PKCζ-dependent upregulation of p27kip1 contributes to oxidative stress induced retinal pigment epithelial cell multinucleation

oxPOS activates PKC and PKC activation is involved in RPE multinucleation

Western blot showed that PKC proteins are constitutively expressed in ARPE19 cells (Fig. 1A). PMA, a PKC activator, and POS or oxPOS did not affect the expression of total PKC protein (Fig.. 1A-1C), although the expression was suppressed by a cocktail of PKC isoform inhibitors (Fig. 1A, 1B). Interestingly, the PKC kinase activity was significantly enhanced by PMA (100 nM, Fig. 1D) and oxPOS in a concentration-dependent manner (Fig 1E). Both PMA and oxPOS increased the percentage of multinucleate ARPE19 cells from 7% (under non-stimulatory conditions) to approximately 33%, an effect that was abolished by the PKC inhibitor cocktail (Fig.. 1F-J). Comparable results were observed when the experiment was performed in human RPE cells derived from primary culture (Fig. S1). Our results suggest that PKC activation can induce RPE multinucleation.

Figure 1. Role of PKC in RPE cell multinucleation and oxPOS induced PKC activation in RPE cells. (A) Representative Western blots showing total PKC protein expression in RPE cells following PMA, total PKC blockade, no treatment and with different concentrations of POS or oxPOS treatments (RPE: POS/oxPOS 1:2, 1:5, 1:10) for 48h. (B-C) quantification of total PKC expression in different treatment groups. The data are represented as relative fold change to the control. (D) Active PKC levels in RPE cells following PMA, total PKC blockade treatments, or untreated control for 48h. (E) Active PKC protein levels in RPE cells following POS or oxPOS treatment at 1:10 and 1:2 (RPE: POS/oxPOS) for 48h. Cells from each treatment group was homogenised and protein levels were evaluated by active PKC ELISA kit. *, P < 0.05; ***, P < 0.001 compared to the control untreated group. One-way ANOVA followed by Dunnett’s multiple comparison test. N = 3. (F-I): histochemical staining of propidium iodide (PI) and Phalloidin in control untreated ARPE19 (F), PMA treated ARPE19 (G), PKC blocked ARPE19 (H) and oxPOS (I) treated ARPE19 cells. Arrows indicate multinucleate RPE cells. (J) histogram showing the percentage of binucleated and multinucleate RPE cells, ***, P < 0.001 compared to control untreated. One-way ANOVA followed by Dunnett’s multiple comparison test. 50 cells were counted from three wells for each group.

PKC isoform expression in POS and oxPOS treated ARPE19 cells

To understand which isoforms of PKC may be involved in oxPOS mediated RPE multinucleation, we examined the expression profile of different PKCs in ARPE19 cells. Of the 11 PKC isoforms from the three PKC sub-families, mRNAs for PKCα β1, δ, ε, ζ, η, ι, and μ isoforms were detected in ARPE19 cells (Fig. 2A). The other isoforms including PKC βII, γ and θ were not detected (Fig. S2), though they were detected in positive controls (human serum cDNA, data not shown). The treatment of ARPE19 cells with oxPOS significantly upregulated the expression of PKC α, δ, ζ, ι and μ compared to untreated controls (Fig. 2B), whereas POS treatment only enhanced the expression of PKC μ (Fig. 2B). Immunocytochemistry further confirmed the expression of PKC α, δ, ζ, ι and μ isoforms in ARPE19 cells under control, POS and oxPOS treated conditions (Fig. 2C).

Figure 2AB. Expression of PKC isoforms in ARPE19 cells. (A) Conventional PCR gel image showing expression of eight PKC isoforms-PKCα, PKCβI, PKCδ, PKCε, PKCη, PKCζ, PKCι and PKCμ in ARPE19 cells. (B) Real-time RT-PCR analysis of PKC isoforms in ARPE19 cells following POS, oxPOS treatments and untreated control. *, P < 0.05; **, P < 0.01, ***, P < 0.001 compared to the control untreated group. One-way ANOVA followed by Dunnett’s multiple comparison test. N = 3.

Figure 2C. Expression of PKC isoforms in ARPE19 cells.(C) Immunofluorescent staining of PKCα, PKCδ, PKCζ, PKCι and PKCμ isoforms in RPE cells following POS or oxPOS treatments. The isoforms were in green, nuclei were labelled by PI in red.

The role of PKCα in oxPOS-mediated RPE multinucleation

PKCα is constitutively expressed by RPE cells at high levels (Fig. 2A, Fig. S2). PKCα belongs to the classic PKC family and its activation is calcium dependent, whereas the novel and the atypical family isoforms are calcium independent [19]. Intracellular calcium labelling using Fluo-4 revealed intense fluorescence in control untreated and oxPOS-treated ARPE19 cells (Fig. S3A and Fig. S3B). The treatment with BAPTA-AM (a Ca2+ selective chelator) significantly reduced Fluo-4 staining in oxPOS-treated cells and almost completed abolished the staining in untreated control cells (Fig. S3), suggesting that BAPTA-AM satisfactory blocked intracellular calcium in ARPE19 cells. Chelation of intracellular Ca2+ using BAPTA-AM 48h before oxPOS treatment led to a 33% reduction in the number of multinucleate cells (Fig.. 3A-D). Similar effects were observed following treatment of the cells with a PKCα inhibitor (Fig. 3E-F). The results suggest that PKCα is partially responsible for oxPOS-induced ARPE19 multinucleation.

Figure 3. Involvement of Ca2+ signalling in RPE cell multinucleation. Intracellular calcium was blocked in ARPE-19 cells using BAPTA-AM. (A) calcium blocked cells. (B) calcium blocked + oxPOS treated cells. (C) oxPOS treated cells. Arrows indicate multinucleate RPE cells. (D) histogram showing the percentage of binucleate and multinucleate RPE cells following calcium chelation *, P < 0.05, **, P < 0.01, ***, P < 0.001 compared to oxPOS treated group. One-way ANOVA followed by Dunnett’s multiple comparison test. 50 cells were counted from three wells for each group. (E) PKCα inhibitor-treated RPE cells. (F) histogram showing the percentage of binucleate and multinucleate RPE cells after PKCα blockade **, P < 0.01 compared to oxPOS treated group. 50 cells were counted from three wells for each group.

The role of PKC δ, ζ, ι and μ in oxPOS-induced RPE multinucleation

To further explore the role of different PKC isoforms on oxPOS-induced RPE cell multinucleation, the activities of other isoforms including PKC δ/ε, ζ, and ι that showed significant upregulation upon oxPOS treatment were blocked using specific inhibitors. PKCι inhibition resulted in 18% reduction in multinucleate cells (P<0.05, Fig. 4A, 4F). Interestingly, PKCζ blockade resulted in 50% reduction in multinucleate cells (Fig. 4B, 4F). Inhibition of PKC δ/ε isoforms did not significantly affect oxPOS-induced ARPE19 cell multinucleation (Fig. 4C, 4F). Our results suggest that PKCζ, a member of the atypical PKC subfamily, plays an important role in oxPOS-induced RPE multinucleation.

Figure 4. The effects of PKC isoform inhibition in RPE cell multinucleation. (A) control RPE cells. (B) oxPOS treated RPE cells. (C) PKCζ inhibited + oxPOS treated RPE cells. (D) PKCι inhibited + oxPOS treated RPE cells. (E) PKC δ/ε inhibited + oxPOS treated RPE cells. Arrows indicate multinucleate RPE cells. (F) histogram showing the percentage of binucleate/multinucleate RPE cells following different treatments *, P < 0.05, ***, P < 0.001 compared to oxPOS treated group. One-way ANOVA followed by Tukey’s multiple comparison test. 50 cells were counted from three wells for each group.

PKC ζ-dependent expression of p27kip1 in ARPE19 cells

PKC activity has been positively associated with phosphorylation of cell cycle regulatory molecules including Cdk inhibitory proteins p21 WAF1 and p27kip1. To further understand the underlying mechanism related to PKC mediated RPE multinucleation, we examined p21 WAF1 and p27kip1 expression in ARPE19 cells. A significant increase in p27kip1 protein expression was observed in oxPOS-treated group as well as the PMA-treated group (Fig. 5A), although the expression of p21 WAF1 was not affected by POS or oxPOS treatment (Fig. S4). The upregulation of p27kip1 in oxPOS treated ARPE19 cells was significantly reduced by PKCζ inhibitor (Fig. 5B, 5C), but not by the blockers specific to PKC α or δ (Fig. 5B, 5C). These results suggest that the upregulation of p27kip1 following oxPOS treatment depends on PKCζ activation.

Figure 5. The effect of PKC inhibition on P27kip1 expression in RPE cells. (A-B) representative Western blots from control, POS, oxPOS, PKCα, ζ, δ and total PKC inhibitor treated RPE cells showing p27kip1 at 28 KDa and β-actin at 40 KDa. (C) quantification of p27kip1 expression by RPE cells in the different groups. Data were expressed as relative fold change to the control. *, P < 0.05; **, P < 0.01, ***, P < 0.001 compared to the control untreated group and ••, P < 0.01 compared to oxPOS treated group. One-way ANOVA followed by Tukey’s multiple comparison test. N = 3. (D) ARPE-19 cells were stained for p27kip1 (green) and PI (red) after 48 h oxPOS treatment. Arrows shows strong p27kip1 staining in cell nucleus. (E) control ARPE-19 cells stained for p27kip1 (green) and PI (red) showing weak, diffused p27kip1 (green) in the cytoplasm and nucleus.

Immunocytochemistry revealed strong p27kip1 expression in both the nucleus and cytoplasm in oxPOS treated ARPE19 cells (Fig. 5D), whereas only weak p27kip1 expression was detected in untreated ARPE19 cells (Fig. 5E).

The role of p27kip1 in RPE cell multinucleation

To further confirm the role of PKCζ - p27kip1pathway in oxPOS-induced RPE multinucleation, a p27kip1 transcriptional inhibitor, Alsterpaullone, 2-Cyanoethyl (A2CE) was used. A2CE inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27kip1 promoter [20]. A2CE at 1μM and 5μM did not show any toxicity to ARPE19 cells (data not shown). However, it dose-dependently suppressed p27kip1 expression (Fig. 6A, 6B), and reduced oxPOS-induced ARPE19 multinucleation (Fig. 6C, 6D). Our results suggest that p27kip1 is critically involved in oxPOS-induced RPE multinucleation.

Figure 6. Inhibition of p27kip1 using Alsterpaullone, 2-Cyanoethyl (A2CE) and its effect on RPE multinucleation. (A) representative western blot from oxPOS, control, 5μM A2CE and 1μM A2CE treated RPE cells showing p27kip1 at 28 KDa and β-actin at 40 KDa. (B) quantification of p27kip1 protein expression by RPE cells in the different treatment groups. Signals for POS treated group were set to one and the data are represented as relative fold change to the POS group. * P <0.05, *** P <0.001 compared to the POS group. One-way ANOVA followed by Tukey’s multiple comparison test. N= 3. C-F: confocal images of multinucleate RPE cells in different treatment groups (C); POS-treated, (D); 1μM A2CE, (E); untreated RPE cells, (F); 5μM A2CE). (G) histogram showing the percentage of binucleate/multinucleate RPE cells following different treatments, * P <0.05, *** P <0.001 compared to oxPOS treated group. Data comprised of percentage of multinucleate cells per well from 5 different well for each group.

In vitro RPE cell culture

Human ARPE19 cells were purchased from ATCC (CRL-2302, Middlesex, UK). Primary human RPE cells were cultured from a donor eye (free of known eye diseases) obtained from the Manchester Eye Bank using the protocol described previously [44]. Briefly, after the removal of vitreous and retina, the eye cup was washed with Hanks’ balanced salt solution (HBSS), and filled with 0.25% (v/v) trypsin (ThermoFisher, Hemel Hempstead, UK) for 2h at 37°C. Cells were then removed from the basement membrane by gentle aspiration. The cells were washed twice in Dulbecco’s Modification of Eagle’s Medium F12 (DMEM F12, GIBCO BRL, Paisley, UK). Both ARPE19 cell and primary human RPE cells were cultured in complete DMEM F12 medium containing 10% fetal calf serum (FCS) and 100 μg/mL Primocin (Invitrogen, San Diego, California, USA). Cells were maintained at 37°C with 5% CO2. The phenotype of RPE cells were confirmed by pan-cytokeratin staining. The use of donated human eye tissue was approved by local Research Ethics Committee. During all cell culture experiments, the RPE cell line used were maintained between passages 3 to 10 to ensure that RPE cell morphology had not changed or transdifferentiated. When inducing multinucleate RPE cells, cell confluency was maintained at approximately 70% to ensure sufficient number of proliferating cells. This also facilitates identifying individual multinucleate cells in culture and omitting false-positive of overlapping cells.

Preparation of photoreceptor outer segments

POS were isolated from bovine eyes using method described previously [45]. Briefly, 10 bovine retinas were placed in 10 ml homogenising solution (20% w/v sucrose, 20 mM Tris acetate, pH 7.2, 2 mM MgCl2, 10 mM glucose and 5 mM taurine). The suspension was shaken gently for 1min and then filtered through a 100 mm cell strainer (BD, Oxford, UK) to remove tissue debris. The suspension was layered on 25~60% w/v continuous sucrose gradients containing 20 mM Tris acetate pH 7.2, 10 mM glucose and 5 mM taurine and centrifuged at 25,000 rpm for 45min at 4ºC. The pink band containing the POS was collected and washed with storage buffer (10 mM sodium phosphate, pH 7.2, 0.1 M NaCl and 2.5% sucrose). For storage, isolated POS aliquots were stored at -80ºC at a concentration of 10⁸ POS/ml. The oxidized POS (oxPOS) were generated by exposing POS to 302-nm ultraviolet light (Ultraviolet Products, Cambridge, UK) in a laminar air-flow box for 12h [45].

In vitro generation of multinucleate RPE cells

Multinucleate RPE cells were generated by incubating the cells with POS or oxPOS as previous described [8]. In brief, ARPE19 and human RPE cells were seeded at a density of 3000 cells per well in 12-well plates and incubated for 5h. The cells were then treated with POS or oxPOS for 48h (RPE: POS = 1:5).

PKC inhibitor experiments

ARPE19 cells incubated with/without oxPOS were treated with PKC inhibitors (Gö 6983, inhibitor of PKCζ at IC50 = 60 nM; Gö 6976, inhibitor of PKCα at IC50 = 10 nM; Aurothiomalate (ATM), inhibitor of PKCε at IC50 = 132 nM; Bisindolylmaleimide I, inhibitor of PKCι at IC50 = 1μM as recommended by manufacturer) for 1h followed by further 48h incubation with POS or oxPOS (RPE: POS = 1:5). The inhibitors were purchased from either Sigma Aldrich, Cheshire, UK (Gö 6983, Gö 6976, Bisindolylmaleimide I) or Cambridge Bioscience, Cambridge, UK (ATM).

Calcium chelator studies

ARPE19 cells were treated with the 10 μM intracellular Ca2+ chelator BAPTA-AM (ThermoFisher, Hemel Hempstead, UK) for 30 min at 37°C. The cells were washed and incubated with POS or oxPOS for 48h. Intracellular Ca2+ staining was performed by incubating cells with 5μM Flou-4 (ThermoFisher, Hemel Hempstead, UK) for 60min at 37°C. The cells were washed thoroughly before imaging with confocal microscope (Eclipse TE2000-U, Nikon, Surrey, UK) at 10X magnification, numerical aperture 0.25.

Western blot

RPE cells were lysed in RIPA buffer with protease inhibitors (Sigma-Aldrich, UK). The protein concentrations were measured using a BCA protein assay kit (PIERCE, Cramlington, UK). 20μg of the total protein was loaded into 10% SDS-PAGE gel. Gels were run at 80V for 30 min followed by 150V for 60min. Proteins were transferred to the Immobilon-FL polyvinylidene difluoride (PVDF) membrane (Millipore, Watford, UK) at 350 mA for 50 min. The membranes were blocked with 5% bovine serum albumin (BSA) in tris-buffer saline with tween 20 (TBS/T) for 1h at room temperature, followed by incubation with primary antibodies at 1:1000 dilutions at 4 °C overnight. After thorough washes, the membranes were incubated with secondary antibodies at 1:5000 dilutions for 2h in dark at room temperature. The following primary antibodies were used for western blot experiments: PKC (1:1000; Cell Signaling), p27kip1 (1:1000; Life Technologies), p21 (1:1000; Life Technologies), β-actin (1:1000; Santa Cruz). The secondary antibodies used were: Goat anti-rabbit IRDye 680 (1:5000), Goat anti-rabbit IRDye 800 (1:5000), Goat anti-mouse IRDye 680 (1:5000), Goat anti-mouse IRDye 800 (1:5000); (LI-COR; Lincoln).

The membrane was then washed in TBS/T 3 times before scanning using Odyssey infrared imaging system (Li-COR Biotechnology, Cambridge, UK). Quantitative western blotting was performed using imageJ software (version 1.45).

PKC kinase activity assay

Active PKC levels in ARPE19 cells were measured using a commercially available kit (ab139437, Abcam, Cambridge, UK) in accordance with the manufacturer's instructions. Briefly, 30μg/30μL protein of each sample was added to an active PKC capturing antibody coated 96-well plate (Abcam) which was then incubated at 30ºC for 90min. 40μL of the phosphospecific substrate antibody was added to each well. After a single wash, 40μL of diluted anti-rabbit IgG: HRP conjugate was added to each well and incubated at room temperature for 30min. Following another single wash, 60μL TMB substrate was added to each well and left for 30min. The assay was terminated by addition of 20μL stop buffer and absorbance measured immediately at 450nm using a microplate reader (BMG Labtech, Ortenberg, Germany).

Real-time polymerase chain reaction (real-time RT-PCR)

Total RNA was extracted from RPE cells using Tri Reagent (Sigma-Aldrich, Cheshire, UK). The quantity and quality of the RNA was determined by Nano-Drop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA synthesis was performed in a reaction of 0.5-2.5μg of total RNA with a random primer using the SuperScript™ II Reverse Transcriptase kit (Invitrogen, Paisley, UK). Real-time RT-PCR was performed in 384-well plates using the LightCycler 480 system (Roche Applied Science, Mannheim, Germany). The reaction mixture contained 6μL of LightCycler 480 SYBR Green Master (Roche Diagnostics GmbH, Mannheim, Germany), 0.5μM primers and diluted cDNA to make a total volume of 12 μL. The primers used were purchased from Integrated DNA Technologies UK, Ltd and are listed in (Table 1). Real-time RT-PCR quantifications were run in triplicates for each sample. PCR products were quantified by the LightCycler 480 software. Expression levels were normalised to 18S. Gene fold changes of treated group against untreated control group were calculated by dividing the gene expression levels of treated samples by control samples. PCR products were electrophoresed on 2% agarose gel in TAE buffer to confirm product size.

Immunofluorescence staining

ARPE19 and human RPE cells derived from primary culture were cultured on cover slips and treated with POS or oxPOS for 48h. Cells on cover slips were washed with cold PBS and fixed with 1% paraformaldehyde (PFA) for 10 min, followed by permeabilisation with 0.1% Triton-X for 5 min. The samples were then blocked with 5% BSA for 30 min at room temperature. Cells were incubated with the desired primary antibody for 4h at 1:100 dilution. After washing with PBS, samples were incubated with the desired secondary antibody at 1:100 dilution and counter stained for Phalloidin (1:100, Sigma Aldrich, Cheshire, UK) and/or PI (Vector Laboratories, Peterborough, UK) in the dark for 1h. After extensive washing, samples were mounted with mounting medium (Vector Laboratories, Peterborough, UK), and examined by confocal microscopy Eclipse TE2000-U, Nikon, Surrey, UK) at 40X magnification, numerical aperture 0.65. Primary and secondary antibodies used in the study are listed in (Table 2).

Data and statistical analysis

Data were expressed as mean ± SEM with P<0.05 deemed statistically significant. Differences between groups were assessed using either an independent t test or one-way analysis of variance with Dunnett’s or Tukey’s post-hoc tests.