Gram-negative septicemia and septic shock are common causes of death in patients who survive traumatic and burn injuries. Stimulation of macrophages by gram-negative bacterial lipopolysaccharide (LPS) is responsible for initiating some of the cellular responses that lead to septic shock. LPS stimulation activates several intracellular signal transduction pathways that induce pro-inflammatory cytokine production. These cytokines, most notably tumor necrosis factor (TNF), are the primary mediators of septic shock. In macrophages infected with HIV-1, LPS stimulation also activates viral gene expression and replication. The investigator has discovered that LPS stimulation leads to the activation of casein kinase II (CKII) and the transcription factor PU.1, molecules which were not previously known to participate in LPS-inducible responses. In turn CKII and PU.1 are required for maximal expression from the HIV-1 and TNF promoters in response to LPS. The investigator's studies have shown that LPS stimulation rapidly upregulates the enzymatic activity of CKII and induces CKII translocation from the cytosol to the nucleus. Nuclear translocation of CKII represents a unique mechanism by which CKII can selectively phosphorylate nuclear substrates following LPS stimulation. One substrate for CKII is PU.1, a member of the Ets family of proto-oncogene transactivating factors. The investigator discovered that PU.1 phosphorylation by CKII upregulates its trans-activation function, but not its DNA-binding activity. While the transcription factor NF-kB is known to be essential for LPS-responsiveness of the HIV-1 long terminal repeat (LTR) and TNF promoter, the investigator found that both NF-kB and PU.1 are required for maximal expression. Genetic studies demonstrate that PU.1 activates HIV-LTR transcription by binding to NF-kB motifs within the viral LTR. This effect was promoter-specific because PU.1 did not activate all NF-kB-dependent promoters. Thus, there appears to be a subset of NF-kB motifs that can interact with PU.1. Most studies which have identified essential NF-kB motifs within LPS-inducible genes have not considered the possibility that other factors can bind to these motifs in vivo. The investigator hypothesizes that LPS-inducible expression of the HIV-LTR, as well as TNF and other genes, is regulated by the binding of PU.1 to NF-kB motifs. The long term objective of this project is to determine how CKII and PU.1 regulate both HIV-1 growth and the production of cytokines that mediate inflammation and septic shock.
The specific aims are to (1) define the mechanisms that control CKII activation and nuclear translocation upon LPS stimulation, (2) determine which LPS-inducible macrophage responses are mediated by PU.1 and CKII, (3) determine which CKII phosphorylation sites on PU.1 confer promotor-specific recognition on this transcription factor, and (4) determine the mechanism by which PU.1 utilizes NF-kB motifs to activate transcription.
| Lodie, T A; Reiner, M; Coniglio, S et al. (1998) Both PU.1 and nuclear factor-kappa B mediate lipopolysaccharide- induced HIV-1 long terminal repeat transcription in macrophages. J Immunol 161:268-76 |
