Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells

Hung-Yun Lin1,2, Faith B. Davis1,2, Jennifer K. Gordinier1,2, Leon J. Martino1, and Paul J. Davis1,2

1 Division of Molecular and Cellular Medicine, Department of Medicine, Albany Medical College and 2 Veterans Affairs Healthcare Network Upstate New York, Albany, New York 12208


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thyroid hormone [L-thyroxine (T4)] rapidly induced phosphorylation and nuclear translocation (activation) of mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the absence of cytokine or growth factor. A pertussis toxin-sensitive and guanosine 5'-O-(3-thiotriphosphate)-sensitive cell surface mechanism responsive to T4 and agarose-T4, suggesting a G protein-coupled receptor, was implicated. Cells depleted of MAPK or treated with MAPK pathway inhibitors showed reduced activation of MAPK and of the signal transducer and activator of transcription STAT1alpha by T4; they also showed reduced T4 potentiation of the antiviral action of interferon-gamma (IFN-gamma ). T4 treatment caused tyrosine-phosphorylated MAPK-STAT1alpha nuclear complex formation and enhanced Ser-727 phosphorylation of STAT1alpha , in the presence or absence of IFN-gamma . STAT1alpha -deficient cells transfected with STAT1alpha containing an alanine-for-serine substitution at residue 727 (STAT1alpha A727) showed minimal T4-stimulated STAT1alpha activation. IFN-gamma induced the antiviral state in cells containing wild-type STAT1alpha (STAT1alpha wt) or STAT1alpha A727; T4 potentiated IFN-gamma action in STAT1alpha wt cells but not in STAT1alpha A727 cells. T4-directed STAT1alpha Ser-727 phosphorylation is MAPK mediated and results in potentiated STAT1alpha activation and enhanced IFN-gamma activity.

thyroxine; signal transducer and activator of transcription 1alpha ; signal transduction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PHYSIOLOGICAL CONCENTRATIONS of thyroid hormone potentiate the antiviral state induced by homologous interferon (IFN)-gamma (21, 22). Antiviral action of IFN-gamma requires tyrosine phosphorylation of the signal transducer and activator of transcription STAT1alpha (7). Maximal antiviral activity of IFN-gamma is obtained when Ser-727 of STAT1alpha is phosphorylated (17), perhaps by mitogen-activated protein kinase (MAPK) (8) or another serine kinase (45). The 44- and 42-kDa MAPK isoforms extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2, respectively) are ubiquitously expressed serine/threonine kinases that are activated by dual-specificity MAPK kinases (MEK1 and MEK2) in response to diverse agonists (28). A number of receptor tyrosine kinases, cytokine receptors, and heterotrimeric G proteins have been shown to activate MEK and MAPK (3, 12).

We have recently shown that thyroid hormone promotes tyrosine phosphorylation and nuclear uptake of STAT1alpha (20) and have speculated that the hormone may activate the MAPK pathway to obtain maximal activation of STAT1alpha and potentiation of the biological activity of IFN-gamma . We report here that thyroid hormone [L-thyroxine (T4) or 3,5,3'-triiodo-L-thyronine (T3)] indeed activates the MAPK cascade in HeLa and CV-1 cells, in both the absence and presence of IFN-gamma . Components of the mechanism by which thyroid hormone activates the MAPK pathway are also described. Because neither HeLa cells (33) nor CV-1 cells (23) contain functional nuclear thyroid hormone receptor (TR), the actions of thyroid hormone on kinase activities in these cell lines are not mediated by TR.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. T4, T3, 3,3',5'-triiodothyronine (rT3), 3,3',5,5'-tetraiodothyroacetic acid (tetrac), T4-agarose, protein A-agarose, and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) were obtained from Sigma Chemical (St. Louis, MO), and recombinant human IFN-gamma was from BioSource International (Camarillo, CA). Pertussis toxin was obtained from Calbiochem (San Diego, CA). CGP-41251 was a gift from Novartis Pharma (Basel, Switzerland), and Lipofectin was obtained from GIBCO BRL (Grand Island, NY). Genistein was obtained from ICN Biochemicals (Costa Mesa, CA), geldanamycin came from the Drug Synthesis and Chemistry Branch, National Cancer Institute (Bethesda, MD), PD-98059 was from Calbiochem (La Jolla, CA), and U-73122 and U-73343 were obtained from Dr. Robert Smallridge (Mayo Clinic, Jacksonville, FL). HeLa cells were obtained from the American Type Culture Collection (Manassas, VA), CV-1 cells were from Dr. Paul M. Yen (National Institutes of Health), and 293T cells were from Dr. Kevin Pumiglia (Albany Medical College, Albany, NY). U3A, STAT1alpha wt, and STAT1alpha A727 cells, derived from human fibroblasts (26, 42), were obtained with the permission of Dr. George Stark (Cleveland Clinic Foundation, Cleveland, OH) from the laboratories of Drs. James E. Darnell, Jr., (Rockefeller University, New York, NY) and Ke Shuai (University of California School of Medicine, Los Angeles, CA). [32P]NAD was obtained from DuPont-NEN (Boston, MA).

Cell culture and preparation of nuclear fractions. Confluent HeLa and CV-1 cells grown in 100-mm culture dishes were treated with 0.25% hormone-depleted fetal bovine serum-containing medium (22) for 48 h. The U3A cell series were grown in the same serum-supplemented medium with 400 µg/ml G418 added. The total and free T4 concentrations in this serum-supplemented medium were 2.3 × 10-11 M and 10-14 M, respectively, and the free T3 concentration was below detectable levels (23). Hormone, hormone analogs, IFN-gamma , and/or inhibitors were then added for different time periods as indicated. Stock solutions (10-4 M) of hormone and analogs were prepared in 0.04 N KOH-4% propylene glycol, and dilutions were made to final concentrations as indicated. In all experiments in which T4 was added to cultures, the total and free T4 concentrations were 10-7 M and 10-10 M, respectively, and total and free T3 levels were below the limits of measurement. The hormone solvent had no effect on signal transduction studies. T4-agarose was provided as a suspension in 0.5 M NaCl containing ~6 mM T4 and was diluted in culture medium to a final T4 concentration of 10-7 M.

After treatment, cells were harvested and nuclear extracts were prepared as follows: the 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 (DTT)] with 0.2% NP-40 on ice for 10 min. After centrifugation at 4°C and 13,000 rpm for 1 min, supernatants were collected as cytoplasmic extracts. Nuclear extracts were prepared according to the method of Wen et al. (42) 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.

Immunoprecipitation and immunoblotting. After normalization of sample protein content, immunoprecipitation was performed using polyclonal anti-phosphotyrosine antibody (Transduction Laboratories, Lexington, KY). After overnight incubation at 4°C with rocking, protein A-agarose was added and samples were rocked for 1 h at 4°C. After two washes with hypotonic buffer containing 0.2% NP-40, immunoprecipitates were eluted with 2× sample buffer, and proteins were separated by discontinuous SDS-PAGE (7.5-9.0%). Proteins were transferred to Immobilon membranes (Millipore, Bedford, MA) by electroblotting. After being blocked with 5% milk in Tris-buffered saline containing 0.1% Tween, membranes were incubated with 1:1,000 monoclonal anti-MAPK antibody (ERK2) (Transduction Laboratories) or with 1:1,000 monoclonal anti-STAT1alpha antibody (Transduction Laboratories) overnight. For selected studies, 1:1,000 polyclonal anti-tyrosine/threonine-phosphorylated MAPK antibody (New England BioLabs, Beverly, MA) was used for immunoblots. In some experiments (see Fig. 5), nuclear extracts were immunoprecipitated with monoclonal anti-STAT1alpha or anti-MAPK antibody (Transduction Laboratories), and the immunoprecipitates were then separated by PAGE and immunoblotted with antibody to MAPK or STAT1alpha , respectively. Polyclonal antibodies to Ser-727-phosphorylated STAT1alpha and to amino acids 73-93 of TRbeta 1 were generously provided by Dr. David Frank (Dana-Farber Cancer Institute, Boston, MA) and Dr. William Chin (Brigham and Women's Hospital, Boston, MA), respectively. The secondary antibodies were rabbit anti-mouse IgG or goat anti-rabbit IgG (1:1,000, DAKO, Carpenteria, CA). Immunoblots were visualized by enhanced chemiluminescence (ECL; Amersham Life Science, Arlington Heights, IL) and quantitated by digital imaging (BioImage, Millipore). Immunoblots shown are representative of two or more experiments.

Cell treatments. Cells were treated with thyroid hormone or analogs and/or IFN-gamma in the concentrations indicated. Different concentrations of the protein kinase C (PKC) inhibitor CGP-41251 (5-100 nM), genistein, a protein tyrosine kinase (PTK) inhibitor (1-100 µg/ml), or pertussis toxin (20-1,000 ng/ml) were added to cultures for 70 min, and T4 (10-7 M) was added for the last 30 min. U-73122, a phospholipase C (PLC) inhibitor (1-10 µM), its inactive analog U-73343, or GTPgamma S (10-8 to 10-5 M) was added for 60 min, and T4 was added for the last 30 min. Geldanamycin (1-10 µM) or PD-98059 (30 µM) was applied to cells for 16 h, and 10-7 M T4 was added for the last 30 min. Cells were harvested, and nuclear proteins were prepared as described. DMSO (0.1%) was the solvent for all inhibitors and had no effect itself on immunoprecipitation, immunoblotting, or antiviral studies.

Oligonucleotide transfection. HeLa cells were treated, as described by Glennon et al. (15), with 2.5 µg/ml Lipofectin for 6 h, and sense or antisense oligonucleotides (Operon Technologies, Alameda, CA) were applied for 48 h in a concentration of 10 µM. Selected cells from each treatment group were then exposed to 10-7 M T4 for 40 min and subsequently harvested for preparation of nuclear extracts, immunoprecipitation, and immunoblotting as described above.

Antiviral studies. Cells were exposed to IFN-gamma (1.0 IU/ml) in the presence or absence of T4 (10-7 M) for the last 24 h of MAPK antisense oligonucleotide transfection. In studies with MAPK pathway inhibitors, cells were treated with T4, with or without geldanamycin or PD-98059, for 24 h; the medium was then replaced, and cells were treated with IFN-gamma (1.0 IU/ml) for 24 h. STAT1alpha wt and STAT1alpha A727 cells were treated with IFN-gamma , with or without T4, for 24 h. After these various treatments, the cells were infected with vesicular stomatitis virus, and an antiviral plaque assay was performed as described previously (22), with results expressed in plaque-forming units (pfu) per milliliter. In our antiviral studies, a maximal effect of IFN-gamma is seen at a cytokine concentration of 1,000 IU/ml, but T4 potentiation of IFN-gamma action is greatest at submaximal IFN-gamma concentrations [1-10 IU/ml (22)]. The plaque assay method does not allow accurate measurement of virus yield below 104 pfu/ml. One-way ANOVA was used to determine statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Thyroxine and analogs cause tyrosine phosphorylation and nuclear translocation of MAPK. Initial studies were performed with T4 alone, in the absence of IFN-gamma . Confluent HeLa cells were treated with 0.25% thyroid hormone-depleted serum-supplemented medium for 2 days, and T4 (10-7 M total, 10-10 M free) was then added to cultures for 10-60 min. This concentration of T4 is physiological. Cells were harvested, and nuclear extracts were immunoprecipitated with anti-phosphotyrosine antibody. Immunoprecipitated proteins were eluted from protein A-agarose, separated by electrophoresis, and immunoblotted with anti-MAPK antibody. Increased nuclear content of tyrosine-phosphorylated MAPK was detected at 10 min after exposure to T4 (Fig. 1A). This transient effect was maximal at 30-40 min and significantly reduced or absent at 60 min. In four experiments, the increase in band density with T4 addition for 30 min, compared with a control sample without hormone, was 25 ± 8-fold (means ± SE). Nuclear tyrosine-phosphorylated MAPK was also detected by immunoblotting of nuclear samples with anti-tyrosine/threonine-phosphorylated MAPK antibody, as shown in Fig. 1B. In the study shown, accumulation of nuclear activated MAPK was maximal at 30 min, and there was a 34-fold increase in combined band intensities of ERK1 and ERK2.


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Fig. 1.   Effect of L-thyroxine (T4) on tyrosine phosphorylation and nuclear translocation of mitogen-activated protein kinase (MAPK). A: nuclear extracts of HeLa cells treated with 10-7 M T4 for 10-60 min were immunoprecipitated with anti-phosphotyrosine antibody; immunoprecipitated proteins were eluted, separated by gel electrophoresis, and immunoblotted with anti-MAPK antibody. Nuclear accumulation of tyrosine-phosphorylated MAPK is seen as early as 10 min, with a maximal level seen at 30 min of T4 treatment. Band shown is 42-kDa extracellular signal-regulated kinase (ERK) 2. B: aliquots of similar nuclear fractions were immunoblotted with antibody to tyrosine-threonine-phosphorylated MAPK, without immunoprecipitation. Again, accumulation of phosphorylated MAPK is seen within 10 min of T4 treatment, reaches a maximum between 30 and 40 min, and is diminished at 60 min. Bands represent ERK1 (44 kDa) and ERK2 (42 kDa).

At a supraphysiological total T3 concentration of 10-7 M, this hormone also increased tyrosine phosphorylation and nuclear translocation (activation) of MAPK in 30 min, but a physiological concentration of T3 (10-10 M) was less effective (Fig. 2A, lanes 2 and 3). Interestingly, rT3 (10-7 M) also increased nuclear translocation of activated MAPK (Fig. 2A, lane 5). Maximal effects of T3 and rT3 were seen at 30 min, as with T4 (results not shown). These results with T3 and rT3 parallel the previously reported relative potentiation by thyroid hormone analogs of the IFN-gamma -induced antiviral state, in that T4 was more effective than T3 at physiological concentrations and rT3 also had a potentiating effect in the antiviral model (21).


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Fig. 2.   Effect of T4, 3,5,3'-triiodo-L-thyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3',5,5'-tetraiodothyroacetic acid (tetrac), and T4-agarose on activation of MAPK. A: HeLa cells were treated with T4 (10-7 M, a physiological concentration), or indicated concentrations of T3 or rT3 for 30 min. Nuclear samples were immunoprecipitated with anti-phosphotyrosine antibody, and precipitates were immunoblotted with anti-MAPK antibody. Neither T3 nor rT3 was as effective as T4 in activating MAPK, although each had some effect at supraphysiological concentrations (10-7 M). Effects that were seen with T3 and rT3 were maximal at 30 min, as shown. B: HeLa cells were treated with T4, with or without tetrac, for 30 min. T4 stimulated tyrosine phosphorylation and nuclear accumulation of MAPK (lane 2), whereas tetrac had minimal effect (lane 3). However, tetrac reduced T4 action in a dose-dependent manner (lanes 4 and 5, compared with lane 2). C: HeLa cells were treated with 10-7 M T4, T4-agarose containing 10-7 M T4 (T4-ag), T4-agarose clarified by 3 washes in PBS (T4-agw), or protein A-agarose (pA-ag) for 30 min, after which nuclear accumulation of activated MAPK was measured. Protein A-agarose (lane 2) had no effect on MAPK activation, whereas T4-agarose (lane 4) was as effective as T4 in activation of MAPK. T4-agarose washed 3 times and reconstituted in same T4 concentration of 10-7 M (lane 5) was as effective as T4. Lane 1, control (con).

Cell surface action of T4. The T4 analog tetrac blocks 1) T4 potentiation of IFN-gamma -induced human leukocyte antigen-DR (HLA-DR) expression (20), 2) T4 potentiation of IFN-gamma -induced antiviral activity (21, 23), and 3) T4-induced activation of STAT1alpha (20). Tetrac also partially inhibited the effect of T4 on tyrosine phosphorylation and nuclear translocation of MAPK, as shown in Fig. 2B (lanes 4 and 5, compared with lane 2). We previously showed that tetrac inhibits binding of T4 to isolated human erythrocyte membranes (10) and therefore postulated that tetrac blocks T4 activation of signal-transducing proteins by inhibition of T4 interaction at the cell surface. To further study this possibility, we treated HeLa cells with T4-agarose. In Fig. 2C, we show that T4, T4-agarose, and T4-agarose clarified with three PBS washes all stimulated tyrosine phosphorylation and nuclear translocation of MAPK (lanes 3-5), whereas protein A-agarose had no effect on MAPK activation (lane 2). These findings further support the action of T4 at the cell membrane as an enhancer of signal transduction. We have also conducted these studies in CV-1 cells, which like HeLa cells lack a traditional TR (23), and have obtained similar results (not shown).

Involvement of a G protein-coupled mechanism of hormone action. Della Rocca et al. (11) reported rapid activation of MAPK by alpha -adrenergic agonists via pathways mediated by the G proteins Gi and Gq. To define the possible role of a G protein-coupled mechanism at the cell surface in the action of T4, GTPgamma S was added to HeLa cells for 70 min, and T4 was added for the last 30 min of GTPgamma S treatment. Nuclear fractions were prepared and immunoblotted with anti-phosphorylated MAPK antibody, and the results are shown in Fig. 3A. There is T4-induced phosphorylation and nuclear accumulation of MAPK and dose-dependent reduction of the T4 effect by GTPgamma S. HeLa cells were also treated with pertussis toxin (20-1,000 ng/ml) for 60 min and with T4 for 30 min; immunoblots with phosphorylated MAPK antibody showed a dose-dependent reduction of the T4 effect by pertussis toxin (Fig. 3B). In the absence of T4, pertussis toxin did not alter MAPK phosphorylation (results not shown). These results suggest a contribution of a pertussis-toxin-inhibitable GTP-binding protein (Gi or Go) to the T4 effect and raise the possibility that thyroid hormone binds to a Gi protein-coupled receptor (GPCR) at the cell membrane.


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Fig. 3.   Effect of guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) and pertussis toxin on T4-induced activation of MAPK. A: HeLa cells were treated with GTPgamma S (10-8 to 10-5 M) for 70 min and T4 (10-7 M) for 30 min. Nuclear samples were immunoblotted with anti-tyrosine-threonine-phosphorylated MAPK. Activation of MAPK by T4 was reduced by GTPgamma S in a dose-dependent manner. GTPgamma S alone had no effect on MAPK activation. B: HeLa cells were treated with pertussis toxin (PT; 20-1,000 ng/ml) for 60 min and T4 (10-7 M) for 30 min. Nuclear samples were immunoblotted as in A. Again, there was a dose-responsive reduction in MAPK activation by T4 with addition of pertussis toxin.

HeLa cells were exposed to pertussis toxin (200 or 1,000 ng/ml) or control solvent for 60 min, after which cell lysates were exposed to [32P]NAD in the presence of pertussis toxin (25 µg/ml) and DTT, as we previously described (9). Control lysates showed abundant labeling of a 39-kDa protein band, whereas lysates from cells treated with 200 and 1,000 ng/ml pertussis toxin showed diminished and absent labeling, respectively, of the same band, thus confirming that these concentrations of the toxin did cause ADP ribosylation during the intact cell incubation in a concentration-dependent manner (results not shown).

Contributions of PKC, PTK, and PLC to the thyroxine effect. We previously reported that T4 potentiation of IFN-gamma -induced HLA-DR expression and antiviral activity is dependent on activities of PKC and PTK (20, 23). Investigation of the effects of kinase inhibitors on T4 activation of MAPK was therefore undertaken. Genistein (1-100 µg/ml), an inhibitor of PTK activity (30), blocked the action of T4 on MAPK phosphorylation and nuclear translocation (Fig. 4A). CGP-41251, an inhibitor of calcium-dependent PKC activity [inhibits PKCalpha , -beta I, -beta II, and -gamma with IC50 values of 24, 17, 32, and 18 nM, respectively (25)], also blocked T4-induced tyrosine phosphorylation of MAPK in a concentration-dependent manner (Fig. 4B). In Fig. 4C, genistein and CGP-41251 are again shown to inhibit the effect of T4 on MAPK phosphorylation and nuclear translocation in nuclear samples immunoblotted with antibody to tyrosine/threonine-phosphorylated MAPK. The two bands representing ERK1 (44 kDa) and ERK2 (42 kDa) are similarly affected by T4 and the inhibitors. The effect of the PLC inhibitor U-73122 (35) and an inactive analog, U-73343, on T4 activation of MAPK was also examined. Figure 4D shows activation of MAPK by T4 in HeLa cells and inhibition of this MAPK activation by U-73122 but not by U-73343. Similar results were obtained with 293T cells, which contain TR, as indicated by immunoblotting with antibody to TRbeta (results not shown).


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Fig. 4.   Role of protein tyrosine kinase, protein kinase C, and phospholipase C (PLC) in activation of MAPK by T4. Detection of phosphorylated MAPK was by anti-phosphotyrosine immunoprecipitation and anti-MAPK immunoblotting in A and B and by anti-phosphorylated MAPK immunoblotting in C and D. In all studies, inhibitor was added for 60 min and T4 (10-7 M) for last 30 min. A: with addition to HeLa cells of T4 and genistein (Gen; 1-100 µg/ml), there was dose-dependent inhibition of T4 effect on tyrosine phosphorylation and nuclear translocation of MAPK. B: CGP-41251 (CGP; 5-100 nM) also caused dose-dependent inhibition of T4-induced MAPK activation in HeLa cells. C: appearance of two activated MAPK bands (42-kDa ERK2 and 44-kDa ERK1) in nuclear fractions of HeLa cells treated with T4 was also demonstrated with use of antibody to phosphorylated MAPK, without prior immunoprecipitation. Inhibition of T4 effect is again seen with both genistein and CGP-41251, similar to findings in A and B. D: in HeLa cells, U-73122 (1 and 10 µM) but not inactive analog U-73343 reduced or completely inhibited T4-induced nuclear accumulation of phosphorylated MAPK, as shown by immunoblotting with anti-phosphorylated MAPK antibody.

Coimmunoprecipitation of MAPK and STAT1alpha . David et al. (8) described coimmunoprecipitation of MAPK and STAT1alpha in extracts of cells treated with IFN-beta . We therefore tested the possibility that a direct interaction between MAPK and STAT1alpha could be detected in cells treated with T4, alone or with IFN-gamma . Nuclear extracts were immunoprecipitated with anti-MAPK antibody, and the solubilized immunoprecipitates were subjected to electrophoresis and immunoblotted with anti-STAT1alpha . Nuclear STAT1alpha appeared in MAPK immunoprecipitates in cells treated with T4 alone (10-7 M), as shown in Fig. 5A. In the study shown in Fig. 5B, cells were treated with T4 with or without IFN-gamma (1-100 IU/ml) for 30 min. The antibody order was reversed, and samples of nuclear extracts were immunoprecipitated with anti-STAT1alpha antibody and the precipitates were immunoblotted with anti-MAPK antibody. Both T4 and IFN-gamma separately caused nuclear complexing of STAT1alpha and MAPK, as reported previously with IFN-beta (8). At each concentration of IFN-gamma , T4 enhanced the cytokine effect. Additional studies using an antibody to Ser-727-phosphorylated STAT1alpha (13) demonstrated increased Ser-727 phosphorylation of STAT1alpha by T4 or IFN-gamma and potentiation of the cytokine effect by thyroid hormone (Fig. 5C). T4 and IFN-gamma (1.0 IU/ml), when applied separately to cells, caused detectable Ser-727 phosphorylation. With addition of hormone together with a low concentration of cytokine (1 IU/ml), there was enhancement of IFN-gamma -induced Ser-727 phosphorylation by T4. With a higher IFN-gamma concentration, there was no further enhancement of the IFN-gamma effect by T4.


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Fig. 5.   Induction by T4, with or without interferon-gamma (IFN-gamma ), of MAPK and STAT1alpha nuclear complexes. A: HeLa cells were treated with T4 (10-7 M) for times indicated. Nuclear extracts were immunoprecipitated with anti-MAPK antibody, and precipitated proteins were separated by PAGE and immunoblotted with anti-STAT1alpha antibody. Shown is accumulation in up to 30 min of nuclear complexed MAPK and STAT1alpha , with subsequent loss of complex by 120 min. B: HeLa cells were treated with IFN-gamma (1-100 IU/ml) for 30 min in presence or absence of T4 (10-7 M). Nuclear fractions were immunoprecipitated with anti-STAT1alpha antibody, and resulting proteins were eluted, separated by PAGE, and immunoblotted with anti-MAPK antibody. Nuclear MAPK coimmunoprecipitated with STAT1alpha was increased in amount in an IFN-gamma dose-dependent manner (lanes 3, 5, and 7). With each IFN-gamma dose, addition of T4 further enhanced MAPK accumulation and STAT1alpha -MAPK complex formation (lanes 4, 6, and 8). C: HeLa cells were treated with IFN-gamma (1 or 100 IU/ml) and/or T4 (10-7 M) for 30 min. Nuclear fractions were immunoblotted with antibody to phosphoserine-727-STAT1alpha . Both T4 and a low concentration of IFN-gamma caused some serine phosphorylation of STAT1alpha , and effect of IFN-gamma (1 IU/ml) was enhanced by cotreatment with T4. No T4 enhancement was seen with a higher concentration of IFN-gamma .

Effect of MAPK pathway inhibition. Studies were undertaken to characterize more proximal steps in the MAPK cascade that might contribute to the T4 effect on activation of MAPK and STAT1alpha and potentiation of IFN-gamma action. Geldanamycin, a MAPK pathway inhibitor that incompletely depletes cellular content of Raf-1 (32, 36), partially inhibited T4-stimulated nuclear uptake of tyrosine-phosphorylated MAPK (Fig. 6A, top, lane 3) and tyrosine-phosphorylated STAT1alpha (Fig. 6A, bottom, lane 3). Similar findings were obtained with an inhibitor of MEK, PD-98059 (27), and are shown in Fig. 6A (lanes 4-6); there was reduction or complete inhibition of T4-stimulated activation of MAPK and STAT1alpha by PD-98059 (lane 6, top and bottom).


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Fig. 6.   Effect of MAPK pathway inhibition on activation and nuclear translocation of MAPK and STAT1alpha and on T4 potentiation of IFN-gamma -induced antiviral activity. A: HeLa cells were treated with T4 (10-7 M) for 30 min after pretreatment with MAPK pathway inhibitors geldanamycin (Gel) or PD-98059 (PD) for 16 h. Anti-phosphotyrosine immunoprecipitates from nuclear samples were immunoblotted with antibody to MAPK or STAT1alpha . Lanes 1 and 2 and lanes 4 and 5 show T4 activation of MAPK and STAT1alpha (top and bottom, respectively); inhibition of T4 effect is seen with both geldanamycin (10 µM) and PD-98059 (30 µM) (lanes 3 and 6, respectively). B: antiviral studies were conducted on HeLa cells treated with geldanamycin (1 or 10 µM) or PD-98059 (3 or 30 µM) for 12 h. Medium was then removed, and fresh medium containing IFN-gamma (1 IU/ml) with or without T4 (10-7 M) was added for 24 h. An antiviral assay was then conducted, with virus yield shown in plaque-forming units (pfu)/ml. Results show means ± SE of 3 studies with each inhibitor. Control virus yield is shown in open bars. IFN-gamma antiviral effect is shown by solid bars, indicating a reduction in virus yield, and significant (P < 0.05 by 1-way ANOVA) potentiation of IFN-gamma effect by T4 is shown by vertical arrows above hatched bars. Both inhibitors blocked T4 potentiation in a dose-dependent manner, but neither had an effect on antiviral action of this IFN-gamma concentration in absence of thyroid hormone, as shown in cross-ruled bars.

To correlate results of signal transduction studies with effects on T4 potentiation of IFN-gamma action, we measured the antiviral activity of IFN-gamma in the presence and absence of T4, geldanamycin, and PD-98059. For these studies, a submaximal concentration of IFN-gamma (1 IU/ml) was used, which permits up to 100-fold potentiation of the antiviral effect of the cytokine by 10-7 M T4 (22). In Fig. 6B, statistically significant T4 potentiation (P < 0.05) is shown by the vertical arrows. Geldanamycin in concentrations of 1 and 10 µM, respectively, partially or totally inhibited T4 potentiation of IFN-gamma action (Fig. 6B, left), without altering the effect of IFN-gamma alone. Similar results were obtained with PD-98059 (Fig. 6B, right), in that there was concentration-dependent inhibition of T4-potentiated IFN-gamma action, without a reduction in the antiviral effect of IFN-gamma alone.

Effect of MAPK antisense oligonucleotide transfection. To further examine the role of the MAPK pathway in T4 potentiation of IFN-gamma action, we reduced the HeLa cell content of MAPK by antisense oligonucleotide transfection (15). Absence of activated MAPK in nuclear fractions of cells treated with the antisense oligonucleotide is shown in Fig. 7A (top, lane 7), and there is little tyrosine phosphorylation or nuclear translocation of MAPK in antisense-treated cells exposed to T4 (top, lane 8), particularly compared with levels in cells treated with Lipofectin alone (lane 4). In the same MAPK-depleted cells, there was a reduction in T4-stimulated nuclear translocation of tyrosine-phosphorylated STAT1alpha , compared with findings in HeLa cells not exposed to the MAPK antisense oligonucleotide (Fig. 7A, bottom, lane 8 compared with lanes 2 and 4). This finding suggests that the presence of stimulable MAPK is a requirement for optimal activation and nuclear translocation of STAT1alpha . In cells treated with sense oligonucleotide, there was more activated MAPK in nuclei of cells not exposed to T4 than in cells exposed to the hormone (Fig. 7A, top, lanes 5 and 6). The basal increase in MAPK and reduced activity of MAPK in response to an inducer in these cells have been described by other investigators in a different cell line (29). To test the effect of cellular MAPK depletion on T4 potentiation of IFN-gamma action, antiviral studies were also conducted on cells transfected with MAPK antisense or sense oligonucleotide or with Lipofectin alone. Significant T4 potentiation of the antiviral action of IFN-gamma (P < 0.05), shown by the vertical arrows in Fig. 7B, was demonstrated in untreated (control) cells and cells treated with sense oligonucleotide or Lipofectin alone but was diminished in MAPK-depleted cells. In the latter cells, however, the antiviral response to IFN-gamma (1.0 IU/ml) in the absence of T4 was not diminished. Taken together, the results of experiments with MAPK pathway inhibition and cellular depletion of MAPK provide evidence that T4-induced activation and nuclear translocation of MAPK and STAT1alpha , and potentiation of the action of IFN-gamma , require activity of Raf-1 and MEK, as well as MAPK. These antiviral experiments with MAPK depletion and MAPK pathway inhibitors were conducted with submaximal concentrations of IFN-gamma .


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Fig. 7.   Effect of MAPK depletion on activation and nuclear translocation of MAPK and STAT1alpha and on T4 enhancement of IFN-gamma -induced antiviral activity. A: HeLa cells were depleted of MAPK as described in MATERIALS AND METHODS and then exposed to T4 or solvent for 30 min. Nuclear extracts of treated cells were immunoprecipitated with anti-phosphotyrosine antibody, and precipitated proteins were immunoblotted with anti-MAPK or anti-STAT1alpha antibody. In cells transfected with MAPK antisense oligonucleotide, there was no nuclear tyrosine-phosphorylated MAPK in cells not treated with T4 (lane 7), and a small amount of nuclear activated MAPK in cells that were treated with T4 (10-7 M; lane 8). Marked T4 activation of MAPK was seen in cells treated with Lipofectin alone (compare lanes 3 and 4). In cells treated with sense oligonucleotide, there was enhancement of nuclear phosphorylated MAPK in control cells (lane 5) and no potentiation in T4-treated cells (lane 6). STAT1alpha in nuclear fractions was minimally increased by T4 in cells treated with antisense oligonucleotide (bottom, lanes 7 and 8) but was markedly increased in control cells (lanes 1 and 2) and in cells treated with Lipofectin alone (lanes 3 and 4). B: antiviral studies were conducted after exposure of cells to oligonucleotides for 24 h. Cells were treated with IFN-gamma (1.0 IU/ml) with or without T4 (10-7 M) for 24 h, followed by antiviral assay. Significant T4 potentiation of IFN-gamma action is highlighted by vertical arrows. Data are means ± SE of 3 experiments. In HeLa cells treated with MAPK antisense oligonucleotide, there was loss of T4 potentiation of IFN-gamma action, whereas hormone effect remained intact in cells treated with sense oligonucleotide or with Lipofectin alone. There was, however, no loss of IFN-gamma antiviral effect in cells exposed to oligonucleotide transfection or Lipofectin.

T4 potentiation of IFN-gamma action requires Ser-727 on STAT1alpha . Maximal antiviral activity of IFN-gamma requires the presence of a serine at position 727 of STAT1alpha (17). It has been suggested that MAPK may catalyze the Ser-727 phosphorylation of STAT1alpha (8, 42), although more recent studies suggest that the Ras-MAPK pathway is not involved, at least in IFN-gamma -induced serine phosphorylation (45). Because we have demonstrated T4-induced nuclear accumulation of Ser-727-phosphorylated STAT1alpha (Fig. 5C), we studied whether T4 potentiation of IFN-gamma action would be seen in cells lacking serine at position 727. U3A cells lack STAT1alpha (26, 42) and on exposure to T4 showed no nuclear accumulation of that protein (Fig. 8A, top). U3A cells with reconstituted wild-type STAT1alpha (STAT1alpha wt) showed stimulation of STAT1alpha tyrosine phosphorylation and nuclear translocation by T4, whereas U3A cells containing STAT1alpha with an alanine-for-serine substitution at position 727 (STAT1alpha A727) showed diminished activation of that protein (Fig. 8A, top). In the same samples, nuclear accumulation of tyrosine-phosphorylated MAPK appeared to be more intense in U3A cells and STAT1alpha A727 cells than in the STAT1alpha wt cells (Fig. 8A, bottom), demonstrating that activation of MAPK does not require the presence of STAT1alpha , whereas activation of STAT1alpha does seem to require the presence of functional MAPK (see Figs. 6 and 7).


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Fig. 8.   Effect of T4 on activation of signal transduction and on IFN-gamma -induced antiviral activity in cells lacking STAT1alpha or lacking Ser-727 of STAT1alpha . A: U3A cells lacking STAT1alpha , wild-type STAT1alpha (STAT1alpha wt) cells, and STAT1alpha -deficient cells transfected with STAT1alpha containing an alanine-for-serine substitution at residue 727 (STAT1alpha A727 cells) were treated with 10-7 M T4 for 30 min, after which nuclear extracts were immunoprecipitated with anti-phosphotyrosine antibody and precipitates were immunoblotted with anti-STAT1alpha or anti-MAPK antibody. In U3A cells, no STAT1alpha is seen, but activated MAPK readily accumulates in T4-treated nuclear fractions. In STAT1alpha wt cells, nuclear accumulation of activated STAT1alpha and MAPK is seen. In STAT1alpha A727 cells, there is reduced tyrosine-phosphorylated STAT1alpha in nuclear fraction, but MAPK accumulation is present. B: antiviral studies in HeLa and STAT1alpha wt cells show a similar dose-dependent antiviral response to IFN-gamma , with progressively less T4 potentiation seen as a maximal concentration of IFN-gamma is reached. In STAT1alpha A727 cells, antiviral effect at each concentration of IFN-gamma is reduced compared with effect in other cells, and there is no T4 potentiation of cytokine action at any IFN-gamma concentration.

Antiviral studies were conducted in HeLa, STAT1alpha wt, and STAT1alpha A727 cells. In HeLa cells, an IFN-gamma dose-response curve is seen in Fig. 8B; as noted above, the greatest T4 potentiation is seen with a submaximal IFN-gamma concentration of 1 IU/ml. The antiviral effect achieved by IFN-gamma (1 IU/ml) together with T4 was the same as the effect that occurs with 100 IU/ml IFN-gamma alone. The STAT1alpha wt cells also demonstrated an IFN-gamma antiviral response and significant T4 potentiation of the effect of 1 IU/ml IFN-gamma . In the STAT1alpha A727 cells, there was a diminished antiviral response to all concentrations of IFN-gamma , as previously shown by other investigators (17), and no significant T4 potentiation at any IFN-gamma concentration.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present observations support a novel role for thyroid hormone as a modulator of signal transduction. We used a cytokine, IFN-gamma , to demonstrate the biological relevance of thyroid hormone action on two signal transduction pathways. We propose that the enhancement by thyroid hormone of IFN-gamma -induced antiviral activity depends on effects of the hormone on the MAPK cascade and, as a result, on the STAT1alpha pathway that is activated by the cytokine. The consequence of the action of thyroid hormone on MAPK phosphorylation and nuclear translocation is viewed by us to be Ser-727 phosphorylation of the STAT1alpha dimer that results in increased binding of the STAT complex to one or more IFN-gamma -responsive genes. Activated STAT1alpha is a critical phosphoprotein in the transduction of IFN-gamma and IFN-alpha /beta signals (7). Activation of STAT1alpha by IFN-gamma requires Tyr-701 phosphorylation by Janus kinases (JAK1 and JAK2); maximal transcriptional activity of STAT1alpha requires, in addition, phosphorylation of Ser-727 (17, 42). MAPK has been implicated in IFN-alpha /beta signal transduction, specifically in the Ser-727 phosphorylation of STAT1alpha (8) and also in Ser-727 phosphorylation of another member of the STAT family, STAT3 (5).

The ability of the two MAPK pathway inhibitors, geldanamycin and PD-98059, to reduce activation of MAPK and STAT1alpha and to block thyroid hormone potentiation of the antiviral action of IFN-gamma further supports our hypothesis that the mechanism of T4 in this potentiation involves the MAPK pathway, principally at the levels of MEK and MAPK. Geldanamycin incompletely depletes cellular content of Raf-1 in MCF7 cells (32) and HeLa cells (36), whereas PD-98059 is regarded as a specific inhibitor of MEK activity at the concentrations we used (4, 27). Tyrosine phosphorylation of STAT1alpha is an integral part of the IFN-gamma -stimulated JAK-STAT pathway. In the absence of IFN-gamma , however, the means by which T4 caused tyrosine phosphorylation of STAT1alpha required clarification. The results obtained with PD-98059 presented in Fig. 6 suggest that, under the direction of thyroid hormone, MEK tyrosine phosphorylates STAT1alpha , in addition to activating its traditional substrate, MAPK. MEK is a dual-specificity tyrosine-threonine kinase. Phosphorylation of threonines at positions 699 and 704 of STAT1alpha (44) may, as described by Cobb and Goldsmith (6), provide an environment suitable for phosphorylation of Tyr-701 by MEK. Our results do not exclude the possibility that an unidentified MEK-dependent tyrosine kinase is responsible for T4-directed tyrosine phosphorylation of STAT1alpha . Depletion of cellular MAPK by antisense oligonucleotide transfection also confirmed that MAPK is required for T4-induced activation of STAT1alpha and for T4 potentiation of the antiviral activity of IFN-gamma .

Fukuda et al. (14) recently reported that the transport of MAPK from cytoplasm to nucleus requires dissociation of the MAPK-MEK complex. It is possible that thyroid hormone may also act at this putative complex to enhance nuclear transfer of MAPK. It should be noted that tyrosine phosphorylation (activation) of MAPK in the present studies was documented by both anti-phosphotyrosine antibody immunoprecipitation with subsequent probing of the immunoprecipitate with anti-MAPK antibody and by the use of antibody to phosphorylated MAPK, which indicated involvement of both ERK1 and ERK2 in the hormone effect. Gorenne et al. (16) recently demonstrated correlation of two measurements of MAPK activation, 1) immunoblotting with antibody to tyrosine/threonine-phosphorylated MAPK and 2) direct measurement of MAPK activity by phosphorylation of myelin basic protein.

The initial step in the mechanism by which thyroid hormone acts on signal transduction is incompletely understood. Several features are clear, however. Because agarose-T4 is as effective as T4 in activating MAPK and STAT1alpha , hormone action must begin at the cell surface. Furthermore, the hormone effect is inhibited by tetrac, an iodothyronine analog that, itself, is not a kinase activator but does inhibit the binding of T4 to human erythrocyte membranes (10). Consistent with this evidence that hormone action requires a putative receptor on the plasma membrane are the observations presented here that T4 action on signal transduction is pertussis toxin and GTPgamma S sensitive. Thus the initial step in the mechanism is interaction of thyroid hormone with a GPCR. We previously described a Gi protein in erythrocyte membranes that may be involved in hormone action (9, 38), but a GPCR responsive to thyroid hormone has not been previously reported in nucleated cells (39). By nondenaturing PAGE of octylglucoside-solubilized plasma membranes, we recently identified two proteins that bind radiolabeled T4 and are candidate GPCRs (M. R. Deziel, F. B. Davis, and P. J. Davis, unpublished observations).

PKCalpha has been shown to activate Raf-1 directly (18), a step that is early in the MAPK cascade (Raf-1/MEK/MAPK). The fact that CGP-41251, an inhibitor of traditional PKC isoform activities (PKCalpha , -beta I, -beta II, and -gamma ), prevented the action of thyroid hormone in our model is consistent with an effect of T4 at the level of Raf-1, although our findings also raise the possibility that activated PKC can mediate the action of T4 through the phosphorylation of MEK, as described by others (34), thus bypassing Raf-1. With the use of CGP-41251, we have effectively ruled out a contribution of calcium-independent, diacylglycerol-dependent novel PKCs and atypical PKCs, as described by Schönwasser et al. (31), some of which phosphorylate Raf-1 without stimulating its kinase activity. We previously reported that physiological concentrations of T4 stimulate erythrocyte cytosol PKC activity (19), also supporting a role for PKC stimulation in the T4 effects we describe in this report. We have elsewhere demonstrated a role for PKC in thyroid hormone potentiation of both antiviral (23) and immunomodulatory (20) actions of IFN-gamma . A role for PLC in iodothyronine action on the MAPK cascade is suggested by results of our studies with the aminosteroid U-73122. That is, hormone activation of PKC (and subsequently of the MAPK pathway) depends on diacylglycerol liberated by PLC.

The proposed sequence of events in the signal transduction pathways acted on by thyroid hormone is shown in Fig. 9. Hormone binding at the cell membrane is followed serially by activation of PLC, PKC, Raf-1, MEK, and MAPK. The possibility that Ras may be involved in the sequence is included in Fig. 9. Although pertussis toxin-sensitive GPCRs have not previously been described to activate Ras through PLC and PKC (39, 40), we have found that thyroid hormone poorly activates MAPK in a dominant negative Ras model (J. Gordinier, F. B. Davis, and P. J. Davis, unpublished observations). Thus Raf-1 may be phosphorylated by PKC in the thyroid hormone-activated cell or PKC may act on Ras (24), thus activating Raf-1.


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Fig. 9.   Proposed mechanism by which thyroid hormone nongenomically activates MAPK signal transduction cascade. Initial step is postulated to be interaction of hormone with a heterotrimeric Gi protein-coupled receptor (GPCR). The beta gamma -subunit of GPCR is depicted as a regulator of PLC activity. Evidence for participation of each of upstream steps leading to MAPK activation is reviewed in DISCUSSION. JAK1 and JAK2, Janus kinases 1 and 2; Ras-GTP, complex of activated Ras and GTP; GAS, IFN-gamma -activated sequences of IFN-responsive genes; MEK, MAPK kinase.

Apparent from the present studies are not only the promotion by T4 of the nuclear uptake of activated STAT1alpha and MAPK but also the recovery of MAPK from nuclear immunoprecipitates made with anti-STAT1alpha antibody and of STAT1alpha from anti-MAPK immunoprecipitates. Thus the nuclear fractions in T4-treated cells contain a STAT1alpha -MAPK complex. Association of MAPK and STAT1alpha has been described by others in response to IFN-alpha /beta (8). We have not detected this complex in cytosol of hormone-treated cells. Ostensibly, the complex reflects the action of MAPK in phosphorylating Ser-727 of STAT1alpha , as suggested by David et al. (8). Documentation of thyroid hormone-potentiated phosphorylation of Ser-727 of STAT1alpha in the presence of IFN-gamma is shown in Fig. 5C. Thus the complexing of STAT1alpha and MAPK under the influence of T4 is not a casual event but is associated with phosphorylation of a specific residue of STAT1alpha . Our studies with the U3A cell series revealed that, in the absence of STAT1alpha , activated MAPK did translocate to the nucleus in the presence of T4, whereas T4-induced nuclear translocation of STAT1alpha was suppressed in the absence of MAPK in antisense oligonucleotide-treated cells.

In the absence of IFN-gamma , thyroid hormone does not induce the antiviral state (22), so that activation of the MAPK pathway by the hormone is insufficient to initiate transcription of antiviral proteins. This is not surprising, since it is the STAT1alpha pathway that is primarily involved in transducing the IFN signal for the antiviral state and MAPK activity has been shown by others to be facilitative (17, 42). We postulate that there is a biological role for thyroid hormone potentiation in the presence of low levels of IFN-gamma ; in fact, we have found that T4 promotes the antiviral action of IFN-gamma at 0.1 IU/ml, a concentration that in itself is not antiviral (22). Thus, through enhanced activation of MAPK and STAT1alpha , the hormone is able to convert an ineffective level of IFN-gamma into an effective concentration.

It is possible that one or more actions of thyroid hormone on kinase cascades are representative of a newly recognized mechanism of hormone signaling, one that 1,25-dihydroxyvitamin D3 also utilizes; the vitamin has been reported by others to stimulate activity and translocation of protein kinases via a nongenomic mechanism in acute promyelocytic NB4 cells (2). Other hormones that activate kinases in the MAPK cascade include gonadotropin-releasing hormone (37), norepinephrine (43), and 17beta -estradiol (41). Inhibition of MEK has been shown to decrease growth hormone-stimulated activation of STAT5 (27) and to decrease insulin stimulation of Ser-727 phosphorylation of STAT3 (4). Thus hormones that have important actions via nuclear receptors, as well as those that act primarily at the cell membrane, can nongenomically alter signal-transducing kinase activities.


    ACKNOWLEDGEMENTS

We thank the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD) for the provision of geldanamycin, Drs. George Stark (Cleveland, OH), James E. Darnell, Jr., (New York, NY), and Ke Shuai (Los Angeles, CA) for provision of U3A, STAT1alpha wt, and STAT1alpha A727 cells, Dr. David Frank (Boston, MA) for the antibody to Ser-727-phosphorylated STAT1alpha , and Dr. William Chin (Boston, MA) for the TRbeta 1 antibody.


    FOOTNOTES

This work was supported in part by funding from the Office of Research Development, Medical Research Service, Department of Veterans Affairs (to P. J. Davis) and by a grant from the Candace King Weir Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. J. Davis, Dept. of Medicine A-57, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: pjdavis{at}albany.net).

Received 20 October 1998; accepted in final form 19 January 1999.


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