Knockout of p47^phox Uncovers a Critical Role of p40^phox in Reactive Oxygen Species Production in Microvascular Endothelial Cells

Abstract

Objective

p40^phox is an important regulatory subunit of NADPH oxidase, but its role in endothelial reactive oxygen species (ROS) production remains unknown.

Methods and Results

Using coronary microvascular endothelial cells isolated from wild-type and p47^phox knockout mice, we found that knockout of p47^phox increased the level of p40^phox expression, whereas depletion of p40^phox in wild-type cells increased p47^phox expression. In both cases, the basal ROS production (without agonist stimulation) was well preserved. Double knockout of p40^phox and p47^phox dramatically reduced (≈65%) ROS production and cells started to die. The transcriptional regulation of p40^phox and p47^phox expressions involves HBP1. p40^phox was prephosphorylated in resting cells. PMA stimulation induced p40^phox swift dephosphorylation (within 1 minute) in parallel with the start of p47^phox phosphorylation. p40^phox was then rephosphorylated, and this was accompanied with an increase in ROS production. Depletion of p40^phox resulted in ≈67% loss in agonist-induced ROS production despite the presence of p47^phox. These were further supported by experiments on mouse aortas stimulated with angiotensin II.

Conclusion

p40^phox is prephosphorylated in resting endothelial cells and can compensate p47^phox in keeping basal ROS production. Dephosphorylation of p40^phox is a prerequisite for agonist-induced p47^phox phosphorylation, and p40^phox through its dynamic dephosphorylation and rephosphorylation is involved in the regulation of agonist-induced ROS production.

It has been well established that endothelial cells (ECs) express constitutively a multi-subunit NADPH oxidase that generates a low level of O₂^.− under basal physiological conditions to modulate redox-sensitive intracellular signaling pathways and to maintain normal EC function.^1,2 The activity of EC NADPH oxidase can be upregulated by agonists such as PMA (PKC activator), TNFα​,³ and angiotensin II (AngII).^4,5 Increased reactive oxygen species (ROS) production causes EC dysfunction, which is involved in the pathogenesis of inflammation and many cardiovascular disorders such as atherosclerosis, hypertension, and diabetes.²

NADPH oxidase contains a cytochrome b₅₅₈ (consisting of 1 member of the Nox family and a p22^phox subunit), and at least 4 regulatory subunits, eg, p40^phox, p47^phox, p67^phox, and rac1. Previous studies have shown that p47^phox plays a pivotal role in promoting agonist-induced EC NADPH oxidase activation, and knockout of p47^phox severely compromises the endothelial ROS response to AngII and TNFα stimulation.⁴ However, the loss of p47^phox did not reduce the basal (without agonist stimulation) ROS production. On the contrary, the basal NADPH-dependent ROS production was significantly higher in coronary microvascular ECs (CMECs) isolated from p47^phox knockout (KO) mice compared to wild-type (WT) controls.^4-6 The mechanism involved is not clear so far.

The p40^phox shares high sequence homology with the p47^phox.^7,8 It contains similar SH3 domains that could interact with proline-rich domains in p67^phox​,⁷ become phosphorylated during NADPH oxidase activation,^9-11 and is able to translocate to the plasma membrane in a similar manner to p47^phox.^12,13 Our hypothesis was that p40^phox might compensate p47^phox and maintain the basal NADPH oxidase activity in p47^phox KO ECs. In this study, we investigated this hypothesis by looking at the relationship between the levels of p40^phox and p47^phox expression and ROS production in CMECs isolated from p47^phox KO and WT mice. We also examined the differences in PMA- and AngII-induced phosphorylation of p40^phox and p47^phox in primary cultures of mouse CMECs as well as in mouse aortas.

Materials and Methods

Cell Culture

CMECs were isolated from 10-week-old wild-type (WT) and p47^phox knockout (KO) mice on a 129sv background.⁵ All studies were performed in accordance with protocols approved by the Home Office under the Animals (Scientific Procedures) Act 1986 UK. Six hearts were used for each CMEC isolation.

Gene Transfection of CMECs

The full-length human p47^phox and p40^phox cDNAs (kindly provided by F. Wientjes, UCL) were subcloned with sense and antisense orientations into a mammalian cell expression vector PcDNA3.1 (Invitrogen) and confirmed by molecular sequencing. Transfection of CMEC was undertaken with Lipofectamine 2000 and Plus reagent.¹⁴ The success of transfection was examined by either real-time PCR or Western blotting.

ROS Production

Three independent complementary methods were used for ROS measurement. NADPH-dependent O₂^.− production by EC homogenates was assessed by lucigenin (5 μmol/L)-enhanced chemiluminescence.¹⁴ The ROS production by living cells was measured using (1) DCF fluorescence visualized and quantified under confocal microscopy; (2) dihydroethidium fluorescence measured by flow cytometer.

In Vitro ³²P Labeling

Cells were labeled with ³²P-orthophosphate (100 μCi/mL) at 37°C overnight in phosphate-free medium.³ Cells were stimulated with PMA (100 ng/mL) and harvested for immunoprecipitation and p40^phox or p47^phox phosphorylation by autoradiography.

Statistics

Data were presented as means±SD from at least 3 experimental results taken from 3 independent cultures for each condition. Comparisons were made by unpaired t test, with Bonferonni correction for multiple testing. P<0.05 was considered statistically significant.

Results

Increased p40^phox and p67^phox Expression in p47^phox KO CMECs

The mRNA expressions of Nox1, Nox2, Nox4, p40^phox, p67^phox, and NoxO1 were examined by quantitative real-time PCR in WT and p47^phox KO CMECs (90% confluence) (Figure 1). Three Nox isoforms, Nox1, Nox2, and Nox4, were all detected in CMECs, and their mRNA expression levels were Nox1​<​Nox2​<​Nox4. Comparing to WT levels, p47^phox KO cells had significantly higher mRNA levels of Nox2 (1.29±0.1-fold), p40^phox (2.3±0.3-fold), p67^phox (1.5±0.2-fold), and NoxO1 (1.24±0.1-fold), and the largest increase was in p40^phox (Figure 1A). We then examined protein levels of the 3 major regulatory subunit, p40^phox, p47^phox, and p67^phox, and found that p47^phox KO cells had significantly higher levels of p40^phox (1.7±0.5-fold) and p67^phox (1.4±0.7-fold) compared to WT controls (Figure 1B). These changes were accompanied by significant increases in basal NADPH-dependent O₂^.− production by p47^phox KO CMEC homogenates (45±4%) detected by lucigenin-chemiluminescence (Figure 1C, left), and in living adherent cells (15±3%) detected by DCF fluorescence in (Figure 1C, right and supplement Figure IIA, available online at http://atvb.ahajournals.org; all P<0.05).

Figure 1.

Expression and activity of NADPH oxidase subunits in WT and p47^phox KO CMEC. WT: wild-type; KO: p47^phox knockout. A) Quantitative real-time PCR. The mRNA levels of Nox1, Nox2, Nox4, p40^phox, NoxO1 and p67^phox were normalized to the levels of β-actin detected in the same samples and expressed as molar/molar ratio to β-actin. B) Western blotting. For quantification, specific protein bands were normalized to α-tubulin detected in the same samples and expressed as arbitrary units. C) ROS production. Right panel: NADPH-dependent lucigenin-chemiluminescence. MLU: mean light units. Left panel: DCF fluorescence detection of ROS production in adherent CMEC (for quantification see supplement Figure IIA). n=3 separate CMEC isolations. *p<0.05 for KO values versus WT values.

Changes in p47^phox Expression Inversely Affected the Expression of p40^phox

We then reexpressed p47^phox into p47^phox KO CMECs. The success of gene transfection was confirmed by real-time PCR (data not shown) and the appearance of p47^phox bands on Western blot (Figure 2A). Equal loading of samples was confirmed by reprobing of the p47^phox membrane for α-tubulin. Reexpression of p47^phox into p47^phox KO cells decreased (40±3%) the levels of p40^phox expression (Figure 2A) and was accompanied by a reduction in basal O₂^.− production (Figure 2D; all P<0.05). This observation was further confirmed by depletion of p47^phox in WT CMECs using a full length antisense p47^phox cDNA. This technique had proven to be very successful in our previous studies^6,14 and avoided possible cross-silencing of genes that have the high sequence similarity with p47^phox and p40^phox. Transient depletion of p47^phox, as confirmed by the Western blot, was accompanied by a reciprocal increase (60±6%) in the p40^phox expression (Figure 2B) and a substantial increase in basal ROS production (Figure 2D; all P<0.05), which mimicked what we observed in p47^phox KO cells. However, overexpression of p47^phox into WT cells had no significant effect on the p40^phox expression (Figure 2C) and ROS production (data not shown). The p67^phox expression was unchanged (Figure 2A through 2C). The enzymatic source of ROS production in WT CMECs after depletion of p47^phox was examined using enzyme inhibitors (supplemental Figure IIB). The ROS production was virtually abolished in the presence of a flavoprotein inhibitor, diphenyleneiodonium (DPI), or a cell-permeable O₂⁻ scavenger, tiron, and was significantly inhibited (≈70%) by superoxide dismutase (SOD) or an NADPH oxidase inhibitor (apocynin). However, the inhibitors of xanthine oxidase (oxypurinol), NOS (L-NAME, N-ω-nitro-l-arginine methyl ester), or mitochondrial complex I enzymes (rotenone) had no significant effect. Similar results were obtained for p47^phox KO CMECs (data not shown).

Figure 2.

Genetic manipulation of p47^phox expression in WT and p47^phox KO CMEC. WT: wild-type; KO: p47^phox knockout; vec: Vector controls. A-C) Western blotting. For quantification, specific protein bands were normalized to α-tubulin detected in the same samples and expressed as arbitrary units. D) NADPH-dependent lucigenin-chemiluminescence. n=3 separate transfections of independent CMEC isolations. *P<0.05 for indicated values versus vector control values.

The Effects of Transient Overexpression or Depletion of p40^phox in WT Cells

Overexpression of p40^phox in WT CMECs, as shown by Western blot, had no significant effect on p47^phox expression (Figure 3A), but significantly increased (30±2%) the basal ROS production detected by lucigenin-chemiluminescence in cell homogenates and by dihydroethidium (DHE) flow cytometry (right shift) in living cells (Figure 3B). In contrast, transient depletion of p40^phox expression in WT CMECs caused a reciprocal significant increase (49±4%) in p47^phox protein expression (Figure 3A), which maintained the basal ROS production without significant change compared to the vector controls (Figure 3B). The protein levels of p67^phox were not affected (Figure 3A). Compared to vector transfected controls, increased basal ROS production attributable to overexpression of p40^phox in WT cells was accompanied by a 22±11% increase in cell number and 46±5% increase in proliferation (MTS assay) (Figure 3C, P<0.05). However, depletion of p40^phox in WT cells did not cause significant change in basal ROS production and in cell proliferation, which might be attributable to the presence of p47^phox.

Figure 3.

Genetic manipulation of p40^phox in CMEC. WT: wild-type (A-C); KO: p47^phox knockout (D-F); vec: Vector controls. p40over: overexpression of p40^phox; p40depl: depletion of p40^phox. A) and D): Western blotting. For quantification, specific protein bands were normalized to α-tubulin detected in the same samples and expressed as arbitrary units. B) and E): ROS production detected in cell homogenates by NADPH-dependent lucigenin-chemiluminescence (left panel) and in living cells by DHE flow cytometry (right panel). C) and F) Cell viability and proliferation detected by counting living cell numbers and expressed as % of vector controls (left panel) and MTS cell proliferation assay expressed as proliferation index (right panel). n=3 separate transfections of independent CMEC isolations. *P<0.05 for indicated values versus vector control values.

The Effects of Transient Overexpression or Depletion of p40^phox in p47^phoxKO CMECs

In p47^phox KO cells that had already a higher level of p40^phox (see Figure 1), transfection of p40^phox cDNA failed to further increase the p40^phox expression (Figure 3D), and there was no significant change in basal O₂^.− production (Figure 3E) or in cell proliferation (Figure 3F). In contrast, depletion of p40^phox expression in p47^phox KO cells (double knockout), as shown by the Western blot (Figure 3D), resulted in a dramatic 64±9% reduction (P<0.05) in basal O₂⁻ production by cell homogenates and in living cells (Figure 3E). Cells stopped proliferating and died, with the cell number dropping to 35±11%, and the proliferation index (MTS assay) reduced to 27±7% of the vector controls (Figure 3F).

The Kinetics of PMA-Induced p40^phox Dephosphorylation and Rephosphorylation and ROS Production

The p40^phox was already prephosphorylated in resting cells (time 0). PMA stimulation induced a swift dephosphorylation of p40^phox within the fist minute, followed by a progressive rephosphorylation up to 30 minutes of PMA stimulation (Figure 4A). In contrast, p47^phox had very little prephosphorylation in resting cells. PMA induced p47^phox phosphorylation, which started in the first minute, peaked at ≈15 minutes, and then remained constant (Figure 4A). We then examined the ROS production in the same cells. In WT cells, PMA significantly increased the O₂^.− production with a time course curve similar to the curve of p47^phox phosphorylation (Figure 4B). However, in p47^phox KO cells, PMA initially reduced the ROS production in parallel to the kinetic of p40^phox dephosphorylation. Although the ROS production in p47^phox KO cells recovered slowly afterward, it was only 52±7% of the WT level at 30 minutes of PMA stimulation. Depletion of p40^phox in WT cells so severely damaged PMA-induced ROS production such that it was only 47±8% of the WT level at 30 minutes of PMA stimulation (Figure 4B). The relationship between p40^phox and p47^phox phosphorylation in response to PMA (30 minutes) stimulation was further examined by immunoprecipitation of p40^phox and p47^phox followed by immunoblotting using phos-serine specific antibodies. We found that a full phosphorylation response of p40^phox and p47^phox to agonist stimulation requires the presence of both subunits. Knockout of p47^phox significantly reduced (≈42%) PMA (30 minutes)-induced p40^phox serine-phosphorylation, and depletion of p40^phox also severely compromised (≈50%) PMA-induced p47^phox serine-phosphorylation (Figure 4C).

Figure 4.

Agonist-induced p40^phox and p47^phox phosphorylation and ROS production.: CMEC (A-C); Aortic rings (D-F). WT: wild-type; KO: p47^phox knockout; vec: Vector controls. p40depl: depletion of p40^phox. P-p40: phosphorylated p40^phox; T-p40: Total p40^phox. For the detection of phosphorylation, p40^phox or p47^phox were immunoprecipitated down and the loading control of IP products was calculated to the equal levels of total p40^phox or p47^phox in different samples. The phosphorylations of p40^phox or p47^phox were detected by ³²P autoradiography (A) or immunoblot using phos-serine specific monoclonal antibody (B and D lower panel). Total p40^phox or p47^phox were detected by immunoblot. For quantification, the levels of phosphorylation bands were normalized to the bands of the total protein detected in the same sample. A) Time course of PMA stimulation. B) Time course of PMA-induced NADPH-dependent ROS production detected by lucigenin-chemiluminescence. C) Differences in serine phosphorylation after 30 min. of PMA stimulation. n=3 separate experiments (A-C). D) Upper panel: Immunoblot for the difference in p40^phox expression between aortas isolated from WT and p47^phox KO mice. Lower panel: Differences in Ang II-induced p40^phox serine phosphorylation between WT and p47^phox KO aortas. E) Differences in NADPH-dependent ROS production between WT and p47^phox KO aortas stimulated with or without Ang II. The results were expressed as mean light units (MLU) per mg protein. F) Endothelial-dependent vessel relaxation to acetylcholine (Ach) with or without Ang II stimulation. n=6 mice (D-F). *P<0.05 for indicated values versus WT values or vector values. †P<0.05 for indicated values versus values without AngII in WT group.

Effects of Acute Ang II Treatment on Aortas Isolated From WT and p47^phox KO Mice

Compared to WT controls, aortas from p47^phox KO mice had higher levels of p40^phox expression (Figure 4D, upper panels) and ROS production (Figure 4E) and reduced endothelium-dependent vessel relaxation to acetylcholine (Ach; Figure 4F, left). Acute Ang II (200 nmol/L, 30 minutes) treatment of WT vessels increased p40^phox serine phosphorylation (Figure 4D, lower) and the ROS production (Figure 4E), and these were accompanied by a reduced endothelium-dependent vessel relaxation (Figure 4F, right). However, these Ang II effects were absent in p47^phox KO vessels.

The Roles of Transcription Factor HBP1 in the Regulation of p40^phox and p47^phox Expression

Compared to WT cells, the levels of HBP1 (HMG box-containing protein 1) were significantly higher (≈2.3 folds) in p47^phox KO cells (Figure 5A, left), and were significantly lower in p40^phox depleted WT CMECs (Figure 5A, right). The role of HBP1 in the transcriptional regulation of p40^phox and p47^phox expression was further examined by transient in vitro knockdown of HBP1 using shRNA¹⁵ in human microvascular ECs (HMEC1). Knockdown of HBP1, as shown by the Western blot, resulted in a significant increase in p47^phox expression and this was accompanied with a significant reduction in p40^phox expression (Figure 5B)

Figure 5.

The role of HBP1 in the regulation of p40^phox and p47^phox expression and p40^phox regulation of EC ROS production. WT: wild-type; KO: p47^phox knockout; vec: Vector controls. p40depl: depletion of p40^phox. A) HBP1 expression in CMEC. For quantification, HBP1 bands were normalized to α-tubulin detected in the same samples and expressed as arbitrary units. B) The effect of knockdown HBP1 on the levels of p40^phox and p47^phox expression in HMEC1. *P<0.05 for indicated values versus WT value or vector values of the same protein. n=3 independent experiments. C) Schematic diagram of the proposed concept of p40^phox regulation of NADPH oxidase activity through its dynamic phosphorylation. Pre-phosphorylated p40^phox contributes to the basal ROS production and inhibits p47^phox phosphorylation. PMA stimulation causes p40^phox dephosphorylation, which allows p47^phox phosphorylation to happen. Once the p47^phox is phosphorylated, the rephosphorylation of p40^phox synergizes p47^phox effects and results in the full activation of NADPH oxidase.

Discussion

The Relationship Between the Levels of p40^phox and p47^phox Expressions in ECs

NADPH oxidase contains at least 4 major regulatory subunits: p40^phox, p47^phox, p67^phox, and rac. The canonical view of the NADPH oxidase activation is that it requires the association of all essential regulatory subunits, ie, p47^phox, p67^phox, and Rac1 with cytochrome b₅₅₈.¹⁶ However, this view has been changed by reports that in a cell-free system, NADPH oxidase could be activated in the total absence of p47^phox if high concentrations of p67^phox and rac were present, albeit at a lower maximal rate than in the presence of p47^phox.^17,18 Previously, we have also shown that p47^phox KO CMECs had a slightly but significantly higher basal NADPH-dependent O₂⁻ production, but the underlying mechanisms were not clear at that time.⁶ Here we report that the levels of p40^phox and p67^phox are indeed elevated at both mRNA and protein levels in p47^phox KO CMECs.

It is known that p40^phox has high sequence homology with p47^phox.⁷ Based on molecular similarity, it seemed possible for p40^phox to replace p47^phox and to maintain the basal O₂⁻ production in p47^phox KO ECs. This hypothesis was confirmed by gene transfection experiments showing that in vitro depletion of p47^phox in WT CMECs increased p40^phox expression and the basal ROS production, whereas reexpression of p47^phox into p47^phox KO CMECs reduced p40^phox expression, which decreased the basal ROS production back to the WT level. The physiological significance of our findings is not only to ascribe a molecular consequence to the loss of p47^phox, but also to define an important compensatory mechanism involved in the regulation of NADPH oxidase activity in ECs.

The Role of p40^phox in EC Basal ROS Production and Cell Proliferation

p40^phox was originally discovered in the complex copurified and coimmunoprecipitated down with p47^phox and p67^phox.¹⁹ Despite extensive studies describing the interaction of p40^phox with p67^phox and p47^phox in cell-free assays,^13,16,19,20 the role of p40^phox in living cells has not been well defined. Paradoxically there are virtually as many reports on an inhibitory role^20,21 as on an activating role^9,10 of p40^phox on NADPH oxidase activation. Our current study, using a series of experiments of in vitro overexpression or depletion of p40^phox, provides evidences that p40^phox is critically involved in the regulation of EC basal ROS production required for EC growth. Overexpression of p40^phox in WT CMECs increased basal levels of ROS production, which was accompanied by an increase in cell proliferation. However, depletion of p40^phox in WT ECs had no significant effect on both basal levels of ROS production and cell proliferation, presumably because of the compensatory effect from increased p47^phox expression. Double knockout of both subunits ie, depletion of p40^phox in p47^phox KO ECs caused a massive drop in basal ROS production, which was accompanied by cell proliferation arrest and cell death. Different from Nox2, Nox4 does not exhibit a binding site for p47^phox, and the known regulatory subunits are not required for its activity.²² Although Nox4 has been found to be involved in mediating insulin-induced cell differentiation in preadipocytes,²³ its levels were not affected by the p47^phox knockout. The mRNA of a newly discovered homologue of p47^phox, NoxO1,²⁴ was also detected in CMECs and was slightly increased in p47^phox KO ECs. However, its expression levels were very low compared to the levels of p40^phox, and it was not able to compensate the loss of p47^phox. Put together, our results strongly suggest that as long as 1 subunit, p40^phox or p47^phox, is present in ECs, that is enough to maintain the basal ROS production from NADPH oxidase and to keep ECs alive and proliferating. However, the loss of both p40^phox and p47^phox severely compromises the basal ROS production and EC survival.

Dynamic Phosphorylation and Dephosphorylation of p40^phox in ECs

An important discovery from the current study is the prephosphorylation of p40^phox in resting ECs, and the dynamic dephosphorylation and rephosphorylation of p40^phox during PMA stimulation. Interestingly, one recent study reported that p40^phox was also in a basal phosphorylated state in resting neutrophils and undergoes further phosphorylation on multiple sites by PKC stimulation. They found in a cell-free assay that both phosphorylated and nonphosphorylated p40^phox were able to interact with p47^phox and p67^phox, but only phosphorylated p40^phox inhibited NADPH oxidase activation if added before full activation of the enzyme.²¹ Based on this previous report and our experimental results, we suggest that prephosphorylated p40^phox interacts with unphosphorylated p47^phox in resting ECs to keep the NADPH oxidase activity at low but optimized basal levels.

PMA is often used to bypass cell surface receptors and directly causes PKC-dependent phosphorylation of p47^phox. Interestingly, p40^phox has also been shown to be phosphorylated by PKC at its serines.¹¹ In the present study, we have extended this knowledge by showing a rapid dephosphorylation followed by a progressive rephosphorylation of p40^phox when ECs were stimulated with PMA. The biphasic phosphorylation of p40^phox has never been reported, and this may contribute to the complexity of its roles in NADPH oxidase activation and the discrepancy between previous studies.^9,10,20,21 Although the mechanisms of p40^phox in EC Nox2 regulation requires further investigation, our hypothesis (Figure 5C) is that prephosphorylated p40^phox inhibits p47^phox phosphorylation. Agonist stimulation induces a rapid p40^phox dephosphorylation which allows p47^phox phosphorylation to occur and to bind to p22^phox. However, once the p47^phox is phosphorylated, rephosphorylation of p40^phox (maybe at different sites) synergizes the action of phosphorylated p47^phox to further promote EC ROS production. The optimal NADPH oxidase response to agonist stimulation requires the presence of both p47^phox and p40^phox, and either depletion of p40^phox or knockout of p47^phox severely compromises agonist-induced O₂^.− production. The pathophysiological significance of p40^phox in the regulation of vascular ROS production and endothelial function was further demonstrated by experiments on WT and p47^phox KO aortic vessels stimulated with or without Ang II. In p47^phox KO vessels, acute Ang II stimulation failed to induce p40^phox phosphorylation, and there was no increase in O₂^.− production and the endothelium-dependent vessel relaxation to Ach was well preserved as compared to WT controls. Superoxide anions are dismutated to H₂O₂. Recently, endothelial H₂O₂ production and H₂O₂-mediated hyperpolarization response to Ach have been found to be closely coupled to nitric oxide synthases (NOSs) system.²⁵ The potential relationship between p40^phox and NOSs system requires further investigation.

The HBP1 is a member of the HMG box family transcriptional factors²⁶ and has been found to play a role in transcriptional repression of p47^phox gene.²⁷ The promoter of p47^phox gene contains 6 adjacent tandem high-affinity HBP1 binding sites, which are required for the transcriptional repression.²⁷ HBP1 has also been reported to enhance promoter activity of genes with lower affinity or a single HBP1 binding site.^28,29 Interestingly, the promoter region of p40^phox has also a HBP1 binding site, which makes p40^phox a possible candidate for HBP1 to activate.²⁷ We propose that HBP1 may play a dual roles (ie, to repress p47^phox and in the mean time to promote p40^phox) and to regulate ROS production by NADPH oxidase. This hypothesis is supported by our results that knockdown of HBP1 significantly increased the p47^phox expression and reduced p40^phox expression. There is also a feedback mechanism from p47^phox to HBP1 because knockout of p47^phox remarkably increased HBP1 expression. Although HBP1 may represent a transcriptional mechanism involved in the regulation of ROS production by NADPH oxidase in ECs, more detailed investigations are required to fully address this question.

In summary, we have reported, for the first time, a critical role of p40^phox in the regulation of basal and agonist-induced ROS production by NADPH oxidase in ECs. The p40^phox is prephosphorylated in resting ECs and plays an inhibitory role to keep the basal ROS production, and is able to substitute for p47^phox in p47^phox KO ECs. p40^phox undergoes rapid dephosphorylation and then rephosphorylation when ECs are stimulated with PMA, and the biphasic phosphorylation of p40^phox is involved in the regulation of NADPH oxidase activity. Depletion of p40^phox resulted in a loss in agonist-induced EC ROS production despite the presence of p47^phox. Inhibition of p40^phox phosphorylation may have a potential therapeutic application in treating EC dysfunction-related diseases.