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.

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beta-Thymosins are the currently favored candidates for maintaining the large actin monomer (G-actin) pool in living cells. To determine if beta-thymosin behaves like a simple G-actin buffering agent in the complex environment of a cell, we overexpressed thymosin beta10 (Tbeta 10) in NIH3T3 cells and determined the effect on the monomer/polymer equilibrium. Tbeta 10 is the predominant beta-thymosin isoform in the NIH3T3 cell line, and it is present in approximately equal molar ratio to profilin and cofilin/actin depolymerizing factor, two other well characterized actin monomer binding proteins. Clonal cell lines that overexpressed three times more Tbeta 10 had 23-33% more polymerized actin than control cells, and the filaments appeared thicker after staining with fluorescent phalloidin. There was no change in total actin, profilin, and cofilin/actin depolymerizing factor content. The overexpressing cells were more motile; they spread faster and had higher chemotactic and wound healing activity. Assuming that there is no compensatory inactivation of the other classes of monomer binding proteins, our paradoxical observation can be accounted for quantitatively by a parallel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y., and Pantaloni, D., (1996) J. Biol. Chem. 271, 9231-9239). beta-Thymosin at levels comparable with that found in the overexpressing cells binds actin filaments and decreases the critical concentration (C(c)) for actin polymerization. This reduces the monomer buffering ability of beta-thymosin, so that above a certain threshold an incremental increase in thymosin does not lead to a corresponding increase in G-actin. Furthermore, the decrease in C(c) reduces the buffering capacity of the other actin monomer binding proteins. As a consequence, an increase in beta-thymosin does not necessarily result in a proportionate increase in actin monomer content in a complex environment containing other actin monomer binding proteins. The outcome depends on the level of beta-thymosin expression relative to the composition of the other actin monomer binding protein. Our results suggest that beta-thymosins are not simple actin buffering proteins and that their biphasic action may have physiological significance.

Abstract-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 Fc␥ receptors, causes parallel okadaic acid-sensitive loss of eNOS function, Fc␥RIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires Fc␥RIIB. In Fc␥RIIB ϩ/ϩ mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in Fc␥RIIB Ϫ/Ϫ mice. Thus Fc␥RIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction. (Circ Res. 2005;97:1124-1131.)Key Words: C-reactive protein Ⅲ endothelial NO synthase Ⅲ Fc␥ receptor Ⅲ PP2A C -reactive protein (CRP) is an acute-phase reactant and a member of the pentraxin family of proteins. Its hepatic synthesis is stimulated by interleukin-6 to yield levels that can rise 500-fold within 24 to 48 hours of the initiation of an inflammatory process. CRP serves as an opsonin and activates complement by binding to C1q. [1][2][3][4] In addition to participating in immune response, CRP has received considerable attention as a risk factor for cardiovascular disease. Although the relative predictive value of CRP versus other risk factors has been variable, the finding that CRP levels correlate with cardiovascular disease has been remarkably consistent across populations. [5][6][7][8][9] CRP is also a risk factor for the progression of subclinical vascular disease and for hypertension. 10,11 Furthermore, a primary effect of CRP on endothelium is plausible because elevated levels are associated with endothelial dysfunction, as evidenced by blunted forearm vascular responses to acetylcholine (Ach), which activates endothelial NO synthase (eNOS) to generate NO on L-arginine conversion to L-citrulline. 12 Potentially consistent with these clinical observations, CRP transgenic mice have exaggerated thrombosis, 13 and CRP blunts eNOS expression and function in cultured endothelial cells. 14,15 However, it has yet to be determined whether CRP has direct effects on vascular endothelium in vivo, and the basis for such effects is unknown.In the present study, we investigated the mechanisms underlying CRP actions on endothelium by testing the hypothesis that CRP attenuates eNOS activation in cultured endothelial cells. The resulting effect on monocyte adhesion was also determined. Because eNOS activation entails ...

SUMMARYC-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/EBPb -mediated IL-6-induced transcription and that Rel p50 acted synergistically with C/ EBPb, in the absence of p65, to enhance CRP transcription. Neither a requirement nor a binding site for the classic nuclear factor (NF)-kB 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/EBPb or STAT3 synergistically enhanced CRP expression. Maximal expression was observed with overexpression of all four transcription factors; comparable effects were observed with IL-1b treatment of cells overexpressing STAT3 C/EBPb. Data from the Human Genome Project revealed 13 potential kB sites in the ®rst 4000 bases of the CRP promoter, only one of which, centred at À2652, bound nuclear p50/p65 heterodimer activated by IL-1b. Our ®ndings indicate that classical NF-kB activation can participate in endogenous CRP induction, and that activated NF-kB may synergistically enhance the effects of C/EBPb and STAT3. They raise the possibility, not as yet established, that NF-kB activation may be responsible for the synergistic effect of IL-1b on IL-6-induced CRP expression.

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 κB 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/EBPβ-containing complexes in nuclei of IL-6-treated cells, indicating that ternary complexes containing C/EBPβ and p50 are formed on the CRP promoter. Depletion of free Rel proteins by pretreatment of nuclear extracts with a κB 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/EBPβ overexpression caused an 18-fold increase; simultaneous overexpression of both transcription factors increased luciferase activity ∼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 κB site enhances and stabilizes binding of C/EBPβ to the CRP promoter and that binding of both C/EBPβ and p50 to their overlapping cognate sites is required for induction of CRP expression by IL-6.

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the rat was isolated from a recombinant library containing the rat genome in phage A Charon 4A. The isolated clone, APCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and -7 kilobases (kb) of flanking sequence at the 5' end and 1 kb at the 3' terminus. Restriction endonuclease mapping, R-loop mapping, and partial DNA sequence assay indicate that the gene is -6.0 kb in length (coding for a mRNA of 2.8 kb) and contains eight introns. Southern blotting of rat DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of APCK1. A control region at the 5' end of the gene contained in a 1.2-kb restriction fragment was isolated and subcloned into pBR322. This segment of the gene contains the usual transcription start sequences and a 24-base sequence virtually identical to the sequence found in the 5'-flanking region of the human proopiomelonocortin gene, which is known to be regulated by glucocorticoids. The 1.2-kb fragment of the phosphoenolpyruvate carboxykinase gene can be transcribed into a unique RNA fragment of predicted size by an in vitro transcription assay.Phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32; P-enolpyruvate carboxykinase] is a key regulatory enzyme in hepatic gluconeogenesis, and its synthesis rate is controlled by a number of hormones including glucagon (acting through cAMP) (1, 2), cAMP (1), insulin (2, 3), epinephrine (4), glucocorticoids (5), and thyroid hormone (6). Of particular importance are: cAMP, which can induce the synthesis rate of P-enolpyruvate carboxykinase from rat liver cytosol 8-fold in 90 min (7); insulin, which causes an equally rapid deinduction of enzyme synthesis when administered to diabetic animals (2); and glucocorticoids, which also stimulate the synthesis of the enzyme (5). These rapid changes in the synthesis of hepatic P-enolpyruvate carboxykinase are accompanied by equally rapid changes in the sequence abundance of the mRNA coding for the enzyme (8,9

C-reactive protein (CRP), the prototypic acute-phase reactant in humans, is synthesized in liver in response to a wide variety of inf lammatory 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 ␣ (TNF-␣) plus interleukin 1␤, but not with TNF-␣ 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 CRPexpressing 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.