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(Investigative Ophthalmology and Visual Science. 2006;47:397-404.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0290
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Selective Relocalization and Proteasomal Downregulation of PKC{alpha} Induced by Platelet-Activating Factor in Retinal Pigment Epithelium

Zahra Faghiri and Nicolas G. Bazan

From the LSU Neuroscience Center of Excellence and Department of Ophthalmology, Louisiana State University Health Sciences Center School of Medicine, New Orleans, Louisiana.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Protein kinases C (PKCs) are key cell-signaling mediators in retinal physiology and pathophysiology. The cellular localization of PKC isoforms is important in defining their activity and specificity; the present study investigated the modulatory potential of the proinflammatory mediator platelet-activating factor (PAF) on the subcellular distribution of PKC{alpha}, ß, and {delta} isotypes.

METHODS. This study used real-time visualization of green fluorescent protein fused to PKC{alpha}, ß, or {delta} in the human retinal pigment epithelial (RPE) cell line ARPE-19.

RESULTS. In PAF-stimulated ARPE-19 cells, PKC{alpha} translocated to the plasma membrane and then colocalized with Golgi markers p230 and GM130; PKCß translocated to the plasma membrane but not to the Golgi; and PKC{delta} translocated to the Golgi. Pretreatment with PKC inhibitor calphostin C abolished the PAF-induced translocation of PKC{alpha} to the plasma membrane or to the Golgi, but the Golgi inhibitor Brefeldin A only prevented the accumulation of PKC{alpha} in Golgi, without affecting its membrane relocalization. PAF promoted depletion of PKC{alpha} and {delta} isoforms but not that of PKCß. Proteasome inhibitors lactacystin and MG-132 prevented the PAF-induced depletion of PKC{alpha}, but the inhibitor of lysosomal proteolysis E-64d was ineffective in rescuing PKC{alpha}.

CONCLUSIONS. These results suggest that the PAF-induced downregulation of PKC{alpha} occurs principally through the proteasomal pathway. This remarkable PAF-mediated diversity in PKC translocation and downregulation highlights the significance of isotype-specific PKC activation in signaling pathways in ARPE-19 cells. These signaling events may be critical during RPE responses to oxidative stress, inflammation, and retinal degenerations, when PAF production is enhanced.

The retinal pigment epithelial (RPE) cells, derived from the neuroepithelium, comprise a monolayer of mitotically inactive cells lying between the choroidal circulation and the outer segments of the photoreceptors in retina. RPE cells contribute to the maintenance of the blood-retinal barrier, retinal adhesion, and transport of trophic factors and nutrients to the retina. In addition, these cells phagocytize the shed distal disks of photoreceptor outer segments.1 2 RPE cells play an active role in inflammatory diseases (e.g., proliferative vitreoretinopathy after retinal injury or detachment) and in age-related macular degeneration. The potent phospholipid messenger platelet-activating factor (PAF) is involved in cell functions and pathology, including the inflammatory response, oxidative stress, and apoptosis.3 PAF actions are mediated through a G-protein–coupled transmembrane receptor4 that is linked to diverse signal-transduction pathways such as phospholipases C and D.5

Protein kinases C (PKCs), a family of serine/threonine kinases, participate in cell signaling, neurotransmission, gene expression, and cell growth and differentiation.6 7 8 The PKC family is classified into classical PKCs ({alpha}, ßI, ßII, {gamma}), which are Ca2+-dependent and diacylglycerol (DAG)-responsive; novel PKCs ({delta}, {epsilon}, {eta}, {theta}), which are Ca2+-independent and DAG-responsive; atypical PKCs ({zeta}, {lambda}/{iota}), which are both Ca2+- and DAG-independent; and the PKCµ/PKD subfamily (PKD1 and -2), which possess unique structural features including a nonhomologous kinase domain. Classical and novel PKCs are cellular targets for tumor-promoting phorbol esters (e.g., phorbol myristate acetate [PMA]).6 9 Acute exposure to PMA induces the translocation to the plasma membrane and activation of cytosolic PMA-responsive PKCs, whereas prolonged incubation results in proteolytic degradation of the responsive PKCs and their depletion from the cell.10 11 Modulation of both activation and downregulation of PKCs is important for cell function, including differentiation and carcinogenesis.12 13

Expression of PKC{alpha}, -ßI, -ßII, and -{delta} in human RPE cells has been linked to both physiologic and pathophysiologic responses.14 15 PKC-mediated signal transduction may be involved in RPE cell migration,16 and activation of PKC by phorbol esters inhibits rod outer segment phagocytosis by rat RPE cells in culture.17 The specificity and biological activity of PKC isoforms are regulated by their subcellular localization. Thus, after activation, PKC isoforms are often translocated to other compartments. Translocation of PKC is isoform-, cell type-, and activator-specific, and is tightly regulated by various cofactors.18 19 20 Each PKC may, therefore, display a distinct subcellular localization and bind to intracellular proteins that serve as substrates and/or carriers, such as receptors for activated C kinases, receptors for inactive C kinases, myristoylated alanine-rich C-kinase substrate, annexins, and cytoskeletal components.21

In the present study, by expressing the fusion proteins green fluorescent protein (GFP) and PKC{alpha}, -ß, or -{delta} in ARPE-19 cells, we studied the modulatory potential of the proinflammatory mediator PAF by real-time visualization. We showed that PKC{alpha} translocated to the plasma membrane and later accumulated within the Golgi and colocalized with both cis- and trans-Golgi markers. PKCß translocated only to the plasma membrane, while PKC{delta} translocated to the Golgi. We also studied how the lipid mediator activates degradation of PKC{alpha}. Using specific inhibitors, we showed that PKC{alpha} downregulation mediated by PAF is proteasome-dependent in ARPE-19 cells. Both diversity in translocation targeting of PKC isoforms and the mechanism of PKC{alpha} degradation may be critical during RPE responses to oxidative stress, when PAF production is enhanced.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Antibodies and Reagents
PAF, U73122, and calphostin C were purchased from Sigma (St. Louis, MO). Monoclonal antibodies for PKC{alpha}, -ß, and -{delta}, and for p230 and GM130, as well as peroxidase-conjugated goat anti-mouse IgG, were obtained from Transduction Laboratories (Lexington, KY). Alexa 546-conjugated goat anti-mouse antibody and BODIPY TR ceramide were purchased from Molecular Probes (Eugene, OR). Proteasome inhibitors MG-132 and lactacystin, and calpain inhibitor E-64d, were obtained from Calbiochem (La Jolla, CA). Golgi inhibitor Brefeldin-A was obtained from Epicenter (Madison, WI). PAF antagonist BN50730 was obtained from Biomol (Plymouth Meeting, PA).

Cell Culture and Transfections
Cultures of ARPE-19 cells were maintained at 37°C in DMEM-F12 supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO2. For live-cell analysis, ARPE-19 cells were grown in 35-mm Petri dishes (Corning, Corning, NY) to 60% to 80% confluence. Cells were transfected overnight with 2 µg plasmid DNA of choice and 5 µL transfection reagent (FuGene 6; Roche, Mannheim, Germany) in 2 mL tissue culture medium. All experiments were performed after 18-hour starvation of cells in DMEM-F12 containing 0.5% FBS.

Real-Time Cell Imaging
To maintain a constant temperature of 37°C during the experimental procedures, an open-perfusion microincubator (Model PDMI-2; Medical System Corp., Greenvale, NY) was attached to the microscope stage. Cells were grown in 35-mm Petri dishes and placed in the microincubator system set at 37°C. Images were recorded at different time points on a confocal microscope (Nikon Diphot 200; Nikon, Tokyo, Japan) with a color-chilled three–charge coupled device camera (ORCA-285 IEEE 1394; Hamamatsu, Bridgewater, NJ) driven by imaging software (Metamorph Imaging Series 4.6; Universal Imaging Corporation, Downington, PA). Movies 1 to 3, available online at http://www.iovs.org/cgi/content/full/47/1/397/DC1, were made using the same software.

Hoechst Staining
ARPE-19 cells were grown in 35-mm Petri dishes, transfected with PKC{alpha}-GFP, and serum-starved for 18 hours. Before addition of the stimulant, cells were stained for nuclei with 2 mL Hoechst 33258 (Sigma), 10 µM in PBS, and incubated at 37°C in darkness for at least 45 minutes. The staining solution was then replaced with 2 mL fresh PBS, and the cells were examined under the microscope.

Golgi Staining
ARPE-19 cells expressing PKC-GFP were treated for 10 to 15 minutes at 37°C with BODIPY TR ceramide (5 nmol/mL) to stain for the Golgi apparatus. Cells were then washed with and incubated in DMEM-F12 supplemented with 0.5% FBS at 37°C for 30 minutes before addition of PAF.

Immunochemistry
ARPE-19 cells were grown in 35-mm Petri dishes, stimulated according to the experimental design, washed once with PBS, and then fixed with methanol for 6 minutes at –20°C. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes. Blocking was performed with 10% bovine serum albumin (BSA) and 1% goat serum in PBS for 30 minutes at room temperature. Cells were incubated with p230 or GM130 mouse antibody (1:200) for 1 hour at room temperature. After being washed three times with 1% BSA in PBS, cells were incubated with an Alexa 546-labeled anti-mouse IgG antibody (1:500) for 30 minutes at room temperature. Cells were then washed three times, mounted in fluorescent mounting medium (Vector, Burlingame, CA), and examined under the confocal microscope.

Preparation of Cell Lysates
ARPE-19 cells were grown in six-well plates to 100% confluence, serum-starved for 18 hours, and stimulated according to the experimental design. Cells were washed once with ice-cold PBS and collected by scraping into 100 to 300 µL cytosolic lysis buffer (20 mM Tris, pH 7.5; 150 mM NaCl; 10 mM EDTA; 200 µM Na3VO4;10 mM NaF; 5 µg/mL leupeptin; 1 mM phenylmethylsulfonylfluoride, and 10% glycerol). Cells were further lysed with 20 strokes of a Dounce homogenizer (Kimble-Kontes, Vineland, NJ), and supernatants were collected as a cytosolic fraction after centrifugation for 30 minutes at 50,000 rpm and 4°C. The pellets were washed once with ice-cold PBS and homogenized in membrane lysis buffer (cytosolic lysis buffer containing 1% NP-40) using 20 strokes of a Dounce homogenizer. After incubation on ice for 20 minutes with interval shaking and centrifugation for 30 minutes at 50,000 rpm and 4°C, the supernatants were collected as the membrane fraction. The whole-cell lysate was prepared by scraping the cells into 100 to 300 µL membrane lysis buffer, incubation on ice for 20 minutes with shaking every 5 minutes, and centrifugation for 30 minutes at 50,000 rpm and 4°C.

Western Blot Analysis
The protein content was estimated with protein assay reagent (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Equal amounts of cell extract protein from different conditions (10–30 µg) were loaded onto and separated by 8% to 16% SDS-PAGE ready-made gels (Invitrogen; Carlsbad, CA). On fractionation, proteins were transferred to polyvinylidene fluoride membranes (Invitrogen) in a transblot apparatus (Novex, San Diego, CA). Membranes were blocked with 5% skim milk (Bio-Rad) in phosphate-buffered saline containing 0.1% Tween 20 (PBST) for 1 hour at room temperature or overnight at 4°C. Membranes were incubated with monoclonal antibody against PKC{alpha} (1:1000), PKCß (1:200), PKC{delta} (1:200), or GAPDH (1:106) for 1 hour at room temperature or overnight at 4°C followed by three washes with PBST and incubation with HRP-conjugated anti-mouse antibody for 20 minutes at room temperature. Protein bands were visualized by ECL (Amersham; Little Chalfont, Buckinghamshire, UK) and autoradiography on X-OMART AR films (Kodak, Rochester, NY). The bands were scanned and quantified by the Gel Doc system using Quantity One software (Bio-Rad).


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
PAF-Stimulated Translocation of PKC Isoforms to Different Subcellular Sites
To evaluate the role of PAF in translocation of PKC isoenzymes in ARPE-19 cells, PKC{alpha}-, -ß-, and -{delta}-GFP were transiently transfected into ARPE-19 cells. As a control, the GFP plasmid without the PKC insert was also transfected in parallel. In the absence of stimuli, all three PKCs were present in the cytoplasm, and the GFP alone appeared both in the cytoplasm and nucleus. Addition of PAF (100 ng/mL) did not change the subcellular localization of GFP (data not shown), but PKC{alpha}-GFP translocated to the plasma membrane (Figs. 1A 1B) and later accumulated in a single spot in the perinuclear region (Fig. 1B , arrows; Movie 1). PKCß-GFP translocated to the plasma membrane after treatment with PAF without accumulating in the perinuclear area (Fig. 1C ; Movie 2). PKC{delta}-GFP translocated to the perinuclear area (Fig. 1D , arrows; Movie 3). Thus, stimulation with PAF resulted in distinctive translocation patterns for these PKC isoenzymes. To further confirm PKC{alpha} translocation to the membrane, cytosolic and membrane expression of PKC{alpha} was investigated by Western blot analysis. In unstimulated cells, PKC{alpha} appeared as a single band at 82 kDa, mainly in the cytosol, and could only be detected as a faint band in the membrane fraction, suggesting that it is present in a very low abundance in membranes. With PAF stimulation (100 ng/mL) there was a gradually decreasing PKC{alpha} expression in the cytosolic fraction, while it greatly increased in the membrane fraction (Fig. 1F) .



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  		FIGURE 1. Differential subcellular translocation of PKC{alpha}, -ß, and -{delta} by PAF in ARPE-19 cells. Cells were transfected with either PKC{alpha}- (A, D), -ß- (B), or -{delta}-GFP (C) and then serum-starved for 18 hours before being stimulated with 100 ng/mL PAF (A–C) or 100 nM lyso-PAF (D). Representative images were recorded immediately before and at different time points after stimulation. In a separate series of experiments, nontransfected ARPE-19 cells were serum-starved for 18 hours and then stimulated with 100 ng/mL PAF (E) or 100 nM lyso-PAF (F) as a control. Cytosol and membrane fractions were prepared at different time points and tested by Western blotting for the presence of PKC{alpha} compared with those of untreated cells (U). Bar diagrams (F, G) represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.

 
The PAF precursor, lyso-PAF, which is the biologically inactive form of PAF, does not activate the PAF receptor.22 We used lyso-PAF to define the specificity of PAF-induced translocation of PKC{alpha} to the membrane. Lyso-PAF did not stimulate translocation of PKC{alpha} to the plasma membrane or Golgi apparatus (Fig. 1E) . To confirm this finding, we also performed Western blot analysis on the cytoplasmic and membrane fractions of lyso-PAF-treated cells. There was no translocation of PKC{alpha} in the cells treated with lyso-PAF (Fig. 1G) at the same time points at which PAF induced a remarkable movement of PKC{alpha} from the cytoplasm to the membrane (Fig. 1A and 1B) .

Colocalization of PKC{alpha} to the Golgi
To confirm that the final accumulation of PKC{alpha}-GFP (30 minutes after stimulation with PAF) was not in the nucleus, we first performed Hoechst staining, which clearly showed the accumulation of PKC{alpha} in the perinuclear area (Fig. 2A) .



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  		FIGURE 2. (A, B) PAF activation promotes the accumulation in the Golgi of PKC{alpha} and -{delta} but not -ß. ARPE-19 cells were transfected with PKC{alpha}-GFP, serum-starved for 18 hours, and stained with Hoechst reagent before being stimulated with 100 ng/mL PAF (A). ARPE-19 cells were also transfected with PKC{alpha}-, -ß-, or -{delta}-GFP and serum-starved for 18 hours, before being stained for Golgi with BODIPY TR ceramide and then stimulated with 100 ng/mL PAF (B). Images were recorded 30 minutes after stimulation. Dual illumination was recorded by sequential exposure using the two filters for green and red fluorescence. Data are representative of three experiments. (C) Colocalization of PKC{alpha} with p230 and GM130 (trans- and cis-Golgi region, respectively). Cells were transfected with PKC{alpha}-GFP and then serum-starved for 18 hours before stimulation. After 30-minute treatment with 100 ng/mL PAF, cells were fixed and immunostained for either p230 or GM130 using an Alexa 546–labeled secondary antibody. Dual illumination was used to show the colocalization of PKC{alpha} (green) and the Golgi markers (red). Data are representative of three experiments. (D) Golgi inhibitor Brefeldin A (BFA) prevented the translocation of PKC{alpha}-GFP to the Golgi but not to the membrane. ARPE-19 cells were transfected, serum-starved for 18 hours, and pretreated with BFA (10 µg/mL) for 10 minutes before stimulation with 100 ng/mL PAF for 30 minutes. Images were recorded immediately before (uninduced) and after stimulation and compared with those recorded from cells that didn’t receive any pretreatment (control). BODIPY TR ceramide (red) staining was performed to show that, unlike the control, PKC{alpha} is not localized in the Golgi area in pretreated cells. Data are representative of three experiments.

 
To identify the perinuclear compartment to which PKC{alpha}- and -{delta}-GFP were localized after PAF treatment, cells were pretreated with a red fluorescent probe, BODIPY TR ceramide, to visualize the Golgi apparatus. As shown in Figure 2B , the colocalization of accumulated PKC{alpha}- and -{delta}-GFP was most apparent when the Golgi was visualized by dual illumination of the cells and sequential placement of green and red fluorescence filters. PKCß did not colocalize with the Golgi (Fig. 2B) . To further elucidate the colocalization of PKC{alpha} with the Golgi, PKC{alpha}-GFP-expressing ARPE-19 cells were stimulated with PAF for 30 minutes and immunostained for Golgi-specific markers p230 and GM130. Immunostaining showed Golgi compartment association of PKC{alpha}-GFP with both cis- and trans-Golgi markers (Fig. 2C) .

PKC{alpha} Translocation and Golgi Structural Integrity
Brefeldin A (BFA) causes disassembly of the Golgi in a variety of cells,23 24 25 blocks membrane export out of the endoplasmic reticulum in vivo,23 24 and inhibits vesicle formation both in vivo26 and in vitro.27 To investigate the effect of BFA on translocation of PKC{alpha}-GFP, ARPE-19 cells were pretreated with BFA and stimulated with PAF. Translocation of PKC{alpha} was then compared with that of the cells stimulated in the absence of BFA. BODIPY TR ceramide staining was also used to visualize the Golgi compartment. Pretreatment with BFA prevented the PAF-induced accumulation of PKC{alpha}-GFP in the Golgi without affecting its membrane translocation (Fig. 2D) . This suggests that disruption of the Golgi apparatus by BFA blocks the translocation of PKC{alpha}-GFP to the Golgi.

PAF Exertion through G-Protein–Linked Cell-Surface Receptor and Phospholipase C
PAF action is mediated by a G-protein–coupled receptor.4 To confirm that PAF-induced translocation of PKC{alpha} in ARPE-19 cells is receptor specific, PKC{alpha}-GFP–expressing cells were pretreated with PAF-receptor antagonist, BN50730, before PAF stimulation. Results were compared to those obtained from controls that were either untreated or stimulated with PAF only (Fig. 3) . PAF antagonist prevented PKC{alpha}-GFP translocation to the plasma membrane and its accumulation in the Golgi. This indicates the involvement of the PAF receptor in the pathway of PKC{alpha} translocation in ARPE-19 cells.



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  		FIGURE 3. Phospholipase C (PLC) inhibitor U73122, PKC inhibitor calphostin C, and PAF antagonist BN50730 prevent the PAF-induced translocation of PKC{alpha}-GFP. ARPE-19 cells were transfected with PKC{alpha}-GFP, serum-starved for 18 hours, and pretreated with U73122 (10 µM), calphostin C (100 nM), or PAF antagonist, BN50730 (10 µM), for 10 minutes before being stimulated with 100 ng/mL PAF. Images were recorded 30 minutes after stimulation with PAF and compared with controls that were either untreated (uninduced) or stimulated in the absence of inhibitors (PAF only). Data are representative of three experiments.

 
Activated phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate to produce diacylglycerol (DAG), which mobilizes calcium from intracellular stores and activates PKC.10 To elucidate the involvement of the PLC-DAG pathway in the PAF-induced translocation of PKC, PKC{alpha}-GFP–expressing cells were pretreated with PLC inhibitor U73122 before being stimulated with PAF. As shown in Figure 3 , stimulation of the cells with PAF in the presence of the PLC inhibitor U73122 caused the translocation of PKC{alpha}-GFP neither to the plasma membrane nor to the Golgi. This suggests that the mechanism of action of the PAF receptor is through the PLC-DAG pathway (Fig. 4) .



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  		FIGURE 4. PAF regulates intracellular trafficking of PKC{alpha} in ARPE-19 cells. PAF induces the activation and translocation of PKC{alpha} to the plasma membrane and Golgi through a G-protein–coupled receptor-specific mechanism involving the PLC-DAG pathway. Proteasome-dependent downregulation of PKC{alpha} is independent of its activation and translocation to Golgi.

 
PKC{alpha} Activity Requirement for PAF-Induced Translocation
To investigate whether the activation of the PKC{alpha} isoenzyme is associated with its translocation from the cytosol to the plasma membrane and then to the Golgi, we studied the effect of PKC-specific inhibitor calphostin C, a perulenequinone that acts on the phorbol ester-binding site of PKC. PKC{alpha}-GFP–transfected ARPE-19 cells were pretreated with calphostin C and then induced with PAF. No translocation was observed in pretreated cells compared with the cells that were not treated with calphostin C (Fig. 3) , suggesting that the activation of PKC{alpha} is required for its translocation to the plasma membrane and Golgi.

PAF-Induced Downregulation of PKC{alpha} and PKC{delta}
To investigate any effect of PAF on degradation of PKC{alpha}, -ß, or -{delta} isotypes, ARPE-19 cells were incubated with PAF (100 ng/mL), and whole-cell lysates were prepared at different times up to 18 hours to be tested by Western blotting for the presence of PKC{alpha}, -ß, and -{delta} isotypes. PKC{alpha} was downregulated starting a few hours after stimulation (4–8 hours) and was hardly detectable by 18 hours (Fig. 5A) . There was no downregulation of PKCß even after 18 hours of stimulation (Fig. 5B) . The decrease in PKC{delta} expression was observed after only 15 minutes and was more prominent at later time points (Fig. 5C) , suggesting that chronic exposure of ARPE-19 cells to PAF downregulates PKC{alpha} and PKC{delta} but not the ß isotype.



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  		FIGURE 5. PAF induces downregulation of PKC{alpha} and -{delta}, but not -ß, in ARPE-19 cells. Cells were serum-starved for 18 hours and then incubated for different times up to 18 hours in the presence of PAF (100 ng/mL). Whole-cell lysates were prepared and tested for the presence of PKC{alpha} (A), -ß (B), and -{delta} proteins (C) by Western blotting along with those of untreated cells (U). Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.

 
Proteasome Inhibitor Suppression of PAF-Induced Depletion of PKC{alpha}
To identify the pathways involved in the downregulation of PKC{alpha} in response to PAF, we first studied the effect of proteasome inhibitors on this depletion. MG132, an inhibitor of proteasome function,28 29 and lactacystin, which is a highly selective inhibitor of proteolysis by the proteasome,30 almost completely prevented the PAF-induced depletion of PKC{alpha} (Figs. 6A 6B , respectively). We then examined the effect of E-64d, which also inhibits calpain protease, but not the proteasome. Pretreatment with this lysosomal inhibitor had no effect on PAF-induced PKC{alpha} depletion (Fig. 6C) . These findings suggest that the proteasomal pathway is involved in the PAF-induced downregulation of PKC{alpha}.



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  		FIGURE 6. Proteasome inhibitors, but not lysosomal, PKC, or Golgi inhibitors, preserve PKC{alpha} protein from downregulation by PAF. ARPE-19 cells were serum-starved for 18 hours and then incubated for 10 minutes with 25 µM proteasome inhibitors MG-132 (A) or lactacystin (B), 25 µM lysosomal inhibitor E-64d (C), 10 µg/mL Golgi inhibitor BFA (D), or 100 nM PKC inhibitor calphostin C (D) before the addition of PAF (100 ng/mL). Whole-cell lysates were prepared at different time points and tested for the presence of PKC{alpha} compared with those of untreated (first lane) and PAF-treated only (second lane) controls. Bar diagrams represent data quantitated by densitometry. Values are means of three separate experiments; error bars represent SD; *P < 0.05.

 
To determine the requirements for the activation of proteasome degradation, we first asked whether PKC activity was important for its degradation. Therefore, we investigated the effect of the PKC inhibitor calphostin C on PAF-induced PKC{alpha} downregulation. We found that calphostin C did not prevent the downregulation of PKC{alpha} (Fig. 6D) . We also investigated the effect of BFA, which reversibly disrupts the Golgi apparatus.31 BFA pretreatment did not affect the downregulation of PKC{alpha} (Fig. 6D) . These findings suggest that the activation of PKC{alpha} and its Golgi localization are not required for its PAF-induced degradation.


   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In the present study, we demonstrated that PAF induced differential subcellular translocation of PKC isoforms. The proinflammatory lipid mediator induced the translocation of PKC{alpha} from cytosol to the plasma membrane and then to the Golgi complex in human RPE cells, ARPE-19. PAF also induced a translocation of PKC{delta} to the Golgi. On the other hand, PAF caused translocation of PKCß to the plasma membrane only, without accumulation in the Golgi. Although both PKC{alpha} and PKC{delta} were translocated to the Golgi complex, their patterns of terminal localization on PAF activation were different. PKC{alpha} was concentrated in a well-defined area within the Golgi complex, while PKC{delta} accumulated diffusely around the nucleus. We also showed that the integrity of the Golgi complex was required for translocation of PKC{alpha} to the Golgi. Taken together, these differences indicate the differential PAF-induced trafficking of PKC isoforms {alpha}, ß, and {delta}. A diversity in the pattern of translocation has been reported for PKC{epsilon} in other cells.32 Thus, members of the PKC family play subtype- and cell-specific roles in signal-transduction pathways regulated by certain activators.

Our findings also provided evidence that PAF induced PKC{alpha} translocation through the G-protein-coupled receptor, since PAF antagonist blocked the PKC-translocating actions. Moreover, the addition of lyso-PAF, the biologically inactive form of PAF, failed to induce PKC{alpha} translocation. PAF-induced nitric oxide release via PKC{alpha} translocation in other cells is also inhibited by PAF-receptor antagonists.33

Focusing on isotype-specific activation of PKC in the RPE cells allowed us to discern activation, targeting, and degradation. Questions to be addressed here included whether translocation of PKC{alpha} to the Golgi is necessary for its degradation, and whether PKC{alpha} degradation depends on its activation. Experiments with PKC inhibitor calphostin C34 and Golgi inhibitor BFA indicate that activation and translocation of PKC{alpha} are independent of its degradation, and support the hypothesis that the proteasome-mediated downregulation of PKC{alpha} is stimulated through a different pathway. Moreover, PKC{alpha} activity and translocation are independently regulated after TNF-{alpha} stimulation in other cells.35 Furthermore, translocation of PKC{delta} to the Golgi is independent of its phosphorylation.36 Translocation of PKC{delta} also precedes tyrosine phosphorylation, which is essential for its activation.37 It may, therefore, be postulated that accumulation of PKC{alpha} in the Golgi of ARPE-19 cells is associated with functions not yet defined, and is not directly related to its downregulation. It is noteworthy that PKC participates in the constitutive transport of protein through the Golgi apparatus, since calphostin C, a DAG antagonist, is a potent inhibitor of export from the endoplasmic reticulum in vivo and in vitro.38 39 Our present results further revealed that, on stimulation, the PAF receptor activated the PLC-DAG pathway, since pretreatment of ARPE-19 cells with either calphostin C or PLC inhibitor U73122 resulted in the inhibition of PKC{alpha} translocation to the plasma membrane or Golgi apparatus (Fig. 3) .

The proteasome degrades many short-lived proteins when it is triggered by external stimuli. The ubiquitin-proteasome pathway is mainly responsible for the disappearance of PKC{alpha} and -{epsilon} isoforms provoked by PMA or bryostatin 1 in human fibroblasts.40 The cellular processes as well as molecular mechanisms involved in this regulation need to be further investigated. Moreover, we showed that treatment of the ARPE-19 cells with PAF resulted in the activation and then depletion of PKC{alpha} and -{delta} but not -ß. Our results also suggest that the downregulation of PKC{alpha} occurs principally via the proteasomal pathway. This remarkable diversity in PKC translocation and downregulation in response to PAF indicates the importance of isotype-specific functions of PKC in signal-transduction pathways in ARPE-19 cells. A subsequent step of our studies will be to define the PAF-induced PKC-specific changes in the context of oxidative stress and pathoangiogenesis. In fact, a recent study demonstrated that a novel PAF-receptor antagonist exerts neuroprotection when the retina is confronted with light-induced damage.41 Pharmacologic modulation of specific steps mediated by the inflammatory mediator PAF may lead to novel therapeutic developments.


   Footnotes
 
Supported by a Macular Degeneration Research Award from the American Health Assistance Foundation and by U.S Public Health Service Grant R01EY05121 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland.

Submitted for publication March 8, 2005; revised June 23, 2005; accepted November 18, 2005.

Disclosure: Z. Faghiri, None; N.G. Bazan, None

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

Corresponding author: Nicolas G. Bazan, Louisiana State University Neuroscience Center of Excellence, 2020 Gravier Street, Suite D, New Orleans, LA 70112; nbazan{at}lsuhsc.edu.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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