Biomedical subjects

David Samols

Publications and source records attributed to David Samols.

10 recordsLinked to original sources

PubMed2006

What does minor elevation of C-reactive protein signify?

Reports of the predictive value of minor elevation of serum C-reactive protein (CRP) levels (between 3 and 10 mg/L) for atherosclerotic events have generated considerable interest, as well as a degree of controversy and confusion. CRP concentrations in this range are found in about one third of the American population. To better understand the mechanisms underlying minor elevation of CRP, we have surveyed its reported associations with a variety of states and conditions. It has become clear that even minimal environmental irritants and inflammatory stimuli elicit a minor CRP response. Minor CRP elevation has been found associated with a number of genetic polymorphisms, with membership in different demographic and socioeconomic groups, with a variety of dietary patterns and with many medical conditions that are not apparently inflammatory. Finally, minor CRP elevation bears negative prognostic implications for many conditions, particularly age-related diseases, and predicts mortality in both diseased and apparently healthy individuals. In sum, minor CRP elevation is associated with a great many diverse conditions, some of which are, or may prove to be, causal. Many of these reported associations imply a mild degree of tissue stress or injury, suggesting the hypothesis that the presence of distressed cells, rather than a resulting inflammatory response, is commonly the stimulus for CRP production.

Biomarkers↗

PubMed2005

FcgammaRIIB mediates C-reactive protein inhibition of endothelial NO synthase.

C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fcgamma receptors, causes parallel okadaic acid-sensitive loss of eNOS function, FcgammaRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcgammaRIIB. In FcgammaRIIB(+/+) mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcgammaRIIB(-/-) mice. Thus FcgammaRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.

Acetylcholine↗

PubMed2005

An intact phosphocholine binding site is necessary for transgenic rabbit C-reactive protein to protect mice against challenge with platelet-activating factor.

C-reactive protein (CRP), an acute phase protein in humans and rabbits, is part of the innate immune system. The role of CRP in host defense has been thought to be largely due to its ability to bind phosphocholine, activate complement, and interact with IgGRs (FcgammaRs). We have shown previously that transgenic rabbit CRP (rbCRP) protects mice from lethal challenges with platelet-activating factor (PAF). To investigate the mechanism of this protection, we created additional lines of transgenic mice that express either wild-type rbCRP, a variant of rbCRP with altered complement activation activity (Y175A), or a variant of rbCRP unable to bind phosphocholine (F66Y/E81K). In the current study, these lines were challenged with a single injection of PAF and their survival monitored. Mice expressing wild-type and Y175A rbCRP were protected against challenge by PAF whereas mice expressing F66Y/E81K rbCRP were not. Treatment with cobra venom factor did not affect survival, confirming the results with the Y175A rbCRP variant and indicating that complement activation was not required to mediate protection. Both wild-type rbCRP and Y175A rbCRP were capable of binding PAF in vitro whereas F66Y/E81K rbCRP was not. Although other interpretations are possible, our results suggest that the protective effect of rbCRP against PAF is due to sequestration of PAF.

Animals↗

PubMed2005

Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer?

OBJECTIVE: Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied. METHODS AND RESULTS: Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein-activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP expression, whereas rbCRP-positive males had significantly higher serum cholesterol levels than the rbCRP-negative counterparts. All mice exhibited extensive atherosclerotic lesions, as studied en face, and no differences were noted between rbCRP-negative and rbCRP-positive animals. Atherosclerotic luminal obstruction of aortic arch and first-order neck branches did not differ significantly between rbCRP-positive and rbCRP-negative mice. There was no correlation between rbCRP levels and atherosclerotic lesion formation. CONCLUSIONS: No marked effect of rbCRP on the formation of moderately advanced atherosclerotic lesions could be discerned in the apoE knockout mouse. Because of the oddities of the mouse complement system, however, this may not be a good model to investigate the role of CRP in human atherosclerosis.

Animals↗

PubMed2004

C-reactive Protein.

C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein, with homologs in vertebrates and many invertebrates, that participates in the systemic response to inflammation. Its plasma concentration increases during inflammatory states, a characteristic that has long been employed for clinical purposes. CRP is a pattern recognition molecule, binding to specific molecular configurations that are typically exposed during cell death or found on the surfaces of pathogens. Its rapid increase in synthesis within hours after tissue injury or infection suggests that it contributes to host defense and that it is part of the innate immune response. Recently, an association between minor CRP elevation and future major cardiovascular events has been recognized, leading to the recommendation by the Centers for Disease Control and the American Heart Association that patients at intermediate risk of coronary heart disease might benefit from measurement of CRP. This review will largely focus on our current understanding of the structure of CRP, its ligands, the effector molecules with which it interacts, and its apparent functions.

Animals↗

PubMed2004

SREBP-1c and Sp1 interact to regulate transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) in the liver.

The sterol regulatory element-binding protein-1c (SREBP-1c), as well as SREBP-1a and SREBP-2, inhibit transcription of the gene encoding the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C). There are two SREBP regulatory elements (SREs) in the PEPCK-C gene promoter (-322 to -313 and -590 to -581). The SRE at -590 overlaps an Sp1 site on the opposite strand of the DNA. These SREs bound SREBP-1a and SREBP-1c with low affinity but the addition of purified upstream stimulatory activity enhanced the binding of SREBP-1 to both of these sites. Mutating these SREs increased both unstimulated (5-fold) and protein kinase A-stimulated transcription (8-27-fold) from the PEPCK-C gene promoter; this was lost when both SREs were mutated. The SRE at -590 differs by a single base pair from the SRE in the low density lipoprotein (LDL) receptor gene (T in the PEPCK-C gene promoter at -582, compared with an A in the SRE of the gene for the LDL receptor promoter). Introduction of the LDL receptor SRE into the PEPCK-C gene promoter increased SREBP-1c binding and caused a 10-fold enhancement of basal transcription from the promoter, rather than an inhibition as observed with the SRE in the PEPCK-C gene promoter. The T/A change does not alter the binding of Sp1 to its site on the opposite strand of the DNA. Sp1 bound to the promoter independently of SREBP-1c but competed with SREBP-1c for binding. Sp1 does not bind to the SRE at -322. Chromatin immunoprecipitation analysis, using rat hepatocytes, demonstrated that SREBP-1 and Sp1 were associated in vivo with putative regulatory regions corresponding to the SREs in the PEPCK-C gene promoter. We propose that insulin represses transcription of the gene for PEPCK-C by inducing SREBP-1c production in the liver, which interferes with the stimulatory effect of Sp1 at -590 of the PEPCK-C gene promoter.

Animals↗

PubMed2003

The phosphocholine and the polycation-binding sites on rabbit C-reactive protein are structurally and functionally distinct.

C-reactive protein (CRP) is an acute phase protein in humans and rabbits that has the ability to bind a number of biologically important ligands including phosphocholine (PCh), histones, and polycations. In addition to this recognition function, ligand-complexed or aggregated CRP is capable of activating the classical complement pathway. We have generated two strains of transgenic mice in order to study CRP-binding to PCh and consequent complement activation. Based on crystallographic and mutagenesis studies in human CRP (huCRP), we mutated Phe66 and Glu81 in the rabbit CRP (rbCRP) gene and generated a strain of transgenic mice (F66Y/E81K), which expressed this variant form of rbCRP. We also mutated Tyr175 in rbCRP to generate transgenic mice which expressed a variant form of rbCRP (Y175A). In vitro, F66Y/E81K rbCRP purified from serum had dramatically reduced binding to PCh. Additionally F66Y/E81K rbCRP not only maintained its ability to bind polycations and histones, but also bound more avidly to specific histones and lysine polymers than wild type (wt) rbCRP. Y175A rbCRP was not able to activate complement when bound to pneumococcal C-polysaccharide (PnC), but was, along with F66Y/E81K and wild type rbCRP, able to activate complement when bound to a small lysine polymer or when directly adsorbed to a solid phase. This complement activation presumably occurs through the classical complement pathway as the three rbCRPs, adsorbed to a solid phase, bound C1q. Taken together, our results demonstrate that the PCh-binding and the polycation-binding sites on rbCRP are distinct but possibly overlapping. The conformational changes in the C1q-binding site of CRP to activate complement depend on the nature of the ligand and on the location of the ligand-binding site.

Amino Acid Substitution↗

PubMed2003

Transcription factor c-Rel enhances C-reactive protein expression by facilitating the binding of C/EBPbeta to the promoter.

Induction of C-reactive protein (CRP) synthesis in hepatocytes by cytokines occurs at the transcriptional level. In Hep3B cells, the transcription factors C/EBPbeta, STAT3, and Rel p50 have been shown to participate in this process. A C/EBP binding site centered at -53 and an overlapping nonconsensus kappaB site on the promoter are critical for CRP expression. We have previously found that an oligonucleotide containing a kappaB site diminished binding of C/EBPbeta to the C/EBP site, suggesting that unidentified Rel proteins present in Hep3B nuclei facilitate the formation of C/EBPbeta-complexes. The current studies were undertaken to determine which of the five Rel proteins, p50/p65/p52/c-Rel/RelB, play such a role. Mutation of the nonconsensus kappaB site did not abolish binding of C/EBPbeta to its binding site, indicating that this site was not necessary for the formation of C/EBPbeta-complexes. Depletion of Rel proteins from Hep3B nuclei led to decreased formation of C/EBPbeta-complexes on a CRP promoter-derived oligonucleotide that contained only the intact C/EBP binding site but not the nonconsensus kappaB site. This finding indicates that Rel proteins are involved in the binding of C/EBPbeta to its binding site by a kappaB site-independent mechanism. Electrophoretic mobility shift assays (EMSAs) revealed that it was c-Rel that facilitated formation of C/EBPbeta-complexes and that c-Rel bound directly to C/EBPbeta-complexes formed on the C/EBP site. Cotransfection of c-Rel enhanced the induction of CRP promoter-driven luciferase activity and enhanced endogenous CRP expression in cells transfected with C/EBPbeta. We conclude that c-Rel regulates CRP expression without the requirement of binding to a kappaB site, and binds directly to C/EBPbeta to facilitate the binding of C/EBPbeta to the CRP promoter.

Animals↗

PubMed2003

Overexpressed nuclear factor-kappaB can participate in endogenous C-reactive protein induction, and enhances the effects of C/EBPbeta and signal transducer and activator of transcription-3.

C-reactive protein (CRP), the prototypical human acute phase protein, is produced primarily by hepatocytes. Its expression is modestly induced by interleukin (IL)-6 in Hep3B cells while IL-1, which alone has no effect, synergistically enhances the effects of IL-6. In previous studies of the proximal CRP promoter, we found that signal transducer and activator of transcription-3 (STAT3) and C/EBPbeta -mediated IL-6-induced transcription and that Rel p50 acted synergistically with C/EBPbeta, in the absence of p65, to enhance CRP transcription. Neither a requirement nor a binding site for the classic nuclear factor (NF)-kappaB heterodimer p50/p65 were found. The current studies were undertaken to determine whether similar novel transcription factor interactions might regulate the endogenous CRP gene. Transiently overexpressed p50 or p65 induced CRP mRNA accumulation in Hep3B cells. The heterodimer p50/p65 was markedly more effective than p50 or p65 homodimers. Co-overexpression of p50 or p65 with C/EBPbeta or STAT3 synergistically enhanced CRP expression. Maximal expression was observed with overexpression of all four transcription factors; comparable effects were observed with IL-1beta treatment of cells overexpressing STAT3 + C/EBPbeta. Data from the Human Genome Project revealed 13 potential kappaB sites in the first 4000 bases of the CRP promoter, only one of which, centred at -2652, bound nuclear p50/p65 heterodimer activated by IL-1beta. Our findings indicate that classical NF-kappaB activation can participate in endogenous CRP induction, and that activated NF-kappaB may synergistically enhance the effects of C/EBPbeta and STAT3. They raise the possibility, not as yet established, that NF-kappaB activation may be responsible for the synergistic effect of IL-1beta on IL-6-induced CRP expression.

C-Reactive Protein↗

PubMed2002

A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide.

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.

Amino Acid Substitution↗