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Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:49-55
Published online before print October 20, 2005, doi: 10.1161/01.ATV.0000191639.08082.04
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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:49.)
© 2006 American Heart Association, Inc.

Vascular Biology

Dynein–Dynactin Complex Mediates Protein Kinase A–Dependent Clustering of Weibel-Palade Bodies in Endothelial Cells

Mariska G. Rondaij; Ruben Bierings; Astrid Kragt; Karina A. Gijzen; Erica Sellink; Jan A. van Mourik; Mar Fernandez-Borja; Jan Voorberg

From the Departments of Plasma Proteins (M.G.R., R.B., A.K., K.A.G., E.S., J.A.v.M., J.V.) and Molecular Cell Biology (M.F-B.), Sanquin Research and Landsteiner Laboratory, and the Department of Vascular Medicine (J.A.v.M.), Academic Medical Centre, University of Amsterdam, The Netherlands.

Correspondence to Jan Voorberg, Department of Plasma Proteins, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. E-mail j.voorberg{at}sanquin.nl


*    Abstract
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 *Abstract
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 down arrowResults
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Objective— Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell–specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor (vWF) secretion. In this study, we investigated this phenomenon of WPB clustering in more detail.

Methods and Results— Our results demonstrate that the clustered WPBs are localized at the microtubule organizing center and that cluster formation depends on an intact microtubule network. Disruption of the microtubules by nocodazole completely abolished clustering, whereas treatment with the actin depolymerizing compound cytochalasin B had no effect on WPB clustering. Interfering with the dynein–dynactin interaction by overexpression of the p50 dynamitin subunit or the CC1 domain of the p150glued subunit of the dynactin complex completely inhibited perinuclear clustering of WPBs, suggesting that dynein activity mediates this process. Furthermore, we found that inhibition of dephosphorylation resulted in an increase in clustering, whereas inhibition of protein kinase A (PKA) markedly reduced WPB clustering.

Conclusions— These results suggest that perinuclear clustering of WPBs involves PKA-dependent regulation of the dynein–dynactin complex. Endothelial cell stimulation with epinephrine results in retrograde movement of a subset of WPBs to the microtubule organizing center. This minus-end directed transport requires an intact microtubular network and is mediated by the motor protein dynein. Together, our results suggest that epinephrine-induced clustering of WPBs involves PKA-dependent regulation of the dynein–dynactin complex.

Perinuclear clustering is observed for several different organelles and illustrates dynamic regulation of the secretory pathway and organelle distribution. Previously, we observed that a subset of Weibel-Palade bodies (WPBs), endothelial cell–specific storage organelles, undergo centralization when endothelial cells are stimulated with cAMP-raising agonists of von Willebrand factor secretion. In this study, we investigated this phenomenon of WPB clustering in more detail.


Key Words: organelle trafficking • dynein • Weibel-Palade bodies • PKA • endothelial cells


*    Introduction
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 *Introduction
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Bidirectional transport is a common phenomenon observed for many different organelles, such as melanosomes,1,2 mitochondria,3 phagosomes,4 endosomes,5 lysosomes,6,7 and secretory vesicles,8,9 but also viruses10,11 are known to display bidirectional motion. For this transport, cells use molecular motors and are equipped with 2 sets of directional tracks: microtubules and actin filaments. Evidence from different studies suggests that microtubules are used for rapid, long-distance transport, whereas actin may be used for local transport or anchorage of cargo to specific locations once delivered there via microtubules.9,12 In nondividing cells, microtubules are arranged in a polarized fashion with their minus ends clustered around a microtubule organizing center (MTOC) near the nucleus and their plus ends directed toward the periphery of the cell. Motor proteins recognize the polarity of the microtubules and move exclusively toward the plus end or the minus end. Plus-end–directed movement is mediated by motor proteins of the kinesin family, whereas dynein is responsible for minus-end–directed transport. Previously, it has been shown that Weibel-Palade bodies (WPBs), which are secretory, endothelial cell–specific storage organelles for von Willebrand factor (vWF), move in an apparently random manner in resting cells.9,13 Some WPBs appeared motionless, whereas others showed movement to both the periphery and the center of the cell. Intriguingly, on stimulation with several distinct VWF secretagogues, differences in WPB dynamics were observed depending on the type of stimulus. Exocytosis of WPBs is induced by Ca2+-raising agonists, such as thrombin and histamine,14,15 as well as by the cAMP-mediated agonists epinephrine and vasopressin.16,17 Plus-end–directed movement of WPBs was observed on stimulation with both Ca2+- and cAMP-mediated agonists of vWF secretion, whereas a marked increase in minus-end–directed transport was observed only after stimulation with epinephrine or the cAMP-raising compound forskolin. These minus-end–directed WPBs formed a star-like cluster at the perinuclear region.13 In this study, we investigated this perinuclear clustering phenomenon of WPBs in more detail. Our results demonstrate that clustering of WPBs is mediated by the dynein/dynactin complex. Because the direction and timing of organelle movement needs to be tightly controlled by the cell, the activity of these motor proteins must be regulated. A variety of data point to phosphorylation as a general means to regulate motor protein activity or cargo interaction.18–23 To investigate whether the observed clustering of WPBs is regulated by phosphorylation, we investigated the effect of phosphatase inhibition and protein kinase A (PKA) inhibition on WPB clustering. Inhibition of phosphatases by okadaic acid resulted in an increase in WPB clustering, whereas the inhibition of PKA resulted in a marked reduction in cAMP-dependent clustering. Together, these data suggest that PKA mediates dynein/dynactin-dependent perinuclear clustering of WPBs at the MTOC.

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*    Methods
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 *Methods
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Please see http://atvb.ahajournals.org for details on the methods used in this study.


*    Results
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 *Results
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cAMP Induces WPB Clustering at the MTOC in a Microtubule-Dependent Manner
We first assessed whether a rise in intracellular cAMP levels is sufficient to induce WPB clustering. Human umbilical vein endothelial cells were incubated for 1 hour with thrombin, a Ca2+-mediated secretagogue (Figure 1B), the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (Figure 1C), epinephrine/IBMX (Figure 1D), forskolin/IBMX (Figure 1E), the cAMP analogue dibutyryl-cAMP (db-cAMP; Figure 1F), or serum-free (SF) medium (Figure 1A). To quantitatively assess clustering of WPBs, 3D images of the distribution of WPBs in endothelial cells were assembled, essentially as described previously.13 The relative position of WPBs is expressed by pseudocolor coding in which red-green-blue WPBs represent basolaterally-centrally-apically localized WPBs. In response to cAMP-raising compounds, the presence of star-like WPB clusters at the perinuclear region was observed in maximally &50% of the cells (Figure 1C through 1F, arrows). The remainder of the cells consists of a heterogeneous population that showed partial WPB clustering or even completely dispersed WPBs (Figure 1C through 1F, open arrowheads). An increase in cAMP levels was sufficient to induce WPB clustering (Figure 1C through 1G), whereas no clustering was observed in unstimulated (Figure 1A and 1G) or in thrombin-stimulated cells (Figure 1B and 1G). In addition, in cells stimulated with cAMP-raising compounds, the average number of residual WPBs was reduced compared with unstimulated cells demonstrating that a rise in cAMP not only results in clustering but also exocytosis of WPBs (data not shown). Subsequently, we expressed a green fluorescent protein (GFP)-tagged version of the vasopressin V2 receptor (V2R) in HUVECs. V2R present on lung endothelial cells has been implicated in the transient rise in plasma levels of VWF after administration of desmopressin to patients with von Willebrand disease and mild hemophilia A. Vasopressin-induced, cAMP-dependent release of WPBs is observed after the introduction of recombinant V2R in HUVECs.17 WPB clustering was also observed in vasopressin- and desmopressin-stimulated HUVECs expressing GFP-V2R (Figure 1G). Next, we investigated whether cAMP-mediated WPB clustering is reversible. Endothelial cells were incubated with either forskolin/IBMX or forskolin/IBMX followed by a 2-hour recovery incubation with fresh SF medium. Our results show that removal of forskolin reverses the clustering of WPBs. Moreover, WPBs in these cells were dispersed throughout the cell in a manner that was reminiscent of unstimulated cells indicating that forskolin-induced WPB-clustering is a reversible process (Figure I, available online at http://atvb.ahajournals.org).



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  		Figure 1. WPB clustering is induced by an increase in cAMP levels. HUVECs were grown to confluence on glass coverslips. Cells were incubated with SF medium, thrombin (1 U/mL), IBMX (100 µmol/L), epinephrine/IBMX (10 µmol/L/100 µmol/L), forskolin/IBMX (10 µmol/L/100 µmol/L), or db-cAMP (1 mmol/L) for 1 hour. Cells were analyzed for WPB clustering as described in Methods. Clustering of WPBs on stimulation with different stimuli was compared with clustering in control cells. Representative 3D confocal images of HUVECs incubated with (A) SF medium, (B) thrombin, (C) IBMX, (D) epinephrine, (E) forskolin, and (F) db-cAMP. Arrows indicate clustering WPBs. Open arrowheads indicate stimulated cells showing partial clustering or complete dispersion. Bars correspond to 10 µm. (G) Analysis of WPB-clustering in response to various stimuli. HUVECs expressing the vasopressin-2 receptor (V2R) were incubated with SF medium, 0.1 µmol/L vasopressin [arginine vasopressin (AVP)] or 0.1 µmol/L desmopressin (DDAVP). Bars, ±SEM. *P<0.05, **P<0.005, ***P<0.0005 by Student t test.

To study the localization of WPB clusters in relation to the actin cytoskeleton and microtubules, HUVECs were incubated for 1 hour with SF medium or forskolin/IBMX. After stimulation, the cells were fixed and vWF, F actin, and microtubules were visualized by immunostaining with polyclonal anti-VWF phalloidin, and monoclonal antityrosinated tubulin, and then analyzed by fluorescence confocal microscopy. Unstimulated cells showed WPBs dispersed throughout the cytoplasm codistributing with actin filaments at the cell periphery (Figure 2A, detail). Forskolin stimulation induced WPB clustering at the perinuclear region. These clustered WPBs never associated with the actin cytoskeleton (Figure 2B, detail). In unstimulated cells, WPBs were localized along microtubules (Figure 2C, detail) confirming previous findings.9 On stimulation with forskolin, clustered WPBs appeared to be localized to areas at the perinuclear region where microtubules emerge, corresponding to the MTOC (Figure 2D, right panel arrow). To investigate the role of the microtubules and the actin cytoskeleton in WPB clustering in more detail, we analyzed forskolin-induced WPB clustering in the absence and presence of either the microtubule-disrupting agent nocodazole or the actin-depolarizing compound cytochalasin B. Microtubule disruption by nocodazole completely abolished forskolin-induced WPB clustering, whereas cytochalasin B had no affect on forskolin-induced clustering of WPBs (Figure 2E). These results indicate that cAMP induces microtubule-dependent minus-end–directed transport of WPBs to the MTOC.



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  		Figure 2. cAMP-induced perinuclear clustering of WPBs occurs at the MTOC. HUVECs were grown to confluence on glass coverslips. Cells were incubated with SF medium or forskolin/IBMX (10 µmol/L/100 µmol/L) for 1 hour. Representative confocal images of WPBs in relation to the actin cytoskeleton, and microtubules are shown as projections of optical sections (Z-stacks). (A and B) VWF, shown in red, and actin, shown in green, in unstimulated and forskolin-stimulated cells. Right panels show detailed images. (C and D) VWF, shown in red, and microtubules, shown in green, in control cells and forskolin-stimulated cells. Right panels show a detailed image of WPBs localized along microtubules in unstimulated cells and microtubule staining showing the MTOC (indicated by arrow). Bars correspond to 10 µm. (E) HUVECs were preincubated with SF medium, 5 µg/mL nocodazole (noc) or 5 µg/mL cytochalasin B (cytoB) for 30 minutes and then stimulated with forskolin for 1 hour. Bars, ±SEM. ***P<0.0005 by Student t test.

Disruption of the Dynein–Dynactin Complex Abolishes Perinuclear Clustering of WPBs
The increase in minus-end–directed transport on epinephrine and forskolin stimulation suggests a role for the minus-end–directed motor protein dynein in this process. Because dynein is associated with vesicles via the anchoring complex dynactin, we hypothesized that disruption of this dynactin complex would result in the inhibition of minus-end–directed transport of WPBs, thereby reducing forskolin-induced cluster formation at the MTOC. To test this hypothesis, we overexpressed GFP-tagged dynamitin, the 50-kDa subunit of the dynactin complex (GFP-p50), which is known to lead to dissociation of the p150glued subunit from the rest of the complex resulting in inhibition of dynein-dependent vesicle transport.24,25 GFP-expressing cells, which were taken as controls, showed perinuclear clustering on forskolin stimulation similar to untransfected cells (Figure 3A through 3D and 3I). In contrast, in GFP-p50–expressing cells, the clustering of WPBs was completely abolished (Figure 3E through 3I). In cells that do not express GFP-p50, WPB clustering was still observed on forskolin stimulation (Figure 3G and 3H, arrows). Furthermore, the residual number of WPBs in GFP-p50–expressing cells stimulated with forskolin was reduced compared with unstimulated GFP-p50–expressing cells, suggesting that exocytosis of WPBs in response to forskolin is unaffected by expression of GFP-p50. Moreover, no difference in the residual number of WPBs on forskolin stimulation was observed in GFP-p50–expressing cells compared with GFP-expressing cells suggesting that exocytosis of WPBs is not increased when clustering is blocked (data not shown). To exclude the possibility that the inhibitory effect of p50 is attributable to interference with pathways other than the dynein–dynactin pathway,26 we expressed RFP-CC1, a fusion protein of DsRed1 and the CC1 part of the dynein-interactive domain of p150glued, in HUVECs. Expression of this construct competitively inhibits dynein–dynactin binding and, thus, prevents dynein-mediated transport.27 Consistent with the GFP-p50 data, RFP-CC1 markedly inhibited WPB clustering in response to forskolin stimulation (Figure 3J). In contrast, overexpression of GFP-KLC2-TPR, which is based on the tetra-trico peptide repeats (TPR) cargo–binding domain of kinesin light chain 2 and was shown previously to block conventional kinesin-dependent movement,28 does not impair forskolin-induced WPB clustering (Figure 3K). These results demonstrate that disruption of the dynein–dynactin complex inhibits WPB cluster formation at the MTOC, indicating that the clustering of WPBs is mediated by the dynein–dynactin complex.



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  		Figure 3. Disruption of the dynactin complex inhibits WPB clustering. HUVECs were transfected with pEGFPc1 (control), pEGFP-p50, pRFP-CC1, or pEGFP-KLC2-TPR as described in Methods; 48 hours after transfection, cells were incubated with forskolin/IBMX (10 µmol/L/100 µmol/L) or SF medium alone. Representative confocal microscopy images of GFP- (A through D) and GFP-p50–positive cells (E through H) and 3D images, showing WPBs (B, D, F, and H). Cells that do not express GFP-p50 show normal clustering on forskolin stimulation (G and H, indicated by arrows). Bars correspond to 10 µm. Analysis of WPB-clustering in (I) GFP- and GFP-p50–postive cells, (J) GFP- and RFP-CC1–positive cells, and (K) GFP- and GFP-KLC2-TPR–positive cells. Bars, ±SEM. *P<0.05, **P<0.005; ns: not significant by Student t test.

WPB Clustering Is Controlled by Protein Phosphorylation Events
Phosphorylation events have been implicated in the regulation of vesicular, bidirectional transport. To determine whether phosphorylation events play a role in the minus-end–directed movement of WPBs, we investigated whether okadaic acid, a broad-spectrum inhibitor of protein phosphatases, affected perinuclear clustering of WPBs. HUVECs were preincubated with okadaic acid for 1 hour and then incubated in SF medium with or without forskolin. The inhibition of dephosphorylation by okadaic acid resulted in WPB clustering in nonstimulated HUVECs. Moreover, the clustering of WPBs induced by forskolin was additionally enhanced by the presence of okadaic acid (Figure II, available online at http://atvb.ahajournals.org). The observation that inhibition of dephosphorylation results in a clustering of WPBs suggests that in resting HUVECs, proteins involved in clustering are continuously phosphorylated and dephosphorylated with the majority of target proteins in the dephosphorylated state. We hypothesize that inhibition of dephosphorylation and stimulation with cAMP-raising agonists shifts this equilibrium toward phosphorylation and promotes the clustering of WPBs. The most common effector of cAMP is PKA, which is known to play a role in vesicle transport. A number of observations support a role for PKA in the regulation of motor proteins in melanophores, the pigment cells of fish and amphibians.1 To test whether the observed clustering of WPBs in human endothelial cells is PKA dependent, HUVECs were incubated for 1 hour with SF medium or forskolin/IBMX in the absence or presence of increasing concentrations of the PKA inhibitor H89 or the cAMP-analogue Rp-8CPT-cAMPS. The presence of H89 inhibited perinuclear WPB clustering in a concentration-dependent manner (Figure 4A), and also Rp-8CPT-cAMPS markedly reduced forskolin-induced WPB clustering (Figure 4B). In addition, we used a genetic approach to establish the involvement of PKA in WPB clustering. Overexpression of pNP210 encoding a recombinant PKA inhibitor prevented forskolin-induced WPB clustering, whereas an inactive variant encoded by plasmid pNP211 did not affect WPB clustering (Figure 4B). In combination with our data on phosphatase inhibition by okadaic acid, these results suggest that PKA-dependent phosphorylation of currently unknown target proteins induces the formation of WPB clusters.



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  		Figure 4. PKA dependence of WPB clustering. HUVECs were preincubated for 30 minutes with different concentrations of the PKA inhibitors H89 (10 to 100 µmol/L) or 500 µmol/L Rp-8CPT-cAMPS (Rp-8) and subsequently incubated with forskolin/IBMX (10 µmol/L/100 µmol/L) for 1 hour. (A) Analysis of the effect of H89 on WPB-clustering. (B) WPB clustering in the presence of Rp-8 and recombinant PKA inhibitors pNP211 (inactive, used as negative control) or pNP210 (active). Bars, ±SEM. ***P<0.0005 by Student t test.


*    Discussion
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 *Discussion
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The bidirectional movement of WPBs in resting HUVECs and the plasma membrane-directed movement of WPBs ultimately resulting in exocytosis of these organelles were found previously to require an intact microtubular network.9,29,30 In this study, we have shown that cAMP-induced perinuclear clustering of WPBs in HUVECs also required the integrity of the microtubular network. Microtubule-dependent clustering of WPBs was also observed in human aortic endothelial cells.31 However, in contrast to our observations in HUVECs, the clustering of WPB in human aortic endothelial cells was evoked by thrombin stimulation. This suggests that WPB clustering is mediated by different signaling pathways in endothelial cells of different vascular origin. The fact that intact microtubules seem to be required for WPB clustering suggests that microtubular motor protein complexes are involved in the observed retrograde movement of WPBs. This conclusion is supported by our findings that disruption of the dynactin complex by overexpression of the p50 dynamitin subunit or the CC1 dynein-interactive domain of the p150glued subunit results in inhibition of forskolin-induced WPB clustering. Most of the active transport in cells is driven by the microtubular motors kinesin and dynein and the actin-mediated myosin motors. Studies in a variety of cell types have indicated that although the basic process of bidirectional movement is essentially the same in these cells, the regulation of directional transport varies in the different cell types. Bidirectional trafficking has been thoroughly studied in melanophores, the pigment cells of fish and amphibians. In melanophores, trafficking of pigment granules is regulated by intracellular cAMP levels. A rise in cAMP results in the dispersion of pigment granules, whereas a decrease in cAMP leads to the aggregation of these vesicles.32 In contrast, our observations suggest that in endothelial cells, a rise in cAMP results in aggregation of a subset of WPBs at the perinuclear region, which could be explained by a difference in regulation of the bidirectional transport machinery in endothelial cells compared with melanophores. Although there is large variety in the way vesicle transport is regulated, the common mechanism of organelle transport regulation seems to be stimulus-induced signaling to accessory proteins, such as the p150glued subunit of dynactin or the motor proteins themselves. Differences in the regulation of these motor proteins are exemplified by the finding that in melanophores, inhibition of PKA resulted in the activation of dynein-dependent transport,33 whereas, in endothelial cells, a dose-dependent inhibition of dynein-mediated transport of WPBs was observed on PKA inhibition. Moreover, in melanophores, dispersion of pigment granules, suggesting inhibition of minus-end transport, was induced by protein kinase C (PKC) activation,34,35 whereas in Mel JuSo cells, inhibition of minus-end transport of lysosomal vesicles was the result of PKC inhibition.6 In endothelial cells, however, forskolin-induced WPB clustering was not affected by PKC inhibition, suggesting that the clustering of WPBs is not PKC dependent (data not shown). High concentrations of inhibitors of PKA34 or PKC6 were required to affect vesicle transport in melanophores and Mel JuSo cells, which corresponds with our observation that a relative high concentration of the PKA inhibitor Rp-8CPT-cAMPs was needed, and WPB clustering was only completely inhibited by H89 when concentrations of 75 µmol/L or higher were used (Figure 4A). Furthermore, incubation of endothelial cells with okadaic acid induced perinuclear clustering of WPBs, whereas it inhibited aggregation of pigment granules in melanophores.36 Taken together, these results indicate that PKA plays a different role in vesicular transport regulation in endothelial cells than in melanophores. Differences in the regulation of vesicle transport between melanophores and endothelial cells might be a consequence of the difference in the physiological function of vesicle trafficking in these cells. The transport of pigment granules in fish and amphibian melanophores provides a mechanism by which these animals can rapidly change color. Stimulus-induced clustering of WPB in endothelial cells likely occurs to limit the exocytosis of WPBs. WPBs provide a reservoir for inflammatory components, such as P-selectin, which, on stimulus-induced membrane exposure, is able to bind leukocytes.37,38 Moreover, under proinflammatory conditions, the synthesis of the chemotactic peptides interleukin 8 (IL-8) and eotaxin 3 are upregulated, resulting in storage of these chemokines in newly formed WPBs, thereby enabling the endothelium to attract neutrophils and eosinophils on activation.39–42 Consequently, stimulated release of WPBs not only contributes to maintaining vascular hemostasis but also mediates inflammatory responses (reviewed in ref 43). Different stimuli are likely to induce different WPB responses, depending on the physiological need.44 In the case of vascular damage, thrombin induces a rapid, local response leading to exocytosis of most of the WPBs present in the cell.14,45 From earlier studies, it is known that the concentration of vWF in blood is raised in response to epinephrine, which is released, for example, during physical exercise.46–48 This rise in vWF levels in the circulation is also the result of exocytosis of WPBs; however, the gradual epinephrine-induced WPB release is markedly different compared with the rapid thrombin-induced release.49 This difference in release pattern and the fact that different stimuli, such as thrombin and epinephrine, induce vWF release via distinct mechanisms16,45,49 enables the cell to regulate the exocytosis of WPBs, and possibly the release of specific WPB-constituents, in such a way that it meets the physiological requirements induced by a certain trigger. Our observation that on epinephrine stimulation only a part of the WPBs undergo exocytosis is supported by Vischer et al,30 who have reported that on stimulation with epinephrine, only the peripheral WPBs are exocytosed, whereas on thrombin stimulation, peripheral, as well as central, WPBs are released. The observed, cAMP-induced transport of a subset of WPBs to the MTOC could be a mechanism by which the cell reduces the amounts of vWF, P-selectin, or other bioactive compounds released on stimulation with cAMP-raising agonists. It is tempting to speculate that a distinct subset of WPBs, carrying a specific marker, is redirected to the MTOC after incubation with cAMP-raising agonists. Alternatively, intracellular localization and/or maturation state might determine whether WPBs are exocytosed or transported to the MTOC. In view of the physiological relevance of differential WPB exocytosis in response to different stimuli, it will be interesting to identify and characterize the possible markers for distinct WPB subsets in future research.


*    Acknowledgments
 
This study was supported by grants from the Netherlands Heart Foundation (grants 2000.097 and 2002B187) and the Netherlands Thrombosis Foundation (grant 2001.1).

Received May 23, 2005; accepted October 5, 2005.


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 *References
 

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