Involvement of PKC{delta} and PKD in pulmonary microvascular endothelial cell hyperpermeability

John H. Tinsley, Nicole R. Teasdale, and Sarah Y. Yuan

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional ({alpha}, {beta}I, {beta}II, and {gamma}), novel [{delta}, {epsilon}, {eta}, and µ (also known as PKD),{theta}], and atypical ({zeta} and {iota}/{lambda}), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms {alpha}, {beta}I, and {epsilon} and complete translocation of PKC{delta} and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKC{delta} and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms {alpha}, {beta}I, and {epsilon} were dispensable with regard to these same phenomena.

signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium


AN INTRICATE MONOLAYER of closely opposed cells, the microvascular endothelium operates as a semipermeable barrier to regulate the transvascular passage of solutes, fluid, and blood cells. In addition to this barrier function, the endothelium participates in homeostatic mechanisms such as antithrombogenesis, angiogenesis, regulation of leukocyte dynamics, and control of tissue perfusion. Inflammatory agonists such as activated neutrophils, histamine, thrombin, and the PKC activators phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG) are capable of educing intracellular signaling events the end result of which is the alteration of normal endothelial operation, i.e., hyperpermeability across the cell monolayer (11, 12, 22, 23, 32).

There are currently 12 known isoforms of PKC: conventional (cPKC) isoforms ({alpha}, {beta}I, {beta}II, and {gamma}), novel (nPKC) isoforms ({delta}, {epsilon}, {eta}, µ, and {theta}), and atypical (aPKC) isoforms ({zeta}, {iota}, and {lambda}) (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 {alpha}, {delta}, and {epsilon} have been shown to be involved in the regulation of barrier function and tight junction permeability in epithelial cells (14, 20). PKC{alpha}, -{beta}I, and -{delta} have been implicated in lipopolysaccharide-induced nitric oxide synthase expression in macrophages (6). Furthermore, studies in neuronal cells show that PKC{zeta} and PKC{delta} 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{alpha} 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{beta} attenuated endothelium-dependent vasodilation caused by experimental hyperglycemia (2). Our recent studies indicate a preferential upregulation of PKC{beta} gene expression in porcine cardiovascular tissues during the early development of diabetes (9). Pharmacological inhibition of PKC{beta} attenuates hyperglycemia-induced increases in coronary venular permeability (31). PKC{zeta} in human pulmonary artery endothelial cells (HPAE) and PKC{delta} in HUVEC have been shown to regulate TNF-{alpha}-induced NADPH oxidase (8) and VEGF-induced proliferation (18). In addition, overexpression of PKC{alpha} apparently augments the hyperpermeability response to thrombin, although PKC{delta} 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 {delta} and PKD were necessary for alterations in endothelial barrier function as measured by albumin flux across the monolayer. Furthermore, the cPKC isoforms {alpha} and {beta}I, as well as nPKC{epsilon}, 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{delta} and PKD were the only two found to fully translocate from the cytosol to the membrane in response to PMA. Finally, elimination of PKC{delta} 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 {alpha}, {beta}I, and {epsilon} 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 {delta} and PKD are both required for pulmonary microvascular endothelial barrier disruption in response to PMA and DAG stimulation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. The chemicals used were PMA, bisindolylmaleimide I (BIM), Rottlerin, Gö6976, and Gö6983 from Calbiochem (La Jolla, CA); dioctanoylglycerol (DOG, a cell-permeable diacylglycerol) was from Sigma.

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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Table 1. Sequences of antisense oligonucleotides

 

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{alpha} (sc-208), -{beta}I (sc-209), -{delta} (sc-213), and -{epsilon} (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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pharmacological inhibition of PMA-induced hyperpermeability across RLMEC monolayer. The phorbol ester PMA is known to activate both cPKC and nPKC isoforms. Furthermore, activation of PKC has been shown to result in endothelial barrier dysfunction (11, 27). RLMEC were treated with increasing concentrations of PMA for 15 min and assessed for albumin flux across the monolayer. As shown in Fig. 1, a significant permeability increase occurs at 10 nM PMA and continues to increase up to 1 µM. At concentrations above 1 µM, PMA elicits a toxic effect on the Transwell membrane-grown monolayer as seen by cell detachment. To determine which PKC isoform(s) is responsible for PMA-induced hyperpermeability, four different pharmacological inhibiters were employed in combination with PMA. None of these inhibitors had a significant effect on basal permeability (Fig. 2A). As expected, BIM, a general PKC inhibitor at 10 nM, blocked the permeability increase elicited by PMA (Fig. 2B). Rottlerin, which selectively inhibits PKC{delta} at 5 µM, and Gö6983, which inhibits all PKC isoforms but not PKD at 1 µM, both significantly attenuated PMA-induced hyperpermeability (Fig. 2B). However, PMA-induced permeability increases were not attenuated by pretreatment with Gö6976, a PKC{alpha} and PKC{beta}I inhibitor at 10 nM (Fig. 2B). These results suggest that it is the novel PKC isoform {delta} and possibly others that are playing prominent roles in PMA-induced hyperpermeability.



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Fig. 1. Permeability of rat lung microvascular endothelial cell (RLMEC) monolayers in response to phorbol 12-myristate 13-acetate (PMA). Cells were grown to confluence on Transwell membranes and treated with PMA for 15 min at the concentrations shown. FITC-albumin was then added to the luminal chamber, and permeability of albumin (Pa) was calculated after 30 min as described in MATERIALS AND METHODS. Pa values are expressed as percentages of basal level. For each treatment, n = 5. *P < 0.05.

 


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Fig. 2. Effects of PKC inhibitors on PMA-induced RLMEC monolayer hyperpermeability. Cells were grown to confluence on Transwell membranes and treated with bisindolylmaleimide I (BIM, 10 nM), Rottlerin (5 µM), Gö6976 (10 nM), or Gö6983 (1 µM) for 15 min (A) or inhibitors followed by PMA (1 µM) for 15 min (B). FITC-albumin was added to the luminal chamber, and Pa was calculated after 30 min. Pa values are expressed as percentages of basal level. For each treatment, n = 5. *P < 0.05 vs. basal; #P < 0.05 vs. PMA.

 

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{alpha}, -{beta}I, -{delta}, -{epsilon}, and PKD. It should be pointed out that we were not able to detect PKC{beta}II, -{gamma}, -{eta}, or -{theta} 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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Fig. 3. Antisense depletion of PKC isoforms and PKD. RLMEC were grown to confluence on 60-mm dishes and depleted of PKC{alpha}, -{beta}I, -{delta}, -{epsilon}, or PKD by treating with antisense phosphorothioate oligonucleotides for 48 h as described in MATERIALS AND METHODS. After lysis, 10 µg of cellular protein were subjected to Western analysis for total PKC isoform content. Antibodies for immunoblotting were PKC{alpha} (A), PKC{beta}I (B), PKC{delta} (C), PKC{epsilon} (D), and PKD (E). Each panel depicts control cell lysate and 3 different antisense treatment lysates to show the inhibition specificity of each oligonucleotide. The experiments were repeated 3 times, and representative Westerns are shown.

 

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{alpha}, -{beta}I, and -{epsilon} did not result in attenuation of the permeability increases elicited by PMA. However, when cells were depleted of PKC{delta} or PKD before PMA treatment, hyperpermeability responses were blocked (Fig. 4B).



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Fig. 4. Effects of PKC isoform-specific antisense oligonucleotides on PMA-induced monolayer permeability. RLMEC were grown to confluence on Transwell membranes and transfected with antisense oligonucleotides for 48 h as described in MATERIALS AND METHODS. The effects of antisense oligonucleotides on basal permeability are shown in A. In B, transfected cells were treated with PMA (1 µM) for 15 min before permeability measurements. Pa was calculated after 30 min as described in MATERIALS AND METHODS. Pa values are expressed as percentages of basal level. For each treatment, n = 5. *P < 0.05 vs. basal; #P < 0.05 vs. PMA.

 

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{delta} or PKD before PMA treatment resulted in attenuation of MARCKS protein phosphorylation, i.e., PKC activity (Fig. 6). Furthermore, PKC{alpha}, -{beta}I, and -{epsilon} depletion had little, if any, effect on PMA-induced PKC activity (Fig. 6).



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Fig. 5. PMA-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation. RLMEC were grown to confluence on 60-mm dishes and treated with increasing concentrations of PMA for 15 min. After lysis, 10 µg of total protein were subjected to Western analysis for detection of phosphorylated MARCKS. The experiments were repeated 3 times, and a representative Western is shown.

 


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Fig. 6. Inhibition of PMA-induced MARCKS phosphorylation using PKC isoform-specific antisense oligonucleotides. RLMEC were transfected with oligonucleotides for 48 h as described, followed by PMA (1 µM) treatment for the times shown. After lysis, 10 µg of total protein were subjected to Western analysis for detection of phosphorylated MARCKS. Protein bands were scanned and quantified using NIH software. The graph depicts the intensity of bands in arbitrary units.

 

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{alpha}, -{beta}I, -{delta}, -{epsilon}, and PKD exist for the most part in the cytosolic fraction (Fig. 7). After PMA treatment for 5–60 min, we saw incomplete translocation of PKC{alpha}, -{beta}I, and -{epsilon} from the cytosol to the membrane (Fig. 7). However, the translocation of PKC{delta} 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{delta} and PKD as the major kinases responsible for cellular activity after phorbol ester treatment.



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Fig. 7. Cytosolic/membrane localization of PKC isoforms after PMA treatment. Confluent RLMEC were treated with PMA (1 µM) for 5, 15, 30, and 60 min, followed by cellular fractionation as described in MATERIALS AND METHODS (b = basal). Ten micrograms of protein from cytosolic (c) and membrane (m) fractions was analyzed for the presence of the 5 PKC isoforms. The experiments were repeated 3 times, and a representative Western is shown.

 

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{delta} and PKD significantly attenuated the DAG-induced hyperpermeability responses, whereas depletion of PKC{alpha}, -{beta}I, and -{epsilon} 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{delta} and PKD. It should also be pointed out that depletion of PKC{alpha} 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{alpha} may play a role in DAG-induced cellular responses.



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Fig. 8. Diacylglycerol (DAG)-induced hyperpermeability and MARCKS phosphorylation. Cells were grown to confluence on Transwell membranes and treated with DAG for 15 min at the concentrations shown. In addition, RLMEC were transfected with antisense oligonucleotides for 48 h as described in MATERIALS AND METHODS, followed by DAG (10–5M) treatment for 15 min. FITC-albumin was then added to the luminal chamber, and Pa was calculated after 30 min as described in MATERIALS AND METHODS. Pa values are expressed as percentages of basal level. For each treatment, n = 5. *P < 0.05 vs. basal; #P < 0.05 vs. DAG 10–5 M. The same treatments were applied to RLMEC to assess the effects of DAG (10–5 M, 15 min) on MARCKS phosphorylation, and a representative Western is shown below the graph.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PKC is known to play an important role in the signaling mechanisms of many different cell types (16, 19, 26, 30). Specifically, the role of PKC in the mediation of microvascular hyperpermeability has been widely established. Activation and inhibition of PKC generally results in increases and decreases, respectively, in permeability (1, 6, 11, 17). We found that both PMA and DAG induced hyperpermeability across RLMEC monolayers in a dosage-dependent manner. Furthermore, PMA and DAG showed the same effects on MARCKS phosphorylation, an accepted measure of PKC activity. However, little was known with regard to which PKC isoforms are involved in the regulation of microvascular endothelial cell barrier function. This study, for the first time, implicates two isoforms required for barrier dysfunction in pulmonary microvascular endothelium, namely PKC{delta} and PKD.

We were able to detect four PKC isoforms ({alpha}, {beta}I, {delta}, and {epsilon}) 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{delta} and PKD to the membrane fraction upon PMA stimulation. PKC{alpha} and PKC{beta}I partially relocated from cytosol to membrane, whereas PKC{epsilon} 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{delta} 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{alpha}, -{beta}I, or -{epsilon} 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{alpha}. This suggests that perhaps PKC{alpha} 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{delta} and PKD. These findings suggest that PKC{delta} 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{epsilon} and PKD. First, in experiments on COS-7 cells, evidence of a direct PKC{epsilon}-PKD phosphorylation cascade in which PKD is phosphorylated at Ser744 and Ser748 of its activation loop was shown (28). Second, PKC{epsilon} 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{eta} 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{delta}-dependent fashion (21). Our study is further evidence of the involvement and possible interaction between PKD and PKC{delta}, 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 {alpha}, {beta}I, and {epsilon} 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{delta} and PKD, whereas the other isoforms play something of a supportive role. In other words, partial activation of PKC{alpha}, -{beta}I, and -{epsilon} may itself induce downstream activation of PKC{delta} and PKD. Depletion of PKC{alpha}, -{beta}I, or -{epsilon} 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{epsilon} and PKD support this line of reasoning. Perhaps PKC{alpha} and -{beta}I play a similar role to PKC{epsilon} in subsequent regulation of PKC{delta} and PKD. Although there is redundancy in the signaling cascade upstream of PKC{delta} 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{delta} and PKD as being required for PMA- and DAG-induced PKC activity and hyperpermeability responses in pulmonary microvascular endothelial cells.


    ACKNOWLEDGMENTS
 
GRANTS

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).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Tinsley, Dept. of Surgery, Texas A & M Univ., System Health Science Center, 702 SW HK Dodgen Loop, Temple, TX 76504 (E-mail: jht{at}tamu.edu).

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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