6 Leu17)-VIP (PACAP type II receptor antagonist) (10-6 mol/L; Bachem, Torrance, CA); phorbol 12-myristate 13-acetate (PMA) (10-7 mol/L); and forskolin (10-6 mol/L) (Sigma Chemical Co., St. Louis, MO). After 24 hours of treatment, the HP75 cells were harvested, cytocentrifuged onto glass slides, fixed with 4% paraformaldehyde for 20 minutes, and used for analysis of apoptosis and immunocytochemistry.

Oligonucleotide Primers and Probes

Oligonucleotide primers and Southern hybridization probes were synthesized with a DNA oligonucleotide synthesizer (Applied Biosystems, Foster City, CA) (Table 1) . Both primers and probes for human PACAP30 and PACAP receptors, including PVR-1,31 PVR-2,32,33 and PVR-3,34 were synthesized on the basis of published sequences as previously reported.10,11


View this table:
[in this window]
[in a new window]
  		Table 1. Sequences of Primers and Hybridization Probes for Human PACAP and PVR mRNAs

 
Solution Polymerase Chain Reaction and Southern Hybridization

Total RNA extraction was performed by single-step methods (TRIzol Reagent Kit; Life Technologies) from HP75 cells.29,35

First-strand complementary DNA (cDNA) was prepared from total RNA with a first-strand synthesis kit (Stratagene, La Jolla, CA). The reverse transcriptase (RT) reaction was performed at 37°C for 60 minutes in a final volume of 50 µl with 5 µg total RNA, 300 ng oligo-dT primer, 1x RT buffer, 1.0 mmol/L of each deoxyribonucleotide (dATP, dCTP, dGTP), 40 U ribonuclease (RNase) inhibitor, and 50 U Moloney murine leukemia virus RT. The reaction product was then heated at 95°C for 5 minutes and immediately placed on ice.

The polymerase chain reaction (PCR) was performed in 100-µl final reaction volumes containing 5 µl RT reaction product as template DNA, corresponding to cDNA synthesized from 500 ng total RNA; 1x PCR buffer (Promega, Madison, WI); 1.5 mmol/L MgCl2; 0.2 mmol/L of each deoxynucleotide (Boehringer Mannheim, Indianapolis, IN); 300 ng of each sense and antisense primer for PACAP and PVR-1, -2, and -3; and 2.5 U Taq DNA polymerase (Promega). Programmed temperature cycling (Perkin Elmer/Cetus 480, Norwalk, CT) was performed with the following cycle profile: 95°C for 5 minutes, followed by 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes (30 cycles) for GAPDH and PACAP, and 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes (40 cycles) for PVR-1, -2, and -3, respectively. After the last cycle, the elongation step was extended at 72°C for 10 minutes.

A 20-µl aliquot of PCR product was analyzed by gel electrophoresis, using a 2% agarose gel, and was stained with ethidium bromide. PH0174 DNA/HaeIII digest (Boehringer Mannheim) was used as the molecular size marker. The separated PCR products were transferred to nylon membrane filters. Southern hybridization, with a single internal probe that hybridized to regions within the amplified sequences, was performed. Hybridization was performed with 1 x 106 cpm/ml [33P]deoxyadenosine diphosphate-labeled probes at 42°C for 18 hours. After washing with 6x SSC–0.1% SDS at 23°C for 20 minutes and at 42°C for 20 minutes, autoradiography was performed at -70°C with Kodak Omat-AR film (Eastman Kodak, Rochester, NY) with intensifying screens. In the RT-PCR experiments, total RNA from the hypothalamus was included as a positive control for PACAP and PVR-1, -2, and -3.

Apoptotic Cell Detection

For analysis of apoptosis, an in situ cell death detection kit with terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) (Boehringer Mannheim) was used. The reaction product was visualized by reaction with nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP) (Life Technologies). Cells were counterstained with nuclear fast red dye.

Negative controls consisted of omission of the TdT in the TUNEL reaction, which resulted in no staining. Positive cells were enumerated by counting a minimum of 500 cells/slide, and the results were expressed as an apoptotic index (AI) (number of apoptotic cells per 100 cells). Ultrastructural studies were done to confirm the presence of apoptotic cells. Cells were fixed in 2% formaldehyde in phosphate-buffered glutaraldehyde and processed for electron microscopy as previously described.29

Detection of Phospho-ERKs

HP75 cells were treated with TGF-ß1, PACAP-38, and TGF-ß1 plus PACAP-38 for 10 minutes, 30 minutes, 60 minutes, and 24 hours, followed by protein extraction and Western blotting. Aliquots of control, TGF-ß1-, PACAP-38-, and TGF-ß1 plus PACAP-38-treated cells were analyzed by Western blotting with antibodies against phospho-specific MAP kinase (phosphorylated ERKS) (1:1000) (Promega, Madison, WI) ERK1, ERK2 (1/500 each; Santa Cruz Biotechnology, Santa Cruz, CA), and actin (1:1500; Sigma Chemical Co.). The reaction product was detected by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL), and the density of the bands was quantified by densitometry as previously reported.21,29

p27 Immunocytochemistry

Immunostaining for p27 on HP75 cells was performed as previosly reported, using the avidin-biotin-peroxidase (Vector Kit; Vector, Burlingame, CA) method.36 Monoclonal antibody to p27 (Transduction Laboratory, Lexington, KY) was used at a 1:1000 dilution. The slides were developed with diaminobenzidine chromogen. Positive cells were enumerated by counting a minimum of 500 cells per slide, and the results were expressed as the percentage of cells with nuclear staining.

Statistical Analysis

Each experiment was performed three to four times. Results were expressed as the mean ± SEM. Duncan’s multiple-range test and Student’s t-test were used for statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT-PCR and Southern Hybridization Analysis

Results of RT-PCR and Southern hybridization studies are shown in Figure 1 . Analysis of PACAP mRNA and PVR-1, -2, and -3 mRNAs demonstrated the expected PCR products in the HP75 cell line and hypothalamus. Both hip (303 bp) and hop (387 bp) isoforms of PVR1 were detected in hypothalamus and in HP75 cells. PACAP mRNA was detected in the hypothalamus, which was used as a positive control, but not in the HP75 cells (Figure 1) . PVR-1, -2, and -3 mRNAs were detected in the hypothalamus and in HP75 cells (Figure 1) .



View larger version (53K):
[in this window]
[in a new window]
  		Figure 1. RT-PCR analysis of PACAP (lanes 1–3, 317 bp) and PVR 1 (lanes 4–6). The hip (303 bp) and hop (387 bp) isoforms are present, PVR2 (lanes 7–9, 324 bp) and PVR 3 (lanes 10–12, 584 bp), in HP75 cells (lanes 2, 5, 8, 11, and 14). Human hypothalamus was used as a positive control (lanes 1, 4, 7, 10, and 13). Negative control without reverse transcriptase was used for HP75 cells (lanes 3, 6, 9, and 12). GAPDH was used as a control to test the integrity of the mRNAs for hypothalamus (lane 13) and HP75 cells (lane 14). Top: Ethidium bromide-stained gel. Bottom: Southern hybridization with an internal probe.

 
Apoptosis in the HP75 Cell Line

PACAP and TGF-ß1 Treatment

Apoptotic cells were readily identified by TUNEL staining (Figure 2) . The negative controls without Tdt were consistently negative (Figure 2) . Ultrastructural studies identified apoptotic cells in control dishes, and these apoptotic cells were increased after TGF-ß1 treatment (Figure 3) .



View larger version (74K):
[in this window]
[in a new window]
  		Figure 2. Apoptosis of HP75 cells identified by blue nuclear staining in the TUNEL reaction. A: control cells; B: PACAP-38-treated cells; C: TGF-ß1-treated cells; D: TGF-ß1 + PACAP-38-treated cells; E: negative control without Tdt. Fast red nuclear counterstaining, x200.

 


View larger version (151K):
[in this window]
[in a new window]
  		Figure 3. Ultrastructural analysis of apoptotic cells after TGF-ß1 treatment shows a cell with two apoptotic bodies, x 4000.

 
Control cells and cells treated with PACAP-38 or PACAP-27 had similarly low apoptotic indices (AIs) (Figure 4) . TGF-ß1 treatment resulted in a two- to threefold increase in the AIs. Combined treatment with TGF-ß1 and PACAP-38 or PACAP-27 treatment decreased the AIs to control levels (Figure 4) .



View larger version (34K):
[in this window]
[in a new window]
  		Figure 4. Apoptotic index in HP75 cells after treatment with various chemicals. TGF-ß1 treatment increased apoptotic cells more than twofold above control levels. PACAP-38 or PACAP-27 treatment blocked the TGF-ß1-induced apoptosis. Data are from four experiments with triplicate slides. a: Compared to control cells (P < 0.01). b: Compared to TGF-ß1-treated cells (P < 0.01). CON, control; PA, PACAP.

 
PACAP Antagonist Treatment

To examine the specificity of the PACAP effect we used PACAP antagonists in combination with TGF-ß1 and PACAP (Figure 5) . PACAP 6–38 (PACAP type I antagonist) and (p-chloro-D-Phe,6 Leu17)-VIP (PACAP type II antagonist) partially blocked the effects of PACAP in reducing TGF-ß1-induced apoptosis (Figure 5) .



View larger version (41K):
[in this window]
[in a new window]
  		Figure 5. The specificity of PACAP-38 in inhibiting TGF-ß1-induced apoptosis in HP75 cells was analyzed by using PACAP receptor antagonist. Data are from three experiments with triplicate slides. a: Compared to control cells (P < 0.01). b: Compared to TGF-ß1 + PACAP38-treated cells (P < 0.01). PA6–38, PACAP6–38 (PACAP type I receptor antagonist); p-VIP, (p-chloro-D-Phe6, Leu17)-VIP (PACAP type II receptor antagonist).

 
The PKA agonist forskolin partially inhibited TGF-ß1-induced apoptosis in HP75 cells (Figure 6) , whereas PMA treatment had no effect on TGF-ß1-stimulated apoptosis (Figure 7) .



View larger version (40K):
[in this window]
[in a new window]
  		Figure 6. Examination of TGF-ß1-induced apoptosis in HP75 cells shows inhibition by the PKA agonist forskolin. Data are from three experiments with triplicate slides. a: Compared to control cells (P < 0.01). b: Compared to TGF-ß1-treated cells (P < 0.05). Forsk, forskolin.

 


View larger version (44K):
[in this window]
[in a new window]
  		Figure 7. Analysis of the PKC agonist PMA showed no effect on TGF-ß1-induced apoptosis in HP75 cells. Data were from three experiments with triplicate slides. Significant difference, a: P < 0.01 compared to control cells.

 
MAP Kinase Analysis

Western blot and densitometric analyses for MAP kinase showed that TGF-ß1 induced phosphorylation of ERK1 and ERK2 at levels twofold above control levels after 24 hours of treatment with an antibody specific for phosphorylated ERKs (Figure 8 , A and B). PACAP-38 did not cause any significant changes in phosphorylated ERKs compared to controls after 10, 30, or 60 minutes of treatment.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 8. Western blot analysis of ERKs and activated ERKs in HP75 cells after 24 hours of treatment. A: Lane 1, control; lane 2, TGF-ß1; lane 3, PACAP-38; lane 4, TGF-ß1 and PACAP-38 treatment. Top, phosphorylated ERK1 and ERK2; center, ERK1 and ERK2; bottom: ß-Actin to check for equal loading in top panel. B: Densitometric analysis of the Western blot analysis. There is a twofold increase in phospho-ERK after TGF-ß1 treatment.

 
p27 Protein Expression

The percentage of cells showing positive nuclear staining for p27 in control cells ranged from 30% to 37% in different experiments. After treatment with TGF-ß1, forskolin, and PMA, the percentage of p27-positive cells increased significantly above control levels (Figure 9) . PACAP-38 and PACAP-27 treatments also significantly increased p27 expression in HP75 cells in three separate experiments (control, 25.8 ± 1.9% positive cells; PACAP-38, 35.7 ± 1.3%* positive cells; PACAP 27, 37.5 ± 1.7%* positive cells, with *P < 0.05).



View larger version (47K):
[in this window]
[in a new window]
  		Figure 9. Analysis of p27 expression in HP75 cells after treatment with TGF-ß1, forskolin, and PMA. Data are from four experiments with triplicate slides. **P < 0.01 compared to control cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We studied the expression of PACAP and PVR mRNAs using a human pituitary adenoma cell line, to determine the effects of PACAP and TGF-ß1 on apoptosis in this cell line. Our results showed expression of PVR mRNAs in the HP75 human pituitary adenoma cells, indicating that these cells were capable of responding to PACAP. PVR-1, -2, and -3 mRNAs were detected by RT-PCR and Southern hybridization, but PACAP mRNA was not detected in HP75 cells. Our recent study in normal and neoplastic human pituitaries demonstrated a differential distribution of PACAP-binding sites and PVR-1, -2, and -3 mRNA expression in human pituitary adenomas.10 The highest levels of PVR were found in gonadotroph and null-cell adenomas, indicating the importance of the cAMP regulatory system in these tumors. PRL tumors did not express PVR1 and PVR2 mRNAs.10 This study, using a human pituitary adenoma cell line derived from a nonfunctional (null cell) adenoma, showed findings similar to those of the previous in vivo studies of human gonadotroph and null-cell adenomas.10

PACAP-27 or -38 did not have any direct effects on apoptosis, but TGF-ß1 stimulated apoptosis more than twofold above control levels. These observations agree with other model systems in which TGF-ß induces apoptosis in cultured cells.37-40 To determine whether PACAP could prevent TGF-ß1 apoptosis, we treated cells with PACAP-38 or PACAP-27 and TGF-ß1. Both PACAP-38 and PACAP-27 inhibited TGF-ß1-induced apoptosis in the HP75 cells. Further analysis with the PACAP receptor antagonists PACAP 6–38 (type I receptor antagonist) and (p-chloro-D-Phe,6 Leu17)-VIP (type II receptor antagonist) showed that the effects of PACAP were highly specific in inhibiting TGF-ß1-induced apoptosis.

The second messenger molecule cAMP regulates the activity of PKA or PKA-related enzymes1 and a wide range of cellular processes. cAMP binds to the regulatory subunit of the inactive cytoplasmic PKA complex and releases the catalytic subunit, which can rapidly and reversibly translocate into the nucleus.14 Recent studies reported that PACAP had a role in preventing apoptosis at high PACAP concentrations7-9 while inducing apoptosis at lower concentrations.7 Both forskolin, which is a direct activator of adenylyl cyclase, as well as PACAP, has been shown to protect cerebellar granule neurons from cell death.36-38 These observations in the brain agree with our results in the HP75 pituitary cell line, in which forskolin and PACAP prevented TGF-ß-induced apoptosis.

PMA, which is an activator of the PKC pathway, has been shown to stimulate apoptosis in renal carcinoma cells.41 However, PMA did not influence TGF-ß1-induced apoptosis in the present study. The human pituitary adenoma cell line HP75 has been shown to respond to PMA and to forskolin by increasing chromogranin A expression.29 These findings indicate that both functional PKC and PKA pathways are present in these immortalized cells. Although PACAP activates a dual-signal transduction mechanism in some cultured cells, the antiapoptotic effect is most likely mediated by cAMP stimulation, inasmuch as it was mimicked by forskolin but not by PMA. In recent studies with rat pituitary cells, the PKC inhibitor hypericin stimulated apoptosis in cultured rat pituitary cells, suggesting that PMA has an antiapoptotic effect in the pituitary.42 Because hypericin is not a specific inhibitor of PKC in the concentrations used in this study,42 it is not known if this effect was due to direct inhibition of PKC.

Recent studies have shown that PACAP protects cerebellar granule neurons from apoptosis by activating the MAP kinase pathway.43 In our studies, analysis of active MAP kinase and total MAP kinase proteins showed that TGF-ß1 but not PACAP-38 increased phosphorylation of MAP kinase, suggesting that PACAP did not inhibit apoptosis by activation of MAP kinase in the HP75 pituitary cell line. Villaba et al43 used a model involving primary culture of rat cerebellar granule neurons in which apoptosis was induced by potassium deprivation to show that PACAP-38 induced activation of MAP kinase through a cAMP-dependent pathway. In our model TGF-ß1 stimulated apoptosis, but PACAP-38 did not alter the phosphorylation of MAP kinase. Although pituitary cells and neurons are part of the diffuse neuroendocrine system, the difference between our studies and that of Villaba et al may be due to the fact that they studied primary cultures, compared to an established pituitary cell line in our model. The effects of TGF-ß1 in the activation of MAP kinase agree with earlier reports.44 More experiments are needed to elucidate the mechanisms involved in the PACAP inhibition of TGF-ß-induced apoptosis.

Cyclin-dependent kinases (CDKs) that control G1- and S-phase progression in mammalian cells are regulated by two families of CDK inhibitors, including the Ink4 family and the Cip/Kip family. Various studies have characterized some of the cell cycle-inhibitory proteins regulated by TGF-ß, including p27 and p15.26-29 p27 functions as a negative regulator of G1 cell cycle progression and may mediate TGF-ß1-induced G1 arrest. To determine the effects of PACAP, forskolin, PMA, and TGF-ß on the regulation of p27 expression in pituitary cells, we analyzed p27 expression in HP75 cells after specific treatments. The results showed that treatment with PACAP-38, PACAP-27, forskolin, PMA, and TGF-ß1 stimulated p27 expression in HP75 cells. The relationship between p27 expression and PKA inhibition of apoptosis is still unclear. Recent studies of breast carcinoma cell lines have shown that overexpression of p27 induced apoptosis in these cells, although this effect was not seen in the total cell population and may not occur in cells arrested in G1.45 Because chemicals that stimulated or blocked apoptosis in HP75 cells all led to an increase in immunoreactive p27 in HP75 cells, the effects of p27 on apoptosis in HP75 cells may occur by mechanisms different from that of TGF-ß1-induced apoptosis.

In summary, our studies show that PACAP is a highly specific inhibitor of TGF-ß-1-induced apoptosis in the HP75 pituitary cell line. This cell line can be used as a model to study the regulation of apoptosis in human pituitary cells.


   Acknowledgements
 
The encouragement of Prof. Toru Kameya, M.D., Department of Pathology, and Prof. Kiyotaka Fujii, M.D., Department of Neurosurgery, Kitasato University School of Medicine, in the performance of these studies is greatly appreciated.


   Footnotes
 
Address reprint requests to Dr. Ricardo V. Lloyd, Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, MN 55905. E-mail: lloyd.ricardo{at}mayo.edu

Supported in part by National Institutes of Health grant CA 42951; by a Grant-in-Aid for Scientific Research (07670219 and 08671611) from the Ministry of Education, Science and Culture; and by a Parents’ Association grant from Kitasato University, School of Medicine, Japan.

Accepted for publication August 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Miyata A, Arimura D, Dahl H, Minamino N, Uehara A, Jiang LM, Culler D, Coy DH: Isolation of a novel 38 residue hypothalamic peptide which stimulates adenylate cyclase in pituitary cells. Biochem Biophys Res Commun 1989, 164:567-574[Medline]
  2. Miyata A, Dahl DH, Jiang L, Kitada C, Kubo K, Fujino M, Minamino N, Arimura A: Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP 38). Biochem Biophys Res Commun 1990, 170:643-648[Medline]
  3. Hart GR, Gowing H, Burrin JM: Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats. J Endocrinol 1992, 134:33-41[Abstract]
  4. Goth MI, Lyons CE, Canny BJ, Thoner MO: Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors. Endocrinology 1992, 130:939-944[Abstract]
  5. Tatuno I, Somogyvari-Vigh A, Mizuno K, Gottschall PE, Hidaka H, Arimura A: Neuropeptide regulation of interleukin-6 production from the pituitary stimulated by pituitary adenylate cyclase-activating polypeptide and calcitonin gene-related peptide. Endocrinology 1991, 129:1797-1804[Abstract]
  6. Propato-Mussafiri R, Kanse MM, Ggatel AS, Bloom SR: Pituitary adenylate-cyclase activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3. J Endocrinol 1992, 132:107-113[Abstract]
  7. Arimura A, Somogyvari-Vigh A, Weill C, Fiore RC, Tatsuno I, Bay V, Brenneman DE: PACAP functions as a neurotrophic factor. Ann NY Acad Sci 1994, 739:228-243[Medline]
  8. Cavallaro S, Copani A, D’Agata V, Musco S, Petralia S, Ventra C, Stivala F, Travali S, Canonico PL: Pituitary adenylate cyclase activating polypeptide prevents apoptosis in cultured cerebellar granule neurons. Mol Pharmacol 1996, 50:60-66[Abstract]
  9. Chang JY, Korolev VV, Wang JZ: Cyclic AMP and pituitary adenylate cyclase-activating polypeptide (PACAP) prevent programmed cell death of cultured rat cerebellar granule cells. Neurosci Lett 1996, 206:181-184[Medline]
  10. Oka H, Jin L, Reubi JC, Qian X, Scheithauer BW, Fujii K, Kameya T, Lloyd RV: Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/Vasoactive intestinal polypeptide (VIP) receptor (PVR) expression in human pituitary adenomas. Am J Pathol 1998, 153:1787-1796[Abstract/Free Full Text]
  11. Rawlings SR, Hezareh M: Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior gland. Endocr Rev 1996, 17:4-29[Medline]
  12. Dowd DR, Miesfeld RL: Evidence that glucocorticoid and cyclic AMP-induced apoptotic pathways in lymphocytes share distal events. Mol Cell Biol 1992, 12:3600-3608[Abstract/Free Full Text]
  13. Taylor SS: cAMP-dependent protein kinase. Model for an enzyme family. J Biol Chem 1989, 264:8443-8446[Free Full Text]
  14. Nigg EA, Hilz H, Eppenberger HM, Dutly F: Rapid and reversible translocation of the catalytic subunit of cAMP-dependent protein kinase type II from Golgi complex to the nucleus. EMBO J 1985, 4:2801-2806[Medline]
  15. Alvaro V, Touraine P, Raisman Vozari R, Bai-Grenier F, Birman P, Joubert D: Protein kinase C activity and expression in normal and adenomatous human pituitaries. Int J Cancer 1992, 50:724-730[Medline]
  16. Jin L, Maeda T, Chandler WF, Lloyd RV: Protein kinase C (PKC) activity and PKC messenger RNAs in human pituitary adenomas. Am J Pathol 1993, 142:569-578[Abstract]
  17. Delidow BC, Billis WM, Agarwal P, White BA: Inhibition of prolactin gene transcription by transforming growth factor-ß in GH3 cells. Mol Endocrinol 1991, 5:1716-1722[Abstract]
  18. Burns G, Sarkar DK: Transforming growth factor ß1-like immunoreactivity in the pituitary gland of the rat: effect of estrogen. Endocrinology 1993, 133:1444-1449[Abstract]
  19. Pastoric M, De A, Boyadjieva N, Vale W, Sarkar DK: Reduction in the expression and action of transforming growth factor ß1 on lactotropes during estrogen-induced tumorigenesis in the anterior pituitary. Cancer Res 1995, 55:4892-4898[Abstract/Free Full Text]
  20. Qian X, Jin L, Lloyd RV: Expression and regulation of transforming growth factor ß1 in cultured normal and neoplastic rat pituitary cells. Endocr Pathol 1996, 7:77-90[Medline]
  21. Qian X, Jin L, Grande JP, Lloyd RV: Transforming growth factor-ß and p27 expression in pituitary cells. Endocrinology 1996, 137:3051-3060[Abstract]
  22. Fujiwara K, Ikeda H, Yoshimoto J: Immunohistochemical demonstration of TGF-ß-receptor type II in human pituitary adenomas. Acta Histochem 1995, 97:445-454[Medline]
  23. Jin L, Qian X, Kuling E, Sanno N, Scheithauer BW, Kovacs K, Young WF, Jr, Lloyd RV: Transforming growth factor-ß, transforming growth factor-ß receptor II, and p27kip1 expression in nontumorous and neoplastic human pituitaries. Am J Pathol 1997, 151:509-519[Abstract]
  24. Massague J, Polyak K: Mammalian antiproliferative signals and their targets. Curr Opin Genet Dev 1995, 5:91-96[Medline]
  25. Hunter T, Pines J: Cyclins and cancer II: cyclin D and CDK inhibitors come of age. Cell 1994, 79:573-582[Medline]
  26. Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massague J: Cloning of p27kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 1994, 78:59-66[Medline]
  27. Toyoshima H, Hunter T: p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell 1994, 78:67-74[Medline]
  28. Ravitz MJ, Yan S, Herr KD, Wenner CE: Transforming growth factor ß-induced activation of cyclin E-cdk2 kinase and down-regulation of p27kip1 in C3H10T 1/2 mouse fibroblasts. Cancer Res 1995, 55:1413-1416[Abstract/Free Full Text]
  29. Jin L, Kulig E, Qian X, Scheithauer BW, Eberhardt NL, Lloyd RV: A human pituitary adenoma cell line proliferates and maintains some differentiated functions following expression of SV40 large T antigen. Endocr Pathol 1998, 9:169-184
  30. Hosoya M, Kimura C, Ogi K, Ohkubo S, Miyamoto Y, Kugoh H, Shimizu M, Onda H, Oshima M, Arimura A, Fujino M: Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene. Biochim Biophys Acta 1992, 1129:199-206[Medline]
  31. Ogi K, Miyamoto Y, Masuda Y, Habata Y, Hosoya M, Ohtaki T, Masuo Y, Onda H, Fujino M: Molecular cloning and functional expression of a cDNA encoding a human pituitary adenylate cyclase activating polypeptide receptor. Biochem Biophys Res Commun 1993, 196:1511-1521[Medline]
  32. Sreedharan SP, Huang JX, Cheung MC, Goetzl EJ: Structure, expression, and chromosomal localization of the type I human vasoactive intestinal peptide receptor gene. Proc Natl Acad Sci USA 1995, 92:2939-2943[Abstract/Free Full Text]
  33. Solano RM, Carmena MJ, Carrero I, Cavallaro S, Roman F, Hueso C, Travali S, Lopez-Fraile N, Guijarro LG, Prieto JC: Characterization of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptors in human benign hyperplastic prostate. Endocrinology 1996, 137:2815-2822[Abstract]
  34. Svoboda M, Tastenoy M, Van Rampelbergh J, Grossens JF, DeNeef P, Waelbroeck M, Robberecht P: Molecular cloning and functional characterization of a human VIP receptor from SUP-T1 lymphoblasts. Biochem Biophys Res Commun 1994, 205:1617-1624[Medline]
  35. Chomczynski P, Sacchi N: Single step method of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal Biochem 1987, 162:156-159[Medline]
  36. D’Mello SR, Galli C, Ciotti T, Calissano P: Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad Sci USA 1993, 90:10989-10993[Abstract/Free Full Text]
  37. Galli C, Meucci O, Scorziello A, Werge TM, Calissano P, Schettini G: Apoptosis in cerebellar granule cells is blocked by high KCL, forskolin, and IGF-1 through distinct mechanisms of action: the involvement of intracellular calcium and RNA synthesis. J Neurochem 1995, 54:1619-1625[Medline]
  38. Yan GM, Lin SZ, Irwin RP, Paul SM: Activation of G proteins bidirectionally affects apoptosis of cultured cerebellar granule neurons. J Neurochem 1995, 65:2425-2431[Medline]
  39. Lin J, Chou C: In vitro apoptosis in the human hepatoma cell line induced by transforming growth factor ß1. Cancer Res 1992, 52:385-388[Abstract/Free Full Text]
  40. Khosla S, Oursler MJ, Schroeder MJ, Eberhardt NL: Transforming growth factor-ß1 induces growth inhibition of a human medullary thyroid carcinoma cell line despite an increase in steady state c-myc messenger ribonucleic acid levels. Endocrinology 1994, 135:1887-1893[Abstract]
  41. Woo KR, Shu WP, Kong L, Liu BCS: Tumor necrosis factor mediated apoptosis via Ca++/Mg++ dependent endonuclease with protein kinase C as a possible mechanism for cytokine resistance in human renal carcinoma cells. J Urol 1996, 155:1779-1783[Medline]
  42. Hamilton HB, Hinton DR, Law RE, Gopalakrishna R, Su YZ, Chen Z-H, Weiss MH, Couldwell WT: Inhibition of cellular growth and induction of apoptosis in pituitary adenoma cell lines by the protein kinase C inhibitor hypericin: potential therapeutic application. J Neurosurg 1996, 85:329-334[Medline]
  43. Villalba M, Bockaert J, Journot L: Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. J Neurosci 1997, 17:83-90[Abstract/Free Full Text]
  44. Harsough MT, Mulder KM: Transforming growth factor ß activation of p44mapk in proliferating cultures of epithelial cells. J Biol Chem 1995, 270:7117-7124[Abstract/Free Full Text]
  45. Katyatose Y, Kim M, Rakker ANS, Li Z, Cowan KH, Seth P: Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor with the cyclin-dependent kinase inhibitor p27. Cancer Res 1997, 57:5441-5445[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
L. Grumolato, A. G. Elkahloun, H. Ghzili, D. Alexandre, C. Coulouarn, L. Yon, J.-P. Salier, L. E. Eiden, A. Fournier, H. Vaudry, et al.
Microarray and Suppression Subtractive Hybridization Analyses of Gene Expression in Pheochromocytoma Cells Reveal Pleiotropic Effects of Pituitary Adenylate Cyclase-Activating Polypeptide on Cell Proliferation, Survival, and Adhesion
Endocrinology, June 1, 2003; 144(6): 2368 - 2379.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. Morgan, D. Conklin, A. J. Pawson, R. Sellar, T. R. Ott, and R. P. Millar
A Transcriptionally Active Human Type II Gonadotropin-Releasing Hormone Receptor Gene Homolog Overlaps Two Genes in the Antisense Orientation on Chromosome 1q.12
Endocrinology, February 1, 2003; 144(2): 423 - 436.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol PatholHome page
J. Yamate, M. Maeda, S. J. Benn, J. E. Laithwaite, A. Allan, M. Ide, M. Kuwamura, T. Kotani, S. Sakuma, and J. Lamarre
Differential Effects of Transforming Growth Factor-{beta}1, a Fibrogenic Factor, on Macrophage-Like Cells (HS-P) and Myofibroblastic Cells (MT-9) In Vitro
Toxicol Pathol, June 1, 2001; 29(4): 483 - 491.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oka, H.
Right arrow Articles by Lloyd, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oka, H.
Right arrow Articles by Lloyd, R. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS