Regulation of Protein Kinase C in Escherichia coli K1 Invasion of Human Brain Microvascular Endothelial Cells

Abstract

Escherichia coli is one of the most important pathogens involved in the development of neonatal meningitis in many parts of the world. Traversal of E. coli across the blood-brain barrier is a crucial event in the pathogenesis of E. coli meningitis. Our previous studies have shown that outer membrane protein A (OmpA) expression is necessary in E. coli for a mechanism involving actin filaments in its passage through the endothelial cells. Focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) have also been activated in host cells during the process of invasion. In an attempt to elucidate the mechanisms leading to actin filament condensation, we have focused our attention on protein kinase C (PKC), an enzyme central to many signaling events, including actin rearrangement. In the current study, specific PKC inhibitors, bisindolmaleimide and a PKC-inhibitory peptide, inhibited E. coli invasion of human brain microvascular endothelial cells (HBMEC) by more than 75% in a dose-dependent manner, indicating a significant role played by this enzyme in the invasion process. Our results further showed that OmpA؉ E. coli induces significant activation of PKC in HBMEC as measured by the PepTag nonradioactive assay. In addition, we identified that the PKC isoform activated in E. coli invasion is a member of the conventional family of PKC, PKC-␣, which requires calcium for activation. Immunocytochemical studies have indicated that the activated PKC-␣ is associated with actin condensation beneath the bacterial entry site. Overexpression of a dominant negative mutant of PKC-␣ in HBMEC abolished the E. coli invasion without significant changes in FAK phosphorylation or PI3K activity patterns. In contrast, in HBMEC overexpressing the mutant forms of either FAK or PI3K, E. coli-induced PKC activation was significantly blocked. Furthermore, our studies showed that activation of PKC-␣ induces the translocation of myristoylated alanine-rich protein kinase C substrate, an actin cross-linking protein and a substrate for PKC-␣, from the membrane to cytosol. This is the first report of FAK-and PI3K-dependent PKC-␣ activation in bacterial invasion related to cytoskeletal reorganization.

Interleukin-1 beta Induces MUC2 Gene Expression and Mucin Secretion via Activation of PKC-MEK/ERK,and PI3K in Human Airway Epithelial Cells

“…And it has been known that PKC or MAPKs cascades require PI3K activation in several signal transduction pathway (23)(24)(25)(26)(27). In this study, LY294002, an inhibitor of PI3K, suppressed the IL-1 -induced MUC2 gene expression and mucin secretion, but not the phosphorylation of ERK.…”

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C, Focal Adhesion Kinase, and Ezrin to Promote Traversal across the Blood-Brain Barrier

“…PKC is known as a downstream effecter of intracellular Ca 2ϩ mobilization and plays a significant role in the signaling cascades leading to actin cytoskeleton rearrangements, involved in cell-cell junction modulation (45) and host cell invasion of E. coli, Neisseria gonorrhea, and Legionella pneumophila (10,46). Our results derived from transmigration assays in the presence of calphostin C further support the previous report that the activation of PKC␣ by C. neoformans contributes to the fungal transmigration across the HBMEC monolayer.…”

Interaction of Cryptosporidium hominis and Cryptosporidium parvum with Primary Human and Bovine Intestinal Cells

“…Like Cryptosporidium, E. coli induces host actin condensation at the site of infection when it invades host cells. Immunocytochemical studies indicated that activated PKC␣ is associated with actin condensation beneath the bacterial entry site (25). Further studies are required to investigate whether a similar situation occurs upon invasion of host cells by Cryptosporidium species.…”