Figure 1.
DUOX proteins are expressed in brain and are induced by PDGF in SK-N-BE cells.
(A) Western blot analysis of DUOX proteins in the human oligodendrocyte cell line M03-13, human neuroblastoma (SK-N-BE) cells and rat brain total extract (total) and membrane fraction (M). (B) Confocal microscopic images displaying DUOX immunoreactivity in coronal rat brain slices. The negative control incubated with secondary antibody alone is shown in a; x10 (b and c) and x20 (d and e) magnifications are shown; in the inset of image e is shown a further magnification of the cell indicated by the white arrow to highlight the staining of both cell body and extensions. (C) SK-N-BE cells were incubated for 18h in medium containing 0.2% FBS and were then stimulated with increasing doses of PDGF for 15min before harvesting them for immunoblot analysis for DUOX protein. (D) SK-N-BE cells were incubated for 18h in medium containing 0.2% FBS and were then stimulated with 15ng/ml of PDGF for the indicated times before harvesting them for immunoblot analysis for DUOX protein. The histograms show the mean +/- SEM values relative to control obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. The same blots were also probed with antibodies directed against superoxide dismutase (SOD) 1 enzyme to point out the specificity of the effect of PDGF on DUOX proteins. * p<0.01 and ** p<0.05 vs Ctr.
Figure 2.
PDGF induction of DUOX levels is mediated by ROS. SK-N-BE cells were incubated 18h in medium containing 0.2% FBS, preincubated 1h with 50µM Apocynin (Apo) and then stimulated with 15ng/ml of PDGF (A) or 10% FBS (B) for 15min before harvesting them for immunoblot analysis of DUOX.
The histograms show the mean +/- SEM values relative to control obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr; ** p< 0.05 vs PDGF (A); ***p< 0.01vs FBS (B); (C) SK-N-BE cells were incubated 18h in medium containing 0.2% FBS and were then stimulated with 15μM H2O2 15min before harvesting them for immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to control obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. (D) RT- PCR analysis of DUOX 1 and 2 mRNA levels of SK-N-BE cells incubated 18h in medium containing 0.2% FBS, preincubated 1h in the presence or absence of 50µM Apocynin (Apo) and then stimulated with 15ng/ml of PDGF, 15μM H2O2 or 10% FBS for 15min. The histograms show the mean +/- SEM values relative to control of three independent experiments. * p< 0.01 vs Ctr; ** p< 0.05 vs PDGF; *** p< 0.01 vs H2O2. **** p< 0.05 vs FBS.
Figure 3.
Silencing of p22phox inhibits the induction of DUOX 1 and 2 protein and mRNA levels by PDGF.
(A) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see Materials and Methods) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. (B) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22phox (siRNA p22phox) or control, scrambled siRNA (scramble) as described in Materials and Methods. 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22phox. The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22phox bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. (C) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. (D, E) 24h after transfection with a mix of the two p22phox siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in Materials and Methods. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.
Figure 4.
Silencing of NOX2 inhibits the induction of DUOX 1 and 2 protein and mRNA levels by PDGF.
(A-F) SK-N-BE were transfected by electroporation with siRNA to NOX2 (05 and 06) (siRNA NOX2) or control, scrambled siRNA (scramble) as described in Materials and Methods. (A) 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of NOX2. The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of NOX2 bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. (B) 48h after transfection with a mix of the two NOX2 siRNA, mRNA was extracted and NOX2 mRNA levels were analyzed by RT-PCR as described in Materials and Methods. The histograms show the mean +/- SEM values relative to scramble of three independent experiments. * p< 0.01 vs scramble. (C) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. (D-F) 24h after transfection with a mix of the two NOX2 siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 (D), DUOX2 (E) and DUOXA1 (F) mRNA levels were analyzed by RT-PCR as described in Materials and Methods. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p< 0.01 vs Ctr; **p< 0.01vs PDGF stimulated scramble.
Figure 5.
PDGF increases DUOX1 and 2 mRNAs stability.
(A, B) RT- PCR analysis of DUOX 1 and 2 mRNA levels of SK-N-BE cells incubated 18h in medium containing 0.2% FBS, and then treated with of 1µg/ml of actinomycin D for the indicated times in absence or presence of 15ng/ml of PDGF and 50µM apocynin as indicated. Values are mean +/- SEM relative to control of three independent experiments. (B) Western blotting analysis of DUOX protein levels of SK-N-BE cells treated as in (A). Values are mean +/- SEM relative to control obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. § p<0.05 and §§ p<0.01 vs Ctr; * p<0.01 vs the corresponding time point of ActD curve; ** p<0.01 and *** p<0.05 vs the corresponding time point of ActD + PDGF curve.
Figure 6.
ROS levels in PDGF stimulated SK-N-BE cells.
(A) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in Materials and Methods. ROS levels were measured fluorimetrically at the time intervals indicated. Ca++-independent ROS were measured in presence of BAPTA-AM. Ca++-dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. (B) Cells were transfected by electroporation with siRNA to p22phox (siRNA p22phox) or control, scrambled siRNA (scramble) as described in Materials and Methods. 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H2O2 levels as described in Materials and Methods. The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.
Figure 7.
Regulation of DUOX by PDGF receptor.
Schematic diagram showing the circuitry induced by PDGF leading to DUOX induction. PDGF activates the specific receptor, which stimulate NADPH oxidase. This sets off a loop that amplifies ROS cascade [32], [38]. ROS stabilize DUOX mRNAs and increase the level of DUOX proteins in plasma membrane.