| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Endocrinology, Medical Research Center, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland2 Department of Biochemistry and Molecular Biology, Medical Center of Postgraduate Education, Marymoncka 99, 01-813 Warsaw, Poland
(Correspondence should be addressed to M Puzianowska-Kuznicka; Email: monika{at}amwaw.edu.pl)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
1 isoform. However, this increase is not a result of direct activation via the thyroid hormone-response element, TRE-DR4, located at the –998 to –983 position in this promoter; furthermore, the presence of 9-cis-retinoic acid receptor is not required. The promoter's activation is abolished in the presence of phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin. The –295 to –107 promoter fragment contains all sequences involved in T3-dependent activation of the MCL-1 promoter, and cAMP-responsive element located at the –262 to –255 position is a major mediator in this process. Therefore, MCL-1 expression is activated by T3, which increases its promoter activity by a non-genomic mechanism using the PI3-K signal transduction pathway. We propose that this is another mechanism by which T3 regulates apoptosis.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
1, TR2, TRβ1, and TRβ2, and a number of other isoforms, are encoded by the TRA and TRB genes (Gosden et al. 1986, Drabkin et al. 1988, Cheng 2000, Weiss & Ramos 2004). Upon binding to TRE, TRs either activate or inhibit the transcription of these genes; whether activation or inhibition occurs depends on the presence or absence of T3 (Eckey et al. 2003, Yen et al. 2006, Oetting & Yen 2007), and on their interaction with coactivators, with corepressors (Chen & Evans 1995, Onate et al. 1995, McKenna et al. 1999, Weiss & Ramos 2004), and with other proteins such as 9-cis-retinoic acid receptors (RXRs) (Rastinjead 2001, Szanto et al. 2004). The non-genomic mode of action of T3 is more complex and, in some cases, is mediated by extra-nuclear fraction of TRs. For example, transcription from mitochondrial DNA is regulated by T3 bound to the truncated form of TR1—p43 (Casas et al. 1999). The interaction of liganded cytoplasmic fractions of TRs with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) induces the kinase activity and generation of phosphatidylinositol-3,4,5-triphosphate. Subsequently, Akt kinases are phosphorylated and they activate their downstream targets (Lei et al. 2004, Cao et al. 2005, Kuzman et al. 2005, Moeller et al. 2005, 2006, Kenessey & Ojamaa 2006, Storey et al. 2006, Verga Falzacappa et al. 2006, 2007). Depending on the developmental stage of the cell, its pathophysiological state and its type, T3 either inhibits or activates apoptosis. For example, T3 serves as a survival factor for human pancreatic β cells (Verga Falzacappa et al. 2006), but stimulates apoptosis in rat hepatic cells (Upadhyay et al. 2004), optic lobe cells of the chick embryo (Ghorbel et al. 1997), and muscle cells of Xenopus laevis tadpole tail undergoing metamorphosis (Sachs et al. 1997). Molecular mechanisms of apoptosis regulation by T3 are obscure. In part, the hormone may act via the members of BCL-2 family of proteins, as T3 treatment increases the level of BCL2-associated X protein (Bax) mRNA in the optic lobe of the chick embryo (Ghorbel et al. 1997) and in the caudal muscle of Xenopus tadpole tail (Sachs et al. 1997), and results in mitochondrial depolarization, the increase of mitochondrial pro-apoptotic Bax and Bak and in the decrease of anti-apoptotic BCL-2 in Jurkat cells (Yehuda-Shnaidman et al. 2005).
Myeloid cell leukemia (MCL-1) is an anti-apoptotic member of the BCL-2 family (van Delft & Huang 2006). It resides in the outer mitochondrial membrane (Yang et al. 1995) where it binds and prevents pro-apoptotic BCL-2 antagonist killer (BAK) from oligomerization and the formation of the channels necessary for cytochrome C release and for the triggering of apoptosis (Nijhawan et al. 2003, Leu et al. 2004, Willis et al. 2005). MCL-1 interacts with pro-apoptotic BCL-2 homology 3 (BH3)-only BCL-2 family members BCL-2-interacting mediator of cell death (BIM) and truncated BH3 interacting domain death agonist (tBID), blocking their function (Clohessy et al. 2006, Han et al. 2006). Multiple interactions of MCL-1 with other BCL-2 family members, its short half-life time, rapid down-regulation in response to stress signals, and multiple levels of expression control indicate that MCL-1 plays an important role in the regulation of apoptosis (Nijhawan et al. 2003, Michels et al. 2005, Yang-Yen 2006).
In this work, the molecular mechanism by which T3 regulates the expression of one of the members of the BCL-2 family is presented. We provide evidence for the activation by T3 of the MCL-1 promoter via TRβ1-dependent activation of PI3-K signaling pathway, and its influence on the increase of MCL-1 protein.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
The 1826 bp human MCL-1 promoter fragment (fragment A) including its transcription start site (Townsend et al. 1999) was cloned from 200 ng human genomic DNA with Taq polymerase and 5'MCL1 5'-AATCCCGGGTATGTCTCTCAGCACCTTG-3' and 3'MCL1 5'-ACGAAGCTTACTGGAAGGAAGCGGAAG-3' forward and reverse primers (SmaI and HindIII restriction sites in bold) respectively. After a 3-min initial denaturation at 94 °C, 3 cycles of 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 3 min, and then 37 cycles of 94 °C for 30 s, 58 °C for 1 min, and 72 °C for 3 min were performed, followed by a final extension at 72 °C for 10 min. The amplified DNA was cloned into the pGEM-T vector (Promega), restricted with SmaI and HindIII and recloned into the pGL2 luciferase reporter vector (Promega) to make the pGL2-MCL-1 reporter vector. The correctness of the promoter sequence was verified by sequencing. In silico search for putative binding sites for thyroid hormone receptors was performed with Transcription Element Search Software (TESS, Technical Report CBIL-TR-1997-1001-v0.0). The search was performed with a consensus TRE hexamer sequence (AGGTCA), with a maximum of one mismatch allowed.
MCL-1 promoter mutants
Deletion mutant E lacked 1515 bp from the 5' end of the pGL2-MCL-1 promoter (restriction with SacI and SmaI, blunting SacI end with Klenow, and re-ligation) and therefore, contained the –295 to +16 part of this promoter (pGL2-MCL-1E). Fragment EE (–107 to +16, pGL2-MCL-1EE) was generated by PCR with proofreading Platinum Pfx DNA polymerase (Invitrogen), with the pGL2-MCL-1 vector as a template, with 5'MCL1EE 5'-CATCCCGGGCCCTTTTATGGGAATACTTTTTT-3' and 3'MCL1 5'-ACGAAGCTTACTGGAAGGAAGCGGAAG-3' forward and reverse primers (incorporated SmaI and HindIII restriction sites in bold) respectively. After a 3-min denaturation at 94 °C, 35 cycles of 94 °C for 30 s, 56 °C for 30 s, and 68 °C for 40 s were performed, and followed by a final extension at 68 °C for 5 min. The PCR product was cloned into the pGL2-basic vector.
To obtain the pGL2-MCL-1TREmut reporter vector with mutated putative TRE, point mutagenesis was performed using the QuikChange multi site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), strictly following the manufacturer's instructions. The mutagenic primer TREmut 5'-AGGCCGAGACAGGCAAAAAACTTGAGGCCATGAGTTC-3' was used in the reaction with the pGL2-MCL-1 vector as a template. PCR was carried out as follows: 95 °C for 1 min, and 30 cycles of 95 °C for 1 min, 55 °C for 1 min, and 65 °C for 15 min. The PCR product was treated with DpnI, and 1.5 µl of the reaction mix was used to transform XL10-Gold ultracompetent Escherichia coli cells. To obtain the pGL2-MCL-1Emut reporter vector containing the –295 to +16 MCL-1 promoter fragment with mutated putative cAMP-responsive element (CRE), another point mutagenesis was performed. The mutagenic primer CREmut 5'-CTCGGAGCCGCCGCACTACGGCCGGCACTCAG-3' was used in the reaction with the pGL2-MCL-1E vector as a template. The PCR was carried out as detailed above. The correctness of the mutated promoter fragments was verified by sequencing.
Cell culture and transcription regulation assay
HK2 cells were cultured in a 24-well dish in Dulbecco Modified Eagle's (DME)/Ham's medium (Sigma–Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS), according to the protocol of Fraser et al. (2002), in a 37 °C humidified incubator with 5% CO2. Just before the experiment, cells were washed with PBS, and 0.5 ml serum-free BIO-MPM-1 multi-purpose serum-free medium for adherent cells (Biological Industries, Kibbutz Beit Haemek, Israel) supplemented with 2 mM glutamine were added. Cells were incubated for 6 h either in the presence or absence of 100 nM T3. Following the incubation, cells were washed with PBS, and used for production of whole-cell protein extracts.
HEK 293 cells were cultured in high glucose DMEM (Sigma–Aldrich) containing 10% FBS. Twenty-four hours before transfection, cells were seeded 1:2 onto a 24-well dish and maintained in DMEM supplemented with 10% FBS. Directly before transfection, cells were washed once with PBS and 0.5 ml serum-free BIO-MPM-1 medium supplemented with 2 mM glutamine were added to each well. Cells were transfected with metafectene (Biontex Laboratories Gmbh, Munich, Germany) according to the manufacturer's directions with 1.5 µl metafectene, 150 ng pGL2-MCL-1 vector containing firefly luciferase reporter gene driven by different MCL-1 promoter fragments, 100 ng pEGFP-C1 expression vector (Clontech Laboratories) or the same vector encoding wild-type TRβ1 or TR1, 100 ng pcDNA3.1(+) encoding wild-type human RXR, and 20 ng phRL-CMV internal control vector (Promega). After 24-h incubation without T3, or in the presence of 100 nM of this hormone, the cells were washed with PBS and lysed with 100 µl passive lysis buffer (Dual-Luciferase Reporter Assay System, Promega). Firefly and Renilla luciferase activities were measured in a microplate luminometer (BMG Labtech, Offenburg, Germany). All experiments were repeated six to nine times. Certain incubations were performed in the presence of the PI3-K inhibitor, wortmannin, according to the protocol of Wang et al. (2003) with the following modifications: after transfection was performed as described above, the cells were incubated for 3 h in BIO-MPM-1 serum-free medium, then wortmannin was added to a final concentration of 100 nM. T3 was added 30 min later to a final concentration of 100 nM and the incubation lasted for an additional 24 h.
Apoptosis detection and flow cytometric analysis
HK2 cells were seeded onto a 60 mm Petri dish and cultured in DMEM supplemented with 10% heat-inactivated FBS. After 24 h, cells were washed with PBS and 3 ml DMEM without FBS were added. Cells were cultured for 48 h either in the presence of 100 nM T3 or without this hormone. The detection of apoptotic cells was performed with ApoTarget Annexin-V FITC Apoptosis Kit (BioSource International, Inc., Camarillo, CA, USA) according to the manufacturer's protocol. Cell viability was determined by propidium iodide exclusion. Cells were analyzed by flow cytometry using FACSCalibur (Becton Dickinson Biosciences, San Jose, CA, USA).
RNA isolation and semi-quantitative RT-PCR
HK2 cells were seeded onto a 60 mm Petri dish and cultured in DMEM supplemented with 10% heat-inactivated FBS. After 24 h, cells were washed with PBS and 3 ml DMEM without FBS were added. Cells were cultured for 6 h either in the presence of 100 nM T3 or without this hormone, collected, and subjected to RNA isolation with TRIzol Reagent (Invitrogen) following the manufacturer's protocol. About 250 ng of each total RNA were used as a template in RT-PCRs performed with SuperScript One-Step RT-PCR with Platinum Taq System (Invitrogen). Each reaction was supplemented with 40 U RNasin ribonuclease inhibitor (Promega). Initial RT-PCR was carried out with control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene primers GAPDH-U 5'-CGCTGAGTACGTCGTGGAGTC-3' and GAPDH-L 5'-TGGCAGTGATGGCATGGAC-3' (product size 278 bp) as follows: one cycle of 30 min at 50 °C and 2 min at 94 °C, 40 cycles of 94 °C for 20 s, 58 °C for 30 s, 68 °C for 1 min. Ten microliters of aliquots were removed from the reaction tube after 20, 25, 30, 35, and 40 cycles, and GAPDH product was analyzed onto a 2% agarose gel to ensure that the main reaction will be stopped while in the exponential growth phase. Next, a main RT-PCR was performed as above with GAPDH and MCL-1 primers (MCL-1F 5'-GCTGCATCGAACCATTAGCAG-3' and MCL-1R 5'-GCCATAATCCTCTTGCCACTTG-3', product size 344 bp), and stopped after 30 amplification cycles. PCR products were resolved onto a 2% agarose gel.
Whole-cell protein extracts, nuclear protein extracts
To make a whole-cell protein extract, the cells (2x105) were resuspended in 70 µl lysis buffer consisting of 75 mM Tris–HCl (pH 8.0), 2% SDS, 15% glycerol, and boiled for 5 min. The extracts were flash-frozen in liquid nitrogen and stored at –80 °C.
To obtain nuclear protein extract, the cells (5x106) were resuspended in 500 µl buffer A consisting of 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, supplemented with a protease inhibitors mix (Complete Protease Inhibitor Cocktail, Roche Applied Science) and phenylmethylsulphonyl fluoride up to 400 µM, and incubated on ice for 1 h. After incubation, the cells were homogenized in an ice-cold glass–teflon homogenizer. The resulting homogenate was transferred to an Eppendorf tube and centrifuged at 400 g for 5 min at 4 °C. The pellet was resuspended in an equal volume of buffer A, centrifuged as before, and again resuspended in two volumes of buffer B consisting of 20 mM HEPES (pH 7.9), 10% glycerol, 420 mM NaCl, 1.5 mM MgCl2, and 0.2 mM EDTA, supplemented with protease inhibitors as above. After a 30-min incubation on ice, the sample was centrifuged at 15 000 g for 20 min at 4 °C, then the supernatant was transferred to a new tube, and supplemented with an equal volume of buffer C consisting of 20 mM HEPES (pH 7.9), 30% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, and protease inhibitors. The sample containing soluble nuclear proteins was aliquoted into pre-chilled Eppendorf tubes, flash-frozen in liquid nitrogen, and stored at –80 °C.
Immunoblot
Forty micrograms of whole-cell protein extract supplemented with β-mercaptoethanol up to 5% and with bromophenol blue up to 0.01% were boiled, and loaded onto a 10% polyacrylamide gel. Proteins were transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc.) and processed following the protocol supplied by the ECL kit manufacturer (Amersham Biosciences UK Limited). The membrane was probed with a mouse monoclonal anti-MCL-1 antibody (1:250, Becton Dickinson), a rabbit polyclonal anti-TR1 antibody (1:1000, Affinity BioReagents, Golden, CO, USA), a rabbit monoclonal anti-TRβ1 antibody (1:5000, Santa Cruz Biotechnology, Inc., Santa Cruz, CO, USA), and a mouse monoclonal anti-β-actin antibody (1:5000, Sigma–Aldrich). Following incubation and washes, a goat anti-rabbit or a goat anti-mouse horseradish peroxidase-conjugated polyclonal secondary antibodies (1:10 000; Calbiochem, San Diego, CA, USA) were applied. Specific bands were visualized by a chemiluminescent reaction performed with an ECL kit. When necessary, the amount of the specific protein was estimated from densitometry measurements after normalization against the β-actin band.
Electrophoretic mobility shift assay (EMSA)
RXR, TR1, and TRβ1 proteins were overexpressed in reticulocyte lysates (T7 Quick Coupled TNT Reticulocyte Lysate System, Promega). One microgram of each pcDNA3.1(+) expression vector (Invitrogen) encoding these receptors were used as templates in 25 µl reactions. The reactions were performed according to the manufacturer's instructions. EMSA was performed with the mix of all reticulocyte lysates (2 µl each) overexpressing the aforementioned receptors. The probe was made of the two primers that were hybridized to each other to make double-stranded DNA containing putative TRE-DR4 from the MCL-1 promoter: 5'DR4 5'-GAGACAGGCAGGTCACTTGAGGCCATGA-3' and 3'DR4 5'-CGAACTCATGGCCTCAAGTGACCTGCCT-3'. A mutated version of the putative TRE-DR4 was also made with 5'DR4mut 5'-GAGACAGGCAAAAAACTTGAGGCCATGA-3' and 3'DR4mut 5'-CGAACTCATGGCCTCAAGTTTTTTGCCT-3' primers. The probes were labeled by fill-in reaction with Klenow enzyme and [-32P]dCTP. Reticulocyte lysates were incubated for 20 min at RT in a binding buffer containing 20 mM Tris–HCl (pH 7.5), 50 mM KCl, 2 mM DTT, 0.1% Triton X-100, 6% glycerol, in the presence of 250 ng dIdC, and 1 ng probe. To verify binding specificity, a 25-fold excess of the specific or non-specific competitor (5'-CCTGCTGATCTATCAGCACAGATTAG-3') was added to certain samples. In the supershift experiments, 0.5 µg anti-TR antibody recognizing both TR1 and TRβ1 isoforms (C4, Santa Cruz Biotechnology, Inc.) was added to the binding reaction. In this case, the mix of reticulocyte lysates, binding buffer, dIdC and antibody was incubated on ice for 50 min, then the probe was added and the incubation was extended for an additional 20 min at RT. The samples were then loaded onto a 5% native gel and electrophoresed at 150 V for 2.5 h at RT. The gel was dried and exposed against the film (Biomax MS, Eastman Kodak Company) for 24 h.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
T3 either inhibits or activates apoptosis depending on the type of cell. Therefore, the initial experiment was aimed at establishing whether T3 had an impact on the rate of apoptosis of HK2 cells (renal tubular epithelial cells) expressing endogenous TR proteins. The cells were incubated in a serum-free medium in the presence of 100 nM T3 or without this hormone. After the incubation, the percentage of apoptotic cells was determined using flow cytometry analysis. We found that in HK2 cells T3 acted as apoptosis-protective factor: after 48-h incubation in the presence of this hormone the frequency of apoptosis of cells was only 1%, while in the absence of this hormone it was 7.5% (Fig. 1A).
|
1 and of TRβ1 remained unaltered (Fig. 1C).
T3 activates transcription from the MCL-1 promoter in the presence of TRβ1, but not TR1
Having shown that T3 treatment resulted in an increase of the MCL-1 protein amount, it became pertinent to determine the mechanism underlying this phenomenon. To establish whether this was a result of increased transcription from the MCL-1 promoter, transcription regulation assays were performed in HEK 293 cells containing biologically insignificant amounts of endogenous TRs (Turowska et al. 2007). The cells were transfected with the pGL2-MCL-1 reporter vector containing firefly luciferase reporter gene driven by the 1.82 kb MCL-1 promoter fragment (–1810 to +16 respective to the transcription start site (Townsend et al. 1999)), with expression vectors encoding wild-type TR1 or TRβ1 fused to EGFP tag by their N-terminal ends, and with the expression vector encoding their heterodimerization partner, RXR. Initially, it was demonstrated that EGFP-TR proteins were efficiently expressed in HEK 293 cells (Fig. 2A), that EGFP did not change their nuclear localization (Fig. 2B), and that the activity of the MCL-1 promoter did not change in the presence of 100 nM T3 and pEGFP-C1 vector encoding EGFP protein (Fig. 2C). Transcription regulation assays performed in the presence of 100 nM T3 showed that the activation of the MCL-1 promoter required the presence of TRβ1, but not of TR1 (Fig. 2D). Under our experimental conditions the MCL-1 promoter was activated twofold. This indicates that TRβ1, but not TR1, is crucial for MCL-1 activation by T3.
|
In silico analysis of the MCL-1 promoter sequence revealed the presence of putative TRE-DR4 (AGGTCActtgAGGCCA, the only mismatch in bold). To establish whether this sequence might mediate T3-dependent activation of the MCL-1 promoter, EMSA was performed with a probe identical to this putative TRE, and with reticulocyte lysate containing overexpressed TR1, TRβ1, and RXR. It revealed that the overexpressed receptors formed complexes with the probe. The complexes were specific, as demonstrated by the results of control experiments. The intensities of the specific bands decreased in the presence of the specific competitor and remained unaltered in the presence of non-specific competitor, the supershifted band was present in lanes containing samples supplemented with antibody recognizing both TR1 and TRβ1, and there was no specific binding in the samples containing mutated MCL-1 TRE-DR4 probe (Fig. 3A).
|
MCL-1 promoter activation by T3 does not require the presence of RXR
RXRs are most important heterodimerization partners of TRs, enhancing their affinity for DNA. Since DNA binding by TRs is not necessary for the MCL-1 promoter activation by T3, we decided to determine if RXR molecule is needed for this activation. We performed transcription regulation assays with or without overexpressed RXR. The sole overexpression of TRβ1 resulted in the activation of the MCL-1 promoter by T3 to the same extent, as in the presence of both TRβ1 and RXR, i.e. twofold (Fig. 4A). This shows that RXR is not necessary for T3-dependent activation of the MCL-1 promoter. Maximal, 2.2-fold activation of the MCL-1 promoter was achieved at 500 nM T3. The increase of T3 concentration to 1000 nM did not further change the activity of the MCL-1 promoter (Fig. 4B).
|
As it had been previously shown by other authors that T3 treatment results in the induction of PI3-K activity (Cao et al. 2005, Kuzman et al. 2005, Moeller et al. 2005, 2006, Kenessey & Ojamaa 2006, Storey et al. 2006, Verga Falzacappa et al. 2007), we decided to establish whether T3-dependent activation of the MCL-1 promoter might be mediated by the PI3-K pathway. To do so, transcription regulation assays were performed as described above, in the presence of 100 nM of PI3-K inhibitor, wortmannin. They revealed that wortmannin completely abolished T3-dependent activation of the MCL-1 promoter observed in the presence of TRβ1 (Fig. 5). This indicated that PI3-K pathway is clearly involved in the regulation of the MCL-1 promoter by this hormone.
|
We then attempted to find the promoter fragment that mediates T3-dependent activation. Transcription regulation assays were performed with the reporter vectors containing the 1.82 kb MCL-1 promoter fragment, the –295 to +16 deletion mutant E, and the –107 to +16 deletion mutant EE. The assays revealed that in the presence of 100 nM T3, the 1.82 kb and E fragments of the MCL-1 promoter were similarly activated, while fragment EE was resistant to T3 action (Fig. 6). This shows that T3-dependent activation of the MCL-1 promoter is mediated by the sequences located in the –295 to –108 part of this promoter.
|
Deletion mutant E, but not EE, contains, among others, a sequence similar to that of the consensus CRE, located at the –262 to –255 position. To determine the role of this putative CRE in the activation of the human MCL-1 promoter by T3, transcription regulation assays were performed with promoter deletion mutant E containing either wild-type or mutant CRE. While 100 nM T3 activated the wild-type fragment E of the MCL-1 promoter twofold, the mutation of putative CRE resulted in the decrease of this activation to 1.3-fold (Fig. 7). This is interpreted as indicating that CRE is a major mediator of T3-dependent activation of the MCL-1 promoter.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
A putative TRE that differed from the consensus TRE-DR4 only by a single nucleotide has been found in the MCL-1 promoter at the –998 to –983 position. Transcription regulation assays showed, however, that it is not involved in promoter's regulation by T3. The –295 to –107 fragment, mediating T3-dependent activation, did not contain other TRE-like sequences, making direct activation of the MCL-1 promoter by T3 unlikely.
It had been previously shown by other authors that the up-regulation of the human MCL-1 protein in basal cell carcinoma cells (Jee et al. 2002) and in Hep3B hepatic cells (Kuo et al. 2001) in response to interleukin-6 administration was due to PI3-K/Akt pathway activation, and resulted in the inhibition of apoptosis. As mentioned in the introduction, the cytoplasmic fraction of TRs, when bound to T3, also activates the PI3-K pathway (Lei et al. 2004, Cao et al. 2005, Kuzman et al. 2005, Moeller et al. 2005, 2006, Kenessey & Ojamaa 2006, Storey et al. 2006, Verga Falzacappa et al. 2006, 2007). Therefore, we examined a new hypothesis stating that the MCL-1 promoter is activated by T3 in a non-genomic mechanism involving this pathway. We indeed demonstrated that this is so, as the activation of this promoter by T3 was completely abolished by wortmannin, a PI3-K inhibitor. We also showed that the induction of MCL-1 expression required the presence of TRβ1, but not TR1. This is in agreement with the results of other authors who showed that T3-dependent activation of Akt kinases and certain target genes by PI3-K was specifically mediated by different TR isoforms, with TRβ1 usually indicated as a go-between molecule (Moeller et al. 2006, Storey et al. 2006, Verga Falzacappa et al. 2007).
While analyzing the details of murine Mcl-1 promoter activation by interleukin-3 and by PI3-K pathway, Wang et al. (1999) showed that the CRE-2 sequence played a role in this process. Similarly, the –295 to –108 fragment of the human MCL-1 promoter, mediating the T3-dependent activation, has been shown to contain putative CRE. Upon its mutation, T3-dependent activation of this promoter decreased from 2- to 1.3-fold. This demonstrates that CRE is a major mediator in this process.
To sum up, the activation of the human MCL-1 by T3 is a result of the non-genomic action of TRβ1 that activates the PI3-K signaling pathway. We propose that this is one of the mechanisms by which T3 regulates apoptosis.
|
Declaration of interest |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionDeclaration of interestFundingReferences |
|---|
Casas F, Rochard P, Rodier A, Cassar-Malek I, Marchal-Victorion S, Wiesner RJ, Cabello G & Wrutniak C 1999 A variant form of the nuclear triiodothyronine receptor c-ErbA1 plays a direct role in regulation of mitochondrial RNA synthesis. Molecular and Cellular Biology 19 7913–7924.
Chen JD & Evans RM 1995 A transcriptional co-repressor that interacts with nuclear hormone receptors. Nature 377 454–457.[CrossRef][Web of Science][Medline]
Cheng SYMultiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptorsD LeRoith Reviews in Endocrine and Metabolic Disorders ,2000New York:Kluwer Academic:9–18.
Clohessy JG, Zhuang J, de Boer J, Gil-Gomez G & Brady HJ 2006 Mcl-1 interacts with truncated Bid and inhibits its induction of cytochrome c release and its role in receptor-mediated apoptosis. Journal of Biological Chemistry 281 5750–5759.
van Delft MF & Huang DC 2006 How the Bcl-2 family of proteins interact to regulate apoptosis. Cell Research 16 203–213.[CrossRef][Web of Science][Medline]
Drabkin H, Kao FT, Hartz J, Hart I, Gazdar A, Weinberger C, Evans R & Gerber M 1988 Localization of human ERBA2 to the 3p22-3p24.1 region of chromosome 3 and variable deletion in small cell lung cancer. PNAS 85 9258–9262.
Eckey M, Moehren U & Baniahmad A 2003 Gene silencing by the thyroid hormone receptor. Molecular and Cellular Endocrinology 213 13–22.[CrossRef][Web of Science][Medline]
Fraser D, Wakefield L & Phillips A 2002 Independent regulation of transforming growth factor-β1 transcription and translation by glucose and platelet-derived growth factor. American Journal of Pathology 161 1039–1049.
Ghorbel M, Seugnet I, Ableitner AM, Hassan A & Demeneix BA 1997 T3 treatment increases mitosis, then bax expression and apoptosis in the optic lobe of the chick embryo. Neuroscience Letters 231 127–130.[CrossRef][Web of Science][Medline]
Gosden JR, Middleton PG, Rout D & Angelis C 1986 Chromosomal localization of the human oncogene ERBA2. Cytogenetics and Cell Genetics 43 150–153.[Web of Science][Medline]
Han J, Goldstein LA, Gastman BR & Rabinowich H 2006 Interrelated roles for Mcl-1 and BIM in regulation of TRAIL-mediated mitochondrial apoptosis. Journal of Biological Chemistry 281 10153–10163.
Harvey CB & Williams GR 2002 Mechanism of thyroid hormone action. Thyroid 12 441–446.[CrossRef][Web of Science][Medline]
Jee SH, Chiu HC, Tsai TF, Tsai WL, Liao YH, Chu CY & Kuo ML 2002 The phosphotidyl inositol 3-kinase/Akt signal pathway is involved in interleukin-6-mediated Mcl-1 upregulation and anti-apoptosis activity in basal cell carcinoma cells. Journal of Investigative Dermatology 119 1121–1127.[CrossRef][Web of Science][Medline]
Kenessey A & Ojamaa K 2006 Thyroid hormone stimulates protein synthesis in the cardiomyocyte by activating the Akt-mTOR and p70S6K pathways. Journal of Biological Chemistry 281 20666–20672.
Kuo ML, Chuang SE, Lin MT & Yang SY 2001 The involvement of PI3-K/Akt-dependent up-regulation of Mcl-1 in the prevention of apoptosis of Hep3B cells by interleukin-6. Oncogene 20 677–685.[CrossRef][Web of Science][Medline]
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.
Leu JI, Dumont P, Hafey M, Murphy ME & George DL 2004 Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nature Cell Biology 6 443–450.[CrossRef][Web of Science][Medline]
McKenna NJ, Lanz RB & O'Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocrine Reviews 20 321–344.
Michels J, Johnson PW & Packham G 2005 Mcl-1. International Journal of Biochemistry and Cell Biology 37 267–271.[CrossRef][Web of Science][Medline]
Moeller LC, Dumitrescu AM & Refetoff S 2005 Cytosolic action of thyroid hormone leads to induction of hypoxia-inducible factor-1 and glycolytic genes. Molecular Endocrinology 19 2955–2963.
Moeller LC, Cao X, Dumitrescu AM, Seo H & Refetoff S 2006 Thyroid hormone mediated changes in gene expression can be initiated by cytosolic action of the thyroid hormone receptor β through the phosphatidylinositol 3-kinase pathway. Nuclear Receptor Signaling 4 e020
Nijhawan D, Fang M, Traer E, Zhong Q, Gao W, Du F & Wang X 2003 Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation. Genes and Development 17 1475–1486.
Oetting A & Yen PM 2007 New insights into thyroid hormone action. Best Practice and Research. Clinical Endocrinology and Metabolism 21 193–208.[CrossRef]
Onate SA, Tsai SY, Tsai MJ & O'Malley BW 1995 Sequence and characterization of coactivator for the steroid hormone receptor superfamily. Science 270 1354–1357.
Puzianowska-Kuznicka M, Pietrzak M, Turowska O & Nauman A 2006 Thyroid hormones and their receptors in the regulation of cell proliferation. Acta Biochimica Polonica 53 641–650.[Web of Science][Medline]
Rastinjead F 2001 Retinoid X receptor and its partners in the nuclear receptor family. Current Opinion in Structural Biology 11 33–38.[CrossRef][Web of Science][Medline]
Sachs LM, Abdallah B, Hassan A, Levi G, De Luze A, Reed JC & Demeneix BA 1997 Apoptosis in Xenopus tadpole tail muscles involves Bax-dependent pathways. FASEB Journal 11 801–808.[Abstract]
Storey NM, Gentile S, Ullah H, Russo A, Muessel M, Erxleben C & Armstrong DL 2006 Rapid signaling at the plasma membrane by a nuclear receptor for thyroid hormone. PNAS 103 5197–5201.
Szanto A, Narkar V, Shen Q, Uray IP, Davies PJ & Nagy L 2004 Retinoid X receptors: X-ploring their (patho)physiological functions. Cell Death and Differentiation 11 S126–S143.[CrossRef][Web of Science][Medline]
Tata JR 2007 A hormone for all seasons. Perspectives in Biology and Medicine 50 89–103.[CrossRef][Web of Science][Medline]
Townsend KJ, Zhou P, Qian L, Bieszczad CK, Lowrey CH, Yen A & Craig RW 1999 Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. Journal of Biological Chemistry 274 1801–1813.
Turowska O, Nauman A, Pietrzak M, Poplawski P, Master A, Nygard M, Bondesson M, Tanski Z & Puzianowska-Kuznicka M 2007 Overexpression of E2F1 in clear cell renal cell carcinoma: a potential impact of erroneous regulation by thyroid hormone nuclear receptors. Thyroid 17 1039–1048.[CrossRef][Web of Science][Medline]
Upadhyay G, Singh R, Kumar A, Kumar S, Kapoor A & Godbole MM 2004 Severe hyperthyroidism induces mitochondria-mediated apoptosis in rat liver. Hepatology 39 1120–1130.[CrossRef][Web of Science][Medline]
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 β-cells undergoing apoptosis. Journal of Cellular Physiology 206 309–321.[CrossRef][Web of Science][Medline]
Verga Falzacappa C, Petrucci E, Patriarca V, Michienzi S, Stigliano A, Brunetti E, Toscano V & Misiti S 2007 Thyroid hormone receptor TRβ1 mediates Akt activation by T3 in pancreatic β cells. Journal of Molecular Endocrinology 38 221–233.
Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ & Yang-Yen HF 1999 The antiapoptotic gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Molecular and Cellular Biology 19 6195–6206.
Wang JM, Lai MZ & Yang-Yen HF 2003 Interleukin-3 stimulation of mcl-1 gene transcription involves activation of the PU.1 transcription factor through a p38 mitogen-activated protein kinase-dependent pathway. Molecular and Cellular Biology 23 1896–1909.
Weiss RE & Ramos HE 2004 Thyroid hormone receptor subtypes and their interaction with steroid receptor coactivators. Vitamins and Hormones 68 185–207.[Web of Science][Medline]
Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI, Adams JM & Huang DC 2005 Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins. Genes and Development 19 1294–1305.
Yang T, Kozopas KM & Craig RW 1995 The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2. Journal of Cell Biology 128 1173–1184.
Yang-Yen HF 2006 Mcl-1: a highly regulated cell death and survival controller. Journal of Biomedical Science 13 201–204.[CrossRef][Web of Science][Medline]
Yehuda-Shnaidman E, Kalderon B & Bar-Tana J 2005 Modulation of mitochondrial transition pore components by thyroid hormone. Endocrinology 146 2462–2472.
Yen PM, Ando S, Feng X, Liu Y, Maruvada P & Xia X 2006 Thyroid hormone action at the cellular, genomic and target gene levels. Molecular and Cellular Endocrinology 246 121–127.[CrossRef][Web of Science][Medline]
Received in final form 4 June 2008
Accepted 13 June 2008
Made available online as an Accepted Preprint 13 June 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
