Involvement of Protein Kinase C in Rickettsia rickettsii -Induced Transcriptional Activation of the Host Endothelial Cell

Abstract

ABSTRACT Our laboratory has reported on a biphasic pattern of nuclear factor κB (NF-κB) activation in cultured human umbilical vein endothelial cells during infection with Rickettsia rickettsii , an obligate, intracellular bacterium, and the etiologic agent of Rocky Mountain spotted fever. Transcriptional activation of the tissue factor (TF) gene during this infection has been shown to involve NF-κB. To further understand the signal transduction events underlying these phenomena, we studied the role of protein kinase C (PKC), a ubiquitous family of phospholipid-dependent enzymes implicated in the regulation of a variety of cell signaling pathways. Two inhibitors of PKC, namely, bisindolylmaleimide I hydrochloride (BM-1) and calphostin C, which exhibit different inhibitory properties towards various isozymes of PKC, were used. Infection of cells with R. rickettsii in the presence of BM-1 (50 nM) did not significantly affect NF-κB, whereas calphostin C (2.5 μM) completely blocked the early phase of NF-κB activation. The late, sustained phase also was not affected by treatment with BM-1. Downregulation of phorbol ester-sensitive PKCs by long-term treatment with phorbol 12-myristate 13-acetate (PMA) did not inhibit NF-κB activation. Likewise, this downregulation had no effect on induction of TF activity. The activity of TF was, however, sensitive to BM-1 and calphostin C, whereas expression of TF mRNA was inhibited only by calphostin C. Overall, these results suggest the lack of involvement of classical PKC pathways in R. rickettsii -induced NF-κB activation but the possible involvement of a non-PMA-responsive PKC isoform in the posttranscriptional control of TF expression.