PMC:2889865 / 5341-8631 JSONTXT

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To determine the involvement of associated pathways, we exposed cells to Ca2+ ionophore with or without PMA and observed a modest involvement of Ca2+ in PMA-dependent AP-1 activation (figure 1c) while Ca2+ alone did not alter AP-1 activity (data not shown). Furthermore, AP-1 activity decreased in a TCR-deficient Jurkat cell line when exposed to PMA compared to the parent cell line indicating that regulation of AP-1 was only partially T-cell receptor dependent (figure 1d).\nFigure 1 AP-1 activation following long-term exposure of Jurkat T-cells to PMA. Jurkat T-cells were transfected with luciferase reporter plasmids containing the AP-1 cis-elements. (A) Time-dependent AP-1 activation in response to PMA. (B) HK E. coli does not activate AP-1. (C) Dose-dependent activation of AP-1 were performed using PMA alone (grey bars) and in combination with calcium ionophore (CaI 955 μM, black bars). (D) TCR-/- deficient cells responded to PMA (162 nM) by up-regulating AP-1 activity. Statistical significance from the control was determined using Student's t-test. (n = 4). Controls were arbitrarily set to 1. NF-κB levels showed a transient increase at 1 min after exposure to PMA (figure 2a). However, 1 h after exposure the NF-κB levels began to drop reaching the lowest levels by 6 h, after which they increased again by 24 h. Exposure of Jurkat T-cells to HK E. coli resulted in a dose-dependent NF-κB activation, with the highest activity observed at a relative concentration of 5 × 107 CFU/ml (figure 2b). The time-dependent activation of NF-κB by HK E. coli was assessed further using the optimal concentration obtained from figure 2b and showed that the NF-κB activity increased after 3 h of exposure (figure 2c). Furthermore, increased intracellular Ca2+ reversed the PMA dependent NF-κB inhibition (figure 2d) and reduced the HK E. coli -dependent NF-κB activation (figure 2e).\nFigure 2 NF-κB down-regulation by PMA and up-regulation by heat killed E. coli MG1655 following long-term stimulation. Transfection of Jurkat T-cells was performed using luciferase reporter plasmids containing NF-κB cis-elements. (A) Time-dependent stimulation of Jurkat T-cells using PMA. NF-κB activation was evaluated using HK E. coli in a (B) dose- and (C) time-dependant manner. Calcium ionophore increased NF-κB activity following PMA exposure (E) and resulted in a negative regulation in response to HK E. coli stimulation (D). Statistical significance from the control was determined using Student's t-test. (n = 4). 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