Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells

Abstract

The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

Figure 1. Effects of CDT on LDH release (A), DNA fragmentation (B), and caspase 3 activity (C).

HIGK were plated 48 h before infection. After 4 hours of co-culture with A. actinomycetemcomitans strains VT1169, D7S-SA or CHE001 (ΔcdtABC) at a MOI of 3000:1 or untreated, cells were washed and incubated for an additional 20, 44, and 68 h with gentamicin (200 µg mL⁻¹) to obtain 24, 48, and 72 h time course data. Parameters were measured as detailed in Materials and Methods. The results are expressed as the mean ± SD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001 versus untreated controls.

Figure 2. Attenuation of caspase 9 activity in cells infected by CDT⁻ A. actinomycetemcomitans.

Cells were cultured as described in Materials and Methods and mock-infected, infected with A. actinomycetemcomitans strains D7S-SA, CHE001 (ΔcdtABC) (MOI 3000:1), or treated with camptothecin (2 µg mL⁻¹). After 24, 48, and 72 hours, Caspase 9 activity was assessed for all infection conditions in duplicate and normalized to total protein. Figure is representative of two experiments. i p = 0.40 versus untreated controls; ii p = 0.89 versus untreated controls and p = 0.42 versus wild-type D7S-SA; iii p = 0.21 versus untreated controls by Student's T-Test.

Figure 3. Modulation of the ATM pathway in CDT-mediated apoptosis.

Panel A. Cells were cultured as described in Materials and Methods and infected with A. actinomycetemcomitans strains VT1169, CHE001 (ΔcdtABC) (MOI 3000:1) or P. gingivalis (MOI 100:1). After 24 h, Western blot was performed using antibodies for total ATM and β-actin. Panel B. HIGK were pretreated with or without caffeine (5 mM) or LY294002 (20 µM) for 2 h followed by incubation with A. actinomycetemcomitans strain VT1169 for 4 h. The cells were then washed and further incubated with gentamicin (200 µg mL⁻¹) in the presence or absence of caffeine and LY294002. After a total incubation time of 48 h, cells were harvested and analyzed for caspase 3 activation. Capase 3 data presented are from two separate experiments each performed in duplicate, and combined results are expressed as the mean ± SD. **p<0.01; ***p<0.001 versus A. actinomycetemcomitans VT1169 infected.

Figure 4. Phosphorylation and activation of Chk2 in HIGK exposed to wild type (CDT⁺) A. actinomycetemcomitans.

Panel A. Cells were co-cultured with A. actinomycetemcomitans VT1169, D7S-SA, or CHE001 (ΔcdtABC) (MOI 3000:1) for 4 h, washed and incubated for an additional 20 h with gentamicin (200 µg mL⁻¹), or treated with camptothecin (2 µg mL⁻¹) for 4 h, as a positive control. Cell lysates were analyzed by Western blotting using antibodies specific for phospho-(Thr⁶⁸)-Chk2, Chk2 protein and β-actin. The results are representative of two separate experiments and were analyzed by densitometry. Panel B. HIGK were pretreated with or without caffeine (5 mM) or LY294002 (20 µM) for 2 h followed by incubation with A. actinomycetemcomitans strain VT1169 for 4 h. The cells were then washed and further incubated with gentamicin (200 µg mL⁻¹) in the presence or absence of caffeine and LY294002. After a total incubation time of 48 h, cells were harvested and cell lysates were prepared and analysed by Western blotting using phospho-(Thr⁶⁸)-Chk2 and β-actin antibodies. The Western blot data are representative of two independent experiments and were analyzed by densitometry.

Figure 5. siRNA-mediated knockdown of Chk2 protein attenuates caspase 3 activation by CDT.

HIGK were transfected with 40 nM Chk2 or control siRNAs for 72 h. Panel A. Cells were harvested and processed for Western blot analysis using Chk2 antibody to demonstrate the efficiency of Chk2 siRNA, and analyzed by densitometry. The results are representative of two separate experiments. Panel B. Transfected cells were co-cultured with A. actinomycetemcomitans VT1169 (MOI 3000:1) for 4 h, washed and incubated for an additional 48 h with gentamicin (200 µg mL⁻¹). Cells were then collected and caspase 3 activity was assessed. The data are representative of three separate experiments.