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Activation of Phospholipase D in FRTL-5 Thyroid Cells by Forskolin and Dibutyryl-Cyclic Adenosine Monophosphate¹

Signal Transduction Laboratories, Departments of Medicine (J.G., W.C.M., S.G.), Biochemistry (D.N.B.), and Oral Health Sciences (L.K.), University of Alberta, Edmonton, Alberta, Canada

Address all correspondence and requests for reprints to: J. Ginsberg, Division of Endocrinology, Department of Medicine, 362 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta T6G 2S2 Canada. E-mail: Jody.Ginsberg{at}UAlberta.Ca

	Abstract

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We demonstrated previously that TSH activates phospholipase D (PLD) via stimulation of protein kinase C (PKC) in Fischer rat thyroid line (FRTL)-5 thyroid cells. To examine the role of the cAMP pathway in the regulation of PLD, we studied the effects of forskolin (0–100 µM; 30 min) and dibutyryl cAMP (dbcAMP;0–1 mM; 30 min) on PLD activation. FRTL-5 thyroid cells were labeled mainly in phosphatidylcholine with [³H]myristate followed by incubation with 200 mM ethanol before the addition of agonist. PLD was assessed by the measurement of [³H]phosphatidylethanol. Forskolin (100 nM to 100 µM) and dbcAMP (100 pM to 100 µM) increased PLD activity significantly. Maximal responses to forskolin and dbcAMP exceed the PLD responses produced by 100 µU/ml of TSH. To determine whether the effects of forskolin and dbcAMP on PLD occurred as a consequence of PKC activation, FRTL-5 thyroid cells were preincubated for 10 min with the PKC inhibitors, chelerythrine (1 µM) or calphostin C (1 µM), or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to down-regulate PKC. Unlike TSH-mediated PLD activation, these treatments had no effect on PLD activation by cAMP agonists. Forskolin (10 µM; 30 min) had no effect on the subcellular distribution of PKC -, -, or -isoforms, confirming the lack of involvement of PKC. The protein kinase A (PKA) inhibitors, H-89 (10 µM; 30 min) and dideoxyadenosine (5 nM;10 min) significantly decreased the forskolin- and dbcAMP-mediated PLD activation without any effect on the phorbol ester-mediated PLD response. Following pretreatment with H-89 or dideoxyadenosine, the TSH-mediated PLD response was also significantly reduced. These studies indicate that forskolin and dbcAMP stimulate PLD in FRTL-5 thyroid cells directly via PKA without involvement of PKC. Studies of cells in the presence and absence of ethanol revealed approximately 60% of the phosphatidate plus diacylglycerol produced via TSH occurs via PLD activation. Although TSH-mediated inositol phosphate generation occurred with similar concentrations of TSH that led to PLD activation, 10-fold higher TSH concentrations were required to increase intracellular Ca²⁺. These results and the lack of a rapid Ca²⁺ transient following physiological TSH concentrations suggest that alternatives to conventional hydrolysis of phosphatidylinositol 4,5-bisphosphate may initiate PKC activation. Thus, the two major signal transduction systems in the FRTL-5 thyroid cell (PKA and PKC) appear to converge on PLD activation. Stimulation of both of these pathways by TSH may be required for optimal physiological activation of PLD.

	Introduction

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THERE is increasing evidence that TSH mediates signal transduction systems in the thyroid via cAMP and also by a phosphatidylinositol (PI) cascade involving protein kinase C (PKC) activation (1). TSH can stimulate phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis in human thyroid slices (2), FRTL-5 thyroid cells (3), and in Chinese hamster ovary (CHO) cells transfected with human TSH receptor complementary DNA (4). Mutation of alanine 623 in the third cytoplasmic loop of the rat TSH receptor results in the loss of the PIP₂ but not the cAMP signal (5). PKC isoenzymes have been identified in Fischer rat thyroid line (FRTL)-5 thyroid cells (6) and shown to be regulated by TSH (7) probably as a result of increased intracellular calcium (Ca²⁺) and diacylglycerol (DAG) concentrations. Activation of the PKC signal by phorbol ester leads to inhibition of TSH-mediated cAMP production (8) demonstrating cross-talk between the signaling pathways. Although 3–10 times the concentration of TSH capable of activating cAMP has been demonstrated to be required to activate the PIP₂ cascade, equivalent concentrations of TSH activate both pathways in FRTL-5 thyroid cells in the presence of an adenosine A1 receptor activator (9).

Recently, we demonstrated that TSH could activate phospholipase D (PLD) in FRTL-5 thyroid cells via stimulation of PKC (10) at TSH concentrations capable of activating cAMP. Similar concentrations of TSH also cause membrane association of PKC in porcine thyroid cells (11) and FRTL-5 thyroid cells (7). PLD activation results in phosphatidylcholine hydrolysis to phosphatidate acid (PA), which is converted to DAG by the subsequent action of phosphatidate phosphohydrolase (12). Previous work demonstrated an increase in DAG production in FRTL-5 thyroid cells treated chronically with TSH or cAMP agonists (13). These investigators could not identify a phospholipid source for the DAG, and speculated whether it was produced via phosphatidylcholine hydrolysis.

The purpose of the present study was to determine whether cAMP agonists could modify PLD activity in FRTL-5 thyroid cells, and whether this activity was related to PKC activation as previously observed for TSH. These experiments indicate a novel stimulation of PLD by cAMP agonists that does not involve PKC. TSH-mediated PLD activation was inhibited by either protein kinase A (PKA) or PKC inhibitors. Thus, physiological PLD activation by TSH may require the activation of both the PKA and PKC cascades.

	Materials and Methods

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Materials
FRTL-5 thyroid cells were kindly provided by Dr. Leonard Kohn from the Interthyroid Research Foundation, Baltimore, MD (patent numbers 4,609,622 and 4,608,341). They are continuous functional epithelial cells derived from normal rat thyroid as originally described by Ambesi-Impiombato et al. (14). Coon’s modified Ham’s F-12 medium, human transferrin, bovine insulin, cortisol, somatostatin, glycyl-L-histidyl-L-lysine, BSA (fatty acid poor), forskolin, dibutyryl cAMP, Nonidet P40, fura-2, and phorbol myristate acetate (PMA) were obtained from Sigma Chemical Co (St. Louis, MO). Sphingosine-1-phosphate was synthesized as previously described (15). Newborn calf serum (heat inactivated) and rabbit peptide-specific antibodies (IgG fraction) that recognize PKC -, -, and - were obtained from Gibco BRL (Grand Island, NY). [³H]Myristate and ³[H]inositol were from Amersham Life Sciences (Arlington Heights, IL). Bovine TSH (bTSH; Thytropar) was purchased from Armour Pharmaceuticals Ltd. (Kankakee, IL). Chelerythrine, N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89), and dideoxyadenosine (DDA) were obtained from Calbiochem (La Jolla, CA). Thin-layer chromatography plates of silica gel 60 without fluorescent indicator were from Merck (Rahway, NJ). All other chemicals were obtained from commercial sources and were of analytical reagent grade.

Cell culture
FRTL-5 thyroid cells were cultured in Coon’s modified Ham’s F-12 medium supplemented with 5% newborn calf serum and six hormone mixture (6H) comprised of bovine insulin (10 µg/ml), cortisol (10 nM), human transferrin (5 µg/ml), glycyl-L-histidyl-L-lycine acetate (10 µg/ml), somatostatin (10 µg/ml), and TSH (10 mU/ml). Cells were maintained at 37 C in an atmosphere saturated with water and containing 95% air-5% C0₂. When confluence was reached, cells were trypsinized and subcultured in 60-mm tissue culture dishes. After the cells approached confluence, the medium was changed to 6H medium devoid of TSH (termed 5H medium), and the cells were incubated for a further 4 days. The culture medium was changed twice weekly. On the day of the experiment, the medium was replaced with Coon’s modified Ham’s F-12 medium without any additional hormones.

Assay of PLD
PLD activity was measured through the formation of phosphatidylethanol from radioactive phospholipid as described previously (16, 17, 18). FRTL-5 thyroid cells were washed three times with modified Ham’s F-12 medium and then incubated in serum-free modified Ham’s F-12 medium containing 0.1% albumin and 1 µCi [³H]myristate/ml for 2 h. The reaction medium was then aspirated, and the cells washed twice with nonradioactive Ham’s medium-albumin solution. The FRTL-5 thyroid cells were then incubated for a further 2 h in medium containing 0.5% albumin with three changes of medium. This procedure removes unesterified myristate thus stopping further labeling but allowing the turnover of prelabeled phospholipids (75% of which was phosphatidylcholine) to be determined. The medium was then replaced with medium containing 0.1% albumin and 200 mM ethanol. PMA (100 nM), bTSH (100 µU/ml, Thytropar), forskolin, or dbcAMP at the concentrations indicated were then added after a 5-min incubation, and the cells were incubated for 30 min as determined previously (10). In some experiments the cells were treated with PMA (100 nM) for 24 h to down-regulate PKC before labeling with [³H]myristate (19). To determine the effects of PKA or PKC inhibitors on TSH, PMA, forskolin, and dbcAMP-mediated PLD activity, FRTL-5 thyroid cells were incubated with the PKA or PKC inhibitor at the concentration and times indicated before the addition of ethanol. The cells were washed twice with ice-cold PBS and scraped in 0.5 ml methanol. The dishes were washed with a further 0.5 ml methanol, and the lipids were extracted and analyzed by thin-layer chromatography (17). Radioactive lipids were detected with a Bioscan System 200 Imaging Scanner (Bioscan, Inc., Washington, DC) and then quantitated by liquid scintillation counting. Agonist-stimulated phosphatidylethanol formation was calculated after subtraction of background radioactivity (average 400 dpm) that was obtained from incubations in the absence of ethanol.

Assay of PKC
PKC isoform mass was assessed by Western blot analysis as described previously (7). On the day of the experiment, the medium was replaced with Coon’s modified Ham’s F-12 medium in the presence or absence of TSH (100 µU/ml), PMA (100 nM), or forskolin (10 µM) for different time intervals before removal of the medium followed by the addition of ice-cold Earle’s Balanced Salt Solution (EBSS). The cells were detached from their dishes by scraping in ice-cold EBSS and centrifuged at 2000 x g for 3 min. The cells were resuspended and sonicated in Tris-HCl (20 mM, pH 7.6), EDTA (2 mM), phenylmethylsulfonylfluoride (2 mM), EGTA (0.5 mM), and sucrose (0.33 M) (which we designated TEPES buffer) and centrifuged for 115,000 x g for 1 h at 4 C. The supernatant was used as the cytosolic fraction, and the pellet (membrane fraction) was resuspended in TEPES buffer containing 1% (vol/vol) Nonidet P40. Protein concentrations were determined using the Bio-Rad assay system (Bio-Rad Labs., Hercules, CA).

SDS-PAGE was performed overnight using 50 µg protein per lane with a 10% acrylamide separating gel and 3% stacking gel, at 9 mA and 200 V followed by 15 mA for 1 h before transfer. The gels were then removed, and the protein samples were transferred onto nitrocellulose membranes (5–7 h at 210 mA). The membranes were removed and blocked with 5% dried skim milk in Tris-buffered saline containing 1% Triton X-100 (BLOTTO) for a minimum of 15 min. The samples were then washed twice with BLOTTO. The membranes were incubated with monoclonal antibodies that recognized PKC -, -, and - isoforms at 1:500 dilution in BLOTTO overnight at 4 C. The membranes were exposed for different periods (5–60 sec) to Kodak X-AR5 film (Eastman Kodak, Rochester, NY) to ensure that the Western blots were quantitated within the linear range of the film. The experiments were repeated on three occasions with similar results, and a representative immunoblot is shown.

Assay of inositol phosphates
Cells were labeled with 2 µCi [³H]inositol in inositol-free DMEM containing 0.5% BSA for 48 h. The cells were subsequently washed twice with HEPES buffer containing: 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgS0₄, 1.2 mM CaCl₂, 5.6 mM dextrose, 20 mM HEPES (pH 7.4), 1 mM inositol, and 10 mM LiCl. After a further 10 min of incubation, agonist was added at the indicated concentration and time. The reaction was terminated by treating the cells with ice-cold HClO₄. The suspension was removed from the plates and centrifuged at 3000 x g for 5 min. The pellet was resuspended in 1 M HClO₄ and centrifuged again. The supernatant was combined with the first fraction and neutralized with 1 M KOH. After centrifuging the supernatant was then loaded onto a Dowex-1-formate column (Bio-Rad) (1 ml), and the column was washed with deionized water until the counts achieved background levels. The inositol phosphates were eluted sequentially with 0.1 M formic acid/0.2 M ammonium formate (inositol phosphate), 0.1 M formic acid/0.4 M ammonium formate (inositol bisphosphate), and 0.1 M formic acid/1.0 M ammonium formate (inositol trisphosphate). The majority of radioactivity eluted in the inositol phosphate and inositol bisphosphate fractions.

Assay of intracellular Ca²⁺
FRTL-5 cells were grown on coverslips in 35-mm dishes as described above, but the time in 5H medium was increased to 7 days. On the day of the experiment, the cells were loaded with 1 µM fura-2 (acetoxymethyl ester) for 30 min in 5H medium. The coverslip was then transferred to a special housing unit warmed by a water circulator to maintain a temperature of 37 C. The medium was then changed to HEPES buffer of the following composition: 134 mM NaCl, 4.5 mM KCl, 2.5 mM NaHCO₃, 1.2 mM MgSO₄, 1.2 mM CaCl₂, 5 mM dextrose, and 20 mM HEPES (pH 7.4). The cells were allowed to equilibrate for 10–15 min before the addition of test agonists at the concentrations and times indicated. The cells were then monitored for intracellular Ca²⁺ for up to 600 sec using a SPEX system (SPEX Industries, Inc., Edison, NJ).

All experiments were performed on at least three separate occasions, and the results were pooled where appropriate. All statistics were performed using a one-way ANOVA (Epistat Computer Program, Dr. Tracy Gustafson, Round Rock, TX) followed by Fisher’s protected least significant differences test (PLSD) except where indicated.

	Results

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The effects of various concentrations of forskolin and dbcAMP on PLD activation in FRTL-5 thyroid cells is shown in Fig. 1. PLD activity was significantly increased by 10^-7–10^-4 M forskolin. PLD activation was also observed with 10^-10–10^-4 M dbcAMP. DDA inhibited the stimulation of PLD by forskolin and dbcAMP by 71% and 75%, respectively, and the equivalent values for H-89 were about 50% and 40%, respectively (Fig. 2). By contrast, DDA and H-89 had no significant effect on the stimulation of PLD by PMA. Pretreatment of thyroid cells with the PKA inhibitors, DDA, or H-89 substantially blocked the stimulation of PLD by TSH by 36% and 60%, respectively.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effects of forskolin and dbcAMP on PLD activation. FRTL-5 thyroid cells were labeled with [3H]myristate and then treated in presence of ethanol with concentrations of forskolin (a) or dbcAMP (b) as indicated for 30 min. Relative PLD activity was determined by measurement of [3H]phosphatidylethanol formation expressed as a percentage of 3H in phosphatidylcholine. Results are means ± SD of three independent experiments. Statistics were determined using one-way ANOVA followed by Fisher’s PLSD. *, P < 0.01 vs. control values.

View larger version (23K): [in this window] [in a new window]   Figure 2. Effects of PKA inhibitors, DDA and H-89 on activation of PLD by TSH, PMA, forskolin, or dbcAMP. FRTL-5 thyroid cells were labeled with [3H]myristate and then incubated in presence (solid bars) or absence (open bars) of DDA (5 nM, 10 min) (a) or H-89 (10 µM, 30 min) (b) before measurement of agonist-stimulated PLD activity by TSH (100 µU/ml), PMA (100 nM), forskolin (1 µM), or dbcAMP (100 nM). Relative PLD activity was determined by measurement of [3H]phosphatidylethanol formation expressed as a percentage of 3H in phosphatidylcholine. Results are means ± SD of three independent experiments. Statistics were determined using one-way ANOVA followed by Fisher’s PLSD. *, P < 0.01.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effects of PKC inhibitor, chelerythrine, on activation of PLD by TSH, PMA, forskolin, or dbcAMP. FRTL-5 thyroid cells were labeled with [3H]myristate and then incubated in presence (solid bars) or absence (open bars) of 1 µM chelerythrine for 10 min before measurement of agonist-stimulated PLD activity by TSH (100 µU/ml), PMA (100 nM), forskolin (1 uM), or dbcAMP (100 nM). Relative PLD activity was determined by measurement of [3H]phosphatidylethanol formation expressed as a percentage of 3H in phosphatidylcholine. Results are means ± SD (where large enough to be shown) of three independent experiments. Statistics were determined using one-way ANOVA, followed by Fisher’s PLSD. *, P < 0.01 vs. control values.

View larger version (18K): [in this window] [in a new window]   Figure 4. Effects of PKC down-regulation on activation of PLD by TSH, PMA, forskolin, or dbcAMP. FRTL-5 thyroid cells were treated in absence (open bars) or presence (solid bars) of 100 nM PMA for 24 h to down-regulate PKC. Cells were then labeled with [3H]myristate before treatment with TSH (100 µU/ml), PMA (100 nM), forskolin (1 µM), or dbcAMP (100 nM) for 30 min in presence of ethanol. Relative PLD activity was determined by measurement of [3H]phosphatidylethanol formation expressed as a percentage of 3H in phosphatidylcholine. Results are ± SD of three independent experiments. Statistics were determined using one-way ANOVA followed by Fisher’s PLSD. *, P < 0.01 vs. control values.

View larger version (44K): [in this window] [in a new window]   Figure 5. Effect of forskolin on PKC isoform redistribution. FRTL-5 cells were incubated with forskolin (10 µM: 30 min) before preparation of cytosol and membrane fractions. Western blotting for PKC -, -, and -isoforms is described in Materials and Methods. TSH (100 µU/ml) and PMA (100 nM) served as positive controls.

View larger version (13K): [in this window] [in a new window]   Figure 6. Effects of TSH on generation of inositol phosphates. FRTL-5 thyroid cells were labeled with 2 µCi [3H]inositol for 48 h before addition of forskolin (1 µM) or TSH at concentrations indicated for 30 min in presence of LiCl, before assessment of total inositol phosphate formation. Results are mean ± SD of quadruplicate determinations for a single batch of cells. Results expressed are a representative of three closely agreeing experiments. Statistics were determined using a one-way ANOVA followed by Fisher’s PLSD. , P < 0.05, *, P < 0.01 vs. basal values.

View larger version (14K): [in this window] [in a new window]   Figure 7. Effects of TSH (1 mU/ml) on intracellular Ca2+ concentrations. FRTL-5 thyroid cells were loaded with 1 µM fura-2 before stimulation with TSH (as indicated by arrow) and measurement of intracellular Ca2+ as described in Materials and Methods. Results are from a single experiment that was confirmed in three independent experiments (see Table 2).

	Discussion

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In many cell systems, there is a transient DAG rise secondary to PIP₂ hydrolysis followed by a sustained DAG peak that prolongs the PKC signal (22). In porcine thyroid cells, TSH transiently stimulates DAG production (23). DAG production is increased synergistically with insulin or IGF-1 in FRTL-5 thyroid cells chronically stimulated with TSH or cAMP agonists (13). Those authors speculated that DAG synthesis could have occurred as a consequence of PLD activation. Although short-term exposure to cAMP agonists was used in the present study, the findings of PLD activation and DAG production would confirm and extend these initial observations. We estimated using incubations in the presence or absence of ethanol that approximately 60% of the PA plus DAG produced following exposure to TSH occurred via PLD activation. The equivalent values for PMA, forskolin, and dbcAMP were 64%, 46%, and 25% respectively. Although we used a concentration (200 mM) of ethanol that produced maximum inhibition of DAG and PA formation, our results might underestimate the contribution of PLD if not all of the reaction were diverted to the production of phosphatidylethanol, or if there are mammalian forms of PLD that do not participate in the transphosphatidylation reaction (24). There is also a rapid turnover of PA and DAG in the presence and absence of ethanol that may also be modified by the agonists that we used. Thus, we measured the steady state accumulation of DAG and PA and could not account for the degradation of these lipids or their conversions to phospholipids and triacylglycerol.

Our results shown in Table 1 indicate the production of DAG by pathways independent of conventional PLD activation. This conclusion is compatible with the need for DAG production for TSH to activate PKC, which in turn stimulates PLD (10). This portion of DAG production could be catalyzed by a phosphatidylcholine-specific phospholipase C, although the contribution of this enzyme to mammalian signal transduction remains controversial. DAG production could also result from the degradation of PIs, and we demonstrated this effect in response to 100 µU/ml TSH. The products were mainly isolated as inositol mono- and bisphosphates even in the presence of LiCl, and this occurred progressively up to 30 min. Similarly, Field et al. (3) reported a relatively long-term generation of inositol phosphates only after 15 min of TSH exposure in FRTL-5 thyroid cells. This indicates that this lower concentration of TSH (100 µU/ml), which activates PKC (7, 11), does not stimulate a rapid breakdown of PIP₂ to generate 1,4,5-inositol trisphosphate (IP₃). In agreement with this concept, concentrations of TSH below 10 mU/ml were unable to cause a rapid Ca²⁺ transient (after 30 sec). However, a slow and sustained increase in intracellular Ca²⁺ was observed at 1 and 10 mU/ml of TSH. Singh et al. (25) also concluded that TSH could stimulate the production of inositol phosphates, but that this did not involve the rapid generation of inositol IP₃. In previous studies using human thyroid slices or CHO cells transfected with human TSH receptor complementary DNA, 1–10 mU/ml of prolonged TSH exposure was required to observe increases in IP₃ generation (2, 4). This is similar to that observed in studies using FRTL-5 thyroid cells (3). The details of which lipids are involved in the production of DAG by different concentrations of TSH remains to be explored.

In FRTL-5 thyroid cells, certain aspects of differentiated thyroid function appear to be regulated by either the cAMP or PI cascades. Iodine uptake, thyroid peroxidase, and thyroglobulin synthesis are regulated by the cAMP cascade, whereas the PI cascade is involved in iodide efflux, hydrogen peroxide generation, and iodination (1). The ability of TSH to induce the cAMP and PI cascades and different roles for each of the signaling pathways has also been described in human thyrocytes (1). PA is produced as a consequence of PLD activation. PA can act as a second messenger in many cell systems (16, 26), including generating superoxide in neutrophils (26) and decreasing adenylyl cyclase in fibroblasts (27). Many of the observed effects of the PI cascade in the thyroid were made following use of the PKC activator, phorbol ester (28). It remains unclear whether these observed effects occur as a consequence of PKC activation directly or subsequent PLD involvement and the generation of PA.

In summary, the present study demonstrates that the cAMP agonists, forskolin and dbcAMP activate PLD in FRTL-5 thyroid cells, and that these effects are independent of PKC activation. The activation of PLD by PMA is also not blocked by inhibition of PKA. These combined results indicate that in the FRTL-5 thyroid cell, independent activation of the PKA and PKC signaling pathways both lead to PLD activation. However, because inhibition of either PKA or PKC can diminish TSH-mediated PLD activation, the physiological activation of PLD by TSH may require simultaneous stimulation and interactions between both of these pathways.

	Acknowledgments

 
The authors thank Ms. Elaine Maisonneuve and Mrs. Isabel McAndry for excellent secretarial assistance.

	Footnotes

 
¹ This work was sponsored by an operating grant from the Medical Research Council of Canada (MT-11019).

Received May 5, 1997.

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