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(Journal of Leukocyte Biology. 2001;69:161-168.)
© 2001 by Society for Leukocyte Biology

Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells

David J. Samuelson*,{dagger}, Marianne-B Powell{ddagger}, Maria Lluria-Prevatt* and Donato F. Romagnolo*,{dagger},§,||

* Interdisciplinary Cancer Biology Program
§ Arizona Cancer Center
|| Southwest Environmental Health Sciences Center
{dagger} Laboratory of Mammary Gland Biology, Department of Nutritional Sciences, The University of Arizona, Tucson, Arizona
{ddagger} Division of Radiation Biology, Stanford University, Stanford, California

Correspondence: Donato F. Romagnolo, 303 Shantz Bldg., The University of Arizona, Tucson, AZ 85721-0038. E-mail: donato{at}ag.arizona.edu


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ABSTRACT
 
The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 µM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 µM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 µM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.


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INTRODUCTION
 
The superoxide anion O2•- is produced normally during respiratory burst of phagocytic leukocytes as a defense mechanism against invading pathogens or in response to a variety of agents that activate the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. Dismutation of O2•- by superoxide dismutases generates hydrogen peroxide, which is a source of hydroxyl radicals [1 ]. Although reactive oxygen species (ROS) may play a normal role in regulation of apoptosis, differentiation [2 ], and in growth control [3 ], copious production of ROS by phagocytic cells may initiate and promote the development of cancer in target tissues [4 ]. For example, the accumulation of ROS as a result of insufficient antioxidant activities or scavenging enzymes, as well as the exposure to radiation and metabolites of a variety of xenobiotics, may damage nucleic acids, proteins, tissue, and cellular organelles [5 ]. Therefore, understanding the development and expression of the phagocyte oxidase in macrophages may aid in unraveling functionally similar mechanisms in nonphagocytic cells. Moreover, the availability of cell-assay systems and biomarkers of ROS production may be useful in assessing exposure to environmental toxicants [6 ] and carcinogens [7 8 9 10 ].

Activation of the NADPH oxidase, a multicomponent enzyme involved in the respiratory burst of leukocytes, requires the coordinate recruitment to the phagocytic membrane of several cytosolic subunits [11 ] and their assembly with the membrane-bound cytochrome b558. The latter comprises the NADPH binding subunit, gp91phox [12 ], which catalyzes the transfer of electrons from NADPH to molecular oxygen, thus generating the superoxide anion. Mutations in gp91phox impair superoxide production, thus contributing to the development of chronic granulomatous disease [13 ]. Conversely, epigenetic effectors that augment the expression or recruitment of components of the NADPH oxidase may lead to increased ROS production. For example, the tumor promoting agent 12-O-tetradeconylphorbol-13-acetate (TPA) contributes to differentiation of HL-60 promyelocytic leukemia cells into ROS-producing macrophage-like cells [14 ] through activation of ß-isozyme of protein kinase C (PKC) [15 ].

In this study, we hypothesized that TPA may stimulate superoxide production in promyelocytic leukemia HL-60 cells through the coordinate induction of differentiation and expression of gp91phox. We demonstrate that stimulation by TPA of ROS production was preceded by upregulation of gp91phox mRNA because of activation of transcription from the proximal gp91phox promoter.


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MATERIALS AND METHODS
 
Materials and cell culture
HL-60 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Fetal bovine serum (FBS) was from Hyclone Laboratories, Inc. (Logan, UT). The 2',7'-dichlorofluorescein diacetate (DCFH-DA) was from Molecular Probes, Inc. (Eugene, OR). Oligonucleotides used for polymerase chain reaction (PCR) amplification of gp91phox and p47phox were obtained from Genosys (The Woodlands, TX). Vent DNA polymerase was from New England Biolabs (Beverly, ME). All other chemicals and cell-culture media were from Sigma-Aldrich Chemical Co. (St. Louis, MO). The Reverse Transcription (RT) and Hybspeed kits, ribosomal 18S primers, and transcription vectors Triplescript and pTRI-cyclophilin were all from Ambion, Inc. (Austin, TX).

Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% FBS. HL-60 cells were grown in suspension at a density of 2.5 x 105 cells/ml media for RNA collection, lucigenin, and luciferase (LUC) assays, whereas a total of 1.0 x 106 cells were used for the DCFH-DA assay.

Lucigenin and DCFH-DA assays
The lucigenin assay was performed as previously described [9 ]. Briefly, cells in suspension were pelleted and washed with 2 ml HEPES-buffered saline solution (HBSS, pH 7.0). After washing, 2 ml of 250 µM lucigenin in HBSS was added to the cell cultures for immediate reading in a Turner Designs 20/20 luminometer. All chemiluminescence readings were an average of a 60-sec integrate time after a 5-sec delay and were expressed as relative light units (RLU).

For detection of intracellular ROS, cells were resuspended for 30 min in DMEM/F12, 0.5% FBS containing DCFH-DA diluted to a final concentration of 20 µM. DCFH-DA solutions were always handled under dim lighting. Immediately prior to fluorescence reading, the cells were washed once with 1 ml HBSS as previously described [10 ]. Cells were resuspended at a final density of 3 x 105 in 0.5 ml HBSS, and fluorescence was read using a Cambridge Technologies 7620 fluorescence plate reader equipped with 485 nm excitation and 530 nm emission filters. Readings were expressed as relative fluorescence units (RFU). In preliminary experiments, we ascertained that increasing the cell number up to 2 x 106 resulted in a linear increase in RFU (unpublished results). Because the RFU data presented in this study were generated from 1 x 106 cells, we concluded that the concentration of 20 µM DCFH-DA was not limiting for efficient detection of intracellular ROS.

Semiquantitative RT-PCR
Total cellular RNA was extracted using a guanidinium thiocyanate procedure [16 ]. RT was performed using total RNA incubated with random hexamer primers, Moloney murine leukemia virus RT, RNase inhibitor (Life Technologies/Gibco BRL, Gaithersburg, MD), and RT buffer at 42°C for 1 h. For semiquantitative PCR amplification, cDNA representative of 500 ng total RNA was used to monitor the expression of gp91phox and p47phox. Oligonucleotides used to amplify gp91phox (773 bp) were (forward) 5'-caacaagagttcgaagacaa-3' (exon 4) and (reverse) 5'-ggatgtcagtgtaaaagggt-3' (exon 9). Oligonucleotides used to amplify p47phox (541 bp) were (forward) 5'-cagacatcaccggccccatca-3' (exon 5/6) and (reverse) 5'-cggacgctgttgcggcgata-3' (exon 10). The PCR products were of the expected size, and their authenticity to the sequences deposited in the GeneBank (gp91phox, accession no. X04011; p47phox, AF184614) was verified by direct DNA sequencing. The 18S ribosomal RNA (488 bp) was used as an internal standard for equal loading and monitoring of PCR conditions. Preliminary experiments with increasing concentrations of 18S competimer primers to 18S primers indicated that ratios of 1.5:1–2.5:1 allowed for linear amplification of 18S (unpublished results). Thus, subsequent PCR amplifications of 18S as internal control were carried out using a ratio of 1.75:1. Relative expression levels of gp91phox and p47phox were estimated by Alpha Imager (Alpha Innotech, Inc., San Diego, CA) analysis and expressed as arbitrary densitometric units (ADU) corrected for the 18S control.

Ribonuclease protection assay of gp91phox
Levels of gp91phox mRNA were measured in total RNA (10 µg) by ribonuclease protection assay (RPA) using the Hybspeed RPA kit (Ambion). The cyclophilin mRNA was used as an internal standard. The ribonucleotide probe designed to target gp91phox mRNA was amplified using the forward 5'-ggtcccatgtttctgtatct-3' and reverse 5'-ggatgtcagtgtaaaagggt-3' oligonucleotides, which spanned a 210-bp region homologous to the gp91phox cDNA region from 838 to 1047 bp. This sequence was cloned in the antisense orientation into the transcription vector Triplescript (Ambion). Direct DNA sequencing confirmed authenticity of the cloned fragment to the gp91phox sequence. The cyclophilin ribonucleotide probe transcribed from pTRI-cyclophilin (Ambion) protected a fragment of 103 bp. Quantitation of gp91phox mRNA was performed by phosphorimager analysis and expressed as arbitrary units corrected for the levels of cyclophilin mRNA (gp91phox/cyclophilin).

The stability of gp91phox mRNA was examined in time-course experiments by culturing HL-60 cells in basal DMEM/F12 plus 10% FBS or DMEM/F12 plus 10% FBS containing 0.1 µM TPA for 6 h, followed by treatment with actinomycin D (5 µg/ml) or actinomycin D plus TPA. Cells were then harvested for extraction of total RNA. Temporal decay of gp91phox mRNA was expressed as percentage of gp91phox mRNA remaining at each time point.

Transient transfection assay
A plasmid containing the -1542 to +12 region of the gp91phox promoter was a gift from Dr. David Skalnik, Department of Pediatrics, Indiana University School of Medicine (Bloomington, IN). The gp91phox promoter fragment was subcloned into the pGL3 basic vector (Promega, Madison, WI) to direct transcription of the LUC reporter system. Transient transfection of HL-60 cells with the gp91phox-LUC construct was carried out using a cationic lipid DMRIE-C (1,2-dimyristoyloxypropyl-3-dimethyl-hydroxyethyl) suspended in Opti-MEM1 medium (Life Technologies/Gibco BRL), according to the manufacturer’s instructions for transfection of cells growing in suspension. Then, cells were treated with 0.1 µM TPA for 24 h, after which LUC activity was detected using a Turner Designs 20/20 luminometer.

Statistical analysis
RLU and RFU are presented as means ± SD. Comparisons of means following a significant (p<.05) analysis of variance (ANOVA) were performed by Fisher’s protected least significant difference test.


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RESULTS
 
TPA-dependent induction of ROS production
Based on the property that the lucigenin substrate cannot be taken up by the cells, it has been used in previous studies to monitor extracellular production of superoxide anion [9 ]. In contrast, the DCFH-DA probe has been adopted to assess intracellular ROS levels [10 ]. The cell membrane is permeable to DCFH-DA but not to the DCFH intermediate, which can be oxidized rapidly by ROS generated from superoxide. In this study, changes in ROS production were expressed as RLU and RFU, as described in Materials and Methods, utilizing the lucigenin and DCFH-DA substrate, respectively. The results depicted in Figure 1A indicate that, in the absence of any other stimulants, 0.1 µM TPA induced a time-dependent increase in RLU. At 48 h, TPA sustained a tenfold elevation (2.4 vs. 0.24) in RLU compared with control cells. Further evidence that TPA enhanced the intracellular levels of ROS was obtained by monitoring the kinetics of DCFH-DA oxidation (Fig. 1B) . This effect was time-dependent, as documented by a 2.5-fold induction in RFU after 24 h compared with untreated cells, although a significant increase in fluorescence was monitored already at 6 h. The DCFH-DA probe detected a burst in intracellular ROS production between 6 and 24 h. This preceded, by approximately 24 h, the increase in extracellular superoxide detected with lucigenin.



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  		Figure 1. A) Detection of superoxide anion by lucigenin. HL-60 cells were cultured in DMEM or DMEM plus 0.1 µM TPA. At the end of the incubation periods, RLU were calculated from chemiluminescence readings obtained with a luminometer. B) TPA induces oxidation of dichlorofluorescin. HL-60 cells were cultured for various periods of time in DMEM or DMEM plus 0.1 µM TPA. RFU were obtained by fluorescence plate reading. Data are RLU and RFU ± SD and are representative of three independent experiments.

As expected, the exposure to TPA induced differentiation from cells in suspension into attached cultures forming protruding pseudopodia and clumps (Fig. 2 ). These cumulative data indicated that treatment with 0.1 µM TPA stimulated morphological changes and the production of ROS effectively, typical of the promyelolytic respiratory burst.



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  		Figure 2. TPA induces differentiation of HL-60 cells. Cells were cultured in control DMEM or DMEM plus 0.1 µM TPA. Phase-contrast miscroscopies were taken after 48 h. The treatment with TPA induced cell attachment and formation of clumps.

In preliminary dose-response experiments, we found that at higher doses (1.0 µM), TPA did not stimulate further ROS production (unpublished results), compared with chemiluminescence induced with 0.1 µM TPA for 48 h. We concluded that concentrations of 0.1 µM were not limiting for long-term induction of superoxide. Nevertheless, we questioned whether short-term treatment of HL-60 cells with TPA might stimulate ROS production, independent of the effects of TPA on cellular differentiation. Therefore, we treated undifferentiated and differentiated HL-60 cells with 0.1 µM TPA for 15 min and compared RLU in resting (DMEM) and TPA-induced cells. The data depicted in Figure 3 show that treatment of undifferentiated cells with 0.1 µM TPA for 15 min produced a modest but significant increase in RLU. We attributed this increase in ROS to the presence of a small population of spontaneously differentiated cells in the cultures. As expected, the resting levels of RLU in differentiated cells were approximately tenfold higher than those detected in undifferentiated cultures. However, chemiluminescence from oxidation of lucigenin was increased further by 20% upon stimulation of attached cultures with TPA for 15 min. We discounted the likelihood that the additional burst in ROS production in undifferentiated and attached cultures was a result of transition into a differentiated phenotype. A possibility is that in resting and differentiated cells, TPA-dependent activation of the PKC may have stimulated phosphorylation of cytosolic components of the NADPH oxidase, thus contributing to their recruitment to the phagocytic membrane [15 ].



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  		Figure 3. Stimulation of ROS production with TPA in undifferentiated and differentiated HL-60 cells, which were precultured in DMEM (undifferentiated) or DMEM plus 0.1 µM TPA (differentiated) for 48 h. Then, cells were cultured in control DMEM medium (resting) or treated (induced) with 0.1 µM TPA for 15 min. At the end of the incubation period, cells were harvested, and ROS production was estimated based on relative lucigenin units as described in Materials and Methods. Data are average RLU ± SD from three independent wells.

TPA induces gp91phox mRNA levels
To gain better insight into the regulation of the NADPH oxidase, we questioned whether, in addition to stimulating maturation to a differentiated phenotype, TPA may induce changes in expression of gp91phox, which encodes for the catalytic subunit of the NADPH oxidase enzyme complex. Using semiquantitative RT-PCR, we ascertained linear accumulation of gp91phox mRNA during a 36-PCR cycle amplification reaction with an input of cDNA corresponding to 500 ng total RNA obtained from HL-60 cells (Fig. 4A ), whereas gp91phox products reached a plateau beyond 37-PCR cycle reactions (Fig. 4B) . In preliminary experiments, we determined that in a 36-PCR cycle reaction, a molar ratio of 18S competimers (18S primers=1.75) allowed for linear amplification of the control 18S ribosomal subunit (unpublished results). These PCR conditions were used to assess the relative changes in expression of gp91phox mRNA in control and TPA-treated HL-60 cells. The results depicted in Figure 5 indicate that stimulation with TPA sustained a 1.6-fold accumulation of gp91phox mRNA at 2 h, which was followed by a further 5.5-fold increase at 6 h. Overall, TPA triggered an 8.8-fold increase in gp91phox mRNA compared with HL-60 cells cultured in control medium.



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  		Figure 4. Semiquantitative RT-PCR analysis of gp91phox. A) PCR amplification was carried out starting from an input cDNA corresponding to 500 ng total RNA from HL-60 cells. B) Effects of number of cycles on amplification of gp91phox PCR products. Data represent arbitrary densitometry units of gp91phox PCR products.



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  		Figure 5. TPA induces gp91phox mRNA. A) HL-60 cells were cultured in DMEM or DMEM plus 0.1 µM TPA. Total RNA was collected at 2 and 6 h posttreatment. Amplifications of gp91phox and 18S were carried out in a 36-PCR cycle reaction. Amplification of ribosomal 18S RNA was performed using a molar ratio of 18S competimers (18S=1.75). In preliminary experiments (unpublished results), these conditions resulted in linear amplification of the control 18S ribosomal subunit. B) Arbitrary densitometry units represent levels of gp91phox corrected for the internal control 18S mRNA (gp91phox/18S).

Further evidence that TPA induced the accumulation of gp91phox mRNA was obtained by ribonuclease protection assay of total RNA extracted from HL-60 cells. As shown in Figure 6 , the exposure to TPA elicited a time-dependent accumulation of gp91phox mRNA, which at 24 h was increased 11.5-fold (Fig. 6C) . Conversely, in control cells (Fig. 6A) or cells treated with the dimethyl sulfoxide (DMSO) vehicle (Fig. 6B) , levels of gp91phox mRNA remained nearly constant. Only at 72 h did the vehicle DMSO stimulate a slight accumulation of gp91phox mRNA. Cumulative analysis of the temporal profiles of gp91phox expression by RT-PCR and ribonuclease protection assay entailed that accumulation of gp91phox mRNA preceded the explosive increase in superoxide production detected at 48 h (Fig. 1A) .



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  		Figure 6. Ribonuclease protection assay of gp91phox mRNA. HL-60 cells were cultured in A) DMEM, B) DMEM plus vehicle (0.006% DMSO), or C) DMEM plus 0.1 µM TPA for various periods of time. At the end of the incubation periods, levels of gp91phox mRNA were analyzed in 10 µg total RNA by RPA as described in Materials and Methods. Cyclophilin mRNA was used as internal standard for equal loading and monitoring of RPA conditions. Data are representative of five independent experiments.

Because the NADPH oxidase is a multicomponent enzyme system, we verified whether TPA elicited the expression of the cytosolic p47phox gene, which is also transcriptionally regulated [17 ]. Using the conditions described in Materials and Methods and Figure 4 , we found that the treatment with 0.1 µM TPA induced a time-dependent increase in p47phox transcripts (Fig. 7 ). Early accumulation of p47phox mRNA was detectable at 6 h and increased up to threefold at 24 h. The cellular levels of p47phox mRNA peaked at 48 h but declined to basal levels by 72 h. In contrast, no measurable changes in the content of p47phox mRNA were recorded in cells cultured in control medium (DMEM) or medium supplemented with the vehicle (DMSO). Overall, these data confirmed that the treatment with TPA was effective in stimulating expression of p47phox, although gp91phox was increased more dramatically than was p47phox.



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  		Figure 7. Semiquantitative RT-PCR analysis of p47phox mRNA. HL-60 cells were cultured in DMEM, DMEM plus vehicle (0.006% DMSO), or DMEM plus 0.1 µM TPA for various periods of time. At the end of the incubation periods, levels of p47phox mRNA were analyzed from an input cDNA corresponding to 500 ng total RNA, as described in Materials and Methods. Ribosomal 18S RNA was used as internal control for equal loading and monitoring of RT-PCR conditions. Data are representative of two independent experiments.

TPA induces transcription from the gp91phox promoter
The RT-PCR and ribonuclease protection assay data suggested the existence of a temporal association between upregulation of gp91phox mRNA and activation of ROS production. However, we considered that the stimulatory effects of TPA on gp91phox expression could be a result of increased stability of the gp91phox mRNA or an effect of TPA on transcriptional regulation of gp91phox. Therefore, in association with TPA, we used actinomycin D to inhibit transcription and monitored the rate of gp91phox mRNA decay. These experiments revealed that the TPA treatment did not influence the stability of gp91phox mRNA because the rate of degradation of the gp91phox transcript in cells challenged with TPA was similar to that monitored in control HL-60 cells (Fig. 8 ). The half-life of the gp91phox mRNA was approximately 5 h in control and TPA-treated cells. These results prompted further investigations into the regulation of the gp91phox promoter by TPA. We transfected HL-60 cells transiently with an expression vector containing a 1.5-kb fragment of the gp91phox proximal promoter (-1542/+12) linked to a firefly-LUC reporter system (Fig. 9 ). Upon treatment with TPA, activity from the gp91phox promoter was increased threefold (Fig. 9A) . Conversely, no changes in reporter activity were detected upon treatment with TPA in nontransfected cells or cells transfected with the pGL3 basic (empty) vector, nor did TPA influence expression from an internal SV40 pGL3-control vector (Fig. 9B) . We concluded that TPA elicited the accumulation of gp91phox mRNA through transcriptional activation of the gp91phox promoter.



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  		Figure 8. gp91phox mRNA stability. HL-60 cells were cultured in basal DMEM or DMEM containing 0.1 µM TPA for 6 h. Then cells were cultured in DMEM medium containing actinomycin D (5 µg/ml) or DMEM/F12 with actinomycin D plus TPA (0.1 µM). Decay of gp91phox mRNA was expressed as the percentage of gp91phox mRNA remaining at each time point.



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  		Figure 9. Transcriptional activation of gp91phox by TPA. A) Transient transfection of HL-60 cells with a 1.5-kb gp91phox-LUC construct. After transfection, cells were treated with 0.1 µM TPA for 24 h. LUC activity in cell extracts was detected using a Turner Designs 20/20 luminometer. B) LUC activity directed from a SV40 control plasmid (pGL3Control) containing the SV40 promoter and enhancer. Bars represent average LUC units ± SD from three independent experiments.


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DISCUSSION
 
The primary objective of this investigation was to examine the relationships between ROS production and regulation of gp91phox gene expression by TPA in promyelocytic leukemia HL-60 cells. Elucidating the mechanisms that lead to activation of the NADPH oxidase may have broad implications in understanding the contribution of ROS in environmental toxicology and carcinogenesis. Recurrent stimulation of phagocytic cells in target tissues may cause exposure to supraphysiological ROS levels. The latter are suspected to participate in the initiation and promotion of carcinogenesis in chronically inflamed tissue via increased rates of DNA damage and alterations in expression of oncogenes and tumor-suppressor genes [18 ]. For example, stimulation of ROS production by TPA has been associated with promotion of neoplastic transformation [5 ].

The superoxide anion is produced in phagocytic cells primarily by the NADPH oxidase enzyme upon attack by pathogens or stimulation with various agents [1 ]. The NADPH oxidase is a multicomponent enzyme system comprised of the membrane-bound cytochrome b558, which includes the gp91phox and p22phox subunits and an array of cytosolic proteins (p40phox, p47phox, and p67phox) [19 ]. Regulation of the NADPH oxidase is complex in structure and mechanism and involves the guanosine 5'-triphosphate (GTP)-binding proteins p21rac and rap1A, along with the guanosine 5'-diphosphate (GDP)-dissociation inhibitor rho [4 , 20 ].

The efficacy of the TPA treatment was documented in this study by the striking increase in RLU and RFU detected from the lucigenin and DCFH substrate, respectively. Although the magnitude of the respiratory burst may be underestimated by the extracellular lucigenin assay [10 ], enhanced oxidation of the intracellular DCFH probe provided confirmatory but important evidence that TPA increased the respiratory activity of HL-60 cells. Furthermore, upon treatment with TPA, cells differentiated from cells growing in suspension into nonproliferating attached cultures.

A general assumption has been that ROS production by neutrophils and macrophages is a direct consequence of activation of the NADPH oxidase system [21 ]. Nevertheless, a functional relationship between ROS production and regulation of gp91phox expression by TPA has not been detailed yet. In particular, we documented that the rapid accumulation of gp91phox transcripts (8.8- to 11.5-fold) preceded, by approximately 6–12 h, the surge in superoxide anion detected with lucigenin. Our data complement earlier studies documenting that the levels of gp91phox mRNA rose 13-fold in differentiated cells, with a twofold increase detectable within 3 h of exposure to retinoic acid [22 ]. In this study, examination of the changes in transcript levels of the cytosolic component p47phox confirmed the efficacy of the TPA treatment, which induced peak accumulation of p47phox mRNA at 12–24 h. Similar detection intervals were demonstrated in previous studies [23 ], in which p47phox was first detected at 16 h of differentiation and increased thereafter.

When studying the expression profiles of gp91phox, we can conclude that the accumulation of gp91phox mRNA preceded maximum production of ROS by approximately 24 h. These findings entail that upregulation of the gp91phox gene is a requirement for activation of the NADPH oxidase complex in HL-60 cells. Regarding the mechanisms responsible for this induction, we tested whether TPA affected the rate of degradation of gp91phox mRNA. Using actinomycin D to inhibit transcription, we obtained evidence that TPA did not influence the stability of gp91phox transcripts because the decay of the gp91phox mRNA was similar in control and TPA-treated cells. In other studies, the half-life of gp91phox transcripts was not affected by treatment with the differentiation inducers interferon (IFN)-{gamma} or tumor necrosis factor (TNF), thus suggesting transcriptional regulation [24 ]. In addition, we ascertained in transient transfection experiments that TPA enhanced transcription of a LUC reporter gene directed from a 1.5-kb gp91phox promoter fragment. Our findings are in accord with earlier studies documenting that an increase in transcription rate accounted for most, if not all, of the accumulation in gp91phox mRNA following treatment with the differentiating agent 1,25-(OH)2-D3 and the cytokines IFN-{gamma} and TNF-{alpha} [25 26 27 ]. Conversely, transcriptional repression of gp91phox by glucocorticoids correlated with decreased NADPH oxidase activity [17 ]. Therefore, our promoter studies of gp91phox add to the current knowledge regarding the molecular mechanisms responsible for regulation of superoxide production by the NADPH oxidase in macrophages [28 ]. We envision that stimulation of gp91phox expression by TPA may contribute to increasing the number of catalytic sites available for electron transfer from NADPH to molecular oxygen, thus favoring the production of superoxide anion. However, the fact that short-term exposure (15 min) to TPA stimulated superoxide production by undifferentiated and TPA-differentiated cultures suggested that some activation of the NADPH oxidase may occur through mechanisms independent of TPA-increased expression of gp91phox or p47phox. This notion is in keeping with evidence that rapid activation by TPA of PKC-dependent phosphorylation contributed to differentiation of HL-60 cells [15 ].

In summary, we present evidence that in addition to stimulating morphological differentiation, TPA elicited the coordinate expression of gp91phox and p47phox and the production of ROS. One broad implication of these studies is that changes in expression of gp91phox may be used as a biomarker of exposure to environmental xenobiotics that stimulate the NADPH oxidase. Furthermore, we document that one component of TPA-dependent activation of the NADPH oxidase is transcriptional activation of the gp91phox promoter. These pleiotropic responses may be elicited directly by TPA or mediated by other factors regulated by TPA. One of the features of the proximal 5' promoter region of gp91phox is that it contains two CCAAT domains, which confer transcriptional repression through binding by a CCAAT displacement factor (CDP) [29 ], whereas transcriptional repression of gp91phox is removed in terminally differentiated neutrophils and B-lymphocytes [30 ]. Whether TPA contributes to removal of repression by CDP warrants further investigation. Our laboratory is currently investigating whether TPA may stimulate transcription through transactivation of an array of TPA-responsive elements comprised in the proximal promoter region of the gp91phox gene (unpublished results). If correct, this hypothesis may provide new clues for understanding the contribution of tumor-promoting agents to the regulation of the NADPH oxidase complex.


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ACKNOWLEDGEMENTS
 
This work was supported in part by a grant to D. F. R. from the Arizona Elks Comprehensive Program in Transplantation Research and Education, The University of Arizona, Tucson, and a graduate scholarship from the Cowden Foundation to D. J. S.

Received March 19, 2000; revised July 13, 2000; accepted July 14, 2000.


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