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Selective Modulation of Protein Kinase A I and II Reveals Distinct Roles in Thyroid Cell Gene Expression and Growth

Department of Medical Sciences (D.C., P.B.-P., L.P.) and Institute of General Pathology (F.M., M.L.), University of Milan, 20100 Milan, Italy; Laboratory of Experimental Endocrinology (D.C., T.d.F., S.L. P.P., R.T., L.P.), Istituto Auxologico Italiano Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), 20095 Cusano Milanino, Italy; Istituto Clinico Humanitas IRCCS (F.M., M.L.), 20089 Rozzano, Italy; and Fondazione Ospedale Maggiore Policlinico IRCCS (P.B.-P.), 20122 Milan, Italy

Address all correspondence and requests for reprints to: Luca Persani, M.D., Ph.D., Laboratory of Experimental Endocrinology, Istituto Auxologico Italiano IRCCS, Via Zucchi 18, 20095 Cusano Milanino (MI), Italy. E-mail: luca.persani{at}unimi.it.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
A global gene expression profiling of TSH stimulation on differentiated (FRTL5) and partially dedifferentiated [FRT/TSHR (TSH receptor)] rat thyroid cells was performed. A total of 123 TSH-regulated genes (95 newly described) were identified in FRTL5, whereas no significant transcriptional modifications were seen in FRT/TSHR cells. Because regulatory subunit IIß (RIIß) of protein kinase A (PKA), a key element downstream of cAMP, was expressed in FRTL5 but not in cAMP-refractory FRT/TSHR cells, we hypothesized that this gene may play an important role in TSH signaling. We therefore performed a series of experiments to investigate the involvement of RIIß and the different PKA isoforms. A positive effect of PKA II- but not of PKA I-selective activation on gene transcription and proliferation in FRTL5 cells, as well as an impairment of TSH nuclear effects after RIIß silencing were observed, suggesting that PKA II plays an essential role in TSH signaling. This view was supported by the restoration of TSH nuclear effects after reexpression of RIIß in FRT/TSHR cells. Because PKA I stimulation could increase iodide uptake in FRTL5 cells without affecting gene transcription, PKA I may mediate TSH actions at posttranscriptional levels. Analyses on three human cancer cell lines confirmed the possible loss of RIIß expression and antiproliferative activity of PKA I-selective cAMP analogs (60% at 200 µM in BRAF-mutated cells). The inhibitory effect of PKA I apparently required constitutive MAPK activation and was associated with an inhibition of ERK phosphorylation. These findings may open new therapeutic perspectives in patients with thyroid cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
TSH IS THE key modulator of thyroid cell growth, function, and differentiation (1, 2, 3). Binding of TSH to its receptor (TSHR) results in an increase of cAMP intracellular concentration, which mediates most of TSH effects. In nonendocrine cells, cAMP increases are generally associated with differentiation and growth inhibition (4, 5, 6). Conversely, in thyrocytes and other neuroendocrine cells, such as somatotrophs, Sertoli, and adrenocortical cells, hormonal activation of the cAMP pathway can induce both mitosis and function (5, 6, 7, 8). Numerous studies have investigated cAMP signaling in the thyroid, but the transcriptional and posttranscriptional events linking cAMP increases to proliferation and differentiation are only partially characterized. Indeed, a comprehensive description of these molecular events retains particular importance, given the frequent involvement of cAMP pathway in a plethora of human pathologies, including thyroid diseases (9).

The major intracellular signaling event triggered by cAMP is the activation of protein kinase A (PKA). PKA is a tetramer composed of two catalytic (C) and two regulatory (R) subunits. Upon cAMP binding to R subunits, active C subunits are released. In mammalian cells, two major types of PKA (PKA I and II) are present, which differ upon the presence of a given regulatory subunit (RI or RII). In addition, four different isoforms, two of RI (RI and RIß) and two of RII (RII and RIIß), each encoded by a separate gene (PRKAR1A, PRKAR1B, PRKAR2A, and PRKAR2B), have been identified. All of these PKA isoforms are characterized by different subcellular localization and tissue distribution (10). The observation that the relative abundance of RI and RII varies considerably during embryonic development, cell differentiation, and neoplastic transformation led us to hypothesize that PKA I and II may have a distinct biological function. Indeed, several studies in nonendocrine cells support a model in which PKA I stimulates cell proliferation, whereas PKA II promotes differentiation (4, 8). However, this model is compromised by the finding that inactivating PRKAR1A mutations are responsible for Carney complex, a familial multiple neoplasia syndrome characterized by skin pigmentation, cardiac myxomas, and tumors of several endocrine glands, including the thyroid (11). Based on this finding, PRKAR1A should be rather regarded as a tumor-suppressor gene, at least in endocrine cells (8, 11, 12). Consistent with this view is the recent finding by our group that a low RI to RII ratio promotes proliferation of transformed somatotrophs (13).

Indeed, the role of PKA isoenzymes in thyroid cells is controversial. Both PKA isoforms were reported to mediate TSH effects on dog thyroid cell function (i.e. iodide uptake and thyroid hormone production), whereas PKA I was reported to be more effective on cell proliferation (14). A major role for PKA I in mediating TSH proliferative effects was later on supported by results of experiments conducted with antisense oligonucleotide against Prkar1a on a Prkar2b-negative clone of a rat thyroid cell line (FRTL5 cells) (15). However, this view is challenged by more recent findings of the same group. Overexpression of v-Ki-Ras oncogene in FRTL5 cells was found to cause RIIß delocalization and eventually loss of its expression. This was accompanied by reduced nuclear localization of the catalytic subunit and down-regulation of cAMP-dependent genes (16). In addition, mouse fibroblasts stably expressing TSHR, and endowed with endogenous expression of RI but not RIIß, were reported to acquire TSH-dependent growth only after transfection of RIIß (6). These recent findings appear to be in contrast with the previous ones, pointing to a major role of PKA II in TSH signaling.

This study was aimed at probing further into the signaling pathways and transcriptional events that link cAMP to thyrocyte proliferation and function and to provide a description of the molecular events associated with thyroid cell dedifferentiation. To this purpose we performed a comparative microarray analysis of the transcription profiles of two rat thyroid cell lines (FRTL5 and FRT/TSHR) stimulated with TSH. FRTL5 cells are a widely used model of well-differentiated cells that require TSH for growth and retain many of the functional properties of thyrocytes. FRT/TSHR cells were generated in our laboratory by reexpressing TSHR in a TSH-independent and dedifferentiated clone of FRTL5 (FRT) and were used as a model of dedifferentiated thyroid cells. These two cell lines share a common genetic background, which make them particularly suited for comparative microarray analysis. Differences in the expression profiles of PKA regulatory subunits in FRTL5 and FRT/TSHR cells prompted us to further investigate the role of PKA I and II in normal and dedifferentiated thyroid cells of rat and human origin.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Microarray Analysis of FRTL5 and FRT/TSHR Cells
A time course analysis of TSH stimulation in two rat thyroid cell lines (FRTL5 and FRT/TSHR) was performed. The expression levels of 8784 transcripts, representing about 30% of rat transcriptome, were measured at three different time points, i.e. before (0), 30 min (30m) and 17 h (17h) after TSH stimulation, by means of Affymetrix RG-U34A arrays. A statistical algorithm [Significance Analysis of Microarrays (SAM)] was applied to identify differentially expressed genes. SAM provides an estimate of the false discovery rate (FDR), i.e. the percentage of gene expression variations identified by chance, which is a relevant issue when performing thousands of statistical tests as in the analysis of microarray experiments. Estimated FDRs were between 1 and 2% in all comparisons. A total of 216 transcripts varied their expression levels after TSH treatment in FRTL5 cells. Despite similar cAMP increases after TSH stimulation in both cell lines at the conditions used for microarray experiments (927 ± 62% and 645 ± 57% over baseline, in FRTL5 and FRT/TSHR, respectively), no significant variations resulted from SAM analysis in FRT/TSHR cells. In some cases, more than one probe set was present for the same gene. Not considering these redundant probes and expressed sequence tags, 123 unique genes were regulated by TSH in FRTL5 cells. Of these, 95 genes had not been previously described to be regulated by TSH at the transcriptional level in different thyroid cellular models (for further details see supplemental Table I published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). Full gene expression profiling data can be found at the Gene Expression Omnibus web site (GEO: http://www.ncbi.nlm.nih.gov/geo/, accession no. GSE5115). Figure 1 shows the expression profiles in FRT/TSHR and FRTL5 cells of genes belonging to five different functional categories. Early genes are reported in panel A. With the exception of Myc, these genes appear to have similar basal expression levels in FRT/TSHR and FRTL5, but are induced at 30m only in FRTL5 cells. Panel B refers to genes involved in cell cycle/apoptosis. Cyclin E (Ccne) and the cyclin-dependent kinase 1 (cell division cycle 2 homolog A, Cdc2a), two key positive regulators of cell cycle, have higher expression levels in FRT/TSHR compared with FRTL5 cells. TSH addition has no effect on the expression levels of these and the other cell cycle genes in FRT/TSHR, but leads to their transcriptional modification in FRTL5 cells. In particular, Ccne and Cdc2a are induced at 17 h. By contrast, B cell translocation gene 1 (Btg1) and growth arrest and DNA damage-inducible 45 (Gadd45a), two antiproliferative genes, are down-regulated at 17 h. Other genes, myeloid differentiation primary response gene 116 (Myd116), immediate early response 3 (Ier3), and B cell translocation gene 2 (Btg2) are transiently induced at 30m, but return to basal levels at 17 h. Panel C shows the expression profiles of genes involved in DNA replication. These genes have higher basal expression levels in FRT/TSHR and are induced at 17 h only in FRTL5 cells. Genes involved in cholesterol biosynthesis, which is also required for thyroid cell proliferation (17, 18), have expression profiles similar to those of DNA replication genes (panel D). These data reveal that in FRTL5 cells, TSH addition induces the sequential transcription of early genes and genes that are required for progression through the cell cycle and DNA replication. The finding that genes involved in DNA replication and cholesterol biosynthesis are expressed at higher levels in FRT/TSHR cells at basal conditions may reflect the higher proliferative rate and TSH-independent growth that characterizes this partially dedifferentiated cell line. Well-characterized TSH-regulated genes were included among the 28 previously described ones. The expression profiles of three of them, the sodium-iodide cotransporter (Slc5a5, alias NIS) (19), phosphodiesterase 4b (Pde4b) (20), and heat-shock protein a5 (Hspa5, alias Bip) (21) are reported in panel E. Slc5a5/NIS, an essential element in the transport of iodide, which is required for thyroid hormone synthesis, is expressed at high levels only in FRTL5 cells, where it is induced by TSH at 17 h. Similarly, thyroid peroxidase (Tpo) and thyroglobulin (Tg), two other well-known genes involved in thyroid-differentiated function, were expressed at high levels and induced by TSH only in FRTL5 cells, but with a fold-change below the threshold of 1.8. These gene expression variations are somewhat smaller than found with other more quantitative methods (Northern blot, real-time PCR), probably reflecting the general tendency of microarrays to underestimate fold-changes (22). The lack of expression and of regulation by TSH of Slc5a5/NIS, Tpo, and Tg in FRT/TSHR cells suggest that the reintroduction of TSHR is not sufficient to restore the expression and regulation of genes involved in thyroid differentiated function. The expression variations of 16 genes resulting from the microarray analysis were validated by real-time PCR, with a confirmation rate of 100% (Fig. 2).

View larger version (69K): [in this window] [in a new window]   Fig. 1. Expression Plot of a Selection of TSH-Regulated Genes in FRTL5 Cells Early genes (A) as well as genes involved in cell cycle/apoptosis (B), DNA replication (C), cholesterol metabolism (D), and exemplary well-characterized TSH-regulated genes (E) are reported. Unsupervised hierarchical clustering analysis was performed within each group. Each row represents one probe set corresponding to one gene. Different colors in the rectangles represent different levels of expression after per chip and per gene normalization (see color bar at bottom).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Validation by Real-Time PCR of Transcription Profiles in FRTL5 Cells The modulation of the expression levels of a subset of genes was evaluated by real-time PCR after stimulation with bTSH (0.1 U/liter). A, Genes regulated at 30 min. B, Genes up-regulated at 17 h. C, Genes down-regulated at 17 h. mRNA levels are expressed relative to basal condition. *, Statistically significant (P < 0.05) vs. basal condition. GHR, GH receptor.

Expression of PKA-Regulatory Subunits and Effect of PKA I or II Modulation on Growth and Function of FRTL5 and FRT/TSHR Cells
The expression of PKA-regulatory subunits in FRTL5 and FRT/TSHR cells was evaluated by real-time PCR (Fig. 3A) and Western blot (Fig. 3B). Prkar1a and Prkar2a were expressed in FRTL5 and FRT/TSHR cells at both mRNA and protein level. Prkar1b mRNA levels were low in both cell lines (data not shown). Prkar2b was clearly expressed in FRTL5 cells, whereas it was undetectable both at mRNA and protein levels in FRT/TSHR cells, confirming the data of the microarray experiment. Based on this finding and on the lack of significant gene expression modifications after TSH stimulation in FRT/TSHR cells, we hypothesized that Prkar2b could be required for the propagation of cAMP signal in thyroid cells. Therefore, we performed a series of experiments aimed at elucidating the involvement of the different PKA isoforms in TSH signaling.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Expression of PKA-Regulatory Subunits in FRTL5 and FRT/TSHR Cells A, Real-time PCR. y-Axis shows mRNA expression levels relative to FRTL5 cells. B, Western blot analysis. ß-Actin was used as control.

View larger version (42K): [in this window] [in a new window]   Fig. 4. PKA I or II Activation in FRTL5 and FRT/TSHR Cells A and B, Effect of cAMP analog pair selective for PKA I (PKA I) or PKA II (PKA II) on the growth of FRTL5 and FRT/TSHR cells, respectively. y-Axis shows the relative growth as percent of control (cells treated with DMSO), measured 72 h after stimulation. *, P < 0.03 difference between PKA I and PKA II. C, Effect of PKA I or II inhibition on TSH- or PKA II-dependent FRTL5 cell growth. Cells were preincubated with the indicated concentrations of PKA inhibitors for 30 min before addition of bTSH or PKAII-selective analogs. y-Axis shows the relative growth as percent of control (cells treated with DMSO), measured 72 h after stimulation. , P < 0.02 vs. bTSH (0.1 U/liter). , P < 0.02 vs. PKA II (100 µM). D, Effect of PKA I or II activation on iodide uptake in FRTL5 cells. Cells were incubated in the presence of selective cAMP analogs for 48 h. y-Axis indicates iodide uptake as percent of control (cells treated with DMSO). *, P < 0.04 vs. control. E, Effect of PKA I- or II-selective analogs on PKA activation. FRTL5 cells were stimulated with bTSH-, PKAI-, or PKAII-selective analogs for 15 min. y-Axis reports PKA activation, expressed as ratio between the activity of free catalytic subunit (C-PKA) and total PKA activity. *, P < 0.02 vs. control (cells treated with DMSO). Experiments on FRTL5 cells were performed after TSH starvation for 5 d. Statistical analysis were performed by two-way (A and B) or one-way (C, D, and E) ANOVA, followed by Bonferroni’s post hoc test. inhib., Inhibition.

We then evaluated the effect of PKA I or II activation on the expression level of a subset of TSH-regulated genes resulting from our microarray analysis. These included early genes, such as early growth response 1 (Egr1), nuclear receptor 4A1 (Nr4a1), 4A2 (Nr4a2), and 4A3 (Nr4a3), genes involved in cell cycle, such as Ccne and Cdc2a, DNA replication, such as proliferating cell nuclear antigen (Pcna), DNA primase (Prim1), and DNA topoisomerase 2 (Top2a), and well-characterized genes required for thyrocyte differentiated function, such as Slc5a5/NIS and Hspa5. In addition, we checked the expression levels of a short Tg transcript (Tg2) and of Tpo, which were induced by TSH in the microarray experiment, but with a fold-change below the cutoff value of 1.8. An induction of all these genes, similar to that obtained with TSH stimulation, was observed only after treatment with PKA II-selective agonists (Fig. 5). We also evaluated the mRNA levels of thyroid transcription factor 1 (Titf1), forkhead box E1 (Foxe1), and Pax8, three thyroid-specific transcription factors involved in thyroid differentiation. In our conditions, TSH and PKA I- and PKA II-selective analogs failed to cause significant modifications of the expression levels of these genes (data not shown).

View larger version (51K): [in this window] [in a new window]   Fig. 5. Effect of PKA I or II Activation on Gene Expression in FRTL5 Cells FRTL5 cells were cultured in the absence of TSH for 5 d and treated for 30 min (A) or 17 h (B) with bTSH or equimolar concentrations of cAMP analog pairs selective for PKA I (PKA I) or PKA II (PKA II). The expression levels of a subset of early genes (A) as well as of proliferative and differentiative genes (B) was evaluated by real-time PCR. y-Axis shows mRNA expression levels relative to FRTL5 cells treated with DMSO (control). *, P < 0.04 vs. control.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Effect of PKA I or II Activation on CREB Phosphorylation in FRTL5 Cells FRTL5 cells were cultured in the absence of TSH for 5 d before stimulation. Reported are the images of a representative Western blot with an antibody specific for CREB phosphorylated at Ser133 (P-CREB) or total CREB and the densitometric analysis of three independent experiments. A, Cells treated with 100 µM of cAMP analog pairs selective for PKA I (PKA I) or PKA II (PKA II). B, Cells treated with bTSH alone (0.1 U/liter) or together with a PKA II inhibitor (0.25 mM), added 30 min before stimulation. *, P < 0.03 vs. basal conditions (time = 0). , P < 0.01 vs. PKA I (15 min). , P < 0.05 vs. TSH (15 min).

View larger version (45K): [in this window] [in a new window]   Fig. 7. Prkar2a and Prkar2b Silencing in FRTL5 Cells Cells were transfected with a siRNA for Prkar2a and two different siRNAs for Prkar2b (siRNA1 and siRNA2). A, Prkar2a and Prkar2b mRNA levels, measured over time by real-time PCR. y-Axis shows mRNA expression levels relative to cells transfected with control (scrambled) siRNAs. Reported are data from a representative experiment. B, Western blot analysis for RI, RII, RIIß, C, and ß-actin (control) 96 h after transfection. C, Effect of bTSH stimulation on CREB phosphorylation after Prkar2a and Prkar2b silencing. After transfection, FRTL5 cells were cultured in the absence of bTSH for 96 h and then stimulated with 0.1 U/liter bTSH for 15 min. CREB phosphorylated at Ser133 (P-CREB) and total CREB levels were evaluated by Western blot analysis. The reported images are representative of three experiments. D and E, effect of bTSH stimulation on the expression of a subset of early genes (D) and differentiative genes (E) in Prkar2b-silenced (siRNA1) FRTL5 cells. After transfection, FRTL5 cells were cultured in the absence of bTSH for 96 h and then stimulated for 30 min (D) or 17 h (E) with 0.1 U/liter bTSH. Relative expression was determined by real-time PCR. *, P < 0.03. Similar results were obtained with siRNA2 (data not shown). F, Effect of Prkar2b silencing on the expression of a subset of proliferative genes. FRTL5 cells were transfected and cultured in the presence of bTSH. The expression levels of these genes were measured 96 h after transfection by real-time PCR. *, P < 0.05 vs. control. y-Axis in panels D–F shows expression levels relative to control (cells transfected with scrambled siRNA at basal conditions).

PKA I and II have different subcellular localizations. PKA I is generally cytosolic, whereas PKA II is targeted to cell membranes by interaction with A-kinase anchoring proteins (AKAPs) (10). To evaluate whether PKA II membrane targeting is required for propagation of cAMP signal, we treated FRTL5 cells with a permeable peptide that disrupts PKA II anchoring (HT31). Preincubation with HT31, but not with a mutated control peptide (HT31P), was associated with an impairment of TSH-induced phosphorylation of CREB (Fig. 8).

View larger version (19K): [in this window] [in a new window]   Fig. 8. Effect of HT31 Treatment on CREB Phosphorylation in FRTL5 Cells Cells were preincubated with 10 µM HT31 or HT31P (control peptide) for 30 min before addition of 0.1 U/liter bTSH for 15 min. CREB phosphorylated at Ser133 (P-CREB) and total CREB levels were evaluated by Western blot analysis. The densitometric analysis of three independent experiments is reported. y-Axis shows variations of P-CREB/total CREB ratio as percentage of the value in cells treated with HT31P only. *, P < 0.03.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Prkar2b Transfection in FRT/TSHR Cells A, Representative Western blot for RIIß and ß-actin (control) of nontransfected cells (NT) or cells transfected with Prkar2b (+ RIIß). B, Effect of RIIß reexpression on TSH-dependent CREB phosphorylation. Cells were stimulated for 15 min with bTSH before Western blot analysis for CREB phosphorylated at Ser133 (P-CREB) or total CREB. A representative of three Western blot analyses is reported. C, Effect of RIIß reexpression on TSH-dependent induction of early genes. Cells were stimulated with bTSH for 30 min. y-Axis shows mRNA expression of three independent experiments relative to NT cells. *, P < 0.03 vs. NT cells.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Expression of PKA-Regulatory Subunits in Human Thyroid Cancer Cell Lines and Effect of PKA I or II Activation on Cell Growth A, comparison of PKA-regulatory subunit mRNA levels by real-time PCR. y-Axis shows mRNA expression levels relative to WRO cells. B, Western blot analysis of RI, RII, and RIIß expression. ß-Actin was used as control. C, MTT assay after treatment with equimolar concentrations of cAMP analog pair selective for PKA I. y-Axis shows the relative growth as % of control (cells treated with DMSO), measured 72 h after stimulation.

Because NPA cells were found to express Prkar2b at reasonable levels, we evaluated the effect of PKA II-selective analogs on gene transcription in this cell line. A significant induction of early genes (Egr1, Nr4a1) was observed at 30 min (data not shown). Thyroid-specific genes were expressed at very low levels either at basal conditions or after PKA II activation for 17 h (data not shown).

A possible mechanism for the antiproliferative effect of PKA is represented by inhibition of the MAPK cascade (5). We therefore evaluated the effect of PKA I activation on ERK phosphorylation in FRT/TSHR and human thyroid cancer cell lines. PKA I activation was associated with a pronounced reduction of ERK phosphorylation in FRT/TSHR, NPA, and ARO cells, whereas only a slight effect was observed in WRO cells (Fig. 11).

View larger version (52K): [in this window] [in a new window]   Fig. 11. Effect of PKA I Activation on ERK 1/2 Phosphorylation in FRT/TSHR and Human Thyroid Cancer Cell Lines Cells were treated with 100 µM of cAMP analog pair selective for PKA I. ERK phosphorylated at Thr202/Tyr204 was detected with a specific antibody. Western blot analysis of total ERK was used as control. One representative experiment of three is reported.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
In this work we used the microarray technology to investigate global transcriptional changes induced by TSH stimulation in a well-differentiated (FRTL5) and in a partially dedifferentiated (FRT/TSHR) thyroid cell line. Microarray data revealed substantial differences in the expression profiles of the two cell lines. In FRTL5 cells, 123 TSH-regulated genes (95 newly described) were identified, including several early genes and factors involved at different steps in cell proliferation. Several of the identified early genes are cAMP responsive (26, 27, 28, 29) and therefore may mediate some of the transcriptional effects of TSH. Interestingly, many of the genes involved in cell replication, i.e. Ccne, Cdc2a, proliferating cell nuclear antigen (Pcna), mini chromosome maintenance deficient 6 (Mcmd6), Mcmd7, DNA primase (Prim1), topoisomerase 2 (Top2a), DNA polymerase (Pola1), DNA polymerase (Pold1), and thymidylate synthase (Tyms), were constituted by E2F-regulated transcripts that are typically induced at G1/S checkpoint and throughout the S phase of cell cycle (30, 31, 32, 33, 34, 35). The induction of genes involved in cholesterol metabolism may also contribute to the proliferative effect of TSH because de novo cholesterol synthesis is required for thyroid cell replication (17, 18). A recent microarray study has evaluated the transcriptional effect of TSH stimulation on human thyrocytes, showing that TSH induces several repressors of cAMP, e.g. cAMP response element modulator (CREM) or Pde4b, and MAPK, e.g. dual specificity phosphatase 2 and 6 (Dusp2 and 6), pathways (36). Our microarray data are in agreement with these results, confirming that TSH stimulation results in the transcriptional activation of negative feedbacks, e.g. up-regulation of Dusp 1, 5, and 6 as well as Pde4b in FRTL5 cells. Despite cAMP responses to TSH stimulation similar to those observed in FRTL5 cells, transcriptional changes were under the cutoff limit in FRT/TSHR cells. This finding suggests that the reintroduction of a functional TSHR is not sufficient to restore an efficient signaling pathway in these dedifferentiated cells. In the search for an explanation, we then compared the expression levels of key elements of the cAMP cascade in the two cell lines and found Prkar2b to be expressed only in FRTL5 cells. Based on these findings, additional experiments were performed to elucidate the role of PKA I and II in thyroid cells.

Our data obtained with isoform-selective PKA activators and inhibitors indicate that PKA II, rather than PKA I, mediates the proliferative effect of TSH in FRTL5 cells. However, PKA I-selective analogs could induce iodide uptake, as previously reported for a similar cAMP analog pair (25), and activate PKA at levels comparable to those obtained with TSH or PKA II-selective analogs. These findings suggest that PKA I may also mediate some of TSH effects, but acting at a posttranscriptional level. Indeed, TSH induces NIS expression to the plasma membrane, and NIS contains consensus sites for PKA phosphorylation (37). Although the relevance of NIS phosphorylation is not presently known, it may constitute a mechanism for a direct regulation of iodide uptake by PKA. We then looked at gene transcription. PKA II, but not PKA I, activation induced the transcription of genes involved in cell proliferation and thyroid function (Slc5a5/NIS, Tg2, Tpo), as well as CREB phosphorylation. These results differ from those of a previous study by Van Sande et al. (14), in which activation of PKA I or II had similar effects on dog thyroid cell differentiation and function, whereas PKA I activation was reported to be more potent in inducing mitosis. This discrepancy may be explained by species-specific differences between rat and dog thyrocytes (1, 2, 3). In addition, the results of Van Sande et al. may be complicated by the low selectivity of the cAMP analog pairs available at that time, which makes it difficult to discriminate between PKA I and II actions. In fact, based on the relative affinity for sites A and B of type I or II PKA-regulatory subunits, the selectivity for PKA I:II obtained in their study is expected to be 2:1 for the PKA I analog pair and 1.28:1 for the PKA II analog pair (38, 39). Instead, the analog pairs used in our study have an estimated selectivity of 5:1 and 1:60, respectively (38, 39, 40, 41).

To corroborate the results obtained with selective cAMP analog pairs and rule out the role of the two PKA II isoforms, we knocked down Prkar2a or Prkar2b expression in FRTL5 cells by gene silencing with small interfering RNAs (siRNAs). Prkar2b and, only to a minor extent, Prkar2a silencing inhibited TSH-dependent CREB phosphorylation. Furthermore, Prkar2b silencing was associated with an impairment of TSH induction of early genes (Nr4a1, Nr4a3) and genes involved in thyroid differentiated function (Slc5a5/NIS, Tpo) and with the down-regulation of the proliferative genes identified by the microarray analysis. These data suggest a major role for Prkar2b in mediating nuclear TSH signaling and transcription of genes involved in both function and proliferation. Noticeably, Prkar2a and Prkar2b silencing was not associated with any modification of the expression levels of C or the other R subunits. This is an interesting paradox, because the drop in R subunit content may be expected to lead to an increase of the amount of free and active C subunit. However, several data indicate that C subunit compartmentalization and membrane targeting through interaction with RII subunits and AKAPs would be required for effective PKA-dependent induction of gene transcription (10). This view is consistent with other findings of our study, i.e. impairment of TSH nuclear signaling by treatment with a disruptor of PKA II/AKAP interaction and the reconstitution of TSH-dependent phosphorylation of CREB and gene transcription upon reexpression of RIIß in FRT/TSHR cells. These findings are also in agreement with the results of two previous studies in FRTL5 cells. Feliciello et al. (42) investigated R subunit subcellular localization during the cell cycle. RII and RIIß were found to be cytoplasmic in G₀ and to redistribute to the Golgi centrosome area in G₁. Interestingly, PKA redistribution to membrane-bound compartments in G₁ was associated with an increase of PKA sensitivity to cAMP. In another paper, v-Ki-Ras transformation was reported to result in RIIß dissociation from membrane-bound compartments, which impaired cAMP-dependent nuclear signaling. Consistently, the effect of v-Ki-Ras transformation could be mimicked by overexpression of an AKAP75-defective mutant (16).

Our data indicate that Prkar2b expression may be lost in dedifferentiated cell lines such as FRT-TSHR and ARO, thus suggesting that Prkar2b expression may be a characteristic of well-differentiated thyroid cells. In agreement with this view are the findings that Prkar2b expression is increased by treatment with a differentiative agent such as vitamin D (25) and decreased by v-Ki-Ras transformation (16). Interestingly, the dedifferentiation process was accompanied by a change in the response to PKA modulation. In fact, PKA stimulation, which had a mitogenic effect on differentiated thyroid cells, was associated with a dose-dependent growth inhibition in dedifferentiated cells (FRT/TSHR, NPA, ARO). Because PKA I stimulation resulted in growth inhibition of NPA cells that express Prkar2b, the effect of PKA I-selective analogs does not appear to be related to a loss of Prkar2b expression. Rather, it correlated well with the presence of a BRAF activating mutation (V600E) in NPA and ARO, but not WRO cells (43) that responded poorly to PKA I activation. Indeed, activating mutations of key elements of the MAPK cascade (BRAF, RET-PTC, Ras) are the most frequent genetic lesions in thyroid carcinomas, leading to constitutive MAPK activation (43, 44), and PKA has a well-established inhibitory effect on the MAPK cascade in other cell types (5). We therefore evaluated the effects of PKA I stimulation on ERK phosphorylation. The results indicate that PKA I activation is associated with a long-lasting inhibition of the MAPK cascade. The effect was more pronounced in NPA and ARO than in WRO cells, correlating well with the antimitotic activity of PKA I-selective analogs. Because PKA has been reported to inhibit Akt activity in rat PCCL3 thyroid cells (3), we also evaluated the effect of PKA I stimulation on Akt phosphorylation. Small modifications of Akt phosphorylation that did not parallel the different response to PKA I stimulation were observed. Interestingly, an antiproliferative effect of cAMP has been previously reported in some de- differentiated thyroid cell lines (45, 46). Moreover, there is some evidence that TSH may have an antiproliferative effect in vitro (47, 48) as well as in vivo (49) on dedifferentiated thyroid cancer cell lines expressing either endogenous or exogenous TSHR.

Modulation of PKA expression/activity is a potential target for cancer therapy (4). A number of studies have documented that PKA I is overexpressed in several human cancer cells. Moreover, RI antisense oligonucleotides and 8-chloro-cAMP, a cAMP analog that down-regulates RI, inhibits the growth of a wide range of cancer cells of nonendocrine origin (4, 8, 50, 51). Based on these results, phase I and II clinical trials have been carried out to assess safety and efficacy of RI antisense oligonucleotides and 8-chloro-cAMP (52, 53, 54). Our data indicate that the modulation of PKA activity retains a therapeutic potential also in the case of thyroid cancer. At variance with results obtained in other cell types, however, stimulation, rather than inhibition, of PKA I has an antimitotic effect on thyroid cancer cell lines.

Based on all these findings, we propose that PKA I and II carry out different biological functions in thyroid cells. In differentiated cAMP-dependent cells (i.e. FRTL5), PKA II appears to be the principal mediator of cAMP on gene transcription and cell growth. Given the crucial role of Prkar2b, it may represent a novel candidate gene for thyroid proliferative disorders and TSH resistance. PKA I may be involved, instead, in posttranscriptional regulations (e.g. iodide uptake) and cross-talk with other signaling pathways (e.g. MAPK). During in vitro dedifferentiation or carcinogenesis, cells accumulate somatic mutations and other events that endow them with autonomous growth. In such cases (e.g. FRT/TSHR and thyroid carcinoma cell lines), the expression of Prkar2b may be lost and PKA I activation may be associated with an antimitotic effect, likely through MAPK inhibition. Constitutive activation of the MAPK pathway, as caused by mutations of BRAF and possibly other elements of this cascade, appears to sensitize thyroid carcinoma cells to the antiproliferative effects of PKA I stimulation. PKA I may thus represent a new pharmacological target for the therapy of poorly differentiated thyroid cancers, where conventional therapies are frequently ineffective.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Chemicals
Cell culture reagents, Trizol, Superscript Double Stranded cDNA synthesis kit, SuperScript First-Strand Synthesis System for RT-PCR, Stealth RNA oligonucleotides, Lipofectamine²⁰⁰⁰, and Cellfectin were purchased from Invitrogen (San Giuliano Milanese, Italy). RNeasy spin columns were purchased from QIAGEN (Milan, Italy). BioArray high-yield RNA transcript labeling kit was purchased from Enzo Diagnostic (New York, NY). RG-U34A genechips were purchased from Affymetrix (Santa Clara, CA). SYBR Green PCR Master Mix was purchased from Applied Biosystems (Warrington, UK). 8-Piperidinoadenosine-cAMP, 8-hexylaminoadenosine-cAMP, N⁶-mono-tert.-butylcarbamoyladenosine-cAMP, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate Sp-isomer, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate Rp-isomer (RP-8-CPT-cAMPS), and 8-bromoadenosine-3',5'-cyclic monophosphorothioate Rp-isomer (RP-8-Br-cAMPS) were purchased from BIOLOG Life Science Institute (Bremen, Germany). BCA kit and nitrocellulose membranes were purchased from Pierce Chemical Co. (Rockford, IL). PKA_RI, PKA_RII, PKA_RIIß, and ß-actin antibodies were purchased from BD Biosciences Pharmingen (Milan, Italy). Phospho-CREB, phospho-ERK, phospho-AKT, CREB, ERK, AKT, and PKA C antibodies were purchased from Cell Signaling Technology (Beverly, MA). Horseradish peroxidase-conjugated mouse and rabbit IgG antibodies were purchased from Chemicon International (Temecula, CA). ECL-plus kit was purchased from Amersham Biosciences Europe (Freiburg, Germany). Stressxpress PKA kinase activity assay kit was purchased from Stressgen Bioreagents (Victoria, British Columbia, Canada). InCELLect St-HT31 and HT31P were purchased from Promega Corp. (Madison, WI). ¹²⁵I was purchased from PerkinElmer (Boston, MA). RIANEN cAMP assay kit was purchased from NEN Life Science Products (Boston, MA). All other reagents were purchased from Sigma-Aldrich (Milan, Italy).

Cell Lines and Transfections
FRTL5 cells derive from a nontransformed clone of Fisher rat thyroid cells adapted to grow in the presence of low serum (55). FRTL5 cells represent a unique and diffuse model of differentiated thyroid cells because they retain TSH-dependent growth and express the major thyroid-specific transcription factors (i.e. Titf1, Foxe1, Pax8). They were cultured in Coon’s modified Ham’s F12 medium supplemented with 5% Donor Calf Serum, 1% penicillin, 1% streptomycin, and a mixture of five hormones (5H) or five hormones and bovine TSH (bTSH) (6H), as previously described (56). FRT/TSHR cells were generated in our laboratory by introducing human TSHR cDNA in FRT cells. FRT cells derive from a TSH-independent and partially dedifferentiated FRTL5 clone that lost the expression of TSHR and other thyroid-specific genes, but retains epithelial polarized phenotype (57). They express thyroid transcription factor Pax8, but not Titf-1 and Foxe1 (58, 59). FRT cells were transfected with pT-TSHR vector (60) by CaCl₂ method, followed by G418 selection. Clones were then screened on the basis of positive immunohistochemical staining for TSHR and presence of a cAMP response to TSH. The clone with the highest cAMP response was selected. FRT/TSHR cells were cultured in Coon’s modified Ham’s F12 medium supplemented with 5% fetal calf serum, 1% penicillin, 1% streptomycin, and 100 mg/liter G418 (FRT medium).

A plasmid encoding human Prkar2b under cytomegalovirus promoter was purchased from Origene (Rockville, MD), and Prkar2b sequence was verified by direct sequencing. This plasmid was transfected into FRT/TSHR cells by lipofection with Cellfectin. FRT/TSHR cells were plated in 35-mm Petri dishes and transfected with 1 µg DNA and 5 µl Cellfectin in medium without serum and antibiotics. After 5 h, medium was replaced with FRT medium. Analyses were performed 48 h after transfection.

Three human thyroid carcinoma cell lines, originating from anaplastic (ARO) (61), papillary (NPA) (62), and follicular (WRO) (63) carcinomas, were used. They were maintained in DMEM supplemented with 10% fetal calf serum, 1% penicillin, and 1% streptomycin.

cAMP Assay
The culture medium was removed and replaced with Krebs-Ringer-HEPES buffer, 0.5% BSA, for 30 min at 37 C. Then cells were incubated at 37 C for 60 min in fresh Krebs-Ringer-HEPES buffer, 0.5% BSA, supplemented with 0.5 mmol/liter isobutylmethylxanthine and the indicated concentrations of bTSH. The medium was removed and the reaction stopped by adding 0.1 mol/liter HCl. Samples were dried and cAMP was measured using a commercial RIA (RIANEN).

Microarray Experiments
FRTL5 and FRT/TSHR cells were seeded on 100-mm Petri dishes and cultured with appropriate medium (6H or FRT medium, respectively). Upon reaching 70% confluence, FRTL5 cells were switched to TSH-free medium (5H) for 5 d. Twelve hours before bTSH stimulation, both cell lines were starved in serum-free media. Cells were then stimulated with 0.1 U/liter (FRTL5 cells) or 1 U/liter (FRT/TSHR cells) bTSH. These bTSH concentrations were predetermined to generate similar increases of cAMP intracellular levels in the two cell lines. Microarray analysis was performed on three independent RNA samples for each experimental point. In brief, total RNA samples were isolated using Trizol reagent and purified on RNeasy spin columns. Double-stranded cDNA was synthesized from 10 µg RNA using Superscript Double Stranded cDNA synthesis kit, phenol/chloroform was extracted, and ethanol was precipitated. Biotinylated cRNAs were synthesized using the BioArray high-yield RNA transcript labeling kit and fragmented. RG-U34A Genechips were hybridized and stained by standard procedures. Images were acquired with the Affymetrix Genearray scanner. Cel files were then quantile normalized and processed using the Robust Microarray Analysis algorithm, available at http://www.bioconductor.org, under R statistical environment (64). A statistical algorithm, SAM (65) was applied to identify differentially expressed genes. This analysis assigns a score to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. For genes with scores greater than an adjustable threshold (), SAM uses permutations of the repeated measurements to estimate the percentage of genes identified by chance, i.e. the FDR. A of 0.45 and a fold-change of at least 1.8 were used, corresponding to a FDR between 1% and 2% in all comparisons. Hierarchical clustering was performed by TIGR Multiexperiment Viewer (MeV) 3.1 tool, after median centering spots (66).

Real-Time PCR
RNA samples were isolated using Trizol reagent. Single-stranded cDNA was synthesized from 1 µg of total RNA using SuperScript First-Strand Synthesis System for RT-PCR. PCR primers were designed with Primer3 software (67). PCR was performed with SYBR Green PCR Master Mix. The reaction conditions were as follows: 2 min at 50 C (one cycle), 10 min at 95 C (one cycle), 15 sec at 95 C and 1 min at 60 C (50 cycles). Gene-specific PCR products were continuously measured with ABI PRISM 7900 sequence detection system (Applied Biosystems). Samples were normalized using a housekeeping gene (ß-actin for rat cells or HGPRT for human cells). Triplicates were performed for each experimental point. The CT method was used to calculate relative expression levels.

Selective Activation or Inhibition of PKA I/II
Selective activation of PKA I/II was obtained by using pairs of site-selective cAMP analogs as previously described (23, 24). 8-Piperidinoadenosine-cAMP + 8-hexylaminoadenosine-cAMP were used for PKA I activation, N⁶-mono-tert.-butylcarbamoyladenosine-cAMP + 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate Sp-isomer were used for PKA II activation. These analog pairs present a PKA I:II selectivity of 5:1 for the PKA I analog pair and 1:60 for the PKA II analog pair (38, 39, 40, 41). Selectivity for PKA I:II was calculated by (AI x BI)^1/2: (AII x BII)^1/2 (38, 68) where AI, BI, AII, and BII are the relative affinities for site A and B of PKA I and II. For inhibition of PKA, cells were preincubated for 30 min with the indicated concentration of a PKAI-selective (RP-8-Br-cAMPS) or PKAII-selective (RP-8-CPT-cAMPS) inhibitor before addition of TSH or PKAII-selective analog pair.

Proliferation Assay
Cell proliferation was evaluated by 3-(4,5-dimetylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded onto 96-well plates and treated with different combinations of TSH, cAMP analog pairs selective for PKAI or PKA II, and inhibitors of PKA I (RP-8-Br-cAMPS) or PKA II (RP-8-CPT-cAMPS). MTT (0.5 mg/ml) was added to the cells for 3 h at 37 C. Formazan crystals, formed by mitochondrial reduction of MTT, were solubilized in dimethylsulfoxide (DMSO)/ethanol (1:1), and the absorbance was read at 550 nm. Relative growth was calculated by the following equation: Relative growth = (OD_550nm of treated wells/OD_550nm of control wells) x 100%. The experiments were repeated at least three times and each determination was done in sextuple.

Iodide Uptake
FRTL5 cells were plated at a density of 100,000 cells per well in 24-well tissue culture plates and cultured in 5H medium for 5 d. Cells were then stimulated with the indicated concentration of TSH-, PKAI-, or PKAII-selective analogs for 48 h. One hour before the end of the 48-h incubation, 10 µM NaI and 0.1 µCi/well Na¹²⁵I were added, and cells were incubated 1 h at 37 C. Cells were then washed three times with ice-cold HEPES-buffered saline solution and treated with 500 µl/well of ethanol 100% for 30 min at room temperature. The extracts were centrifuged at maximum speed to remove cell debris and counted in a -counter. The radioactivity was normalized to cell count.

PKA Activity
PKA activity was measured with the nonradioactive Stressxpress PKA activity assay kit on 50 ng of cell extract, according to manufacturer’s protocol. The assay is based on a solid-phase ELISA that uses a specific synthetic peptide (kempeptide) as a substrate for PKA and a polyclonal antibody that recognizes only the phosphorylated substrate. PKA activity was measured in the presence or absence of 20 µmol/liter cAMP. Unspecific activity was measured by performing the assay in the presence of the protein kinase inhibitor PKI (10 µmol/liter). Calculations were performed according to Feliciello et al. (16). The activity of free catalytic PKA subunit (C-PKA) was obtained by subtracting values in the presence of PKI from those in the absence of cAMP. Total PKA activity was calculated by subtracting values in the presence of PKI from those in the presence of cAMP. The ratio between C-PKA and total PKA was used as a measure of PKA activation.

siRNA Knockdown of Prkar2a and Prkar2b
Stealth RNA oligonucleotides, 25-bp double-stranded RNA oligonucleotides with proprietary chemical modifications, were used for Prkar2a and Prkar2b silencing. These oligonucleotides were designed utilizing Invitrogen Web tool (https://rnaidesigner.invitrogen.com) that ensures targeting of unique (i.e. gene-specific) sequences. Sequence specificity was also checked by BLAST analysis against rat mRNAs and the sequences of the remaining PKA-regulatory subunits. FRTL5 cells were seeded at a density of 2 x 10⁵ cells per well in six-well plates. Transfections were performed with 100 pmol of each oligonucleotide and Lipofectamine²⁰⁰⁰ in the absence of antibiotics and serum. Culture medium was replaced 5 h after transfection.

Western Blot
For detection of PKA-regulatory subunits, cells were lysed in modified RIPA buffer [10 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P40, 1% Na deoxycholate, 2 mM EDTA, 2 mM Na₂VO₄, 2 mM Na₄P₂O₇, 2 mM NaF]. For detection of CREB, ERK, and Akt, cells were lysed directly with SDS sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 50 mM dithiothreitol; 0.01% bromophenol blue), immediately heated for 5 min at 95 C, and sonicated. Protein concentration was determined by BCA assay. Protein extracts (20 µg) were electrophoresed on 10% SDS polyacrylamide gel and electrotransfered to a nitrocellulose membrane. Membranes were blocked with TBS-T + 5% milk, probed with primary antibody overnight at 4 C, and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Detection was performed utilizing ECL-plus Kit.

Statistical Analysis
Values are expressed as mean ± SE. One-way or two-way ANOVA followed by Bonferroni’s post hoc test were used, as appropriate, to evaluate differences between means. P < 0.05 was considered statistically significant.

	ACKNOWLEDGMENTS

 
We thank Dr. L. Nitsch (Naples, Italy) for the kind gift of FRT cells; Dr. I. Bongarzone (Milan, Italy) for WRO, NPA, and ARO cells; and Dr. H.-G. Genieser (Bremen, Germany) for technical advice on cAMP analogs.

	FOOTNOTES

 
This work was partially supported by Funds of Italian Ministry of Public Health (FSN 2003; project 08M301) and by Funds of Italian Ministry for Instruction, University and Research (PRIN 2004; project 2004052155_005). D.C., T.d.F., S.L., and L.P. were supported by Research Funds of Istituto Auxologico Italiano IRCCS (project 05C107).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 3, 2006

¹ D.C., T.d.F., and S.L. contributed equally to this work.

Abbreviations: AKAP, A-kinase anchoring protein; CREB, cAMP response element-binding protein; DMSO, dimethylsulfoxide; FDR, false discovery rate; MTT, 3-(4,5-dimetylthiazole-2-yl)-2,5-diphenyltetrazolium bromide; NIS, sodium iodide transporter; PKA, protein kinase A; RIIß, regulator subunit IIß; RP-8-Br-cAMPS, 8-bromoadenosine-3',5'-cyclic monophosphorothioate Rp-isomer; RP-8-CPT-cAMPS, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate Rp-isomer; SAM, Significance Analysis of Microarrays; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; TSHR, TSH receptor.

Received for publication December 6, 2005. Accepted for publication July 26, 2006.

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