Thioredoxin-interacting protein stimulates its own expression via a positive feedback loop.

ORIGINAL

RESEARCH

Thioredoxin-Interacting Protein Stimulates Its Own Expression via a Positive Feedback Loop Junqin Chen, Gu Jing, Guanlan Xu, and Anath Shalev Comprehensive Diabetes Center and Department of Medicine, Division of Endocrinology, Diabetes and Metabolism, University of Alabama at Birmingham, Birmingham, Alabama 35294

Thioredoxin-interacting protein (TXNIP) has emerged as a key regulator of important cellular processes including redox state, inflammation, and apoptosis and plays a particularly critical role in pancreatic ␤-cell biology and diabetes development. High glucose and diabetes induce TXNIP expression, whereas inhibition of TXNIP expression or TXNIP deficiency protects against pancreatic ␤-cell apoptosis and diabetes. We now have discovered that TXNIP stimulates its own expression by promoting dephosphorylation and nuclear translocation of its transcription factor, carbohydrate response element-binding protein (ChREBP), resulting in a positive feedback loop as well as regulation of other ChREBP target genes playing important roles in glucose and lipid metabolism. Considering the detrimental effects of elevated TXNIP in ␤-cell biology, this novel pathway sheds new light onto the vicious cycle of increased TXNIP, leading to even more TXNIP expression, oxidative stress, inflammation, ␤-cell apoptosis, and diabetes progression. Moreover, the results demonstrate, for the first time, that TXNIP modulates ChREBP activity and thereby uncover a previously unappreciated link between TXNIP signaling and cell metabolism. (Molecular Endocrinology 28: 674 – 680, 2014)

hioredoxin-interacting protein (TXNIP) has emerged as a key regulator of important cellular processes including redox state (1–5) and apoptosis (6 –10), is involved in inflammasome activation (11), inflammation, and endoplasmic reticulum stress (12, 13), and plays a particularly critical role in pancreatic ␤-cell biology (6 –9). Initially identified as the top glucose-induced gene in a human islet microarray study, we found that ␤-cell expression of TXNIP is increased in diabetes (7, 8, 14). Moreover, TXNIP overexpression induces ␤-cell apoptosis and is essential for glucotoxicity-induced ␤-cell death (7), whereas lack of TXNIP promotes endogenous ␤-cell survival and prevents type 1 and type 2 diabetes (8, 9, 15). Glucose-induced TXNIP expression is mediated via a nonpalindromic E-box motif consisting of a repeat of CACGAG sequences in the TXNIP promoter that serves as the binding site for the carbohydrate response element-binding protein (ChREBP) (16). The basic helix-loop-helix transcription factor ChREBP has been recognized as the main transcription

T

factor mediating glucose-induced gene expression in liver (17) as well as in pancreatic ␤-cells (16), whereas its paralog, MondoA, has been shown to do the same in muscle (18), and a recently discovered isoform, ChREBP-␤, has been described in adipocytes (19). To date, ChREBP/ MondoA remains the only bona fide nutrient/glucoseresponsive transcription factor known to control various target genes involved in glucose and lipid metabolism (13, 14). The activity of ChREBP is regulated by its phosphorylation status and cellular localization and, in particular, dephosphorylation of Ser196 (as observed in response to glucose) allows ChREBP to enter the nucleus and transactivate its target genes such as liver-type pyruvate kinase (L-PK) or TXNIP (17, 20, 21). Surprisingly, we now have discovered that TXNIP can induce its own transcription, and the present studies were aimed at identifying the mechanisms involved. In addition to revealing the processes controlling this positive feedback loop and promoting TXNIP expression, we discov-

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received February 4, 2014. Accepted March 5, 2014. First Published Online March 14, 2014

Abbreviations: AMPK, AMP-activated protein kinase; ChIP, chromatin immunoprecipitation; ChoRE, carbohydrate response element; ChREBP. carbohydrate response elementbinding protein; L-PK, liver-type pyruvate kinase; PP2A, protein phosphatase 2A; TXNIP, thioredoxin-interacting protein.

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Mol Endocrinol, May 2014, 28(5):674 – 680

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doi: 10.1210/me.2014-1041

ered that TXNIP enhances ChREBP dephosphorylation and nuclear localization and induces ChREBP target gene expression, uncovering a novel cross talk mechanism between these two key signaling pathways.


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(20 ng/well; Promega Corp) using DharmaFECT Duo. Cells were harvested 24 hours after transfection, and luciferase activity was determined by Dual Luciferase assay kit (Promega Corp).

Chromatin immunoprecipitation (ChIP)

Materials and Methods Cell culture and islet isolation INS-1 cells were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and 0.05 mM 2-mercaptoethanol. The INS-hTXNIP cell line stably transfected with a human (h) TXNIP expression plasmid and the control INS-LacZ cell line overexpressing LacZ have been described previously (9). Mouse islets were isolated from male 8-week-old, wild-type C57BL/6 mice (The Jackson Laboratory) or from obese, insulinresistant, and diabetic BTBRlepob/ob (BTBRob/ob) and control BTBRlep⫹/⫹ (BTBRlean) mice (8) by collagenase digestion as described previously (7, 9). All mouse studies were approved by the University of Alabama at Birmingham Animal Care and Use Committee.

Quantitative real-time RT-PCR Total RNA was extracted using RNeasy kit (QIAGEN) according to the manufacturer’s instructions. RNA (1 ␮g) was reverse transcribed to cDNA using the first strand cDNA synthesis kit (Roche). Quantitative real-time PCR was performed on a Prism 7000 Sequence Detection System using Sybergreen (Applied Biosystems). The various species-specific TXNIP primers (9) are listed in Supplemental Table 1, and their specificities are shown in Supplemental Table 2. (Of note, rat and mouse primers were not able to detect endogenous or transfected in, hTXNIP. In contrast, specially designed rat/human primers detected both endogenous rat and transfected in hTXNIP.) All samples were corrected for the 18S ribosomal subunit (Applied Biosystems) run as an internal standard.

Plasmid construction and transient transfection assays Construction of the various TXNIP promoter and simian virus 40-carbohydrate response element (ChoRE) reporter plasmids were described previously (9) as were the TXNIP and ChREBP overexpression plasmids (9, 16). C57BL/6 mouse islets (⬃200) were transfected with hTXNIP expression or control LacZ plasmids (1 ␮g/mL) using DharmaFECT Duo transfection reagent (5 ␮L/2 mL medium in a suspension tissue culture dish) and harvested 48 hours later for RNA extraction. INS-hTXNIP and INS-LacZ cells were grown in 12-well plates and transfected with the full-length TXNIP promoter reporter plasmid or control simian virus 40 plasmid (0.4 ␮g/well) using DharmaFECT Duo transfection reagent 1 (1 ␮L/well; Dharmacon/Thermo Scientific). To determine the TXNIP promoter region responsible for the TXNIP effect, INS-1 cells were transfected with different TXNIP promoter deletion reporter plasmids (0.4 ␮g/well) together with pRL-TK control plasmid

ChIP assays were performed as previously detailed (16). In brief, 500 ␮g of cross-linked INS-1 protein extracts (by BCA protein assay) were incubated overnight at 4°C with 4 ␮g of goat anti-ChREBP (sc-21189) antibodies or normal goat IgG (sc2028) (Santa Cruz Biotechnology). Immune complexes were captured with 50 ␮L of 50% protein A-Sepharose agrose slurry (Sigma). DNA fragments were purified using a Qiagen PCR purification kit and quantified by real-time PCR with primers as described previously (16) and listed in Supplemental Table 1.

RNA interference INS-1 cells were grown in 6-well plates and transfected with 3 specific small interfering RNA oligos for rat TXNIP (siTXNIP; Dharmacon; siGENOME SMAPRTpool gene identification no. 117514) rat ChREBP (siChREBP; Invitrogen; MlxxiplRSS301430) or scrambled oligo (0.1 ␮M) (Dharmacon, D-001810 – 01–20) using DharmaFECT transfection reagent (Dharmacon/Thermo Scientific) (5 ␮L per well) as described previously (16, 22). The final concentration of oligos used was 25 nM. Cells were harvested after 48 hours for RNA and protein extraction.

Cell fractionation, Western blotting, and immunohistochemistry Nuclear and cytoplasmic protein extracts were prepared as described previously (7, 23). The rabbit anti-ChREBP IgG (1: 200; sc-33764), and goat antirabbit IgG (1:5000, sc-2004) antibodies were used (Santa Cruz Biotechnology). Phosphorylated ChREBP was detected as described previously (24) using a specific phospho-ChREBP antibody detecting only ChREBP phosphorylated at Ser196, the site critical for controlling nuclear translocation (25) (generous gift of Dr C. Postic, Paris, France). Phosphorylated 5⬘-AMP-activated protein kinase (phosphoAMPK) and total AMPK were detected using the phosphoAMPK (1:1000; Cell Signaling Technology; catalog no. 2535) and total AMPK (1:1000; Cell Signaling Technology; catalog no. 5831) antibodies, respectively. ␤-Actin (1:5000; Abcam; antibody 3280) was run as a control. Bands were visualized by ECL Plus (Amersham GE Health) and quantified by ImageQuant. Immunohistochemistry for insulin and ChREBP was performed as previously described (8, 23).

Statistical analysis Student’s t tests were used to calculate the significance of a difference between 2 groups. For data sets of more than 2 groups, we performed one-way-ANOVA calculations.

Results and Discussion TXNIP induces its own transcription Using our rat INS-1 ␤-cells stably transfected with a hTXNIP expression plasmid (INS-hTXNIP) we were able


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TXNIP Self-Induction via ChREBP

Figure 1. TXNIP effects on ␤-cell TXNIP expression. A, Rat INS-1 ␤cells overexpressing hTXNIP or control (LacZ) were assessed for endogenous rat TXNIP expression using quantitative RT-PCR and species-specific rat primers. B, Isolated, primary wild-type (C57BL/6J) mouse islets were transfected with hTXNIP or LacZ control plasmid and 48 hours later endogenous mouse TXNIP was measured by quantitative RT-PCR and mouse-specific primers. Bars represent means ⫾ SEM; n ⫽ 3– 4.

to achieve effective overexpression (Supplemental Figure 1, A and B) with an increase in total TXNIP mRNA and TXNIP protein levels comparable to the approximately 4-fold increase observed in diabetes (22) assuring physiological relevance of this model. Taking advantage of this unique model and the ability to differentially measure human, rat, or mouse TXNIP mRNA in a species-specific manner (Supplemental Tables 1 and 2), we discovered that TXNIP overexpression induces endogenous TXNIP mRNA expression (Figure 1A). Transient transfection of hTXNIP into native rat INS-1 ␤-cells or rat H9C2 cardiomyocytes also resulted in a significant increase in endog-


enous rat TXNIP (Supplemental Figure 2, A and B) demonstrating that the effect was not restricted to our stable cell lines or to ␤-cells and also occurred in other cell types. Moreover, we confirmed these findings in primary mouse islets transiently transfected with our human TXNIP expression plasmid (Figure 1B). In addition, we found that this TXNIP induction in response to TXNIP overexpression occurred at the transcriptional level as demonstrated by the increase in TXNIP promoter-driven luciferase activity (Figure 2A), but not of a heterologous promoter (Figure 2B). Because TXNIP has not been found to bind to DNA and act as a transcription factor, the observed effects likely required involvement of other factors, and we therefore set out to identify the cis- and trans-acting factors conferring TXNIP-induced TXNIP transcription. An E-box ChoRE motif in the TXNIP promoter is required for TXNIP-induced TXNIP transcription To identify the promoter region responsible for TXNIP-induced TXNIP transcription, we performed promoter deletion studies and found a highly significant induction of the hTXNIP promoter unless the E-box motif of the ChoRE was mutated or deleted (mutD4, D5) (Figure 2C). (Although the small effects of TXNIP on mutD4 and D5 were not significant, we cannot exclude the possibility that TXNIP may also have some minor effects on basal transcription.) Moreover, this ChoRE was able to confer TXNIP responsiveness to a heterologous promoter demonstrating that this E-box motif is not only necessary but also sufficient for the observed TXNIP effects (Supplemental Figure 2C). Of note, this is the same E-box repeat we previously identified to be responsible for glucose-induced TXNIP expression and to serve as the binding site for ChREBP (9, 16). Obviously, this raised the possibility that ChREBP might also be mediating the TXNIP effects.

Figure 2. Analysis of TXNIP promoter activity and cis-acting element conferring TXNIP effects. TXNIP effects on hTXNIP promoter activity (panel A) or simian virus 40 (SV40) promoter activity (panel B) as assessed by luciferase activity in INS-LacZ and INS-hTXNIP cells transfected with the respective reporter plasmids. C, Promoter deletion study using the full-length hTXNIP promoter (FL) and a number of deletions/mutations (D1–D5) transfected into INS-hTXNIP and INS-LacZ cells. Black boxes represent E-box motif ChoRE. Bars represent mean fold change in luciferase activity ⫾ SEM of at least 3 independent experiments as compared with INS-LacZ control cells. mut, mutant; N.S., nonsignificant.

TXNIP increases ChREBP binding to the TXNIP (and L-PK) promoters Indeed, we found that TXNIP overexpression increases ChREBP occupancy of the TXNIP promoter in vivo as demonstrated by ChIP assays, whereas no binding was observed to the glyceraldehyde 3-phosphate dehydrogenase internal con-


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TXNIP knockdown (Supplemental Figure 3A), but also led to a dramatic reduction in nuclear ChREBP and its nuclear/cytoplasmic ratio (Supplemental Figure 3, B and C), further confirming the important role TXNIP plays in regulating nuclear ChREBP localization. Translocation of ChREBP into the nucleus has been shown to require a number of dephosphorylation steps (20, 21), in particular dephosphorylation of Ser196 (25). We therefore also investigated whether TXNIP decreased ChREBP phosphorylation and indeed found a clear decline in Ser196 phosphorylation in response to TXNIP (Figure 4D and Supplemental Figure 4B). The phosphorylation status of Figure 3. ChREBP binding to target gene promoters in response to TXNIP. INS-LacZ and INSChREBP has been shown to be hTXNIP cells were maintained in regular growth medium and cross linked, and ChIP assays were mainly regulated by protein phosperformed as described in Materials and Methods. TXNIP effects on ChREBP occupancy of the TXNIP (panel A) and L-PK (panel B) promoter. C, IgG negative control ChIP. D, TXNIP effects on phatase 2A (PP2A) (27) and by L-PK mRNA level as measured by quantitative RT-PCR. Bars represent mean fold change ⫾ SEM AMPK (28, 29). We therefore first of at least 3 independent experiments. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. used the protein serine/threonine phosphatase inhibitor okadaic acid trol, and the IgG control showed no enrichment confirming the specificity of these ChIP assays (Figure 3). and indeed found that it was able to counteract the TXFurthermore, TXNIP also induced ChREBP binding to NIP-induced expression of endogenous TXNIP as well as the promoter of the bona fide ChREBP target gene, L-PK of L-PK (Supplemental Figure 5, A and B) raising the (17, 26) (Figure 3B) and increased L-PK expression (Fig- possibility that activation of PP2A was involved in the ure 3D) demonstrating that the effects are not restricted to observed TXNIP effects. However, analysis of PP2A acTXNIP, but also include other ChREBP target genes. tivity revealed no difference in response to TXNIP overThese findings provide additional support for the idea expression (Supplemental Figure 5C) making it very unthat ChREBP is mediating the observed downstream ef- likely that PP2A was mediating TXNIP-induced TXNIP fects of TXNIP on gene expression (in addition to serving expression. We therefore next investigated the possibility as the main upstream regulator of ␤-cell TXNIP that AMPK might be involved. In fact, we found that the AMPK activator 5-amino-1-␤-D-ribofuranosyl-imidaexpression.) zole-4-carboxamide completely blunted TXNIP-induced TXNIP inhibits ChREBP phosphorylation and TXNIP expression and L-PK expression (Figure 5, A and promotes nuclear ChREBP B). Moreover, if TXNIP were conferring its effects via The transcriptional activity of ChREBP is primarily AMPK, we would expect that TXNIP would lead to deregulated by its cellular localization, nuclear entry, and creased phosphorylation and activation of AMPK and posttranslational modification (17, 20, 21, 25). There- that is exactly what we observed (Figure 5C and Supplefore, we next investigated whether TXNIP affected nu- mental Figure 4C). These findings suggest that TXNIP clear ChREBP levels. Indeed, TXNIP promoted nuclear promotes ChREBP-mediated transcription (including localization of ChREBP as shown by the increased TXNIP and L-PK) through inhibition of AMPK phosChREBP levels in nuclear cell fractions, the increased nu- phorylation/activation and thereby provide mechanistic clear/cytoplasmic ratio, and immunohistochemistry insight into this novel pathway. However, because TXshowing increased nuclear staining for ChREBP in INS- NIP is not a phosphatase, its inhibitory effects on AMPK hTXNIP cells (Figure 4A–C and Supplemental Figure phosphorylation are likely to be indirect and to involve 4A). Furthermore, transfection of INS-1 cells with TX- yet additional factors (eg, inhibition of upstream AMPKNIP small interfering RNAs not only resulted in effective kinase (30), activation of protein phosphatases such as


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(Supplemental Figure 6). This further supports the notion of ChREBP activity playing an important role in the observed TXNIP effects on TXNIP transcription. Furthermore, we also explored the in vivo effects of increased TXNIP on ChREBP using diabetic BTBRob/ob mice. We have previously shown that BTBRob/ob islets have a 4-fold increase in TXNIP expression as compared with control BTBRlean islets (22). Now we found that this is accompanied by a significant increase in ChREBP in BTBRob/ob islets (Figure 4, E and F). These results are consistent with a recent report of increased ChREBP Figure 4. TXNIP effects on ChREBP phosphorylation and nuclear localization. INS-LacZ and INShTXNIP cells were maintained in regular growth medium prior to fractionation into nuclear and in diabetic human ␤-cells (32) as cytoplasmic protein extracts and measurement of ChREBP protein levels by Western blotting. TXNIP well as with the suggested role of effects on nuclear ChREBP protein levels (panel A) and nuclear to cytoplasmic ChREBP ratio (panel B). ChREBP in our newly discovered C, Visualization of ChREBP (green) and insulin (red) in INS-LacZ and INS-hTXNIP cells by fluorescent pathway and provide an intriguing immunohistochemistry (⫻100). D, Assessment of phosphorylated ChREBP (p-Ser196-ChREBP) in INSLacZ and INS-hTXNIP cell extracts and quantification of the ratio of phosphorylated ChREBP to total pathophysiological link to diabetes. ChREBP. E, Islets of diabetic and obese BTBR ob/ob mice with a known increase in TXNIP levels were In peripheral tissues such as musassessed for ChREBP expression and compared with islets of control BTBR lean mice using quantitative cle and adipose tissue, TXNIP has RT-PCR. F, Immunohistochemistry of pancreatic cross sections and visualization of ChREBP protein (blue) in a control BTBR lean islet and a diabetic BTBR ob/ob islet (⫻40); higher magnification of respective nuclei been shown to inhibit glucose up(insets). Bars represent means ⫾ SEM of at least 3 independent experiments. take via inhibition of glucose transPP1-R6 (31) and/or allosteric modulation (30)). Interest- porter 1 (GLUT1) (33–35), but, as such, is less likely to ingly, the level of TXNIP-induced TXNIP expression and have any major suppressive effects on glucose uptake and L-PK expression was also significantly reduced in re- flux in pancreatic ␤-cells. In fact, if it did, one would sponse to siChREBP as compared with scrambled control expect a negative feedback on TXNIP expression because glucose is a strong stimulus for TXNIP transcription. However, our data demonstrate that TXNIP overexpression results in increased ChREBP-mediated TXNIP transcription in pancreatic ␤-cells suggesting a positive feedback loop. These results indicate that TXNIP overexpression can bypass and override any potential minor effects from decreased intracellular glucose, most likely via the observed inhibition of AMPK and activation of ChREBP-mediated transcription. Together, these findings suggest that TXNIP induces its own expression by promoting dephosphorylation, nuclear translocation, and DNA binding Figure 5. Role of AMPK in TXNIP-induced gene expression. Effects of the AMPK activator 5amino-1-␤-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) (1 mM for 24 hours), on TXNIPof its transcription factor ChREBP. induced TXNIP expression (panel A) and TXNIP-induced L-PK expression (panel B) as assessed by Moreover, the results demonstrate, quantitative RT-PCR in INS-LacZ and INS-hTXNIP cells. C, TXNIP effects on phosphorylated AMPK for the first time, that TXNIP inhibits as assessed by Western blotting and compared with total AMPK; ␤-actin is shown as a loading control. Bars represent means ⫾ SEM of 3 independent experiments. N.S., nonsignificant. AMPK phosphorylation/activation,


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modulates ChREBP activity and thereby regulates other ChREBP target genes such as L-PK, revealing a previously unappreciated cross talk between these important signaling pathways. In summary, we have discovered the existence of a positive feedback loop regulating TXNIP expression that is active in pancreatic ␤-cells and primary islets as well as in other cell types (Figure 1 and Supplemental Figure 2). Taking into account the crucial role that elevated TXNIP levels play in ␤-cell glucose toxicity (7), endoplasmic reticulum stress (12, 13), apoptosis (6 –9), and the pathogenesis of diabetes (8), these findings support the notion that TXNIP levels rise over time not only as a result of elevated blood glucose levels and/or endoplasmic reticulum stress, but also as part of a vicious cycle by which increased TXNIP levels lead to more TXNIP expression and thereby amplify the associated detrimental effects on ␤-cell biology including oxidative stress, inflammation, and ultimately ␤-cell death and disease progression. Moreover, we have identified the mechanism conferring this feedback loop and found that TXNIP promotes the activity of its own transcription factor, ChREBP, which in turn induces transcription of other ChREBP target genes playing important roles in glucose and lipid metabolism. This suggests that this novel pathway has implications even beyond TXNIP expression and reveals a thus-far unrecognized link between TXNIP signaling and cell metabolism.

Acknowledgments Address all correspondence and requests for reprints to: Anath Shalev, MD, Professor of Medicine, Director, Comprehensive Diabetes Center, University of Alabama at Birmingham, 1825 University Boulevard, SHEL 1206, Birmingham, AL 35294 – 2182. E-mail: [email protected]. This work was supported by grants to A.S. from the US National Institutes of Health (R01DK-078752), the American Diabetes Association (7–12-BS-167), and the Juvenile Diabetes Research Foundation and JNJSI (40 –2011–1). Disclosure Summary: The authors have nothing to disclose.


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15-Lipoxygenase promotes chronic hypoxia-induced phenotype changes of PASMCs via positive feedback-loop of BMP4.

Endothelin-1 stimulates its own synthesis in human endothelial cells.

GATA4 autoregulates its own expression in mouse gonadal cells via its distal 1b promoter.

The positive feedback action of vasopressin on its own release from rat septal tissue in vitro is receptor-mediated.

A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle.

A Positive Feedback Loop Governed by SUB1A1 Interaction with MITOGEN-ACTIVATED PROTEIN KINASE3 Imparts Submergence Tolerance in Rice.

Intronic promoter-mediated feedback loop regulates bean PvSR2 gene expression.

High glucose forces a positive feedback loop connecting Akt kinase and FoxO1 transcription factor to activate mTORC1 kinase for mesangial cell hypertrophy and matrix protein expression.

STAT3 positive feedback loop in hepatocellular carcinoma (HCC) cells.

c-KIT Feedback Loop Drives Tumor Progression in Advanced Primary Liver Cancer.

A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis.

TCF7L2 Positive Feedback Loop That Promotes Cancer Cell Stemness and Metastasis.

A positive feedback loop linking enhanced mGluR function and basal calcium in spinocerebellar ataxia type 2.

Completing the feedback loop.