Pancreatic and duodenal homeobox gene-1(PDX-1) is a transcription factor which regulates the insulin gene expression. In this study, we tried to elucidate the role of PDX-1 in the glucose-induced transcriptional activation of the human insulin gene promoter in MINE cells. Electrophoretic mobility shift assay (EMSA) and chloramphenicol acetyltransferase (CAT) assay demonstrated that both DNA-binding activity and transcriptional activity of PDX-1 were increased with 20mmol/l glucose more than with 2mmol/l glucose. The DNA-binding activity of PDX-1 induced by high glucose was blocked by phosphatase treatment, suggesting the involvement of PDX-1 phosphorylation in this event. In an in vitro phosphorylation study, PDX-1 was phosphorylated by protein kinase C (PKC), but not by cAMP dependent protein kinase (PKA) or mitogen-activated protein kinase (MAPK). Furthermore, increased PDX-1 function induced by high glucose was blocked by calphostin C, an inhibitor of all PKC isoforms, but unaffected by phorbol 12-myristate 13-acetate (PMA), an activator of classical and novel PKC, or Gö 6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 in MINE cells was atypical PKC. Western blot and immunocytochemical studies with anti-PKCζ antibody confirmed the presence of PKC ζ, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKC ζ activity was significantly increased by glucose stimulation. These results suggest that high glucose increased DNA-binding activity of PDX-1 by activating atypical PKC including PKC ζ, resulting in transcriptional activation of the human insulin gene promoter.
Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and M209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and M209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in [3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and M209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and M209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and M209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.
Insulin receptor substrate 1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream. We have previously shown that the levels of IRS-1 mRNA varied in different tissues. To elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and transacting factors in CHO and HepG2 cells. Using the chloramphenicol acetyltransferase (CAT) assay with the various deletion mutants of the IRS-1 promoter–CAT fusion plasmids, several regions responsible for positive or negative regulation in each cell line were identified. A region from −1645 to −1585 bp, which regulated expression negatively in CHO cells and positively in HepG2 cells, was further analyzed. Within this region sa fragment from −1645 to −1605 bp upregulated the IRS-1 promoter only in HepG2 cells, whereas a fragment from −1605 to −1585 bp downregulated only in CHO cells. In the gel mobility shift assay, several nuclear proteins that bind to these fragments were detected, and among them, two nuclear proteins that bind to a potential E box (nucleotide [nt] −1635 to −1630) and two nuclear proteins that bind to a potential C/EBP binding site (nt −1599 to −1591) were identified in HepG2 and CHO cells, respectively. CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell-specific regulation of the IRS-1 gene. We therefore concluded that the two nuclear proteins that bind to the E box regulate IRS-1 gene expression positively in HepG2 cells and the two nuclear proteins that bind to the C/EBP binding site regulate it negatively in CHO cells.
We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5′-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from −97 to −59 and from −59 to −37 and one negative regulatory site from −120 to −97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from −225 to −120 and one negative responsive site from −97 to −59, which contained the NF-κB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from −169 to −160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-κB binding site. Supershift assay showed that nuclear proteins bound to NF-κB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from −169 to −160 was a positive responsive element to Ox-LDL and that the NF-κB binding site from −91 to −82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription. We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5′-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from −97 to −59 and from −59 to −37 and one negative regulatory site from −120 to −97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from −225 to −120 and one negative responsive site from −97 to −59, which contained the NF-κB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from −169 to −160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-κB binding site. Supershift assay showed that nuclear proteins bound to NF-κB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from −169 to −160 was a positive responsive element to Ox-LDL and that the NF-κB binding site from −91 to −82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription. colony-stimulating factor granulocyte/macrophage colony-stimulating factor oxidized low density lipoprotein protein kinase C electrophoretic mobility shift assay enzyme-linked immunosorbent assay polymerase chain reaction reverse transcriptase-PCR Atherosclerosis is an inflammatory fibroproliferative process involving a complex set of interconnected events, including endothelial cell injury; smooth muscle cell migration and phenotypic changes; accumulation of monocytes, macrophages, and T lymphocytes; and formation of lipid-laden foam cells (1Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar). 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Matsuda H. Anami Y. Sasahara T. Kobori S. Shichiri M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1726-1733Crossref PubMed Scopus (30) Google Scholar). This finding strongly suggests that Ox-LDL acts as a growth inducer to macrophages by inducing certain intracellular signaling pathways. Subsequent studies from our laboratory identified the exact intracellular signaling pathways in Ox-LDL-induced macrophage growth. These included a rise in intracellular calcium ion and uptake of lysophosphatidylcholine through the scavenger receptors, which resulted in activation of protein kinase C (PKC) (17Matsumura T. Sakai M. Kobori S. Biwa T. Takemura T. Matsuda H. Hakamata H. Horiuchi S. Shichiri M. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3013-3020Crossref PubMed Scopus (93) Google Scholar). Moreover, expression of GM-CSF at the mRNA level was located downstream the signaling pathway from PKC activation to macrophage growth (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). GM-CSF is a glycoprotein produced by many cells including lymphocytes (18Wrong G.G. Witek J.S. Temple P.A. Wilkens K.M. Leary A.C. Luxenberg D.P. Jones S.S. Brown E.L Kay R.M. Orr E.C. Shoemaker C. Golde D.W. Kaufman R.J Hewick R.M. Wang E.A. Clark S.C. Science. 1985; 228: 810-815Crossref PubMed Scopus (762) Google Scholar), fibroblasts (19Munker R. Gasson J. Ogawa M. Koeffler H.P. Nature. 1986; 323: 79-82Crossref PubMed Scopus (365) Google Scholar), vascular endothelial cells (20Broudy V.C. Kaushansky K. Harlan J.M. Adamson J.W. J. Immunol. 1987; 139: 464-468PubMed Google Scholar), eosinophils (21Kita H. Ohnishi T. Okubo Y Weiler D. Abrams J.S. Gleich G.J. J. Exp. Med. 1991; 174: 745-748Crossref PubMed Scopus (280) Google Scholar), keratinocytes (22Kupper T.S. Lee F. Birchall N. Clark S. Dower S. J. Clin. 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Int. Immunol. 1991; 8: 807-817Crossref Scopus (30) Google Scholar) demonstrated that two cis-acting DNA elements on the GM-CSF promoter region, CLE2/GC, were required for the induction of GM-CSF by phorbol 12-myristate 13-acetate and calcium ionophore. Moreover, Tsuboiet al. (29Tsuboi A. Muramatsu M. Tsutsumi A. Arai K. Arai N. Biochem. Biophys. Res. Commun. 1994; 199: 1064-1072Crossref PubMed Scopus (46) Google Scholar) demonstrated that cooperation among AP-1-, NF-κB-, and NF-AT-binding sequences was required for the induction of GM-CSF, which was located downstream of PKC- and Ca2+-signaling pathways in T lymphocytes. Furthermore, Areset al. (30Ares M.P.S. Kallin B. Eriksson P. Nilsson J. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1584-1590Crossref PubMed Scopus (84) Google Scholar) reported that Ox-LDL could induce the activation of AP-1 in smooth muscle cells. Therefore, it is reasonable to speculate that induction of GM-CSF by Ox-LDL is also regulated by the activation of certain cis-acting DNA element(s) and nuclear transcription factor(s) after PKC activation in macrophages. However, the mechanism of GM-CSF production by Ox-LDL in macrophages remains unknown at present. In this study, we examined the promoter activity of GM-CSF in Ox-LDL-induced GM-CSF production by macrophages. Calphostin C was purchased from Sigma and dissolved in Me2SO. The final concentrations of Me2SO were <0.1% in the culture medium, which did not affect cell viability and cell growth. [γ-32P]ATP was from NEN Life Science Products. Antibodies for NF-κB p50 and p65, AP-2α, AP-2β, and AP-2γ were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Other chemicals were of the best grade available from commercial sources. Human LDL (d = 1.019–1.063 g/ml) was isolated by sequential ultracentrifugation from the plasma of consented normolipidemic subjects obtained after overnight fasting (31Hakamata H. Miyazaki A. Sakai M. Suginohara Y. Sakamoto Y. Horiuchi S. Arterioscler. Thromb. 1994; 14: 1860-1865Crossref PubMed Scopus (57) Google Scholar). LDL was dialyzed against 0.15 mol/liter NaCl and 1 mmol/liter EDTA, pH 7.4. Acetyl-LDL was prepared by chemical modification of LDL with acetate anhydride (32Miyazaki A. Sakai M. Suginohara Y. Hakamata H. Sakamoto Y. Horiuchi S. J. Biol. Chem. 1994; 269: 5264-5269Abstract Full Text PDF PubMed Google Scholar). Ox-LDL was prepared by incubation of LDL with 5 μmol/liter CuSO4 for 20 h at 37 °C followed by the addition of 1 mmol/liter EDTA and cooling (33Sakai M. Miyazaki A. Sakamoto Y. Shichiri M. Horiuchi S. FEBS Lett. 1992; 314: 199-202Crossref PubMed Scopus (57) Google Scholar, 34Ohta T. Takata K. Horiuchi S. Morino Y. Matsuda I. FEBS Lett. 1989; 257: 435-438Crossref PubMed Scopus (122) Google Scholar). The concentration of proteins was determined by BCA protein assay reagent (Pierce) using bovine serum albumin as a standard (35Miyazaki A. Rahim A.T.M.A. Araki S. Morino Y. Horiuchi S. Biochim. Biophys. Acta. 1991; 1082: 143-151Crossref PubMed Scopus (41) Google Scholar). Endotoxin levels associated with these lipoproteins were <1 pg/mg protein measured by a commercially available kit (Toxicolor system; Seikagaku Corp., Tokyo, Japan). Moreover, growth and viability of Raw 264.7 cells were not affected by endotoxin at a concentration of <1 ng/ml in our experimental system. The oligonucleotides used for electrophoretic mobility shift assay contained the following sequences (only one strand is shown): the element of the region from position −173 to −147 of the mouse GM-CSF promoter region 5′-AAA CCC CCA AGC CTG ACA ACC TGG GG-3′ (fragment A) and the region from position −95 to −70 of the mouse GM-CSF promoter region 5′-CTC AGG TAG TTC CCC CGC CCC CCT GG-3′ (fragment B). Competitors corresponding to a putative AP-2 and NF-κB binding sites were designed from their consensus sequences (competitor A, 5′-GAT CGA ACT GAC CGC CCG CGG CCC GT-3′; competitor C, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′). Mutated competitors corresponding to a putative AP-2 and NF-κB binding sites were designed from their consensus sequences (competitor B, 5′-GAT CGA ACT GAC CGC TTG CGG CCC GT-3′; competitor D, 5′-AGT TGA GGC GAC TTT CCC AGG C-3′). Raw 264.7 cells were maintained in suspension at a density of 2 × 105 to 1 × 106cells/ml in RPMI 1640 (Life Technologies, Inc.) containing heat-inactivated 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin and 100 μg/ml streptomycin (Life Technologies) (medium A). For experiments, the cells were incubated in 100-mm tissue culture dishes (5 × 106 cells/dish) or plated at a density of 1 × 106 cells/well in culture dishes (35-mm diameter; Falcon). All cell experiments were performed in a humidified atmosphere under 5% CO2 in air at 37 °C. When Raw 264.7 cells reached approximately 80% confluence in 100-mm plates in medium A, cells were washed twice with prewarmed phosphate-buffered saline (pH 7.4, 37 °C) and then incubated for 3 or 24 h with 20 μg/ml of Ox-LDL. Nuclear extract was purified as described previously by Dignamet al. (36Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). Protein concentrations were determined by the Micro BCA Protein Assay Reagent (Pierce). Double-stranded oligonucleotides were used as radiolabeled probes or unlabeled competitors. Probes were end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. EMSA employing [γ-32P]ATP-labeled probes, competitors, and nuclear extract was performed as described previously (37Matsuda K. Araki E. Yoshimura R. Tsuruzoe T. Furukawa N. Kaneko K. Motoshima H. Yoshizato K. Kishikawa H. Shichiri M. Diabetes. 1997; 46: 354-362Crossref PubMed Scopus (22) Google Scholar). Five μg/μl firefly luciferase reporter plasmids (pGL3 Basic; Promega, Madison, WI) containing the 5′-upstream regions of the GM-CSF gene was transiently transfected into Raw 264.7 cells by the DEAE-dextran method using a commercially available kit (Stratagene, La Jolla, CA), with 5 μg/μl Renilla luciferase control plasmid (pRL-SV40; Promega). After a 24-h incubation with medium A alone, cells were incubated for 24 h in the presence or absence of 20 μg/ml Ox-LDL. A plasmid lacking the 5′-upstream region of the GM-CSF gene was used as a negative control (pGL3 Basic, Promega). After incubation, cells were washed twice by phosphate-buffered saline and then lysed with 1× passive lysis buffer (Promega) for 15 min at room temperature. The luciferase activity in the resulting protein lysates was measured using the Dual Luciferase Reporter Assay system (Promega). The results were expressed as normalized firefly luciferase activity divided by Renilla luciferase activity, to adjust any differences in transfection efficiency. Raw 264.7 cells (5 × 106 cells/plate, 100 mm in diameter; Falcon) were cultured in 15 ml of medium A with or without 20 μg/ml Ox-LDL. During incubation for 24 h, 300 μl of the medium were collected at various time intervals and immediately centrifuged at 10,000 × g for 1 min to remove any particulate material. The supernatant was stored at −80 °C immediately. After completion of all culture experiments, the frozen culture supernatants were quickly thawed to determine GM-CSF levels in the medium. The concentration of GM-CSF protein was determined according to the instructions provided by the manufacturer of the GM-CSF-specific ELISA system (Amersham Pharmacia Biotech) using recombinant murine GM-CSF as a standard (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Standard molecular biology techniques were used (38Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). After incubation of Raw 264.7 cells (2 × 106 cells/well in a six-well plate, 35 mm in diameter; Nunc) with or without Ox-LDL (20 μg/ml) for different time intervals (0–5 h), total RNA was extracted with TRIzol (Life Technologies, Inc.). The first strand cDNA synthesis containing 1 μg of total RNA was primed with oligo(dT). Primers used for PCR amplification of GM-CSF and β-actin were designed on the basis of murine GM-CSF cDNA (39Miyake S. Otsuka T. Yokota T. Lee T. Arai K. EMBO J. 1988; 4: 2561-2568Crossref Scopus (160) Google Scholar) and murine β-actin cDNA (40Alonso S. Minty A. Bourlet Y. Buckingham M. J. Mol. Evol. 1986; 23: 11-22Crossref PubMed Scopus (604) Google Scholar) sequences as follows: for GM-CSF, forward primer was TGT GGT CTA CAG CCT CTC AGC AC (nucleotides 64–86 of murine GM-CSF coding sequence), and reverse primer was CAA AGG GGA TAT CAG TCA GAA AGG T (nucleotides 407–431 of murine GM-CSF coding sequence) (39Miyake S. Otsuka T. Yokota T. Lee T. Arai K. EMBO J. 1988; 4: 2561-2568Crossref Scopus (160) Google Scholar); for β-actin, forward primer was GTG GGC CGC TCT AGG CAC CAA (nucleotides 25–45 of murine β-actin coding sequence), and reverse primer was CTC TTT GAT GTC ACG CAC GAT TTC (nucleotides 541–564 of murine β-actin coding sequence) (40Alonso S. Minty A. Bourlet Y. Buckingham M. J. Mol. Evol. 1986; 23: 11-22Crossref PubMed Scopus (604) Google Scholar). The sizes of RT-PCR products of GM-CSF and β-actin were expected to be 368 and 540 base pairs, respectively. The cycling conditions in the GeneAmp 9600 System consisted of a first step of 94 °C denaturation for 10 min, followed by 35 cycles of annealing at 54 °C for 60 s, extension at 75 °C for 90 s, and denaturation at 94 °C for 30 s, with a final elongation step at 75 °C for 10 min. Amplification products were analyzed by 1.5% agarose gel electrophoresis. To verify that the amplification products were consistent with the reported sequences of murine GM-CSF and β-actin, they were ligated into pGEM-T (Promega), transfected intoEscherichia coli XL1-Blue, and sequenced by using 373A DNA sequencer (Applied Biosystems, Foster City, CA) (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). All data were expressed as mean ± S.D. Differences between groups were examined for statistical significance using Student's t test. A p value less than 0.05 denoted the presence of a statistically significant difference. We previously demonstrated that mouse peritoneal macrophages could produce GM-CSF in response to Ox-LDL (12Biwa T. Hakamata H. Sakai M. Miyazaki A. Suzuki H. Kodama T. Shichiri M. Horiuchi S. J. Biol. Chem. 1998; 273: 28305-28313Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 16Sakai M. Biwa T. Matsumura T. Takemura T. Matsuda H. Anami Y. Sasahara T. Kobori S. Shichiri M. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1726-1733Crossref PubMed Scopus (30) Google Scholar). To confirm whether Raw 264.7 cells also respond to Ox-LDL, we investigated the Ox-LDL-induced GM-CSF production in Raw 264.7 cells at protein and mRNA levels by using ELISA and RT-PCR, respectively. Fig. 1 A shows that LDL or acetyl-LDL did not induce GM-CSF release into the medium. However, the addition of 20 μg/ml Ox-LDL to Raw 264.7 cells significantly induced GM-CSF release into the medium, with the peak release occurring at 4 h after the addition of Ox-LDL (Fig. 1 A). RT-PCR analysis showed that GM-CSF mRNA was increased by Ox-LDL with the peak level occurring at 3 h (Fig. 1 B), whereas both LDL and acetyl-LDL had no effect on GM-CSF mRNA expression in these cells (data not shown). These results demonstrated that Ox-LDL also induced GM-CSF expression in Raw 264.7 cells. Previous studies demonstrated that several nuclear factor binding sites existed in GM-CSF gene 5′-flanking region from sequence −133 to −30, which positively regulated GM-CSF expression in response to PKC activation in T lymphocytes (27Sugimoto K. Tsuboi A. Miyake S. Arai K. Arai N. Int. Immunol. 1990; 2: 787-794Crossref PubMed Scopus (29) Google Scholar, 28Tsuboi A. Sugimoto K. Yodoi J. Miyatake S. Arai K. Arai N. Int. Immunol. 1991; 8: 807-817Crossref Scopus (30) Google Scholar, 29Tsuboi A. Muramatsu M. Tsutsumi A. Arai K. Arai N. Biochem. Biophys. Res. Commun. 1994; 199: 1064-1072Crossref PubMed Scopus (46) Google Scholar). Thus, the GM-CSF gene 5′-flanking region from −225 to +26 was cloned and inserted into the promoterless luciferase reporter plasmid (pGL3-basic) (Fig. 2 A) and transiently transfected into Raw 264.7 cells. Incubation with Ox-LDL significantly increased luciferase activity (Fig. 2 B), and luciferase activity reached a plateau level at 24 h (Fig. 2 C). In contrast, both LDL and acetyl-LDL had no effect on luciferase activity (Fig. 2 B). These results suggested that Ox-LDL-induced GM-CSF mRNA expression might be regulated, at least in part, by the transcriptional activation of GM-CSF promoter from −225 to +26. To identify the regulatory elements in the mouse GM-CSF promoter, we constructed a series of plasmids containing 5′-deletions of GM-CSF promoter fused to the luciferase reporter gene (Fig. 3 A). As shown in Fig. 3 B, in the unstimulated state, luciferase activity in pGL3GM120-transfected cells was almost equal to that in pGL3GM225-transfected cells. However, deletion extending to position −97, which removed the CLE1 region, resulted in increased luciferase activity. In contrast, deletion extending to −59 and to −37 resulted in a reduction of luciferase activity (Fig. 3 B). These results suggested that a negative regulatory site existed in the region extending from −120 to −97 and that two positive regulatory sites existed in the regions extending from −97 to −59 and from −59 to −37 in the unstimulated state. To confirm this notion, we constructed two plasmids containing mutations in a region from −120 to −97 and a region from −97 to −59 (Fig. 3 A). Fig. 3 B also showed that luciferase activities in pGL3GM120mt- or pGL3GM97mt-transfected cells were significantly higher or lower than those in wild type plasmid-transfected cells, respectively, demonstrating that a region from −120 to −97 was a negative regulatory site and that a region from −97 to −59 was a positive regulatory site for GM-CSF expression under unstimulated states. On the other hand, when cells transfected with pGL3GM225 were incubated with Ox-LDL, luciferase activity increased, whereas luciferase activity in pGL3GM120-transfected cells was not changed by Ox-LDL-stimulation (Fig. 3 B). These results suggested that the region from −225 to −120 contained a positive responsive site for Ox-LDL stimulation. In contrast, luciferase activity was significantly decreased by Ox-LDL in pGL3GM97-transfected cells, which contained a positive regulatory site under unstimulated state, suggesting that a negative responsive site for Ox-LDL existed in the 5′-flanking region from −97 to −59. We performed a computer analysis of the region extending from −225 to −120, which showed the presence of a putative AP-2 binding site from sequence −169 to −160 in the mouse GM-CSF promoter region (Fig. 4). Moreover, the NF-κB binding site was reported to exist from −91 to −82 (41Schreck R. Baeuerle P.A. Mol. Cell. Biol. 1990; 10: 1281-1286Crossref PubMed Google Scholar). We next performed a functional analysis using a luciferase reporter plasmid containing mutations in a putative AP-2 binding site from −169 to −160 (Fig. 5 A). As shown in Fig. 5 B, mutation in the putative AP-2 binding site reduced basal promoter activity by 20% and completely diminished Ox-LDL-induced promoter activity. To confirm that the putative AP-2 binding site is a positive responsive site for Ox-LDL stimulation, we constructed pGL3GM177 plasmid, which contained the sequence spanning position −177 of GM-CSF promoter region, and pGL3GM177 tandem plasmid, which had two more copies of the putative AP-2 binding site (Fig. 6 A). Cells transfected with pGL3GM177 plasmid induced 2.5-fold expression of luciferase in response to Ox-LDL, relative to unstimulated cells (Fig. 6 B). Transfection of the pGL3GM177 tandem plasmid increased basal level luciferase activity at 1.5-fold and Ox-LDL-induced luciferase activity at 4.4-fold, relative to unstimulated cells (Fig. 6 B). These results demonstrated that Ox-LDL-induced GM-CSF expression was required for a putative AP-2 binding site.FIG. 5Mutation in a putative AP-2 binding site reduces transcriptional activation by Ox-LDL. A, structure of the mutated GM-CSF promoter-luciferase reporter (pGL3GM225mt) that was generated from pGL3GM225 by introducing the mutation using PCR mutagenesis. The position of the mutation in a putative AP-2 binding site is underlined. B, pGL3GM225 and pGL3GM225mt (5 μg) were transfected into Raw 264.7 cells (5 × 106) by DEAE-dextran method. After a 24-h incubation with medium A alone, cells were treated with 20 μg/ml Ox-LDL and then harvested 24 h later. Luciferase activity was determined as described under "Experimental Procedures." Data represent the mean ± S.D. of four separate experiments. †,p < 0.05, compared with unstimulated pGL3GM225; ††,p < 0.01, compared with unstimulated pGL3GM225 (Student's t test).View Large Image Figure ViewerDownload (PPT)FIG. 6Tandem repeat of AP-2 binding site enhances the transcriptional activation of GM-CSF by Ox-LDL. A, structure of the GM-CSF promoter-luciferase reporter construct, which has two more copies of putative AP-2 binding site (pGL3GM177tandem).B, pGL3GM225, pGL3GM177, and pGL3GM177 tandem plasmids (5 μg) were transfected into Raw 264.7 cells (5 × 106) by DEAE-dextran method. After a 24-h incubation with medium A alone, cells were treated with 20 μg/ml Ox-LDL and then harvested 24 h later. Luciferase activity was determined as described under "Experimental Procedures." Data represent the mean ± S.D. of four separate experiments. †, p < 0.05, compared with unstimulated pGL3GM225, ††, p < 0.01, compared with unstimulated pGL3GM225, †††, p < 0.001, compared with unstimulated pGL3GM225 (Student's ttest).View Large Image Figure ViewerDownload (PPT) To elucidate whether nuclear protein(s) would bind to the promoter region of GM-CSF gene, the nuclear protein specific for binding to cis-acting elements from −173 to −147, containing a putative AP-2 binding site (Fig. 7 A), and from −95 to −70, containing an NF-κB binding site (Fig. 8 B), were analyzed by EMSA. As shown in Fig. 7 B using a putative AP-2 binding site as a probe, the nuclear proteins from unstimulated cells produced two faint bands, which became prominent bands by Ox-LDL. These bands were completely diminished by cold excess unlabeled fragment A but not completely competed by cold excess unlabeled AP-2 consensus oligonucleotides. Moreover, supershift analysis using anti-AP-2α, AP-2β, and AP-2γ antibodies (Santa Cruz Biotechnology) did not affect the position and density of the bands (data not shown). These results suggested that the binding of nuclear factor(s) to a putative AP-2 binding site was different from AP-2α, AP-2β, or AP-2γ but might be a nuclear protein highly homologous to the AP-2 family. As shown in Fig. 8 B, when the NF-κB binding element was used as a probe, a strong band was detected in unstimulated cells. Interestingly, this band was decreased by incubation with Ox-LDL for 24 h (Fig. 8 B). Cold excess NF-κB consensus oligonucleotides diminished this band. However, cold excess mutated NF-κB consensus oligonucleotides completely failed to compete for nuclear protein binding to fragment B. Moreover, the density of this band was reduced by both anti-p50 and anti-p65 antibodies but not by nonimmune IgG (Fig. 8 C), suggesting that this band contained, at least, NF-κB p50 and p65.FIG. 8Binding of NF-κB to a NF-κB bind
Several important structures, such as the carotid artery, oculomotor nerve, trochlear nerve, abducent nerve and trigeminal nerve are located in the cavernous sinus. This sinus is also in contact with the lateral wall of the sphenoidal sinus, and various lesions arising in the sphenoidal sinus extend beyond its confines, giving rise to diplopia and visual disturbances. Between October 1995 and February 2001, we experienced 7 cases of cavernous sinus involvement: 2 cases of sphenoidal sinusitis, one case of sphenoidal mucocele, two case of sphenoidal carcinoma, and two cases of nasopharyngeal carcinoma. These cases were analyzed, and the regional anatomies and variety of lesions are reported.
Oxidized low density lipoprotein (Ox-LDL) can induce macrophage proliferation in vitro. To explore the mechanisms involved in this process, we reported that activation of protein kinase C (PKC) is involved in its signaling pathway (Matsumura, T., Sakai, M., Kobori, S., Biwa, T., Takemura, T., Matsuda, H., Hakamata, H., Horiuchi, S., and Shichiri, M. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 3013–3020) and that expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) and its subsequent release in the culture medium are important (Biwa, T., Hakamata, H., Sakai, M., Miyazaki, A., Suzuki, H., Kodama, T., Shichiri, M., and Horiuchi, S. (1998) J. Biol. Chem.273, 28305–28313). However, a recent study also demonstrated the involvement of phosphatidylinositol 3-kinase (PI3K) in this process. In the present study, we investigated the role of PKC and PI3K in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced macrophage proliferation was inhibited by 90% by a PKC inhibitor, calphostin C, and 50% by a PI3K inhibitor, wortmannin. Ox-LDL-induced expression of GM-CSF and its subsequent release were inhibited by calphostin C but not by wortmannin, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by wortmannin by 50% but not by calphostin C. Ox-LDL activated PI3K at two time points (10 min and 4 h), and the activation at the second but not first point was significantly inhibited by calphostin C and anti-GM-CSF antibody. Our results suggest that PKC plays a role upstream in the signaling pathway to GM-CSF induction, whereas PI3K is involved, at least in part, downstream in the signaling pathway after GM-CSF induction.
We present 32 cases of orbital complication caused by sinusitis. Clinical parameters such as age, gender, affected sinus, eye symptoms, and treatment were evaluated. Cases involved 25 males and 7 females with a mean age of 32 years ranging from 3 to 71 years. On the first visit, He subjects were seen in ophthalmology in 16, internal medicine in 6, and otolaryngology in 6, and other in 4. Ethmoid sinus was most frequently encountered. Presenting eye symptoms consisted of combination, such as ocular pain in 26, diplopia in 10, proptosis in 8, and deterioration of visual acuity in 4. Of these 20 were treated conservatively and 10 surgically. Hospitalization lasted 4 to 30 days (mean 12.2 days). CT and MRI were useful in diagnosis. Cases with visual disorders need immediate surgical intervention.
