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Curcumin alters EpRE and AP-1 binding complexes and elevates glutamate-cysteine ligase gene expression ¹

Department of Environmental Health Sciences, School of Public Health, and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA; and
^* Lady Davis Institute for Medical Research, Department of Medicine, McGill University, Montréal, Québec H3T 1E2, Canada

²Correspondence: Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 1665 University Blvd., Ryals 534, Birmingham, AL 35294-0022, USA. E-mail: hforman{at}uab.edu

SPECIFIC AIMS

Curcumin has long been used in Eastern medicine and is gaining attention in Western medicine, primarily as a nonsteroidal anti-inflammatory drug but also for its chemopreventative properties; however, the mechanisms of action are only beginning to be investigated rigorously. These studies were designed to investigate the molecular mechanisms by which curcumin protects cells against stress, using the glutathione (GSH) biosynthetic genes as representative phase II genes.

PRINCIPAL FINDINGS

1. Curcumin causes transcription of the Gcl genes leading to increased GCL and GSH content
To investigate the mechanism by which curcumin affects GSH content, content of the GCL enzyme, mRNAs for both Gclc and Gclm and intracellular GSH were measured. We noted that the intracellular GSH content showed dose- and time-dependent increases, with no significant change in GSSG at any dose or time. Even at 24 h, GSH content had not yet begun to decline, but appeared to have stabilized relative to 12 h for 10 and 15 µM curcumin; cells exposed to 20 µM curcumin continued to increase GSH content. In other studies where agents increased GSH, GSH returned to control levels by 24 h and often by 12 h postexposure. The sustained increase in GSH content in response to curcumin may provide an increased capacity for removal of toxicants relative to other inducing agents.

Equally important, the ability for rapid replenishment of intracellular GSH rose as curcumin exposure increased the content of both GCL subunits, which was sustained to 24 h, providing the potential for continuously enhanced GCL activity. Increased GCL content correlated with an increase in Gclc and Gclm mRNA, which was inhibited by actinomycin D, suggesting that curcumin increased GSH biosynthesis via increased expression of the Gcl genes.

2. Curcumin causes increased AP-1 and EpRE binding activity but did not affect NF-B activity
The transcription of Gcl genes has been studied extensively over the last decade. Much work has focused on the EpRE consensus elements as well as TRE and TRE-like elements, all found in both promoters. NF-B is present only in Gclc. The involvement of these putative cis-acting elements in the Gcl promoters in response to curcumin was assessed using the electrophoretic mobility shift assay (EMSA). AP-1 DNA binding activity (Fig. 1 ) increased with time, reached a maximum at 30 min (40% increase compared to control), and was sustained up to 3 h. These data demonstrated that curcumin can mediate an increase in AP-1 DNA binding activity, in contrast to many other reports. A smaller but still significant increase in EpRE DNA binding activity occurred at 15 and 30 min (25% increase vs. control cells), which was sustained for 3 h. NF-B binding activity does not change with curcumin exposure for as long as 3 h with doses of curcumin of up to 30 µM, contrary to reports that curcumin suppresses NF-B binding activity.

View larger version (50K): [in this window] [in a new window]   Figure 1. Changes in DNA binding activities of AP-1, EpRE and NF-B in response to curcumin. HBE1 cells were treated with 10 µM curcumin or vehicle alone. Vehicle-alone-treated cells were normalized to 0% increased binding activity (no induction); curcumin-mediated changes in DNA binding activity are expressed relative to this control. Top: pixel images generated by InstantImager® from one typical experiment. Bottom: semiquantitative summary based on electronic autoradiographic values generated by InstantImager® analysis over at least 3 independent experiments. Values plotted are relative means ± RE. Statistically significant differences from vehicle control are indicated with an asterisk.

3. Curcumin exposure leads to changes in the composition of the AP-1 and EpRE binding complexes
The AP-1 complex composition in control and curcumin-exposed cells was examined using immunodepletion (gel shift) with specific antibodies to members of the Jun and Fos families, typical AP-1 components. The basal AP-1 binding complex contained c-Fos, JunB, Fra1, and JunD. No c-Jun was detectable in the basal complex. By contrast, the AP-1 binding complex in cells exposed to curcumin contained c-Jun. Thus, a change in the composition of the AP-1 complex, along with increased AP-1 binding, occurred in response to curcumin.

Using the immunodepletion assay for EpRE complexes, we demonstrated that the basal EpRE binding complex consisted of c-Jun, Nrf2, and MafG/MafK with a trace of JunD. In contrast, curcumin exposure led to a distinct appearance of JunD in the EpRE binding complex and a subtle decrease in MafG/MafK. Thus, although EpRE binding increased only a small but significant amount, the composition of the complex was markedly altered.

4. Curcumin exposure leads to changes in the phosphorylation of c-Jun, the content of JunD, and the nuclear content of Nrf2 and MafG/MafK
The immunodepletion assay is a powerful tool to describe which proteins comprise a transcription factor complex. It is, however, a semiquantitative method highly dependent on the binding affinities of the antibodies used. To strengthen these findings, we confirmed the results using Western analysis and found changes consistent with results of the immunodepletion analyses. Western analysis was used to assess the content, phosphorylation, and/or nuclear translocation of the transcription complex components in the AP-1 and EpRE complexes. Although binding of c-Jun in the AP-1 complex is dependent on curcumin exposure, to be transcriptionally active in the AP-1 complex, c-Jun must be phosphorylated. Phosphorylation of c-Jun increased twofold in response to curcumin exposure whereas total c-Jun did not change. The cellular content of JunD was found to increase 2.5-fold upon exposure to curcumin, which correlated with increased JunD in the AP-1 and EpRE binding complexes in cells exposed to curcumin.

The small Maf proteins, including MafG and MafK, have been reported to be associated with negative regulation of EpRE-regulated genes. Whereas MafG/MafK was a component of basal and curcumin-treated EpRE binding complexes, the proportion of MafG/MafK in the curcumin-treated EpRE complex was somewhat decreased. The nuclear content of MafG/MafK was reduced 40% with curcumin exposure at 30 min compared with control cells. Nrf2 can heterodimerize with members of the Jun family to form EpRE binding complexes. Nrf2 is maintained in the cytosol through interaction with Keap1, a cytoskeleton binding protein that is dissociated from Nrf2 upon stimulation, allowing Nrf2 translocation to the nucleus. We demonstrated that with curcumin exposure, the cytosolic content of Nrf2 decreased 60% whereas nuclear content increased 40%.

CONCLUSIONS

The molecular mechanism by which curcumin, the most widely consumed dietary spice in the world, affects changes in the GSH biosynthetic enzymes was investigated. A sustained increased in GSH content, which can provide increased resistance against subsequent stress, was mediated by an increase in the expression of the Gcl genes that led to a sustained increase in the GCL subunit proteins, which comprise the active enzyme.

The expression of the Gcl genes is often found to be related to EpRE and TRE elements in their promoters. Using EMSA analysis, AP-1 complexes (which bind TRE) and EpRE binding complexes were both found to increase in response to curcumin with the increase sustained for at least 3 h. NF-B did not change. Previous results are in conflict as to whether NF-B is repressed or activated by curcumin. Curcumin is often referred to as a specific inhibitor of AP-1 activation, although this seems a misinterpretation of the original literature. Instead of making assumptions, the effects of curcumin on transcription factor content, phosphorylation, and DNA binding in HBE1 cells were examined directly. The results show that curcumin can increase AP-1 activity and does not affect NF-B binding activity.

The increases in AP-1 and EpRE binding activities are consistent with what is generally believed about the cis-acting elements responsible for Gcl expression. Immunodepletion was then used to examine changes in the composition of these DNA binding complexes as a function of curcumin. Curcumin exposure led to a remodeling of these complexes to include c-Jun in the AP-1 complex, substantially increasing JunD whereas modestly decreasing MafG/MafK in the EpRE complex.

Although whole cell c-Jun content did not change, there was a dramatic increase in the content of phosphorylated c-Jun, the post-translational modification necessary for its transcriptional activity in the AP-1 complex. The whole cell JunD content increased dramatically with curcumin, as did the nuclear content of Nrf2, which was correlated with decreased cytosolic content. MafG/MafK nuclear content decreased with curcumin.

These results suggest that curcumin acts by changing the nuclear content and/or activation of transcription factors, specifically MafG/MafK, Nrf2, JunD and phosphorylated c-Jun, leading to alterations in the composition of the DNA binding complexes recognizing TRE, TRE-like, and EpRE consensus elements in gene promoters. The expression of many phase II genes, such as the Gcl genes studied here, are regulated by EpRE elements, with TRE and TRE-like elements having roles in many protective genes. This suggests a potential molecular mechanism of dietary curcumin action in preventive medicine.

View larger version (30K): [in this window] [in a new window]   Figure 2. Schematic diagram of curcumin-mediated changes on transcription factor proteins. The basal AP-1 complex contains c-Fos, Fra1, JunB, and a small amount of JunD, but notably lacks c-Jun. The basal EpRE complex contains JunD, the small Maf proteins MafG and MafK, Nrf2, and, unlike the AP-1 complex, a small amount of c-Jun. Much of the Nrf2 is bound in the cytosol to Keap1. Upon stimulation with curcumin, Keap1 dissociates and allows Nrf2 to migrate to the nucleus. With curcumin stimulation, there is a dramatic increase in the content of phosphorylated c-Jun, which is now a component of the AP-1 complex. The content of JunD increases with curcumin stimulation and becomes more prominent in EpRE and AP-1 complexes. The small Maf proteins MafG and MafK, often repressors, decrease in nuclear abundance with curcumin. These changes in transcription factor complex composition are a component of the curcumin-mediated expression of the phase II genes Gclc and Gclm, leading in part to increased DNA binding activity.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0566fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0566fje

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