Zhixing K. Pan

Bradykinin stimulates NF-kappaB activation and interleukin 1beta gene expression in cultured human fibroblasts.

Bradykinin (BK), a pluripotent nonameric peptide, is known for its proinflammatory functions in both tissue injury and allergic inflammation of the airway mucosa and submucosa. To understand the mechanisms by which BK serves as an inflammatory mediator, the human lung fibroblast cell line WI-38 was stimulated with BK and the expression of IL-1beta gene was examined. BK at nanomolar concentrations induced a marked increase in immunoreactive IL-1beta, detectable within 2 h in both secreted and cell-associated forms. BK-induced IL-1beta synthesis was inhibited by a B2-type BK receptor antagonist and by treatment of the cells with pertussis toxin, indicating the involvement of a BK receptor that couples to the G(i)/G(o) class of heterotrimeric G proteins. Whereas cycloheximide and actinomycin D both inhibited BK-induced IL-1beta synthesis, results from Northern blot and nuclear run-on assays suggested that BK acted primarily at the transcription level which led to the accumulation of IL-1beta message in stimulated cells. Gel mobility shift assays were used with nuclear extracts from stimulated WI-38 cells to examine the transcription mechanism for BK-induced IL-1beta expression. A DNA binding activity specific for the decameric kappaB enhancer was detected and was found to contain the p50 and p65 subunits of the NF-kappaB/rel protein family. BK-induced NF-kappaB activation correlated with IL-1beta message upregulation with respect to agonist concentration, time course, sensitivity to bacterial toxins, and blockade by the B2 receptor antagonist. After BK stimulation, a significant increase in the activity of chloramphenicol acetyltransferase was observed in WI-38 cells transfected with a reporter plasmid bearing the kappaB enhancers from the IL-1beta gene. Deletion of the kappaB enhancer sequence significantly reduced BK-induced chloramphenicol acetyltransferase activity. These findings suggests a novel function of BK in the activation of NF-kappaB and the induction of cytokine gene expression.

Activation of Raf/MEK/ERK/cPLA2 Signaling Pathway Is Essential for Chlamydial Acquisition of Host Glycerophospholipids

Chlamydiae, a diverse group of obligate intracellular pathogens replicating within cytoplasmic vacuoles of eukaryotic cells, are able to acquire lipids from host cells. Here we report that activation of the host Raf-MEK-ERK-cPLA2 signaling cascade is required for the chlamydial uptake of host glycerophospholipids. Both the MAP kinase pathway (Ras/Raf/MEK/ERK) and Ca2+-dependent cytosolic phospholipase A2 (cPLA2) were activated in chlamydia-infected cells. The inhibition of cPLA2 activity resulted in the blockade of the chlamydial uptake of host glycerophospholipids and impairment in chlamydial growth. Blocking either c-Raf-1 or MEK1/2 activity prevented the chlamydial activation of ERK1/2, leading to the suppression of both chlamydial activation of the host cPLA2 and uptake of glycerophospholipids from the host cells. The chlamydia-induced phosphorylation of cPLA2 was also blocked by a dominant negative ERK2. Furthermore, activation of both ERK1/2 and cPLA2 was dependent on chlamydial growth and restricted within chlamydia-infected cells, suggesting an active manipulation of the host ERK-cPLA2 signaling pathway by chlamydiae.

Extracellular Signal–Regulated Kinase Signaling Pathway Regulates Breast Cancer Cell Migration by Maintaining slug Expression

Cell migration is a critical step in cancer cell invasion. Recent studies have implicated the importance of the extracellular signal-regulated kinase (ERK) signaling pathway in cancer cell migration. However, the mechanism associated with ERK-regulated cell migration is poorly understood. Using a panel of breast cancer cell lines, we detected an excellent correlation between ERK activity and cell migration. Interestingly, we noticed that a 48-hour treatment with U0126 [specific mitogen-activated protein/ERK kinase (MEK)-1/2 inhibitor] was needed to significantly inhibit breast cancer cell migration, whereas this inhibitor blocked ERK activity within 1 hour. This observation suggests that ERK-dependent gene expression, rather than direct ERK signaling, is essential for cell migration. With further study, we found that ERK activity promoted the expression of the activator protein-1 (AP1) components Fra-1 and c-Jun, both of which were necessary for cell migration. Combination of U0126 treatment and Fra-1/c-Jun knockdown did not yield further reduction in cell migration than either alone, indicating that ERKs and Fra-1/c-Jun act by the same mechanism to facilitate cell migration. In an attempt to investigate the role of Fra-1/c-Jun in cell migration, we found that the ERK-Fra-1/c-Jun axis regulated slug expression in an AP1-dependent manner. Moreover, the occurrence of U0126-induced migratory inhibition coincided with slug reduction, and silencing slug expression abrogated breast cancer cell migration. These results suggest an association between ERK-regulated cell migration and slug expression. Indeed, cell migration was not significantly inhibited by U0126 treatment or Fra-1/c-Jun silencing in cells expressing slug transgene. Our study suggests that the ERK pathway regulates breast cancer cell migration by maintaining slug expression.

Crosstalk Between BCR/ABL Oncoprotein and CXCR4 Signaling through a Src Family Kinase in Human Leukemia Cells

Stromal-derived factor (SDF)-1 and its G protein–coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein–coupled receptor signaling and function of mammalian precursors.

Lysophosphatidic Acid Stimulates Ovarian Cancer Cell Migration via a Ras-MEK Kinase 1 Pathway

Lysophosphatidic acid (LPA) is present at high concentrations in ascites and plasma of ovarian cancer patients. Studies conducted in experimental models demonstrate that LPA promotes ovarian cancer invasion/metastasis by up-regulating protease expression, elevating protease activity, and enhancing angiogenic factor expression. In this study, we investigated the effect of LPA on ovarian cancer migration, an essential component of cancer cell invasion. LPA stimulates both chemotaxis and chemokinesis of ovarian cancer cells and LPA-stimulated cell migration is GI dependent. Moreover, constitutively active H-Ras enhances ovarian cancer cell migration, whereas dominant negative H-Ras blocks LPA-stimulated cell migration, suggesting that Ras works downstream of Gi to mediate LPA-stimulated cell migration. Interestingly, H-Ras mutants that specifically activate Raf-1, Ral-GDS, or phosphatidylinositol 3′-kinase are unable to significantly enhance ovarian cancer cell migration, suggesting that a Ras downstream effector distinct from Raf-1, Ral-GDS, and phosphatidylinositol 3′-kinase is responsible for LPA-stimulated cell migration. In this article, we demonstrate that LPA activates mitogen-activated protein kinase kinase 1 (MEKK1) in a Gi-Ras-dependent manner and that MEKK1 activity is essential for LPA-stimulated ovarian cancer cell migration. Inhibitors that block MEKK1 downstream pathways, including MEK1/2, MKK4/7, and nuclear factor-κB pathways, do not significantly alter LPA-stimulated cell migration. Instead, LPA induces the redistribution of focal adhesion kinase to focal contact regions of the cytoplasm membrane, and this event is abolished by pertussis toxin, dominant negative H-Ras, or dominant negative MEKK1. Our studies thus suggest that the Gi-Ras-MEKK1 signaling pathway mediates LPA-stimulated ovarian cancer cell migration by facilitating focal adhesion kinase redistribution to focal contacts.

The G12/13-RhoA signaling pathway contributes to efficient lysophosphatidic acid-stimulated cell migration.

The membrane redistribution and phosphorylation of focal adhesion kinase (FAK) have been reported to be important for cell migration. We previously showed that Lysophosphatidic acid (LPA) induced FAK membrane redistribution and autophosphorylation in ovarian cancer SK-OV3 cells and the signaling pathway consisting of Gi-Ras-MEKK1 mediated LPA-induced FAK membrane redistribution but not FAK autophosphorylation. We also showed that the disruption of the Gi-Ras-MEKK1 pathway led to a significant reduction in LPA-stimulated cell migration. These findings raised the question of whether LPA-induced FAK autophosphorylation was required for LPA-stimulated cell migration and what signaling mechanism was involved in LPA-induced FAK autophosphorylation. In this study, we expressed the membrane anchored wild-type FAK (CD2-FAK) in SK-OV3 cells and found that the expression of CD2-FAK greatly rescued LPA-stimulated cell migration in Gi or Ras-inhibited cells. However, Gi inhibitor pertussis toxin or dominant-negative H-Ras still significantly inhibited LPA-stimulated cell migration in cells expressing the membrane anchored FAK containing a mutation in the autophosphorylation site [CD2-FAK(Y397A)]. These results suggest that FAK autophosphorylation plays a role in LPA-stimulated cell migration. With the aid of p115RhoGEF-RGS, G12 and G13 minigenes to inhibit G12/13, we found that the G12/13 pathway was required for LPA-induced FAK autophosphorylation and efficient cell migration. Moreover, LPA activated RhoA and Rho kinase (ROCK) in a G12/13-dependent manner and their activities were required for LPA-induced FAK autophosphorylation. However, Rho or ROCK inhibitors displayed no effect on LPA-induced FAK membrane redistribution although they abolished LPA-induced cytoskeleton reorganization. Our studies show that the G12/13-RhoA-ROCK signaling pathway mediates LPA-induced FAK autophosphorylation and contributes to LPA-stimulated cell migration.

cDNA cloning of a novel G protein-coupled receptor with a large extracellular loop structure.

A cDNA designated as AZ3B has been isolated from a differentiated HL-6 0 cell cDNA library with a probe derived from the N-formyl peptide receptor gene. The 1.97-kb cDNA encodes a novel G protein-coupled receptor (GPCR) with 482 amino acids. In addition to the predicted 7 transmembrane domains common to all GPCRs, the protein encoded by AZ3B contains a large extracellular loop of approximately 172 amino acids between the fourth and the fifth transmembrane domains, a feature unique among the hundreds of GPCRs identified to date. High sequence homology exists between the AZ3B protein and a number of chemoattractant receptors in the amino-terminal 170 residues and the carboxyl-terminal 150 residues. Northern and flow cytometric analyses suggested that the AZ3B message and protein are widely expressed in several differentiated hematopoietic cell lines, in the lung, placenta, heart, and endothelial cells. We postulate that the AZ3B protein defines a distinct group of receptors within the GPCR superfamily.

Rac1-MKK3-p38-MAPKAPK2 Pathway Promotes Urokinase Plasminogen Activator mRNA Stability in Invasive Breast Cancer Cells

We reported previously that down-regulating or functionally blocking αv integrins inhibits endogenous p38 mitogen-activated protein kinase (MAPK) activity and urokinase plasminogen activator (uPA) expression in invasive MDA-MB-231 breast cancer cells whereas engaging αv integrins with vitronectin activates p38 MAPK and up-regulates uPA expression (Chen, J., Baskerville, C., Han, Q., Pan, Z., and Huang, S. (2001) J. Biol. Chem. 276, 47901–47905). Currently, it is not clear what upstream and downstream signaling molecules of p38 MAPK mediate αv integrin-mediated uPA up-regulation. In the present study, we found that αv integrin ligation activated small GTPase Rac1 preferentially, and dominant negative Rac1 inhibited αv integrin-mediated p38 MAPK activation. Using constitutively active MAPK kinases, we found that both constitutively active MKK3 and MKK6 mutants were able to activate p38 MAPK and up-regulate uPA expression, but only dominant negative MKK3 blocked αv integrin-mediated p38 MAPK activation and uPA up-regulation. These results suggest that MKK3, rather than MKK6, mediates αv integrin-induced p38 MAPK activation. Among the potential downstream effectors of p38 MAPK, we found that only MAPK-activated protein kinase 2 affects αv integrin-mediated uPA up-regulation significantly. Finally, using β-globin reporter gene constructs containing uPA mRNA 3′-untranslated region (UTR) and adenosine/uridine-rich elements-deleted 3′-UTR, we demonstrated that p38 MAPK/MAPK-activated protein kinase 2 signaling pathway regulated uPA mRNA stability through a mechanism involving the adenosine/uridine-rich elements sequence in 3′-UTR of uPA mRNA.

Serum Amyloid A Is an Endogenous Ligand That Differentially Induces IL-12 and IL-23

The acute-phase proteins, C-reactive protein and serum amyloid A (SAA), are biomarkers of infection and inflammation. However, their precise role in immunity and inflammation remains undefined. We report in this study a novel property of SAA in the differential induction of Th1-type immunomodulatory cytokines IL-12 and IL-23. In peripheral blood monocytes and the THP-1 monocytic cell line, SAA induces the expression of IL-12p40, a subunit shared by IL-12 and IL-23. SAA-stimulated expression of IL-12p40 was rapid (≤4 h), sustainable (≥20 h), potent (up to 3380 pg/ml/106 cells in 24 h), and insensitive to polymyxin B treatment. The SAA-stimulated IL-12p40 secretion required de novo protein synthesis and was accompanied by activation of the transcription factors NF-κB and C/EBP. Expression of IL-12p40 required activation of the p38 MAPK and PI3K. Interestingly, the SAA-induced IL-12p40 production was accompanied by a sustained expression of IL-23p19, but not IL-12p35, resulting in preferential secretion of IL-23, but not IL-12. These results identify SAA as an endogenous ligand that potentially activates the IL-23/IL-17 pathway and present a novel mechanism for regulation of inflammation and immunity by an acute-phase protein.

Up-Regulation of Functional Kinin B1 Receptors in Allergic Airway Inflammation

B1 receptors are known to be induced during allergic airway inflammation in animal models. However, little is known regarding in vivo B1 receptor expression in humans. We examined B1 receptor mRNA expression in nasal tissue samples from allergic rhinitis and normal subjects. Allergic rhinitis subjects displayed significantly higher expression of B1 receptor mRNA than did the normal subjects, and nasal allergen challenge increased B1 receptor mRNA expression at 8 to 24 h time points in allergic rhinitis subjects. No significant difference was found in B2 receptor expression. To confirm B2 and B1 receptor functional activity, subjects were challenged with kinin agonists. Nasal challenge with the B1 receptor ligand, Lys-des-Arg-bradykinin (BK), activated extracellular signal-regulated kinase in allergic rhinitis, but not normal, subjects. Nasal challenge with the B2 receptor ligand, BK, activated extracellular signal-regulated kinase in both allergic rhinitis and normal subjects. The consequences of B1 receptor activation were investigated using the human airway epithelial cell lines A549 and BEAS-2B. We demonstrated that Lys-des-Arg-BK activates the transcription factor AP-1. Taken together, these results show that functional B1 receptors are induced in the airway during allergic inflammation and suggest that they participate in the regulation of gene expression.