No abstract

Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphorylation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with 32P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of β-catenin, resulting in nuclear accumulation and transcriptional activity of β-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear β-catenin in human alveolar macrophages and expression of genes that require nuclear β-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of β-catenin in the nucleus of any cell, including alveolar macrophages.

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC ζ, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC ζ, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC ζ partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC ζ to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC ζ. PKC ζ has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC ζ/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC ζ activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC ζ.

No abstract

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide. Because ceramide exposure induces apoptosis in other cell types and the PI 3-kinase pathway is known to inhibit apoptosis, we determined the relationship between LPS-induced ceramide and PI 3-kinase activation in alveolar macrophages. We found that ceramide exposure activated PI 3-kinase and Akt. When we blocked LPS-induced ceramide with the inhibitor D609, we blocked LPS-induced PI 3-kinase and Akt activation. Evaluating cell survival after ceramide or LPS exposure, we found that blocking PI 3-kinase induced a significant increase in cell death. Because these effects of PI 3-kinase inhibition were more pronounced in ceramide- vs LPS-treated alveolar macrophages, we also evaluated NF-κB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-κB translocation to the nucleus. As a composite, these studies suggest that the effects of ceramide exposure in alveolar macrophages may be very different from the effects described for other cell types. We believe that LPS induction of ceramide results in PI 3-kinase activation and represents a novel effector mechanism that promotes survival of human alveolar macrophages in the setting of pulmonary sepsis.