The DNA sequence requirements of chicken U1 RNA gene expression have been examined in an oocyte transcription system. An enhancer region, which was required for efficient U1 RNA gene expression, is contained within a region of conserved DNA sequences spanning nucleotide positions -230 to -183, upstream of the transcriptional initiation site. These DNA sequences can be divided into at least two distinct subregions or domains that acted synergistically to provide a greater than 20-fold stimulation of U1 RNA synthesis. The first domain contains the octamer sequence ATGCAAAT and was recognized by a DNA-binding factor present in HeLa cell extracts. The second domain (the SPH domain) consists of conserved sequences immediately downstream of the octamer and is an essential component of the enhancer. In the oocyte, the DNA sequences of the SPH domain were able to enhance gene expression at least 10-fold in the absence of the octamer domain. In contrast, the octamer domain, although required for full U1 RNA gene activity, was unable to stimulate expression in the absence of the adjacent downstream DNA sequences. These findings imply that sequences 3' of the octamer play a major role in the function of the chicken U1 RNA gene enhancer. This concept was supported by transcriptional competition studies in which a cloned chicken U4B RNA gene was used to compete for limiting transcription factors in oocytes. Multiple sequence motifs that can function in a variety of cis-linked configurations may be a general feature of vertebrate small nuclear RNA gene enhancers.
Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contribute to periprosthetic osteolysis. The particle-induced activation of macrophages and monocytes has been extensively studied, but only limited information is available on the response of osteoblasts to particulate wear debris. This study examines the effects of particulate wear debris, proinflammatory cytokines, and growth factors on osteoblast functions.MG-63 osteoblasts were treated with metal particles (titanium, titanium alloy, and chromium orthophosphate) or polymeric particles (polyethylene and polystyrene) of phagocytosable sizes or were treated with exogenous cytokines and growth factors. The kinetics of particle phagocytosis and the number of engulfed particles were assessed with use of fluoresceinated particles. Cell proliferation was determined according to [3H]-thymidine incorporation, and cell viability was determined by either fluorescein diacetate uptake or trypan blue exclusion. Expressions of osteoblast-specific genes were quantified with Northern blot hybridization, and the secretions of osteoblast-specific proteins and cytokines were analyzed by enzyme-linked immunosorbent assays.MG-63 osteoblasts phagocytosed particles and became saturated after twenty-four hours. A maximum of forty to sixty particles per cell were phagocytosed. Each type of particle significantly suppressed procollagen alpha1[I] gene expression (p<0.05), whereas other osteoblast-specific genes (osteonectin, osteocalcin, and alkaline phosphatase) did not show significant changes. Particle-stimulated osteoblasts released interleukin-6 (p<0.05) and a smaller amount of transforming growth factor-beta1. Particles reduced cell proliferation in a dose-dependent manner without affecting cell viability (p<0.05). Exogenous tumor necrosis factor-alpha also enhanced the release of interleukin-6 (p<0.01) and transforming growth factor-beta1 (p<0.05), whereas the secretion of transforming growth factor-beta1 was increased by insulin-like growth factor-I and prostaglandin E2 as well. Insulin-like growth factor-I and transforming growth factor-beta1 significantly increased procollagen alpha1[I] gene expression in osteoblasts (p<0.05), while tumor necrosis factor-alpha and prostaglandin E2 significantly suppressed procollagen alpha1[I] gene expression (p<0.01). In contrast, neither exogenous nor endogenous interleukin-6 had any effect on other cytokine secretion, on proliferation, or on procollagen alpha1[I] gene expression. The transcription inhibitor actinomycin D reduced both procollagen alpha1[I] transcription and interleukin-6 production. Inhibitors of protein synthesis (cyclohexamide) and intracellular protein transport (brefeldin A and monensin) blocked the release of interleukin-6, but none of these compounds influenced the suppressive effect of titanium on procollagen alpha1[I] gene expression.MG-63 osteoblasts phagocytose particulate wear debris, and this process induces interleukin-6 production and suppresses type-I collagen synthesis. Osteoblast-derived interleukin-6 may induce osteoclast differentiation and/or activation, but the resorbed bone cannot be replaced by new bone because of diminished osteoblast function (reduced type-I collagen synthesis). Exogenous cytokines (tumor necrosis factor-alpha and interleukin-1beta), growth factors (insulin-like growth factor-I and transforming growth factor-beta1), and prostaglandin E2 can modify particulate-induced alterations of osteoblast functions.
Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R. Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R. The balance between bone formation and bone resorption is controlled at least in part by different osteotrophic hormones and soluble mediators such as various pro- and anti-inflammatory cytokines. Recently, interleukin-6 (IL-6) 1The abbreviations used are: IL-6interleukin-6IL-6Rinterleukin-6 receptorgp130glycoprotein 130JAKJanus kinaseSTATsignal transducer and activator of transcriptionMAPKmitogen-activated protein kinasesIL-6Rsoluble interleukin-6 receptorFBSfetal bovine serumAPalkaline phosphataseTNF-αtumor necrosis factor-α, LPS, lipopolysaccharidePMAphorbol myristate acetatePI3Kphosphatidylinositol 3-kinaseMEKmitogen-activated protein kinase kinasePKCprotein kinase CPTKprotein-tyrosine kinasePKAprotein kinase ATAPItumor necrosis factor-α protease inhibitorRPARNase protection assayLIFleukemia inhibitory factorGM-CSFgranulocyte-macrophage colony-stimulating factor has attracted special attention because a strong correlation has been found between serum and/or local levels of IL-6 and bone resorption in various diseases (1.Jilka R.L. Hangoc G. Girasole G. Passeri G. Williams D.C. Abrams J.S. Boyce B. Broxmeyer H. Manolagas S.C. Science. 1992; 257: 88-91Crossref PubMed Scopus (1287) Google Scholar, 2.Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Crossref PubMed Scopus (797) Google Scholar, 3.Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (686) Google Scholar, 4.Manolagas S.C. Ann. N. Y. Acad. Sci. U. S. A. 1999; 840: 194-204Crossref Scopus (156) Google Scholar, 5.Jones S.A. Horiuchi S. Topley N. Yamamoto N. Fuller G.M. FASEB J. 2001; 15: 43-58Crossref PubMed Scopus (533) Google Scholar). IL-6 is a pleiotropic cytokine with multiple and diverse effects on various cell types (2.Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Crossref PubMed Scopus (797) Google Scholar, 4.Manolagas S.C. Ann. N. Y. Acad. Sci. U. S. A. 1999; 840: 194-204Crossref Scopus (156) Google Scholar, 6.Taga T. Ann. Med. 1997; 29: 63-72Crossref PubMed Scopus (66) Google Scholar, 7.Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. 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A. 1999; 840: 194-204Crossref Scopus (156) Google Scholar, 11.Adebanjo O.A. Moonga B.S. Yamate T. Sun L. Minkin C. Abe E. Zaidi M. J. Cell Biol. 1998; 142: 1347-1356Crossref PubMed Scopus (75) Google Scholar), processes that depend on the activation of IL-6-induced signaling mechanisms in osteoblasts (8.Bellido T. Borba V.Z.C. Roberson P. Manolagas S.C. Endocrinology. 1997; 138: 3666-3676Crossref PubMed Scopus (147) Google Scholar, 9.Nishimura R. Moriyama K. Yasukawa K. Mundy G.R. Yoneda T. J. Bone Miner. Res. 1998; 13: 777-785Crossref PubMed Scopus (87) Google Scholar,12.Udagawa N. Takahashi N. Katagiri T. Tamura T. Wada S. Findlay D.M. Martin T.J. Hirota H. Taga T. Kishimoto T. Suda T. J. Exp. Med. 1995; 182: 1461-1468Crossref PubMed Scopus (330) Google Scholar, 13.O'Brien C.A. Gubrij I. Lin S.C. Saylors R.L. Manolagas S.C. J. Biol. Chem. 1999; 274: 19301-19308Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). interleukin-6 interleukin-6 receptor glycoprotein 130 Janus kinase signal transducer and activator of transcription mitogen-activated protein kinase soluble interleukin-6 receptor fetal bovine serum alkaline phosphatase tumor necrosis factor-α, LPS, lipopolysaccharide phorbol myristate acetate phosphatidylinositol 3-kinase mitogen-activated protein kinase kinase protein kinase C protein-tyrosine kinase protein kinase A tumor necrosis factor-α protease inhibitor RNase protection assay leukemia inhibitory factor granulocyte-macrophage colony-stimulating factor IL-6 exerts its effect through the IL-6 receptor complex, which is composed of a ligand binding domain (IL-6 receptor (IL-6R or gp80)) and the signal-transducing molecule glycoprotein 130 (gp130). IL-6 binds to its cognate receptor, and the IL-6/IL-6R forms a complex with a gp130 homodimer. This receptor-ligand interaction activates Janus kinases (JAKs). JAKs phosphorylate the tyrosine residues of the cytoplasmic tail of gp130, which then activates various members of the signal transducer and activator of transcription (STAT) family and also the mitogen-activated protein kinase (MAPK) pathway (2.Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Crossref PubMed Scopus (797) Google Scholar, 6.Taga T. Ann. Med. 1997; 29: 63-72Crossref PubMed Scopus (66) Google Scholar, 7.Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1749) Google Scholar, 8.Bellido T. Borba V.Z.C. Roberson P. Manolagas S.C. Endocrinology. 1997; 138: 3666-3676Crossref PubMed Scopus (147) Google Scholar, 14.Kishimoto T. Akira S. Taga T. Int. J. Immunopharmacol. 1992; 14: 431-438Crossref PubMed Scopus (65) Google Scholar, 15.Murakami M. Hibi M. Nakagawa N. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. 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We also investigated the mechanism of sIL-6R generation and analyzed distinct functions of the cell surface IL-6R and the osteoblast-derived sIL-6R by monitoring the IL-6-induced tyrosine phosphorylation of gp130 in human osteoblasts. Human osteoblast-like cell lines MG-63, SaOS-2, and HOS and human monocytic cell line THP-1, a positive control for cell surface IL-6R expression (23.Jones S.A. Horiuchi S. Novick D. Yamamoto N. Fuller G.M. Eur. J. Immunol. 1998; 28: 3514-3522Crossref PubMed Scopus (59) Google Scholar), were purchased from the American Type Culture Collection (Rockville, MD). The cells were cultured in monolayers in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., Logan, UT) in a standard tissue culture condition (41.Glant T.T. Jacobs J.J. Molnár G. Shanbhag A.S. Valyon M. Galante J.O. J. Bone Miner. Res. 1993; 8: 1071-1079Crossref PubMed Scopus (300) Google Scholar, 42.Yao J. Glant T.T. Lark M.W. 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Orthop. Res. 1997; 15: 546-557Crossref PubMed Scopus (273) Google Scholar, 45.Muschler G.F. Boehm C. Easley K. J. Bone Joint Surg. 1998; 79A: 1699-1709Google Scholar). Briefly, buffy coat-separated nucleated bone marrow cells (2 × 107/T75 tissue culture flasks; Corning Inc., Corning, NY) were cultured in α-minimal essential medium (Invitrogen) containing 10% FBS, 10 nm dexamethasone, 50 μg/ml ascorbic acid, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B, and 50 μg/ml gentamicin, all purchased from Sigma. The first medium change was performed on day 7, at which time the medium was supplemented with 5 μmβ-glycerophosphate (Sigma). Dense colonies of cells were trypsinized and 1 × 105 cells plated in 10-cm Petri dishes (Corning). Cells were then cultured to obtain a confluent monolayer culture. All experiments with bone marrow-derived osteoblasts were carried out using these first passage cultures. At the time of this first passage, aliquots of cells were also seeded in 24- and 96-well plates (Corning) for viability and cell proliferation tests, and alkaline phosphatase (AP) activity assays. AP activity was measured by Alkaline Phosphatase Colorimetric End point assay (Sigma) in cell lysates of first-passaged osteoblasts. Confluent cultures were stainedin situ for AP positivity using Naphthol-AX and Fast Blue reagents (Sigma) following the manufacture's instructions. Osteoblast cultures showing higher than 80–85% AP positivity were used in these experiments. Semiconfluent cultures of cells were subjected to serum starvation (0.3% FBS) for 24 h prior to treatment. Culture media were then replaced with fresh media containing 0.3% FBS and various compounds. Proliferation and viability assays, flow cytometry analysis, and total RNA and protein extractions were carried out on untreated and treated cells. Tissue culture media were collected at various time points, centrifuged, and stored at −80 °C. All experiments were performed in duplicate or triplicate in at least five independent experiments for osteoblast cell lines and in at least three independent experiments for primary osteoblasts. Reagents listed below were purchased from Calbiochem (La Jolla, CA), R&D Systems (Minneapolis, MN), or Sigma. All concentrations were selected after serial dilutions of each compound tested in either MG-63 or primary osteoblast cell cultures. Only the viable range and the most effective concentrations were used for further experiments. Tumor necrosis factor-α (TNF-α, 20 ng/ml), IL-6 (50 ng/ml), sIL-6R (200 ng/ml), lipopolysaccharide (LPS, O127:B8, 1 mg/ml), phorbol myristate acetate (PMA, 20 ng/ml), actinomycin D (an inhibitor of transcriptional events, 1 μg/ml), cycloheximide (an inhibitor of protein synthesis, 10 μm), brefeldin A (an inhibitor of protein transport from endoplasmic reticulum to Golgi, 1 μm), monensin (an inhibitor of protein transport from Golgi, 10 μm), tunicamycin (an inhibitor of N-glycosylation, 2 μg/ml), wortmannin (an inhibitor of phosphatidylinositol 3-kinase (PI3K), 0.1 μm), SB203580 (an inhibitor of p38 MAPK, 10 μm), UO126 (an inhibitor of MAPK kinase 1 and MAPK kinase 2 (MEK1 and MEK2), 1 μm), staurosporine (0.01 μm), calphostin C (0.1 μm), and bisindolylmaleimide I (1 μm) (all inhibitors of protein kinase C (PKC)), genistein (an inhibitor of protein-tyrosine kinases (PTK), 20 μm), H-89 (a potent inhibitor of protein kinase A (PKA), 30 μm), TAPI-1 (a hydroxamate-based metalloproteinase inhibitor, 50, 100, and 150 μm), and Galardin (GM-6001, a potent metalloproteinase inhibitor, 10, 50, and 100 μm) were added either alone or in different combinations. As calphostin C requires photoactivation (46.Bruns R.F. Miller F.D. Merriman R.L. Howbert J.J. Heath W.F. Kobayashi E. Takahashi I. Tamaoki T. Nakano H. Biochem. Biophys. Res. Commun. 1991; 176: 288-293Crossref PubMed Scopus (358) Google Scholar) to inhibit PKC, experiments with calphostin C were carried out in an incubator with a 5-watt light source located 15 cm above culture dishes. The trypan blue exclusion test was used to assess the viability of cells. Viability tests were performed in duplicate, and at least 200 cells were counted. Determination of cell viability was used to select the concentrations of each compound in which the cells remained viable (>95%), during the indicated time period. Cytokine concentrations in supernatants of osteoblast cultures were measured by sandwich enzyme-linked immunosorbent assays in 96-well microtitration plates following the manufacturer's instructions. High sensitivity assay kits for IL-6 (sensitivity range from 3 to 200 pg/ml) and sIL-6R (range from 31 to 2000 pg/ml) were purchased from R&D Systems and BIOSOURCE (Camarillo, CA), respectively. Confluent layers of cells were either untreated or treated with different compounds for various times. Cells were harvested with enzyme-free cell dissociating buffer (Invitrogen) and then washed three times in washing buffer (phosphate-buffered saline, pH 7.4, containing 1% bovine serum albumin (Sigma)). Cells were resuspended in 100 μl of washing buffer and incubated with 10 ng/μl mouse anti-human IL-6R monoclonal antibody (IgG1, clone B-R6, BIOSOURCE) or with 10 ng/μl mouse anti-human-gp130 monoclonal antibody (IgG2a, clone B-R3, BIOSOURCE) for 1 h at 4 °C, followed by biotin-labeled polyclonal anti-mouse Ig antibody (10 ng/μl; BD PharMingen, San Diego, CA). The reaction was developed with streptavidin-phycoerythrin (Invitrogen). Samples were fixed in 2% formalin (Sigma) and then analyzed by FACScan (BD PharMingen) using Cell Quest software (BD PharMingen). Isotypic control antibodies corresponding to the primary antibodies were used to determine nonspecific background levels in all experiments. All compounds were tested for autofluorescence. Calphostin C- and bisindolylmaleimide I-treated cells exhibited red fluorescence necessitating the use of streptavidin-fluorescein isothiocyanate (BD PharMingen) in experiments whenever these two compounds were applied. Treated and untreated cells were lysed in ice-cold lysis buffer (50 mmTris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, and 1% Nonidet P-40) containing protease inhibitors (1 mmphenylmethylsulfonyl fluoride and 1 unit/ml aprotinin), phosphatase inhibitors (50 mm NaH2PO4, 10 mm sodium pyrophosphate, 50 mm KF, and 1 mm Na3VO4), and 0.01% NaN3 for 1 h at 4 °C. Cell lysates were ultrasonicated (Virtis, Gardina, NY) for 10 s at 4 °C and cleared by centrifugation, after which 700 μg of cell lysate protein was incubated with either 3 μg of anti-human-IL-6R antibody (rabbit IgG, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or 3 μg of anti-human-gp130 antibody (rabbit IgG, Santa Cruz) for 2 h at 4 °C. Immunocomplexes were collected with Protein G-Sepharose (Amersham Biosciences) during an overnight incubation at 4 °C. Protein G-bound complexes were washed in lysis buffer, and then proteins of boiled samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in reducing condition as described (43.Vermes C. Roebuck K.A. Chandrasekaran R. Dobai J.G. Jacobs J.J. Glant T.T. J. Bone Miner. Res. 2000; 15: 1756-1765Crossref PubMed Scopus (102) Google Scholar). Samples were transferred onto a nitrocellulose membrane (Bio-Rad), and the free binding capacity of the membrane was blocked with 5% skimmed milk in phosphate-buffered saline for 2 h at room temperature. Membranes were immunoblotted with anti-human IL-6R (1 μg/ml), anti-human gp130 (1 μg/ml), or biotinylated anti-phosphotyrosine (clone 4G10, 1 μg/ml; Upstate Biotechnology, Inc., Lake Placid, NY) antibodies. The reaction was developed by enhanced chemiluminescence (Amersham Biosciences) after using appropriate second step reagents conjugated with horseradish peroxidase-labeled reagents (Zymed Laboratories Inc., San Francisco, CA). N-Glycosidase F (New England Biolabs, Beverly, MA) was used to remove N-linked oligosaccharide chains from both IL-6R and gp130. The immunoprecipitated proteins were digested with N-glycosidase F according to the manufacturer's protocol and then analyzed using
Activation of HIV-1 requires the binding of host cell transcription factors to cis elements in the proviral long terminal repeat (LTR). This study identifies c-fos-responsive sequence motifs in the U5 transcribed noncoding leader sequences downstream of the viral transactivator responsive (TAR) element. These DNA sequence motifs are the most downstream regulatory elements described thus far in the HIV-1 LTR. Functional studies, using human colon epithelial cell lines, demonstrate that the downstream elements are transactivated by expression of the c-fos protooncogene and can transmit PMA and TNF alpha activation signals to the viral LTR. Moreover, the c-fos-responsive elements mediate HIV-1 LTR transcription independent of Tat and the NF kappa B-binding enhancer element. Nuclear extracts of colon epithelial cells form distinct gel mobility shift complexes with the c-fos-responsive elements. These complexes comigrate with a gel shift complex formed on a classical CRE oligonucleotide and are competed by CRE oligonucleotides. These data indicate that the HIV-1 LTR contains previously unrecognized functional DNA cis-regulatory elements downstream of TAR in the transcribed noncoding 5' leader sequence and suggest that early response genes such as c-fos play a role in the activation of HIV-1 gene expression.
The transcriptional enhancer of a chicken U1 small nuclear RNA gene has been shown to extend over approximately 50 base pairs of DNA sequence located 180 to 230 base pairs upstream of the U1 transcription initiation site. It is composed of multiple functional motifs, including a GC box, an octamer motif, and a novel SPH motif. The contributions of these three distinct sequence motifs to enhancer function were studied with an oocyte expression assay. Under noncompetitive conditions in oocytes, the SPH motif is capable of stimulating U1 RNA transcription in the absence of the other functional motifs, whereas the octamer motif by itself lacks this ability. However, to form a transcription complex that is stable to challenge by a second competing small nuclear RNA transcription unit, both the octamer and SPH motifs are required. The GC box, although required for full enhancer activity, is not essential for stable complex formation in oocytes. Site-directed mutagenesis was used to study the DNA sequence requirements of the SPH motif. Functional activity of the SPH motif is spread throughout a 24-base-pair region 3' of the octamer but is particularly dependent upon sequences near an SphI restriction site located at the center of the SPH motif. Using embryonic chicken tissue as a source material, we identified and partially purified a factor, termed SBF, that binds sequence specifically to the SPH motif of the U1 enhancer. The ability of this factor to recognize and bind to mutant enhancer DNA fragments in vitro correlates with the functional activity of the corresponding enhancer sequences in vivo.
Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-kappaB. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier function and promotion of leukocyte adhesion. Particular emphasis is placed on the regulation of cell-cell and cell-surface adhesion molecules, the actin cytoskeleton, key protein kinases, and signal transduction events.
