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. 2020 Apr-Jun;16(2):129–135. doi: 10.4183/aeb.2020.129

TRIIODOTHYRONINE ACTIVATES GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 3 VIA AGGTCA-LIKE-DIRECT-REPEAT-4 TYPE THYROID HORMONE RESPONSE ELEMENT

LF Zhao 1,*, Y Iwasaki 2, BL Han 1, J Wang 1, Y Zhang 1, J Han 1, GY Liu 1, X Jiang 1
PMCID: PMC7535892  PMID: 33029227

Abstract

Background

Thyroid hormone participates in lipid metabolism regulation. However, the effects on triacyleride or triacylglycerol metabolism are complex and not fully clarified yet. In this study, we try to identify novel thyroid hormone-targeting lipogenic metabolic genes and analyze their molecular regulative mechanism.

Method

Thirty-five promoters of twenty-nine human lipogenic regulative enzyme genes were constructed into pXP1 luciferase reporter plasmid (PFK2/FBP2-luc) and transfected into HeGP2 cells, respectively. Gene expression induced by triiodothyronine (T3) was detected by luciferase assay. The T3-activated gene promoter was then analyzed by sequence analysis, deletion and mutation, and electrophoretic mobility shift assay (EMSA).

Results

After 10 nM T3 stimulation for 36 h, phosphogluconate dehydrogenase, malic enzyme, Glycerol-3-phosphate acyltransferase (GPAT) 3, and 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 were significantly activated, respectively. A AGGTCA-like-direct-repeat-4 consensus thyroid hormone response element (DR4-TRE)-like sequence was found in the GPAT3 promoter, which was then verified to be necessary for T3-induced GPAT3 activation by gene deletion and mutation analysis. EMSA further identified that T3-thyroid receptor (TR) α-retinoid-X receptor (RXR) complex directly bound on the GPAT3 promoter.

Conclusion

Triiodothyronine could activate the GPAT3 through DR4-TRE-like sequence binding to participate in lipogenic regulation. AGPAT2 may be another thyroid hormone target enzyme.

Keywords: triiodothyronine, lipid metabolism

INTRODUCTION

Thyroid hormones are primarily responsible for metabolic regulation (1). Their physiological regulative effect on cholesterol metabolism is well characterized (2, 3). However, their physiological effects on triacyleride or triacylglycerol metabolism are complex and not fully clarified yet. Interestingly, two well known proteins, malic enzyme (ME) and thyroid hormone-responsive protein Spot 14 (THRSP, Spot 14) are lipogenic proteins which are positively regulated by thyroid hormone (4-7). Indeed, thyroid hormone increases both the activity and expression of ME (4), which promotes lipogenesis through NADPH supplement (8). Spot 14 is verified to participate in lipid synthetic regulation (7). In addition, glucose-6-phosphate dehydrogenase (G6PD), that takes part in fatty acid biosynthesis through providing NADPH, is reported as a thyroid hormone-responsive gene as well (9). These results indicate that triiodothyronine is profoundly involved in triglyceride (TG) synthesis.

Thyroid hormones target gene regulation is initiated by association of thyroid hormone receptor (TR), thyroid hormone response element (TRE), and retinoid X receptor (RXR). TR-TRE-RXR complex without triiodothyronine is in an inactive state. T3 binding to the complex will change its conformation to recruit RNA polymerase, which then transcribes the target gene expression (10). Interestingly, TRE is found in the promoter region of ME gene. The induction or repression of the ME gene transcription is directly affected by the binding state of ME and TRE (6). Campbell et al. verified, as well, that AGGTCA-like-direct-repeat-4 consensus (DR-4) TRE is in the promoter fragment (-2774 to -2000bp) of human Spot 14 by site-directed mutagenesis (11).

Since all of the investigations concerning the thyroid hormone-regulated TG metabolic enzyme genes are single-point studies so far, it is still not revealed that how many other TG regulatory enzymes are actually controlled by T3, and the biological function of thyroid hormone mainly acts through gene transcriptional regulation, especially mediates through DR-4 TRE, we hypothesize that there might be other T3 targeting TG regulatory genes and DR-4 TRE factor is one of the mechanisms of TG enzyme regulation by thyroid hormone. In this study, we cloned 35 promoters of 29 human sugar and lipid metabolism related enzyme genes, detected the activity of these promoters induced by thyroid hormone in a luciferase report system, and further analyzed the action of DR-4 TRE in the target genes, trying to identify new triiodothyronine targeting TG regulatory genes and their molecular regulative mechanism.

MATERIALS AND METHODS

Cell culture

Human liver hepatocellular carcinoma cell, HePG2 (ACTT, Manassas, VA) was cultured with Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Adrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; Gibco, EI Paso, Texas, USA) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 37°C incubator with 5% CO2. The passaged well-established HePG2 cells were detached and dispensed into 24-well Petri dishes for subsequent transfection.

Gene clone and transfection

DNA fragment (6-8 kb) of the 35 promoters of the 29 human sugar and lipid metabolism related enzyme genes were isolated from a human genomic library in lambda phage as previously described (12). Their promoter sequence was amplified by the PCR with Taq DNA polymerase (Perkin-Elmer, Norwalk, CT) using forward and reverse primers containing XhoI and KpnI restriction sites, respectively and constructed into a luciferase reporter vector, pXP1, respectively (12). After identification by enzyme digestion and DNA sequence, the plasmids were prepared by Midi QIAGEN Plasmid Kits (Quiagen; Valencia, CA). In each assay, cells were plated in 24-well dishes until 50% confluent, and transient transfected into HePG2 cells with the target plasmids using FuGENE® HD Transfection Reagent (Roche, Indianapolis, IN). On the next day, the medium was exchanged to basal DMEM. The test reagents were supplied directly into the culture medium. The cells were incubated at indicated time interval.

Detection of the triiodothyronine (T3)-induced target promoter activation by luciferase assay

Luciferase assays were performed using Dual-Glo® Luciferase Assay System (Promega) under the instruction of kit manual. Briefly, the HePG2 cells were stimulated with 10 nM 3,3’,5 Triiodothyronine (T3) (Sigma-Aldrich, St Louis, MO) for 36 h after the target promoter region containing pXP1 transfection and lysed by lysis buffer (included in the kit). After removing the culture medium, the cells were harvested for the luciferase activity assay by a Berthold Luminometer (Berthold Lumat LB9507, Bad Wildbad, Germany). The ratio of Renilla over firefly luciferase activities was determined as the relative luciferase activity value. Each detection was repeated at least three times. DR4-pXP1 plasmid was employed as positive control because DR4 was verified to be activated by T3 (13).

Sequence analysis

Sequence analysis of the promoter fragments of the influential enzyme genes was carried out. Using the sequence analysis software, DNA Strider (https://sourceforge.net/projects/dnastrider/), we analyzed the sequence of the influencing promoter peptide to find the matched TRE sequence.

PGAT3 Gene mutagenesis

The four base pairs of the oligonucleotides (AGGT) before GC box of the DR4 in human PGAT3 gene were replaced by TCCA (PGAT3-m1; Fig. 3). Other six base pairs of the oligonucleotides (AGGGCA) after GC box of the DR4 in human PGAT3 gene were replaced by TCCGGT (PGAT3-m2; Fig. 3). Both of the PGAT3 mutations (PGAT3-m1 and PGAT3-m2) were sub-cloned into pXP1 vector, transfected into HePG2 cells for the T3-induced PGAT3 promoter activation assay as above described. Wild type PGAT3-pXP1 plasmid was as control.

Figure 3.

Figure 3.

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Luciferase assay detects the activation of mutated human GPAT3 promoters. Three human GPAT mutations, GPAT3-450m1, GPAT3-450m2, and GPAT3-430, were constructed. The mutated code area of the head of human GPAT3 was shown in the light grey box and that of the end of the GPAT3 was shown in the dark grey box. The sequence comparison of the wild type of GPAT3 and mutation of GPAT3-450m1 and GPAT3-450m2 were listed in the left-up corner. In the construct of GPAT3-430, all the DR4-TRE codes were deleted. GPAT3-587 was the original GPAT3 promoter. The luciferase activity of the GPAT3-587-, GPAT3-450m1-, GPAT3-450m2-, and GPAT3-430-pGL4.72 in HeGP2 cells was then detected after stimulation with 10nM R3. Each data was from three independent assays. * indicates p < 0.05.

Electrophoretic mobility shift assay (EMSA)

The EMSA assay of the GPAT3 DNA was performed using a commercially available non-RI EMSA kit (LightShift Chemiluminescent EMSA kit; Pierce, Rockford, IL). Briefly, nuclear fraction of the detecting HeGP2 cells (5 × 105-7) was extracted using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). The supernatant of the extracted nuclear protein was then determined by Bicinchoninic Acid Kit (BCA) (Sigma-Aldrich). 1 × 106 cells could get 50-75g nuclear protein. Isotope labeling and purification of GPAT3 oligonucleotides probe. 6μL [γ-32P] ATP (PerkinElmer; Waltham, MA), 5μL 10 × Enzyme Buffer (in the kit), 2μL ddH2O, and 1μL T4 polynucleotide kinase (6-10U; from the kit) were mixed in a sterilized Eppendorf tube and kept at 37°C for 1h. The labeled GPAT3 probe (5’-AGCTGAGGAGGTGGGGCGAGGG CAGCGC-3’) was purified by a TE buffer (10 mM Tris-Cl, pH 7.5. 1 mM EDTA)-equilibrated Sephadex G-25 column. To prepare binding reaction mixture, 6μg nuclear extract, 4 μL 5 × Binding buffer (in the kit), 2 μL 10% BSA (in the kit), and 1μg poly (dI-dC) (in the kit) in 6 μL distilled water without (negative control) or with TRβ, RXRα, anti-TRβ, and/or cold T3 (non-labeled) were mixed and kept at room temperature for 15 min as manufacturer’s instruction. The mixture was added with 2 μL of the [γ-32P] ATP-labeled GAPT3 oligonucleotide probe, mixed, and reacted at room temperature for 20 min. The reacted mixture was then separated on a 6 % non-denaturing polyacrylamide gel (160 V for 4h). The electrophoresed gel was dried and detected with a digital imaging (LightCapture, ATTO, Tokyo, Japan).

Statistical analysis

The experimental data (triplicate or quadruplicate) were presented as mean ± SEM. The data were compared by one-way analysis of variance with Fisher’s PLSD test, and P values below 0.05 were considered as statistically significant.

RESULTS

T3-induced PGDHase, ME, GPAT3, and AGPAT2 promoter activation by luciferase assay

To investigate the regulation mechanism of TG metabolism by triiodothyronine at gene inserted them into a luciferase reporter vector, pXP1, respectively. The target gene promoter- pXP1-transfected HePG2 cells were then stimulated with 10 nM T3 for 36 h. The luciferase activity in the cellular lysate was detected by Dual-Glo® Luciferase Assay System. In the series of glycolytic pathway [glucokinase (GK), fructokinase (FK), phosphofructokinase (PFK) 1] and fatty acid synthesis pathway [acetyl-CoA carboxylase (ACC)1, ACC2, fatty acid synthase (FAS), fatty acid elongases 6 (Elovl 6)] enzymes, eleven promoters of the seven target genes were not significantly activated by T3 compared to the positive control of DR4 (14) (Fig. 1A). The results indicated that triiodothyronine activates in the glycolytic and fatty acid synthesis regulation. Next, the nicotinamide adenine dinucleotide phosphate-oxidase (NAPDH) production-related enzymes including pentose phosphate pathway [Glucose-6-phosphate dehydrogenase (G6PD), hexose-6-phosphate dehydrogenase (H6PD), polygalacturonase (PGLase), and phosphogluconate dehydrogenase (PGDHase)] and malate-pyruvate and –oxaloacetate shutters [malic enzyme (ME), malate dehydrogenase (MDH) 1, and MDH2] were detected. Among them, PGDHase and ME promoters were significantly activated by T3 (p < 0.05) (Fig. 1B), indicating that triiodothyronine controls the NAPDH synthesis through PGDHase and ME regulation. Finally, the TG synthetic enzymes Glycerol-3-phosphate acyltransferase (GPAT), 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), lipin, monoacylglycerol acyltransferase (MGAT), lysophosphatidylglycerol acyltransferase (LPGAT), and diglyceride acyltransferase (DGAT) were analyzed. Interestingly, two (GPAT3 and AGPAT2) of the 17 promoters were significantly activated by T3 treatment (p < 0.05) (Fig. 1C). These results suggested that thyroid hormone adjusts the TG synthesis through GPAT3 and AGPAT2 enzymes regulation.

Figure 1.

Figure 1.

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Luciferase assay detects the activation of TG synthesis related enzyme promoters induced by T3. 11 promoters of 7 glycolytic pathway (GK, FK, and PFK1) and fatty acid synthesis pathway (ACC1, ACC2, FAS, and Elovl 6) enzymes (A), 7 promoters of 7 pentose phosphate pathway (G6PD, H6PD, PGLase, and PGDHase) and malate-pyruvate and –oxaloacetate shutters (ME, MDH1, and MDH2) synthesis enzymes (B), and 17 promoter of five TG synthetic enzymes (GPAT, AGPAT, MGAT, LPGAT, and DGAT) (C) were inserted into pXP1 vector, and transfected HePG2 cells, respectively. The luciferase activity of the cellular lysate was detected by Dual-Glo® Luciferase Assay System after stimulation with 10nM T3 for 36h. Each data was from three independent assays. * indicates the p < 0.05.

Human GPAT3 gene has an AGGTCA-like-direct-repeat-4 consensus thyroid hormone response element (DR4-TRE) sequence at -447--432 bp

PGDHase and ME have been verified to be up-regulated by triiodothyronine before (5, 6, 15). Thus, we focus on the GPAT3 gene to analyze whether the GPAT3 promoter sequence contains any TR regulate element. It is interesting that using DNA Strider analyzing tool, we found out a DR4-TRE-like sequence in the human GPAT promoter 3 at -447 to -432 bp (Fig. 2).

Figure 2.

Figure 2.

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Human GPAT3 gene promoter contains a DR4-TRE like sequence at -447--432 bp. The GPAT3 gene promoter sequence was analyzed by using DNA Strider analyzing tool. A DR4-TRE-like sequence (AGGTGGGGCGAGGGCA) was found out at -447 to -432 bp of the human GPAT promoter 3 (yellow heighted box). The consensus comparison of the DR4-TRE like sequence of the human GPAT3 to the DR4 sequence was shown in the right box. ATG code is the start code of the human GPAT3.

DR4-TRE involves in the T3-induced GPAT activation

DR4-TRE directly involves in thyroid hormone-TR binding-induced target gene expression (14). To analyze whether DR4-TRE involves in the T3-induced GPAT3 activation, we constructed three human GPAT3 mutations. They are GPAT3-450m1, GPAT3-450m2, and GPAT3-430 (Fig.3). In GPAT3-450m1 construct, the heat codes of the DR4-TRE (AGGT) were replaced with TCCA. In GPAT3-450m2 construct, the end codes of the DR4-TRE (AGGGCA) were replaced with TCCGGT. In the construct of GPAT3-430, all the DR4-TRE codes were deleted. GPAT3-587 was the original GPAT3 promoter. In addition, a standard DR4-TRE promoter sequence (AGGTCANNNNAGGGCA) was constructed as a positive control. These constructs were then ligated into pXP1 vector, transfected into HeGP2 cells, and underwent luciferase assay, respectively. pXP1 alone was used as negative control. Compared with DR4, all three mutations, including GPAT3-450m1, GPAT3-450m2, and GPAT3-430, were not significantly activated by 10 nM T3 stimulation. However, full DR4-TRE-like sequence containing GPAT3 promoter (GPAT3-587) was significantly activated by T3 compared with either positive control of DR4 or negative control pXP1 (p < 0.05) (Fig. 3). This result indicated that full DR4-TRE-like sequence (AGGTGGGGCGAGGGCA) is necessary for T3-induced GPAT3 activation.

T3 directly bound to GPAT3 promoter

Paquette et al. have shown that both the TR homodimer and TR/RXR heterodimer could interact with DR4-TRE to induce luciferase reporter activity in the presence or absence of thyroid hormone (16). To explore in detail the mechanism of the GPAT3 activation by T3, we performed an EMSA assay to analyze whether T3 could directly bind to GPAT3 promoter. Oligonucleotides 5-AGCTGAGGAGGTGGGGCGAGGGCAGCGC-3 of GPAT3 promoter, which contains the DR4-TRE-like sequence of human GPAT3 promoter, were synthesized and labelled with [γ-32P] ATP as probe. In the basal reaction mixture which contains the HePG2 nuclear sample, T3, and [γ-32P]-GAPT3 prober, the thyroid receptor (TR) β and retinoid-X receptor (RXR) α (a member of the TR superfamily transcription factors) supplied reaction (4th lane of Fig. 4) showed a clear shift up band (RXRα/TRβ) that corresponds to that of the DR4-RXRα-TRβ reaction (9th lane of Fig. 4), indicating that T3-TRβ-RXRα directly bound to the GPAT3 promoter. This binding was further verified by TRβ antibody supplied reaction (5th lane of Fig. 4) which created another higher shift band (Super shift band) because of specific binding of the T3-TRβ-RXRα and TRβ antibody. Conversely, high concentration (× 10 or × 20 times) cold probe (non-[γ-32P] ATP-labelled probe) obviously deleted the RXRα/TRβ band (6th and 7th lanes of Fig. 4). These results suggested that T3-TRβ-RXRα complex directly bound to the PGAT3 promoter to activate PGAT3 activity.

Figure 4.

Figure 4.

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EMSA assay of GPAT3 promoter and TRβ-RXRα. The basal reaction mixture (first line containing HePG2 nuclear sample, T3, and [γ-32P]-GAPT3 prober) with thyroid receptor (TR) retinoid-X receptor (RXR) anti antibody, and/or (x 10 or × 20) concentrated cold promoter were loaded on a 5% PAGE gel, separated by electrophoresis, dried, and exposed on an X-ray film. Standard DR4 was employed as positive control. The shift bands were indicated on the right side.

DISCUSSION

To explore novel thyroid hormone-targeting lipogenic metabolic genes and their corresponding regulatory molecular mechanism, we cloned thirty-five promoters of twenty-nine human lipogenesis-related enzyme genes into pXP1 and transfected them into HeGP2 cells, respectively. The activation of the promoter induced by T3 was detected using luciferase assay, showing that PGDHase, ME, GPAT3, and AGPAT2 were significantly activated by T3, respectively. A DR4-TRE-like sequence was found in human GPAT3 promoter. Mutation or deletion of the DR4-TRE-like sequence significantly attenuated the T3-induced GPAT3 activation. In addition, in an EMSA assay, TRβ-RXRα complex showed it directly interacts with the GPAT3 promoter.

Strait et al. have found that EM contained a TR binding domain, cis-regulatory element at -281 to -261 bp (4). Thyroid hormone regulated ME through interaction with ME’s TRE binding and its expression is directly correlated with thyroid hormone level (4). Thyroid hormone up-regulates ME (17) to promote lipogenesis through NADPH supplement (6). Another thyroid hormone-responsive gene, G6PD, may involve in the lipogenesis regulation through providing a different source of NADPH (9). Moreover, thyroid hormone up-regulated Spot 14 (5) and PGDHase expression (9). These data indicated that thyroid hormone is profoundly involved in TG synthesis. In this study, we not only reconfirmed that ME is up-regulated by thyroid hormone, but also revealed that GPAT3, AGPAT2, and PGDHase were the thyroid hormone targeting enzymes by a T3-inducing luciferase activation report system (Fig. 1B). However, these findings need to be further verified in an in vivo system to provide solid evidence.

GPATs and AGPATs participate in the synthesis of phosphatidic acid (PA). PA is an intermediate product of the TG and glycerophospholipids synthesis. Dephosphorylation of PA produces diacylglycerol (DAG), which is then acylated to take part in the TG synthesis (18). Indeed, GPATs and AGPATs contribute the most to fatty acids synthesis. GPAT catalyzes the esterification of long-chain acyl-CoA to glycerol-3-phosphate, which is believed to be the initial step of phospholipid synthesis (19). Four isoforms of GPTA are found so far, among which, two isoforms (GPAT3 and GPAT4) are verified as N-ethylmaleimide (NEM) sensitive isoforms that distribute in the endoplasmic reticulum (20). However, questions like “why do those GPAT isoforms exist” and “how are they different functionally” have been confused for long to researchers and have not been fully understood (20). Here, our study clearly showed that GPAT3, but not GPAT1, GPAT2, and GPAT4, is directly regulated by thyroid hormone to involve in the thyroid hormone-mediated lipogenesis regulation. A GPAT3 gene deletion study indicated that total GPAT activity in white adipose decreased 80% in GPAT3-deficient [Gpat3(-/-)] mice, suggesting that GPAT3 is the predominant GPAT to participate in white adipose synthesis (21). In this point, the finding of thyroid hormone-GPAT3 regulation axis may provide a clue to interfere with some adipose synthetic abnormal diseases, clinically, such as obesity. Interestingly, Ma et al. (22) have reported that thyrotropin (TSH), but not thyroid hormone, directly triggered GPAT3 activity in vivo. TSH receptor (TSHR) or peroxisome proliferator-activated receptor γ (PPARγ) KO mice, or constituted AMPK could block this TSH-induced GPAT3 activity. Reversely, TSHR or PPARγ expression, or AMPK dominant negative mutation, could reconstitute the TSH-induced GPAT3 activity and adipogenesis in vivo. These results indicate that TSH can directly, not via thyroid hormone, up-regulate adipogenesis through TSHR/AMPK/PPARγ/GPAT3 pathway. Noticeably, in this study, we revealed another possible pathway that TSH may indirectly up-regulate GPAT3 through thyroid hormone. In addition, we also showed that AGPAT2 was upregulated by T3 in the luciferase report system (Fig. 1C). This is a novel finding, which provides an evidence to distinguish the physio - and pathological activity of the AGPAT isoforms, it means that AGPAT2, but not AGPAT 1u or 1d, participates in the thyroid hormone-controlling lipogenesis. Interestingly, the DNA analysis of the AGPAT2 did not show any TRE codes in the promoter area, suggesting that another molecular regulation mechanism by thyroid hormone may be involved. Further investigation about it is valuable. The findings that GPAT3 and AGPAT2 upregulation by thyroid hormone further provide strong evidence to support that thyroid hormone is a fundamental factor to regulate lipogenesis, which provide another clue to further study in obesity, diabetes, hypertension, neutral lipid storage disease etc. TRE is the critical element that targets gene regulation by triiodothyronine, which could bind to TR to control the target gene expression, T3 TR to associate with RXR and TRE to drive the target gene expression. TREs are composed with two hexamer half-sites (AGGTCA). Based on the orientation and spacing of the half-sites, TRE is classified as i) direct repeat 4; ii) inverted repeat 0; and iii) everted repeat 6 (23). The most frequently TRE structure contains two direct repeats of AGGTCA codes (half site) with a 4bp spacer (DR4) between the half sites (24, 25). Zavacki et al. did a mutation analysis of a type iii TRE (an everted repeat with a 6bp spacer or tail to tail organization), F2 (26). They altered the orientation of either the first, secondary half site, both half sites, space codes, or even space deletion to find out that any changes of the half-site orientation or sequence will affect the sensitivity of the F2 TRE to TR. In this study, we mutated a type-i TRE (DR4-TRE in GPTA3) by replacing the AGGT of the first half site with TCCA (PGAT3-m1; Fig. 3) and AGGGCA of the secondary half site with TCCGGT (PGAT3-m2; Fig. 3). In T3-induced luciferase activity report system, any mutation of either the first half site or the secondary half site, or the DR4-TRE deletion obviously obligated the T3-induced luciferase activity (Fig. 3). These results revealed the molecular regulatory mechanism of thyroid hormone-GPAT3 that all the DR4-TRE codes are necessary for thyroid hormone recognition, which is consistent with the Zavacki’s finding that any alteration of the half-site will significantly disturb the sensitivity of the TRE to TR.

In conclusion, thyroid hormone activates the ME, G6PDHase, GPAT3, and AGPAT2 activities. Thyroid hormone activates GPAT3 through interaction with DR4-TRE-like sequence and TR-RXR to participate in lipogenic regulation.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgment

This work was supported by the Kochi University.

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