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• W2040984041 abstract "Antioxidants are important candidate agents for the prevention of disease. However, the possibility that different antioxidants may produce opposing effects in tissues has not been adequately explored. We have reported previously that (–)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol antioxidant, stimulates expression of the keratinocyte differentiation marker, involucrin (hINV), via a Ras, MEKK1, MEK3, p38δ signaling cascade (Balasubramanian, S., Efimova, T., and Eckert, R. L. (2002) J. Biol. Chem. 277, 1828–1836). We now show that EGCG activation of this pathway results in increased CCAAT/enhancer-binding protein (C/EBPα and C/EBPβ) factor level and increased complex formation at the hINV promoter C/EBP DNA binding site. This binding is associated with increased promoter activity. Mutation of the hINV promoter C/EBP binding site eliminates the regulation as does expression of GADD153, a dominant-negative C/EBP factor. In contrast, a second antioxidant, curcumin, inhibits the EGCG-dependent promoter activation. This is associated with inhibition of the EGCG-dependent increase in C/EBP factor level and C/EBP factor binding to the hINV promoter. Curcumin also inhibits the EGCG-dependent increase in endogenous hINV levels. The curcumin-dependent suppression of C/EBP factor level is inhibited by treatment with the proteasome inhibitor MG132, suggesting that the proteasome function is required for curcumin action. We conclude that curcumin and EGCG produce opposing effects on involucrin gene expression via regulation of C/EBP factor function. The observation that two antioxidants can produce opposite effects is an important consideration in the context of therapeutic antioxidant use. Antioxidants are important candidate agents for the prevention of disease. However, the possibility that different antioxidants may produce opposing effects in tissues has not been adequately explored. We have reported previously that (–)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol antioxidant, stimulates expression of the keratinocyte differentiation marker, involucrin (hINV), via a Ras, MEKK1, MEK3, p38δ signaling cascade (Balasubramanian, S., Efimova, T., and Eckert, R. L. (2002) J. Biol. Chem. 277, 1828–1836). We now show that EGCG activation of this pathway results in increased CCAAT/enhancer-binding protein (C/EBPα and C/EBPβ) factor level and increased complex formation at the hINV promoter C/EBP DNA binding site. This binding is associated with increased promoter activity. Mutation of the hINV promoter C/EBP binding site eliminates the regulation as does expression of GADD153, a dominant-negative C/EBP factor. In contrast, a second antioxidant, curcumin, inhibits the EGCG-dependent promoter activation. This is associated with inhibition of the EGCG-dependent increase in C/EBP factor level and C/EBP factor binding to the hINV promoter. Curcumin also inhibits the EGCG-dependent increase in endogenous hINV levels. The curcumin-dependent suppression of C/EBP factor level is inhibited by treatment with the proteasome inhibitor MG132, suggesting that the proteasome function is required for curcumin action. We conclude that curcumin and EGCG produce opposing effects on involucrin gene expression via regulation of C/EBP factor function. The observation that two antioxidants can produce opposite effects is an important consideration in the context of therapeutic antioxidant use. IntroductionThe human epidermis is a stratifying squamous epithelium comprised of a single, basally located layer of proliferating keratinocytes and multiple suprabasal layers of differentiating keratinocytes (1Green H. Harvey Lect. 1980; 74: 101-139Google Scholar, 2Eckert R.L. Crish J.F. Robinson N.A. Physiol. Rev. 1997; 77: 397-424Google Scholar, 3Eckert R.L. Rorke E.A. Environ. Health Perspect. 1989; 80: 109-116Google Scholar, 4Fuchs E. Byrne C. Curr. Opin. Genet. Dev. 1994; 4: 725-736Google Scholar). During differentiation, keratinocytes undergo a choreographed series of morphological and biochemical changes that result in the assembly of a protective cornified envelope (5Steinert P.M. Cell Death Differ. 1995; 2: 33-40Google Scholar). Involucrin is a component of this structure that is specifically expressed in the epidermal suprabasal layers (6Steinert P.M. Marekov L.N. J. Biol. Chem. 1997; 272: 2021-2030Google Scholar, 7Yaffe M.B. Murthy S. Eckert R.L. J. Investig. Dermatol. 1993; 100: 3-9Abstract Full Text PDF Google Scholar). Activation of involucrin gene expression is controlled by a p38 MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; EGCG, (–)-epigallocatechin-3-gallate; hINV, human involucrin; C/EBP, CCAAT/enhancer-binding protein; KSFM, keratinocyte serum-free medium; EBS, ets factor binding site; caRas, constitutively active Ras. signaling cascade. This cascade includes novel protein kinase C, Ras, MEKK1, MEK3, and p38 (8Balasubramanian S. Efimova T. Eckert R.L. J. Biol. Chem. 2002; 277: 1828-1836Google Scholar), and it targets various transcription factors (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar, 10Banks E.B. Crish J.F. Welter J.F. Eckert R.L. Biochem. J. 1998; 331: 61-68Google Scholar, 11Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Google Scholar). These factors, in turn, bind to the hINV promoter upstream regulatory region (nucleotides –2473 to –1) to activate hINV gene expression (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar, 12Efimova T. Deucher A. Kuroki T. Ohba M. Eckert R.L. J. Biol. Chem. 2002; 277: 31753-31760Google Scholar). In the present manuscript, we utilize this system to study the role of antioxidants in regulating keratinocyte function.Antioxidants comprise a collection of agents, derived from various sources, that are potential disease-preventive agents (13Kelloff G.J. Crowell J.A. Steele V.E. Lubet R.A. Boone C.W. Malone W.A. Hawk E.T. Lieberman R. Lawrence J.A. Kopelovich L. Ali I. Viner J.L. Sigman C.C. Ann. N. Y. Acad. Sci. 1999; 889: 1-13Google Scholar, 14Mukhtar H. Ahmad N. Proc. Soc. Exp. Biol. Med. 1999; 220: 234-238Google Scholar, 15Dong Z. Biofactors. 2000; 12: 17-28Google Scholar). We have reported recently that (–)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol antioxidant, increases hINV promoter activity and endogenous hINV expression in normal epidermal keratinocytes (8Balasubramanian S. Efimova T. Eckert R.L. J. Biol. Chem. 2002; 277: 1828-1836Google Scholar). An important question is whether individual antioxidants produce similar or opposing effects in cells. This is important because these agents are being considered for use in treating a host of diseases, and the possibility exists that simultaneous treatment with structurally dissimilar antioxidants may produce opposing effects. To investigate this possibility, we have examined the interplay between EGCG and a second antioxidant, curcumin, in regulating involucrin gene expression.EGCG is the major bioactive polyphenol component of green tea and has been reported to regulate a wide range of processes in a variety of cell types (16Ahmad N. Mukhtar H. Nutr. Rev. 1999; 57: 78-83Google Scholar, 17Chung J.Y. Park J.O. Phyu H. Dong Z. Yang C.S. FASEB J. 2001; 15: 2022-2024Google Scholar, 18Chung J.Y. Huang C. Meng X. Dong Z. Yang C.S. Cancer Res. 1999; 59: 4610-4617Google Scholar). Curcumin (diferuloylmethane), commonly called turmeric, is a polyphenol derived from the plant Curcuma longa (19Bonte F. Noel-Hudson M.S. Wepierre J. Meybeck A. Planta Med. 1997; 63: 265-266Google Scholar, 20Aggarwal B.B. Kumar A. Bharti A.C. Anticancer Res. 2003; 23: 363-398Google Scholar, 21Leu T.H. Maa M.C. Curr. Med. Chem. Anti-Canc. Agents. 2002; 2: 357-370Google Scholar). Both agents are effective cancer therapeutic and prevention agents. However, because of the varying chemical structure of these agents, some of their effects may be unrelated to their antioxidant properties. Thus, it is important to evaluate the effects of individual antioxidants to determine whether they produce contributing or opposing effects. In the present study, we show that curcumin opposes the EGCG-dependent activation of involucrin gene expression by inhibiting the EGCG-dependent increase in C/EBP transcription factor levels. These finding indicate that not all antioxidants produce parallel changes in human keratinocyte function and indicate that potential opposing effects of these agents must be considered when antioxidants are proposed for therapeutic use.MATERIALS AND METHODSChemicals and Reagents—(–)-Epigallocatechin-3-gallate and curcumin were purchased from Sigma. EGCG was prepared as 1000-fold stock in sterile distilled water. MG132 (Z-Leu-Leu-Leu-CHO) was purchased from Biomol. Keratinocyte serum-free medium (KSFM), gentamicin, trypsin, and Hanks' balanced salt solution were obtained from Invitrogen. Dispase was from Roche Applied Science. The pGL2-Basic plasmid and chemiluminescent luciferase assay system were purchased from Promega. Chemiluminescence was measured using a Berthold luminometer, and synthetic oligonucleotides were synthesized using an Applied Biosystems DNA synthesizer. [γ-32P]ATP was purchased from PerkinElmer Life Sciences. C/EBP transcription factor-selective rabbit polyclonal antibodies specifying C/EBPα (sc-61X, 1:500), C/EBPβ (sc-150X, 1:2000), C/EBPδ (sc-636X, 1:500), and GADD153 (sc-793, 1:500) were obtained from Santa Cruz Biotechnology. An antibody specific for β-actin from Sigma (A5441) was used for immunoblot at a dilution of 1:10,000.Plasmids—The structure of the hINV promoter reporter plasmid, pINV-241, has been described previously (11Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Google Scholar). To create pINV-241(C/EBPm), the C/EBP site mutant, the fragment containing the wild type C/EBP site (5′-GCTGCTTAAG-3′), was released as part of a larger fragment by digestion of pINV-241 with ApaI/PstI and replaced with the identical segment containing a mutated C/EBP site (5′-GCTGAGATCT-3′) (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar). The modified nucleotides are underlined. C/EBPα was kindly provided by Dr. David Samols (Case Western Reserve University School of Medicine) (22Hendricks-Taylor L.R. Darlington G.J. Nucleic Acids Res. 1995; 23: 4726-4733Google Scholar). C/EBPβ (CRP2, rat) and C/EBPδ (CRP3, mouse) were expressed using pMEX and provided by Dr. Peter Johnson (23Williams S. Cantwell C. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Google Scholar). GADD153 and pCMV-neo were provided by Dr. Nikki Holbrook (24Fawcett T.W. Eastman H.B. Martindale J.L. Holbrook N.J. J. Biol. Chem. 1996; 271: 14285-14289Google Scholar, 25Bartlett J.D. Luethy J.D. Carlson S.G. Sollott S.J. Holbrook N.J. J. Biol. Chem. 1992; 267: 20465-20470Google Scholar). Constitutively active Ras (RasG12V) was obtained from Dr. Michael Simonson (26Herman W.H. Simonson M.S. J. Biol. Chem. 1995; 270: 11654-11661Google Scholar). Wild type MEKK1, cloned in pEECMV, was provided by Dr. Dennis Templeton (27Yan M. Templeton D.J. J. Biol. Chem. 1994; 269: 19067-19073Google Scholar).Cell Transfection and Luciferase Assay—Normal human foreskin keratinocytes were isolated and cultured as described previously (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar). Keratinocytes (60% confluent, third passage) were transfected in 9.5-cm2-area dishes. FuGENE 6 transfection reagent was added to KSFM at a final concentration of 4% and incubated at 25 °C for 5 min. The mixture was then added to 2 μg of involucrin promoter reporter plasmid or, for co-transfection experiments, with 1 μg of involucrin reporter plasmid and 1 μg of a second plasmid. The mixture was incubated at 25 °C for 15 min and then added directly to the cultures in 2 ml of KSFM. The final DNA concentration in all groups was adjusted to 2 μg of DNA/4 μlof FuGENE 6 reagent/9.5-cm2 dish by addition of empty expression vector. After 24 h, the cells were incubated with KSFM in the presence or absence of EGCG and/or curcumin. After an additional 24 h, the cells were washed with phosphate-buffered saline, dissolved in 200 μl of cell culture lysis reagent (Promega), and harvested by scraping. Luciferase activity was assayed immediately using Promega luciferase assay kit and a Berthold luminometer. All assays were performed in triplicate, and each experiment was repeated a minimum of three times. Luciferase activity was normalized per μg of protein. Transfection efficiency was normalized using a green fluorescent protein-encoding expression vector.Preparation of Nuclear Extracts and Gel Mobility Shift Analysis— Cultured keratinocytes (80% confluent) were treated for 24 h with KSFM or with KSFM containing 40 μm EGCG and/or 20 μm curcumin prior to preparation of nuclear extracts (8Balasubramanian S. Efimova T. Eckert R.L. J. Biol. Chem. 2002; 277: 1828-1836Google Scholar). The protein content was determined using Bradford reagent (Bio-Rad). Binding of transcription factors to the hINV promoter C/EBP site was detected using gel mobility shift assay (8Balasubramanian S. Efimova T. Eckert R.L. J. Biol. Chem. 2002; 277: 1828-1836Google Scholar). Five μg of nuclear extract was incubated for 10 min at room temperature in a total volume of 20 μl containing 40 mm HEPES (pH 7.6), 10% glycerol, 200 mm KCl, 10 mm dithiothreitol, 2 μg/ml poly(dI-dC), and 100,000 cpm of radioactive double-stranded DNA oligonucleotide. The oligonucleotide, 5′-GGTTTGCTGCTTAAGATGCCTG-3′, that encodes the hINV C/EBP binding site (C/EBP site is underlined) was end-labeled using polynucleotide kinase and [γ-32P]ATP. For competition studies, radioinert DNA competitor was added to the DNA binding reaction at 10- or 100-fold molar excess. Protein-DNA complexes were resolved on a 6% nondenaturing polyacrylamide gel using 0.5× Tris/borate/EDTA running buffer. The gel was dried, and the radioactivity was detected by autoradiography.Immunoblot Analysis—Total cell extracts were prepared from cultured human keratinocytes as described previously (8Balasubramanian S. Efimova T. Eckert R.L. J. Biol. Chem. 2002; 277: 1828-1836Google Scholar). Equivalent amounts of protein were electrophoresed on 10% denaturing polyacrylamide gels and transferred to nitrocellulose. The membranes were blocked, incubated with a specific primary antibody, washed, and exposed to an appropriate horseradish peroxidase-conjugated secondary antibody. Chemiluminescent detection (Amersham Biosciences) was used to visualize secondary antibody binding.RESULTSC/EBP Site Is Required for EGCG-dependent hINV Promoter Regulation—We first examined the effects of EGCG treatment on hINV promoter activity. Fig. 1A shows a schematic of the hINV promoter constructs. Promoter activity assays, shown in Fig. 1B, indicate that EGCG treatment produces a 4-fold increase in activity of the intact hINV promoter construct, pINV-241. Mutation of the C/EBP site, pINV-241(C/EBPm), results in a loss of the EGCG-dependent regulation. As a control, we examined the effects of EGCG treatment on a promoter construct in which an ets factor binding site (EBS), pINV-241(EBS-2m), is mutated. This mutation results in an overall increase in basal and EGCG-stimulated promoter activity, but the 4-fold EGCG-dependent increase is still observed. These results suggest that the C/EBP site is specifically required for EGCG-dependent gene expression. Fig. 1C confirms relevance of the response by showing that endogenous hINV gene expression is increased by 4-fold as measured by increased presence of hINV protein. Additional studies reveal a parallel increase in hINV mRNA (not shown).EGCG Increases C/EBPα-dependent hINV Promoter Activation—The above results suggest that C/EBP factors may be important mediators of the EGCG-dependent response. To test this further, we evaluated whether C/EBP factors influence hINV promoter activity. Cells were transfected with pINV-241 in the presence of expression vectors encoding C/EBPα, -β, or -δ. Fig. 2A shows that hINV promoter activity is markedly increased in the presence of C/EBPα, with a lesser increase observed in the presence of C/EBPβ and -δ. In contrast, GADD153, a dominant-negative C/EBP family member that suppresses function of other C/EBP proteins by inhibiting their interaction with DNA (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar), completely inhibits the EGCG-dependent increase (Fig. 2B). Basal activity is also inhibited, indicating that basal transcription also relies on C/EBP factors. Fig. 2C confirms that C/EBPα, C/EBPβ, C/EBPδ, and GADD153 are each expressed when cells are treated with the corresponding expression vector. Longer exposure of these films revealed the presence of endogenous C/EBPα and C/EBPβ; however, C/EBPδ was not detected (not shown).Fig. 2C/EBP factor activity is required for EGCG-dependent regulation of hINV gene expression. A, keratinocytes were co-transfected with 1 μg of pINV-241 reporter construct and 1 μg of empty expression vector (EV) or vectors encoding C/EBPα, -β, or -δ. After 48 h, the cells were harvested and assayed for the luciferase activity. Activity was normalized per μg of protein. B, GADD153 suppressed EGCG-dependent gene expression. Cultured keratinocytes were transfected with 1 μg of pINV-241 and 1 μg of either empty vector or GADD153 expression vector. After 24 h, the cells were treated for 24 h with 40 μm EGCG. After an additional 24 h, the cells were harvested and assayed for luciferase activity. The errors bars represent the S.E. This experiment was repeated three times with similar results. C, cells were transfected in the absence (–) or presence (+) of vector expressing the indicated protein as described above. Total cell extracts were prepared and immunoblotted with antibodies specific for each transcription factor. β-Actin was immunoblotted to assure appropriate loading. Longer exposure revealed the presence of endogenous C/EBPα and C/EBPβ but not C/EBPδ (not shown).View Large Image Figure ViewerDownload (PPT)The above results suggest that EGCG may control hINV promoter activity by regulating C/EBP factor level. To determine whether EGCG regulates endogenous C/EBP factor levels, nuclear extracts were prepared from keratinocytes after treatment for 24 h with 40 μm EGCG. C/EBP factor levels were then assayed by immunoblot. As shown in Fig. 3A, EGCG treatment substantially increases C/EBPα and C/EBPβ level. C/EBPδ could not be detected. To determine whether the increased C/EBP level correlates with increased C/EBP binding to the hINV promoter C/EBP DNA element, we performed gel mobility shift studies. Cells were treated with vehicle or 40 μm EGCG. After 24 h the cells were harvested, and nuclear extracts were incubated with 32P-labeled C/EBP oligonucleotide (32P-C/EBP). As shown in Fig. 3B, basal protein binding to the hINV C/EBP element is markedly increased following EGCG treatment. Moreover, addition of radioinert competitor oligonucleotide causes a reduction in DNA binding, suggesting that the binding is specific. In addition, this binding is not competed by an unrelated oligonucleotide, Sp1c, that encodes a consensus Sp1 site (11Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Google Scholar).Fig. 3EGCG regulation of C/EBP factor level and DNA binding. A, nuclear extracts were prepared from keratinocytes after treatment for 24 h in the absence (–) or presence (+) of 40 μm EGCG. Equal quantities of protein were electrophoresed, transferred to nitrocellulose, and immunoblotted with a C/EBPα- or C/EBPβ-specific antibody. Binding of the primary antibody was detected by incubation with an anti-rabbit IgG secondary antibody with chemiluminescence-dependent visualization. Similar results were obtained in each of three experiments. B, sequence of oligonucleotides used in gel mobility shift experiments (11Welter J.F. Crish J.F. Agarwal C. Eckert R.L. J. Biol. Chem. 1995; 270: 12614-12622Google Scholar). The C/EBP sequence is derived from the hINV C/EBP site (bold) and flanking regions. C, nuclear extracts (NE) were prepared from near-confluent keratinocyte cultures following treatment for 24 h with (+) or without (–) 40 μm EGCG. Nuclear extracts were then incubated with 32P-labeled C/EBP double-stranded DNA oligomer containing the hINV C/EBP binding site (32P-C/EBP). Specific binding was demonstrated by including a 10- and 100-fold molar excess of homologous competitor oligonucleotide (C/EBP) or Sp1c during the binding reaction. DNA-protein complexes were separated by electrophoresis on a nondenaturing acrylamide gel and visualized by autoradiography. C/EBP indicates the migration position of the C/EBP oligonucleotide complex, and FP indicates migration of the free probe.View Large Image Figure ViewerDownload (PPT)Curcumin Antagonizes the EGCG-dependent Regulation— The above studies indicate that EGCG increases hINV gene expression, both the endogenous gene and the promoter, and suggest that this increase is dependent upon a functional C/EBP site and also elevated C/EBP levels. We next assessed the role of another antioxidant, curcumin. As shown in Fig. 4, unlike EGCG, curcumin treatment does not increase hINV promoter activity. In addition, incubation of curcumin with EGCG results in inhibition of the EGCG-dependent increase in promoter activity. To assess the concentration dependence of this response, cells were treated with or without an optimal concentration of EGCG (40 μm) and challenged with increasing concentrations of curcumin. As shown in Fig. 5A, curcumin suppresses the EGCG-dependent increase in promoter activity. Complete suppression is observed at curcumin concentrations ≥15 μm. To determine whether the endogenous gene is regulated in a like manner, cells were treated with optimal concentrations of each agent, and involucrin protein level was measured in total cell extracts. Fig. 5B shows that EGCG increases the level of endogenous hINV by 3.3-fold and that this increase is inhibited by curcumin.Fig. 4Curcumin inhibits the EGCG-dependent increase in promoter activity. Human keratinocytes were grown in KSFM until 70% confluent and then transfected with the pINV-241 luciferase reporter construct (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar). At 24 h after transfection, the cells were treated for 24 h in the presence or absence of 40 μm EGCG and/or 20 μm curcumin. The cells were then harvested for extract preparation and measurement of luciferase activity (28Efimova T. LaCelle P. Welter J.F. Eckert R.L. J. Biol. Chem. 1998; 273: 24387-24395Google Scholar). The errors bars represent the S.E. (n = 3).View Large Image Figure ViewerDownload (PPT)Fig. 5Curcumin inhibition is concentration-dependent. A, human keratinocytes were grown in KSFM until 70% confluent and then transfected with the pINV-241 luciferase reporter construct (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar). At 24 h after transfection, the cells were treated for 24 h in the presence or absence of 40 μm EGCG in the presence of increasing curcumin concentration. Promoter activity was monitored by assaying luciferase activity (28Efimova T. LaCelle P. Welter J.F. Eckert R.L. J. Biol. Chem. 1998; 273: 24387-24395Google Scholar). The errors bars represent the S.E. (n = 3). B, curcumin inhibits the EGCG-dependent increase in endogenous hINV protein levels. Cells were treated with the indicated concentration of EGCG or curcumin for 24 h, and extracts were prepared to monitor hINV and β-actin levels by immunoblot. The -fold change was determined by laser densitometric scanning after normalizing to the β-actin signal. This experiment is representative of three separate experiments.View Large Image Figure ViewerDownload (PPT)Curcumin Inhibits Ras- and MEKK1-dependent Activation of hINV Promoter Activity—Previous studies indicate that EGCG activates a Ras, MEKK1, MEK3, p38δ signaling cascade to increase hINV gene expression (8Balasubramanian S. Efimova T. Eckert R.L. J. Biol. Chem. 2002; 277: 1828-1836Google Scholar). As outlined in Fig. 6A, our present studies show that C/EBP factors are a downstream target of this cascade. To identify potential sites of curcumin action in this cascade, hINV promoter activity was activated by kinases positioned at various levels within this cascade, and the effect of curcumin treatment was monitored. Constitutively active Ras (caRas) and wild type MEKK1 (MEKK1) were utilized, as these kinases are known to increase hINV promoter activity (28Efimova T. LaCelle P. Welter J.F. Eckert R.L. J. Biol. Chem. 1998; 273: 24387-24395Google Scholar). As shown in Fig. 6, B and C, treatment with curcumin inhibits both caRas- and MEKK1-dependent promoter activation. This suggests that the site of curcumin action is downstream of these kinases.Fig. 6Curcumin inhibits caRas- and MEKK1-dependent hINV promoter activity. A, EGCG regulates hINV gene expression via a MAPK cascade. B and C, human keratinocytes were grown in KSFM until 70% confluent and then transfected with the 1 μg of pINV-241 luciferase reporter construct (9Agarwal C. Efimova T. Welter J.F. Crish J.F. Eckert R.L. J. Biol. Chem. 1999; 274: 6190-6194Google Scholar) in the presence or absence of 1 μg of empty vector (EV) or caRas or MEKK1 expression vector. At 24 h after transfection, the cells were treated for 24 h in the presence of the indicated concentration of curcumin. The cells were then harvested for extract preparation and measurement of luciferase activity (28Efimova T. LaCelle P. Welter J.F. Eckert R.L. J. Biol. Chem. 1998; 273: 24387-24395Google Scholar). The error bars represent the S.E. (n = 3).View Large Image Figure ViewerDownload (PPT)Curcumin Treatment Reduces C/EBP Transcription Factor Levels—One possibility is that curcumin acts to reduce C/EBP transcription factor level. To test this, we monitored the effect of curcumin on the level of endogenous C/EBPα and -β. As shown in Fig. 7A, treatment with EGCG results in an increase in nuclear C/EBPα and C/EBPβ level, and this increase is inhibited by curcumin treatment. As shown in Fig. 7B, the reduction in C/EBP factor level is associated with reduced binding at the hINV promoter C/EBP site.Fig. 7Curcumin inhibits EGCG-dependent increase in the C/EBP factor level and also C/EBPα binding to the hINV promoter C/EBP site. A, cells were treated with EGCG and curcumin for 24 h, and extracts were prepared to monitor C/EBPα, C/EBPβ, and β-actin levels by immunoblot. The -fold change was determined by laser densitometric scanning after normalizing to the β-actin signal. This experiment is representative of three separate experiments. B, curcumin reduces EGCG-dependent C/EBP factor binding to hINV promoter C/EBP site. Cells were treated for 24 h with the indicated concentration of EGCG or curcumin. Nuclear extracts (NE) were prepared for gel mobility shift analysis. The left-most lane shows migration of free 32P-C/EBP probe in the absence of nuclear extract. FP and C/EBP indicate migration of the free probe and the C/EBP band, respectively.View Large Image Figure ViewerDownload (PPT)Proteasome Inhibition Reverses Curcumin-dependent Reduction in C/EBP Factor Level—We next investigated the mechanism of this regulation. Several studies suggest that C/EBP factor level may be regulated by proteasome-dependent mechanisms (29Hungness E.S. Robb B.W. Luo G.J. Pritts T.A. Hershko D.D. Hasselgren P.O. Biochem. Biophys. Res. Commun. 2002; 290: 469-474Google Scholar, 30Shim M. Smart R.C. J. Biol. Chem. 2003; 278: 19674-19681Google Scholar). To assess the impact of proteasome function on the curcumin-dependent reduction of C/EBPα and -β level, cells were incubated with curcumin in the absence or presence of the proteasome inhibitor MG132. As shown in Fig. 8, treatment with curcumin results in a reduction in endogenous C/EBPα and C/EBPβ levels. This reduction is reversed by treatment with the proteasome inhibitor MG132.Fig. 8MG132 inhibits the curcumin-dependent reduction in C/EBP factor level. Keratinocytes were incubated in the presence of curcumin and/or MG132 for 24 h prior to preparation of nuclear extracts. Equivalent amounts of nuclear extract were electrophoresed on parallel lanes of a 10% denaturing acrylamide gel and then incubated with anti-C/EBPα or anti-C/EBPβ followed by incubation with an appropriate secondary antibody. Secondary antibody binding was then visualized using chemiluminescent detection reagents.View Large Image Figure ViewerDownload (PPT)We next assessed the effect of C/EBPα overexpression and proteasome activity on hINV promoter activity. As shown in the upper panel in Fig. 9A, C/EBPα overexpression increases hINV promoter activity, and this increase is inhibited by curcumin treatment. The lower panel in Fig. 9A shows that the curcumin-dependent reduction in promoter activity is associated with a parallel reduction in C/EBPα level. The upper panel in Fig. 9B shows that the curcumin-dependent reduction in hINV promoter" @default.
• W2040984041 created "2016-06-24" @default.
• W2040984041 creator A5000749676 @default.
• W2040984041 creator A5013153315 @default.
• W2040984041 date "2004-06-01" @default.
• W2040984041 modified "2023-09-29" @default.
• W2040984041 title "Green Tea Polyphenol and Curcumin Inversely Regulate Human Involucrin Promoter Activity via Opposing Effects on CCAAT/Enhancer-binding Protein Function" @default.
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