C/EBPβ, 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 myeloid cell differentiation, and the acute phase response. Three C/EBPβ isoforms (i.e. LAP*, LAP, and LIP) were known to arise from differential translation initiation and display different functions in gene regulation. C/EBPβ is known to induce interleukin (IL)-6 gene when P388D1 cells are treated with lipopolysaccharide (LPS). Exactly how the transcriptional activities of C/EBPβ isoforms are involved in the regulation of the IL-6 gene remains unclear. Here we report that LPS-induced expression of IL-6 gene in P388D1 cells is mediated by a redox switch-activated LAP*. The intramolecular disulfide 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 intramolecular disulfide bonds that form the basis for redox switch regulation. The DNA binding activity and transcriptional activity of LAP* are enhanced under reducing 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/EBPβ that regulates, through a redox switch, the LPS-induced expression of the IL-6 gene. C/EBPβ, 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 myeloid cell differentiation, and the acute phase response. Three C/EBPβ isoforms (i.e. LAP*, LAP, and LIP) were known to arise from differential translation initiation and display different functions in gene regulation. C/EBPβ is known to induce interleukin (IL)-6 gene when P388D1 cells are treated with lipopolysaccharide (LPS). Exactly how the transcriptional activities of C/EBPβ isoforms are involved in the regulation of the IL-6 gene remains unclear. Here we report that LPS-induced expression of IL-6 gene in P388D1 cells is mediated by a redox switch-activated LAP*. The intramolecular disulfide 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 intramolecular disulfide bonds that form the basis for redox switch regulation. The DNA binding activity and transcriptional activity of LAP* are enhanced under reducing 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/EBPβ that regulates, through a redox switch, the LPS-induced expression of the IL-6 gene. Members of the C/EBP 1The abbreviations used are: C/EBPCCAAT/enhancer binding proteinLPSlipopolysaccharideILinterleukinTNFtumor necrosis factorPDTCpyrrolidine dithiocarbamateNACN-acetylcysteineCATchloramphenicol acetyltransferaseDTTdithiothreitolHPLChigh pressure liquid chromatographyChIPchromatin immunoprecipitationIGFinsulin-like growth factorIGFBPIGF-binding protein. family of proteins have been implicated in the differentiation of myelomonocytic cells and the regulation of gene expression during activation of macrophages (1Lekstrom-Himes J. Xanthopoulos K.G. J. Biol. Chem. 1998; 273: 28545-28548Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 2Poli V. J. Biol. 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The promoter regions of the genes for IL-6, TNFα, IL-1, IL-8, and granulocyte colony-stimulating factor contain C/EBPβ-binding sites (3Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar, 5Pope R.M. Leutz A. Ness S.A. J. Clin. Invest. 1994; 94: 1449-1455Crossref PubMed Scopus (166) Google Scholar, 6Zhang Y. Rom W.N. Mol. Cell. Biol. 1993; 13: 3831-3837Crossref PubMed Google Scholar, 7Shirakawa F. Saito K. Bonagura C.A. Galson D.L. Fenton M.J. Webb A.C. Auron P.E. Mol. Cell. Biol. 1993; 13: 1332-1344Crossref PubMed Google Scholar). The stable expression of C/EBPβ in a murine P388 lymphoblast cell line is sufficient to confer LPS-inducible IL-6 expression (8Bretz J.D. Williams S.C. Baer M. Johnson P.F. Schwartz R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7306-7310Crossref PubMed Scopus (75) Google Scholar, 9Hu H.M. Tian Q. Baer M. Spooner C.J. Williams S.C. Johnson P.F. Schwartz R.C. J. Biol. Chem. 2000; 275: 16373-16381Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Conversely, inhibition of endogenous C/EBPβ expression by antisense RNA interference blocks LPS induction of IL-6 expression in the murine P388D1(IL1) macrophage-like cells (8Bretz J.D. Williams S.C. Baer M. Johnson P.F. Schwartz R.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7306-7310Crossref PubMed Scopus (75) Google Scholar). C/EBPβ can also activate the proximal promoter of the human TNF-α gene in RAW264.7 monocytic cells, whereas overexpression of a dominant negative C/EBPβ inhibits LPS-induced activation (10Pope R. Mungre S. Liu H. Thimmapaya B. Cytokine. 2000; 12: 1171-1181Crossref PubMed Scopus (38) Google Scholar). In the murine P388D1(IL1) cell line, C/EBPβ is constitutively expressed, and IL-6 and TNF-α can readily be induced by agents such as LPS. The exact molecular mechanisms of LPS stimulation of IL-6 and TNF-α gene expression and how this relates to C/EBPβ-mediated activation of these genes remain unclear. CCAAT/enhancer binding protein lipopolysaccharide interleukin tumor necrosis factor pyrrolidine dithiocarbamate N-acetylcysteine chloramphenicol acetyltransferase dithiothreitol high pressure liquid chromatography chromatin immunoprecipitation insulin-like growth factor IGF-binding protein. Structurally, members of C/EBP family are bZIP proteins with sequence-specific DNA binding activity when homo- or heterodimerized. The DNA-binding and transactivation domains are located in the C- and N-terminal regions, respectively (11Williams S.C. Baer M. Dillner A.J. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar). The intronless c/ebpβ 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/EBPβ are differentially translated from the same mRNA (12Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar). The expression of these isoforms is developmentally and hormonally regulated (12Descombes P. Schibler U. Cell. 1991; 67: 569-579Abstract Full Text PDF PubMed Scopus (862) Google Scholar, 13Diehl A.M. Michaelson P. Yang S.Q. Gastroenterology. 1994; 106: 1625-1637Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 14Raught B. Liao W.S. Rosen J.M. Mol. Endocrinol. 1995; 9: 1223-1232PubMed Google Scholar). The functional differences between the two activator isoforms, LAP* and LAP, have been addressed in two examples (15Lee Y.M. Miau L.H. Chang C.J. Lee S.C. Mol. Cell. Biol. 1996; 16: 4257-4263Crossref PubMed Google Scholar, 16Kowenz-Leutz E. Leutz A. Mol. Cell. 1999; 4: 735-743Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). The 21 amino acids of the N terminus region of LAP* are responsible for its functional interaction with NF-κB and for recruiting to the SWI-SNF complex involved in chromatin remodeling. Both NF-κB and C/EBPβ can activate the inflammatory cytokine genes, and conversely, these cytokines can induce NF-κB and C/EBPβ (17Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10193-10197Crossref PubMed Scopus (880) Google Scholar). These results suggest that the mechanisms for activating C/EBPβ and NF-κB 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 Cys11 involved in the tertiary structure 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 thioredoxin, thioredoxin reductase, and NADPH, as well as the reactive oxygen species, may be elevated by LPS treatment (18Ejima K. Koji T. Nanri H. Kashimura M. Ikeda M. Placenta. 1999; 20: 561-566Crossref PubMed Scopus (39) Google Scholar, 19Rosenspire A.J. Kindzelskii A.L. Petty H.R. J. Immunol. 2002; 169: 5396-5400Crossref PubMed Scopus (44) Google Scholar, 20Bochkov V.N. Kadl A. Huber J. Gruber F. Binder B.R. Leitinger N. Nature. 2002; 419: 77-81Crossref PubMed Scopus (328) Google Scholar). Redox-based transcriptional regulation has been studied extensively in prokaryotes (21Bauer C.E. Elsen S. Bird T.H. Annu. Rev. 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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 importance and the biological significance of this discovery are discussed. 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 mml-glutamine (Invitrogen). Both cultures were supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin. 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, 1 μg of AGP-CAT, 0.3 μg of pSV-β-galactosidase, and various constructs of pCMV-C/EBPβ (detailed in the legend of figures). pCMV plasmid DNA was used to give a final amount of 2 μg of DNA for each transfection. 20 h post-transfection, the cultures were replaced with fresh medium and treated with either 10 μm pyrrolidine dithiocarbamate (PDTC) or 1 mmN-acetylcysteine (NAC) for 16 h. The cells were harvested, and CAT activity was determined and normalized with β-galactosidase activity. At least two independent, duplicate experiments were performed to each assay. Plasmids—The AGP-CAT and pCMV-C/EBPβ plasmids were obtained as described elsewhere (38Lee Y.M. Tsai W.H. Lai M.Y. Chen D.S. Lee S.C. Mol. Cell. Biol. 1993; 13: 432-442Crossref PubMed Scopus (28) Google Scholar). 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. Cys → Ser) were generated with the M13mp18 site-directed mutagenesis system (Promega) using the following synthetic primers: C11S, 5′-ACGCAGCAAGCCTCCCG-3′; C33S, 5′-AGCCCGACAGCCTGGAA-3′; C123S, 5′-CCGCCCGCAAGCTTTCCG-3′; C143S, 5′-CCGCGGACAGCAAGCGC-3′; C201S, 5′-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 hybridomas generated by fusing splenocytes from BALB/c mouse immunized with MHRLLAWDAACLPPPAAFPKLH with myelomas. 1H7 monoclonal antibody was obtained by immunizing BALB/c mice with recombinant LIP. A16 and other antibodies were described previously (15Lee Y.M. Miau L.H. Chang C.J. Lee S.C. Mol. Cell. Biol. 1996; 16: 4257-4263Crossref PubMed Google Scholar). Chromatin Immunoprecipitation Assays—Chromatin immunoprecipitation assays were modified from previously described methods (39Duong D.T. Waltner-Laws M.E. Sears R. Sealy L. Granner D.K. J. Biol. Chem. 2002; 277: 32234-32242Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Briefly, P388D1(IL-1) cells were cross-linked with 1% formaldehyde for 10 min at 37 °C. The nuclei were isolated and sonicated into oligonucleosomes of ∼600 bp in length. The sheared chromatin was immunoprecipitated with 18F8 and 1H7 antibodies overnight, followed by 1 h of incubation with protein G-agarose, cross-linking reversal, and deproteination. The presence of immunoprecipitated DNA was detected by PCR with the following primers: IL-6, sense, 5′-GCTTCTTAGGGCTAGCCTCA-3′, and antisense, 5′-AGCTACAGACATCCCCAGTC-3′; TNF-α, sense, 5′-TTCCGAGGGTTGAATGAGAGCT-3′, and antisense, 5′-TTTCTGTTCTCCCTCCTGGCTA-3′; and C/EBPβ, sense, 5′-GTAGCTGGAGGAACGATC3′, and antisense, 5′-TCGGGAACACGGAGGAGC-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 1 μg/ml LPS (Escherichia coli 0111:B4; Sigma). The total RNA was extracted with TRIZOL reagent (Invitrogen). Approximately 10 μg 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 generated by reverse transcription-PCR from P388D1(IL1) RNA with the following primers: IL-6 forward primer, 5′-ATGAAGTTCCTCTCTGCAAGAG-3′, and IL-6 reverse primer, 5′-CTAGGTTTGCCGAGTAGATCTCA-3′. Full-length cDNA was used to probe C/EBPβ RNA as described previously (4Chang C.J. Chen T.T. Lei H.Y. Chen D.S. Lee S.C. Mol. Cell. Biol. 1990; 10: 6642-6653Crossref PubMed Scopus (200) Google Scholar). Alkylation of Free Thiol Groups with Iodoacetamide in Vivo—The alkylation method was performed as described previously (40Mancini R. Fagioli C. Fra A.M. Maggioni C. Sitia R. FASEB J. 2000; 14: 769-778Crossref PubMed Scopus (89) Google Scholar). P388D1(IL1) cells were stimulated with 1 μg/ml LPS in the presence of 0.1 mm iodoacetamide (Sigma) to prevent free thiol groups from oxidizing to disulfide. Direct lysates were prepared at the indicated times with SDS sample dye containing 200 mm iodoacetamide at room temperature. The lysates were then resolved by nonreducing SDS-PAGE followed by Western blot analysis. Electrophoretic Mobility Shift Assay—The specific probe for C/EBPβ was prepared by annealing the oligonucleotides 5′-GATCATTTTGTGTAAGAC-3′ and 5′-GATCGTCTTACACAAAAT-3′. The probe was labeled with T4 polynucleotide kinase and [γ-32P]ATP. Nuclear extract (5-10 μg) 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, 1-2 μl of anti-C/EBPβ antibody was added, and the incubation continued at room temperature for additional 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/EBPβ 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/EBPβ 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-μm 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% aqueous formic acid (solution A) and 0.1% formic acid in acetonitrile (solution 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. LAP* Is Recruited to IL-6 Gene Promoter after LPS Treatment—Schematic representation of the C/EBPβ 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 in LIP (i.e. Cys201 and Cys296). Monoclonal and polyclonal antibodies 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 monoclonal antibodies are indicated at the top of this figure). To investigate the differential recruitment of C/EBPβ isoforms to target gene promoters, 18F8 and 1H7 were employed for chromatin immunoprecipitation (ChIP) experiments of IL-6 and C/EBPβ 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/EBPβ 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/EBPβ is undetectable as demonstrated by ChIP assay with 18F8, but the time course of 1H7 binding to the C/EBPβ promoter remains unchanged upon LPS treatment (Fig. 1B, 18F8 and 1H7 of right panels). NF-κB, known as the predominant factor responsible for the induction of inflammatory cytokine genes (41Sweet M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (713) Google Scholar), was also tested in ChIP experiment with anti-p50 antibody. Indeed, NF-κB was also recruited to the IL-6 promoter, albeit with different kinetic pattern from that of LAP* (Fig. 1B, α p50 of left panels). The kinetics of LAP* recruitment to the IL-6 promoter correlates well with the expression of IL-6 mRNA (Fig. 1B, 18F8 and IL-6 of left panels). C/EBPβ is constitutively expressed in P388D1(IL1) cells (Fig. 1B, C/EBPβ of right panels and data not shown). Neither LAP* nor NF-κB was recruited to the C/EBPβ promoter upon LPS treatment of P388D1(IL1) cells (Fig. 1B, 18F8 and α 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/EBPβ gene. In addition to, or in conjunction with LAP*, NF-κB could also play important roles in LPS-mediated IL-6 gene expression. LAP* Activation Correlates with the Reduction of Intramolecular Disulfide Bonds—We next aimed to examine the potential mechanisms that govern the LPS-induced recruitment of LAP* to the IL-6 gene promoter. A number of different pathways have been described to activate C/EBPβ, including transcriptional up-regulation, post-translational modifications, and nuclear translocation (42Bradley M.N. Zhou L. Smale S.T. Mol. Cell. Biol. 2003; 23: 4841-4858Crossref PubMed Scopus (100) Google Scholar). The Northern data in Fig. 1B (C/EBPβ panel) exclude the possibility of transcriptional up-regulation in the present assay system. Immunological analyses demonstrated that both protein level and subcellular localization of C/EBPβ 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 alignment shows that the cysteine residues in C/EBPβ 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 during LPS treatment, we performed Western blot analysis to LPS-treated P388D1(IL1) under nonreducing conditions. Iodoacetamide was used to trap the free thiol groups during LPS treatment. At least two species of LAP* displaying slower mobility were observed 1 h after LPS treatment (Fig. 2B), suggesting 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 analyzed 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 migration form was also observed for LAP from the same extracts (Fig. 2C, lower panel), whereas no mobility change was detected in LIP (data not shown). The same migration behavior was also observed for FLAG-tagged recombinant C/EBPβs expressed 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 Cys123 and Cys143, and between Cys201 and Cys296 of 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 endogenous 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 residue. Analysis of this mutant FLAG-LAP* (C11S) reveals that only Cys123 and Cys201, and not Cys11 and Cys33 nor Cys201 and Cys296, were linked by disulfide bond formation. When Cys11 is 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 Cys11 and Cys33 is disrupted by point mutation, 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* undergoes 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 published elsewhere, LAP is more active than LAP* when tested by transient transfection experiments (15Lee Y.M. Miau L.H. Chang C.J. Lee S.C. Mol. Cell. Biol. 1996; 16: 4257-4263Crossref PubMed Google Scholar). When LAP* transfectants were treated with the reducing agents PDTC or NAC, the activa
Aims: This study was aimed at elucidating whether arecoline-induced ROS production is significantly correlated with the secretion of mtDNA D-loop and PD-L1 in EVs, whether mtDNA D-loop attenuates T cell immunity and is a prognostic biomarker for OSCC patients, and whether orally administered WGP β-glucan reduces Treg cell numbers and promotes oral cancer cell apoptosis.Main methods: ROS production and cytosolic mtDNA D-loop were analyzed using fluorescent DCF and qPCR in OSCC cell lines. mtDNA D-loop and PD-L1 in EVs, serum IFN-γ, and Treg cells were identified using qPCR, ELISA, and flow cytometry for 60 OSCC patients.Key findings: We demonstrated that arecoline stimulated ROS production in a dose-dependent manner to significantly induce cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. Higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. However, WGP β-glucan could mitigate Treg cell numbers and promote oral cancer cell apoptosis.Significance: Arecoline induced ROS to elicit cytosolic mtDNA D-loop leakage and immune suppression. Upregulated EV mtDNA D-loop and serum Treg cell numbers were independent poor prognostic biomarkers, but WGP β-glucan could enhance dual effects on T cell immunity and cancer cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.
Insulin-like growth factor II mRNA-binding protein 3 (IMP3) is an RNA-binding protein expressed in embryonic tissues and multiple cancers. To investigate the role of IMP3 in hepatocellular carcinoma (HCC), its protein expression in the surgically resected unifocal tumors of 377 HCC patients (296 men and 81 women) with ages ranging from 7 to 88 years (mean, 55.49 years) was analyzed by immunohistochemistry. IMP3 was expressed in 255 (67.6%) of 377 resected unifocal primary HCCs. IMP3 protein was predominantly expressed in tumor border and invasive front, and it was more abundant in the satellite nodules and tumor thrombi than in the main tumors. The expression correlated with high α-fetoprotein (>200 ng/mL, P < 1 × 10−7), larger tumor size (>5 cm, P = 0.006), high tumor grade (P < 1 × 10−7), and high tumor stage with vascular invasion and various degrees of intrahepatic metastasis (P < 1 × 10−7). IMP3 expression predicted early tumor recurrence (P < 1 × 10−7) and was a strong indicator of poor prognosis (P < 0.0001). Depletion of IMP3 with RNA interference in HCC cell line HA22T caused a decrease in cell motility, invasion, and transendothelial migration. Microarray analysis revealed that IMP3 depletion was associated with downregulation of multiple genes involved in tumor invasion. Conclusion: Our results indicate that IMP3 plays an important role in tumor invasion and metastasis and is a strong prognostic factor for patients with HCC. (HEPATOLOGY 2008.)
Oral squamous cell carcinoma (OSCC) is a common malignancy often associated with poor prognosis due to chemoresistance. In this study, we investigated whether arecoline, a major alkaloid in betel nuts, can stimulate aldo-keto reductase family 1 member B10 (AKR1B10) levels in OSCC, promoting cancer stemness and leading to resistance to cisplatin (CDDP)-based chemotherapy. Gain- and Loss- of AKR1B10 functions were analyzed using WB and q-PCR of OSCC cells. Stemness, epithelial mesenchymal transition (EMT) markers, and CDDP drug resistance in overexpressed AKR1B10 were also identified. Upregulated AKR1B10 in OSCC significantly increased cell motility and aggregation. The results also showed that the canonical TGF-β1-Smad3 pathway was involved in arecoline-induced AKR1B10 expression, further increasing cancer stemness with CDDP resistance via the Snail-dependent EMT pathway. Moreover, oleanolic acid (OA) and ROS/RNS (reactive oxygen/nitrogen species) inhibitors effectively reversed AKR1B10-induced CDDP-resistance. Arecoline-induced ROS/RNS to hyper-activate AKR1B10 in tumor sphere cells via the TGF-β1-Smad3 pathway. Furthermore, AKR1B10 enhanced CDDP resistance in OSCC cells via EMT-inducing markers. Finally, Finally, OA may efficiently target CDDP resistance, reverse stemness in OSCC cells, and have the potential as a novel anticancer drug.
Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis.This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice.Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining.Scardovia wiggsiae (S. wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S. wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model.Our metagenome results suggested that S. wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S. wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S. wiggsiae and other periodontopathic bacteria are warranted.
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