Nuclear run-on experiments were conducted to determine if R. rickettsii
infection of EC increased the rate of transcription of the TF gene. Nuclei were extracted from uninfected and infected EC, in vitro transcription was conducted with 32
P-labeled UTP, and then radiolabeled products were hybridized with TF and γ-actin cDNA probes and with plasmid vector alone. The intensity of labeled actin mRNA in infected and uninfected EC was equivalent, yet intensity of labeled TF mRNA was increased (2.5-fold higher than the control value in this representative experiment), indicating an enhanced rate of transcription of the TF gene (Fig. ). This observation is consistent with the increased steady-state level of TF mRNA observed at 4 h following R. rickettsii
). The transcription inhibitor actinomycin D (10 μg/ml), when present during infection, completely blocked R. rickettsii
-induced expression of TF activity as measured by a two-stage clotting assay (not shown), providing further evidence that a transcriptional event even is necessary for R. rickettsii
-induced expression of TF.
FIG. 1 Nuclear run-on assay for TF mRNA transcription rate in control and R. rickettsii (RR)-infected EC. In vitro transcription was carried out on nuclei isolated from 3-h-infected EC, using 32P-labeled UTP, and then radiolabeled RNA was hybridized with immobilized (more ...)
The half-life of TF mRNA in infected EC was measured to determine if mRNA stability was prolonged at the times of peak increases in steady-state mRNA levels. EC were infected for 3 h, and then actinomycin D (10 μg/ml) was added to inhibit further transcription. Total cellular RNA was harvested following further incubation for various time intervals and subjected to Northern blot analysis (Fig. A). TF mRNA content was quantitated by densitometric scanning and normalized to the amount of 18S rRNA. Scanning data, shown plotted on a linear scale in Fig. B, were subsequently linearized on semilog plots and subjected to regression analysis to calculate half-life values. TF mRNA in infected EC (0 h) was highly unstable, with a calculated half-life of approximately 1.6 h, based on a regression line with an r value of −0.993. Stability of TF mRNA in uninfected EC could not be determined since this mRNA was present at undetectable levels. R. rickettsii infection did not alter that half-life of the housekeeping mRNA species γ-actin, which was determined to be 9.5 h (data not shown). Therefore, change in stability of mRNA species was not a general cellular phenomenon.
FIG. 2 Stability of TF mRNA in R. rickettsii-infected and uninfected EC. Actinomycin D (10 μg/ml) was added to EC 4 h after infection to inhibit further transcription, and then TF mRNA (2.2 kb) was analyzed by Northern blotting following incubation times (more ...)
The requirement for de novo host cell protein synthesis in R. rickettsii
-induced expression of TF mRNA was explored by using the eukaryotic protein synthesis inhibitor CHX (10 μg/ml) (Fig. ). EC were infected in the presence and absence of CHX, treatment which does not inhibit entry of the organisms into EC (31
). Consistent with the previously reported observation (8
), Northern blot analysis of TF mRNA at 4 h demonstrated appearance of TF factor mRNA with CHX treatment alone (lane 2), whereas untreated EC contained nearly undetectable levels (lane 1). This effect of CHX treatment may derive from stabilization of this highly labile mRNA species or from enhanced transcription rate. R. rickettsii
infection alone resulted in the appearance of TF mRNA (lane 3); however, superinduction occurred when CHX was present during infection (lane 4). This result indicated that induction of TF expression during infection occurred independently of de novo host cell protein synthesis, and thus host cell signalling involved in this response did not involve synthesis of a protein intermediate.
FIG. 3 Effect of CHX on EC expression of TF mRNA during R. rickettsii infection. The eukaryotic protein synthesis inhibitor CHX (10 μg/ml) was added to EC cultures during infection with R. rickettsii (RR) for 4 h, and then mRNA levels were determined (more ...)
TF expression in cultured EC induced by various soluble agonists is dependent on activation of members of the NF-κB/Rel family of transcription factors (20
), and it was recently reported that infection of cultured EC with R. rickettsii
results in activation of NF-κB (33
). To explore involvement of NF-κB activation in R. rickettsii
-induced expression of TF, we used two inhibitors of NF-κB activation, the antioxidant PDTC (25 μM) (4
) and the proteasome inhibitor TPCK (50 μM) (17
). Neither agent affected the initial rate of infection (Table ) or resulted in any obvious changes in morphology of the infected cells (Fig. ). Both agents were effective at blocking R. rickettsii
-induced activation of NF-κB (Fig. A), which appears as two gel-shifted complexes representing a p50 homodimer (C1) and a p50-p65 heterodimer (C2) (24
), as determined by gel shift assay using a 32
P-labeled oligonucleotide probe corresponding to the κB binding domain of the murine kappa light-chain gene enhancer and as measured at 3 h following the initiation of infection. These compounds specifically inhibited NF-κB activation, as there was no inhibitory effect on the level of activation of the unrelated transcription factor, AP-1 (not shown). TPCK (Fig. B) and PDTC (not shown), when present during infection, blocked R. rickettsii
-induced NF-κB activation and prevented R. rickettsii
-induced increases in steady-state levels of EC IκBα mRNA, as determined by RT-PCR analysis. Increased IκBα mRNA is a sensitive indicator of NF-κB activation since four NF-κB sites are present in its promoter region (17
FIG. 4 Immunofluorescence staining of R. rickettsii-infected EC. EC cultured on plastic coverslips were infected for 6 h in the absence (a) or presence (b) of the proteasome inhibitor TPCK. Coverslips were then fixed and stained by fluorescence using polyclonal (more ...)
FIG. 5 Effect of PDTC and TPCK on R. rickettsii-induced activation of the transcription factor, NF-κB. (A) Activated NF-κB (C1 and C2) was assayed by gel shift assay of nuclear extracts prepared from uninfected EC (C), R. rickettsii-infected (more ...)
NF-κB inhibition by PDTC and TPCK was used to determine if activation of this transcription factor participated in R. rickettsii-induced expression of TF mRNA and activity. PDTC and TPCK treatment during infection resulted in abrogation of R. rickettsii-induced expression of TF mRNA, as measured by RT-PCR analysis (Fig. A). A two-stage clotting assay was used to measure levels of TF procoagulant activity present in uninfected and infected cell lysates. While R. rickettsii infection alone resulted in a nearly 10-fold increase in TF activity, such expression was nearly completely inhibited by PDTC or TPCK (Fig. B).
FIG. 6 Effects of PDTC and TPCK on R. rickettsii-induced expression of TF mRNA and TF activity. (A) Levels of TF mRNA (398-bp product) and GAPDH mRNA (588-bp product) were analyzed in total RNA samples prepared from uninfected EC (C), R. rickettsii-infected (more ...)