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*LEVOTHYROXINE
Endocrinology Vol. 145, No. 4 1708-1717
Copyright © 2004 by The Endocrine Society

Disparate Effects of Thyroid Hormone on Actions of Epidermal Growth Factor and Transforming Growth Factor-{alpha} Are Mediated by 3',5'-Cyclic Adenosine 5'-Monophosphate-Dependent Protein Kinase II

Ai Shih, Shenli Zhang, H. James Cao, Heng-Yuan Tang, Faith B. Davis, Paul J. Davis and Hung-Yun Lin

Research Service, Stratton Veterans Affairs Medical Center, the Ordway Research Institute, Inc., and the Wadsworth Center, New York State Department of Health, Albany, New York 12208

Address all correspondence and requests for reprints to: Paul J. Davis, M.D., Ordway Research Institute, 150 New Scotland Avenue, Albany, New York 12208. E-mail: pjdavis{at}albany.net.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epidermal growth factor (EGF) and TGF{alpha} share the same plasma membrane receptor. In the present studies in HeLa cells, both EGF and TGF{alpha} caused MAPK (ERK1/2) activation and expression of the immediate-early gene c-fos. Thyroid hormone (T4) nongenomically enhanced EGF- and TGF{alpha}-induced MAPK activation. This T4 action was duplicated by T4-agarose and blocked by tetraiodothyroacetic acid, which inhibits binding of T4 to plasma membranes. TGF{alpha}-induced MAPK activation was potentiated by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) but not 8-chloro-cyclic adenosine monophosphate. TGF{alpha}, T4, and 8-Br-cAMP each caused protein kinase A (PKA) II serine phosphorylation, whereas phosphorylation of PKA-II was not seen in cells treated with EGF or 8-chloro-cyclic adenosine monophosphate. In a PKA activity assay, the enzyme was stimulated by T4, EGF, and TGF{alpha}; T4 enhanced the effect of TGF{alpha} but not that of EGF. T4, although it potentiated c-fos gene expression in EGF-treated cells, suppressed this effect in cells treated with TGF{alpha}. Cells exposed to 8-Br-cAMP also inhibited TGF{alpha}-stimulated c-fos expression. Studies of cell proliferation indicated that T4 potentiated EGF action but inhibited that effect in TGF{alpha}-treated cells. The disparate effects of T4 on actions of EGF and TGF{alpha}, which share the same cell surface receptor, are mediated by hormone phosphorylation and activation of PKA-II.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE MODULATES transduction of several cytokine and growth factor signals in human cell lines (1, 2, 3, 4, 5, 6). This hormone effect is in part nongenomic in mechanism and involves a cell surface G protein-coupled receptor for iodothyronines (1) and subsequent stimulation of the MAPK cascade (1, 3). Cross-talk between the thyroid hormone-activated MAPK pathway and the signal transducer and activator of transcription (STAT) proteins (7, 8) is relevant to potentiation by thyroid hormone of the actions of interferon-{gamma} (1, 2, 4, 5) and epidermal growth factor (EGF) (3). Activation of the MAPK cascade by T4 causes translocation of activated (phosphorylated) ERKs 1 and 2 (pERK1/2, MAPK) to the cell nucleus in which the kinases interact with and serine phosphorylate the nuclear thyroid hormone receptor TRß1 (9) and other nucleoproteins (10). These effects of T4 on intracellular signaling are blocked by inhibitors of traditional protein kinase C (PKC)-{alpha}, -ß, and -{gamma} (1, 2, 3, 9) and cAMP-dependent protein kinase A (PKA) (9). These effects of thyroid hormone are obtained with physiologic concentrations of T4 (1, 2, 3, 9) or supraphysiologic levels of T3 (1, 9).

EGF interacts with its plasma membrane receptor (EGFR), causing tyrosine phosphorylation and nuclear translocation of MAPK and STAT proteins (8, 11, 12, 13), resulting in activation of immediate-early genes (3, 14). In addition, EGF also activates adenyl-cyclase activity and results in increased cAMP levels (15), which activate cAMP-dependent protein kinase (PKA). PKA-I and PKA-II are holoenzyme subtypes of PKA that are formed by the combination of regulatory (RI or RII) with catalytic (CI or CII) subunits.

Whereas the type I PKA holoenzymes are predominantly cytosolic, the type II holoenzymes-{alpha} and -ß are found associated with subcellular structures such as the actin cytoskeleton, Golgi apparatus, and the perinuclear area (16, 17). Binding of RII subunits to site-specific A kinase-anchoring proteins mediates this selective subcellular localization and membrane anchoring, thus controlling cAMP signaling to the nucleus (16, 17). Furthermore, translocation of RII subunits back to the cytosol is associated with down-regulation of cAMP signaling (16). When stimulated with EGF, the EGFR can form either homo- or heterodimers with other members of this receptor family, including ErbB2, -3 and -4 (18). Transforming growth factor-{alpha}, TGF{alpha} also activates both the EGFR and ErbB-2 in an in vivo animal model (19).

T4 enhances the effects of EGF on MAPK activation and protooncogene expression (3). Because TGF{alpha} also uses the plasma membrane EGFR (20), we postulated that transduction of the TGF{alpha} signal would also be potentiated by thyroid hormone. In the studies reported here, we show that T4 indeed enhances the TGF{alpha} signal leading to MAPK activation, but the hormone inhibits, rather than stimulates, TGF{alpha}-induced c-fos expression. Studies of the mechanism of this novel action of thyroid hormone on TGF{alpha}-induced immediate-early gene expression reveal that the effect of the hormone depends on stimulation by T4 of cAMP-dependent protein kinase (PKA) activity, specifically the activity of the PKA-II holoenzyme.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
T4, T3, tetraiodothyroacetic acid (tetrac), T4-agarose, 8-bromo-cAMP (8-Br-cAMP) and 8-chloro-cAMP (8-Cl-cAMP) were obtained from Sigma Chemical Co. (St. Louis, MO). EGF and TGF{alpha} were purchased from BioSource (Camarillo, CA), and KT5720 from the Kamiya Biomedical Co. (Thousand Oaks, CA). NIH3T3 and stably transfected dominant-negative Ras cells (N17) were generously provided by Dr. Geoffrey M. Cooper (Boston University School of Medicine, Boston, MA) (21) and are in use in our laboratory (10). These cells express an Asn-17 ras gene that has a significantly reduced affinity for GTP and has been shown to inhibit cellular ras activity in vivo without reducing cellular ras content (21). HeLa cells are maintained in the laboratory (1).

Cell culture and preparation of nuclear fractions
Cells were cultured and nuclear fractions prepared as we have previously described (1, 2, 3). HeLa cells were grown to confluence and then exposed for 48 h to DMEM supplemented with 0.25% fetal bovine serum that was depleted of thyroid hormone (1). Endogenous thyroid hormone contributed by hormone-depleted serum was at or below the lower limits of detection (10, 22). NIH3T3 and N17 cells were cultured in the same manner, with G418, 400 µg/ml, added to the culture medium for maintenance of the N17 ras dominant-negative transfectant. G418 is an aminoglycoside antibiotic that is used in the selection of eukaryotic expression vectors carrying the bacterial neor/kanr genes (21). The preparation of stock thyroid hormone solutions was as previously reported (1). L-T4 was added to cells in a final total hormone concentration of 10-7 M for the indicated time periods, resulting in a free T4 concentration of 0.7 x 10-10 M as measured in the Clinical Chemistry Laboratory of the Albany Medical Center Hospital (10). The cells were harvested and nuclear proteins prepared as previously reported (1): cells were washed twice with ice-cold PBS and lysed in hypotonic buffer [20 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM Na3VO4, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 3 µg/ml aprotinin, 1 mg/ml pepstatin, 20 mM NaF, and 1 mM dithiothreitol] with 0.2% Nonidet P-40 on ice for 10 min. After centrifugation at 4 C and 13,000 rpm for 1 min, supernatants were collected as cytosolic extracts. Cytosols were prepared for PKA-II activation studies. Nuclear extracts were prepared by resuspension of the crude nuclei in high-salt buffer (hypotonic buffer with 20% glycerol and 420 mM NaCl) at 4 C with rocking for 30 min. The supernatants were collected after centrifugation at 4 C and 13,000 rpm for 10 min.

Immunoblotting and immunoprecipitation
The techniques are standard and have been previously described (1, 2, 3). In brief, 25-µg aliquots of each protein sample were separated on discontinuous SDS-PAGE (9% gels) and then transferred by electroblotting to Immobilon membranes (Millipore, Bedford, MA). After blocking with 5% milk in Tris-buffered saline containing 0.1% Tween, the membranes were incubated with various antibodies overnight. Antibodies used in our studies include monoclonal anti-c-Fos, anti-proliferating cell nuclear antigen (PCNA), and anti-actin from Santa Cruz Biotechnology (Santa Cruz, CA). Also used were polyclonal goat anti-PKA-II [Upstate Biotechnology (UBI), Lake Placid, NY], rabbit anti-phospho-MAPK (anti-pERK1/pERK2, Cell Signaling, Beverly, MA), and rabbit anti-phospho-serine (Research Diagnostics, Flanders, NJ). Goat antimouse PKA-RII{alpha}/ß, (UBI, lot 13454), and rabbit antimouse serine-96-phosphorylated PKA-RII (UBI, lot 18375) were also used. Secondary antibodies were goat antirabbit (1:1000), rabbit antimouse (1:1000), or rabbit antigoat IgG (1:2000) (Dako, Carpenteria, CA), depending on the origin of the primary antibody. Immunoreactive proteins were detected by chemiluminescence (Amersham, Piscataway, NJ), and the integrated ODs (IODs) of bands compared by scanning (Bio-Image, Millipore, Billerica, MA). For immunoprecipitation, 200 µg protein per sample were used, followed by SDS-PAGE and immunoblotting of the precipitated proteins, as previously described (1, 3, 9, 10). Results presented are representative of three or more experiments, each with a separate set of control and treated cells. Results of immunoblots are presented as mean ± SEM of the IOD of each band normalized to a value of 1 in untreated cells in three or more experiments.

PKA assay
Measurement of PKA activity in cell lysates was performed using a PKA assay kit (UBI). Cells were rinsed with PBS, placed on ice, and lysed in PKA assay buffer (25 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM ß-mercaptoethanol, 0.5% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride). The PKA assay was performed in the presence of 5 µl cell lysate, 83 µM kemptide substrate (LRRASLG; UBI), 0.33 µM PKC inhibitor peptide (RFARKGALRQKNV; UBI), 3.33 µM calmodulin-dependent protein kinase inhibitor (R24571), and 83.3 µM ATP with 5 µCi [{gamma}-32P]-ATP per sample. To demonstrate specificity for phosphorylation by PKA, reactions were also performed in the presence of both kemptide substrate and 1 µM of PKA inhibitor peptide (TYADFIASGRTGRRNAI-NH2; UBI). After incubation for 10 min at 30 C, 15-µl samples were blotted onto phosphocellulose P81 paper, washed four times in 0.75% phosphoric acid and once in acetone, and radioactivity then measured in a scintillation counter. Values were normalized to protein concentrations of the lysates and to specific radioactivity (counts per minute per picomole) of the reaction mix.

RT-PCR
Total RNA was isolated as described previously (2). First-strand complementary DNAs were synthesized from 1 µg total RNA using oligo dT and AMV reverse transcriptase (Promega, Madison, WI). First-strand cDNA templates were amplified for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs by PCR using a hot start (Ampliwax, PerkinElmer, Foster City, CA). Primer sequences were as previously described (23). The PCR cycle was an initial step of 95 C for 3 min followed by 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, and then 25 cycles and a final cycle of 72 C for 8 min. PCR products were electrophoresed through 2% agarose gels containing 0.2 mg/ml ethidium bromide. Gels were visualized under UV light and photographed with Polaroid film (Polaroid Co., Cambridge, MA). Photographs were scanned under direct light (BioImage, Millipore) for quantitation and illustration, and results from PCR products were normalized to the GADPH signal.

EMSA
Nuclear extracts (10 µg protein) were incubated in a 25-µl total reaction volume that contains 10 mM Tris (pH 7.5), 50 mM NaCl, 1.0 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 4% glycerol, and 0.05 µg/µl poly(dI-dC) (Promega). [32P]-dCTP-labeled oligo-nucleotide was added to the total reaction mixture, which was then incubated for 20 min at room temperature. Samples were loaded on 4% polyacrylamide gels in low-ionic-strength buffer (22.3 mM Tris, 22.2 mM borate, 0.5 mM EDTA) and run at 15 V/cm with cooling. The gel was then dried, exposed to x-ray film, and the radioautograph analyzed. Activator protein-1 (AP-1) and stimulatory protein-1 (SP-1) oligonucleotides were obtained from Promega.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of MAPK (ERK1/2) by T4, TGF{alpha}; potentiation by T4 of effect of TGF{alpha} on activation of MAPK
By a nongenomic action at the plasma membrane of cultured cells, T4 causes phosphorylation and translocation to nuclei of activated MAPK, specifically of ERKs 1 and 2. This hormone action has been shown to require activities of Ras (10), Raf-1 (1), and MAPK kinase (1, 9, 10). In the present studies, HeLa cells were treated with T4 for 40 min and nucleoproteins prepared and immunoblotted with antibody to phosphorylated MAPK (pERK1/2). A representative study (Fig. 1AGo) shows increased phosphorylation and nuclear accumulation of ERK1/2 in response to T4, and a reduction in the T4 effect with the addition of tetrac, an inactive deaminated analog of T4 that is known to block T4 binding to plasma membranes (1, 9). TGF{alpha} also caused activation of MAPK, and the addition of T4 to TGF{alpha} enhanced this effect. Again, tetrac blocked the added effect of T4 in cells treated with TGF{alpha}. In Fig. 1BGo, the effects of TGF{alpha} and T4 on MAPK activation are again seen. In addition, T3, 10-7 M, activated MAPK, but the effect of a more physiologic concentration of T3 (10-10 M) was not comparable with that of T4, as we have previously shown (1). Agarose-T4 was as effective as T4, demonstrating that the T4 effect does not require entry of the hormone into the cell. In the presence of a small concentration of TGF{alpha} (1 ng/ml), which was not very effective alone, there was potentiation of the growth factor’s effect on MAPK activation by 10-7 M T4 and T4-agarose but less potentiation by T3.



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  		FIG. 1. Effect of TGF{alpha}, T4, T3, and tetrac on MAPK activation. A, HeLa cells were treated with TGF{alpha} (1 ng/ml) for 10 min, with or without pretreatment with T4 (10-7 M) and/or tetrac (10-7 M) for 30 min. Nuclear fractions were then prepared as described. MAPK activation, shown by immunoblotting with anti-pERK1/2 (upper panel), was evident with T4 (lane 2), and this effect was blocked by tetrac (lane 4). TGF{alpha} (1 ng/ml, 10 min) also activated MAPK (lane 5), and T4 enhanced that effect (lane 6). With TGF{alpha} present, tetrac had no potentiating effect (lane 7) but blocked the potentiating effect of T4 (lane 8, compared with lane 6). A graphic representation of the mean ± SE of normalized band IOD, as shown by the representative blot in the upper panel, is indicated in the lower panel (n = 3 experiments). A ß-actin blot served as a gel-loading control for the blot shown. B, HeLa cells were exposed to T4, T4-agarose (T4-ag), or T3 at the concentrations indicated for 45 min, and MAPK activation is seen in lanes 3–5. TGF{alpha} (1 ng/ml for 15 min) caused little activation alone (lane 6), but in the presence of T4, T4-ag, or T3 (10-7 M), there was enhancement of the TGF{alpha} effect (lanes 8–10, compared with lane 6). The graph shows normalized results from three experiments. C, NIH3T3 cells and N17 dominant-negative ras cells were treated with TGF{alpha} (1 ng/ml) for 10 min, with or without pretreatment with T4 (10-7 M) for 30 min. T4 caused potentiation of MAPK activation by TGF{alpha} in NIH3T3 cells; the N17 cells did not show activation of MAPK with either TGF{alpha} or T4.

 
The study shown in Fig. 1CGo demonstrates T4 potentiation of MAPK activation by TGF{alpha} in NIH3T3 cells, which contain the signaling protein Ras and are controls for N17 cells containing a dominant-negative ras construct. The latter cells were unresponsive to T4 and TGF{alpha} with respect to MAPK kinase activation, indicating that the effects of T4 and TGF{alpha} require Ras for effective signaling.

Action on PKA-II of T4, TGF{alpha}, and cAMP analogs
Possible contributions of PKA and cAMP analogs to the effects of T4 and TGF{alpha} were identified by the studies shown in Fig. 2Go. In cells treated with T4, TGF{alpha}, or both, nuclei were immunoprecipitated with antiphosphoserine, and the precipitates immunoblotted with antibody to PKA-II. Both T4 and TGF{alpha} caused serine phosphorylation of PKA-II, and the two agents had a small additive effect (Fig. 2AGo). KT5720, an inhibitor of PKA activity, is shown in Fig. 2BGo to inhibit TGF{alpha} activation of MAPK, suggesting that this effect of TGF{alpha} on MAPK is dependent on activation of PKA. Measurement of PKA activity was then carried out using [{gamma}-32P]-ATP. Total PKA activity was enhanced by both growth factors in the absence of T4. T4, alone, activated PKA, and there was a potentiating effect of T4 on TGF{alpha}-induced PKA activation (Fig. 2CGo). In contrast, there was no enhancement by T4 of the EGF effect (Fig. 2CGo). That PKA-II specifically contributed to the increase in overall PKA activity caused by T4 and TGF{alpha} was suggested by the parallel increases in serine-phosphorylated PKA-II and PKA activity in Fig. 2Go. Serine and threonine phosphorylation of the regulatory subunit of PKA II has been shown by others to be associated with activation of the enzyme (24, 25, 26).



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  		FIG. 2. TGF{alpha} and T4 serine-phosphorylate and activate PKA-II. A, HeLa cells were treated with TGF{alpha} (1 ng/ml) for 10 min, with or without 10-7 M T4 preincubation for 30 min. Cytosolic extracts were immunoprecipitated (IP) with anti-phosphoserine antibody and the precipitates immunoblotted with anti-PKA-II antibody. Both TGF{alpha} and T4 caused serine phosphorylation of PKA-II, seen in this representative immunoblot from three experiments and the accompanying graph. The effect of TGF{alpha} was potentiated by T4. B, TGF{alpha} (1 ng/ml) was added to HeLa cells for 10 min with or without preincubation with the PKA inhibitor, KT5720 (KT, 10 nM to 1 µM) for 30 min. TGF{alpha}-induced MAPK activation was inhibited by KT in a concentration-dependent manner. The graph shows normalized results from three experiments. C, HeLa cells were treated with TGF{alpha} (1 ng/ml) for 10 min, with or without 10-7 M T4 preincubation for 30 min. PKA activity assays were performed as described in Materials and Methods, and the combined, normalized results of three assays are shown in the graph. Both growth factors and T4 activated PKA. The effect of TGF{alpha} was potentiated by T4, whereas the effect of EGF was not enhanced by the hormone.

 
The effects of two cAMP analogs, 8-Br-cAMP and 8-Cl-cAMP, on MAPK activation in response to TGF{alpha} and EGF, were compared. In Fig. 3Go it is evident that both analogs caused modest activation of MAPK and that MAPK activation by TGF{alpha} was enhanced by 8-Br-cAMP, but not by 8-Cl-cAMP. On the other hand, MAPK activation by EGF was enhanced by 8-Cl-cAMP but not by 8-Br-cAMP.



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  		FIG. 3. Effect of cAMP analogs on MAPK activation induced by growth factors. TGF{alpha} (1 ng/ml) or EGF (10 ng/ml) was added to HeLa cells for 10 min with or without 8-Br-cAMP or 8-Cl-cAMP (100 µM) preincubation for 30 min. Both cyclic nucleotides activated MAPK (lanes 2 and 3). 8-Br-cAMP enhanced TGF{alpha}-induced MAPK activation (lane 5, compared with lane 4), but 8-Cl-cAMP appeared to inhibit the TGF{alpha} effect (lane 6). 8-Br-cAMP decreased EGF-induced MAPK activation (lane 8), whereas 8-Cl-cAMP enhanced EGF-induced MAPK activation (lane 9, compared with lane 7). The graph displays the results of three similar experiments.

 
Effects of cAMP analogs on MAPK activation and PKA activities
To further investigate the relationships of these two growth factors to PKA-I and -II, measurements were first made of phosphorylated PKA-II by immunoblot, along with immunoblots of activated ERK1/2, in cells treated with 8-Br-cAMP or 8-Cl-cAMP in the presence or absence of KT5720. 8-Cl-cAMP did not cause appearance of phosphorylated PKA-II but did cause phosphorylation (activation) of MAPK (Fig. 4AGo). In contrast, 8-Br-cAMP caused both MAPK activation and the appearance of phosphorylated PKA-II. The inhibitor KT5720 blocked the effects of both cAMPs. These results are consistent with the function of 8-Br-cAMP as an activator of PKA-II.



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  		FIG. 4. Effect of cAMP analogs on phosphorylation of PKA-II. A, HeLa cells were treated with either 8-Br-cAMP or 8-Cl-cAMP (100 µM) for 30 min in the absence or presence of KT5720 (1 µM). The appearance of phospho-PKA-II, indicated in the upper immunoblot, occurred in response to 8-Br-cAMP, and this effect was inhibited by KT5720. There was no phosphorylation of PKA-II seen with 8-Cl-cAMP. The lower immunoblot shows MAPK activation (phosphorylation) by both 8-Br-cAMP and 8-Cl-cAMP and partial inhibition of each effect by KT5720. The graphsshow the normalized results from three experiments. B, HeLa cells were treated with either 8-Br-cAMP or 8-Cl-cAMP (100 µM) for 30 min in the absence or presence of TGF{alpha} (1 ng/ml) or EGF (10 ng/ml) for 15 min. The upper blotshows the presence of two bands, representing phosphorylated and unphosphorylated PKA-II, most evident in the sample treated with both 8-Br-cAMP and TGF{alpha} and to a lesser extent in samples treated with 8-Br-cAMP or TGF{alpha} alone. 8-Br-cAMP and TGF{alpha} both caused an increase in serine-phosphorylated PKA-II and had some additive effect, as seen in the lower blot. 8-Br-cAMP enhanced the effect of TGF{alpha} on PKA phosphorylation, whereas 8-Cl-cAMP did not enhance that effect. EGF did not stimulate PKA-II phosphorylation in either the presence or absence of the cAMP analogs. The graphsdemonstrate the normalized results from three experiments.

 
The effects on PKA serine phosphorylation by both cAMPs together with either EGF or TGF{alpha} were then examined. TGF{alpha} caused the appearance of phospho-PKA-II, which was enhanced by 8-Br-cAMP, as seen in both panels of Fig. 4BGo. In contrast, the effects of 8-Br-cAMP and EGF on PKA-II phosphorylation were negligible. 8-Cl-cAMP caused less phosphorylation of PKA-II than 8-Br-cAMP and caused inhibition of the effect of both EGF and TGF{alpha} on PKA-II phosphorylation.

The studies of MAPK and PKA-II phosphorylation and activation of PKA presented above can be summarized as follows: 1) T4 enhances the action of TGF{alpha} on MAPK activation; 2) this action of T4 is duplicated by 8-Br-cAMP; 3) both T4 and 8-Br-cAMP cause serine phosphorylation of PKA-II; and 4) a general PKA inhibitor, KT5720, blocks the activation of MAPK by TGF{alpha}, T4, and 8-Br-cAMP, and blocks the phosphorylation of PKA-II by 8-Br-cAMP.

Actions of TGF{alpha}, EGF, and T4 on immediate-early gene expression; mediation of effect of T4 by PKA-II
Both EGF and TGF{alpha} are known to stimulate expression of c-Fos and c-Jun. T4 has previously been shown to enhance c-Fos expression by EGF (3), and results of such an experiment are shown in Fig. 5AGo. EGF stimulates accumulation of c-Fos, and this effect is enhanced by T4 (upper panel). Levels of c-fos mRNA are also increased by EGF and further enhanced by addition of T4 (lower panel). In contrast, c-Fos protein and c-fos mRNA are both enhanced by TGF{alpha} alone, but in the presence of T4, the TGF{alpha} effect is reduced (Fig. 5BGo).



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  		FIG. 5. Effect of T4 on growth factor-induced immediate-early gene expression. A, EGF, 10 ng/ml, was added to HeLa cells for 60 min in the presence or absence of T4, 10-7 M, for the last 30 min. Cellular c-Fos was then measured by immunoblotting (upper panel). EGF increased c-Fos expression, and this was further enhanced by T4. In similar experiments cells were incubated with EGF (10 ng/ml) for 15 min, either without T4, or after treatment with T4, 10-7 M, for 30 min; c-fos and GAPDH RNA levels were measured by RT-PCR (lower panels). EGF induced expression of c-fos, which was enhanced by T4. The bar graphs show the changes in c-Fos protein (upper graph) and c-fos RNA band density corrected for values of GAPDH (lower graph) and normalized to a value of 1 in untreated cells in three similar experiments. B, TGF{alpha} (1 or 10 ng/ml) was added to HeLa cells for 15 min, with or without T4, 10-7 M, for 45 min; c-Fos protein and c-fos RNA were then measured as in A. TGF{alpha} caused expression of c-Fos protein and transcription of c-fos, both of which were inhibited by T4. The bar graphs show fold change in c-Fos and c-fos RNA corrected for GAPDH values, normalized as in Fig. A. C, A representative electrophoretic mobility gel shift assay of HeLa cell samples treated with T4 and/or TGF{alpha} is shown in the left panel. T4, 10-7 M, was applied to selected cell samples for 45 min and TGF{alpha} (1 ng/ml) added for the last 15 min of T4 treatment or in the absence of T4. Using a radiolabeled AP-1 probe, no shift in band mobility with T4 alone was seen in this representative experiment (lane 2). There was increased AP-1 binding with TGF{alpha} treatment (lane 3), which was diluted by excess unlabeled AP-1 (lane 4) but not by excess unlabeled stimulatory protein-1 (SP-1, lane 5). T4 inhibited AP-1 binding induced by TGF{alpha} (lane 6, compared with lane 3). A similar representative experiment with EGF, 1 ng/ml (right panel), showed that T4 enhanced EGF-induced AP-1 binding (lane 10, compared with lane 9), in contrast to the T4 effect on TGF{alpha}.

 
The effect of T4 on binding of c-Fos to an AP-1 oligonucleotide was also studied in the presence of TGF{alpha} or EGF (Fig. 5CGo). TGF{alpha} caused binding of protein to AP-1, which was inhibited by T4 (left panel). In contrast, EGF also caused protein binding to AP-1, but this effect was enhanced by T4. These results are consistent with those of c-Fos expression studies shown above.

Further evidence that the T4 effect on c-Fos expression described above is mediated by activation of PKA-II was provided by studies of the effects of TGF{alpha}, EGF, and 8-Br-cAMP and 8-Cl-cAMP on c-Fos protein expression. Cellular c-Fos levels increased after treatment with TGF{alpha} (Fig. 6AGo), and this effect was reduced by cotreatment with 8-Br-cAMP. The effects of 8-Br-cAMP and 8-Cl-cAMP were compared, and results in Fig. 6BGo show that whereas 8-Br-cAMP inhibited TGF{alpha}-induced c-Fos expression, 8-Cl-cAMP potentiated the effect of TGF{alpha}. In contrast, immediate-early gene expression induced by EGF (Fig. 6CGo) was inhibited by 8-Cl-cAMP but potentiated by 8-Br-cAMP.



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  		FIG. 6. Role of PKA in TGF{alpha}-induced c-Fos expression. A, TGF{alpha} (1 ng/ml) was added to HeLa cell cultures for 60 min, with or without different concentrations of 8-Br-cAMP (0.1 nM to 100 µM) preincubated for 30 min. 8-Br-cAMP, at concentrations of both 1 and 100 µM, inhibited TGF{alpha}-induced c-Fos expression. The bar graph shows fold change in c-Fos band ODs, compared with those of the corresponding untreated samples normalized to a value of 1, from data in three experiments. B, TGF{alpha} (1 ng/ml) was added to HeLa cell cultures for 60 min with or without 8-Cl-cAMP (100 µM) or 8-Br-cAMP (100 µM) preincubation for 30 min. In the blot shown, representative of three experiments, 8-Br-cAMP, but not 8-Cl-cAMP, inhibited TGF{alpha}-induced c-Fos expression. C, EGF (10 ng/ml) was added to HeLa cell cultures for 60 min with or without 8-Br-cAMP (100 µM) or 8-Cl-cAMP (100 µM) preincubation for 30 min. 8-Br-cAMP enhanced EGF-induced c-Fos expression in this representative experiment, whereas 8-Cl-cAMP reduced c-Fos expression in the presence of EGF. The bar graph summarizes results from three similar experiments.

 
Effects of TGF{alpha} and EGF on cell proliferation
The expression of PCNA induced by EGF and TGF{alpha} in the presence or absence of T4 is shown in Fig. 7Go. Treatment with either growth factor increased PCNA expression. T4 had no effect alone but enhanced the action of EGF on cell proliferation. In contrast, the hormone inhibited the action of TGF{alpha}.



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  		FIG. 7. Effect of T4 on induction of PCNA by EGF or TGF{alpha} in HeLa cells. Cells were incubated for 24 h with either 10 ng/ml EGF or 1 ng/ml TGF{alpha}, in the presence or absence of T4, 10-7 M, and nuclear PCNA was measured by immunoblot. Both growth factors increased PCNA (lanes 3 and 5). The effect of EGF was significantly enhanced by T4 (lane 4). The effect of TGF{alpha}, however, was inhibited by T4 (lane 6). The blot shown, with an actin blot as a control for protein-loading, is representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although both EGF and TGF{alpha} bind to the cell surface EGFR (19, 20), the present studies indicate that HeLa cells can distinguish between the ligands, resulting in subtly different intracellular signals for EGF and TGF{alpha}. By a nongenomic action generated at the plasma membrane, T4 treatment permits detection of these different intracellular signals. In the absence of thyroid hormone, EGF and TGF{alpha} both activated MAPK (ERK1/2) and caused, downstream of MAPK, expression of the immediate-early gene, c-fos. T4 is known to enhance activation of MAPK by EGF (3); T4 enhanced MAPK activation by TGF{alpha} in this study. The TGF{alpha} effect on c-fos expression was, however, inhibited by T4, whereas the EGF effect on immediate-early gene expression was enhanced by T4. The T4 effects on TGF{alpha}-induced MAPK activation and immediate-early gene expression were similar to those of the cAMP analog, 8-Br-cAMP. We have previously shown that enhancement by T4 of the antiviral action of interferon-{gamma} (4, 6) and HLA-DR expression (5) induced by the cytokine was dependent on activation of PKA by T4, and that inhibition of T4-induced nuclear complexing of MAPK with the nuclear receptor TRß1 is seen with the PKA inhibitor, KT5720 (9), again implicating T4 in PKA activation.

MAPK activation by TGF{alpha} was potentiated by a low concentration of 8-Br-cAMP (10 nM) but not by 8-Cl-cAMP. Induction of immediate-early gene expression by TGF{alpha}, however, was inhibited by 8-Br-cAMP but enhanced by 8-Cl-cAMP. TGF{alpha}-induced MAPK activation and immediate-early gene expression were both inhibited by the PKA inhibitor KT5720. On the other hand, EGF-induced MAPK activation was inhibited by 8-Br-cAMP (10 nM), as has been shown by other groups (27), and was slightly enhanced by 8-Cl-cAMP (Fig. 3Go). Together, these findings suggest that MAPK activation by TGF{alpha} is enhanced by PKA-II but that immediate-early gene induction by TGF{alpha} is inhibited by PKA-II activity. Results presented here infer that 8-Br-cAMP activates PKA-II in HeLa cells; 8-Cl-cAMP, described elsewhere as a site-specific analog that acts to decrease PKA-I abundance in several cell lines (28, 29, 30), had a spectrum of actions largely, but not completely, different in HeLa cells from those of 8-Br-cAMP. The mechanisms of actions of these cAMP analogs may thus vary among cell lines.

PKA that is activated in cultured thyrocytes by 8-Br-cAMP has been reported by Heinrich and Kraiem (31) to inhibit immediate-early gene expression when cells are also exposed to EGF. In the present studies, PKA activation by thyroid hormone in HeLa cells enhanced the action of EGF on immediate-early gene expression but inhibited the action of TGF{alpha} on c-fos expression. Thus, PKA activation in the context of growth factor action at the EGFR has signal transduction effects that are cell type and growth factor specific.

PKA has been shown to serine phosphorylate the EGFR in cells exposed to a nonhydrolyzable cAMP analog (15). This action decreases the intrinsic tyrosine kinase activity of the receptor and EGFR-generated signal transduction. In contrast, stimulation of PKA activity by T4 is not likely to reflect a short loop effect of PKA on EGFR because the signals of both EGF and TGF{alpha} upstream of MAPK activation are enhanced by thyroid hormone.

The results of studies of T4 on the actions of EGF and TGF{alpha} on immediate-early gene expression, obviously downstream of MAPK, were the initial indication of the ability of EGFR to differentiate between the two growth factors. However, we also examined cell proliferation as an index of the significance of the hormone’s ability to disclose discrimination by EGFR between EGF and TGF{alpha}. Using PCNA measurement to monitor proliferation, we found that T4 enhanced the effect of EGF on cell growth but decreased the action of TGF{alpha} on cell proliferation. Thus, the discrimination by EGFR between the growth factors, unmasked by thyroid hormone, can be expressed at the level of a fundamental cellular function.

In our initial studies of the effects of cAMP analogs on MAPK activation in HeLa cells, we used 100-µM analog concentrations that are conventionally applied to cAMP-responsive models (32, 33, 34). Such concentrations appeared to decrease MAPK activity, as others have reported (33). Depending on the model and experimental conditions, however, high concentrations of cAMP analogs may stimulate MAPK activity (35). Low concentrations of cAMP analogs have also been reported to be biologically active. For example, 1–5 µM 8-Cl-cAMP is known to reduce growth in myeloma cells (36) and down-regulate RI{alpha} subunit of cAMP protein kinase and up-regulate PKA-II in HL-60 cells (28). Against this background of apparent effectiveness of lower concentrations of cAMP analogs, we carried out studies with 8-Br-cAMP concentrations from 0.1 nM to 100 µM and found in HeLa cells that even submicromolar concentrations may alter the effect of TGF{alpha} on c-Fos expression (Fig. 6AGo).

We have reported previously that T4 activates the MAPK pathway via a G protein-sensitive receptor at the cell surface (1). Hormone stimulation of the MAPK cascade requires activation, upstream of MAPK, of PKC and Raf-1 (1). We (1) and others (38, 39) have demonstrated that the action of T4 not unexpectedly involves phospholipase C and PKC. Evidence is presented here that T4 in addition stimulates serine phosphorylation of PKA isoform-II in HeLa cells and stimulates PKA activity; phosphorylation of regulatory subunit II of PKA has been shown by others to be associated with activation of PKA (24, 25, 26). TGF{alpha} also stimulates PKA-II serine phosphorylation and stimulates PKA activity. These effects of TGF{alpha} appear to be potentiated by T4. However, when cells are exposed to T4 and TGF{alpha}, T4-activated PKA-II appears to inhibit TGF{alpha}-induced c-Fos expression. We have shown above that T4 enhances the effect of TGF{alpha} on MAPK activation and have seen in unpublished studies that CGP41251, an inhibitor of traditional PKC{alpha}, -ß1, -ß2, and -{gamma}, inhibits T4 potentiation of MAPK activation by EGF and TGF{alpha}. The same PKC inhibitor suppresses T4 potentiation of c-Fos expression by EGF (3). Thus, the actions of T4 on MAPK and PKA-II activation support the existence of cross-talk between the PKC and PKA pathways, as described by others (14).

In a model of prostaglandin action, rather than growth factor action, Tokuda et al. (40) showed that T3 inhibited both PKA and PKC pathways to impair prostaglandin-induced IL-6 synthesis in osteoblasts. Others have shown that physiological concentrations of T3 may inhibit PKC activity in isolated liver cells, reducing an adrenoceptor-mediated increase in intracellular free Ca2+ (41). Thus, depending on the cell system and the particular activator(s) acting on the cell, thyroid hormone can activate or inhibit PKA and PKC pathways to modulate transduction of signals.

From results of the studies reported here, it is proposed that physiologic levels of thyroid hormone may enhance the autocrine/paracrine effects of EGF on cells but block the effects of TGF{alpha}. This may be relevant to tumor cell growth. In this report, we have presented evidence that T4 suppresses cell proliferation induced by TGF{alpha}. It has been shown by others that genomic cellular actions of thyroid hormone include increased expression of the EGF gene (42, 43). It is therefore likely that in the euthyroid organism, many cells are elaborating these growth factors in response to physiologic levels of thyroid hormone.

The actions of T4 reported here, however, are nongenomic. That is, the actions do not primarily require the participation of the nuclear thyroid hormone receptor (44) and occur in cells, such as HeLa cells, which do not possess a nuclear thyroid hormone receptor (10). We have shown elsewhere that T4 nongenomically potentiates EGF action through cross-talk of MAPK with STAT3 in HeLa cells (3). In the present studies, the nongenomic nature of the effect of T4 was substantiated by: 1) the action of tetrac, a hormone analog that blocks the actions of T4 at the cell surface (1, 9), and T4-agarose, which does not permeate the plasma membrane (1); 2) the participation of the MAPK pathway, including Ras, in the actions of the hormone; and 3) the rapid onset of the effects of T4 on signal transduction induced by TGF{alpha} and EGF.

Whereas we show here that the cell surface EGFR is able to distinguish between its two polypeptide ligands, EGF and TGF{alpha}, the basis for this capacity of the receptor is not known. We propose that the physical interactions of the two growth factors with EGFR are different and that the distinctive protein-protein interactions cause a change in EGFR conformation that may alter the access of the transmembrane segment of the receptor to lipid rafts that contain signal transducing proteins (45).


   Footnotes
 
This work was supported by the Office of Research Development, Medical Research Service, Department of Veterans Affairs (to P.J.D. and H.-Y.L.) and the Charitable Leadership Foundation, Candace King Weir Foundation, and Beltrone Foundation.

Abbreviations: AP-1, Activator protein-1; 8-Br-cAMP, 8-bromo-cAMP; 8-Cl-cAMP, 8-chloro-cAMP; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IOD, integrated OD; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; pERK, phosphorylated ERK; PKA, cyclic adenosine monophosphate-dependent kinase; PKC, protein kinase C; STAT, signal transducer and activator of transcription; tetrac, tetraiodothyroacetic acid.

Received June 12, 2003.

Accepted for publication December 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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