Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors.

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1alpha and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation.

Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration.

CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane-anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up-regulated in endothelial cells by proinflammatory cytokines such as IL-1 or TNF-alpha. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time- and concentration-dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT-PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease-activated receptor 1 (PAR1) as demonstrated by the use of PAR1-activating peptide and the PAR1-specific antagonist SCH 79797. The thrombin-induced CX3CL1 expression was NF-kappaB-dependent, as shown by EMSA, ELISA, and by inhibition of the NF-kappaB signaling pathway by the IkappaB kinase inhibitor acety-11-keto-beta-boswellic acid or by transient overexpression of a transdominant-negative form of IkappaBalpha. Upon cocultivation of human monocytes with HUVEC, the thrombin-dependent induction of membrane-anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP-1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin-induced monocyte/endothelial cell cross-talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas.

Azole antimycotics differentially affect rifampicin-induced pregnane X receptor-mediated CYP3A4 gene expression.

Azole antifungal drug ketoconazole has recently been demonstrated as an inhibitor of a ligand-induced pregnane X receptor (PXR)-mediated transcriptional regulation of the CYP3A4 gene through disruption of PXR interaction with steroid receptor coactivator (SRC)-1. In contrast, other clotrimazole-derived antifungal agents are known as potent inducers of CYP3A4 through PXR. In the present study, we examined effects of azole antimycotics clotrimazole, ketoconazole, econazole, oxiconazole, miconazole, fluconazole, and itraconazole on PXR-mediated expression of CYP3A4. We investigated individual effects of the tested azoles as well as their action on rifampicin-induced PXR-mediated transactivation and expression of CYP3A4 in LS174T cell line and primary human hepatocytes, their interactions with PXR ligand-binding domain, and azole-mediated recruitment of SRC-1 to PXR. In addition, applying the pharmacodynamic approach and dose-response analysis, we aimed to describe the nature of potential interactions of tested azole antimycotics coadministered with a prototypical PXR ligand rifampicin in transactivation of CYP3A4 gene. We describe additive and antagonistic interactions of partial and full agonists of PXR nuclear receptor in the therapeutic group of azole antimycotics in rifampicin-mediated transactivation of CYP3A4. We show that oxiconazole is a highly efficacious activator of CYP3A4 transactivation, which could be antagonized by rifampicin in a competitive manner. In addition, we show that activation of the CYP3A4 promoter is a complex process, which is not exclusively determined by azole-PXR interactions, and we suggest that the ability of some azoles to affect recruitment of SRC-1 to PXR modulates their net effects in transactivation of CYP3A4 both in the absence or presence of rifampicin.

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

Identification of the annexin A2 heterotetramer as a receptor for the plasmin-induced signaling in human peripheral monocytes.

We have previously demonstrated that plasmin acts as a potent proinflammatory activator of human peripheral monocytes. Here we identify the annexin A2 heterotetramer, composed of annexin A2 and S100A10, as a receptor for the plasmin-induced signaling in human monocytes. Monocytes express the annexin A2 heterotetramer on the cell surface as shown by flow cytometry, fluorescence microscopy, and coimmunoprecipitation of biotinylated cell surface proteins. Binding of plasmin to annexin A2 and S100A10 on monocytes was verified by biotin transfer from plasmin labeled with a trifunctional cross-linker. Antibodies directed against annexin A2 or S100A10 inhibited the chemotaxis elicited by plasmin, but not that induced by fMLP. Further, down-regulation of annexin A2 or S100A10 in monocytes by antisense oligodeoxynucleotides impaired the chemotactic response to plasmin, but not that to fMLP. Antisense oligodeoxynucleotides similarly decreased the TNF-alpha release by plasmin-stimulated, but not by LPS-stimulated, monocytes. At the molecular level, stimulation with plasmin, but not with catalytically inactivated plasmin, induced cleavage of annexin A2 and dissociation of the heterotetramer complex. Substitution of lysine to alanine in position 27 abolished the cleavage of recombinant annexin A2 in vitro. Together, these data identify the annexin A2 heterotetramer as a signaling receptor activated by plasmin via proteolysis.

The serine protease plasmin triggers expression of MCP-1 and CD40 in human primary monocytes via activation of p38 MAPK and janus kinase (JAK)/STAT signaling pathways.

The mechanism of proinflammatory activation of human monocytes by plasmin is unknown. Here we demonstrate that in human primary monocytes, plasmin stimulates mitogen-activated protein kinase (MAPK) signaling via phosphorylation of MAPK kinase 3/6 (MKK3/6) and p38 MAPK that triggers subsequent DNA binding of transcription factor activator protein-1 (AP-1). The AP-1 complex contained phosphorylated c-Jun and ATF2, and its DNA binding activity was blocked by the p38 MAPK inhibitor SB203580. In addition, plasmin elicits Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, as detected by phosphorylation of JAK1 tyrosine kinase and STAT1 and STAT3 proteins. Plasmin-induced DNA binding of STAT1 and STAT3 was blocked by SB203580 and AG490, inhibitors of p38 MAPK and JAK, respectively, but not by U0126, an inhibitor of MKK1/2. DNA binding of NF-kappaB remained unaffected by any of these inhibitors. The plasmin-induced signaling led to expression of monocyte chemoattractant protein-1 (MCP-1) and CD40, which required activation of both p38 MAPK and JAK/STAT signaling pathways. Additionally, signaling through both p38 MAPK and JAK is involved in the plasmin-mediated monocyte migration, whereas the formylmethionylleucylphenylalanine-induced chemotaxis remained unaffected. Taken together, our data demonstrate a novel function of the serine protease plasmin in a proinflammatory signaling network.

Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells.

Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain, unmasking a new N-terminus acting as tethered ligand. Whereas the role of PARs in platelets is well known, their presence and function in human monocytes and other antigen-presenting cells has not been characterized. Here it is demonstrated that human peripheral monocytes and monocyte-derived macrophages and dendritic cells differentially express PARs. Human monocytes express mainly PAR1 and less PAR3. Differentiation of monocytes into macrophages by either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) elicits enhanced expression of PAR1, PAR2, and PAR3. In contrast, dendritic cells differentiated from monocytes by GM-CSF and interleukin-4 (IL-4) strongly down-regulated PAR1, PAR2, and PAR3, both at the mRNA and the protein level. Down-regulation of the PAR expression was apparently due to IL-4, because treatment of macrophages with IL-4 caused down-regulation of PAR1, PAR2, and PAR3. PAR4 mRNA expression remained undetectable in any of the cell types investigated. Stimulation of PAR1, PAR2, and PAR3 with thrombin, trypsin, or established receptor-activating peptides (PAR-APs) triggered cytosolic Ca2+ responses, indicating functionally active PARs. Further, stimulation of monocytes or macrophages with thrombin or PAR1-AP, but not with PAR2-or PAR4-AP, triggers expression of monocyte chemoattractant protein-1 (MCP-1) both at the mRNA and the protein level. These data demonstrate that differentiation of human monocytes is associated with differential expression of functionally active PARs that mediate distinct regulatory functions in inflammation and atherogenesis.

Intracellular interleukin-1alpha functionally interacts with histone acetyltransferase complexes.

Interleukin-1alpha (IL-1alpha) is an inflammatory cytokine acting extracellularly via membrane receptors. Interestingly, a significant portion of synthesized IL-1alpha is not secreted; instead, it is actively translocated into the cell nucleus. IL-1alpha was indeed shown to be involved in certain intracellular processes, such as control of proliferation, apoptosis, or migration, however, the mechanisms of such actions are not known. Here we show that intracellular IL-1alpha fused to the Gal4p DNA-binding domain (Gal4BD) possesses strong transactivation potential that can be boosted by overexpression of the transcriptional coactivator p300. We demonstrate that the IL-1alpha precursor interacts via its N-terminal peptide (IL-1NTP) with histone acetyltransferases p300, PCAF, Gcn5 and with the adaptor component Ada3, and that it integrates into the PCAF.p300 complex in a non-destructive manner. In analogy with known acidic coactivators, yeast strains expressing Gal4BD/IL-1NTP display a toxic phenotype that can be relieved by depletion of various components of the SAGA complex. Our data provide the first solid evidence for the nuclear target of the IL-1alpha precursor and suggest its novel function in transcriptional control.