| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Endocrinology |
1 Institute of Endocrine Sciences, Ospedale Maggiore, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS); 2 Pathology Unit, Department of Medicine, Surgery and Dentistry, Azienda Ospedaliera San Paolo and Ospedale Maggiore; and 3 Department of Neurosurgery, Ospedale Maggiore IRCCS; University of Milan, Milan, Italy
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
subunit of the stimulatory G protein gene (the so-called gsp oncogene) have been found in 
30 to 40% of growth hormone-secreting pituitary adenomas and in subsets of thyroid, adrenocortical, ovarian, and testicular stromal Leydig cell tumors (1
, 2)
. More recently, genetic defects downstream of cAMP production and affecting PKA complex have been identified in endocrine disorders associated with benign and malignant neoplasia. In mammalian cells there are two types of PKA, PKA1 and PKA2, which share common catalytic subunits but possess different regulatory subunits, R1 and R2 (3) . Through gene cloning, four genes coding for different R isoforms, R1A, R1B, R2A, and R2B, that differ in tissue distribution, subcellular localization and biological properties, have been identified (3) . Dramatic changes in the proportion of R1 and R2 during embryonic development, differentiation processes, and neoplastic transformation indicate distinct roles for these isoenzymes in growth control (4 , 5) . In particular, previous studies supported the view that R1 was related to cell proliferation whereas R2 was primarily involved in tissue differentiation (4, 5, 6) . Accordingly, in a variety of human cancer cell lines, transformation coincides with a sharp increase in R1, whereas R2 overexpression reverts the malignant phenotype into a nontransformed phenotype (7, 8, 9) . However, the involvement of R1 in promoting cell proliferation and transformation has been challenged by the recent identification of R1A gene (PRKAR1A) mutations causing the loss of R1 expression and function in patients with Carney complex, a familial multiple neoplasia syndrome characterized by the association of skin pigmentation, cardiac myxomas and different endocrine tumors, including growth hormone (GH)-secreting pituitary tumors (10 , 11) . On the basis of this evidence, it has been suggested that the impact of unbalanced R1 and R2 expression on cell growth may depend on the cell type (4) .
The aim of this study was to evaluate the relative expression of the different PKA regulatory subunits in pituitary tumors and to examine the effect of their selective activation on proliferation of somatotroph-lineage cells, the pituitary cell type in which cAMP promotes proliferation.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
PRKAR1A Sequencing Analysis.
Genomic DNA was extracted with the phenol-chloroform method from adenomatous tissues (Nucleon-Amersham Life Science Europe, Milan, Italy). The 12 exons and flanking intronic sequences of the PRKAR1A gene (GenBank accession no. NM 002734) were amplified by polymerase chain reaction (PCR; primers and amplification conditions available on request). Direct sequencing of the amplified fragments was then performed with the AmpliTaq BigDye Terminator kit and 310 Genetic Analyzer (Perkin-Elmer Corp., Applied Biosystems, Foster City, CA). G protein stimulatory subunit (Gs) analysis was performed in GH-omas, as described previously (12)
.
Real-time Reverse Transcription-PCR.
Total RNA was isolated from tissue specimens with a commercial kit, Trizol (Invitrogen S.R.L., Milan, Italy) according to the manufacturer’s instructions and 200 ng RNA was reverse transcribed (Applied Biosystems). PKA R1A, PKA R2A, and PKA R2B mRNA levels in pituitary adenomas were evaluated by real-time quantitative reverse transcription-PCR based on TaqMan methodology, with the ABI Prism 7700 Sequence Detection System (Applied Biosystems).
PKA R1A, PKA R2A and PKA R2B mRNA expression were determined applying the 
Ct method, as described previously (13)
. We identified a calibrator sample that represents the unitary amount of the target of interest. The other samples express n-fold mRNA relative to the calibrator. As calibrator, we used one of the pituitary adenomas of the series under study. To normalize the amount of total RNA added to each reaction mixture, we quantified as internal RNA control the ß-actin (ACTB) mRNA. Final amounts of target were determined as follows: target amount = 2–Ct, where Ct = [Ct (PKAr) – Ct (ACTB)]sample – [Ct (PKAr) – Ct (ACTB)]calibrator.
The primers and probe nucleotide sequence for ß-actin mRNA were previously reported (13) . For the quantification of PKA R1A, PKA R2A, and PKA R2B mRNA, we used a ready-to-use assay (identification numbers Hs00267597_m1, Hs00177760_m1, and Hs00176966_m1, respectively; Assay-on-Demand Gene Expression Products, Applied Biosystems), according to the manufacturer’s instructions. All of the reactions were performed in duplicate. The thermal cycling conditions included 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. All of the reagents used for PCR were from Applied Biosystems.
Immunohistochemistry.
Sections from paraffin-embedded tissues from 30 surgically removed pituitary adenomas were processed for immunohistochemistry, as reported previously (14)
. Specific monoclonal antibodies for PKA R1A, PKA R2A, and PKA R2B were used under the conditions specified by the manufacturer (BD Transduction Laboratories, Lexington, United Kingdom). Antigen-antibody detection was performed with the DAKO ChemMate En Vision detection kit (DAKO A/S, Glostrup, Denmark) according to the manufacturer’s instructions. Sections were stained with 3,3'-diaminobenzidine substrate and counterstained with Meyer hematoxylin; slides were prepared for light microscopy examination, as reported previously (14)
. As positive controls, normal human adrenal tissues were used. Negative controls were obtained by occulting the primary antibody or by using an unrelated mouse monoclonal antibody. At least two blinded readers graded the specimens for all stainings. Briefly, PKA R1A, PKA R2A, and PKA R2B immunoreactivities were graded 0 to 3, with 0 = absence of immunoreactivity, 1 = <10%, 2 = 10 to 50%, and 3 = >50% in at least 400 cells in the main representative high power field.
PKA R1A Degradation.
To determine the degradation pathway of PKA R1A, we incubated GH3 cells and cells obtained by enzymatic digestion from 4 GH-omas with 10 µmol/L lactacystin or 100 µmol/L chloroquine (Sigma-Aldrich, Milan, Italy) for 20 hours at 37°C. Incubation was stopped by placing the cells on ice; the medium was removed, and cells were treated with 500 µL ice-cold lysis buffer in the presence of protease and phosphatase inhibitors. Western blot analysis of PKA R1A was performed with the same monoclonal antibody used for immunohistochemistry, detected by chemiluminescent method and the resulting bands evaluated by imaging densitometer, as previously reported (12)
. Experiments were repeated at least twice.
Protein Kinase A Activity.
PKA activity was measured with a nonradioactive PKA kinase activity assay kit (Stressgen, Victoria, BC, Canada) in 2 µg of cell extracts from six GH-secreting tumors and from GH3 cells. The assay is based on a solid phase ELISA that uses a specific synthetic peptide as a substrate for PKA (kempeptide) and a polyclonal antibody that recognizes the phosphorylated form of the substrate. PKA activity reflects the enzymatic activity after stimulation with 5 µmol/L cAMP, free PKA activity represents basal activity, in the absence of cAMP stimulation, and total PKA activity is calculated as the difference between cAMP-stimulated PKA and the PKA inhibited by the protein kinase inhibitor PKI (5 µmol/L).
Cell Proliferation.
Cell proliferation was assessed by colorimetric measurement of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation during DNA synthesis in proliferating cells (Cell Proliferation Biotrak Elisa, Amersham, Piscataway, NJ). Briefly, cells were cultured in 96-well plate (20,000 cells per well) in the presence of test substances (5, 10, 100 µmol/L 8-Cl cAMP or 100 µmol/L 8-Br cAMP alone or in combination with 5 µmol/L PKA inhibitor PKI, Sigma-Aldrich, Milan, Italy) for 72 hours at 37°C and then with BrdUrd for 2 hours to allow BrdUrd incorporation in newly synthesized cellular DNA. Proliferation was expressed as relative fluorescence units (RFU). All of the experiments were repeated at least 3 times on 2 different GH3 clones and on 4 GH-secreting adenomas, and each determination was done in quintuple.
Cyclin D1 Expression.
After 24 hours of serum starvation, cells obtained by enzymatic digestion from four GH-omas were incubated with different agents (100 µmol/L 8-Cl cAMP or 100 µmol/L 8-Br cAMP, alone or in combination with 5 µmol/L PKA inhibitor PKI) for 8 hours at 37°C. The determination of Cyclin D1 was performed after immunoprecipitation of cell lysates with a specific monoclonal antibody (Novocastra, Newcastle, United Kingdom) and Western blotting, as reported previously (12)
. The same experiment was repeated in GH3 cells transfected with siRNA. Experiments were repeated at least twice.
Synthesis and Transfection of Small Interfering RNA.
Small interfering RNA (siRNA) for rat R1A gene was synthesized by Ambion, and GH3 cells were transfected with the double-stranded RNA with amine transfection reagent according to the manufacturer’s instructions (Ambion, Austin, TX). Cells were exposed to double-stranded RNA and transfection reagent for 96 hours before performing Western blot analysis with the specific R1A antibody. Corresponding scrambled siRNA for the same regulatory subunit and siRNA for GAPDH were used as internal negative and positive controls, respectively.
Statistical Analysis.
The results are expressed as the mean ± SD. A paired or unpaired two-tailed Student’s t test was used to detect the significance between two series of data. P < 0.05 was accepted as statistically significant
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Real-time PCR.
PKA R1A, R2A, and R2B mRNA expression levels were evaluated by real-time PCR in 18 of 30 pituitary adenomas included in the study. The mean Ct value of the internal control (ACTB) was 20.08 ± 0.79 (range 18.6–21.3) documenting the appropriate quality of RNA. PKA R1A, R2A, and R2B mRNA expression was detected in all of the samples examined. Although variable, PKA R1A mRNA levels were significantly higher than those of R2A and R2B mRNA (4.7 ± 2 versus 2.2 ± 1.2 and 2.3 ± 0.7, respectively; P < 0.005; Fig. 1
). Accordingly, in almost all tumors analyzed, the R1A/R2B ratio was higher than 1 (2.2 ± 1.0). Finally, there was no difference in the levels of expression of the three subunits between GH-omas expressing or not the gsp oncogene (data not shown). Similarly, no correlation with clinical parameters such as age of the patient, size of the tumor, aggressiveness, hormone secretion, and responsiveness to medical treatment was observed (data not shown).
|
. Conversely, all tumors showed a strong positivity for both R2A and R2B subunits, which were detected in >50% of the total cell population in most cases. The R2B staining was exclusively cytoplasmic, whereas immunopositivity for R2A was also localized to perinuclear dots (Fig. 2)
. The same pattern of staining, showing barely detectable R1A and high levels of R2A and R2B, was observed in GH3 cells (data not shown).
|
as well as in GH3 cells (data not shown). By contrast, no effect on R1A protein levels was induced by the lysosomal inhibitor chloroquine (100 µmol/L for 20 hours).
|
. This response was totally abrogated by the addition of the PKA inhibitor PKI (Fig. 4)
, resulting in a high total PKA activity in both GH-omas and GH3 cells.
|
, the maximum effect being observed at 100 µmol/L (362 ± 42% over basal levels). Coincubation with the PKA inhibitor PKI (5 µmol/L) resulted in a significant reduction of this effect (Fig. 5)
. Conversely, a reproducible but nonsignificant increase (58 ± 22% over basal levels) of cell proliferation was induced by 8-Br cAMP only at 100 µmol/L (Fig. 5)
. The same experiments performed on primary cultures from four GH-omas showed a slight increase of cell proliferation induced by 8-Cl cAMP, that did not reach the statistical significance, probably because of the low and variable proliferation rate that characterized these neoplasia (Fig. 5)
.
|
), whereas a moderate response to 8-Br cAMP was observed in one (30% versus basal). The elevation of Cyclin D1 levels induced by 8-Cl cAMP was significantly reduced by coincubation with PKA inhibitor PKI (Fig. 6)
. Similar results were observed in GH3 cells (data not shown).
|
.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This study showed that, consistent with the absence of genetic defects, mRNA of the three subunits was detected in all of the pituitary tumors, R1A being the most represented regulatory subunit in the majority of pituitary samples, independently from the tumor type. R1A, R2A, and R2B mRNA levels were not affected by the presence of the gsp oncogene in GH-omas, suggesting that contraregulatory mechanisms associated with these mutations, such as cAMP degradation due to phosphodiesterase overactivity, prevented the up-regulation of PKA subunits, particularly R2, expression (21) .
In contrast to the pattern of R1A mRNA expression, pituitary tumors were characterized by a dramatic reduction or complete loss of R1A at the protein level, which was clearly detectable in normal adrenal tissue used as positive control. Therefore, because R2A and R2B were expressed at high levels, almost all of the tumors included in the study displayed a strong prevalence of R2 over R1 protein. The discrepancy between R1A mRNA and protein expression suggested the existence of a high rate of protein degradation in pituitary tumors. Indeed, the incubation of primary cell cultures as well as GH3 cells with the potent proteasome inhibitor lactacystin induced a striking elevation of R1A protein. This finding is in accordance with the notion that the physiologic degradation pathway of PKA R1A is proteasome-mediated (22) . Moreover, although R1A is particularly susceptible to degradation when dissociated from the catalytic subunit, a persistent R1A dissociation as cause of low R1A expression seems unlikely, when the cAMP-inducible PKA activity recorded in all pituitary tumors examined is taken into account. However, in this study, it was not possible to ascertain whether the low expression of R1A protein was a tumor-related event. In fact, the precise assessment of R1A expression and degradation as well as PKA activity was unfeasible in the normal autoptic pituitary gland, because this subunit is a soluble cytoplasmic enzyme with a rapid turnover.
In this study, we asked what was the impact of unbalanced R1 and R2 expression on the proliferation of cells of the somatotroph lineage. It is well known that cAMP, through the activation of PKA, may exert different actions on cell proliferation. According to the cell type, it may have no influence, induce cell arrest, or even suppress mitogenic action of growth factors in some cell lines, or, conversely, promote the transition from G0 to G1 phase and stimulate cell growth in others (21) . Evidence from in vitro studies and naturally occurring human diseases (gsp+ GH-secreting adenomas, and carney complex) indicate that somatotrophs belong to the set of cells that recognizes cAMP as a growth factor (21 , 23) . However, no information on the relative expression of PKA R1 and R2 in normal and tumoral somatotrophs is thus far available, although it is known that these isoenzymes are differentially expressed in a variety of human cancers and exert distinct roles in differentiation processes and neoplastic transformation (4 , 6 , 7) . In particular, in contrast to the recent observation that loss of PKRAR1A was associated with multiple neoplasia, previous studies carried out on human cancers and cell lines, such as breast, colon and lung carcinomas, fibrosarcomas, gliomas, and leukemias, indicated that R1 was the major or the sole R subunit detectable and was primarily involved in cell proliferation (6 , 7 , 24, 25, 26) . Conversely, R2 would be related to tissue differentiation and the induction of R2 subunits by 8-Cl-cAMP treatment in malignant cell lines was associated with growth arrest and reversion of the transformed phenotype (5 , 8 , 9 , 27) .
These considerations prompted us to examine the proliferative role of the two different PKA regulatory subunits in transformed somatotrophs, by using two cell models that displayed a strong prevalence of R2 over R1 protein, i.e., the somatomammotroph GH3 cell line and human GH-omas. Increasing concentrations of 8-Cl cAMP, a cAMP analog able to selectively activate R2 subunits, induced a strong proliferation of GH3 and increased the expression of Cyclin D1, a key regulator of G1 phase progression, in GH-omas (28) , both parameters being marginally modified by the activator of all subunits. The effect of changes in the R1/R2 ratio on GH3 cells proliferation was further investigated by silencing R1A RNA expression. In particular, the decrease in the R1/R2 ratio obtained by this manipulation induced a dramatic increase in Cyclin D1 expression, thus confirming the R2-mediated stimulatory effect of 8-Cl cAMP on cell proliferation. Taken together, these data are consistent with previous observations indicating that cAMP-dependent pathway may activate proliferative signals in somatotrophs and provide evidence for a crucial role of the prevalent expression of R2 over R1 protein in this process (21) . The observation that a low R1/R2 ratio promoted cell proliferation in the tumoral pituitary is consistent with the Carney complex model in which inactivating mutations of the R1A subunit further unbalance this ratio in favor of R2 subunits (10 , 11) .
Finally, the unbalanced R1/R2 expression was associated with well-differentiated pituitary neoplasia. This is consistent with the presence of PRKA1A mutations in adrenocortical adenomas and their absence in adrenocortical cancers (16) , but it is the opposite of what is observed in thyroid tumors in which PRKA1A mutations are associated with the malignant phenotype (17) . This suggests that with regard to the role of PKA, diverse tumorigenic processes exist in endocrine cells of different origin.
In conclusion, the results presented here demonstrate that tumoral pituitary cells are characterized by low or absent expression of the PKA R1A subunit protein. Defective expression of R1A at the protein level was not due to reduced transcription but was associated with proteasome-dependent protein degradation. Analysis of the proliferative response to cAMP analogs suggests that, in analogy with the proliferative phenotype resulting from loss-of-function PRKAR1A mutations in the Carney complex, unbalanced expression of the R1 and R2 PKA subunits underlies cAMP-dependent proliferation of somatotroph cells. How R1A or R2 excess insufficiency lead to tumorigenesis, however, remains unclear.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: A. Lania and G. Mantovani contributed equally to this work and should both be considered first authors.
Requests for reprints: Anna Spada, Institute of Endocrine Sciences, Ospedale Maggiore IRCCS-Pad. Granelli, Via F. Sforza, 35, 20122 Milan, Italy. Phone: 39-02-50320613; Fax: 39-02-50320605; E-mail: anna.spada{at}unimi.it
Received 5/26/04. Revised 9/15/04. Accepted 10/12/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C Vincent-Dejean, L Cazabat, L Groussin, K Perlemoine, G Fumey, F Tissier, X Bertagna, and J Bertherat Identification of a clinically homogenous subgroup of benign cortisol-secreting adrenocortical tumors characterized by alterations of the protein kinase A (PKA) subunits and high PKA activity. Eur. J. Endocrinol., June 1, 2008; 158(6): 829 - 839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Bondioni, A. R. Angioni, S. Corbetta, M. Locatelli, S. Ferrero, E. Ferrante, G. Mantovani, L. Olgiati, P. Beck-Peccoz, A. Spada, et al. Effect of 9-cis Retinoic Acid on Dopamine D2 Receptor Expression in Pituitary Adenoma Cells Experimental Biology and Medicine, April 1, 2008; 233(4): 439 - 446. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Yin, L. Williams-Simons, A. F. Parlow, S. Asa, and L. S. Kirschner Pituitary-Specific Knockout of the Carney Complex Gene Prkar1a Leads to Pituitary Tumorigenesis Mol. Endocrinol., February 1, 2008; 22(2): 380 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Calebiro, T. de Filippis, S. Lucchi, F. Martinez, P. Porazzi, R. Trivellato, M. Locati, P. Beck-Peccoz, and L. Persani Selective Modulation of Protein Kinase A I and II Reveals Distinct Roles in Thyroid Cell Gene Expression and Growth Mol. Endocrinol., December 1, 2006; 20(12): 3196 - 3211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Mantovani, S. Bondioni, S. Ferrero, B. Gamba, E. Ferrante, E. Peverelli, S. Corbetta, M. Locatelli, P. Rampini, P. Beck-Peccoz, et al. Effect of Cyclic Adenosine 3',5'-Monophosphate/Protein Kinase A Pathway on Markers of Cell Proliferation in Nonfunctioning Pituitary Adenomas J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6721 - 6724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Nadella and L. S. Kirschner Disruption of Protein Kinase A Regulation Causes Immortalization and Dysregulation of D-Type Cyclins Cancer Res., November 15, 2005; 65(22): 10307 - 10315. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
, P < 0.05 versus PKA activity.
