Endothelial cell confluence regulates Weibel-Palade body formation

Howell, Gareth; Herbert, Shane P.; Smith, Jennifer; Mittar, Shweta; Ewan, Lorna C.; Mohammed, Mudassir; Hunter, Alison R.; Simpson, Nigel; Turner, Anthony J.; Zachary, Ian; Walker, John H.; Ponnambalam, Sreenivasan

doi:10.1080/09687860400011571

Cited by 42 publications

(59 citation statements)

References 26 publications

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“…Thus, it is possible that the overall role of Rab27 in secretion depends on the ratio of mature versus immature granules. This ratio is known to be severely affected by cell density (Howell et al, 2004), and possibly by other factors, such as cellular activation, proliferation state, migration and others. Differences in these conditions, due to different transfection protocols and culturing methods, may account for the apparent differences between our study and the previous report.…”

Section: Discussionmentioning

confidence: 99%

Rab-genome analysis reveals novel insights in Weibel-Palade body exocytosis

Zografou

Basagiannis

Papafotika

et al. 2012

Journal of Cell Science

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SummaryWeibel-Palade bodies (WPBs) are endothelial-cell-specific organelles that, upon fusion with the plasma membrane, release cargo molecules that are essential in blood vessel abnormalities, such as thrombosis and inflammation, as well as in angiogenesis. Despite the importance of WPBs, the basic mechanisms that mediate their secretion are only poorly understood. Rab GTPases play fundamental role in the trafficking of intracellular organelles. Yet, the only known WPB-associated Rabs are Rab27a and Rab3d. To determine the full spectrum of WPB-associated Rabs we performed a complete Rab screening by analysing the localisation of all Rabs in WPBs and their involvement in the secretory process in endothelial cells. Apart from Rab3 and Rab27, we identified three additional Rabs, Rab15 (a previously reported endocytic Rab), Rab33 and Rab37, on the WPB limiting membrane. A knockdown approach using siRNAs showed that among these five WPB Rabs only Rab3, Rab27 and Rab15 are required for exocytosis. Intriguingly, we found that Rab15 cooperates with Rab27a in WPB secretion. Furthermore, a specific effector of Rab27, Munc13-4, appears to be also an effector of Rab15 and is required for WPB exocytosis. These data indicate that WPB secretion requires the coordinated function of a specific group of Rabs and that, among them, Rab27a and Rab15, as well as their effector Munc13-4, cooperate to drive exocytosis.

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Section: Discussionmentioning

confidence: 99%

Rab-genome analysis reveals novel insights in Weibel-Palade body exocytosis

Zografou

Basagiannis

Papafotika

et al. 2012

Journal of Cell Science

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“…To test this hypothesis, we took advantage of the fact that VWF content and WPB numbers in HUVECs increase with time in culture. 32 We determined how cellular levels of the WPB-specific marker Proregion and of Rab27A change with time in culture. HUVEC numbers and total protein content increased with time, reaching a plateau by 96 hours (supplemental Figure 6B).…”

Section: Analysis Of Slp4-a Myrip and Wpb-rab Function In Wpb Exocymentioning

confidence: 99%

“…To test this hypothesis, we took advantage of the fact that VWF content and WPB numbers in HUVECs increase with time in culture. 32 We determined how cellular levels of the WPB-specific marker Proregion and of Rab27A change with time Quantification of mRNA for Slp4-a (Ai) and MyRIP (Bi) after nucleofection with either control (siCTRL) or specific KD oligos as indicated. mRNA was depleted by 65% Ϯ 3% (Ai) or 67% Ϯ 3% (Bi) in KD compared with control.…”

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confidence: 99%

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis

Bierings

Hellen

Kiskin

et al. 2012

Blood

166

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Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/ Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/ Rab27A, or overexpression of EGFPSlp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis. (Blood. 2012;120(13):2757-2767) IntroductionHormone-evoked VWF secretion from endothelial cells (ECs) is mediated by exocytosis of specialized secretory granules (SGs) called Weibel-Palade bodies (WPBs). 1 WPB exocytosis is triggered by increases in intracellular free Ca 2ϩ or cAMP concentrations, and involves a number of molecular components, including the Nethylmaleimide-sensitive factor, VAMP3, SNAP23, syntaxin 4, RalA, the annexin A2/S100A10 complex, and phospholipase D. [2][3][4][5][6][7] In addition, Rab proteins also regulate WPB exocytosis. A subset of Rab proteins, including Rab3A-3D, Rab27A/B, and Rab37, is associated with SGs in different cell types where they regulate SG biogenesis, trafficking, and exocytosis. 8 Secretory cells often express a mixture of these "secretory" Rabs, which may have overlapping or distinct functions. Human ECs are reported to express mRNA for Rab3A, Rab3D, and Rab37,3,9,10 Rab3B protein, 11 and Rab27A mRNA and protein. 12,13 To date, Rab27A is the only endogenous EC Rab protein that has been detected on WPBs. Through its effector MyRIP and Myosin Va, Rab27A is proposed to negatively regulate WPB exocytosis. 13,14 Rab27A can interact with different effector molecules, and many secretory cells express a mixture of these effectors. 8 In these cases, SG exocytosis probably depends on the balance of Rab27A interactions with the complement of Rab effectors in the cell.In addition to MyRIP, ECs contain mRNA for the Rab27A effector Slp4-a (granuphilin). 13 Slp4-a links SGs to the plasma membrane (PM) through SG-associated Rab proteins (principally Rab27A), PM-associated syntaxins (1a, 2, or 3) and soluble Munc18 isoforms. [15][16][17][18][19] Syntaxins exist in open and closed conformations that determine their participation in SNARE complex f...

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“…Cell Culture and Materials-Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords as previously described (22,23). Human dermal microvascular endothelial cells were purchased from PromoCell.…”

Section: Methodsmentioning

confidence: 99%

The Confluence-dependent Interaction of Cytosolic Phospholipase A2-α with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation

Herbert

Odell

Ponnambalam

et al. 2007

Journal of Biological Chemistry

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The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A 2 -␣ (cPLA 2 ␣) cleaves phospholipids in a Ca 2؉ -dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca 2؉-independent association of cPLA 2 ␣ with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA 2 ␣ and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA 2 ␣ is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA 2 ␣ and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA 2 ␣ activity and endothelial cell prostaglandin generation.

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Endothelial cell confluence regulates Weibel-Palade body formation

Cited by 42 publications

References 26 publications

Rab-genome analysis reveals novel insights in Weibel-Palade body exocytosis

Rab-genome analysis reveals novel insights in Weibel-Palade body exocytosis

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis

The Confluence-dependent Interaction of Cytosolic Phospholipase A2-α with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation

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