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RECEPTORS AND SIGNAL TRANSDUCTION

Protein kinase C-dependent and -independent signaling in genotoxic response to treatment of desferroxamine, a hypoxia-mimetic agent

Departments of ¹Molecular Pharmacology and Toxicology and ²Medicine, the ³Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles; and ⁴Department of Clinical and Molecular Pharmacology, City of Hope National Medical Center, Duarte, California; ⁵Department of Craniofacial Biology/Cell and Developmental Biology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado; and ⁶Department of Biology, Universidad Nacional de Colombia, Bogota, Colombia

Submitted 8 August 2006 ; accepted in final form 15 February 2007

	ABSTRACT

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Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKC is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKC by RNAi (sh-PKC) or expression of the kinase-dead (Lys376Arg) mutant of PKC (PKCKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (-H2AX), a hallmark for DNA damage, was altered by sh-PKC, and sh-PKC completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKC-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKC and caspase-3, suggesting that PKCKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKC-dependent and -independent manners. In summary, these findings suggest that the activation of a PKC-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment.

DNA damage; caspase-3; Akt

Hypoxia-reoxygenation has been shown to cause DNA replication fork arrest, followed by DNA damage and stabilization of p53, resulting in the DNA damage response (18, 20). The DNA damage response is usually initiated by the activation of the ataxia telangiectasia mutated (ATM) and the homologous ATM- and Rad3-related (ATR) proteins (46). ATM and ATR share many common substrates and are usually activated with distinct kinetics or by different genotoxins. Once activated, ATM phosphorylates various downstream substrates, including p53, Chkl, Chk2, and the histone H2AX. ATM activation exerts three crucial functions: regulation and stimulation of DNA repair, signaling of cell cycle checkpoints, and signaling of cell death (29). If unrepaired, this DNA damage, especially DNA double-stranded breaks (DSBs), could trigger cell death. Caspases are intracellular cysteine proteases that mediate cell death and inflammation. Caspase-3 is a major mediator of apoptotic cell death (55). Conversely, the protein Akt is a serine-threonine kinase that has a central role in provoking suppression of apoptosis. Inhibition of apoptosis by Akt leads to increased cell survival (1). The substrates of Akt include proapoptotic Bad, caspase-9, and ASK1, leading to a direct suppression of the mitochondrial apoptotic pathway. Since DFO mimics the hypoxic condition in various cells and tissues, it will be of great interests to evaluate the effect of DFO treatment on modulating the DNA damage response and pro/anti-apoptotic signaling and their cross talk.

The protein kinase C (PKC) family of serine-threonine kinases is activated by diverse stimuli and participates in a variety of cellular processes, such as growth, differentiation, apoptosis, and cellular senescence (2, 9, 19, 47, 51, 56). To date, the PKC family comprises 11 isoforms that are subgrouped on the basis of structure and their activation modes into 3 subfamilies: the classical PKCs (-, -, and -), which are activated by diacylglycerol (DAG) and calcium; the novel PKCs (-, -, -, and -), which require DAG, but not calcium, for activation; and the atypical PKCs (- and -/), which can be activated in a DAG- and calcium-independent manner (5, 40, 47). Studies with PKC^–/– mice suggest that PKC plays pivotal roles in the regulation of cell proliferation and apoptosis (2, 21, 30, 47). Mechanistically, PKC is activated in response to numerous cellular stimuli by various mechanisms, including membrane translocation (23, 49), protein-protein interaction (3), tyrosine phosphorylation (11, 25), and proteolytic cleavage (11, 25, 44). The translocation of PKC to different subcellular compartments and/or proteolytic cleavage can be induced by ceramide, TNF-, UV irradiation, ionizing radiation, testosterone, oxidative stress, and etoposide (5, 13, 37, 38, 44).

Although PKC plays a central role in the regulation of responses incurred by a variety of stimuli, a detailed characterization of the function of PKC and its underlying mechanism under a hypoxia- or DFO-induced stress condition is still elusive. Herein, we report that PKC is a key regulator of the salivary adaptive signaling network in response to the genotoxic stress elicited by DFO treatment. We used lentiviral delivery of sh-PKC, wildtype PKC-enhanced green fluorescent protein (EGFP), or Lys376Arg-mutated PKC-EGFP to investigate DFO-mediated PKC activation and examine whether DNA damage and cell survival pathways are differentially regulated in salivary epithelial Pa-4 cells with a number of distinct PKC contexts upon the exposure of cells to DFO. Results from our studies demonstrate a time-dependent proteolytic activation and nuclear translocation of PKC in response to DFO treatment. We further establish that the Lys376 residue of PKC is essential for DFO-induced PKC activation, and that Lys376Arg-mutated PKC-EGFP functionally dysregulates PKC-dependent adaptive responses in a dominant-negative manner. Our results also suggest that DFO treatment induces differential p53, Chk1, and Akt activation profiles in Pa-4, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells. The latter results provide a potential underlying mechanism by which fine tuning of salivary cellular responses to genotoxic responses on treatment with DFO may be accomplished through PKC. A cross talk among the activated PKC, caspase-3, Akt, and ATM signaling pathways is proposed. To the best of our knowledge, this is the first demonstration showing that PKC plays a unique role in mediating adaptive responses to DFO-exerted genotoxicity.

	MATERIALS AND METHODS

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Cell culture. The rat parotid epithelial Pa-4 cell line and all variants generated were cultured on Primaria culture dishes (Falcon) with Dulbecco's modified Eagle's/F-12 (DME/F-12, 1:1 mixture) medium supplemented with 2.4% fetal bovine serum, insulin (5 µg/ml), L-glutamine (2.3 mM), transferrin (5 µg/ml), epidermal growth factor (25 ng/ml), hydrocortisone (1.1 µM), glutamate (5 mM), T₃ (3,3',5-triiodo-L-thyronine) (1.7 nM), kanamycin (94 µg/ml), and fungizone (47 µg/ml) and maintained at 35°C in a humidified atmosphere of 5% CO₂ and 95% air (33, 43).

Design and generation of lentiviral constructs. The lentilox 3.7 expression vector (pLL3.7) (45) was used as the frame for the generation of both gene silencing and overexpression vectors. For gene silencing, the EGFP encoding sequence was deleted from pLL3.7 by restriction enzyme digestion and compatible ends re-ligation, yielding the so-called gene silencing empty vector (pGS-EV). This empty vector was linearized by digestion with HpaI and XhoI restriction enzymes, and a preannealed synthetic short hairpin (sh) DNA fragment was directionally ligated. To facilitate the screening of positive clones, a BamHI restriction site was included in the sh sequence. The sequences of all sh constructs used in this study are summarized in Table 1. Each construct was engineered downstream of the human U6 promoter in the pGS-EV. For overexpression or exogenous expression, the wildtype pPKCWT-EGFP and kinase-dead (KD; a Lys376Arg mutation in the ATP binding site) pPKCKD-EGFP (13) were subcloned downstream of the cytomegalovirus (CMV) promoter in pLL3.7 to generate lenti-PKCWT-EGFP and lenti-PKCKD-EGFP, respectively. As a negative control, the PKCWT-EGFP sequence was deleted by restriction enzyme digestion and compatible ends re-ligated, yielding the so-called overexpression empty vector (pOE-EV). All constructs were verified by DNA sequencing.

Transduction of Pa-4 cells. For lentiviral infection, Pa-4 cells were plated 1 day before infection and cultured overnight to reach 25% confluence. The culture medium was then aspirated, and fresh medium containing concentrated virus was added and incubated for 24 h in the presence of polybrene (9 µg/ml). With the use of fluorescence-activated cell sorter (FACS) analyses to monitor EGFP expression, a dose-dependent transduction was established in Pa-4 cells by increasing the multiplicity of infection (MOI) from 0.1 to 40 to yield 21–99% EGFP-positive cells, respectively, as confirmed in part by immunofluorescence microscopy (data not shown).

Cloning and characterization of transduced cell lines derived from Pa-4 cells. Transduced Pa-4 cells were cloned by serial dilution to obtain single cell-derived clones. For gene silencing studies, clones expressing the lowest PKC protein levels, based on Western analyses with an antibody against PKC, were used. For studies of kinase function of PKC, clones were initially screened for the overexpression of exogenous pPKC-EGFP chimeric constructs by fluorescence microscopy and then further confirmed by Western analyses using an antibody that detects GFP tag. Clones expressing the highest exogenous protein levels were then used. All cells used in this study are summarized in Table 2.

Live cell imaging. Pa-4/PKCWT-EGFP and Pa-4/PKCKD-EGFP cells were seeded at 25% confluence in eight-well cell culture chambers (Lab-Tek) and cultured for 24 h. Cells were then treated without or with 50 µM DFO (time t = 0), and images were acquired at 0, 12, 24, 36, and 48 h. Exposure time under UV light irradiation was kept as short as possible, and cells with no DFO treatment served as a control, using a Nikon epifluorescence inverted microscope. Five randomly selected fields per each specimen were acquired per time point with a total magnification of x600. Acquired images were processed using Metamorph Imaging, Corel Photo-Paint, and LSM 5 Image Browser Software. For confocal images, cells were examined using a Nikon PCM 2000 Confocal System equipped with argon ion and green HeNe lasers attached to a Nikon TE300 Quantum inverted microscope (Center for Liver Disease Research, Univ. of Southern California).

Immunocytochemistry. Cells were seeded and treated as described for live cell images, except for using 16-well cell culture chambers (Lab-Tek), and further processed for immunofluorescence microscopy. Briefly, DFO-treated and control cells were rinsed with phosphate-buffered saline (PBS), fixed with 2% paraformaldehyde in PBS, quenched with 50 mM ammonium chloride in PBS, permeabilized with 0.5% Triton X-100 in PBS, blocked with 1% bovine serum albumin in PBS, and incubated with the corresponding primary antibody. Cells were then washed with PBS, followed by incubation with the corresponding secondary antibody to obtain multicolor staining. The secondary antibodies used were as follows: goat-anti-mouse antibody conjugated to fluorescein isothiocyanate (FITC; ICN Pharmaceuticals, Cappel Products) or conjugated to tetramethyl-rhodamine (TMR; Santa Cruz Biotechnologies) and goat-anti-rabbit antibody conjugated to TMR or FITC. 4'-6-Diamidine-2-phenyl indole (DAPI; Molecular Probes) was used as a counterstain for identification of nuclei.

MTT assays for measurement of cell viability. Pa-4, Pa-4/sh-PKC, Pa-4/PKCWT-EGFP, or Pa-4/PKCKD-EGFP cells were seeded into 24-well plates to reach 35–50% confluence on the day of the experiment. The cells were treated with 100 µM DFO or vehicle for 48 h followed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma) assay according to the manufacturer's recommendations. Absorbance of each well was read at 540 nm in a scanning multiwell spectrophotometer. The results, depicted as percentage of cell viability, are reported as means ± SD of three independent experiments performed in triplicate. P values from two-sidedt-test were calculated, and the difference between two data sets is significant when P < 0.05.

Flow cytometry for cell cycle analyses. Pa-4, Pa-4/sh-PKC, or Pa-4/PKCKD-EGFP cells were seeded at 50–80% confluence in 35-mm dishes and synchronized by serum starvation overnight. After treatment with 100 µM DFO for different time points, as indicated, cells were fixed in 70% ethanol overnight and stained with propidium iodide (PI)-Triton X-100-RNase A in PBS for 30 min at room temperature. Flow cytometry was performed using FACSCaliber (Becton Dickinson) as previously described (41). Statistical analyses were performed by two-way ANOVA, with randomized sample blocks and post hoc tests using Fisher's least-squared difference method of protected t-tests. P < 0.05 was considered statistically significant.

	RESULTS

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PKC is proteolytically activated on DFO treatment. To establish the role of PKC in DFO-induced cellular response(s), we engineered a series of lentivirus constructs expressing wildtype (WT) or KD (Lys376Arg) PKC fused to EGFP (PKCWT-EGFP or PKCKD-EGFP, respectively), or RNAi (sh-PKC) for suppressing the expression of endogenous PKC, as described in MATERIALS AND METHODS. Since salivary Pa-4 cells express a high level of endogenous PKC, they were chosen to be an experimental model for our studies to achieve our goal. Pools of Pa-4 cells transduced with individual lentivirus, harboring either PKCWT-EGFP or PKCKD-EGFP, were used to obtain clones of Pa-4/PKCWT-EGFP or Pa-4/PKCKD-EGFP, respectively (Fig. 1A). Individual clones, expressing the highest protein levels of PKCWT-EGFP or PKCKD-EGFP, were selected and expanded for use in the experiments reported herein. To downregulate PKC expression, four different sh-PKCs were pooled in a simultaneous transduction to obtain 40% PKC gene silencing effect (Fig. 1A). This pool of transduced cells was further used to obtain several individual clones. Two of these clones showed 90% downregulation of endogenous PKC expression (Fig. 1A, clones 5 and 7) and were used for the studies shown herein. A summary of the engineered cell pools and cell lines used in this study is shown in Table 2.

View larger version (46K): [in this window] [in a new window]   Fig. 1. PKC is activated on desferroxamine (DFO) exposure. A: generation of Pa-4/sh-PKC, PKCWT-EGFP, and PKCKD-EGFP cells. Pa-4 cells were transduced as described in MATERIALS AND METHODS with lenti-sh-PKC, lenti-PKCWT-EGFP, and lenti-PKCKD-EGFP, respectively. These pools of transduced cells were used to generate clones and screened by fluorescence microscopy. Positive clones were further screened by Western blot analyses using an anti-PKC antibody. Tubulin level in each lane was immunoblotted as a loading control. B: DFO treatment induces PKC cleavage. Pa-4 cells were treated with increasing concentrations of DFO and harvested at 32 h posttreatment. Equal amounts of whole cell extracts were used for Western analyses with an anti-PKC antibody recognizing the COOH-terminal portion of PKC, while an anti-tubulin antibody was used to quantitate tubulin levels as a loading control. The activation of PKC is indicated by generation of the catalytic fragment (CF) at an apparent molecular mass of 40 kDa. Relative levels of PKC-CF are indicated as italic nos. C: kinase activity of PKC is required for DFO-mediated activation of PKC. Parental Pa-4, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells were treated with 50 µM DFO for the indicated time periods before Western analyses as described in B. Relative levels of PKC-CF are indicated as italic nos. One representative Western blot from three independent experiments is shown. PKC, protein kinase C; EGFP, enhanced green fluorescent protein; sh, short hairpin; WT, wildtype; KD, kinase-dead.

DFO stimulates nuclear translocation of PKC. The activation of classical and novel PKC is often noted with their translocation from the soluble to the particulate cellular fractions through the interaction between the C1 domain and membrane lipids (40). Since subcellular localization change is expected on PKC activation, we performed immunocytochemical analyses to investigate whether DFO treatment induces PKC translocation to different cellular compartments. As shown in Fig. 2A, endogenous PKC was seen throughout the cells before DFO treatment (Fig. 2Aa). Following treatment with DFO, PKC became enriched in the nucleus, where the intensity of signals detected from endogenous PKC reached maximum between 24 and 32 h posttreatment (Fig. 2A, b–d), correlating with time-dependent generation of PKC-CF (Fig. 1C). We next tried to confirm this observation by performing imaging analyses with Pa-4/PKCWT-EGFP cells through monitoring EGFP signals at different post-DFO treatment time points (Fig. 2B). Notably, PKCWT-EGFP exhibited a restricted vesicle-like signal surrounding the nucleus in untreated cells (Fig. 2B, a and b) but translocated to the nucleus (Fig. 2B, j, n, and r, and Fig. 2Cc) in DFO-treated cells. Although the gross intracellular localization of PKC and the trend of nuclear translocation kinetics during the course of DFO treatment were comparable between the endogenous PKC and the stably expressed PKCWT-EGFP, notable differences existed. We cannot, however, rule out the possibility that EGFP tagging perhaps contributes, at least in part, to the localization of PKCWT-EGFP fusion protein and the kinetics of PKCWT-EGFP nuclear translocation on DFO exposure (Fig. 2B) compared with those seen with endogenous PKC (Fig. 2A).

View larger version (45K): [in this window] [in a new window]   Fig. 2. Kinase activity is required for nuclear translocation of PKC on DFO treatment. A: DFO induces nuclear translocation of PKC. Pa-4 cells were seeded on 16-well cell culture chambers, treated with 50 µM DFO, and harvested at the indicated time points. Cells were processed for immunocytochemistry as indicated in MATERIALS AND METHODS. Immunostaining of endogenous PKC (green), DAPI staining for identification of nuclei (blue), and immunostaining for tubulin (red) are shown. Images were acquired using a Nikon epifluorescence inverted microscope and processed using Metamorph Imaging, Corel Photo-Paint, and LSM 5 Image Browser Software. B and C: PKC kinase function is required for its DFO-induced nuclear translocation. Pa-4/PKCWT-EGFP or Pa-4/PKCKD-EGFP cells were seeded in 8-well cell culture chambers and treated either with or without 50 µM DFO in full culture medium. Live cell images were acquired (B) at the indicated time points using a Nikon epifluorescence inverted microscope and processed as in A. C: orthogonal image planes were reconstructed using C-Imaging, Corel Photo-Paint, and LSM 5 Image Browser Software. DFO treatment induces a translocation of PKC-EGFP from cytoplasm to nucleus, whereas PKCKD-EGFP accumulates at the plasma membrane.

Dysregulation of DFO-induced DNA damage response signaling by sh-PKC or PKCKD-EGFP. DFO treatment has been reported, by us and others (10, 41), to affect genome integrity. We next performed biochemical studies to investigate the cross talk between the ATM cascade and PKC signaling during DFO-induced adaptive responses. We first evaluated the phosphorylation status of ATM and its substrate H2AX in Pa-4 cells treated with DFO for different time periods. ATM activation was assessed by ATM phosphorylation at Ser-1981 during the course of DFO treatment (Fig. 3A, top). Moreover, it is likely that manipulation of the PKC level, by use of sh-PKC or PKC function by expression of PKCKD-EGFP, evoked a modest modulation of the fold-of-activation or the onset of maximal induction, as noted by the quantitative analysis of the relative pSer-1981-ATM level in each sample (Fig. 3A, middle and bottom).

View larger version (39K): [in this window] [in a new window]   Fig. 3. A DNA damage response pathway is activated by DFO treatment. A: DFO induces ataxia telangiectasia mutated (ATM) and H2AX phosphorylation. Pa-4, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells were treated with 50 µM DFO for the indicated time periods before harvesting. Equal amounts of whole cell extracts were used for Western analyses with anti-ATM, anti-phospho-Ser-1981-ATM, and anti-phopho-Ser-139-H2AX antibodies, while an anti-tubulin antibody was used for loading control. Activation of ATM is evidenced by its phosphorylation at Ser-1981 and by the phosphorylation of its substrate, H2AX, at Ser-139 (-H2AX). *ATM and pSer-1981-ATM. Relative levels of p-ATM and -H2AX are indicated as italic nos. B: -H2AX accumulates in nuclei of Pa-4 cells in response to DFO treatment. Pa-4 cells were seeded in 16-well cell culture chambers, treated with 50 µM DFO, and harvested at the indicated time points. Cells were processed for immunocytochemistry as indicated in MATERIALS AND METHODS. Immunostaining of endogenous -H2AX (green) and PKC (red) and DAPI (blue) staining for identification of nuclei are shown. Images were acquired using a Nikon epifluorescence inverted microscope and processed using Metamorph Imaging, Corel Photo-Paint, and LSM 5 Image Browser Software. C: DFO treatment induces p53 and Chk1 activation. Pa-4, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells were treated with 100 µM DFO for the indicated time periods before harvesting. Equal amounts of whole cell extracts were used for Western blot analyses with indicated antibodies, while an anti-actin antibody was used for loading control. Relative levels of pSer15-p53 and pSer345-Chk1 are indicated as italic nos. One representative Western blot from three independent experiments is shown.

Phosphorylation of both p53 at Ser-15 and Chk1 at Ser-345 has also been shown to be involved in DNA damage signaling (36, 39, 50). To determine whether different PKC contexts affect DFO-induced activation of a molecule(s) other than ATM, we assessed the phosphorylation profile of p53 at Ser-15 (pSer15-p53) and Chk1 at Ser-345 (pSer345-Chk1) on exposure to DFO in parental and engineered Pa-4 cells. As shown in Fig. 3C, exposure to DFO caused a sustained induction of pSer15-p53 and a temporal increase of pSer345-Chk1 signal in all cells tested. Both the durations and signals of pSer345-Chk1 and pSer15-p53 were somehow altered in both DFO-treated Pa-4/sh-PKC and Pa-4/PKCKD-EGFP cells. Taken together, these data demonstrate that it is likely that DNA damage signaling pathways other than ATM may also contribute to the DFO-mediated signaling events.

Activation of proapoptotic caspase-3 and prosurvival Akt in response to DFO treatment. Because caspase-3 is a key effector component of several apoptotic signaling pathways (4, 42, 48), we next explored the possibility that caspase-3 also participates in DFO/PKC-mediated signaling. The characteristic cleavage of caspase-3, a hallmark of caspase-3 activation, appeared to occur 32 h post-DFO treatment (Fig. 4A, top), correlating with PKC cleavage (Fig. 1C) and -H2AX activation (Fig. 3A). Since PKC has been implicated in a positive activation loop with caspase-3 in other apoptotic contexts (5), we also evaluated the effect of PKC expression on DFO-mediated caspase-3 activation. As expected, the steady-state levels of the cleaved fragment of caspase-3 (caspase-3-CF) in Pa-4/sh-PKC and Pa-4/PKCKD-EGFP cells were barely detectable by Western analyses during the course of DFO treatment (Fig. 4A, two bottom panels), suggesting that PKC is necessary for DFO-induced caspase-3 activation.

View larger version (66K): [in this window] [in a new window]   Fig. 4. Cross talk between PKC, caspase-3, and Akt activation on DFO exposure. A: caspase-3 cleavage in response to DFO treatment. Pa-4, Pa-4/PKCWT-EGFP, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells were treated with 50 µM DFO for the indicated time periods before harvesting. Equal amounts of whole cell extracts were used for Western blot analyses with an anti-caspase-3 antibody, while an anti-tubulin antibody was used to assess equal loading. The proteolytically generated fragment of 17 kDa is indicated as caspase-3-CF. The proteolytic generation of caspase-3-CF in both Pa-4/sh-PKC and Pa-4/PKCKD-EGFP cells is negligible. Relative levels of caspase-3-CF in Pa-4 and Pa-4/PKCWT-EGFP cells are indicated as italic nos. B: DFO induces activation of p53 and Chk1 in a caspase-3-independent manner. MCF7/Neo and MCF7/Caspase-3 cells were treated with 100 µM DFO for the indicated time periods before harvesting. Equal amounts of whole cell extracts were used for Western blot analyses with indicated antibodies, while an anti-actin antibody was used for loading control. Relative levels of PKC-CF, caspase-3-CF, pSer15-p53, and pSer345-Chk1 are indicated as italic nos. C: attenuation of DFO-induced Akt phosphorylations at Ser-473 by PKC. Cells were treated as described in A. Equal amounts of whole cell extracts were used for Western blot analyses with anti-phospho-Ser-473-Akt or anti-phospho-Thr-308-Akt antibodies or an anti-actin antibody to assess equal loading. D: PKCKD downregulates hypoxia-induced PKC and caspase-3 activation. Pa-4 and Pa-4/PKCKD-EGFP cells were treated with 1% O2 for the indicated time periods before harvesting. Equal amounts of whole cell extracts were used for Western analyses with an anti-PKC or an anti-caspase-3 antibody, while an anti-tubulin antibody was used to assess equal loading. Relative levels of PKC-CF and caspase-3-CF in Pa-4 and Pa-4/PKCKD-EGFP cells are indicated as italic nos. One representative Western blot from three independent experiments is shown.

Next, we evaluated the effect of PKC on the Akt activation profile during the DFO-induced responses. As shown in Fig. 4C, Ser-473-Akt was modestly phosphorylated by DFO treatment, while no marked difference in the profile of DFO-induced Ser-473-Akt phosphorylation was noticed throughout the time course we examined using the three cell lines. In some experiments, we observed that pSer-473-Akt was induced at 8 h posttreatment with DFO, followed by downregulation of the pSer-473-Akt signal until approximately 4048 h posttreatment (Fig. 4C, top) or with a higher basal level of pSer-473-Akt signal (data not shown) in Pa-4 cells. In general, the DFO-stimulated Thr-308-Akt phosphorylation signals were more noticeable (also a delayed event) in Pa-4, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells despite their distinct PKC context (Fig. 4C). In a comparison of their DFO-induced pSer-473-Akt and pThr-308-Akt profiles (Fig. 4C), PKC appears to play a fine-tuning role in modulation of DFO-triggered Akt phosphorylation. Last, we examined whether we could extend our observations that PKCKD-EGFP attenuates the DFO-induced proteolytic activation of PKC and caspase-3 in response to hypoxic exposure. As shown in Fig. 4D, it appeared that PKCKD-EGFP also modulated the generation of PKC-CF and caspase-3-CF on prolonged exposure to 1% O₂ (Fig. 4D, bottom), supporting the notion that PKC could play a vital role in modulating the cellular response to adverse conditions, such as exposure to DFO or 1% O₂. However, 1% O₂-evoked PKC-CF began to appear, at a significant level, 8 h earlier in Pa-4/PKCKD-EGFP cells than in Pa-4 cells (Fig. 4D). The exact reason underlying this phenomenon is still unclear.

The involvement of PKC in DFO-induced cellular responses in Pa-4 cells. To further evaluate the contribution of PKC to DFO-mediated signaling cross talk and its biological consequences, these parental and engineered Pa-4 cells were assayed for their sensitivity toward DFO-mediated cell growth inhibition by MTT assays. As shown in Fig. 5A, both Pa-4 cells with decreased expression of endogenous PKC and Pa-4 cells expressing PKCKD-EGFP were less sensitive to DFO treatment than parental Pa-4 cells, the pool of transduced cells with PKCWT-EGFP, and cells transfected with the corresponding empty vector (data not shown). These data support our notion that PKC plays an antagonistic role by suppressing cell survival in response to DFO. The lack of further enhancement in DFO-elicited cell growth inhibition by exogenous PKCWT-EGFP suggests that the endogenous PKC level in Pa-4 cells may be sufficient for conveying the signaling elicited by DFO, or a PKC-independent pathway may counter the effect of an enhanced PKC/caspase-3 pathway. Since DFO treatment is known to arrest Pa-4 cells in S-phase and induce apoptosis (41), we also determined the role of PKC in DFO-elicited cell cycle dysregulation. Neither sh-PKC nor exogenous PKCKD-EGFP alone conveyed a marked effect on modulating cell cycle progression before DFO treatment (data not shown). In Fig. 5B, except for 24 h post-DFO exposure in Pa-4/PKCKD-EGFP cells, significant increases in sub-G₁ cell population were observed in DFO-treated cells compared with vehicle-treated cells in all three cell lines. Moreover, we also observed significant decreases in sub-G₁ cell population for both Pa-4/sh-PKC and Pa-4/PKCKD-EGFP cells compared with Pa-4 cells at both 24 and 48 h post-DFO treatment (Fig. 5B). We concluded that PKC enhances the cytotoxic effect of DFO in Pa-4 cells.

View larger version (29K): [in this window] [in a new window]   Fig. 5. PKC is essential for DFO-mediated cytotoxicity. A: sh-PKC or PKCKD increases Pa-4 cell survival under conditions of DFO-induced cell death. MTT assays were performed as described in MATERIALS AND METHODS. Results from 3 independent assays are shown. Error bars correspond to SD. *Significant difference (P < 0.01) between corresponding cells. B: attenuation of PKC by sh-PKC or PKCKD protects Pa-4 cells against DFO-elicited apoptosis as reflected by a decrease in sub-G1 population. The populations of sub-G1 cells were normalized and expressed as a percentage compared with that of vehicle-treated Pa-4, Pa-4/sh-PKC, and Pa-4/PKCKD-EGFP cells. Data are presented as means ± SD from 3 separate experiments performed in triplicate. **P < 0.01 and *P < 0.05. C: proposed model for DFO-induced activation of PKC, DNA damage responses, and Akt to mediate salivary adaptive responses.

	DISCUSSION

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Several lines of evidence provided herein support the notion that PKC activation is involved in DFO-elicited signaling events. First, DFO treatment induces time-dependent nuclear translocation and proteolytic cleavage of PKC and PKC-dependent caspase-3 activation (Figs. 1, 2, and 4, A and B). Second, PKC maintains the high level of sustained -H2AX signal, a hallmark of DNA damage response, on exposure to DFO (Fig. 3A). Third, the kinase-dead PKC-EGFP is capable of attenuating DFO/hypoxia-induced activation of endogenous PKC and caspase-3 (Figs. 1C and 4, A and D). In addition, PKCKD-EGFP fails to be translocated to the nucleus of DFO-treated cells (Fig. 2, B and C) even though it harbors PKC bipartite nuclear localization signal, as reported by DeVries et al. (13), consistent with the proposed dominant-negative role of PKCKD (34). Last, sh-PKC or PKCKD-EGFP conveys partial resistance to DFO-elicited cytotoxicity (Fig. 5, A and B). We conclude that PKC is an important component of the activated signaling pathway, consequent to DFO treatment. Furthermore, DFO treatment also induces Chk1 and p53 phosphorylation in a PKC-independent manner (Figs. 3C and 4B).

Although the hypoxia-mimetic effect of DFO has been well documented, no direct link between DFO-mediated genotoxic stress and activation of a specific PKC isoform has been described to date. Our present work suggests that PKC is involved in mediating DFO-induced genotoxic responses. First, we showed that DFO-induced accumulation of DNA damage marker -H2AX is modulated by expression of sh-PKC or Lys376Arg-mutated PKC-EGFP. Current thinking predicts that ATM is activated by DNA DSBs and recruits more ATM molecules as well as other repair proteins and sensors, such as MRN, 53BP1 and MDC1, to the DNA lesion (16). Moreover, -H2AX can also be activated by DNA-dependent protein kinase (DNA-PK) and ATR in addition to ATM. While our present work suggests that PKC activation effects a sustained DFO-elicited -H2AX activation (Fig. 3, A and B), the exact molecular mechanism underlying this observation is still elusive. It is possible that H2AX serves as a direct kinase substrate of nucleus-translocated PKC, given that PKC is translocated to the nucleus during DFO treatment (Figs. 2A and 3B). Second, PKC is cleaved by a caspase-3-dependent mechanism to a 40-kDa PKC-CF, analogous to PKC activation in cells treated with genotoxic DNA damaging agents (13, 15), in DFO-treated cells (Figs. 1B and 4B). Because a relatively higher level of caspase-3-CF was observed in Pa-4/PKCWT-EGFP cells treated with DFO for 48 h (Fig. 4A), a feedforward pathway for PKC and caspase-3 reciprocal activation (Fig. 5C) is likely to exist in response to DFO treatment, consistent with previous reports in other apoptotic systems (26, 27, 31, 44). Moreover, overexpression of PKC-CF has been shown to induce chromatin condensation and DNA fragmentation, supporting a role for PKC-CF in mediating cell response to genotoxic stress (17). Last, studies by others have also shown that PKC interacts with p53 DINP1 and p53, leading to p53 phosphorylation on exposure to genotoxicity (53), and that PKC mediates the cytotoxic effect of arsenate-induced cell death (32). In addition, cells derived from PKC^–/– mice were shown to be defective in mitochondria-dependent apoptosis (21, 30). These findings collectively support our proposition of a progenotoxic role of PKC in DFO-dependent signaling in salivary epithelial cells.

Previous studies have reported that treatment of cells with DNA damaging agents is associated with translocation of PKC to the nucleus (13, 54). However, these studies also showed that etoposide elicits PKC proteolytic cleavage and p53 phosphorylation via a rapid induction in salivary cells in vitro and in vivo (13, 21). By contrast, our present results suggest that the induction of PKC and caspase-3 proteolytic cleavage and p53 phosphorylation by DFO treatment occurs in a delayed manner. One possible explanation for this time-dependent action in DFO-treated cells is that DFO, a membrane-impermeant iron chelator, is taken up primarily by fluid-phase endocytosis, travels through various endosomes to late endosomes, and eventually accumulates in the lysosomal compartment (14, 28). It is plausible to assume that the change in the extent of DFO-chelatable irons in extracellular milieu and cytoplasmic and other subcellular compartments and membrane destabilization of these organelles result in the activation of various signaling pathways, including PKC, caspase-3, ATM, p53 and Chk1, in a rather delayed manner. In addition, we have recently reported that hypoxia or DFO treatment leads to a transient activation of the small ubiquitin-like modifier (SUMO)ylation process, and that overexpression of SUMO-1 protects salivary Pa-4 cells against hypoxic injury (41). It is conceivable that SUMOylation alters and/or delays the signaling event(s) activated by DFO treatment.

While PKC plays a role in mediating cell response to DFO treatment, as shown herein, a signaling pathway other than PKC also contributes in part to the effect of DFO in salivary cells. It appears that the PKC-independent signaling pathway is also activated by DFO treatment. For example, the activation of both Chk1 and p53 is also known to be part of genotoxic responses. As shown in Fig. 3C, the DFO-exerted Chk1 phosphorylation at Ser-345 and p53 phosphorylation at Ser-15 were noted in cells lacking a functional PKC pathway, such as Pa-4/sh-PKC and Pa-4/PKCKD-EGFP cells. DFO treatment also induced Chk1 and p53 activation without generating PKC-CF in MCF7/Neo cells (Fig. 4B). Consistent with our results is a report by Humphries et al. (21), who demonstrated the involvement of a signaling pathway other than PKC in governing the steady-state level of p53 and its Ser-15 phosphorylation under genotoxic conditions in cells prepared from PKC^–/– and PKC^+/+ mice. Previously, Gibson et al. (18) reported that hypoxia induces Chk2 activation in an ATM-dependent manner (18). Our results unequivocally showed that DFO not only activates ATM (and presumably its downstream Chk2) but also induces PKC-independent Chk1 stimulation. Although Chk1 functionally shares many substrates with Chk2, we suggest that Chk1 activation may represent an additional pathway to ascertain a proper adaptive response to DFO-induced genotoxic stress.

A complete picture of the signaling pathways and functions of PKC in DFO-treated cells has yet to emerge. The presented studies expanded our understanding of PKC biology as well as perhaps the role of PKC in regulating a DFO- and maybe hypoxia-induced adaptive response. Taken together, our data suggest a role for the cross talk of PKC and DNA damage response signaling pathways in DFO-mediated genotoxicity and also demonstrate that both PKC-dependent and -independent pathways functionally cooperate to mediate salivary adaptive responses against DFO (Fig. 5C).

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported in part by the Hastings Foundation and National Institutes of Health Research Grants R01-DE-10742 and DE-14183 (to D. K. Ann), HL-38658 and HL-64365 (to K.-J. Kim), and DE-15648 (to M. E. Reyland).

	ACKNOWLEDGMENTS

 
We thank members of D. K. Ann's laboratory for sharing reagents and for helpful discussion and Drs. Wei Li (University of Southern California) and Yun Yen (City of Hope) for critical reading of the manuscript and helpful discussions.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. K. Ann, City of Hope National Medical Center, KCRB Rm. 1004, 1500 E. Duarte Road, Duarte, CA 91010 (e-mail: dann{at}coh.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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