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NF-B Activation during Rickettsia rickettsii Infection of Endothelial Cells Involves the Activation of Catalytic IB Kinases IKK and IKKß and Phosphorylation-Proteolysis of the Inhibitor Protein IB

Dawn R. Clifton,^1, Elena Rydkina,² Robert S. Freeman,³ and Sanjeev K. Sahni^2*

Department of Environmental Medicine,¹ Hematology-Oncology Unit, Department of Medicine,² Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York³

Received 22 June 2004/ Returned for modification 27 July 2004/ Accepted 22 September 2004

Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-B (NF-B) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-B subunits, p65 and p50, and IB proteins, IB and IBß. The transcript and protein levels of p50, p65, and IBß remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IB at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IB mRNA. The level of IB mRNA gradually returned toward baseline, whereas that of total IB protein remained lower than the corresponding controls. The activities of IKK and IKKß, the catalytic subunits of IB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-B response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKK and IKKß, leading to attenuation of nuclear translocation of NF-B. Also, increased activity of IKK was evident later during the infection, coinciding with the late phase of NF-B activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-B activation. Since NF-B is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

Editor: J. B. Bliska

Present address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.

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