spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 4 March 2008
doi: 10.1242/jcs.020693

Journal of Cell Science 121, 989-1001 (2008)
Published by The Company of Biologists 2008
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koh, W.
Right arrow Articles by Davis, G. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koh, W.
Right arrow Articles by Davis, G. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling

Wonshill Koh1, Rachel D. Mahan1 and George E. Davis1,2,*

1 Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65212, USA
2 Department of Pathology and Anatomical Sciences, School of Medicine, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65212, USA


Figure 1
View larger version (91K):
[in this window]
[in a new window]

  		Fig. 1. Cdc42 and Rac1 are required for EC tubular morphogenesis and invasion in 3D collagen matrices. (A) ECs transfected with the indicated siRNAs were suspended within collagen matrices for 48 hours (vasculogenesis assay). The culture media contained vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2) and phorbol ester (TPA), as described previously (Davis and Camarillo, 1996Go). Arrows indicate EC tubular networks with a lumenal structure. (B) ECs transfected with the indicated siRNAs were seeded onto collagen matrices (angiogenesis assay) and stimulated to invade for 48 hours in response to 200 ng/ml SDF-1{alpha}, as described previously (Saunders et al., 2006Go). Arrows indicate the invading EC sprouts and tube formation; arrowheads indicate ECs on the monolayer. Bars, 100 µm.

 

Figure 2
View larger version (161K):
[in this window]
[in a new window]

  		Fig. 2. Time-lapse images of ECs treated with individual siRNAs against Cdc42 and luciferase in a 3D collagen gel. Arrows indicate EC tubular structures with lumenal compartments. Bars, 50 µm.

 

Figure 3
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 3. siRNA suppression of Cdc42 or Rac1 markedly blocks EC lumen and tube formation as well as EC invasion as Cdc42 and Rac1 are highly activated during EC tube and lumen formation in 3D collagen matrices. (A-C) ECs were treated with the indicated siRNAs and were suspended within collagen matrices (A) or on the surface of collagen matrices containing either 1 µM S1P (B) or 200 ng/ml SDF-1{alpha} (C). Quantification occurred at either 48 hours (A,C) or 24 hours (B). Data are shown as the mean number of EC lumens per high-power field (HPF) (A) or invading ECs per HPF (B,C) ±s.d. (n=6). *, P<0.01 compared with luciferase control. (D) siRNA-transfected EC lysates were prepared for western blot analysis and probed for Cdc42, Rac1, RhoA or beta-actin (control). (E) ECs were suspended in collagen matrices and extracts were prepared at the indicated time-points. Equal amounts of extracts were incubated with GST-PAK-PBD protein agarose beads or control blank beads. Eluates and starting extracts were analyzed by western blot using antibodies against Cdc42 or Rac1, and levels of activated Cdc42 or Rac1 were compared with their total levels. (F) Relative levels of activated Cdc42 or Rac1 and total Cdc42 or Rac1 were quantified by using Scion imaging software. The data are expressed in arbitrary units (AU)±s.d. (n=3).

 

Figure 4
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 4. Identification of Cdc42 and Rac1 effectors regulating EC tubular morphogenesis and invasion. (A-C) ECs were treated with the indicated siRNAs and were suspended within collagen matrices (A) or on the surface of collagen matrices containing either S1P (B) or SDF-1{alpha} (C). Quantification occurred at either 48 hours (A,C) or 24 hours (B). The data are shown as the mean number of EC lumens per HPF (A) or invading ECs per HPF (B,C) ±s.d. (n=5). *, P<0.01 compared with luciferase control.

 

Figure 5
View larger version (88K):
[in this window]
[in a new window]

  		Fig. 5. siRNA suppression of Pak2 or Pak4 markedly blocks EC lumen and tube formation as well as EC invasion in 3D collagen matrices. (A) ECs transfected with the indicated siRNAs were suspended within collagen matrices for 48 hours before fixation for photography. Bar, 100 µm. (B) ECs transfected with the indicated siRNAs were allowed to invade for 24 hours in response to S1P before fixation for photography. Bar, 100 µm. (C) siRNA-transfected EC lysates were prepared for western blot analysis and probed for Pak2, Pak4 or beta-actin (control).

 

Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 6. Expression of dominant-negative (DN) Pak2 or Pak4 blocks EC lumen formation in 3D collagen matrices. ECs infected with GFP, Pak2 or Pak4 DN mutant adenoviruses (Ad) were suspended within 3D collagen matrices for 24 hours. (A) Graph showing the mean number of EC lumens per HPF±s.d. (n=3). *, P<0.03 compared with GFP control. (B) Representative fields of ECs infected with the indicated adenoviruses. Arrows indicate EC lumen formation. Bar, 50 µm.

 

Figure 7
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 7. Pak2 and Pak4 are strongly activated during EC lumen and tube formation. ECs were suspended in collagen matrices and were allowed to undergo EC tube morphogenesis for 48 hours for time-lapse analysis or western blot analysis. (A) The areas of EC lumens (>10 lumens/field) were traced using Metamorph software from digital images of the indicated time-points and plotted (from two representative movies). (B) Extracts of EC cultures in 3D collagen matrices were prepared at the indicated time-points and probed for phospho-Pak2, Pak2, phospho-Pak4 and Pak4. Beta-actin was used as a loading control. (C) The relative levels of phosphorylated Pak2 or Pak4 and total Pak2 or Pak4 were quantified by using Scion imaging software. The data are expressed in arbitrary units (AU)±s.d. (n=2).

 

Figure 8
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 8. Activation of Cdc42 and its association with downstream targets during EC tube morphogenesis in 3D collagen matrices. ECs were treated with S-GFP-Cdc42 or control GFP-Cdc42 recombinant adenovirus (Ad) before suspension in 3D collagen matrices. Extracts at the indicated time-points were prepared. Equal amounts of extracts were incubated with S-protein agarose beads and probed with antibodies to phospho-Pak2, phospho-Pak4 and Par3 to detect binding interactions.

 

Figure 9
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 9. Par3 and Par6 regulate EC tube and lumen formation in 3D collagen matrices. (A) ECs transfected with the indicated siRNAs were suspended within collagen matrices for 24 hours before fixation for photography. Bar, 50 µm. (B) Quantification of EC vasculogenesis assay at 24 hours, at which point the areas of EC lumens were traced using Metamorph software. The data are shown as the mean EC lumenal area±s.d. (n=3). *, P<0.03 compared with luciferase control. (C) siRNA-transfected ECs lysates were prepared for western blot analysis and probed for Par3 or beta-actin (control). (D) siRNA-transfected ECs were prepared for RNA isolation. Semi-quantitative RT-PCR was performed for Par6a, Par6b or G3PDH-1 (control).

 

Figure 10
View larger version (58K):
[in this window]
[in a new window]

  		Fig. 10. Phorbol ester (TPA) strongly stimulates EC lumen formation in 3D collagen matrices. ECs were suspended in collagen matrices with or without TPA for 24 hours. (A,B) Graphs show the mean number of EC lumens per HPF (A) or the mean EC lumenal area (Metamorph software) (B) ±s.d. in the absence or presence of TPA (n=3). *, P<0.05 compared with TPA. (C) Sequential time-lapse images of the EC fields at the indicated time-points in the presence of TPA. Arrowheads indicate EC vacuoles; arrows indicate EC tubular structures with lumens. (D) Sequential time-lapse images of the EC fields at the indicated time-points in the absence of TPA. Arrowheads indicate EC vacuoles, arrows indicate EC cord-like structures without lumens. Bars, 100 µm.

 

Figure 11
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 11. TPA promotes EC tube morphogenesis through PKC. ECs were suspended in collagen matrices for 24 hours in the absence and presence of PKC inhibitors GF109203X (2.5 µM), Ro-32-0432 (5 µM), Go6983 (5 µM) or Go6976 (5 µM). (A) Quantification of the mean number of EC lumens per HPF±s.d. (n=2). *, P<0.08 compared with the TPA control. (B) Tracing of the mean EC lumenal area (Metamorph software)±s.d (n=2). *, P<0.05 compared with control TPA.

 

Figure 12
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 12. Pak2 and Pak4 are phosphorylated downstream of PKC activation. (A,B) Extracts were prepared at 24 hours from ECs suspended in collagen matrices for 24 hrs in the absence and presence of TPA (A) or GF109203X (2.5 µM), Ro-32-0432 (5 µM), Go6983 (5 µM) or Go6976 (5 µM) (B) for western blot analysis and probed for phospho-Pak2, phospho-Pak4 or beta-actin (control).

 

Figure 13
View larger version (71K):
[in this window]
[in a new window]

  		Fig. 13. siRNA-mediated suppression of PKC{epsilon} or PKC{zeta} inhibits EC tube morphogenesis. ECs were treated with the indicated siRNAs and were suspended in 3D collagen matrices for 24 hours before fixation for photography and quantification. (A) Representative fields of ECs treated with the indicated siRNAs. Bar, 50 µm. (B,C) Quantification of EC vasculogenesis assay at 24 hours. The data are shown as the mean number of EC lumens per HPF (B) or the mean EC lumenal area (Metamorph software) (C) ±s.d. (n=3). *, P<0.05 compared with the luciferase control. (D) EC lysates were prepared for western blot analysis and probed with antibodies against the indicated PKC isoforms or actin (control).

 

Figure 14
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 14. Schematic diagram illustrating Cdc42/Rac1- and PKC-mediated EC lumen and tube formation signaling pathways in 3D collagen matrices. There is an activation of Cdc42 and Rac1 in response to an interaction between collagen matrices and {alpha}2β1 integrins, which leads to the recruitment of the Par3-Par6b-PKC{zeta} polarity complex that regulates EC lumen and tube formation. Cdc42 and Rac1 also promote the phosphorylation of Pak2 and Pak4, which serve as downstream targets of PKC{epsilon} in response to TPA to stimulate EC lumen and tube formation in 3D collagen matrices.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2008