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Molecular Endocrinology 19 (1): 102-112
Copyright © 2005 by The Endocrine Society

Thyroid Hormone Induces Rapid Activation of Akt/Protein Kinase B-Mammalian Target of Rapamycin-p70S6K Cascade through Phosphatidylinositol 3-Kinase in Human Fibroblasts

Xia Cao, Fukushi Kambe, Lars C. Moeller, Samuel Refetoff and Hisao Seo

Research Institute of Environmental Medicine (X.C., F.K., H.S.), Nagoya University, Nagoya 464-8601, Japan; and Departments of Medicine (L.C.M., S.R.), Pediatrics (S.R.), and Committee on Genetics (S.R.), The University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Hisao Seo, Research Institute of Environmental Medicine, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail: hseo{at}riem.nagoya-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated that T3 increases the expression of ZAKI-4{alpha}, an endogenous calcineurin inhibitor. In this study we characterized a T3-dependent signaling cascade leading to ZAKI-4{alpha} expression in human skin fibroblasts. We found that T3-dependent increase in ZAKI-4{alpha} was greatly attenuated by rapamycin, a specific inhibitor of a protein kinase, mammalian target of rapamycin (mTOR), suggesting the requirement of mTOR activation by T3. Indeed, T3 activated mTOR rapidly through S2448 phosphorylation, leading to the phosphorylation of p70S6K, a substrate of mTOR. This mTOR activation is mediated through phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling cascade because T3 induced Akt/PKB phosphorylation more rapidly than that of mTOR, and these T3-dependent phosphorylations were blocked by both PI3K inhibitors and by expression of a dominant negative PI3K ({Delta}p85{alpha}). Furthermore, the association between thyroid hormone receptor ß1 (TRß1) and PI3K-regulatory subunit p85{alpha}, and the inhibition of T3-induced PI3K activation and mTOR phosphorylation by a dominant negative TR (G345R) demonstrated the involvement of TR in this T3 action. The liganded TR induces the activation of PI3K and Akt/PKB, leading to the nuclear translocation of the latter, which subsequently phosphorylates nuclear mTOR. The rapid activation of PI3K-Akt/PKB-mTOR-p70S6K cascade by T3 provides a new molecular mechanism for thyroid hormone action.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CALCINEURIN (Cn) WAS first purified as a calmodulin-binding protein from bovine brain (1). It is the only known serine/threonine protein phosphatase under the control of Ca2+/calmodulin. The discovery that immunosuppressants, cyclosporin A and FK506, inhibit Cn activity through binding with their cognate immunophilins [cyclophilin A and FK506-binding protein (FKBP)] established Cn as a mediator involved in the regulation of multiple biological processes such as T cell activation (2), muscle hypertrophy (3), memory development (4), glucan synthesis (5), ion homeostasis (6), and cell cycle control (7).

Recently, several physiological inhibitors of Cn have been identified, including CBP1/calcipressin (8) and regulator of calcineurin 1 in yeast (9), DSCR1 (Down syndrome candidate region 1) (10), ZAKI-4 (also termed DSCR1L1 by gene nomenclature committee) (11), and DSCR1L2 (12) in human. DSCR1 homolog in hamster was identified as Adapt78 (13) and in mouse as modulatory calcineurin-interacting protein 1 (14). These proteins were shown to bind with Cn and to inhibit Cn activity in vitro and in vivo.

The endogenous Cn inhibitor ZAKI-4 has two isoforms, {alpha} and ß. Both isoforms inhibit Cn activity by binding through the common C-terminal region, whereas only the expression of ZAKI-4{alpha}, but not ß, responds to thyroid hormone in human skin fibroblasts (15). Regulation of gene expression by T3 is mediated through the thyroid hormone receptor (TR), usually acting as a ligand-dependent nuclear transcription factor (16). Liganded TR binds with its cognate cis-element (thyroid hormone-responsive element), present in the regulatory region of target genes, and promotes their transcription. This T3 action is often referred to as genomic action. However, we found that T3-dependent expression of ZAKI-4{alpha} is not mediated by such genomic action, because there is no canonical thyroid hormone-responsive element in the promoter of ZAKI-4{alpha} gene, and because a protein synthesis inhibitor cycloheximide (CHX) abrogated T3-dependent ZAKI-4{alpha} expression, suggesting that de novo protein synthesis is required (11).

On the other hand, it was reported that expression of regulator of calcineurin 1, yeast homolog of ZAKI-4, was increased by intracellular Ca2+ mobilization, and that this increase was sensitive to FK506 (9). More recently, it was reported that forced expression of Cn in the heart of transgenic mice increased expression of modulatory calcineurin-interacting protein 1 (17). These findings suggest a common mechanism regulating the expression of endogenous Cn inhibitors through activation of Cn.

We therefore investigated a possible involvement of Cn in T3-dependent expression of ZAKI-4{alpha} by utilizing a Cn inhibitor FK506 and its structural analog rapamycin, which specifically prevents the activation of a protein kinase, mammalian target of rapamycin (mTOR). Unexpectedly, rapamycin, but not FK506, greatly attenuated T3-induced ZAKI-4{alpha} expression. This finding implicated mTOR in the control of ZAKI-4{alpha} expression by T3.

mTOR is a member of the phosphatidylinositol kinase-related protein kinase family. Its carboxyl-terminal region is highly homologous to lipid kinases. However, evidence supports a role for mTOR as a serine/threonine protein kinase (18). It was discovered biochemically, based on its binding properties to FKBP-rapamycin complex (19). In mammals the complex inhibits mTOR through interaction with the FKBP-rapamycin binding domain in mTOR (20). The minimal FKBP-rapamycin binding domain spans residues 2025–2114 and lies N-terminal to the catalytic kinase domain. mTOR mediates signaling in response to nutrients and growth factors, such as insulin and IGF-I, and controls the mammalian translation initiation machinery through activation of the p70S6K protein kinase (p70 ribosomal S6 kinase) and through inhibition of the eukaryotic initiation factor 4E inhibitor, 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) (21, 22). For mTOR activation, phosphorylation of two serine residues, S2448 and S2481, located in the C-terminal repressor domain, seems to be important. Deletion of this domain renders the mutant constitutively active (23). Because S2481 phosphorylation does not occur in a kinase-inactive mTOR, this site is considered to be autophosphorylated (24). Rapamycin was shown to have no effect on S2481 phosphorylation. In contrast, three stimuli, insulin, amino acids, and muscle loading, have been demonstrated to induce S2448 phosphorylation and to activate mTOR kinase (25, 26). However, significance of this phosphorylation remains to be established because substitution of S2448 with alanine was reported not to affect mTOR kinase activity (23).

In the present study we demonstrate, for the first time, a novel nongenomic action of T3 that phosphorylates S2448 of mTOR and activates the kinase rapidly. This T3 action is initiated by activation of phosphatidylinosityl 3-kinase (PI3K)-Akt/protein kinase B (PKB) signaling by liganded TR, leading to an increased expression of ZAKI-4{alpha}.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
T3-Dependent ZAKI-4{alpha} Expression Is Sensitive to Rapamycin
Human skin fibroblasts cultured in a medium containing T3-depleted serum were incubated with T3 for 12 h in the presence or absence of a Cn inhibitor, FK506, or its analog, rapamycin (Fig. 1AGo). As we previously reported, ZAKI-4{alpha} mRNA was increased by physiological dose of T3. Unexpectedly, pretreatment with FK506 had no effect on the T3-dependent induction, whereas rapamycin attenuated T3-dependent ZAKI-4{alpha} expression. Because both FK506 and rapamycin require FKBP for their function, the effect of rapamycin warrants the presence of FKBP in human fibroblasts, and lack of FK506 effect could not be due to the absence of FKBP. These results therefore demonstrate that T3-dependent ZAKI-4{alpha} expression is mediated by activation of mTOR but not by Cn signaling. The unexpected effect of rapamycin prompted us to investigate the T3-dependent signaling cascade leading to mTOR activation because rapamycin-sensitive signaling pathway has not been described in the T3 action.



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  		Fig. 1. T3 Induces ZAKI-4{alpha} Expression through Activating mTOR

Human skin fibroblasts were cultured in DMEM with 5% T3-depleted FBS for 2 d and were treated with T3 (10–8 M) for 12 h. FK506 (1 µM) and rapamycin (1 µM) were added 30 min before T3 addition. Total RNAs were subjected to Northern blot analysis and whole-cell lysates to Western blot analysis. Panel A shows the image of the Northern blot hybridized with ZAKI-4{alpha}-specific probe. Glyceraldehyde-3 phosphate-dehydrogenase (GAPDH) was used as an internal control. Panel B shows T3-dependent activation of mTOR and p70S6K.

 
T3 Induces mTOR Activation and Its S2448 Phosphorylation
We investigated whether T3 induces mTOR activation by phosphorylating S2448. Activation of mTOR was ascertained by phosphorylation of p70S6K at T389, because it was established that mTOR is an upstream kinase for p70S6K, phosphorylating the residue in a rapamycin-sensitive manner (27).

We first studied the effect of 12 h exposure to T3 on S2448 phosphorylation of mTOR to match the exposure time for Northern blot analysis (Fig. 1AGo). As shown in Fig. 1BGo, an increase in S2448 phosphorylation was detected after 12 h exposure to T3. Note that total mTOR protein levels were not affected by T3. The phosphorylation is associated with a marked increase in T389 phosphorylation of p70S6K. These results demonstrate T3-dependent phosphorylation of S2448 activates mTOR kinase.

T3 Induces Rapid and Persistent Activation of mTOR in a CHX-Insensitive Manner
We next examined the earlier time course of T3 effect on mTOR activation. As shown in Fig. 2Go, A and B, S2448 phosphorylation of mTOR was detected as early as 10 min after T3 addition, followed by a peak level at 25 min and preservation of the phosphorylated form up to 12 h, whereas total protein levels were not altered. This phosphorylation is correlated with T389 phosphorylation of p70S6K, which started 10 min after T3 treatment and gradually increased up to 25 min. These results confirmed the rapid and sustained activation of mTOR by T3. This T3 action did not require de novo protein synthesis, because treatment with a protein synthesis inhibitor CHX did not affect S2448 phosphorylation of mTOR even after 30 min exposure of T3 (Fig. 2CGo). Rapid and CHX-insensitive activation of mTOR strongly suggests that this T3 action is nongenomic, implying that transcriptional activation by TR is not required.



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  		Fig. 2. T3 Induces Phosphorylation of mTOR and p70S6K Rapidly in a CHX-Insensitive Manner

Human skin fibroblasts were cultured in DMEM with 5% T3-depleted FBS for 2 d. They were then treated with T3 (10–8 M) and harvested at intervals (A and B) for Western blot analysis. In panel C, CHX (10 µg/ml) was added 30 min before T3 treatment, and cells were harvested 30 min after T3 addition.

 
T3-Dependent Activation of mTOR Is Mediated by PI3K-Akt/PKB Signaling Pathway
We next examined the possible involvement of PI3K-Akt/PKB signaling pathway in T3-dependent activation of mTOR, because it is known that insulin activates mTOR through this pathway (23). Binding of insulin to the membrane receptor results in the phosphorylation of its receptor and insulin receptor substrate (IRS) proteins. These proteins then interact with Src homology 2 (SH2) domain-containing proteins such as p85{alpha}, the regulatory subunit of PI3K (28). PI3K is subsequently recruited to the membrane where it converts lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], to phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3]. The synthesis of PtdIns(3,4,5)P3 recruits the proteins possessing pleckstrin homology domain, such as Akt/PKB and phosphoinositide-dependent protein kinase 1, from the cytoplasm to the plasma membrane. Then, phosphorylation and activation of Akt/PKB occur near the plasma membrane. Phosphorylations of T308 and S473 in Akt/PKB are required for its full activation (29). Phosphorylation of the former site is directly catalyzed by phosphoinositide-dependent protein kinase 1. However, a kinase responsible for the latter phosphorylation has not yet been identified.

The involvement of Akt/PKB in T3-dependent S2448 phosphorylation of mTOR was determined by Western blot analysis using a specific antibody against Akt/PKB phosphorylated at S473. As shown in Fig. 2BGo, S473 phosphorylation of Akt/PKB was induced by T3 as early as 5 min, whereas total amount of Akt/PKB was not altered by T3. Note that this S473 phosphorylation precedes S2448 phosphorylation of mTOR, suggesting that T3-dependent mTOR activation is mediated by Akt/PKB.

The involvement of PI3K was examined using PI3K-specific inhibitors, wortmannin and LY294002. Human skin fibroblasts cultured in the medium containing T3-depleted serum were exposed to T3 for 30 min in the presence or absence of wortmannin and LY294002. As shown in Fig. 3AGo, T3-dependent S473 phosphorylation of Akt/PKB, S2448 phosphorylation of mTOR, and T389 phosphorylation of p70S6K were all inhibited by wortmannin and by LY294002. These results suggest that PI3K mediates T3-dependent activation of Akt/PKB, mTOR, and p70S6K. More importantly it indicates that T3-dependent PI3K activation is an initial event leading to the activation of Akt/PKB, mTOR, and p70S6K.



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  		Fig. 3. Inhibition of PI3K Abrogates T3-Dependent Activation of mTOR

A, Cells incubated in DMEM with 5% T3-depleted FBS for 2 d were treated with T3 (10–8 M) for 30 min. PI3K inhibitors, wortmannin (wort, 2 µM) and LY294002 (LY, 50 µM), were added 30 min before T3 addition. Whole-cell extracts were subjected to Western blot analysis. B, Adenoviruses expressing either green fluorescent protein (AdGFP, {Delta}p85{alpha} minus) or {Delta}p85{alpha} were infected at a M.O.I of 200 for 1 h. They were then incubated in DMEM with 5% T3-depleted FBS for 2 d and treated with T3 (10–8 M) for 30 min.

 
However, Brunn et al. (30) reported that both wortmannin and LY294002 are able to inhibit mTOR directly. To confirm the contribution of PI3K to T3-dependent activation of mTOR, a mutant p85 subunit of PI3K ({Delta}p85{alpha}) lacking an interacting domain with p110 was overexpressed. {Delta}p85{alpha} has been shown to inhibit insulin-dependent PI3K activation (31). Its inhibitory action is explained by the disruption of interaction between IRS and p85/p110 PI3K due to the predominant association of {Delta}p85{alpha} with IRS.

Human skin fibroblasts infected with a recombinant adenovirus expressing {Delta}p85{alpha} were exposed to T3 for 30 min. As shown in Fig. 3BGo, overexpression of {Delta}p85{alpha} abolished T3-dependent S2448 phosphorylation of mTOR. These results clearly demonstrate the role of PI3K in T3-mediated mTOR activation and indicate the presence of a certain protein(s) associating with {Delta}p85{alpha} is involved in T3 action. The most likely candidate to mediate the action is TR.

TRß1 Complexes with p85{alpha} Subunit of PI3K in a Ligand-Independent Manner
TR belongs to a steroid hormone receptor superfamily also including estrogen receptor, glucocorticoid receptor, progesterone receptor, retinoic acid receptor and so on. The genomic actions of these receptors have been well demonstrated. Recently, their nongenomic actions, outside the nucleus, were also identified. For example, estrogen and retinoic acid activate PI3K rapidly through the nontranscriptional action of their receptors (32, 33, 34, 35). Furthermore, estrogen receptor-{alpha} was demonstrated to activate PI3K through binding with p85{alpha} either in a ligand-dependent manner in endothelial cells (32) or a ligand-independent manner in epithelial cells (33).

We therefore investigated whether TR interacts with p85{alpha} using recombinant adenoviruses expressing wild-type TRß1 or its dominant negative mutant TR G345R. This mutant has been shown to lack T3-binding property, but to preserve dimerization property with wild-type TR and thereby inhibit its transactivation function in a dominant negative manner (36). The cells with overexpression of either wild-type TRß1 or TR G345R were treated with T3 for 30 min. The cell lysate was then immunoprecipitated with anti-p85{alpha} or anti-TRß1 antibody and probed with the antibodies to each other. As shown in Fig. 4AGo, immunoprecipitation with anti-p85{alpha} antibody resulted in pulling down of TRß1 in the presence or absence of T3. In addition, TR G345R without T3-binding ability also associates with p85{alpha}. As shown in Fig. 4BGo, immunoprecipitation with anti-TRß1 antibody confirmed that TRß1 forms complex with p85{alpha} in a ligand-independent manner. Two bands were detected for TRß1 and TR G345R because of alternative initiation.



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  		Fig. 4. TRß1 Complexes with p85{alpha} in a Ligand-Independent Manner

Human skin fibroblasts were incubated in serum-free DMEM containing recombinant adenoviruses expressing wild-type TRß1 (wt) or TR G345R (G345R) at a M.O.I. of 200 for 1 h. They were then cultured in the medium with 5% T3-depleted FBS for 2 d and treated with T3 (10–8 M) for 30 min. Whole-cell extracts were immunoprecipitated with anti-p85{alpha} antibody (panel A) or anti-TRß1 antibody (panel B) and then subjected to immunoblot analysis probed with antibodies to each other. A goat antimouse IgG immobilized on CNBr-activated Sepharose 4 Fast Flow was used to show the nonspecific binding, and the samples without immunoprecipitation were used to confirm the expression of each protein. Our recombinant adenovirus of wild-type TRß1 (wt) or TR G345R (G345R) produces two bands at molecular mass of 52 and 49 kDa because of the alternative initiation.

 
p85{alpha} has been shown to bind with several proteins through its functional domains including SH2, Rho-GAP, and SH3 domain, resulting in subsequent activation of PI3K. Binding of insulin receptor and IRS to SH2 domain (37, 38) or binding of proline-rich protein to SH3 domain (39, 40) or binding of Rho family protein such as Rac and Cdc42 to Rho-GAP domain (41, 42) leads to a stimulation of PI3K activity. Insulin receptor and IRS bind to SH2 domain due to their tyrosine phosphorylation (37, 38). Although TR was shown to be a phosphoprotein, tyrosine phosphorylation was not reported (43, 44). It is thus unlikely that p85{alpha} binds with TRß1 through SH2 domain. It is noteworthy that Rho-GAP domain contains three repeats of LXXLL motif (Swiss-Prot accession no. Q63787), which is considered as a sequence interacting with nuclear receptor directly and found in many nuclear receptor-associating proteins, such as steroid receptor coactivator-1/p160, transcriptional intermediary factor-2/glucocorticoid receptor interacting protein-1, and CBP/p300 (45). It is thus possible that Rho-GAP domain of p85{alpha} might comprise a surface for a direct interaction with TRß1, although we could not exclude a possible involvement of SH3 domain. The interaction between TRß1 and p85{alpha} might take place in the cytosol or near plasma membrane, because the presence of TR in the cytoplasm has been shown even though its amount is less than that in the nucleus (44, 46). Further studies are required to substantiate this speculation.

Activation of PI3K Requires T3 Binding with TRß1-p85{alpha}
Demonstration of T3-independent complex formation between TRß1 and p85{alpha} and T3-dependent phosphorylation of Akt/PKB and mTOR suggested that T3 binding with TRß1 is required for the activation of PI3K. To confirm this hypothesis, PI3K activity in the cells with or without overexpression of wild-type TRß1 or TR G345R was determined by competitive ELISA. As shown in Fig. 5AGo, overexpression of wild-type TRß1 alone did not increase PI3K activity. Further treatment of these cells with T3 resulted in a similar degree of T3-dependent induction of PI3K activity as in the cells infected with control adenovirus (AdGFP), suggesting that a sufficient amount of TR is expressed in human skin fibroblasts. When the cells were infected with the virus expressing TR G345R, T3-dependent activation of PI3K could not be detected, demonstrating the involvement of liganded TRß1 in PI3K activation. These results are compatible with a report by Simoncini et al. (32). They showed the presence of PI3K activity in the anti-TR antibody immunoprecipitate prepared from T3-treated human endothelial cells, whereas little activity was observed in cells not treated with T3.



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  		Fig. 5. Expression of a Dominant Negative TR (G345R) Abrogates T3-Induced Activation of PI3K and mTOR

A, The cell treatment was same as described in Fig. 4Go except for control cells, which were infected with AdGFP. Equal amount of proteins (500 µg) were immunoprecipitated with anti-p85{alpha} antibody at 4 C overnight. The PI3K activity of each precipitate was then measured by ELISA and expressed as the relative production of PI(3,4,5)P3 by each sample. PI(3,4,5)P3 produced by control cells was set as 100, and others were compared with the control. The assay was done in triplicate. B, The whole-cell extracts were subjected to immunoblot analysis probed with antiphospho-mTOR (S2448) antibody.

 
In concordance with T3-dependent PI3K activation, S2448 phosphorylation of mTOR was also similarly regulated (Fig. 5BGo). T3-dependent mTOR phosphorylation was not further enhanced by overexpressing wild-type TRß1, whereas that of TR G345R inhibited it. The inhibition by TR G345R is not due to squelching of T3, because it does not bind T3. Because both wild-type TRß1 and TR G345R form a complex with p85{alpha}, as shown in Fig. 4Go, it indicates that mutant TR competes with endogenous TR to bind with p85{alpha}, and this complex fails to activate PI3K due to the lack of T3 binding activity.

T3 Promotes Nuclear Translocation of S473-Phosphorylated Akt/PKB
A variety of nuclear proteins have been shown to be substrates for Akt/PKB (47, 48, 49). However, a limited number of reports have shown the nuclear translocation of Akt/PKB (50, 51). We therefore investigated T3 effects on subcellular localization of Akt/PKB and mTOR. Human skin fibroblasts maintained in serum-free medium were treated with T3, and nuclear and cytosolic fractions were subjected to Western blot analysis, using antibody directed against Akt/PKB, not against phosphorylated Akt/PKB. As shown in Fig. 6AGo, Akt/PKB was detected in nuclear fractions as early as 5 min after T3, and the amount gradually increased. Wortmannin prevented this nuclear accumulation. In contrast, cytosolic Akt/PKB gradually decreased after T3 treatment. The total amounts of nuclear and cytosolic Akt/PKB appear not to be altered during T3 treatment, in agreement with the result shown in Fig. 2BGo. These results clearly demonstrate that T3 induces nuclear translocation of Akt/PKB in a PI3K-dependent manner. The translocated Akt/PKB is likely to be a S473-phosphorylated, active form, because the time course of appearance of Akt/PKB in nuclear fractions is similar to that of S473 phosphorylation shown by Western blot analysis using whole-cell lysates (Fig. 2BGo). In accordance with these results, Western blot analysis using the antibody against S473-phosphorylated Akt/PKB revealed phosphorylated Akt/PKB in the nucleus was markedly increased by T3, whereas in cytoplasm, the increase was moderate (Fig. 6BGo). The different degree of increase of phospho-Akt/PKB in nucleus and cytoplasm is likely due to nuclear translocation of newly phosphorylated Akt/PKB.



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  		Fig. 6. T3 Induces Activation of Akt/PKB and Its Nuclear Migration

A, Nuclear migration of Akt/PKB activated by T3. After incubation of the fibroblasts in serum-free DMEM for 1 d, they were treated with T3 and harvested at intervals. Wortmannin (wort, 100 nM) was added 30 min before T3. Nuclear and cytosolic fractions were subjected to Western blot (WB) analysis using a polyclonal antibody against Akt/PKB. B, An antibody against phospho-Akt (S473) was used. C, Localization of Akt/PKB after T3 treatment. Human skin fibroblasts were incubated in serum-free DMEM for 1 d and stimulated with T3 (10–10 M) for 30 min. Wortmannin (wort, 100 nM) was added 1 h before T3 addition. Fibroblasts were then fixed and incubated with the rabbit polyclonal antibodies against Akt, phospho-Akt (S473), mTOR, and phospho-mTOR (S2448) at 4 C overnight. The cells were subsequently incubated with Alexa Fluor 488-coupled secondary antibody. The representative laser confocal scanning images were shown at a magnification of x400.

 
To further clarify the intracellular localization of Akt/PKB and mTOR, we performed immunocytochemical analysis (Fig. 6CGo). The serum-starved cells were treated with T3 for 30 min. In unstimulated cells, Akt/PKB was detected mainly in the cytoplasm, and Akt/PKB phosphorylated at S473 was barely detected in the nucleus. T3 treatment resulted in marked translocation of Akt/PKB into the nucleus. This was concordant with an increase in the phosphorylated Akt/PKB in the nucleus. T3-dependent phosphorylation and nuclear translocation of Akt/PKB were abrogated by wortmannin. These observations confirm T3- and PI3K-dependent phosphorylation and nuclear translocation of Akt/PKB.

In contrast, both mTOR and S2448-phosphorylated mTOR were detected only in the nucleus. S2448-phosphorylated mTOR was hardly detected in the unstimulated cells. T3 markedly increased the amount of the phosphorylated mTOR in the nucleus, and this was inhibited by wortmannin. However, there was no change in total mTOR (Fig. 2Go and Fig. 6CGo). The result strongly suggests that Akt/PKB-dependent activation of mTOR occurs in the nucleus. Nuclear localization of mTOR was also reported by Zhang et al. (52). They showed that mTOR is predominantly present in the nucleus of human fibroblasts as well as several cell lines, with human embryonic kidney 293 cells being the only exception. The nuclear mTOR might be an active form, because they used cells cultured with serum not depleted of T3. However, the predominant existence of activated mTOR in the nucleus appears curious, because p70S6K and 4E-BP1, the best characterized downstream targets of mTOR, are present mainly in the cytoplasm. It was shown recently that mTOR is a cytoplasmic-nuclear shuttling protein, and that inhibition of nuclear export of mTOR reduces p70S6K and 4E-BP1 activation (53). Therefore, a part of activated mTOR might be exported from the nucleus and activate p70S6K in the cytoplasm after T3 treatment.

T3-Mediated ZAKI-4{alpha} Regulation Is Initiated by the Sequential Activation of PI3K-Akt/ PKB-mTOR Cascade
Although it has been shown that PI3K, Akt/PKB, and mTOR can be independently activated by a variety of stimuli, in this study we show that T3 activates Akt/PKB, mTOR, and p70S6K only through PI3K, because the activation of the former three kinases is prevented by PI3K inhibitors, wortmannin and LY294002, and by {Delta}p85{alpha}. In addition, time course studies demonstrated sequential phosphorylations of serine and threonine residues of Akt/PKB, mTOR, and p70S6K, which are critical for their kinase activity. These findings clearly indicate that the activation of these kinases by T3 is not a parallel, but hierarchical event, which involves PI3K, Akt/PKB, and mTOR kinase cascade. The present study also demonstrates that activation of mTOR is required for T3-dependent ZAKI-4{alpha} expression. To confirm the involvement of PI3K-Akt/PKB cascade, the effects of PI3K inhibitor LY294002 and the dominant negative mutant {Delta}p85{alpha} on T3-induced ZAKI-4{alpha} expression were examined by Northern blot analysis. Human skin fibroblasts cultured in the medium containing T3-depleted serum were incubated with T3 for 12 h in the presence or absence of LY294002 or with adenovirus expressing {Delta}p85{alpha}. As shown in Fig. 7Go, A and B, both LY294002 and {Delta}p85{alpha} abrogated T3-induced ZAKI-4{alpha} expression, demonstrating the involvement of PI3K-Akt/PKB.



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  		Fig. 7. Inhibition of T3-Dependent ZAKI-4{alpha} Expression by PI3K Inhibitor and {Delta}p85{alpha}

A, Cells were incubated in DMEM with 5% T3-depeleted FBS for 2 d and treated with T3 (10–8 M) for 12 h. PI3K inhibitor LY294002 (LY, 50 µM) was added 30 min before T3 addition. B, Adenovirus expressing either green fluorescent protein (AdGFP, {Delta}p85{alpha} minus) or {Delta}p85{alpha} was infected at a M.O.I of 200 for 1 h. Fibroblasts were then incubated in DMEM with 5% T3-depleted FBS for 2 d and treated with T3 (10–8 M) for 12 h. Total RNAs were subjected to Northern blot analysis and hybridized with ZAKI-4{alpha}-specific probe.

 
The PI3K-Akt/PKB and mTOR signaling pathways may regulate ZAKI-4{alpha} expression by controlling downstream effectors such as p70S6K. However, CHX-sensitive regulation of ZAKI-4{alpha} by T3 suggested requirement of de novo protein synthesis (11), and thus the downstream effectors could not regulate ZAKI-4{alpha} expression directly. The physiological target of p70S6K is the 40S subunit of the S6 ribosomal protein. Phosphorylation of S6 subsequently initiates translation of 5'-terminal oligopyrimidine mRNAs, which encode components of the translational machinery such as elongation factors, ribosomal proteins, and poly(A)-binding protein and thus plays a key role in modulating translational efficiency (27). A certain protein(s) under the control of such mTOR-p70S6K-initiated translational machinery might be involved in ZAKI-4{alpha} expression. A search for such protein(s) will elucidate the mechanism of T3-mediated ZAKI-4{alpha} regulation.

It is noteworthy that T3 inhibits endogenous Cn (protein phosphatase 2B) through regulating ZAKI-4{alpha} (15). The present study demonstrating T3-dependent activation of mTOR raises a possibility that T3 may also regulate protein phosphatase 2A because activated mTOR has been shown to phosphorylate protein phosphatase 2A and to prevent the dephosphorylation of 4E-BP1 and p70S6K (54).

T3 Possibly Modulates Divergent Cellular Functions through Regulation of PI3K-Akt/ PKB-mTOR-p70S6K Cascade
Thyroid hormone plays important roles in growth, development, metabolism, and differentiation. These functions were considered to be mediated mainly by nuclear TR, which regulates the transcription of target genes after T3 binding. On the other hand, nongenomic action of thyroid hormone has recently been recognized at the molecular levels. It was suggested to control Ca2+ entry, intracellular protein trafficking, and regulation of protein kinase C, MAPKs, and cytoskeleton (55). Involvement of PI3K in the nongenomic action was also suggested by using PI3K inhibitors. They inhibited thyroid hormone-dependent activation of Na+/H+ exchange and amino acid transport in chick embryo hepatocytes (56) and activation of certain class of voltage-gated potassium channel in a rat pituitary cell line (57). The present study demonstrates, for the first time, a novel nongenomic action of T3 that requires TRß1 bound to p85{alpha} and causes a rapid activation of PI3K-Akt/PKB-mTOR-p70S6K cascade in human skin fibroblasts.

Activation of this cascade has been shown to be crucial in multiple biological processes such as cell growth (58), neuronal cell survival (59), glucose uptake (60), and cardiac hypertrophy (61). Ubiquitous distribution of TR suggests that the nongenomic action of T3 mediated by this cascade may modulate different cellular functions cooperatively with genomic actions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Antibodies and Reagents
The following rabbit polyclonal antibodies were used: anti-Akt and antiphospho-Akt (S473), anti-mTOR and antiphospho-mTOR (S2448), and antiphospho-p70S6K (T389). An immunohistochemistry-specific antiphospho-Akt (S473) was used for staining Akt (S473). All the antibodies were purchased from Cell Signaling Technology (Beverly, MA). Monoclonal antibody specific to TRß1 isoform was purchased from Affinity BioReagents (Golden, CO). A part of the antibody was immobilized on CNBr-activated Sepharose 4 Fast Flow (Amersham Biosciences Corp., Piscataway, NJ) according to the manufacturer’s instructions and used for immunoprecipitation. An immobilized anti-p85{alpha} antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for immunoprecipitation, whereas a monoclonal anti-p85{alpha} antibody (BD Biosciences, San Jose, CA) was used for immunoblot analysis. Rabbit polyclonal antibody against mTOR (Santa Cruz Biotechnology) was used for immunocytochemistry. Enhanced chemiluminescence detection reagents were obtained from Pierce Biotechnology, Inc. (Rockford, IL). Wortmannin, LY294002, rapamycin and CHX were purchased from Sigma Chemical Co. (St. Louis, MO). FK506 is a gift from Fujisawa Pharmaceutical Co. (Tokyo, Japan).

Recombinant Adenoviruses
Constructions of recombinant viruses expressing wild-type TRß1 and a mutant TRß1 (TR G345R), exhibiting a strong dominant negative action in vitro and in vivo, were described previously (62). TR G345R was identified in a family with resistance to thyroid hormone (63). A recombinant adenovirus expressing green fluorescent protein (AdGFP) was constructed using AdEasy System and was used to determine the suitable multiplicity of infection (M.O.I.). The virus expressing a dominant negative form of p85{alpha}-regulatory subunit of PI3K ({Delta}p85{alpha}) was a gift from Dr. Kasuga (31). Substitution of residues 479–513 with Ser and Arg abolished the binding to the p110 catalytic subunit.

Cell Cultures and Treatments
The source of human skin fibroblasts and their growth conditions have been described previously (15). Adenoviral vectors were delivered to cells grown to 90% confluence by incubation for 1 h in serum-free DMEM. Preliminary experiments revealed that nearly 100% of cells could be infected at a M.O.I. of 200. They were then incubated in the medium with 5% T3-depleted fetal bovine serum (FBS) for 2 d. The inhibitors such as FK506 (1 µM), rapamycin (1 µM), wortmannin (2 µM or 100 nM), LY294002 (50 µM), and CHX (10 µg/ml) were added 30 min before treatment with T3 (10–8 M). Cells were harvested at intervals for determination of mRNA levels, Western blot analysis, or immunoprecipitation.

Northern Blot Analysis
RNA extraction and the construction of specific probes for ZAKI-4{alpha} and GAPDH (glyceraldehyde-3 phosphate-dehydrogenase) were described previously (15). Images were analyzed by BAS2000 bioimage analysis system (Fuji Photo Film Co., Tokyo, Japan). The experiments were performed in duplicate dishes and repeated at least three times.

Western Blot Analysis
The nuclear, cytosol, or whole-cell extracts were separated by 7.5% SDS-PAGE. Equal amounts of protein per lane were loaded and transferred onto polyvinylidene difluoride membrane (Amersham Biosciences Corp.). The blots were probed with the antibodies described above (diluted in Tris-buffered saline, 0.1% Tween 20 with 5% (wt/vol) skim milk at 1:1,000) followed by incubation with horseradish peroxidase-conjugated antirabbit IgG (1:2500 diluted). The protein was then visualized by enhanced chemiluminescence detection reagents.

Immunoprecipitation
Human skin fibroblasts were lysed in the buffer containing 50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40 (NP-40), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF. The lysate was then centrifuged at 4 C for 15 min at 14,000 x g, and the supernatant was incubated with immobilized anti-p85{alpha} antibody or anti-TRß1 antibody. The precipitated proteins were detected by immunoblot analysis with anti-p85{alpha} or with anti-TRß1 antibody.

PI3K Assay
Cell culture and adenovirus infection are the same as described above. PI3K activity was measured using an ELISA kit (Echelon Biosciences, Inc., Salt Lake City, UT) according to the supplier’s protocol. In brief, the cells were rinsed with buffer A [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM MgCl2, 1 mM CaCl2 and 0.1 mM sodium orthovanadate] and harvested in lysis buffer (buffer A plus 1% NP-40 and 1 mM phenylmethylsulfonyl fluoride). After centrifugation at 4 C for 15 min at 13,000 x g, protein concentrations of the supernatants were measured, and equal amounts (500 µg) were incubated with immobilized anti-p85{alpha} antibody overnight. Immune complexes were washed three times with buffer A plus 1% NP-40; three times with buffer containing 100 mM Tris-HCl (pH 7.4), 5 mM LiCl, and 0.1 mM sodium orthovanadate; and twice with buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.1 mM sodium orthovanadate. The complexes were then incubated with a reaction mixture containing PtdIns(4,5)P2 substrate and ATP. The reaction mixtures were first incubated 3 h later with antibody to PtdIns(3,4,5)P3 and then added to the PtdIns(3,4,5)P3-coated microplate for competitive binding. A peroxidase-linked secondary antibody and colorimetric detection are used to detect anti-PtdIns(3,4,5)P3 binding to the plate. The colorimetric signal is inversely proportional to the amount of PtdIns(3,4,5)P3 produced by activated PI3K. Finally, the PtdIns(3,4,5)P3 production was calculated according to the standard curve. PtdIns(3,4,5)P3 produced by the control cells without T3 treatment and infected with AdGFP was set as 100, and those of others were compared with control cells.

Immunocytochemistry
Human skin fibroblasts, incubated in serum-free DMEM for 24 h, were treated with wortmannin (100 nM) and 1 h later were stimulated with T3 (10–10 M) for 30 min. Samples were washed twice in cold PBS and fixed with freshly prepared 4% paraformaldehyde (30 min at room temperature) and permeabilized with 0.3% Triton X-100 in PBS (10 min at room temperature). Nonspecific binding of the antibodies was blocked with 3% BSA before incubation with the antibodies (diluted 1:100 in blocking solution) at 4 C overnight. The cells were subsequently incubated with 1000-fold diluted Alexa Fluor 488-conjugated antirabbit IgG (Molecular Probes, Inc., Eugene, OR). Laser confocal scanning images were obtained using a Zeiss LSM510 Laser Scan Microscope (Carl Zeiss, Tokyo, Japan) under the same conditions.


   FOOTNOTES
 
This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (Monbukagakusho) of Japan (nos. 13470217 and 16390269) (to H.S.); by the 21st Century COE program "Integrated Molecular Medicine for Neuronal and Neoplastic Disorders" of Monbukagakusho (to H.S. and X.C.); and by National Institutes of Health Grant DK15070 (to S.R.). L.C.M. is a recipient of a grant (Mo 1018/1-1) from the Deutsche Forschungsgemeinschaft (DFG).

First Published Online September 23, 2004

Abbreviations: CHX, Cycloheximide; Cn, Calcineurin; DSCR1, Down syndrome candidate region 1; 4E-BP1, eukaryotic initiation factor 4E-binding protein-1; FBS, fetal bovine serum; FKBP, FK506-binding protein; IRS, insulin receptor substrate; M.O.I., multiplicity of infection; mTOR, mammalian target of rapamycin; NP-40, Nonidet P-40; PKB, protein kinase B; PI3K, phosphatidylinositol 3-kinase; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-triphosphate; PtdI ns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; SH2, Src homology 2; TR, thyroid hormone receptor.

Received for publication March 3, 2004. Accepted for publication September 14, 2004.


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NURSA Molecule Pages Link:

Nuclear Receptors:   TRβ
Ligands:   Thyroid hormone



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