Hsien-Yeh Hsu

Ligand Binding to Macrophage Scavenger Receptor-A Induces Urokinase-type Plasminogen Activator Expression by a Protein Kinase-dependent Signaling Pathway

Macrophage scavenger receptor-type A (MSR-A) has been implicated in the transmission of cell signals and the regulation of diverse cellular functions (Falcone, D. J., and Ferenc, M. J. (1988) J. Cell. Physiol. 135, 387–396; Falcone, D. J., McCaffrey, T. A., and Vergilio, J. A. (1991)J. Biol. Chem. 266, 22726–22732; Palkama, T. (1991)Immunology 74, 432–438; Krieger, M., and Herz, J. (1994)Annu. Rev. Biochem. 63, 601–637); however, the signaling mechanisms are unknown. In studies reported here, we demonstrate that binding of both lipoprotein and non-lipoprotein ligands to MSR-A induced protein tyrosine phosphorylation and increased protein kinase C (PKC) activity leading to up-regulated urokinase-type plasminogen activator (uPA) expression. Specifically, the binding of acetylated low density lipoprotein and fucoidan to MSR-A in human THP-1 macrophages triggered tyrosine phosphorylation of many proteins including phospholipase C-γ1 and phosphatidylinositol-3-OH kinase. Inhibitors of tyrosine kinase dramatically reduced MSR-induced protein tyrosine phosphorylation and PKC activity. Moreover, inhibitors of tyrosine kinase and PKC reduced uPA activity expressed by THP-1 macrophages exposed to MSR-A ligands. The intracellular signaling response for tyrosine phosphorylation following ligand binding was further demonstrated by using the stable MSR-transfected Bowes cells that express surface MSR-A. These findings establish for the first time a signaling pathway induced by ligand binding to MSR-A and suggest a molecular model for the regulation of macrophage uPA expression by specific ligands of the MSR-A.

Tumor necrosis factor-alpha -mediated protein kinases in regulation of scavenger receptor and foam cell formation on macrophage.

We previously reported tumor necrosis factor-α (TNF) modulates transcriptional and post-transcriptional down-regulation of macrophage scavenger receptor (MSR) (Hsu, H. Y., Nicholson, A. C., and Hajjar, D. P. (1996) J. Biol. Chem. 271, 7767–7773); however, TNF-mediated signaling mechanisms are unknown. Here, we demonstrate that ligation of TNF receptor stimulates activity of p21-activated protein kinase (PAK) and mitogen-activated protein kinases (MAPK) as follows: ERK, JNK, and p38 in murine macrophage J774A.1 cells. Upon activation of protein kinases (PK), TNF rapidly increases MSR message and protein; later it markedly reduces MSR expression. Studies using PK inhibitors and dominant negative constructs demonstrate phosphatidylinositol 3-kinase/Rac1/PAK/JNK and phosphatidylinositol 3-kinase/Rac1/PAK/p38 pathways contribute to important roles in the late stage of TNF down-regulation of MSR expression and taking up of OxLDL. Alternatively, the PKC/MEK1/ERK pathway in the early stage plays a significant role in up-regulation of the MSR gene. By using anti-TNF-R1 agonist antibody, we further confirm TNF-R1-mediated MAPK in regulation of MSR. Furthermore, in MSR gene promoter-driven luciferase reporter assays with TNF, PKC activator increases, but antioxidantN-acetylcysteine, PK inhibitors, and dominant negative constructs decrease luciferase activity in MSR gene promoter-transfected cells. Our current results show the first evidence of crucial roles for TNF-mediated MAPK pathways in the transcriptional regulation of MSR gene and increase MSR expression; in contrast, with TNF longer treatment the pathways down-regulate MSR and foam cell formation probably via post-transcriptional process.

A Colorimetric DNA Hybridization Method for the Detection of Escherichia coli in Foods

A colorimetric DNA hybridization assay has been developed for the rapid detection of Escherichia coli in foods. The method employs two oligonucleotide probes which are specific for the 16S ribosomal RNA of E. coli . Probes are added to lysates of test cultures and allowed to hybridize to target rRNA if present. The probe-target complex is captured via hybridization to a polystyrene dipstick. The immobilized target is detected using an antibody-horseradish peroxidase conjugate which binds to the immobilized probe-target complex. The probe-target-antibody complex generates a colorimetric signal when exposed to a substrate/chromogen mixture. A total of 233 E. coli isolates representing typical, toxigenic, invasive, hemorrhagic serotype 0157:H7, and other pathogenic strains all resulted in a positive assay signal. Dose-response experiments indicate the sensitivity of the assay is approximately 1 × 106 CFU/ml. Specificity of the assay was determined by testing 207 strains of non- E. coli species at 109 CFU/ml. All of the non- E. coli organisms tested were negative with the exception of Escherichia fergusonii and Shigella species. A total of 345 enriched samples including inoculated, uninoculated, and naturally-contaminated foods was tested for the presence of E. coli by the hybridization assay and a conventional cultural method. The false-negative rate for the hybridization assay was 1.2%. By comparison, the false-negative rate for the culture method in these studies was 23.4%. Based on these data, the DNA hybridization method is significantly more accurate than conventional methods for the detection of E. coli in foods.