Department of Surgery, Cardiovascular Research Institute, Texas A & M University System Health Science Center, Temple, Texas 76504
Submitted 6 August 2003 ; accepted in final form 8 September 2003
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ABSTRACT |
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signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium
There are currently 12 known isoforms of PKC: conventional (cPKC) isoforms (,
I,
II, and
), novel (nPKC) isoforms (
,
,
, µ, and
), and atypical (aPKC) isoforms (
,
, and
) (13, 15). PKCµ is often called protein kinase D (PKD), and we will hereafter refer to it as such. Activation of these three subtypes of PKC occurs under different mechanisms. The cPKC isoforms are dependent on phosphatidylserine, DAG, and Ca2+, and they are activated by PMA. The nPKC isoforms differ in that they become activated in the absence of Ca2+. The aPKC isoforms are both independent of DAG and Ca2+ and are not activated by PMA. The PKC isoforms vary widely with regard not only to cell specificity but also to subcellular localization within distinct cell types. For example, the PKC isoforms
,
, and
have been shown to be involved in the regulation of barrier function and tight junction permeability in epithelial cells (14, 20). PKC
, -
I, and -
have been implicated in lipopolysaccharide-induced nitric oxide synthase expression in macrophages (6). Furthermore, studies in neuronal cells show that PKC
and PKC
are activated by different growth factors and that these processes independently result in mitogen-activated protein kinase activation (7).
Our previous studies and those of others have implicated the involvement of PKC in regulating endothelial function (1, 11, 24, 27). Much of the recent work is being devoted to elucidating the specific isoform(s) of PKC being expressed and operating in endothelial cells of macrovascular origin. For example, PKC is known to increase endothelial permeability and is required for endothelial cell migration and vascular tube formation in human umbilical vein endothelial cells (HUVEC) (16, 29). In vivo experiments on healthy humans determined that inhibition of PKC
attenuated endothelium-dependent vasodilation caused by experimental hyperglycemia (2). Our recent studies indicate a preferential upregulation of PKC
gene expression in porcine cardiovascular tissues during the early development of diabetes (9). Pharmacological inhibition of PKC
attenuates hyperglycemia-induced increases in coronary venular permeability (31). PKC
in human pulmonary artery endothelial cells (HPAE) and PKC
in HUVEC have been shown to regulate TNF-
-induced NADPH oxidase (8) and VEGF-induced proliferation (18). In addition, overexpression of PKC
apparently augments the hyperpermeability response to thrombin, although PKC
may protect against barrier dysfunction (10). This sampling of previous findings makes apparent the diverse functions controlled by various PKC isoforms in the macrovascular endothelium. However, little is known regarding the role(s) of PKC and, more precisely, the mechanisms regulated by distinct PKC isoforms in the microvascular endothelium, the main barrier to blood-stream/tissue exchange of fluids, solutes, and gasses.
This study focuses on the effects of PMA- and DAG-induced PKC activation and subsequent hyperpermeability response across the endothelial monolayer in rat lung microvascular endothelial cells (RLMEC). Using various pharmacological inhibitors, we were able to predict which PKC isoforms were activated by PMA and responsible for permeability increases. By employing more specific means, i.e., antisense oligonucleotides directed against the different isoforms, we found that the nPKC isoform and PKD were necessary for alterations in endothelial barrier function as measured by albumin flux across the monolayer. Furthermore, the cPKC isoforms
and
I, as well as nPKC
, were not required for PMA- and DAG-induced endothelial hyperpermeability. In addition, of the five isoforms we were able to detect in these cells, PKC
and PKD were the only two found to fully translocate from the cytosol to the membrane in response to PMA. Finally, elimination of PKC
and PKD by antisense oligonucleotides attenuated the PMA- and DAG-induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent PKC substrate (5, 25). The PKC isoforms
,
I, and
showed incomplete cytosol-to-membrane translocation and apparently were not required for MARCKS phosphorylation after PMA treatment. This work is the first to show that the specific PKC isoform
and PKD are both required for pulmonary microvascular endothelial barrier disruption in response to PMA and DAG stimulation.
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MATERIALS AND METHODS |
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Cell culture. RLMEC (VEC Technologies, Rensselaer, NY) were maintained on gelatin-coated dishes in complete MCDB-131 medium supplemented with 10% fetal bovine serum and used at passages 37. Cells were incubated at 37°C in 5% CO2. RLMEC exhibited properties characteristic of the endothelial cell, such as typical cobblestone morphology and incorporation of acetylated low-density lipoprotein.
Measurement of endothelial permeability. RLMEC permeability was determined by measuring FITC-albumin flux across the monolayer as previously described (22, 23). Cells were grown on gelatin-coated Costar Transwell membranes (VWR, Houston, TX). All transfection reagents and chemicals were added to the luminal chamber. FITC-albumin (3 µg/ml) was added to the luminal chamber for 30 min, and samples were removed from both luminal and abluminal chambers for fluorometry analysis. The readings were converted with the use of a standard curve to albumin concentration. These concentrations were then used in the following equation to determine the permeability coefficient of albumin (Pa): Pa = ([A]/t) x (1/A) x (V/[L]) where [A] is abluminal concentration, t is time in seconds, A is area of membrane in cm2, V is volume of abluminal chamber, and [L] is luminal concentration.
Oligonucleotide transfection. RLMEC were grown to confluence on either 60-mm dishes for Western analysis or Transwell membranes for permeability experiments. The complete medium was replaced with OPTI-MEM (Invitrogen) containing the antisense phosphorothioate oligonucleotides (5.6 µg/ml) that had been preincubated with Lipofectin (10 µl/ml) (Invitrogen) for 30 min. After 6 h of incubation at 37°C, the cells were washed and returned to complete medium containing oligonucleotide (2.9 µg/ml) for an additional 42 h before experimentation. All antisense oligonucleotides were synthesized by Sigma-Genosys (The Woodlands, TX) and HPLC purified before transfection. Sequences are shown in Table 1.
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Subcellular fractionation and Western analysis. Cells were treated as appropriate before examining PKC isoform translocation. After washes with cold phosphate-buffered saline (PBS), cells were suspended in 600 µl of digitonin buffer [0.55% digitonin, 25 mM Tris·HCl, pH 7.6, 5 mM EGTA, 5 mM EDTA, Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL)]. Endothelial cells were incubated on ice for 30 min and centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was labeled the cytosolic fraction. The remaining pellet was dissolved in 500 µl of digitonin buffer containing 1% Triton X-100, passed through a 26-gauge needle several times, and centrifuged again for 10 min at 10,000 g for 10 min at 4°C. The resulting supernatant was labeled the membrane fraction. For total lysate, cells were suspended in digitonin buffer containing 1% Triton X-100, passed through a needle, and centrifuged as above with the supernatant labeled total protein.
For Western analysis, 10 µg of protein were electrophoresed by SDS-PAGE and blotted to nitrocellulose membrane. Membranes were blocked with 3% bovine serum albumin followed by exposure to primary antibodies to PKC isoforms or MARCKS protein and subsequent secondary antibody conjugated to horseradish peroxidase. Enhanced chemiluminescence (ECL) was employed to detect bands of interest. Primary antibodies (all rabbit polyclonal) used were PKC (sc-208), -
I (sc-209), -
(sc-213), and -
(sc-214) from Santa Cruz Biotechnology (Santa Cruz, CA). PKCµ was from Cell Signaling Technology (Beverly, MA) and phospho-MARCKS was from Proteintech Group (Chicago, IL). Anti-rabbit IgG-HRP secondary antibody was from Cell Signaling Technology.
Data analysis. Analysis of variance was used to evaluate the significance of intergroup differences in the permeability studies. A value of P < 0.05 was considered significant for the comparisons.
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RESULTS |
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Antisense oligonucleotide depletion of PKC isoforms. To make a more specific evaluation of which PKC isoforms are important to PMA-induced activity in microvascular endothelial cells, isoform-specific antisense oligonucleotides were designed against PKC, -
I, -
, -
, and PKD. It should be pointed out that we were not able to detect PKC
II, -
, -
, or -
in the RLMEC. As shown in Fig. 3, antisense treatment was successful in depleting the cells of most, if not all, of the five isoforms we were able to detect in untreated cells. To confirm the specificity of the oligonucleotides, lysate was analyzed for both the isoform which antisense treatment was intended to deplete and two of the other isoforms. In every case, the antisense oligonucleotide was effective against its specific isoform and had no effect on expression of the others (Fig. 3).
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PMA-induced hyperpermeability across RLMEC monolayers after antisense oligonucleotide treatment. Now that we had the means to specifically deplete the cells of individual PKC isoforms, we sought to determine which isoform(s) was responsible for PMA-induced hyperpermeability. The antisense oligonucleotides had no significant effects on basal permeability (Fig. 4A). Figure 4B shows that treatment with antisense oligonucleotides directed against PKC, -
I, and -
did not result in attenuation of the permeability increases elicited by PMA. However, when cells were depleted of PKC
or PKD before PMA treatment, hyperpermeability responses were blocked (Fig. 4B).
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PMA-induced MARCKS phosphorylation. Having deduced which PKC isoforms elicit hyperpermeability responses, we sought to evaluate the actual PKC activity in the cells after PMA treatment. Phosphorylation of MARCKS protein is a widely used indicator of PKC activity in the cell. Both conventional and novel PKC isoforms are known to phosphorylate MARCKS (5, 25). Treatment of RLMEC with increasing concentrations of PMA led to dosage-dependent increases in MARCKS phosphorylation (Fig. 5), reaching maximal levels at 1 µM. To determine which PKC isoform(s) is responsible for PMA-induced MARCKS phosphorylation, we again employed antisense oligonucleotides. PMA-induced MARCKS phosphorylation occurred rapidly (5 min) and stayed at a level approximately fourfold above basal levels for 60 min (Fig. 6). We found that depletion of PKC or PKD before PMA treatment resulted in attenuation of MARCKS protein phosphorylation, i.e., PKC activity (Fig. 6). Furthermore, PKC
, -
I, and -
depletion had little, if any, effect on PMA-induced PKC activity (Fig. 6).
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PMA-induced PKC isoform translocation. We employed another method to evaluate activity levels of the PKC isoforms of interest, cytosol vs. membrane distribution. Under basal conditions, PKC, -
I, -
, -
, and PKD exist for the most part in the cytosolic fraction (Fig. 7). After PMA treatment for 560 min, we saw incomplete translocation of PKC
, -
I, and -
from the cytosol to the membrane (Fig. 7). However, the translocation of PKC
and PKD to the membrane fraction after PMA treatment was complete at 15 and 30 min, with an apparent initial return to the cytosol at 60 min (Fig. 7). This finding further corroborates activity levels measured by MARCKS phosphorylation, as well as the permeability studies implicating PKC
and PKD as the major kinases responsible for cellular activity after phorbol ester treatment.
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DAG-induced hyperpermeability and MARCKS phosphorylation. Although PMA is a widely accepted and utilized PKC activator, we sought to determine which PKC isoforms are responsible for DAG (a natural PKC activator)-induced cellular responses. As shown in Fig. 8, the effect of a 15-min DAG treatment on monolayer hyperpermeability was maximal at 10 µM and resulted in a 38% increase in albumin flux. Furthermore, depletion of PKC and PKD significantly attenuated the DAG-induced hyperpermeability responses, whereas depletion of PKC
, -
I, and -
did not (Fig. 8). To further measure the effect of DAG on cellular responses, we looked at MARCKS phosphorylation and found that maximal phosphorylation was also reached with 10 µM DAG treatment (Fig. 8). In addition, DAG-induced MARCKS phosphorylation was lowered to near basal levels upon depletion of PKC
and PKD. It should also be pointed out that depletion of PKC
apparently reduced the effects of DAG on hyperpermeability and MARCKS phosphorylation (Fig. 8). Although these reductions were not statistically significantly, the results suggest that PKC
may play a role in DAG-induced cellular responses.
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DISCUSSION |
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We were able to detect four PKC isoforms (,
I,
, and
) and PKD by Western blot from RLMEC lysate. Several parameters were employed to assess the activity conferred by the various kinases. One such measure of activity, cytosol-to-membrane translocation, showed complete movement of PKC
and PKD to the membrane fraction upon PMA stimulation. PKC
and PKC
I partially relocated from cytosol to membrane, whereas PKC
was found in approximately equal amounts in the two fractions. These findings coincide with the pharmacological inhibitor analysis, suggesting that it was the novel, not conventional, isoforms responsible for PMA-induced PKC activity and hyperpermeability responses. However, the specificity of pharmacological inhibitors is often called into question, and a more precise technique for determining the role of individual isoforms was sought.
By specifically inhibiting individual isoforms employing antisense technology, we determined that PKC and PKD were both required for PMA- and DAG-induced hyperpermeability responses across the endothelial monolayer. In addition, depletion of either of these isoforms attenuated PMA- and DAG-induced MARCKS phosphorylation. Antisense oligonucleotides directed at PKC
, -
I, or -
showed little or no effect on PMA-induced hyperpermeability or MARCKS phosphorylation. However, we did see an apparent reduction in DAG-induced hyperpermeability and MARCKS phosphorylation upon depletion of PKC
. This suggests that perhaps PKC
plays a role in maximizing DAG-induced hyperpermeability in the microvascular endothelium but is not required for a response, as in the case with PKC
and PKD. These findings suggest that PKC
and PKD are either essential components of a single pathway or perhaps involved in different pathways, both of which participate in the hyperpermeability response. Recent work, which has uncovered apparent PKC kinase cascades, suggests the former. Studies from two independent groups have discovered a link between PKC
and PKD. First, in experiments on COS-7 cells, evidence of a direct PKC
-PKD phosphorylation cascade in which PKD is phosphorylated at Ser744 and Ser748 of its activation loop was shown (28). Second, PKC
has been identified as an upstream kinase for PKD in the regulation of downstream effectors (3). Further evidence of PKC signaling cascades is demonstrated by the discovery that PKC
both colocalizes with and activates PKD via phosphorylation of PKDSer738/742 (4). Most importantly with regard to our work, a recent study has shown that upon thrombin activation of aortic smooth muscle cells, PKD activation is mediated in a PKC
-dependent fashion (21). Our study is further evidence of the involvement and possible interaction between PKD and PKC
, specifically upon activation by phorbol ester. These findings illustrate the newly discovered complexities in PKC signaling cascades, and our work is the first to directly implicate the two kinases with both PKC activity and cellular abnormalities, i.e., hyperpermeability, in the microvascular endothelium.
As mentioned above, there was incomplete translocation from the cytosol to the membrane with the PKC isoforms ,
I, and
upon PMA stimulation of the cells. However, we found these isoforms to be dispensable with regards to PMA- and DAG-induced PKC activity as measured by MARCKS phosphorylation and hyperpermeability responses in this cell line. It could be that in the pulmonary microvasculature, specific activation by PKC or DAG is propagated mainly by PKC
and PKD, whereas the other isoforms play something of a supportive role. In other words, partial activation of PKC
, -
I, and -
may itself induce downstream activation of PKC
and PKD. Depletion of PKC
, -
I, or -
individually may in itself not be sufficient to significantly attenuate PMA- and DAG-induced responses. That is to say, the other two kinases may be able to continue the signaling cascade in the absence of the other. The two studies mentioned above (3, 28) that discovered a link between PKC
and PKD support this line of reasoning. Perhaps PKC
and -
I play a similar role to PKC
in subsequent regulation of PKC
and PKD. Although there is redundancy in the signaling cascade upstream of PKC
and PKD, the continued propagation of PKC activity with respect to hyperpermeability is dependent on both of these enzymes. In summary, our work identifies PKC
and PKD as being required for PMA- and DAG-induced PKC activity and hyperpermeability responses in pulmonary microvascular endothelial cells.
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-61507 and HL-70752 (to S. Y. Yuan) and a Department of Veterans Affairs Veterans Integrated Service Network 17 Grant (to J. H. Tinsley).
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FOOTNOTES |
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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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