L.New and Y.Jiang contributed equally to this workWe have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38α and p38β both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo.

The urokinase plasminogen activator binds to its cellular receptor with high affinity and initiates signaling cascades that are implicated in pathological processes including tumor growth, metastasis, and inflammation. We report the crystal structure at 1.9 angstroms of the urokinase receptor complexed with the urokinase amino-terminal fragment and an antibody against the receptor. The three domains of urokinase receptor form a concave shape with a central cone-shaped cavity where the urokinase fragment inserts. The structure provides insight into the flexibility of the urokinase receptor that enables its interaction with a wide variety of ligands and a basis for the design of urokinase-urokinase receptor antagonists.

Tissue factor (TF) expression is associated with life-threatening thrombosis in a variety of human diseases, including sepsis, cancer, and atherosclerosis. Recently, it was shown that inactivation of the murine TF (mTF) gene results in embryonic lethality. To date, despite extensive studies on the regulation of the TF promoter in vitro, no studies have examined the cis-acting regulatory elements that control TF gene expression in vivo. Here we report that a human TF (hTF) minigene containing the human TF promoter and human TF cDNA directed a low level (approximately 1% relative to mouse TF) of both constitutive and LPS-inducible human TF expression in transgenic mice. Importantly, the human TF minigene rescued the embryonic lethality of murine TF null embryos, suggesting that human TF substituted for murine TF during embryogenesis. Rescued mice (mTF-/-, hTF+), which expressed low levels (approximately 1%) of TF activity, developed normally with no signs of a bleeding diathesis, suggesting that low TF expression can maintain hemostasis compatible with normal survival. These studies establish a novel mouse model system that can be used to examine the regulation of the human TF gene in vivo and the impact of low TF levels on the hemostatic balance in various thrombotic diseases.

Chemokines are potent mediators of cell migration and activation and therefore play an essential role in early events of inflammation. In conjunction with cell adhesion molecules, chemokines help to localize cells to a specific site and enhance the inflammatory reaction at the site. Clinically, elevated levels of chemokines have been found in a variety of inflammatory diseases. The prototype C-C chemokine is monocyte chemoattractant protein-1 (MCP-1) which is synthesized by variety of cell types including endothelial cells in response to a variety of stimuli. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we investigated the factors involved in cytokine-induced MCP-1 gene expression in human endothelial cells. We present evidence that the nuclear factor (NF)-kappa B-like binding site and the AP-1 binding site located 90 and 68 base pairs upstream of the transcriptional start site, respectively, are required for maximal induction of the human MCP-1 promoter by interleukin-(IL)-1 beta. Site-directed mutagenesis or deletion of the NF-kappa B-like site decreased the cytokine-induced activity of the promoter. Site-directed mutagenesis of the AP-1 binding site also decreased the cytokine-induced activity of the promoter. We show that the NF-kappa B-like site located at-90 in the MCP-1 promoter binds to the p50/p65 heterodimer of the NF-kappa B/Rel family in IL-1 beta-stimulated human endothelial cells. Overexpression of p65 results in the transactivation of the MCP-1 promoter as well. The data presented in this study suggest that cytokine-induced MCP-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1.

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Recognition of bacterial endotoxin (LPS) elicits multiple host responses, including activation of cells of the innate immune system. LPS exposure occurs repeatedly during septicemia, making strict regulation of gene expression necessary. Such regulation might prevent, for example, the continuous production of proinflammatory cytokines such as tumor necrosis factor (TNF), which could lead to severe vascular collapse. Tolerance to LPS is characterized by a diminished production of TNF during prolonged exposure to LPS, and is therefore likely to represent an essential control mechanism during sepsis. In the present study, which uses mice with genetic deletions of the proteins of NF-B complex, we provide data demonstrating that increased expression of the p50 subunit of NF-B directly results in the downregulation of LPS-induced TNF production. This contention is supported by the following observations: (1)

Tissue factor (TF) expression by endothelial cells is implicated in thrombotic episodes in patients with a variety of clinical disorders. In a baboon model of lethal sepsis, TF is expressed by endothelial cells in the splenic microvasculature. In vitro, endothelial cells are induced to express TF in response to tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and bacterial endotoxin (lipopolysaccharide [LPS]). Here, we identified cis-acting regulatory elements that control TF gene transcription in primary human endothelial cells. Functional studies showed that the TF promoter contained a 56-bp enhancer (-227 to -172 bp), which included two activator protein-1 (AP-1) sites and a kappa B-like site, that mediated induction by TNF-alpha, IL-1 beta, and LPS. Electrophoretic mobility shift assays demonstrated that endothelial cells contained constitutive AP-1 binding activity, whereas the kappa B-like site, 5'-CGGAGTTTCC-3', bound an inducible nuclear complex composed of c-Rel-p65 heterodimers. Taken together, our data suggest that induction of TF gene transcription in endothelial cells is mediated by functional interactions between Fos-Jun and c-Rel-p65 heterodimers.

The interaction of amyloid-beta (Aβ) and tau in the pathogenesis of Alzheimer's disease is a subject of intense inquiry, with the bulk of evidence indicating that changes in tau are downstream of Aβ. It has been shown however, that human tau overexpression in amyloid precursor protein transgenic mice increases Aβ plaque deposition. Here, we confirm that human tau increases Aβ levels. To determine if the observed changes in Aβ levels were because of intracellular or extracellular secreted tau (eTau for extracellular tau), we affinity purified secreted tau from Alzheimer's disease patient-derived cortical neuron conditioned media and analyzed it by liquid chromatography-mass spectrometry. We found the extracellular species to be composed predominantly of a series of N-terminal fragments of tau, with no evidence of C-terminal tau fragments. We characterized a subset of high affinity tau antibodies, each capable of engaging and neutralizing eTau. We found that neutralizing eTau reduces Aβ levels in vitro in primary human cortical neurons where exogenously adding eTau increases Aβ levels. In vivo, neutralizing human tau in 2 human tau transgenic models also reduced Aβ levels. We show that the human tau insert sequence is sufficient to cause the observed increase in Aβ levels. Our data furthermore suggest that neuronal hyperactivity may be the mechanism by which this regulation occurs. We show that neuronal hyperactivity regulates both eTau secretion and Aβ production. Electrophysiological analysis shows for the first time that secreted eTau causes neuronal hyperactivity. Its induction of hyperactivity may be the mechanism by which eTau regulates Aβ production. Together with previous findings, these data posit a novel connection between tau and Aβ, suggesting a dynamic mechanism of positive feed forward regulation. Aβ drives the disease pathway through tau, with eTau further increasing Aβ levels, perpetuating a destructive cycle.