JME Society for Endocrinology Archive
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Journal of Molecular Endocrinology (2007) 38 221-233    DOI: 10.1677/jme.1.02166
© 2007 Society for Endocrinology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Verga Falzacappa, C.
Right arrow Articles by Misiti, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Verga Falzacappa, C.
Right arrow Articles by Misiti, S.

Thyroid hormone receptor TRß1 mediates Akt activation by T3 in pancreatic ß cells

 Cecilia Verga Falzacappa1,2, Eleonora Petrucci3, Valentina Patriarca1,2, Simona Michienzi1,2, Antonio Stigliano1,2, Ercole Brunetti2, Vincenzo Toscano1,2 and Silvia Misiti1,2

1 Chair of Endocrinology, II Faculty of Medicine, University La Sapienza, Rome, Italy
2 Centro Ricerca AFaR, Ospedale San Pietro FBF, Via Cassia, 600, 00189 Rome, Italy
3 Oncology and Hematology Department, ISS, Rome, Italy

(Requests for offprints should be addressed to S Misiti; Email: silvia.misiti{at}uniroma1.it)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
It has recently been recognized that thyroid hormones may rapidly generate biological responses by non-genomic mechanisms that are unaffected by inhibitors of transcription and translation. The signal transduction pathways underlying these effects are just beginning to be defined. We demonstrated that thyroid hormone T3 rapidly induces Akt activation in pancreatic ß cells rRINm5F and hCM via thyroid hormone receptor (TR) ß1. The phosphorylation of Akt was T3 specific and dependent. Coimmunoprecipitation and colocalization experiments revealed that the phosphatidylinositol 3 kinase (PI3K) p85{alpha} subunit and the thyroid receptor ß1 were able to form a complex at the cytoplasmic level in both the cell lines, suggesting that a ‘cytoplasmic TRß1’ was implicated. Moreover, we evidenced that T3 treatment was able to induce kinase activity of the TRß1-associated PI3K. The silencing of TRß1 expression through RNAi confirmed this receptor to be crucial for the T3-induced activation of Akt. This action involved a T3-induced nuclear translocation of activated Akt, as demonstrated by confocal immunofluorescence. In summary, T3 is able to specifically activate Akt in the islet ß cells rRINm5F and hCM through the interaction between TRß1 and PI3K p85{alpha}, demonstrating the involvement of TRß1 in this novel T3 non-genomic action in islet ß cells.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Thyroid hormone, T3, is classically known for its ability to regulate gene expression via binding with thyroid hormone receptors (TRs) on specific promoter regions of target genes. TRs act as ligand-dependent transcription factors and have been traditionally involved in the genomic action of T3, which have a considerable latency with response times in hours to days. Among TR isoforms, two major functional TRs with specific physiological functions have widely been recognized, TR{alpha}1 and TRß1 (Ribeiro et al. 1998, Yen 2001). Recent findings have elucidated the role of nuclear TRß1 in mediating some T3 action on cell proliferation (Porlan et al. 2004) and in regulation of specific cell survival cascades (Cao et al. 2005), therefore, TRß1 could be considered a good candidate for mediating thyroid hormones (THs) non-genomic action.

Non-genomic actions of thyroid hormones have been described at the level of the plasma membrane, cytoskeleton, cytoplasm, and organelles of mammalian cells (Hennemann et al. 2001). Some of these actions rapidly lead to post-translational modification of nucleoproteins, including thyroid and estrogen nuclear receptors, and are mediated by MAPK ERK 1/2; (Davis et al. 2000, Shih et al. 2001, D’Arezzo et al. 2004, Tang et al. 2004). Upstream mitogen activated protein kinase (MAPK), protein kinase C (PKC), and the phosphatidylinositol pathways may be activated by iodothyronines; these effects have also been shown in cells that lack functional nuclear thyroid hormone receptors (Lin et al. 1999, Shih et al. 2001).

Activation of MAPK by thyroid hormone T3 and rapid modulation of specific cascades imply the existence of membrane receptors for the hormone that may be linked to signal transduction pathways. Membrane-binding sites for thyroid hormones were identified years ago in cell membranes isolated from human and rat hepatocytes (Gharbi & Torresani 1979, Pliam & Goldfine 1997). It has recently been demonstrated (Bergh et al. 2005) that the extracellular domain of a structural membrane protein, integrin {alpha}Vß3, is capable of binding thyroid hormone T4, thus activating the MAPK pathway.

Thyroid hormone T3 is also involved in the PI3K pathway: it has recently been reported to regulate the Na, K-ATPase activity via PI3K in alveolar epithelial cells (Lei et al. 2004) and to activate the protein kinase B via PI3K, in human fibroblasts (Cao et al. 2005). Estrogen and retinoic acid have also been recognized to activate PI3K rapidly through the non-transcriptional action of their receptors (Simoncini et al. 2000, Sun et al. 2001, Lopez-Carballo et al. 2002, Haynes et al. 2003). Furthermore, estrogen receptor {alpha} was demonstrated to activate PI3K through binding with p85{alpha} either in a ligand-dependent manner in endothelial cells (Simoncini et al. 2000) or in a ligand-independent manner in epithelial cells (Sun et al. 2001).

Cellular processes, such as proliferation, survival, and glucose metabolism induced by different hormones and growth factors are dependent on the activation of PI3K generating D3-phosphorylated phosphoinositides that are capable of binding PKB/Akt (Ueki et al. 2002, Liang & Slingerland 2003). Akt has been implicated as a critical mediator of insulin-stimulated glucose uptake, suppression of apoptosis, stimulation of glycolysis, and activation of glycogen and protein synthesis in various cell culture systems (Coffer et al. 1998). It has recently become evident that PKB/Akt activation plays a pivotal role in ß cell survival (Lingor et al. 2003) and growth; moreover, recent evidence reviewed in Elghazi et al.(2006) underscore the importance of Akt for regulation of ß cell mass and function. In pancreatic ß cells, PKB/ Akt can be activated by different factors, such as IGF-I and GLP-1 (Giannoukakis et al. 2000, Buteau et al. 2001), and it is directly activated by glucose (Dickson & Rhodes 2004). When activated, via a cAMP-dependent or -independent mechanism, Akt mediates a large number of cellular processes, including mitogenesis, survival, and differentiation.

We have previously demonstrated that thyroid hormone T3 (10–7 M) protects pancreatic ß cells from a pharmacologically induced apoptosis. Our evidence suggested the involvement of the PI3K pathway in mediating this novel survival action of T3; in particular, we have shown that T3 is able to induce the phosphorylation of Akt in the islet cells hCM and rRINm5F (Verga Falzacappa et al. 2006). Additionally, Cao et al.(2005) have demonstrated that T3 is specifically able to activate Akt in a non-genomic manner. Interestingly, this activation implies the recruitment of the thyroid receptor ß1 by the PI3K, suggesting a mechanism similar to those identified for steroids. The PI3K expression is mainly cytoplasmic, and it might be possible that cytoplasmic or cell membrane-bound TRs are responsible for the recruitment of PI3K. Extranuclear TR expression, indeed, has been described long since (Heery et al. 1997, Zhu et al. 1998, Davis et al. 2000).

To examine the specific pathway via which T3 can activate Akt in pancreatic ß cells and to understand further the possible implication of a non-genomic action of T3 in these cellular systems, we suggest a specific role for a ‘cytoplasmic’ thyroid hormone receptor ß1 involved in the PI3K pathway and able to mediate non-genomic actions of T3 in pancreatic ß cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
3,5,3'-Tri-iodothyronine (T3), LY-294 002 hydrochloride, 3,5,3'-tri-iodoacetic acid (TRIAC), bisphenol A dimethacrylate (BPA), and cycloheximide (CHX) were obtained from Sigma-Aldrich (St Louis, MO, USA).

Cell culture

Human insulinoma cell line CM was characterized from Dr M G Cavallo (Cavallo et al. 1996, Baroni et al. 1999); rat insulinoma cell line RINm5F (Cat. no. CRL-11605) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

rRINm5F cells were cultured in RPMI 1640 (Cambrex Corp., East Rutherford, NJ, USA) complemented with 10% fetal bovine serum (Cambrex Corp.); hCM cells were cultured in RPMI 1640 (Cambrex Corp.) containing 5% FBS; all cell culture media were supplemented with L-glutamine 2 mM, according to manufacturer’s instruction, and penicillin 100 µg/ml and streptomycin 50 µg/ml. Cells were maintained at 37 °C under humidified conditions of 95% air and 5% CO2.

To determine the effects of T3 (10–7 M) on Akt activation in the cell lines, cells were cultured to 60% confluence and exposed to the hormone T3, 10 µM LY-294 002 hydrochloride, 10 µM bisphenol A, 10 µM TRIAC, and 3 mM CHX added only once, at the beginning of the individual experiments. Each inhibitor has been utilized as previously described by Moriyama et al.(2002) and Hui et al.(2003); LY-294 002, bisphenol A, and TRIAC were resuspended in stock solution, according to the manufacturer’s instruction, and stored at –20 °C. Control cultures were grown under the same culture conditions as treated cells but in the absence of drugs. The final concentration of methanol, ethanol, and DMSO were identical, in every culture, irrespective of the particular treatment group.

Western blot analyses

Approximately, 3x106 cells were lysed for 30 min at 4 °C in buffer containing 1% Tween 20, 10% glycerol, 150 mM NaCl, 50 mM Hepes (pH 7.0), 1 mM MgCl2, 1 mM CaCl2, 1 mM NaF, 10 mM Na4P2O7, 2 mM NaVO3, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors. The samples were then centrifuged at 12 000 r.p.m. for 30 min and the total cellular protein content was measured using the Bradford method (Bio-Rad). Cytosol–nuclear protein separation was obtained by nuclear/cytosol fractionation kit (MBL, International Corporation, Woburn, MA, USA) following the manufacturer’s instruction. Forty micrograms total extracts and 30 µg nuclear or cytosol proteins per sample were loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and then blotted onto PVDF membranes (Bio-Rad). As a loading control, proteins were stained with Ponceau. Filters were blocked for non-specific reactivity by incubation for 1 h at RT in 5% non-fat dry milk dissolved in PBS 1x, Tween 20 0.1% and then incubated for 16 h at 4 °C, with: TRß1 (Santa Cruz Biotechnology, Inc., San Diego, CA, USA; 1:500), Akt (Santa Cruz, 1:500), phospho Akt 1/2/3-Ser 473 (Santa Cruz, 1:500), GSK3{alpha} (Santa Cruz, 1:500), pGSK3{alpha}-Ser 21 (Santa Cruz, 1:500), and histone H1 (Santa Cruz, 1:1000), and 1 h at RT with ß-actin (Sigma, 1:1000), and {alpha}-tubulin (Santa Cruz, 1:1000), diluted in 5% milk, and PBS 1x, Tween 20 0.1%. After three washes in PBS 1x (Cambrex Corp.), Tween 20 0.1%, the membranes were incubated with the secondary HRP antibodies (anti-mouse, anti-rabbit; Sigma) 1:4000 in milk 5%, PBS 1x, Tween 20 0.1% for 45 min at RT. Immunoreactivity was visualized by the ECL immunodetection system (Amersham Corp.) following the manufacturer’s instruction.

Immunofluorescence microscopy

Cells were plated onto multichamber slides, (BD Falcon, Franklin Lakes, NJ, USA) cultured to 60% confluence and then exposed to the hormone treatment for the indicated times. Slides were washed in PBS 1x, air-dried, fixed, and permeabilized with acetone/methanol (1:1 (v/v) stored at –20 °) for 10 min, and then rehydrated in PBS 1x. Slides were then incubated with 20% fetal bovine serum in PBS 1xfor 20 min and washed thrice in PBS 1x. Thereafter, slides were stained with primary antibodies (rabbit anti-PI3K, Santa Cruz; mouse anti-TRß1, Santa Cruz; rabbit anti-phospho Akt 1/2/3–Ser 473, Santa Cruz) at a dilution of 1:100, for 45 min at RT in a humid chamber. After three washes in PBS 1x, slides were incubated with secondary antibodies (FITC-conjugated rabbit anti-mouse IgG, TRITC-conjugated swine anti-rabbit IgG, DAKO, Glostrup, Denmark) for 45 min at room temperature in the dark, at a dilution of 1:40. Immunofluorescence analysis of cell slides was carried out using an inverted fluorescence microscope equipped for confocal microscopy (Olympus Flowview FV500, Olympus Mikroskopie, Hamburg, Germany). Negative controls including omission of the primary antibody were also performed.

Immunoprecipitation

Cells were lysed in 1% NP40, 0.2 mM PMSF, 10 mM NaF, 0.7 µg/ml pepstatin, and 25 µg/ml aprotinin in PBS 1x. After 10 min on ice, samples were sonicated and centrifuged at 12 000 g for 15 min. The total cellular protein content was measured using the Bradford method (Bio-Rad). Cell lysate (400 µg) was incubated for 2 h with 30 µl G-protein (Roche Diagnostics). After preclearing, the extracts were incubated overnight at 4 °C with mouse anti-TRß1 antibody (Santa Cruz, 1 µg) and 30 µl freshly prepared G-protein. The immunoprecipitates were electrophoresed onto an 8% SDS-polyacrylamide gel, and blotted onto PVDF membrane (Bio-Rad). The membranes were blocked with 5% non-fat dry milk in PBS 1x–Tween 20 0.1% for 1 h at room temperature and probed with rabbit anti-PI3K p85{alpha} (1:500), and afterwards with mouse anti-TRß1 antibody (Santa Cruz, 1:500) diluted in 5% non-fat dry milk in PBS 1x–Tween 20 0.1% overnight at 4 °C under gentle rocking. After washing in PBS 1x–Tween 20 0.1%, the membranes were incubated with the secondary antibody HRP conjugated (anti-mouse, anti-rabbit; Sigma) 1:4000 in 5% milk, PBS 1x, Tween 20 0.1%, for 1 h at RT. Immunoreactivity was visualized by the ECL immunodetection system (Amersham Corp.) following the manufacturer’s instructions.

PI3K kinase assay

Cells were cultured in 100 mm dishes to 60% confluence and exposed to T3 10–7 M, LY 10 µM or vehicles alone for 30 min. At the end of the treatment, cells were lysed and PI3K activity was estimated using a commercially available PI3K ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA) according to manufacturer’s instructions. Protein G beads were then utilized for the PI3K ELISA (Echelon Biosciences, Inc.) following manufacturer’s instruction. Briefly, cells were lysed with a low stringency buffer and 100 µg lysates were incubated with 1 µg TRß1 antibody for 1 h at 4 °C. Then, protein G agarose beads were added to equal amounts of total protein, and the samples were rocked (4 °C) for 16 h. The immunoprecipitated TRß1 samples were incubated with phosphatidylinositol 4,5-bisphosphate substrate and reaction buffer for 1 h 30 min. The amount of PIP3 formed from phosphatidylinositol 4,5-bisphosphate by PI3K activity was detected using a competitive ELISA. The optical density (OD) values obtained were inversely proportional to the amount of PI(3,4,5)P3 produced by PI3K activity. Enzyme activity was estimated by comparing the values from the samples with those in the standard curve.

RNA interference

Cells were plated onto six multiwells and grown in complete medium after 24 h cells were transfected with siRNA SmartPool THRB (Dharmacon, Lafayette, CO, USA). Transfection was performed by incubating cells with 200 pmol siRNAs in 2 ml serum-free transfection medium using lipofectamine reagent (Invitrogen). After 3 h of incubation, normal medium supplemented with serum replaced transfection medium and cells were grown for 30 h before starting T3 treatment. Subsequently, cells were lysed for western blot analysis and samples were analyzed as described earlier.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
T3 induces rapid activation of Akt in a CHX-insensitive manner

As we previously demonstrated (Verga Falzacappa et al. 2006), hCM and rRINm5F cells exposed to T3 (10–7 M) treatment are able to counteract an apoptotic cascade ongoing. This survival effect is mediated by PI3K and involves an Akt activation by T3 after 24 h of hormone treatment. At first, we decided to examine an earlier time course of Akt activation by T3 treatment. hCM and rRINm5F cells were cultured in the presence or the –7 M) for 10 absence of the hormone treatment (T3 10 and 30 min, and then were T3 deprived for 30 min. In addition, cells in the presence of T3 10–7 M were incubated with the thyroid hormone receptor antagonists, bisphenol A and TRIAC (10 µM). The 10 µM concentration of each inhibitor was selected as the more efficacious after experiments of dose–response performed with different concentrations (100 nM, 1, and 10 µM; data not shown). BPA is a thyroid hormone analog capable of disrupting the action of the said hormone through the thyroid hormone receptor (Moriyama et al. 2002), while TRIAC is a thyroid hormone analog which is specifically able to potentiate T3 effect on transcription through TRß isoforms (Messier & Langlois 2000). A negative control for Akt phosphorylation was obtained by incubating the cells with the PI3K inhibitor LY-294 002 hydrochloride 10 µM. As shown in Fig. 1Go, a significant increase in Ser 473 phosphorylation of Akt was detected as early as 10 min after T3 addition and persisted for up to 30 min in both hCM and rRINm5F cells. This effect appeared to be T3 dependent, since the deprivation of the hormone from cell culture media led to a decrease in the phosphorylation level of Akt after 30 min up to the basal level; moreover the effect which appeared to be T3 dependent as suggested by the ability of BPA to block the phosphorylation, seemed to be independent of gene transcription, since the presence of TRIAC could not augment pAkt levels.


Figure 1
View larger version (40K):
[in this window]
[in a new window]

  		Figure 1 T3 induces rapid activation of pAkt in a CHX-insensitive manner. Cells (a, hCM; b, rRINm5F) were exposed to T3 (10–7 M), and then deprived of the hormone treatment for the indicated times. Cells treated with T3 (30 min) were also exposed to LY (10 µM), BPA (10 µM), and TRIAC (10 µM; upper panels). Additionally, samples were hormone treated and concurrently exposed to 3 mM CHX for 30 min (lower panels). Western blot analyses (WB) were performed as described in Materials and methods and a specific band corresponding to the phosphorylated Akt (Ser 473) was detected. The expression of unphosphorylated Akt was analyzed as a control for gel loading. Densitometric absorbance values from three separate experiments were averaged (±S.D.), after they had been normalized to Akt for equal loading. Data are presented in the histogram as fold of induction (y-axis), calculated as treated sample/control. The different experimental groups are indicated on the x-axis. A comparison of the individual treatment was conducted by using Student’s t-test. *P<0.05; {dagger}P<0.01. P<0.05 was considered significant. At least three different experiments were performed, and a representative one is shown here.

 
The T3 action we observed did not require de novo protein synthesis; the addiction of the protein synthesis inhibitor CHX to the culture medium indeed did not affect the Ser 473 phosphorylation of Akt even after 30 min of exposure to T3. These data support our hypothesis that the T3 activation of Akt is mediated by a non-genomic mechanism.

TRß1 exists in the cytoplasmic region and is able to colocalize with PI3K p85{alpha}

As we observed, the effect of T3 on Akt phosphorylation was rapid and CHX independent. Our results showed that BPA, but not TRIAC can influence the T3 effect on the activation of Akt, moreover, the activation was not influenced by CHX, thus suggesting a non-genomic mechanism for this T3 action, presumably taking place outside the nucleus. The Akt phosphorylation is specifically due to PI3K, which resides at the plasma membrane; since a cytoplasmic location for thyroid receptor has been shown (Zhu 1998, Davis et al. 2000), we decided to investigate whether a thyroid receptor was detectable in a subcellular localization of the examined cells. As shown in Fig. 2a and dGo, both hCM and rRINm5F cells immunostained for TRß1 showed a positivity at the cytoplasmic level, showing the presence of the thyroid receptor in this cell compartment of hCM and rRINm5F cells. Once the cells have been counter-stained for the p85{alpha} subunit of PI3K, images by fluorescence microscopy were merged to evaluate the localization of both the proteins analyzed. Interestingly, the TRß1 and the PI3K p85{alpha} were able to colocalize at a cytoplasmic level. This ability and the expression level of each single protein were not affected by the hormone treatment (data not shown), indicating this to be a ligand-independent effect. The cytosol localization of TRß1 was moreover confirmed by the detection of a specific band in the western blot analyses, Fig. 2bGo (hCM) and e (RINm5F), performed on the cytosolic fractions of the cells. As shown, TRß1 was clearly detectable at the nuclear level in both immunofluorescence and western blot analyses, confirming its typical localization is not altered in the analyzed cells.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		Figure 2 TRß1 is present at a cytoplasmic level and complexes with PI3K p85{alpha}. Immunofluorescence (IF) analysis (hCM, a; rRINm5F, d). Cells were cultured and immunostained for TRß1 (green fluorescence), and subsequently for PI3K p85{alpha} (red fluorescence); nuclei were counterstained with Hoechst. The images were then merged to show the colocalization of the two signals. The images were acquired and analyzed through fluorescence microscopy as described in Materials and methods. Western blot (WB) analyses performed specifically on the cytosol and nuclear fractions of hCM (b) and rRINm5F (e). Cell extracts were analyzed for the presence of TRß1 as described in Materials and methods. Coimmunoprecipitation (IP) experiments (hCM, c; rRINm5F, f). Cells were exposed to T3 and then deprived of the hormone treatment for the indicated times (min), and the same cells were also exposed to BPA (10 µM) or TRIAC (10 µM); immunoprecipitation experiments for TRß1 were performed on total extracts. Western blot analyses for PI3K p85{alpha} and TRß1 were performed as described in Materials and methods. All the data shown are representative of at least three independent experiments.

 
TRB1 complexes with PI3K p85{alpha} in a ligand-independent manner

Thyroid receptors have recently been demonstrated to act non-genomically similarly to other steroid receptors, in particular Cao et al.(2005) and Storey et al.(2006) have shown an interaction between the subunit p85{alpha} of PI3K and the thyroid receptor ß1, similar to that observed for the estrogen receptor {alpha} (Simoncini et al. 2000). Given the results obtained by the immunofluorescence experiments, we decided to analyze the ability of TRß1 and PI3K p85{alpha} to form a complex. As shown in Fig. 2c and fGo, immunoprecipitation with anti-TRß1 antibody resulted in pulling down of the catalytic subunit of PI3K p85{alpha} in the presence or the absence of the hormone T3 and of its analogs BPA and TRIAC. In the same experimental conditions, the presence of the regulatory subunit of PI3K p110 was not detectable (data not shown), indicating that only the p85{alpha} subunit can be pulled down by TRß1. TRß1 and PI3K p85{alpha} can form a complex in a ligand-independent manner, and together with the evidence obtained by fluorescence microscopy and cytosol western blot, these data suggest this complex that is located at the cytosol or at the plasma membrane level.

T3 induces TRß1-associated PI3K activity

To investigate whether T3 was able to affect the PI3K activity, a competitive ELISA assay was performed on TRß1 pulled down samples. Samples were collected from cells (hCM and RINm5F) exposed or not to the hormone treatment for 30 min and exposure to LY 10 µM was used as a negative control. As shown in Fig. 3aGo, the presence of T3 provoked an increase in the kinase activity of about twofold; the PI3K activity was indeed about 90 pmol/mg protein per hour in the control samples of hCM cells, while it reached 200 pmol/mg protein per hour in the hormone-treated samples. The same experiments performed on rRINm5F showed a similar trend (data not shown). These data indicate that, although the hormone treatment does not influence the formation of the complex between p85{alpha} and TRß1, it is able to induce the kinase activity in the TRß1-associated PI3K.


Figure 3
View larger version (34K):
[in this window]
[in a new window]

  		Figure 3 (a) T3 induces TRß1-associated PI3K and Akt activities. PI3K activity: hCM and rRINm5f cells were exposed to T3 (10–7 M) alone or concurrently with T3 and LY (10 µM) and to vehicle alone for 30 min. Then, total extracts were immunoprecipitated for TRß1 and analyzed for PI3K activity as described in Materials and methods. The kinase activity was estimated by comparing the values from the samples with those in the standard curve obtained following manufacturer’s instruction. All the data are presented as means±S.D. and are the results of five different experiments at least in which every sample was run in triplicate. (b) Akt activity: hCM and RINm5F cells were cultured in the presence or not of T3 (10–7 M) and inhibitors for the indicated times (min). Western blot (WB) analyses were performed as described in Materials and methods and a specific band corresponding to the phosphorylated GSK3{alpha} (Ser 21) was detected. The expression of GSK3{alpha} was analyzed as a control for gel loading. Densitometric absorbance values from three separate experiments were averaged (±S.D.), after they had been normalized to GSK3{alpha} for equal loading. Data are presented in the histogram as fold of induction (y-axis), calculated as treated sample/control. The different experimental groups are indicated on the x-axis. A comparison of the individual treatments was conducted by using Student’s t-test. *P<0.05; {dagger}P<0.01. P<0.05 was considered significant. At least three different experiments were performed, and a representative one is shown here. ctrl, control.

 
T3 induces Akt activity

To evaluate if the increase in Akt phosphorylation levels was accompanied by an increase in the Akt kinase activity, activation of a specific Akt substrate was analyzed by western blot analyses. The main targets of Akt activity are the glucogen synthetase kinases {alpha} and ß, which are specifically phosphorylated on the Ser 21 and Ser 9 residues respectively by Akt (Cross et al. 1995); as shown in Fig. 3bGo, western blot analyses performed on the same samples utilized for Akt activation evaluation, were performed for the analysis of GSK3{alpha} phosphorylation levels. Interestingly, the phosphorylation of GSK3{alpha} on Ser 21 was increased by the hormone treatment already after 10 min; moreover, the addition of neither BPA nor TRIAC was able to affect the T3 effect on this phosphorylation suggesting that the thyroid hormone T3 is able to fully activate Akt, thus promoting its kinase activity, in both hCM and rRINm5F cells.

T3 induces the nuclear translocation of activated Akt

To better understand the dynamics of the activation observed, the cells exposed to the hormone treatment (T3) were immunostained for pAkt-Ser 473 and analyzed with a fluorescence microscope equipped for confocal microscopy. After stimulation through PI3K, Akt is recruited to the plasma membrane where it is activated by phosphorylation. Once activated, pAkt is translocated to the different cell compartments, including the nucleus, where its target proteins are.

We have analyzed the T3 effect on the localization of pAkt (Ser 473) in hCM and in rRINm5F cells as shown in Fig. 4aGo (hCM) and c (rRINm5F). As shown, activated pAkt-Ser 473 was rapidly translocated to the nucleus, leading to a stronger fluorescence in this cell compartment. Interestingly, this effect was promptly increased by the presence of the hormone treatment (30 min), suggesting that T3 can promote the activation of Akt and its subsequent nuclear relocalization. To confirm the observed phenomena, western blot analyses were performed on the separate cytosol and nuclear fractions of the cells exposed to the hormone treatment for 10 and 30 min. As shown in Fig. 4bGo (hCM) and d (rRINm5F), the phosphorylated Akt levels were remarkably augmented in the nuclear fraction of treated cells, suggesting that the increase observed in pAkt levels in the whole proteins is mainly due to the nuclear fraction. The quality and the loading of the extracts were examined by histone H1 (nucleus) and {alpha}-tubulin (cytosol) blotting as shown. These data suggest that in our cellular models, T3 is able to increase Akt activation by specific phosphorylation on Ser 473 and promotes its nuclear localization as shown in the cells exposed to the hormone.


Figure 4
View larger version (53K):
[in this window]
[in a new window]

  		Figure 4 T3 induces nuclear translocation of pAkt. hCM (a) and rRINm5F (c) cells were exposed to T3 (10–7 M) or vehicle alone for 30 min and then immunostained for pAkt-Ser 473 as described in Materials and methods, and images were analyzed by confocal microscopy. Additionally, western blot (WB) analyses on the cytoplasmic and nuclear fractions of proteins extracts from hCM (b) and rRINm5F cells (d) exposed to T3 treatment for 30 min were performed and a specific band corresponding to the phosphorylated Akt (Ser 473) was detected. The expression of histone H1 (nuclear) and {alpha}-tubulin (cytosolic) was analyzed as a control for gel loading and to exclude the contamination of the cytosol with the nuclear components and vice versa. At least three different experiments were performed, and a representative one is shown here.

 
The T3 activation of Akt in ß cells depends on TRß1

To confirm the crucial role of the thyroid receptor ß1 for the T3 activation of Akt via PI3K pathway in hCM and rRINm5F cells, we analyzed if the specific silencing of TRß1 could alter T3 ability of the to promote Akt phosphorylation. TRß1 was ‘knocked down’ in hCM and rRINm5F cells by RNAi experiments. The cells were transfected with TRß1 siRNAs and exposed or not to T3 treatment; total extracts were then immunoblotted for TRß1 and pAkt-Ser 473. As shown in Fig. 5a and bGo, when TRß1 was silenced, as demonstrated by western blot analyses, the phosphorylation of Akt induced by T3 was completely abolished; on the other hand, the TRß1 silencing per se was not able to influence the basal activation of Akt in the ß cells analyzed. These data strongly indicated that the ability of T3 to induce Akt activation in the islet cells utilized, is specifically mediated by the thyroid receptor ß1.


Figure 5
View larger version (62K):
[in this window]
[in a new window]

  		Figure 5 TRß1 interference abolishes T3 effect on Akt activation. RNA interference experiments to silence TRß1 expression were performed as described in Materials and methods on hCM (a) and rRINm5F (b) cells exposed or not to T3 (10–7 M) for the indicated times. Western blot analyses were performed as described in Materials and methods and a specific band corresponding to the phosphorylated Akt (Ser 473) and TRß1 was detected. The expression of Akt was analyzed as a control for gel loading. At least three different experiments were performed, and a representative one is shown here.

 
To exclude the possibility that T3 effects on Akt depend on the amount of TRß1, we performed a western blot to analyze TRß1 expression in treated cells (data not shown). Neither hCM nor rRINm5F TRß1 protein levels were affected by T3 treatment, strongly indicating that the effect we observed was completely independent of the receptor amount.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
The thyroid hormone T3 is known to access the cell interior and the cell nucleus, where it binds to its nuclear receptors and transactivates thyroid hormone-regulated genes. This action, classically referred to as genomic, happens in hours to days, which is consistent with the typical hormone effects, including regulation of cell growth, development, and metabolism. On the other hand, a different mechanism has been observed, which induces very fast responses in cells, happening within minutes or even seconds, and it is called non-genomic or extranuclear action. Such effects have actually been known for many years, but their mechanisms still remain unclear. One of the main questions to be answered is which are the mediators of this thyroid hormone action; in particular it is not yet clear whether a new thyroid hormone receptor is involved in the non-genomic action, although no specific membrane-associated TR isoform has been identified yet, or if the well known receptor has novel additional functions, since 10% of TRs are cytoplasmic in the absence of T3 (Baumann et al. 2001).

In this study, we have shown in pancreatic ß cells that a non-genomic T3 action involving the PI3K pathway exists and that the thyroid receptor ß1 is essential in mediating this T3 action.

The factors that drive ß cell proliferation and function under normal or pathological conditions are still unknown. Available data indicate that there exists a plethora of ß cell growth factors acting in genetically heterogeneous and presumably oligogenic fashion (Nielsen et al. 1999). Cellular processes, such as proliferation, survival, and glucose metabolism induced by different hormones and growth factors are dependent on the activation of PI3K; moreover, the involvement of Akt in the regulation of replication and survival of pancreatic ß cells has been demonstrated (Tuttle et al. 2001, Lingor et al. 2003). Multiple Akt substrates are involved in regulating various aspects of ß cell function, in this view augmented protein synthesis represents an important component of the growth response, but not the sole effector. Nevertheless, the activation of protein kinase B can occur within minutes, and is related to non-genomic action of specific activators of the PI3K pathway. The thyroid hormones have recently been demonstrated to induce Akt activation (Cao et al. 2005, Kuzman et al. 2005), and this effect occurs in minutes or hours. In this study, we demonstrated that T3 (10–7 M), whose role in ß cell growth and survival we previously evidenced (Verga Falzacappa et al. 2006), provokes Akt phosphorylation in minutes, this effect being independent of de novo protein synthesis.

Activation of Akt entails a complex series of events involving additional proteins. First, the PI3K-generated lipid products PI(3,4,5)P3 and PI(3,4)P2 recruit Akt to the plasma membrane through their affinity for the PH domain of Akt (Burgering & Coffer 1995, Franke et al. 1997). Once membrane proximal, Akt is phosphorylated on two residues (Thr 308 and Ser 473), then active Akt can rapidly translocate to specific intracellular compartments. This ordered series of events is necessary to generate fully activated Akt and has been demonstrated even in in vivo models (Scheid et al. 2002). In this study, we demonstrated how T3 treatment is able to influence these events, in particular inducing the translocation of active Akt to the nucleus.

Optimal signaling through the PI3K pathway depends on a critical molecular balance between the regulatory and the catalytic subunits; in particular, the p85{alpha} subunit is thought to be the major response pathway for most stimuli (Shepherd et al. 1998). The relationship between Akt activation and T3 action remains to be elucidated; Cao et al.(2005) and Storey et al.(2006) have demonstrated that the thyroid receptor ß1 is able to interact with the regulatory subunit p85{alpha} of the PI3K. This ability of a nuclear receptor of complexing with the regulatory subunit of PI3K has already been observed for the estrogen receptor ER{alpha} (Simoncini et al. 2000), for which controversy exists concerning whether or not it has a role outside the nucleus (Pietras & Szego 1977). It has been suggested that such non-genomic actions might be mediated by membrane-associated isoforms of the classical nuclear receptors with significantly different agonist/antagonist affinities (Losel & Wehling 2003). We demonstrated that the ß1 isoform of the thyroid receptor not only is able to form a complex with the p85{alpha} subunit, but also is clearly detectable in the cytoplasmic area of the cells examined. Interestingly, we show that although the interaction between PI3K and TRß1 is not influenced by the presence of the hormone, T3 treatment can enhance the TRß1-associated PI3K activity. In fact, it has been demonstrated that thyroid receptors can locate outside the nucleus in the absence of T3 (Baumann et al. 2001), thus confirming that the TRß1-PI3K binding does not require the presence of T3. Otherwise, the presence of the hormone does stimulate the kinase activity, when it is complexed to TRß1. These data suggest that the hormone is somehow able to activate the kinase and that this action is TR mediated. In addition, we demonstrated that this T3 action can trigger a cascade of events that are PI3K dependent; in fact, not only Akt is activated by T3, but also its activity is influenced by the presence of the hormone. Our findings showed that phosphorylation of GSK3{alpha}, one of the main Akt targets, is enhanced by the hormone treatment and that even this activation shows a trend similar to the one observed for Akt phosphorylation itself. These data suggest that the full activation of Akt induced by T3 via PI3K activity stimulation, leads to an increment even in Akt kinase activity. Moreover, given the evidence of the cytoplasmic localization of TRß1, our data support the hypothesis that nuclear thyroid receptors might exist also outside the nucleus (Zhu et al. 1998, Davis et al. 2000) playing an important role in non-genomic actions of thyroid hormones.

We also showed that the ‘knockdown’ of the gene encoding TRß1, through experiments of RNA interference, led to an abolishment of the Akt activation induced by T3, and once again the role of TRß1 seems to be essential.

Phosphoinositide 3-kinase plays a pivotal role in the metabolic and mitogenic actions of insulin, in particular the direct binding between IRS-2 and PI3K leads to Akt activation in pancreatic ß cells, modulating the cell survival, the proliferation and the cell size (Accili 2001). Given this crucial role of IRS-2 in PI3K action in pancreatic ß cells, we analyzed its expression after long-term (24 h) treatment with T3; interestingly the protein levels of IRS-2 were upregulated by the hormone treatment (data not shown), suggesting that T3 can interact with the PI3K pathway even at this level. In fact, if all the effects of IRS-2 are mediated by Akt, there must be additional players, which are likely to include also transcriptional factors and additional kinases. In this context, we could speculate that TRß1 plays a specific role directly entering the PI3K pathway.

Baumann et al.(2001) have shown that TRß rapidly shuttles between the nucleus and the cytoplasm. The possible modes of interaction between non-genomic and genomic thyroid hormone signaling are complex, but they appear to act synergistically. The postulated non-genomic signaling pathway may complement such actions by generating second messengers and by activating multiple signaling cascades. For example, the genomic and non-genomic effects of thyroid hormones, in the mitochondria, also appear to be strikingly synergistic (Bassett et al. 2003). These findings reveal the complexity of the TR signaling and suggest that a thyroid receptor with high homology to the nuclear one at least, if not the same itself, might be implicated in the effects we characterized in pancreatic ß cells. Short interfering RNAs, designed to specifically act on nuclear TRß1 mRNA, can silence both cytoplasmic and nuclear TRß1 without distinction (data not shown); in addition, the commercial monoclonal antibody against the nuclear thyroid hormone receptor recognizes something that is responsible for the T3 effect observed and that is located in the cytoplasm, strengthening our hypothesis that a thyroid hormone receptor with high similarities to the nuclear one but with a peculiar localization is involved.

In conclusion, we suggest that a ‘cytoplasmic’ thyroid receptor exists in the pancreatic ß cells RINm5F and CM that is able to mediate the T3 action on the PI3K signaling pathway; this receptor seems to be similar to the nuclear TRß1 form, but with some peculiar characteristics that led its location.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
This work has been supported by a grant from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MIUR Cofin), Rome, Italy, and by the Associazione Fatebenefratelli per la Ricerca (A Fa R). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Accili D 2001 A kinase in the life of the ß cell. Journal of Clinical Investigation 108 1575–1576.[CrossRef][Web of Science][Medline]

Baroni MG, Cavallo MG, Mark M, Monetini L & Stoherer B 1999 Beta-cell gene expression and functional characterization of the human insulinoma cell line CM. Journal of Endocrinology 161 59–68.[Abstract]

Bassett JHD, Harvey CB & Williams GR 2003 Mechanisms of thyroid hormone receptor-specific nuclear and extra nuclear actions. Molecular and Cellular Endocrinology 213 1–11.[CrossRef][Web of Science][Medline]

Baumann CT, Maruvada P, Hager GL & Yen PM 2001 Nuclear cytoplasmic shuttling by thyroid hormone receptors. Multiple protein interactions are required for nuclear retention. Journal of Biological Chemistry 276 11237–11245.[Abstract/Free Full Text]

Bergh JJ, Lin H-Y, Lansing L, Mohamed SN, Mousa S, Davis FB, Mousa S & Davis PJ 2005 Integrin {alpha}vß3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis. Endocrinology 146 2864–2871.[CrossRef][Web of Science][Medline]

Burgering BM & Coffer PJ 1995 Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376 599–602.[CrossRef][Medline]

Buteau J, Foisy S, Rhodes CJ, Carpenter L, Biden TJ & Prentki M 2001 Protein kinase C zeta activation mediated glucagon-like peptide-1 induced pancreatic beta cell proliferation. Diabetes 50 2237–2243.[Abstract/Free Full Text]

Cao X, Kambe F, Moeller LC, Refetoff S & Seo H 2005 Thyroid hormone induces rapid activation of Akt/protein kinase B-mammalian target of rapamycin-p70S6K cascade through phosphatidylinositol 3-kinase in human fibroblasts. Molecular Endocrinology 19 102–112.[Abstract/Free Full Text]

Cavallo MG, Dotta F, Monetini L, Dionisi S, Previti M, Valente L, Toto A, Di Mario U & Pozzilli P 1996 Beta-cell markers and autoantigen expression by a human insulinoma cell line: similarities to native beta cells. Journal of Endocrinology 150 113–120.[Abstract/Free Full Text]

Coffer PJ, Jin J & Woodgett JR 1998 Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochemical Journal 335 1–13.

Cross DA, Alessi DR, Cohen P, Andjelkovich M & Hemmings BA 1995 Inhibition of glycogen synthase kinase-3 by insulin is mediated by protein kinase B. Nature 378 785–789.[CrossRef][Medline]

D’Arezzo S, Incerpi S, Davis FB, Acconcia F, Marino M, Farias RN & Davis PJ 2004 Rapid non-genomic effects of 3,5,3'-triiodo-L-thyronine on the intracellular pH of L-6-myoblasts are mediated by intracellular calcium mobilization and kinase pathways. Endocrinology 145 1660–1668.

Davis PJ, Shih A, Lin HY, Martino LJ & Davis FB 2000 Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR. Journal of Biological Chemistry 275 38032–38039.[Abstract/Free Full Text]

Dickson LM & Rhodes CJ 2004 Pancreatic ß cell growth and survival is the onset of type 2 diabetes: a role for protein kinase B in the Akt? American Journal of Physiology. Endocrinology and Metabolism 287 E192–E198.[Abstract/Free Full Text]

Elghazi L, Balcazar N & Bernal-Mizrachi E 2006 Emerging role of protein kinase B/Akt signalling in pancreatic ß-cell mass and function. International Journal of Biochemistry and Cell Biology 38 157–163.[Web of Science][Medline]

Franke TF, Kaplan DR, Cantley LC & Toker A 1997 Direct regulation of the Akt proto-oncogene product by phosphatidiylinositol-3,4-bisphosphate. Science 275 665–668.[Abstract/Free Full Text]

Gharbi J & Torresani J 1979 High affinity thyroxine binding to purified rat liver plasma membranes. Biochemical and Biophysical Research Communications 88 170–177.[CrossRef][Web of Science][Medline]

Giannoukakis N, Mi Z, Rudert WA, Gambotto A, Trucco M & Robbins P 2000 Prevention of beta-cells dysfunction and apoptosis activation in human islets by adenoviral gene transfer of the insulin-like growth factor I. Gene Therapy 7 2015–2022.[CrossRef][Web of Science][Medline]

Haynes MP, Li L, Sinha D, Russell KS, Hisamoto K, Baron R, Collinge M, Sessa WC & Bender JR 2003 Src kinase mediates phosphatidylinositol 3-kinase/Akt-dependent rapid endothelial nitric-oxide synthase activation by estrogen. Journal of Biological Chemistry 278 2118–2123.[Abstract/Free Full Text]

Heery DM, Kalkhoven E, Hoare S & Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387 733–736.[CrossRef][Medline]

Hennemann G, Docter RE, Friesema CH, de Jong M, Krenning EP & Visser TJ 2001 Plasma membrane transport of thyroid hormones and its role in thyroid hormone metabolism and bioavailability. Endocrine Reviews 22 451–476.[Abstract/Free Full Text]

Hui H, Nourparvar A, Zhao X & Perfetti R 2003 Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A- and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology 144 1444–1455.[Abstract/Free Full Text]

Kuzman JA, Gerdes AM, Kobayashi S & Liang Q 2005 Thyroid hormone activates Akt and prevents serum starvation-induced cell death in neonatal rat cardiomyocytes. Journal of Molecular and Cellular Cardiology 39 841–844.[CrossRef][Web of Science][Medline]

Lei J, Mariash CN & Ingbar DH 2004 3,3' ,5-Triiodo-L-thyronine up-regulation of Na,K-ATPase activity and cell surface expression in alveolar epithelial cells is Src kinase- and phosphoinositide 3-kinase-dependent. Journal of Biological Chemistry 279 47589–47600.[Abstract/Free Full Text]

Liang J & Slingerland JM 2003 Multiple roles of PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle 2 339–345.[Medline]

Lin HY, Davis FB, Gordinier JK, Martino LJ & Davis PJ 1999 Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells. American Journal of Physiology 276 C1014–C1024.

Lingor MK, Dickinson LM, Wrede CE, Briaud I, McCuaig JF, Myers MG Jr & Rhodes CJ 2003 Decreasing IRS-2 expression in pancreatic ß-cells (INS-1) promotes apoptosis, which can be compensated for by introduction of IRS-4 expression. Molecular and Cellular Endocrinology 209 17–31.[CrossRef][Web of Science][Medline]

Lopez-Carballo G, Moreno L, Masia S, Perez P & Barettino D 2002 Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. Journal of Biological Chemistry 277 25297–25304.[Abstract/Free Full Text]

Losel R & Wehling M 2003 Non-genomic actions of steroid hormones. Nature Reviews. Molecular and Cellular Biology 4 46–56.

Messier N & Langlois MF 2000 Triac regulation of transcription is T(3) receptor isoform- and response element-specific. Molecular and Cellular Endocrinology 165 57–66.[CrossRef][Web of Science][Medline]

Moriyama K, Tagami T, Akamizu T, Usui T, Saijo M, Kanamoto N, Hataya Y, Shimatsu A, Kuzuya H & Nakao K 2002 Thyroid hormone action is disrupted by bisphenol A as an antagonist. Journal of Clinical Endocrinology and Metabolism 87 5185–5190.[Abstract/Free Full Text]

Nielsen JH, Svensson C, Galsgaard ED, Moldrup A & Billestrup N 1999 Beta cell proliferation and growth factors. Journal of Molecular Medicine 77 62–66.[CrossRef][Web of Science][Medline]

Pietras RJ & Szego CM 1977 Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 265 69–72.[CrossRef][Medline]

Pliam MB & Goldfine ID 1997 High affinity thyroid hormone binding sites on purified rat liver plasma membranes. Biochemical and Biophysical Research Communications 79 166–172.

Porlan E, Vega S, Iglesias T & Rodriguez-Poena A 2004 Unliganded thyroid hormone receptor beta1 inhibits proliferation of murine fibroblasts by delaying the onset of the G1 cell-cycle signals. Oncogene 23 8756–8765.[CrossRef][Web of Science][Medline]

Ribeiro RC, Apriletti JW, Wagner RL, West BL, Feng W, Huber R, Kushner PJ, Nilsson S, Scanlan T, Fletterick RJ et al. 1998 Mechanisms of thyroid hormone action: insights from X-ray crystallographic and functional studies. Recent Progress in Hormone Research 53 351–394.[Medline]

Scheid MP, Marignani PA & Woodgett JR 2002 Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B. Molecular and Cellular Biology 22 6247–6259.[Abstract/Free Full Text]

Shepherd PR, Withers DJ & Siddle K 1998 Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochemical Journal 333 471–490.

Shih A, Lin H-Y, Davis FB & Davis PJ 2001 Thyroid hormone promotes serine phosphorylation of p53 by mitogen-activated protein kinase. Biochemistry 40 2870–2878.[CrossRef][Medline]

Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW & Liao JK 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407 538–541.[CrossRef][Medline]

Storey NM, Gentile S, Ullah H, Russo A, Muessel M, Erxleben C & Armstrong DL 2006 Rapid signalling at the plasma membrane by a nuclear receptor for thyroid hormone. PNAS 103 5197–5201.[Abstract/Free Full Text]

Sun M, Paciga JE, Feldman RI, Yuan Z, Coppola D, Lu YY, Shelley SA, Nicosia SV & Cheng JQ 2001 Phosphatidylinositol-3-OH kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor {alpha} (ER{alpha}) via interaction between ER{alpha} and PI3K. Cancer Research 61 5985–5991.[Abstract/Free Full Text]

Tang HY, Lin H-Y, Zhang S, Davis FB & Davis PJ 2004 Thyroid hormone causes mitogen-activated protein kinase-dependent phosphorylation of the nuclear estrogen receptor. Endocrinology 145 3265–3272.[Abstract/Free Full Text]

Tuttle RL, Gill NS, Pugh W, Lee JP, Koeberlein B, Furth EE, Polonsky KS, Naji A & Birnbaum MJ 2001 Regulation of pancreatic ß cell growth and survival by the serine/threonine protein kinase Akt1/PKB{alpha}. Nature Medicine 7 1133–1137.[CrossRef][Web of Science][Medline]

Ueki K, Fruman DA, Brachmann SM, Tseng Y-H, Cantley LC & Kahn CR 2002 Molecular balance between the regulatory and catalytic subunits of phosphoinositide 3-kinase regulates cell signalling and survival. Molecular and Cellular Biology 22 965–977.[Abstract/Free Full Text]

Verga Falzacappa C, Panacchia L, Bucci B, Stigliano A, Cavallo MG, Brunetti E, Toscano V & Misiti S 2006 3,5,3'-Triiodothyronine (T3) is a survival factor for pancreatic beta-cells undergoing apoptosis. Journal of Cellular Physiology 206 309–321.[CrossRef][Web of Science][Medline]

Yen PM 2001 Physiological and molecular basis of thyroid hormone action. Physiological Reviews 81 1097–1142.[Abstract/Free Full Text]

Zhu XG, Hanover JA, Hager GL & Cheng SY 1998 Hormone-induced translocation of thyroid hormone receptors in living cells visualized using a receptor green fluorescent protein chimera. Journal of Biological Chemistry 273 27058–27063.[Abstract/Free Full Text]

Received in final form 7 November 2006
Accepted 20 November 2006
Made available online as an Accepted Preprint 13 December 2006




This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Lei, M. Bhargava, and D. H. Ingbar
Cell-specific signal transduction pathways regulating Na+-K+-ATPase. Focus on "Short-term effects of thyroid hormones on the Na+-K+-ATPase activity of chick embryo hepatocytes during development: focus on signal transduction"
Am J Physiol Cell Physiol, January 1, 2009; 296(1): C1 - C3.
[Full Text] [PDF]

Home page
 EndocrinologyHome page
P. de Lange, R. Senese, F. Cioffi, M. Moreno, A. Lombardi, E. Silvestri, F. Goglia, and A. Lanni
Rapid Activation by 3,5,3'-L-Triiodothyronine of Adenosine 5'-Monophosphate-Activated Protein Kinase/Acetyl-Coenzyme A Carboxylase and Akt/Protein Kinase B Signaling Pathways: Relation to Changes in Fuel Metabolism and Myosin Heavy-Chain Protein Content in Rat Gastrocnemius Muscle in Vivo
Endocrinology, December 1, 2008; 149(12): 6462 - 6470.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. J. Holness, G. K. Greenwood, N. D. Smith, and M. C. Sugden
PPAR{alpha} activation and increased dietary lipid oppose thyroid hormone signaling and rescue impaired glucose-stimulated insulin secretion in hyperthyroidism
Am J Physiol Endocrinol Metab, December 1, 2008; 295(6): E1380 - E1389.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
M. Pietrzak and M. Puzianowska-Kuznicka
Triiodothyronine utilizes phosphatidylinositol 3-kinase pathway to activate anti-apoptotic myeloid cell leukemia-1
J. Mol. Endocrinol., September 1, 2008; 41(3): 177 - 186.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
E. Ortega, J. Koska, N. Pannacciulli, J. C Bunt, and J. Krakoff
Free triiodothyronine plasma concentrations are positively associated with insulin secretion in euthyroid individuals
Eur. J. Endocrinol., February 1, 2008; 158(2): 217 - 221.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
S. Michienzi, B. Bucci, C. Verga Falzacappa, V. Patriarca, A. Stigliano, L. Panacchia, E. Brunetti, V. Toscano, and S. Misiti
3,3',5-Triiodo-L-thyronine inhibits ductal pancreatic adenocarcinoma proliferation improving the cytotoxic effect of chemotherapy
J. Endocrinol., May 1, 2007; 193(2): 209 - 223.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Verga Falzacappa, C.
Right arrow Articles by Misiti, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Verga Falzacappa, C.
Right arrow Articles by Misiti, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS