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

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.

Activation of Mechanistic Target of Rapamycin (mTOR) in Human Endothelial Cells Infected with Pathogenic Spotted Fever Group Rickettsiae

“…As a follow-up to confirm this finding, we further assessed the phosphorylation status of protein kinase C (PKC) as yet another substrate of mTORC2 to find a robust increase in cellular levels of phospho-PKC in response to both R. rickettsii and R. conorii infection ( Figure 2 C). This result is in concordance with our previous observations implicating selective involvement of specific isoforms of PKC in transcriptional responses (as measured by activation of NF-κB and induced expression and activity of tissue factor) of host ECs [ 14 ]. Taken together, these results demonstrate the activation of mTORC2 as well consequent to EC infection with spotted fever group rickettsiae.…”

“…As a versatile signaling moiety, mTOR also serves as a critically important determinant of inflammation by virtue of regulation of NF-κB- and STAT-mediated host responses. Previous work from our laboratory has unequivocally established activation of both NF-κB and STAT-3 or STAT-1 as well as expression and secretion of cytokines (IL-1 and interferon-β) and chemokines (IL-8 and MCP-1) by ECs infected with R. rickettsii and R. conorii [ 1 , 2 , 3 , 10 , 11 , 14 ]. Increased cytokine production in response to invading pathogens may serve to sustain the inflammatory immune response by maintaining the recruitment of infiltrating macrophages and/or neutrophils to the foci of infection.…”

Host Defenses to Rickettsia rickettsii Infection Contribute to Increased Microvascular Permeability in Human Cerebral Endothelial Cells

“…Likewise, IL-6 has been shown to induce a loss of TER across human umbilical vein endothelial cells in a protein kinase C (PKC)-dependent manner [38]. Therefore, it is not surprising to find that PKC is also up regulated in R. rickettsii-infected endothelial cells [39]. The exact role of IL-6 and/or PKC activation in rickettsiae-induced permeability is an area of current investigation.…”

Activation of p38 stress-activated protein kinase during Rickettsia rickettsii infection of human endothelial cells: role in the induction of chemokine response

“…Studies using two major pathogenic species of spotted fever group (SFG) rickettsiae, namely R. rickettsii and R. conorii , have established the occurrence of significant changes in the expression of important coagulation proteins, adhesion molecules and regulatory chemokines during in vitro and in vivo infection of host endothelial cells (EC) (Eremeeva et al ., 2000; Valbuena et al ., 2003; Walker et al ., 2003; Clifton et al ., 2005a; Valbuena and Walker, 2005). Our laboratory has further identified activation of nuclear factor‐kappa B (NF‐κB) and involvement of specific isozymes of protein kinase C in the host cell transcriptional responses to R. rickettsii (Sporn et al ., 1997; Sahni et al ., 1999).…”