SILAC analysis of oxidative stress-mediated proteins in human pneumocytes: new role for treacle

Abstract

To better understand lung oxidant stress responses, we examined A549 lung cells exposed to H(2)O(2) using "stable isotope labeling by amino acids." We identified 466 cytosolic and 387 nuclear proteins; H(2)O(2) exposure produced >or=twofold differences in 31, all were downregulations. None were previously reported as oxidant stress response proteins, although they share common functions. One of the responders, treacle, was linked to p53, an important oxidative stress response. The Treacher Collins-Franceschetti syndrome can result from treacle mutation and insufficiency was suggested to cause increased p53 leading to the syndrome. However, results here indicate p53 and treacle responses to H(2)O(2) are independent: treacle remains suppressed after p53 recovery; the threshold for treacle reduction is well above that for p53 induction; and treacle suppression by short interfering RNA does not modify the p53 response. Evidence of treacle antioxidant activity include reduction being driven by proteasome degradation independently of mRNA, typical for oxidant-absorbing proteins, and increased sensitivity to H(2)O(2) consequent to short interfering RNA suppression. Data here show a link between oxidative stress and treacle reduction, demonstrate that treacle does not control p53, provide evidence of a treacle oxidant defense role, support the hypothesis that oxidant stress plays a role in the Treacher Collins-Franceschetti syndrome, and raise the possibility that treacle plays an anti-oxidant role in lungs.

Figure 1. SILAC data showing treacle downregulation in nuclei of A549 cells stressed by H₂O₂ exposure

Cells exposed to H₂O₂ were grown in medium containing amino acids labeled with heavy isotopes allowing separation of peptides in mixed samples containing protein from cells not exposed to H₂O₂ that were grown in medium with light amino acids. The upper panel shows reduced peak heights of one heavy treacle peptide (right side of the panel) indicating reduced expression in response to H₂O₂ exposure. The lower panel shows that expression of the unrelated control protein Lamin-A/C was not altered by H₂O₂ indicating a specific effect.

Figure 2. Immunoblot data showing treacle downregulation and p53 upregulation in A549 cells stressed by H₂O₂

Cells were harvested at 6 hr after exposure to 1 mM H₂O₂, the same time point and condition for SILAC analysis. Treacle and Lamin-A/C control were detectable in nuclear fractions while p53 and β-actin control were detectable in both nuclear and cytoplasmic fractions. Exposure to 1 mM H₂O₂ caused reduced nuclear treacle, some increase in nuclear p53, a greater relative increase in cytoplasmic p53, and no changes in Lamin A/C or β-actin controls.

Figure 3. Time course of H₂O₂ exposure and expression of treacle and p53

Immunoblots show exposure to 1 mM H₂O₂ results in treacle downregulation and p53 upregulation in nuclear and cytoplasmic fractions at 6 hrs. Restoration of p53 to base expression in both fractions occurs more quickly than restoration of nuclear treacle. B-actin and Lamin-A/C controls remain unchanged. The data were representative of two independent experiments.

Figure 4. Dose responses of H₂O₂ and treacle/p53 expression

The threshold for treacle response is 1.0 mM H₂O₂. In contrast, p53 responds to concentrations as low as 0.2 mM H₂O₂ and responds maximally to 0.5 mM H₂O₂. The data were representative of two independent experiments.

Figure 5. siRNA suppression of treacle and effect on p53

(“NS” = transfection with non-specific control siRNA; “Treacle” = transfection with treacle-specific siRNA). a) Treacle-specific siRNA reduced treacle expression at 24 hrs and abolished expression at 48 hrs, but had no effect on expression of p53 or the β-actin control. b) The response of p53 to H₂O₂ was the same for cells transfected with treacle-specific and control siRNA. Neither siRNA nor H₂O₂ affected expression of the β-actin control. The data were representative of three independent experiments.

Figure 6. Human primary bronchial epithelial cells exposed to H₂O₂

Treacle downregulation and p53 upregulation were detected at 6 hrs following exposure to H₂O₂. It should be noted that the dose for the primary cells is about one tenth of the dose used in A549. The data were representative of three independent experiments

Figure 7. Proteasome inhibitor MG-132 and suppression of treacle in A549 cell response to H₂O₂

A549 cells exposed 1 mM H₂O₂ for 6 hr (lane 3) had reduced treacle compared to controls (lane 1). However, treacle did not decline if cells were first incubated with 10 MG-132 μM for 10 min prior to H₂O₂ exposure (lane 4) although the level was less than in cells treated with the inhibitor and not exposed to H₂O₂ (lane 2). These data are representative of three independent experiments.

Figure 8. Treacle suppression by siRNA increases sensitivity to H₂O_2:

48 hrs after transfection, cells were exposed to 1mM H₂O₂ for 4 hr and medium was replaced with fresh medium containing 10% alarmBlue reagent. After additional two hour incubation, the absorbance data for blue and red color were collected. Results were normalized by presenting viability as a percentage of measured viability of similarly transfected control cells not exposed to H₂O₂. Values are means ± SEM (n = 4) and the p value is a two-tailed student t-test result. “NS” = transfection with non-specific control siRNA; “Treacle” = transfection with treacle-specific siRNA