NADPH Oxidase Activation in Pancreatic Cancer Cells Is Mediated through Akt-dependent Up-regulation of p22phox*

We recently showed that Nox4 NADPH oxidase is highly expressed in pancreatic ductal adenocarcinoma and that it is activated by growth factors and plays a pro-survival, anti-apoptotic role. Here we investigate the mechanisms through which insulin-like growth factor I and serum (FBS) activate NADPH oxidase in pancreatic cancer (PaCa) cells. We show that in PaCa cells, NADPH oxidase is composed of Nox4 and p22phox catalytic subunits, which are both required for NADPH oxidase activity. Insulin-like growth factor I and FBS activate NADPH oxidase through transcriptional up-regulation of p22phox. This involves activation of the transcription factor NF-κB mediated by Akt kinase. Up-regulation of p22phox by the growth factors results in increased Nox4-p22phox complex formation and activation of NADPH oxidase. This mechanism is different from that for receptor-induced activation of phagocytic NADPH oxidase, which is mediated by phosphorylation of its regulatory subunits. Up-regulation of p22phox represents a novel pro-survival mechanism through which growth factors and Akt inhibit apoptosis in PaCa cells.

Activation of the phagocytic NADPH oxidase occurs through assembly of a multicomponent complex comprised of the membrane-bound Nox2-p22 phox subunits and the cytosolic subunits. Receptor-induced phosphorylation of the regulatory p47 phox and p67 phox subunits causes their recruitment from cytosol, resulting in conformational changes in the membrane-bound Nox2-p22 phox complex and oxidase activation. In phagocytes, the Akt kinase mediates p47 phox phosphorylation and thus NADPH oxidase activation (1,8). The mechanisms of non-phagocytic NADPH oxidase activation remain poorly defined. Differently from the phagocytic NADPH oxidase, non-phagocytic oxidase activation may not involve the regulatory subunits. For example, various receptors including IGF-I (9), insulin, TGF␤, TNF␣, and TLR4 all activate Nox4 NADPH oxidase without engaging regulatory subunits (10 -13).
We recently showed that Nox4 NADPH oxidase is highly expressed in pancreatic ductal adenocarcinoma (14,15), and further, that it plays an important pro-survival role in pancreatic cancer (9,14,15). Growth factors (GFs) activate NADPH oxidase in PaCa cells, and the resulting ROS promote sustained activation of pro-survival kinases, such as JAK, thus suppressing apoptosis (15). Therefore, activation of NADPH oxidase is an important mechanism through which GFs protect PaCa cells from death. Of note, Nox4 NADPH oxidase was recently shown to promote endothelial tumor growth in mice (16).
The present study describes a novel mechanism whereby growth factors (in particular IGF-I) activate NADPH oxidase in PaCa cells, namely through transcriptional up-regulation of p22 phox . We show that GFs stimulate Akt, which mediates activation of the transcription factor NF-B to up-regulate p22 phox expression. GF-induced p22 phox up-regulation results in increased NADPH oxidase activity, leading to inhibition of apoptosis in PaCa cells.

MATERIALS AND METHODS
Reagents-Antibodies against Akt1, Nox4, and p22 phox were from Santa Cruz Biotechnology (Santa Cruz, CA); antibody against Akt1 phosphorylated at Ser-473 was from Cell Signaling (Beverly, MA). Human IGF-I was from R&D Systems (Minneapolis, MN); Akt inhibitor VIII (isozyme selective Akti-1/2) was from Calbiochem. All other antibodies and chemicals were from Sigma-Aldrich.
Cell Culture-Human pancreatic ductal adenocarcinoma cells, the poorly differentiated MIA PaCa-2 and moderately differentiated PANC-1 cell lines, were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in 1/1 DMEM/F-12 medium (Invitrogen) supplemented with 15% FBS, 4 mM L-glutamine, and antibiotic/antimycotic solution (Omega Scientific, Tarzana, CA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 and used between passages 4 and 12. For the experiments, MIA PaCa-2 and PANC-1 cells were cultured for up to 48 h without and with 100 ng/ml IGF-I or 15% FBS.
Immunoprecipitation-Cells were collected, washed twice in a buffer containing 20 mM Tris (pH 7.5) and 10 mM DTT, and then resuspended in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EGTA, 10 g/ml each of leupeptin and aprotinin, 1 mM PMSF, 1% IGEPAL CA-630), and sonicated for 30 s. The lysates were clarified by centrifugation, and 500 g of protein was subjected to immunoprecipitation with the indicated antibodies at room temperature according to the Catch and Release immunoprecipitation kit (Upstate Biotech Millipore, Temecula, CA). In this and other assays, protein concentration was measured by the Bradford assay (Bio-Rad Laboratories).
Measurement of Apoptosis-Internucleosomal DNA fragmentation was measured by using the Cell Death Detection ELISA Plus kit (Roche Applied Science, Manheim, Germany) according to the manufacturer's instructions (9,14,18).
Measurement of Intracellular ROS Production-Intracellular ROS levels were measured by flow cytometry in cells loaded with the redox-sensitive dye DCFH-DA as described previously (9,14). Approximately 1 ϫ 10 6 cells were incubated in the dark for 30 min at 37°C with 10 M DCFH-DA, harvested, and resuspended in the medium without DCFH-DA. Fluorescence was recorded on the FL-1 channel of a FACScan (BD Biosciences). The data were analyzed with the Cell Quest program.
Measurement of NADPH Oxidase Activity-Superoxide production was measured in total cell homogenates by using lucigenin-derived chemiluminescence (12,19), as described (14). Briefly, cells were collected, washed in PBS, and then lysed in 50 M phosphate buffer containing 1 mM EGTA and 150 mM sucrose and homogenized in a Dounce homogenizer with 100 strokes. Homogenates were centrifuged, and the pellets (membrane fractions) were stored at Ϫ80°C. For superoxide measurements, 50 g of protein was diluted in 500 l of the same lysis buffer. Dark-adapted lucigenin (15 M) was added to the sample, and measurement of chemiluminescence immediately started. Chemiluminescence (in arbitrary units) was measured at 15-s intervals for 1 min in a Turner 20/20 luminometer (Turner Designs, Sunnyvale, CA). NADPH was used as a substrate.
Measurement of NF-B DNA Binding Activity-Preparation of nuclear protein extracts and the electrophoretic mobility shift assay (EMSA) have been as described previously (20,21). Briefly, MIA PaCa-2 cells were lysed in a hypotonic buffer, the nuclear protein was extracted and stored at Ϫ80°C, and EMSA was performed as in Refs. 20 and 21.
For the DNA probes, we used the 5Ј-GCAGAGGGGACTT-TCCGAGA-3Ј oligonucleotide containing consensus B binding motif (underlined), as well as several oligonucleotides containing putative NF-B binding sites in the promoter region of human p22 phox gene, in particular, 5Ј-ACAGAGGGGCTC-CCCTCAAA-3Ј (located 795 nucleotides upstream of the transcription start codon and hence termed "p22-795") and 5Ј-AGCGCAGGAACTCCCCGCCT-3Ј (correspondingly, termed "p22-241"). These sites were identified by analyzing the p22 phox gene sequence (GenBank TM NG_007291) with the use of the TFSEARCH software (version 1.3). After annealing to their respective complementary oligonucleotides, the probes were end-labeled with T4 polynucleotide kinase.
In the cold competition experiment, a 100ϫ molar excess of nonlabeled wild-type or mutated consensus NF-B binding probe was added to the reaction together with the 32 P-labeled probe. In the mutated oligonucleotide, the consensus B binding motif was changed (lowercase) to GGccACTaaCC.
Measurement of NF-B Transcriptional Activity-NF-B transcriptional activity was assessed with the Dual-Luciferase reporter assay system (Promega, Madison WI). Briefly, MIA PaCa-2 cells were simultaneously co-transfected with the pGL3-4B-Luc and pRL-TK plasmids by using the Nucleofector TM II system (Amaxa, Inc.) according to the manufacturer's protocol. pGL3-4B-Luc contains the reporter gene encoding firefly luciferase driven by a promoter region containing four copies of NF-B-responsive element. pRL-TK is a Renilla luciferase driven by a basic thymidine kinase promoter, thereby playing the role of a reference plasmid. To inhibit NF-B, cells were transfected with the pcDNA3.1-IB⌬N plasmid expressing the NF-B inhibitor IB␣ (22). This plasmid expresses IB lacking the N-terminal 36 amino acids and known to be resistant to phosphorylation and degradation.

p22 phox , Nox4, and Akt in Pancreatic Cancer Cell Survival
Statistical Analysis-Results are expressed as means Ϯ S.E. and represent data from at least three independent experiments. Differences between two groups were analyzed using Student's t test. p Ͻ 0.05 was considered statistically significant.

RESULTS
Growth Factors Stimulate NADPH Oxidase Activity in PaCa Cells by Up-regulating p22 phox -With RT-PCR, we assessed the expression of various catalytic subunits of NADPH oxidase in PaCa cell lines (Fig. 1A). Both Nox4 and p22 phox mRNA were highly expressed in all the cell lines tested, whereas the expression of other Nox proteins varied.
To determine the involvement of p22 phox and Nox4 in ROS production, MIA PaCa-2 and PANC-1 cells were transfected with Nox4 or p22 phox siRNA (Fig. 1B). Knocking down of either Nox4 or p22 phox decreased ROS levels in cells cultured with FBS ( Fig. 1C), indicating that both of these catalytic subunits are required for ROS generation.
NADPH oxidase in PaCa cells is activated by growth factors, IGF-I and FBS (9). Transfection with p22 phox or Nox4 siRNA prevented this activation in both MIA PaCa-2 and PANC-1 cells (Fig. 1D). Knocking down p22 phox and Nox4 together decreased NADPH oxidase activity to the same level as the knockdown of either Nox4 or p22 phox alone (Fig. p22 phox , Nox4, and Akt in Pancreatic Cancer Cell Survival 1D), suggesting that both subunits are equally required for NADPH oxidase functional activity in PaCa cells.
We next asked whether the up-regulation of Nox4 and/or p22 phox could mediate the induction of NADPH oxidase activity by growth factors. Both IGF-I and FBS increased protein levels of p22 phox in MIA PaCa-2 and PANC-1 cells at 24 and 48 h of incubation ( Fig. 2A). IGF-I did not affect Nox4 protein level, whereas FBS moderately increased it at later times (48 h) ( Fig. 2A). Similar effects were observed in PANC-1 cells ( Fig. 2A).

p22 phox , Nox4, and Akt in Pancreatic Cancer Cell Survival
Further, IGF-I and FBS significantly increased p22 phox mRNA levels in both MIA PaCa-2 and PANC-1 cells (Fig. 2B). These increases in mRNA were completely abolished in the presence of actinomycin, indicating that the GFs up-regulate the protein level of p22 phox through stimulating its transcription. Neither IGF-I nor FBS up-regulated Nox4 mRNA level (Fig. 2B).
We determined (Fig. 2C) that Nox4 and p22 phox co-immunoprecipitate and that both IGF-I and FBS markedly increase the amount of p22 phox co-immunoprecipitated with Nox4. Complex formation between Nox proteins and p22 phox is known to increase the stability of both proteins (4,23,24). This provides an explanation for our finding (Fig. 2D) that Nox4 siRNA reduced the protein level of not only Nox4 but also p22 phox in MIA PaCa-2 cells. Similarly, p22 phox siRNA decreased somewhat the level of Nox4 (Fig. 2D). Of note, nei-ther Nox4 nor p22 phox siRNAs had any effect on the levels of other Nox proteins tested, i.e. Nox3 and Nox5 (data not shown).
These results indicate that activation of NADPH oxidase by growth factors in PaCa cells is mediated by transcriptional upregulation of p22 phox . In support of this conclusion, overexpression of p22 phox stimulated NADPH oxidase activity (Fig. 2E), mimicking the effect of the growth factors. Further, there was little additional stimulation of NADPH oxidase activity by the GFs in cells overexpressing p22 phox (Fig. 2E).
Akt Kinase Mediates the Up-regulation of p22 phox by Growth Factors-We next assessed the role of Akt kinase, a key mediator of GF receptor signaling, in the activation of NADPH oxidase in PaCa cells. To inhibit Akt, we applied a specific inhibitor of Akt isoforms 1/2, which greatly decreased the active (phosphorylated) Akt (Fig. 3A), and Akt-1 siRNA, which decreased the total Akt-1 level (Fig. 3B). Both the Akt1/2 inhibitor and the  MARCH 11, 2011 • VOLUME 286 • NUMBER 10

JOURNAL OF BIOLOGICAL CHEMISTRY 7783
Akt-1 siRNA prevented the up-regulation of p22 phox protein induced by IGF-I or FBS (Fig. 3, A and B). Further, the Akt1/2 inhibitor prevented the increases in p22 phox mRNA induced by IGF-I or FBS in both MIA PaCa-2 and PANC-1 cells (Fig. 3, C  and D).
The effect of Akt inhibition on p22 phox expression depended on whether cells were cultured with or without GFs. That is, Akt inhibition greatly down-regulated p22 phox protein and mRNA in cells cultured with the GFs, whereas p22 phox downregulation was minimal in cells cultured without GFs (i.e. in cells with a low level of Akt activity). Of note, the GFs significantly increased active (i.e. phosphorylated) Akt in PaCa cells (Fig. 3, A and B) (15). Neither the Akt1/2 inhibitor nor the Akt-1 siRNA affected Nox4 protein (Fig. 3, A and B) nor mRNA (not illustrated) levels.
Akt inhibition prevented the activation of NADPH oxidase by IGF-I and FBS (Fig. 4A) and decreased intracellular ROS (Fig. 4B) in both PaCa cell lines. The combined data in Figs. 3 and 4 indicate that Akt mediates GF-induced transcriptional up-regulation of p22 phox , resulting in NADPH oxidase activation. In contrast, Nox4 expression is not regulated by Akt. Interestingly, the results suggest that Akt is not involved in the regulation of p22 phox expression in cells cultured without growth factors.
NF-B Mediates the Up-regulation of p22 phox Expression Induced by IGF-I and FBS-In search for transcription factors mediating p22 phox up-regulation, we tested the involvement of NF-B because the p22 phox gene has putative NF-B binding sites in its promoter (see below) and because growth factors activate NF-B in PaCa cells (25). Moreover, it has been recently reported that NF-B mediates p22 phox expression in smooth muscle cells (26).
Both IGF-I and FBS stimulated NF-B DNA binding activity in PaCa cells, measured by EMSA both at 48 h and at shorter incubation times (Fig. 5, A and B). Importantly, the increase in NF-B activity was not only on a consensus NF-B binding site but also on DNA sequences containing putative NF-B binding sites in the human p22 phox gene promoter. These sites were identified with the use of the TFSEARCH software (see "Materials and Methods"). Of note, we detected NF-B binding on some, but not all, of the tested putative sites in p22 phox promoter: for example, the p22-241 site (located 241 nucleotides upstream of the transcription start) but not the p22-795 site (Fig. 5, A and B). The specificity of binding was confirmed in a cold competition experiment (Fig. 5C) in which nuclear protein bound to the p22-241 site was competed away by a 100ϫ excess of unlabeled NF-B probe, whereas the binding was not decreased with the same excess of mutated NF-B probe.
GF-induced NF-B binding was abolished by the specific Akt1/2 inhibitor (Fig. 5B). Importantly, the Akt1/2 inhibitor blocked NF-B binding not only to the consensus DNA sequence but also to the p22 phox promoter p22-241 binding site (Fig. 5B).
To further confirm that NF-B mediates p22 phox expression, we measured the effect of NF-B inhibition on p22 phox mRNA and protein levels. To inhibit NF-B, MIA PaCa-2 cells were transfected with pcDNA3.1IB⌬N plasmid encoding for nondegradable IB␣. IBs are a family of physiologic NF-B inhibitory proteins that keep NF-B proteins inactive in the cytosol (27). Upon activation, IB␣ is degraded in the proteasome, thus allowing NF-B translocation to the nucleus. Overexpression of IB␣ inhibited NF-B transcriptional activity in MIA PaCa-2 cells measured with a luciferase reporter assay (Fig. 5D). Importantly, it prevented the up-regulation of p22 phox mRNA and protein levels by IGF-I and FBS (Fig. 5, E and F). The results in Fig. 5 strongly indicate that NF-B mediates the up-regulation of p22 phox transcription induced by GFs in PaCa cells.
p22 phox Mediates the Anti-apoptotic Effect of the Growth Factors-Knocking down p22 phox with siRNA markedly increased apoptosis in cells cultured with growth factors (Fig.  6A), but it had little effect on apoptosis in GF-free conditions (i.e. at a relatively low p22 phox level) (Fig. 6A). Conversely, p22 phox overexpression had little effect on apoptosis in cells cultured with growth factors (i.e. in conditions resulting in a relatively high level of p22 phox ) but greatly decreased apoptosis in cells cultured in GF-free conditions (Fig. 6B).
Although siRNA knockdown of p22 phox stimulated apoptosis in cells cultured with either IGF-I or FBS, this stimulation was much greater for IGF-I, completely reversing its anti-apoptotic effect (Fig. 6A). This suggests that p22 phox -mediated mechanism(s) is largely responsible for the anti-apoptotic action of IGF-I in PaCa cells. In comparison, the effect of p22 phox siRNA on apoptosis in cells cultured with FBS was less pronounced (Fig. 6A), indicating that FBS inhibits apoptosis through both p22 phox -dependent and p22 phox -independent mechanisms.

p22 phox , Nox4, and Akt in Pancreatic Cancer Cell Survival
The anti-apoptotic effect of IGF-I was also abrogated by the Akt inhibitor (Fig. 6C). Interestingly, the stimulation of apoptosis in IGF-I-treated cells by the Akt inhibitor and p22 phox siRNA was non-additive (Fig. 6C). That is, Akt inhibition did not further increase apoptosis in cells in which apoptosis had been stimulated by p22 phox siRNA transfection (Fig. 6C). These results indicate that p22 phox mediates the pro-survival effect of Akt in PaCa cells.
We and others previously showed the anti-apoptotic role for Nox4 in PaCa cells (9,14,15,28). Here, we tested whether Nox proteins other than Nox4 also have an anti-apoptotic effect in PaCa cells. Knockdown of Nox3 or Nox5 with corresponding siRNAs (Fig. 6D) did not reverse the anti-apoptotic effects of IGF-I and FBS in MIA PaCa-2 cells, in contrast to Nox4 siRNA knockdown (Fig. 6E). This indicates that the pro-survival, anti-apoptotic role of Nox proteins is isoform-specific; in PaCa cells, it is mediated by Nox4, but not the other Nox proteins. The combined results in Fig. 6, together with our previous findings (9,14,15), indicate that NADPH oxidase composed of Nox4 and p22 phox mediates the resistance of PaCa cells to death.

DISCUSSION
One reason why pancreatic cancer (i.e. pancreatic adenocarcinoma) is so aggressive and unresponsive to chemo-and radiotherapy is its resistance to apoptosis. Growth factors play a key role in pancreatic tumorigenesis; in particular, IGF-I is overexpressed in PaCa cells, suppressing apoptosis and stimulating growth both in cell lines and in animal models of pancreatic cancer (29,30).
We have recently shown (9,15) that the pro-survival, antiapoptotic effect of IGF-I and FBS in PaCa cells is mediated by Nox4 NADPH oxidase. The present study investigates the mechanisms through which the GFs activate NADPH oxidase.
Our previous findings demonstrated a critical role of Nox4 in ROS production in PaCa cells (9,14,15). The present study shows that both Nox4 and p22 phox catalytic subunits are required for NADPH oxidase activity in PaCa cells. In other cell types, Nox4 similarly requires p22 phox , but not the cytosolic regulatory subunits, for oxidase activity (5-7). Our results show that growth factors transcriptionally up-regulate p22 phox , resulting in increased level of p22 phox protein, complex formation between p22 phox and Nox4, and oxidase activity.
Of note, little is known on transcriptional regulation of p22 phox expression. In phagocytes, p22 phox transcription is likely mediated through blood cell-specific transcription factor PU.1 (31), and NF-B was recently suggested to regulate p22 phox transcription in smooth muscle cells (26). The transcription factors that control p22 phox expression in epithelial cells remain undefined.
Several lines of evidence indicate that in PaCa cells, NF-B mediates transcriptional up-regulation of p22 phox by the GFs.   Akt kinase is an important effector of GF receptors and is activated by both IGF-I and FBS in MIA PaCa-2 and PANC-1 cells (15). In the present study, we show that Akt mediates GFinduced NF-B activation in PaCa cells through which the GFs stimulate p22 phox expression (Fig. 7). Based on findings in other cell types, the effect of Akt on NF-B activity is likely through phosphorylation of the p65 NF-B subunit (32).
Our results show that the mechanism of NADPH oxidase activation in PaCa cells is different from that of phagocytic NADPH oxidase. In PaCa cells, the GF-induced activation of NADPH oxidase is through transcriptional up-regulation of p22 phox , whereas in phagocytes, NADPH oxidase activation requires receptor-induced phosphorylation of its regulatory subunits. Of note, although Akt plays a critical role in NADPH oxidase activation in both PaCa cells and phagocytes, the underlying mechanisms are quite different. In particular, in phagocytes, Akt-mediated phosphorylation of the p47 regulatory subunit is important for NADPH oxidase activation (1, 8).
As we have shown, Nox4 is abundantly expressed in human pancreatic ductal adenocarcinomas (15) and plays a pro-survival role in PaCa cells (Refs. 9 and 15 and this study). Here, we show that Nox4 and p22 phox both contribute to the pro-survival effect of the GFs and that knocking down either of these subunits stimulates apoptosis in PaCa cells.
Our data further indicate that up-regulation of p22 phox , leading to NADPH oxidase activation, is critical for the anti-apoptotic effect of growth factors. Up-regulation of p22 phox fully mediates the anti-apoptotic effect of IGF-I, and to a lesser extent, that of FBS. The fact that overexpressing p22 phox by itself greatly inhibits apoptosis in PaCa cells further demonstrates the anti-apoptotic, pro-survival role of NADPH oxidase and ROS in these cells, supporting our previous findings (9,14,15,18). Moreover, the results show that the pro-survival effect of Akt is mediated, at least in part, through up-regulation of p22 phox (Fig. 7).
In sum, the results establish a novel pathway for receptormediated activation of Nox4 NADPH oxidase. We find that both Nox4 and p22 phox are required for NADPH oxidase activity in PaCa cells. IGF-I and FBS activate NADPH oxi- A, protein levels of p22 phox were measured by immunoblotting; ␤-actin served as loading control. A-C and E, DNA fragmentation was measured using cell death ELISA. D, Nox3, Nox4, and Nox5 mRNA levels were measured using real time PCR. In all panels, values are means Ϯ S.E. (n ϭ 3). *, p Ͻ 0.05 versus cells transfected with control siRNA (or control pcDNA plasmid) and cultured without growth factors. #, p Ͻ 0.05 versus cells cultured in the same condition without inhibitor or siRNA transfection. p22 phox , Nox4, and Akt in Pancreatic Cancer Cell Survival dase by up-regulating p22 phox expression through activation of NF-B, which is mediated by Akt. The p22 phox -mediated activation of NADPH oxidase, in turn, leads to inhibition of apoptosis and thus promotes pancreatic cancer cell survival.