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D Samols

Publications and source records attributed to D Samols.

At least 19 recordsLinked to original sources
PubMed2001

Transactivation of C-reactive protein by IL-6 requires synergistic interaction of CCAAT/enhancer binding protein beta (C/EBP beta) and Rel p50.

We have previously found that overexpression of the Rel protein p50 stimulated C-reactive protein (CRP) expression in Hep 3B cells and that p50 could bind to a nonconsensus kappaB site overlapping the CCAAT/enhancer binding protein (C/EBP) binding site centered at position -53 on the CRP promoter. Accordingly, we employed EMSA to investigate possible cooperation between p50 and C/EBP proteins using an oligonucleotide probe (-63/-41) derived from the CRP promoter and containing both C/EBP and p50 binding sites. Abs to p50, but not to p65, decreased formation of C/EBPbeta-containing complexes in nuclei of IL-6-treated cells, indicating that ternary complexes containing C/EBPbeta and p50 are formed on the CRP promoter. Depletion of free Rel proteins by pretreatment of nuclear extracts with a kappaB consensus oligonucleotide markedly decreased formation of C/EBP complexes, indicating that Rel proteins are required for formation of such complexes. Overexpression of p50 in transient cotransfection studies using the proximal CRP promoter (-125/+9) linked to a luciferase reporter caused a 3-fold increase of luciferase activity, while C/EBPbeta overexpression caused an 18-fold increase; simultaneous overexpression of both transcription factors increased luciferase activity approximately 600-fold. Mutation of either the C/EBP binding site or the p50 binding site drastically reduced the effects of overexpressed transcription factors. Taken together, our findings indicate that binding of Rel p50 to the nonconsensus kappaB site enhances and stabilizes binding of C/EBPbeta to the CRP promoter and that binding of both C/EBPbeta and p50 to their overlapping cognate sites is required for induction of CRP expression by IL-6.

Binding Sites↗
PubMed2000

The Rel family member P50 mediates cytokine-induced C-reactive protein expression by a novel mechanism.

Transcription of C-reactive protein (CRP) in Hep 3B cells is induced by IL-6, acting through C/EBP isoforms and STAT3. IL-1beta, which alone has no effect, greatly enhances IL-6-induced transcription by unknown mechanisms. Because IL-1beta activates the NF-kappaB system, we explored the effects of overexpressed Rel family members on CRP expression. Unexpectedly, transactivation assays in transiently transfected Hep 3B cells showed p50 overexpression to markedly induce CRP transcription, acting in a region 3' to -86. In the presence of overexpressed p50, IL-1beta induced a 3-fold increase in CRP expression, and responses to IL-6 and to IL-6 plus IL-1beta were 4-fold greater than seen in cells without p50 overexpression. In contrast, overexpressed p65 abolished CRP induction by p50 and by cytokines. EMSA studies demonstrated that recombinant p50 bound to a nonconsensus kappaB site overlapping the proximal C/EBP binding site on the CRP promoter. Mutation of a polypyrimidine tract in the p50-binding site inhibited the transactivating effect of cytokines. P50- but not p65-containing dimers were found in nuclei of Hep 3B cells 18 h after stimulation with IL-1beta, when C/EBPbeta is greatly activated, in the presence or absence of IL-6. These findings suggest that IL-1beta induces nuclear translocation of p50-containing dimers and that p50 interacts with C/EBPbeta activated by both IL-6 and IL-1beta to induce CRP expression.

3' Untranslated Regions↗
PubMed1999

Modulation of hepatic acute phase gene expression by epidermal growth factor and Src protein tyrosine kinases in murine and human hepatic cells.

As part of systemic inflammatory reactions, interleukin 6 (IL-6) induces acute phase protein (APP) genes through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Epidermal growth factor (EGF), which contributes to the regenerative process after liver injury and also activates STATs, does not induce but attenuates IL-6-stimulated expression of several APP genes in primary mouse hepatocytes. The APP-modifying action of EGF receptor (EGFR) was characterized in HepG2 cells. Although EGF less effectively engages STAT proteins in these cells, it reduces expression of fibrinogen and haptoglobin, but stimulates production of alpha(1)-antichymotrypsin and induces transcription through the alpha(1)-antichymotrypsin and C-reactive protein promoter. The stimulatory EGFR signal is insensitive to inhibition of JAKs and appears to involve Src kinases and STAT proteins as shown by inhibition through overexpression of C-terminal Src kinase (Csk) and transdominant negative STAT3, respectively. A mediator role of Src is supported by the ability of c-Src and v-Src to activate STATs and induce transcription through APP promoters. Src kinases have been observed in association with the IL-6 receptor; however, inhibition of Src kinases by Csk enhances IL-6-induced transcription. The Csk effect is attributed to prevention of Src kinases from phosphorylating gp130 at the docking site for the signal-moderating protein tyrosine phosphatase SHP-2. The inhibitory EGFR signal on APP expression correlates with the activation of Erk1 and Erk2. The study shows a dual signaling function for EGFR and suggests that the ratio of receptor-activated STATs and Erks influence the level of stimulated or inhibited expression of individual APPs.

Acute-Phase Proteins↗
PubMed1997

Transgenic mice expressing rabbit C-reactive protein are resistant to endotoxemia.

C-reactive protein (CRP), the prototypic acute-phase reactant in humans, is synthesized in liver in response to a wide variety of inflammatory stimuli. We have generated a line of transgenic mice that express rabbit CRP from the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter in response to gluconeogenic signals. Here we show that transgenic mice expressing high levels of CRP were partially protected from a lethal challenge of bacterial lipopolysaccharide compared with littermates in which CRP expression had been suppressed. Similar protection was observed with challenges from platelet-activating factor (PAF) and the combination of tumor necrosis factor alpha (TNF-alpha) plus interleukin 1beta, but not with TNF-alpha alone. We further demonstrate that although PAF was able to bind CRP, the mechanism by which CRP provides protection probably does not involve sequestration of PAF. The biologically inactive precursor of PAF, lyso-PAF, also bound CRP but did not render the transgenic mice sensitive to PAF when CRP-expressing animals were simultaneously challenged with PAF and an excess of lyso-PAF. These results suggest that CRP functions in vivo by modulating host defense systems.

Animals↗
PubMed1996

STAT3 participates in transcriptional activation of the C-reactive protein gene by interleukin-6.

Interleukin-6 (IL-6) is the major cytokine inducing transcription of human C-reactive protein (CRP) during the acute phase response. STAT (signal transducers and activators of transcription) family members, recently shown to be important mediators of the effects of many cytokines including IL-6, generally induce their effects by binding to palindromic sequences with TT(N)5AA motifs. We report an IL-6 responsive element in the proximal region of the human CRP 5'-flanking region that bears a TT(N)4AA motif, which we have termed CRP acute phase response element (CRP-APRE). In Hep3B cells, IL-6 but not interferon-gamma was capable of activating CAT constructs driven by the CRP promoter containing CRP-APRE. Overexpressed STAT3 was able to transactivate CRP-chloramphenicol acetyltransferase constructs through the CRP-APRE and was able to enhance endogenous CRP mRNA accumulation in response to IL-6. STAT3 (or an antigenically related molecule) bound to the CRP-APRE in response to IL-6. Overexpression of STAT3 in the presence of IL-6 was capable of inducing expression of a construct consisting of the CRP-APRE and a minimal thymidine kinase promoter lacking a C/EBP site. Taken together, these findings indicate that STAT3 participates in the transcriptional activation of CRP in response to IL-6.

Acute-Phase Proteins↗
PubMed1996

Cloning, expression, and characterization of polyphosphate glucokinase from Mycobacterium tuberculosis.

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using polyphosphate or ATP as the phosphoryl donor. The M. tuberculosis H37Rv gene encoding this enzyme has been cloned, sequenced, and expressed in Escherichia coli. The gene contains an open reading frame for 265 amino acids with a calculated mass of 27,400 daltons. The recombinant polyphosphate glucokinase was purified 189-fold to homogeneity and shown to contain dual enzymatic activities, similar to the native enzyme from H37Ra strain. The high G+C content in the codon usage (64.5%) of the gene and the absence of an E. coli-like promoter consensus sequence are consistent with other mycobacterial genes. Two phosphate binding domains conserved in the eukaryotic hexokinase family were identified in the polyphosphate glucokinase sequence, however, "adenosine" and "glucose" binding motifs were not apparent. In addition, a putative polyphosphate binding region is also proposed for the polyphosphate glucokinase enzyme.

Adenosine↗
PubMed1996

C-reactive protein and serum amyloid A mRNA stability following induction by cytokines.

We determined the effects of cytokine withdrawal on C-reactive protein (CRP) and serum amyloid A (SAA) mRNA abundance in Hep3B cells following 24 h of preinduction with interleukin 6 plus interleukin 1 beta. After cytokine withdrawal, CRP transcription rate rapidly fell to undetectable levels and mRNA levels fell with a half-disappearance time of about 2.5 h. In view of the relatively small amount of CRP transcription occurring at this time, it is likely that this value closely reflects the actual half-life of CRP mRNA. In contrast, substantial SAA transcription continued for at least 8 h, while SAA mRNA fell with a half-disappearance time of about 8.5 h. It is not possible, under these conditions, to determine SAA mRNA half-life, but it clearly was no greater than 8.5 h. Both Actinomycin D (ActD) and cycloheximide enhanced the stability of SAA mRNA, strongly suggesting that SAA mRNA degradation requires synthesis of a short-lived protein. CRP mRNA stability was also enhanced by ActD, but cycloheximide did not have a protracted stabilizing effect, suggesting complex regulatory processes. These studies provide insight into the stability of CRP and SAA mRNA following induction with [IL-6 + IL-1 beta] and into the mechanisms regulating their degradation.

Blotting, Northern↗
PubMed1996

Transgenic mice expressing rabbit C-reactive protein exhibit diminished chemotactic factor-induced alveolitis.

The acute phase protein, C-reactive protein (CRP), can increase more than a thousandfold during acute inflammatory states, and it is known to modulate neutrophil-mediated inflammatory responses. We have previously shown that CRP inhibits chemotaxis of C5a-stimulated neutrophils in vitro and that rabbits with elevated CRP blood levels exhibit diminished pulmonary vascular permeability and neutrophil infiltration in a model of alveolitis. To study the effect of CRP on alveolitis induced by different chemoattractants, transgenic mice capable of expressing rabbit CRP in a dietary-inducible fashion were treated with inflammatory doses of the chemoattractants. Intratracheal installation of FMLP (8 x 10(-10) mol), LTB4 (2 x 10(-11) mol), or IL-8 (5 x 10(-12) mol) in normal CF1 mice resulted in significant (p<0.05) influx of neutrophils and protein into the alveolar space. Transgenic mice with elevated plasma levels of CRP showed significantly (p<0.05) diminished infiltration of neutrophils into bronchoalveolar lavage fluid (BALF) and significant reduction in BALF protein compared with that in normal mice. Rabbit CRP (10 to 500 micrograms/ml) inhibited in vitro neutrophil chemotaxis in a concentration-dependent fashion when stimulated by the various chemoattractants examined. These data show that rabbit CRP can modify both in vivo and in vitro neutrophil responses to several classes of chemoattractants and that CRP has a significant protective effect in alveolitis by reducing neutrophil influx and protein leakage into the lung.

Animals↗
PubMed1995

The effect of interleukin-1 on C-reactive protein expression in Hep3B cells is exerted at the transcriptional level.

The combination of interleukin 6 (IL-6) and interleukin 1 (IL-1) synergistically induces the human acute-phase reactant, C-reactive protein (CRP) in Hep3B cells. While previous studies have indicated that IL-6 induces transcription of CRP, the mode of action of IL-1 has not been clearly defined. It has been suggested that the effect of IL-1 might be post-transcriptional, exerted through the 5'-untranslated region (5'-UTR). To evaluate the role of IL-1 in CRP gene expression, we studied the effects of interleukin-6 (IL-6) and interleukin-1 beta (IL-1 beta) on both the endogenous CRP gene and on transfected CRP-CAT constructs in Hep3B cells. In kinetic studies of the endogenous CRP gene, IL-1 beta alone had no effect on CRP mRNA levels, but when added to IL-6, synergistically enhanced both CRP mRNA levels and transcription, as determined by Northern-blot analyses and nuclear run-on studies. IL-6 alone and the combination of [IL-1 beta + IL-6] each induced increases in mRNA levels roughly comparable with observed increases in transcription. These findings indicate that the effect of IL-1 beta on CRP expression is exerted largely at the transcriptional level in this system. This conclusion was confirmed by studies in Hep3B cells transiently transfected with CRP-CAT constructs, each containing 157 bp of the CRP 5'-flanking region but differing in the length of the 5'-UTR from 104 bp to 3 bp. All constructs responded in the same way; IL-6, but not IL-1 beta, induced significant chloramphenicol acetyltransferase (CAT) expression which was synergistically enhanced 2- to 3-fold by IL-1 beta. These results indicate that IL-1 beta stimulates transcriptional events in the presence of IL-6 and that the upstream 157 bases of the CRP promoter contain elements capable of both IL-6 induction and the synergistic effect of IL-1 beta on transcription.

C-Reactive Protein↗
PubMed1995

Induction of human serum amyloid A in Hep 3B cells by IL-6 and IL-1 beta involves both transcriptional and post-transcriptional mechanisms.

Previous studies of murine serum amyloid A (SAA) regulation during inflammatory states or following exposure to macrophage-conditioned medium have raised the possibility that both post-transcriptional and transcriptional mechanisms participate in induction of this family of proteins. Since IL-6 and IL-1 have been shown to induce SAA in human hepatoma cell lines, we explored the possibility that these cytokines might induce human SAA through post-transcriptional as well as transcriptional mechanisms. In kinetic studies, we found that continuous exposure of Hep 3B cells to either IL-6 or IL-1 beta alone caused only minimal increases in SAA mRNA and marginal increases in transcription (as measured by nuclear runon). In contrast, the combination of these cytokines led to a 23-fold increase in transcription, maximal at 12 h, with continuing increase in mRNA, achieving levels more than 1,000-fold greater than baseline by 72 h. This massive disparity between increases in mRNA and in transcription rate strongly supports the participation of post-transcriptional mechanisms in SAA induction by (IL-6 + IL-1 beta), whereas the lag between peaks of transcription and mRNA abundance reflects a relatively slow degradation rate of SAA mRNA. As observed by other workers, mean size of SAA mRNA decreased progressively over the course of incubation. Simultaneous kinetic studies of complement factors B and C3, haptoglobin, and alpha-1 protease inhibitor revealed several different patterns of response to IL-6 and IL-1 beta.

Blotting, Northern↗
PubMed1995

Expression of rabbit C-reactive protein in transgenic mice.

C-reactive protein (CRP) is a prototypic acute phase reactant in humans and rabbits whose serum concentration can increase up to 1000-fold following an acute inflammatory stimulus. CRP binds to many phosphate ester-containing compounds including phosphorylcholine, nucleotides, chromatin and snRNP. To examine the in vivo function of this protein, we produced transgenic mice capable of significant CRP synthesis. In contrast to most other vertebrates, mice synthesize CRP in only trace amounts. The transgenic animals express rabbit CRP from either the phosphoenolpyruvate carboxykinase promoter (PEPCK-CRP) or the mouse metallothionein I promoter (MT-CRP). Manipulating the diet in one of the PEPCK-CRP lines led to a rise in serum CRP levels from < 5 mu g/mL to 100-200 mu g/mL over a period of 2 days. The two MT-CRP lines examined expressed CRP constitutively which could be further elevated 2-4-fold following an inflammatory stimulus. Transgenic CRP bound phosphorylcholine was pentameric, had a circulating half-life of 30-60 min and was capable of activating mouse complement when bound to a ligand. We conclude that these transgenic lines express CRP with many of the properties of authentic rabbit CRP, and that the expression of CRP can be controlled to be dependent or independent of the acute phase response.

Acute-Phase Reaction↗
PubMed1995

Kinetic modeling and mathematical analysis indicate that acute phase gene expression in Hep 3B cells is regulated by both transcriptional and posttranscriptional mechanisms.

To evaluate the possible role of posttranscriptional mechanisms in the acute phase response, we determined the kinetics of transcription (by nuclear run-on assay) and mRNA accumulation of five human acute phase genes in Hep 3B cells incubated with conditioned medium from LPS-stimulated monocytes. Increase in mRNA accumulation was comparable to increase in transcription rate for fibrinogen-alpha and alpha-1 protease inhibitor, suggesting largely transcriptional regulation. In contrast, mRNA accumulation was about 10-20-fold greater than transcriptional increase for serum amyloid A, C3, and factor B, suggesting participation of posttranscriptional mechanisms. Since finding a disparity between the magnitudes of increase in mRNA and transcription does not definitively establish involvement of posttranscriptional mechanisms, we subjected our data to modeling studies and dynamic mathematical analysis to evaluate this possibility more rigorously. In modeling studies, accumulation curves resembling those observed for these three mRNAs could be generated from the nuclear run-on results only if posttranscriptional regulation was assumed. Dynamic mathematical analysis of relative transcription rates and relative mRNA abundance also strongly supported participation of posttranscriptional mechanisms. These observations suggest that posttranscriptional regulation plays a substantial role in induction of some, but not all acute phase proteins.

Acute-Phase Reaction↗
PubMed1994

Two carboxylesterases bind C-reactive protein within the endoplasmic reticulum and regulate its secretion during the acute phase response.

We have previously reported that C-reactive protein (CRP) is normally synthesized by rabbit hepatocytes at relatively low rates and is retained in the endoplasmic reticulum (ER), apparently by specific interaction with a 60-kDa lumenal ER protein. During the acute phase response to tissue injury, a marked increase in CRP synthesis is associated with a decrease in the CRP binding capacity of the 60-kDa protein, with accompanying rapid secretion of CRP. In the present studies, we purified two 60-kDa ER lumenal glycoproteins (referred to as gp60a and gp60b) capable of binding CRP. gp60b, though present at only 5% the level of gp60a, was found to account for 80% of the total CRP binding capacity. Amino-terminal amino acid sequence analysis and biochemical characterization identified gp60a and gp60b as two microsomal carboxylesterases previously reported by others to contain COOH-terminal ER retention signals (HIEL and HTEL). The CRP binding activities of gp60a and gp60b were found to be independent of their esterase activities. In animals undergoing the acute phase response, the levels of gp60a and gp60b were diminished by about 50%, but the CRP binding capacities were reduced by 4-6-fold for gp60a and 25-30-fold for gp60b. These findings indicate that CRP is normally retained within the ER via interaction with gp60a and gp60b, while during the acute phase response a decrease in the CRP binding affinity of these proteins, particularly gp60b, results in efficient secretion of CRP.

Acute-Phase Reaction↗
PubMed1994

Inhibition of C5a des Arg-induced neutrophil alveolitis in transgenic mice expressing C-reactive protein.

C-reactive protein (CRP) is the classic acute phase reactant in man with serum levels elevated up to 1,000-fold after the onset of inflammation. CRP inhibits chemotaxis of complement (C5a)-stimulated neutrophils in vitro and rabbits with elevated serum CRP levels exhibit diminished neutrophil infiltration and vascular permeability in a model of C5a-induced alveolitis. To specifically evaluate the effect of CRP on C5a-induced neutrophil inflammation in vivo, experiments were performed in transgenic mice capable of expressing rabbit CRP in an inducible fashion. After direct instillation of a known inflammatory agent (C5a des Arg) into the airways, transgenic mice with high plasma levels of CRP showed significantly diminished infiltration of neutrophils into bronchoalveolar lavage fluid (BALF) and a significant reduction of BALF total protein levels compared with normal mice. These data indicate that CRP can diminish lung injury by a reduction in neutrophil influx and protein leakage into alveoli following complement-induced inflammation.

Animals↗
PubMed1993

Identification and characterization of a factor which is essential for assembly of transcarboxylase.

Transcarboxylase (TC) from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. It is composed of a central, hexameric 12S subunit with six outer, dimeric 5S subunits held in a stable 26S complex by twelve 1.3S biotinyl subunits. Each of these subunits has been cloned from the P. shermanii genome and expressed in Escherichia coli. The purified, expressed recombinant proteins are all indistinguishable from their authentic counterparts except for the recombinant 5S subunit (termed 5S WT), which does not form TC complexes or catalyze the overall transcarboxylase reaction. Circular dichroism and isoelectric focusing suggested differences existed between the authentic and E. coli-expressed 5S proteins. HPLC gel filtration was used to separate the authentic 5S dimer from additional components in the preparation. 5S dimer thus purified was unable to form TC complexes or catalyze the overall reaction, behaving identically to the recombinant 5S WT subunit. Fractions from the HPLC gel-filtration purification of authentic 5S were then added to 5S WT or 5S dimer, and one fraction was identified which catalyzed the assembly of TC complexes with either 5S preparation. This assembly activity was shown to be dependent on the concentration of this HPLC fraction. Assembly-promoting factor (APF) is heat-stable and probably a protein, on the basis of its protease susceptibility. Studies with APF and the other TC subunits demonstrate its ability to promote complex formation with 12S and 1.3S subunits. Since the APF was purified from crystals of 26S TC, we believe it to be a novel, previously unidentified subunit of transcarboxylase.

Biotin↗