Differential Activation of a C/EBP
 Isoform by a Novel Redox
Switch May Confer the Lipopolysaccharide-inducible Expression
of Interleukin-6 Gene*
Received for publication, May 27, 2003, and in revised form, October 3, 2003 Published, JBC Papers in Press, October 6, 2003, DOI 10.1074/jbc.M305501200
Wen-Chi Su‡§, Han-Yi Chou‡§, Ching-Jin Chang¶, Yu-May Lee¶, Wei-Hao Chen‡, Kuo-Hsiang Huang‡, Ming-Yue Lee‡, and Sheng-Chung Lee‡¶储**
From the Institutes of ‡Molecular Medicine and储Clinical Medicine, College of Medicine, National Taiwan University and the¶Institute of Biological Chemistry, Academia Sinica, Taipei 100, Taiwan
C/EBP, a member of the CCAAT/enhancer binding protein (C/EBP) family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which play important roles in innate immunity, inflammatory response, adipocyte and mye-loid cell differentiation, and the acute phase response. Three C/EBP isoforms (i.e. LAP*, LAP, and LIP) were known to arise from differential translation initiation and display different functions in gene regulation. C/EBP is known to induce interleukin (IL)-6 gene when P388D1 cells are treated with lipopolysaccharide (LPS). Exactly how the transcriptional activities of C/EBP iso-forms are involved in the regulation of the IL-6 gene remains unclear. Here we report that LPS-induced ex-pression of IL-6 gene in P388D1 cells is mediated by a redox switch-activated LAP*. The intramolecular disul-fide bonds of LAP* and LAP have been determined. Among the cysteine residues, amino acid 11 (Cys11) of
LAP* plays key roles for determining the overall in-tramolecular disulfide bonds that form the basis for re-dox switch regulation. The DNA binding activity and transcriptional activity of LAP* are enhanced under re-ducing condition. LAP and LIP, lacking 21 and 151 amino acids, respectively, in the N-terminal region, are not regulated in a similar redox-responsive manner. Our results indicate that LAP* is the primary isoform of C/EBP that regulates, through a redox switch, the LPS-induced expression of the IL-6 gene.
Members of the C/EBP1family of proteins have been
impli-cated in the differentiation of myelomonocytic cells and the regulation of gene expression during activation of macrophages (1, 2). Regulation of macrophage gene expression by
lipopoly-saccharide (LPS) is of potential importance in the pathogenesis of infection, inflammation, and septic shock. A detailed under-standing of the molecular basis of such disease requires a thorough analysis of the mechanisms by which the actions of LPS are transduced to the nucleus to alter gene expression. The expression of C/EBP is inducible in monocytes and macro-phages by LPS. C/EBP has been implicated in the regulation of proinflammatory cytokines as well as other genes associated with macrophage activation and the acute phase response (3, 4). The promoter regions of the genes for IL-6, TNF␣, IL-1, IL-8, and granulocyte colony-stimulating factor contain C/EBP-binding sites (3, 5–7). The stable expression of C/EBP in a murine P388 lymphoblast cell line is sufficient to confer LPS-inducible IL-6 expression (8, 9). Conversely, inhibition of endogenous C/EBP expression by antisense RNA interference blocks LPS induction of IL-6 expression in the murine P388D1(IL1) macrophage-like cells (8). C/EBP can also acti-vate the proximal promoter of the human TNF-␣ gene in RAW264.7 monocytic cells, whereas overexpression of a domi-nant negative C/EBP inhibits LPS-induced activation (10). In the murine P388D1(IL1) cell line, C/EBP is constitutively expressed, and IL-6 and TNF-␣ can readily be induced by agents such as LPS. The exact molecular mechanisms of LPS stimulation of IL-6 and TNF-␣ gene expression and how this relates to C/EBP-mediated activation of these genes remain unclear.
Structurally, members of C/EBP family are bZIP proteins with sequence-specific DNA binding activity when homo- or heterodimerized. The DNA-binding and transactivation do-mains are located in the C- and N-terminal regions, respec-tively (11). The intronless c/ebp gene (also known as lap in rat, agp/ebp in mouse, and NF-IL6 in human) encodes three isoforms, including two activators (termed LAP* and LAP, respectively) and a repressor (i.e. LIP). These isoforms of C/EBP are differentially translated from the same mRNA (12). The expression of these isoforms is developmentally and hormonally regulated (12–14).
The functional differences between the two activator iso-forms, LAP* and LAP, have been addressed in two examples (15, 16). The 21 amino acids of the N terminus region of LAP* are responsible for its functional interaction with NF-B and for recruiting to the SWI-SNF complex involved in chromatin remodeling. Both NF-B and C/EBP can activate the inflam-matory cytokine genes, and conversely, these cytokines can induce NF-B and C/EBP (17). These results suggest that the mechanisms for activating C/EBP and NF-B may converge on a post-translational level. There is a cysteine residue, Cys11,
located in the 21-amino acid stretch of the N-terminal region of LAP*. The possibility of Cys11involved in the tertiary structure
* This work was supported by National Science Council Grants NSC90-2321-B002-003 and NSC92-2320-B002-188 and by funds from the Institute of Biological Chemistry, Academia Sinica, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked
“adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed: Institute of Molec-ular Medicine, National Taiwan University, #7, Chung Shan South Rd., NTUH, Taipei 100, Taiwan. Tel.: 886-2-2356-2982; Fax: 886-2-2321-0977; E-mail: slee@ccms.ntu.edu.tw.
1The abbreviations used are: C/EBP, CCAAT/enhancer binding
pro-tein; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; PDTC, pyrrolidine dithiocarbamate; NAC, N-acetylcysteine; CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; HPLC, high pressure liquid chromatography; ChIP, chromatin immunoprecipi-tation; IGF, insulin-like growth factor; IGFBP, IGF-binding protein.
This paper is available on line at http://www.jbc.org
or redox sensing that results in the differential activation of LAP* and LAP deserves to be investigated.
The activities of transcription factors may be regulated in a redox-sensitive manner. LPS has been shown to induce changes in cellular redox states. The cellular levels of thiore-doxin, thioredoxin reductase, and NADPH, as well as the reac-tive oxygen species, may be elevated by LPS treatment (18 – 20). Redox-based transcriptional regulation has been studied extensively in prokaryotes (21–24) and demonstrated in eu-karyotic cells (25–31). Among the redox-sensing proteins, thi-oredoxin, thioredoxin reductase, and other related oxidoreduc-tases are involved in the regulation of activities of many key proteins in species ranging from bacteria to eukaryotic cells. REF-1 has been characterized as a factor that imparts strong DNA binding activity to the AP-1 complex by maintaining the cysteines as reduced sulfhydryl groups (32). The DNA binding activity of transcription factors can be affected by redox modi-fications (25, 27, 29, 33). Cysteine residues of several transcrip-tion factors, although not directly involved in DNA binding, modulate DNA binding activity in response to the cellular redox states (25–27, 33–37).
There are six, five, and two cysteines in LAP*, LAP, and LIP, respectively. To address the potential regulation of LAP* and LAP activity by LPS, we performed detailed biochemical and functional studies on the differential regulation of LAP* and LAP. Here we report that only LAP* is activated by LPS-induced redox switch. Upon reduction, LAP* is activated to up-regulate the transcription of IL-6 gene in P388D1(IL1) cells. We have determined that disulfide bonds formed in LAP* and LAP. A cysteine residue located in the N-terminal 21-amino acid region plays key roles in determining the tertiary structure and the redox-regulated activation of LAP*. The im-portance and the biological significance of this discovery are discussed.
EXPERIMENTAL PROCEDURES
Cell Culture and Transient Transfection—293T cells were cultured in
Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Hyclone). P388D1(IL1) cells were cultured in Iscove’s modified Dulbecco’s medium (Invitrogen) supplemented with 10% fetal calf serum and 2 mM L-glutamine (Invitrogen). Both cultures were supplemented with 100 units/ml penicillin and 100g/ml strep-tomycin. All nuclear extracts from 293T or P388D1(IL1) cells were fresh prepared with nitrogen gas. Transient transfection of 293T cells was performed with a calcium phosphate precipitation method. The calcium phosphate-plasmid DNA precipitate contained, in each well of a 6-well plate, 1g of AGP-CAT, 0.3 g of pSV--galactosidase, and various constructs of pCMV-C/EBP (detailed in the legend of figures). pCMV plasmid DNA was used to give a final amount of 2g of DNA for each transfection. 20 h post-transfection, the cultures were replaced with fresh medium and treated with either 10Mpyrrolidine dithiocarba-mate (PDTC) or 1 mMN-acetylcysteine (NAC) for 16 h. The cells were
harvested, and CAT activity was determined and normalized with -galactosidase activity. At least two independent, duplicate experi-ments were performed to each assay.
Plasmids—The AGP-CAT and pCMV-C/EBP plasmids were ob-tained as described elsewhere (38). FLAG-tagged LAP*, LAP, and LIP constructs were obtained by cloning the full-length and the respective N-terminal deletion constructs into pCMV-tag2 expression vector (Stratagene). Mutants of each of the six cysteine residues (i.e. Cys3 Ser) were generated with the M13mp18 site-directed mutagenesis system (Promega) using the following synthetic primers: C11S, 5 ⬘-AC-GCAGCAAGCCTCCCG-3⬘; C33S, 5⬘-AGCCCGACAGCCTGGAA-3⬘; C123S, 5⬘-CCGCCCGCAAGCTTTCCG-3⬘; C143S, CCGCGGACAGC-AAGCGC-3⬘; C201S, CCCGCCGCAAGCTTCGCG-3⬘; and C296S, 5⬘-CGGGCCACAGCTAGCGCGGCGCG-3⬘. The mutated nucleotides are underlined. The mutated cDNAs were then cloned into pCMV-tag vector.
Antibodies—18F8 monoclonal antibody was produced from
hybri-domas generated by fusing splenocytes from BALB/c mouse immu-nized with MHRLLAWDAACLPPPAAFPKLH with myelomas. 1H7 monoclonal antibody was obtained by immunizing BALB/c mice with
recombinant LIP. A16 and other antibodies were described previously (15).
Chromatin Immunoprecipitation Assays—Chromatin
immunopre-cipitation assays were modified from previously described methods (39). Briefly, P388D1(IL-1) cells were cross-linked with 1% formaldehyde for 10 min at 37 °C. The nuclei were isolated and sonicated into oligonu-cleosomes of⬃600 bp in length. The sheared chromatin was immuno-precipitated with 18F8 and 1H7 antibodies overnight, followed by 1 h of incubation with protein G-agarose, cross-linking reversal, and depro-teination. The presence of immunoprecipitated DNA was detected by PCR with the following primers: IL-6, sense, 5⬘-GCTTCTTAGGGC-TAGCCTCA-3⬘, and antisense, 5⬘-AGCTACAGACATCCCCAGTC-3⬘; TNF-␣, sense, 5⬘-TTCCGAGGGTTGAATGAGAGCT-3⬘, and antisense, 5⬘-TTTCTGTTCTCCCTCCTGGCTA-3⬘; and C/EBP, sense, 5⬘-GTAG-CTGGAGGAACGATC3⬘, and antisense, 5⬘-TCGGGAACACGGAGGAG-C-3⬘. PCR was performed for 25 cycles. The products were resolved by 2% agarose gels and visualized with ethidium bromide staining. UV-illuminated images were photographed and analyzed by AlphaImager 1220 (Alpha Innotech Corp.)
Northern Blot Analysis—P388D1(IL1) cells were cultured to 80%
confluent and stimulated with 1g/ml LPS (Escherichia coli 0111:B4; Sigma). The total RNA was extracted with TRIZOL reagent (Invitro-gen). Approximately 10g of total cellular RNA of each sample was separated by electrophoresis in a 1% agarose gel and analyzed by Northern blotting according to standard protocols. IL-6 probe was gen-erated by reverse transcription-PCR from P388D1(IL1) RNA with the following primers: IL-6 forward primer, 5⬘-ATGAAGTTCCTCTCTGC-AAGAG-3⬘, and IL-6 reverse primer, 5⬘-CTAGGTTTGCCGAGTAGAT-CTCA-3⬘. Full-length cDNA was used to probe C/EBP RNA as de-scribed previously (4).
Alkylation of Free Thiol Groups with Iodoacetamide in Vivo—The
alkylation method was performed as described previously (40). P388D1(IL1) cells were stimulated with 1g/ml LPS in the presence of 0.1 mMiodoacetamide (Sigma) to prevent free thiol groups from
oxidiz-ing to disulfide. Direct lysates were prepared at the indicated times with SDS sample dye containing 200 mMiodoacetamide at room
tem-perature. The lysates were then resolved by nonreducing SDS-PAGE followed by Western blot analysis.
Electrophoretic Mobility Shift Assay—The specific probe for
C/EBP was prepared by annealing the oligonucleotides 5⬘-GAT-CATTTTGTGTAAGAC-3⬘ and 5⬘-GATCGTCTTACACAAAAT-3⬘. The probe was labeled with T4 polynucleotide kinase and [␥-32P]ATP.
Nuclear extract (5–10g) from P388D1(IL1) cells or transfected 293T cells was incubated with the probe (1 ng) and 300 ng poly(dI-dC) in either the absence or presence of DTT, NADPH (Sigma), thioredoxin (Sigma), or the combination of NADPH, thioredoxin, and thioredoxin reductase (Sigma) at 37 °C for 20 min. For supershift assay, after the reaction mixture was incubated, 1–2l of anti-C/EBP antibody was added, and the incubation continued at room temperature for addi-tional 20 min. The reaction mixtures were separated by 5% PAGE in Tris/glycine/EDTA buffer.
Disulfide Bond Determination by Liquid Chromatography Electro-spray Ionization Mass Spectrometry —FLAG-C/EBP isoforms were
expressed in 293T cells, immunoprecipitated by M2 beads, and resolved in SDS-PAGE under nonreducing conditions. The in-gel tryptic digest of C/EBP was dissolved in 0.1% formic acid, separated with an ABI 140D HPLC (Perkin-Elmer, 150 ⫻ 0.5 mm Brownlee reversed phase C18 column packed with 5-m particles with a 300-Å pore), and on-line detected with a Finnigan Mat LCQ ion trap mass spectrometer. The mobile phase of HPLC consisted of various mixing ratios of 0.1% aque-ous formic acid (solution A) and 0.1% formic acid in acetonitrile (solu-tion B). The HPLC analysis was run using a gradient: 5% B for the first 25 min, 5–15% B in 25–30 min, 15–50% B in 30 –100 min, and 50 – 65% B in 100 –105 min; this final isocratic solvent was used until all of the peptides were eluted. The eluted peptides were analyzed under positive ion mass spectrometry scan mode over an m/z range of 395–1605. A table of detected masses was generated from the acquired mass spectra by the SEQUEST Browser software (Finnigan). The acquired ion masses were then manually examined for possible combinations of peptides containing cysteine residues.
RESULTS
LAP* Is Recruited to IL-6 Gene Promoter after LPS Treat-ment—Schematic representation of the C/EBP isoforms is
shown in Fig. 1A. There are six cysteine residues in the mouse LAP* (i.e. Cys11, Cys33, Cys123, Cys143, Cys201, and Cys296), five
in LAP (i.e. Cys33, Cys123, Cys143, Cys201, and Cys296), and two
FIG. 1. LAP* is specifically recruited to IL-6 promoter upon LPS stimulation. A, schematic representation of the C/EBP isoforms. The
associated bars indicate the antigen used to produce the three monoclonal antibodies (18F8, A16, and 1H7) and polyclonal antibody N21. Arrows mark the six highly conserved cysteine residues. TA, transactivation domain; bZIP, basic leucine zipper domain. B, the kinetics of promoter binding
in LIP (i.e. Cys201and Cys296). Monoclonal and polyclonal
an-tibodies that recognize only LAP* (i.e. 18F8 and N21), both LAP* and LAP (i.e. A16), as well as all three isoforms (i.e. 1H7) were generated (Fig. 1A, the regions recognized by these mono-clonal antibodies are indicated at the top of this figure). To investigate the differential recruitment of C/EBP isoforms to target gene promoters, 18F8 and 1H7 were employed for chro-matin immunoprecipitation (ChIP) experiments of IL-6 and C/EBP genes from LPS-treated P388D1(IL1) cells. Schematic representations of these target genes are shown in the top
panels of Fig. 1B. Semi-quantitative analysis of the amounts of
DNA immunoprecipitated by 18F8, 1H7, and anti-p50 antibody is shown in Fig. 1C. LAP* is selectively recruited to the IL-6 gene promoter only after LPS stimulation, whereas the binding of the sum of all three C/EBP isoforms as visualized by ChIP using 1H7 remains constant, independent of the time course of LPS treatment (Fig. 1B, 18F8 and 1H7 of left panels). The binding of LAP* to the promoter region of C/EBP is undetect-able as demonstrated by ChIP assay with 18F8, but the time course of 1H7 binding to the C/EBP promoter remains un-changed upon LPS treatment (Fig. 1B, 18F8 and 1H7 of right
panels).
NF-B, known as the predominant factor responsible for the induction of inflammatory cytokine genes (41), was also tested in ChIP experiment with anti-p50 antibody. Indeed, NF-B was also recruited to the IL-6 promoter, albeit with different kinetic pattern from that of LAP* (Fig. 1B,␣ p50 of left panels). The kinetics of LAP* recruitment to the IL-6 promoter corre-lates well with the expression of IL-6 mRNA (Fig. 1B, 18F8 and
IL-6 of left panels). C/EBP is constitutively expressed in
P388D1(IL1) cells (Fig. 1B, C/EBP of right panels and data not shown). Neither LAP* nor NF-B was recruited to the C/EBP promoter upon LPS treatment of P388D1(IL1) cells (Fig. 1B, 18F8 and␣ p50 of right panels). Taken together, these results suggest that LAP* may play key roles for LPS-induced IL-6 expression but not in the constitutive expression of C/EBP gene. In addition to, or in conjunction with LAP*, NF-B could also play important roles in LPS-mediated IL-6 gene expression.
LAP* Activation Correlates with the Reduction of Intra-molecular Disulfide Bonds—We next aimed to examine the
potential mechanisms that govern the LPS-induced recruit-ment of LAP* to the IL-6 gene promoter. A number of different pathways have been described to activate C/EBP, including transcriptional up-regulation, post-translational modifications, and nuclear translocation (42). The Northern data in Fig. 1B (C/EBP panel) exclude the possibility of transcriptional up-regulation in the present assay system. Immunological analy-ses demonstrated that both protein level and subcellular local-ization of C/EBP isoforms remain unchanged regardless of whether the P388D1(IL1) cells were treated with LPS or not (data not shown). Because LPS treatment is associated with alterations of the redox state, we hypothesized that LAP* could be regulated by a redox switch. Interestingly, amino acid align-ment shows that the cysteine residues in C/EBP are highly conserved among species (Fig. 2A). Moreover, an extra cysteine residue is located in the N-terminal 21-amino acid stretch that is unique to LAP* (Fig. 2A). To assess whether these cysteine residues were involved in disulfide bond rearrangements dur-ing LPS treatment, we performed Western blot analysis to LPS-treated P388D1(IL1) under nonreducing conditions.
Io-doacetamide was used to trap the free thiol groups during LPS treatment. At least two species of LAP* displaying slower mo-bility were observed 1 h after LPS treatment (Fig. 2B), sug-gesting the differential reduction of intramolecular disulfide bonds. The kinetics of these mobility changes in LAP* also correlates with the ChIP pattern of 18F8 antibody and the induction of IL-6 gene depicted in Fig. 1B. However, when the same protein preparations were subjected to a treatment with the reducing agent DTT, no mobility change could be observed (Fig. 2B, upper panel). On the contrary, LAP does not show a similar change of mobility under nonreducing conditions (Fig. 2B, bottom panel). These observations suggest that endogenous LAP* exists in a more compact conformation, which is relaxed by disulfide bond reduction upon LPS stimulation. To further confirm this finding, we treated the nuclear extract of P388D1(IL1) cells with various concentrations of DTT and an-alyzed by Western blot under nonreducing conditions. As shown in Fig. 2C, the N21 antibody detected three different migration forms of LAP* (upper panel). A slightly slower mi-gration form was also observed for LAP from the same extracts (Fig. 2C, lower panel), whereas no mobility change was de-tected in LIP (data not shown). The same migration behavior was also observed for FLAG-tagged recombinant C/EBPs ex-pressed in 293T cells (Fig. 2D).
Identification of the Intramolecular Disulfide Bonds in LAP* and LAP—To determine which cysteine residues are involved
in the intramolecular disulfide bond formation in LAP* and LAP, we performed mass spectrometric analysis. FLAG-LAP* was digested with trypsin, and the resulting peptides were analyzed by mass spectrometer. Disulfide bonds between Cys11
and Cys33, between Cys123and Cys143, and between Cys201and
Cys296of LAP* were identified (Fig. 3A). When FLAG-LAP was
subjected to a similar analysis, only Cys123 and Cys201 were
found to form intramolecular disulfide bond. This result is consistent with the barely detectable mobility shift of endoge-nous LAP shown in Fig. 2B. The major determinant in disulfide bond formation between LAP* and LAP hinges on the Cys11
residue of LAP*. To assess the importance of this residue in the overall disulfide bond formation, we performed site-directed mutagenesis that substituted the cysteine with a serine resi-due. Analysis of this mutant FLAG-LAP* (C11S) reveals that only Cys123and Cys201, and not Cys11and Cys33nor Cys201and
Cys296, were linked by disulfide bond formation. When Cys11is
substituted, the tertiary structure of FLAG-LAP*(C11S) is apparently similar to that of LAP. To correlate these results, we expressed each mutant of the cysteine residues of LAP* and examined their migration pattern under nonreducing SDS-PAGE. As shown in Fig. 3B, all of the mutants migrate more slowly than the wild type protein. The mobility of C123S, C143S, C201S, and C296S was somewhat diffused as compared with that of C11S and C33S. The mobility of C11S and C33S was similar, in accordance to their partnership in disulfide bond formation. Taken together, these data provide evidence that all of the cysteine residues in LAP* are linked by disulfide bond formation. Cys11 plays a central role in
determining the tertiary structure of LAP*. If the disulfide bond between Cys11and Cys33 is disrupted by point
muta-tion, LAP* assumes a conformation that is similar to that of LAP.
Transcriptional Activity of LAP* Is Greatly Enhanced under Reducing Conditions—Having demonstrated that LAP*
under-and the expression profiles of C/EBP target genes. Transcription initiation site on IL-6 and C/EBP gene promoters (arrows) and the PCR fragments amplified (line drawings) are indicated. ChIP experiments were performed with 18F8, 1H7, and anti-NFB p50 antibodies from P388D1(IL1) cells at the indicated times after LPS stimulation (1g/ml). Steady-state RNA levels are monitored by Northern blotting, GAPDH serves as a loading control (boxed panels). C, semi-quantitative analysis of the ChIP results are represented as the mean intensity over the fixed area normalized against background values.
goes reduction of disulfide linkages and activation upon LPS stimulation, we then evaluated the effect of reducing power on the transcription activity of LAP*. The expression vectors of LAP* and LAP were transfected into 293T cells together with AGP-CAT reporter. Consistent with our previous results pub-lished elsewhere, LAP is more active than LAP* when tested by transient transfection experiments (15). When LAP* transfec-tants were treated with the reducing agents PDTC or NAC, the activation of reporter gene was enhanced in a PDTC and NAC concentration-dependent manner. These stimulatory effects were not observed for LAP transfectants (Fig. 4, A, PDTC, and
B, NAC). These reducing agents may exert their effect by
directly reducing the disulfide bonds in LAP*. To explore this possibility, we assayed the transcription activity of the cysteine mutants, which mimic the reduction of their respective disul-fide bonds. For C11S and C33S, the transactivation of AGP-CAT was greatly enhanced as compared with that of wild type LAP* and other cysteine mutants (Fig. 4C). Because the results shown in Fig. 3 implicate that the C11S mutant (and possibly C33S as well) adopt the same disulfide linkage as in LAP, the most reasonable explanation for these observations is that PDTC or NAC also promotes the disengagement of disulfide linkage between Cys11and Cys33, rendering LAP* to acquire
the disulfide pairing as in LAP, which is the most transcrip-tionally active isoform.
Reducing Conditions Enhance the DNA Binding Activity of LAP*—To determine the mechanism by which change in redox
state influences the transcription activity of LAP*, we per-formed electrophoretic mobility shift assays. The DNA binding activity of FLAG-LAP*, but not FLAG-LAP and FLAG-LIP, was enhanced by the addition of DTT (Fig. 5A, compare lanes 3 and 6 for LAP*, lanes 4 and 7 for LAP, and lanes 5 and 8 for LIP). Similarly, the DNA binding activity of endogenous LAP* was detected only in the DTT-treated extracts, as demon-strated by the antibody-specific supershifts (Fig. 5B, compare
lanes 3, 4, 9, and 10; asterisks mark the supershifted
com-plexes). The DNA binding activity of LAP did not appear to be influenced by the presence of DTT (Fig. 5B, compare lanes 5, 6,
11, and 12; supershifted complex detected by A16). One could
argue that DTT may not reflect in vivo conditions. To explore the impact of endogenous redox-sensing systems on LAP*, we tested the effects of NADPH, thioredoxin, and the combination of NADPH, thioredoxin, and thioredoxin reductase in electro-phoretic mobility shift assays. FLAG-LAP* was purified with M2 beads to avoid the consumption of reducing agents from other proteins. As shown in Fig. 5C, DTT (lanes 2 and 3) and
FIG. 2. LPS induces reduction of disulfide bonds in LAP*. A, amino acid sequence alignment of C/EBP from various species. The conserved cysteines are shown in bold type. B, LAP* displays redox-sensitive mobility changes during LPS stimulation. P388D1(IL1) cells were treated with 1g/ml LPS in the presence of 0.1 mMiodoacetamide. The cell lysates harvested at the indicated time were subjected to reducing and nonreducing (boxed panels) SDS-PAGE and visualized by Western blot with the N21 (LAP*) and A16 (LAP) antibodies. C and D, presence of intramolecular disulfide bonds in endogenous and recombinant LAP*. Nuclear extracts from P388D1(IL1) cells (C) and C/EBP-transfected 293T cells (D) were prepared with buffers devoid of DTT. Nuclear extract was incubated with various concentrations of DTT at 37 °C for 20 min. The reaction mixtures were subjected to nonreducing SDS-PAGE and visualized as in B.
thioredoxin (lanes 6 and 7) were effective in enhancing the DNA binding activity of LAP*. The reducing effect of thiore-doxin alone seemed less effective than that of DTT or the combination of thioredoxin, thioredoxin reductase, and
NADPH. This may be attributed to the decreased reducing power by partially oxidized form of thioredoxin. NADPH has an almost undetectable effect (Fig. 5C, lanes 4 and 5). When LAP* was treated with thioredoxin, NADPH, and thioredoxin
reduc-FIG. 4. The transcriptional activity of LAP*, but not LAP, is enhanced under reducing conditions. A and B, treatment with antioxidants stimulates the transactivity of LAP*. 293 T cells were transfected with 1g of AGP-CAT, 0.5 g of pSV--galactosidase, and 50 ng of pCMV-tag-LAP* or pCMV-tag-LAP. The transfected cells were treated with various concentrations of PDTC (A) and NAC (B) for 16 h before harvesting for CAT assays. The CAT activity was normalized with-galactosidase. C, transcriptional activity of LAP* and each cysteine mutants were measured by CAT assay. 293T cells were co-transfected with 0.75g of AGP-CAT, 0.25 g of pSV--galactosidase, and 0.02 g of each corresponding LAP* construct. The CAT assays were performed 36 h post-transfection. Each normalized CAT value represents the means of four independent experiments.
FIG. 3. Mapping of intramolecular
disulfide bond(s) in C/EBP. A,
sche-matic representation of the identified in-tramolecular disulfide bonds of C/EBP isoforms. FLAG-tagged C/EBPs were ex-pressed in 293T cells, immunoaffinity-pu-rified with M2 beads, and recovered from SDS-PAGE. The peptides generated by trypsin digestion were analyzed with LCQ (Finnigan) and MALDI-TOF. B, wild type (WT) and mutant LAP*s migrate dif-ferently in nonreducing SDS-PAGE. FLAG-LAP* and its cysteine mutants (Cys3 Ser) were expressed in 293T cells. Nuclear extract prepared with buffer de-void of DTT was subjected to SDS-PAGE and visualized by Western blot with M2 antibody.
tase, its DNA binding activity was comparable with that of DTT-treated samples (Fig 5C, lanes 8 and 9). Taken together, our findings provide strong evidence that the DNA binding activity of LAP* may be regulated in a redox-sensitive manner. The thioredoxin and thioredoxin reductase are among the can-didate players in the activation of LAP*. In contrast, LAP and LIP are insensitive to the change in redox condition.
DISCUSSION
C/EBP is one of the key players in the regulation of growth, differentiation, metabolism, and inflammation. Understanding the mechanism of differential regulation of C/EBP is an impor-tant issue for basic and applied biological research. Although functional differences have been described between LAP* and
LAP (15, 16), how they are separately regulated remains com-pletely unknown. In the present study, we report for the first time that upon LPS stimulation, the full-length isoform LAP* is differentially activated by a redox switch mechanism, and conse-quently, LAP* gains DNA binding activity and is recruited to the IL-6 promoter for its transcriptional induction.
Because numerous studies revealed that C/EBP regulation is extremely complicated and cell type-specific (42), we focused our studies in the P388D1(L1) cells, in which both inducible (IL-6) as well as constitutive regulation (C/EBP itself) by C/EBP are well documented. The observation that although a bulk of C/EBP is permanently associated with both target promoters, the induction of IL-6 does not occur until the re-cruitment of LAP* strongly suggests that LAP* is the determi-nant isoform in LPS-inducible IL-6 expression. On the other hand, LAP* does not seem to be involved in the regulation of constitutively expressed genes, as exemplified by C/EBP it-self. This is in pretty good agreement with our previous report that LAP* preferentially interacts with NF-B in a synergistic manner, whereas LAP does not. NF-B is known to be a crucial factor in the LPS-inducible expression of inflammatory cyto-kine genes (41). Our ChIP data confirmed its initial recruit-ment to the IL-6 promoter simultaneously with LAP*; never-theless, NF-B binding persisted long after decreased IL-6 mRNA production. This difference in promoter binding kinetics prompted us to examine another LPS-inducible gene, TNF-␣, for which NF-B is considered as the determinant factor of its expression. We found that both C/EBP and NF-B were re-cruited to the TNF-␣ promoter with the same kinetics (data not shown). A possible implication of these results is that although the activation of C/EBP and NF-B are both necessary for the induction of LPS-up-regulated genes, they may differ in play-ing the triggerplay-ing roles for specific genes. In light of the perfect correlation between IL-6 transcription and LAP* recruitment to its promoter, our data strongly suggest that LAP* may play the switch roles for IL-6 transcription, whereas NF-B plays the key role for the induction of TNF-␣ gene.
A previous report described the association of LAP* with the SWI-SNF complex through the 21-amino acid stretch in the N terminus (16). It is then speculated that LAP* may be involved in the activation of those target genes requiring chromatin remodeling activity provided by the SWI-SNF complex. The roles of LAP* on chromatin remodeling of the IL-6 and C/EBP genes would be crucial for further investigation. Moreover, our biochemical studies demonstrated a novel function for the extra amino acids at the N terminus of LAP*. Specifically, Cys11is
clearly involved or affected in the intramolecular disulfide bond formation of LAP*. When cDNA of C/EBP is transfected into cells, proteins of all three isoforms are produced. To obtain any single species of C/EBP isoform, FLAG-tagged cDNA is used for generating recombinant protein by transient transfection into host cells. When we performed SDS-PAGE and Western blot analysis under nonreducing and reducing conditions using P388D1(IL1)-derived and FLAG-tagged proteins, the mobility of endogenous-derived and recombinant proteins are similar. This result suggests that the intramolecular disulfide bond formation for endogenous and recombinant proteins is likely to be the same. Mass spectra analysis indicates that Cys11and
Cys33, Cys123and Cys143, and Cys201and Cys296of LAP* form
three intramolecular disulfide bonds. LAP, lacking the N-ter-minal 21 amino acids and thus Cys11, is different from LAP* in
tertiary structure. The intramolecular disulfide bond of LAP is between Cys123and Cys201. The intramolecular disulfide bond
of LAP*(C11S) is the same as that of LAP. These results indi-cate that Cys11of LAP* is key to the tertiary structure of LAP*.
The Cys11and Cys33disulfide bond could affect the function of
FIG. 5. The DNA binding activity of LAP* is enhanced under
reducing conditions. A, nuclear extracts (5g) from 293T cells
ex-pressing various recombinant C/EBP isoforms were incubated in the absence (lanes 1–5) or presence (lanes 6 – 8) of DTT, reacted with [␣-32P]ATP-labeled probe, and then separated by 5% native PAGE. Lane 1, probe only; lane 2, nuclear extract from mock transfected 293T
cells; lanes 3 and 6, LAP*; lanes 4 and 7, LAP; lanes 5 and 8, LIP. B, 10 g of nuclear extract from P388D1(IL1) cells were treated in the ab-sence or preab-sence of DTT. The reaction mixtures were then incubated with labeled probe in the absence or presence of antibodies specific to C/EBP isoforms. The asterisks indicate the supershifted complexes. Two different dosages of antibodies were used: 1l (⫹) and 2 l (⫹⫹).
C, recombinant FLAG-LAP* was purified from 293T transfected cells
by immunoprecipitation and eluted with FLAG peptide. The purified FLAG-LAP* was incubated with various reducing agents and further reacted with probe. Lane 1, FLAG-LAP* only; lanes 2 and 3, 0.2 and 2 mMof DTT; lanes 4 and 5, 0.2 and 2 mMNADPH; lanes 6 and 7, 10 and 20Mthioredoxin (Txn); lanes 8 and 9, thioredoxin reductase (TxR) and NADPH and 10 and 20MTxn were added to the reaction mixtures.
the transactivation domain of LAP*. Maybe when Cys11gets
reduced under redox switch, the conformation of LAP* changes to facilitate its association with the SWI-SNF complex and hence turning on target gene expression (16). Therefore, the activities of LAP* and LAP are likely to be different, and LAP* may be the primary factor among C/EBP isoforms that confers the redox-sensitive transactivation of target genes.
Closely related family member proteins were known to dis-play different activities because of the difference in their in-tramolecular disulfide bonds (43). For example, insulin-like growth factor (IGF)-binding proteins (IGFBPs 1– 6) are respon-sible for modulating the action of IGFs. IGFBPs 1–5 contain 18 conserved cysteines, but IGFBP-6 lacks two of the 12 N-termi-nal cysteines. The N-termiN-termi-nal intramolecular disulfide link-ages of IGFBP-6 differ significantly from those of IGFBP-1 as determined by mass spectrometry. This difference could con-tribute to the distinctive IGF binding properties of IGFBP-6 (which has the highest affinity for IGF-II). Our present results on the difference of intramolecular disulfide bonds between LAP* and LAP are analogous to those of IGFBPs.
How does LAP* get reduced in LPS-treated P388D1(IL1) cells? The more likely physiological scenario is through oxi-doreductases (e.g. thioredoxin and thioredoxin reductase). There is a report on the elevation of thioredoxin and thiore-doxin reductase levels in placenta of pregnant mice exposed to LPS (18). The LPS-treated P388D1(IL1) cells may generate high levels of thioredoxin, thioredoxin reductase, and NADPH and thus facilitate the activation of LAP*. Alternatively, oxi-doreductase such as protein disulfide isomerase may be acti-vated under LPS-stimulated conditions and may then activate LAP*. Our results demonstrated that LAP* gets reduced when P388D1(IL1) cells are treated with LPS (Fig. 2B). These results suggest that post-translational events, such as the reduction of disulfide bonds are responsible for the activation of LAP*. Consistent with the ChIP results, transient transfection exper-iments using wild type LAP* also demonstrated that LAP*, not LAP, is specifically activated by PDTC or NAC treatment. When treated with 50M PDTC, the transcription activity of LAP* is higher than that of LAP. The transactivation activity of LAP*(C11S) or LAP*(C33S) is much higher than that of wild type LAP*, suggesting that the reduction of intramolecular disulfide bond between Cys11and Cys33of LAP* may be
im-portant for its activation. The intramolecular disulfide bond between Cys201and Cys296may pose a constraint for
dimeriza-tion of LAP*, which must be reduced or disrupted preceding DNA binding. Alternatively, Cys11or Cys33may influence the
overall tertiary structure and conformation during protein translation. That is why when Cys11is mutated, the
intramo-lecular disulfide bond of LAP*(C11S) is the same as LAP. The disulfide bond between Cys123and Cys201may be buried in the
molecule based on the results of our molecular modeling (data not shown). The DNA binding activity of LAP* is activated by reducing agents, whereas LAP is constitutively active. In ad-dition to playing roles of direct disulfide bond formation, Cys11
could influence the folding of LAP* during translation. Muta-tion of cysteine residues other than Cys11and Cys33could have
very different effects on intramolecular disulfide bond forma-tion and folding of LAP*. The results from transient transfec-tion experiments performed with various cysteine mutants clearly define that Cys11and Cys33are functionally different
from other cysteines (Fig. 5C). In addition to dimerization and DNA binding, domain(s) located near the N-terminal region is important for the transactivation of LAP*. Cys11 and Cys33
could influence the disulfide bonds formation mediated by the other four cysteines; however, the other mutants of cysteines (i.e. Cys123, Cys143, Cys201, and Cys296) may or may not
influ-ence the disulfide bond formation between Cys11 and Cys33.
These possibilities are to be addressed in future experiments. Any results derived from the mutation of cysteine must be interpreted with caution. It should also be noted that, because resident genes are embedded in chromatin but the ectopically transfected plasmids are not, the activities of LAP* defined by transient transfection and those derived from the ChIP for are likely to be different.
The results from this study suggest that LAP* may function as a cellular sensor for the sudden surge of reducing potential that is required for switching the key regulatory genes as exemplified by the induction of IL-6 gene. LAP, on the other hand, does not respond to the reducing power but may regulate the transcription of genes with house keeping functions. Taken together, these results demonstrate that redox switch is re-sponsible for the activation of LAP*, but not LAP, for targeting genes encoding key regulatory proteins. This novel type of molecular switch for differential regulation of two transcrip-tional activators resulted from alternative translation of the same mRNA, may be found in other systems, and should have general interest in studying gene regulation.
Acknowledgments—We thank Li-Ping Tu, Ruey Chao, and Han Ting
Chou for excellent technical assistance.
REFERENCES
1. Lekstrom-Himes, J., and Xanthopoulos, K. G. (1998) J. Biol. Chem. 273, 28545–28548
2. Poli, V. (1998) J. Biol. Chem. 273, 29279 –29282
3. Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T. (1990) EMBO J. 9, 1897–1906 4. Chang, C. J., Chen, T. T., Lei, H. Y., Chen, D. S., and Lee, S. C. (1990) Mol.
Cell. Biol. 10, 6642– 6653
5. Pope, R. M., Leutz, A., and Ness, S. A. (1994) J. Clin. Invest. 94, 1449 –1455 6. Zhang, Y., and Rom, W. N. (1993) Mol. Cell. Biol. 13, 3831–3837
7. Shirakawa, F., Saito, K., Bonagura, C. A., Galson, D. L., Fenton, M. J., Webb, A. C., and Auron, P. E. (1993) Mol. Cell. Biol. 13, 1332–1344
8. Bretz, J. D., Williams, S. C., Baer, M., Johnson, P. F., and Schwartz, R. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7306 –7310
9. Hu, H. M., Tian, Q., Baer, M., Spooner, C. J., Williams, S. C., Johnson, P. F., and Schwartz, R. C. (2000) J. Biol. Chem. 275, 16373–16381
10. Pope, R., Mungre, S., Liu, H., and Thimmapaya, B. (2000) Cytokine 12, 1171–1181
11. Williams, S. C., Baer, M., Dillner, A. J., and Johnson, P. F. (1995) EMBO J. 14, 3170 –3183
12. Descombes, P., and Schibler, U. (1991) Cell 67, 569 –579
13. Diehl, A. M., Michaelson, P., and Yang, S. Q. (1994) Gastroenterology 106, 1625–1637
14. Raught, B., Liao, W. S., and Rosen, J. M. (1995) Mol. Endocrinol. 9, 1223–1232 15. Lee, Y. M., Miau, L. H., Chang, C. J., and Lee, S. C. (1996) Mol. Cell. Biol. 16,
4257– 4263
16. Kowenz-Leutz, E., and Leutz, A. (1999) Mol. Cell 4, 735–743
17. Matsusaka, T., Fujikawa, K., Nishio, Y., Mukaida, N., Matsushima, K., Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10193–10197
18. Ejima, K., Koji, T., Nanri, H., Kashimura, M., and Ikeda, M. (1999) Placenta 20, 561–566
19. Rosenspire, A. J., Kindzelskii, A. L., and Petty, H. R. (2002) J. Immunol. 169, 5396 –5400
20. Bochkov, V. N., Kadl, A., Huber, J., Gruber, F., Binder, B. R., and Leitinger, N. (2002) Nature 419, 77– 81
21. Bauer, C. E., Elsen, S., and Bird, T. H. (1999) Annu. Rev. Microbiol. 53, 495–523
22. Pomposiello, P. J., and Demple, B. (2001) Trends Biotechnol. 19, 109 –114 23. Carmel-Harel, O., and Storz, G. (2000) Annu. Rev. Microbiol. 54, 439 – 461 24. Zheng, M., and Storz, G. (2000) Biochem. Pharmacol. 59, 1– 6
25. Abate, C., Patel, L., Rauscher, F. J., III, and Curran, T. (1990) Science 249, 1157–1161
26. Pognonec, P., Kato, H., and Roeder, R. G. (1992) J. Biol. Chem. 267, 24563–24567
27. Hayashi, T., Ueno, Y., and Okamoto T. (1993) J. Biol. Chem. 268, 11380 –11388
28. Markus, M., and Benezra, R. (1999) J. Biol. Chem. 274, 1040 –1049 29. Nakshatri, H., Bhat-Nakshatri, P., and Currie, R. A. (1996) J. Biol. Chem. 271,
28784 –28791
30. Grant, C. M. (2001) Mol. Microbiol. 39, 533–541
31. Toone, W. M., Morgan, B. A., and Jones, N. (2001) Oncogene 20, 2336 –2346 32. Xanthoudakis, S., and Curran, T. (1992) EMBO J. 11, 653– 665
33. Myrset, A. H., Bostad, A., Jamin, N., Lirsac, P. N., Toma, F., and Gabrielsen, O. S. (1993) EMBO J. 12, 4625– 4633
34. Storz, G., Tartaglia, L. A., Farr, S. B., and Ames, B. N. (1990) Trends Genet. 6, 363–368
35. Guehmann, S., Vorbrueggen, G., Kalkbrenner, F., and Moelling, K. (1992)
Nucleic Acids Res. 20, 2279 –2286
36. Matthews, J. R., Wakasugi, N., Virelizier, J. L., Yodoi, J., and Hay, R. T. (1992)
Nucleic Acids Res. 20, 3821–3830
37. McBride, A. A., Klausner, R. D., and Howley, P. M. (1992) Proc. Natl. Acad.
Sci. U. S. A. 89, 7531–7535
38. Lee, Y. M., Tsai, W. H., Lai, M. Y., Chen, D. S., and Lee, S. C. (1993) Mol. Cell.
Biol. 13, 432– 442
39. Duong, D. T., Waltner-Laws, M. E., Sears, R., Sealy, L., and Granner, D. K.
(2002) J. Biol. Chem. 277, 32234 –32242
40. Mancini, R., Fagioli, C., Fra, A. M., Maggioni, C., and Sitia, R. (2000) FASEB
J. 14, 769 –778
41. Sweet, M. J., and Hume, D. A. (1996) J. Leukocyte Biol. 60, 8 –26
42. Bradley, M. N., Zhou, L., and Smale, S. T. (2003) Mol. Cell. Biol. 23, 4841– 4858
