QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 11 March 2008
doi: 10.1242/jcs.020289

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by van Helden, S. F. G. Articles by van Leeuwen, F. N. Search for Related Content PubMed PubMed Citation Articles by van Helden, S. F. G. Articles by van Leeuwen, F. N. Social Bookmarking                         What's this?

PGE₂-mediated podosome loss in dendritic cells is dependent on actomyosin contraction downstream of the RhoA–Rho-kinase axis

¹ Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands
² Laboratory of Pediatric Oncology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands

View larger version (62K): [in this window] [in a new window]   Fig. 1. PGE2 stimulation mediated by EP2 and EP4 receptors or by cAMP elevation leads to podosome dissolution in DCs. (A-D) PGE2 stimulation of iDCs leads to podosome loss and the appearance of FAs. (A,B) iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE2 for 5 minutes (min) or 16 hours (h) and stained with an anti-vinculin mAb (green) and phalloidin-Texas Red (to detect F-actin, red). (A) Representative images are depicted. (B) The number of cells displaying podosomes or FAs was counted in seven images per condition per experiment and an average (with s.e.m.) of six experiments is shown. (C) iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE2 for 5 minutes and stained with an anti-zyxin antibody (green) and phalloidin-Texas Red (to detect F-actin, red). (D) iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE2 for 1-5 minutes and stained with an anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was determined in seven images per condition per experiment and an average (with s.e.m.) of four experiments is shown. Asterisks indicate significant differences (P<0.05).

View larger version (91K): [in this window] [in a new window]   Fig. 2. Podosome loss and FA formation in DCs upon PGE2 stimulation, visualized with IRM. Also see Movies 1, 2 and Fig. S1A,B in the supplementary material. (A) IRM images compared to fluorescent images (detected with anti-vinculin mAb, GaM-ATTO633 and phalloidin-Texas Red, to detect F-actin) identify adhesion structures as podosomes (seen as dark dots with IRM and as actin dots surrounded by vinculin rings in the fluorescent images) and FAs (seen as dark stripes with IRM and as vinculin stripes in the fluorescent images). (B) Images from an IRM movie (Movie 1 in the supplementary material) showing podosomes before stimulation (–1 min) and loss of podosomes in response to PGE2. Note how the dissolution of podosomes is (transiently) preceded by the formation of bright rings surrounding the podosome core. FA formation in response to stimulation can be observed in the upper-right part of the images. (C) Overlay of the fluorescence intensity of the vinculin staining (broken line) and the actin staining (solid line) from straight-line selections with ImageJ through the middle of the podosome. The actin peak flanked by two vinculin peaks reflects the actin core surrounded by the vinculin-containing ring. (D-F) The size of the adhesive contacts observed by live-imaging IRM, the actin core and the vinculin ring were determined in 50 podosomes from ten different cells by making straight-line selections with ImageJ through the middle of the podosome. A representative curve of one podosome is shown and the average size is depicted in the upper-right corner of the curves with errors depicted as s.e.m. (D) The fluorescence intensity of the actin staining from a straight-line selection through the middle of a podosome. The width of the peak at half the height was used to determine the width of the actin core (0.78±0.01 µm). (E) The fluorescence intensity of the vinculin staining from a straight-line selection through the middle of a podosome. The distance from one peak to the other peak was measured to obtain the size of the vinculin ring (0.91±0.02 µm). (F) The light intensity in the IRM image from a straight-line selection through the middle of a podosome. The width of the peak at half the height was used to determine the width of the adhesive contact (0.75±0.02 µm).

View larger version (29K): [in this window] [in a new window]   Fig. 3. PGE2-induced podosome loss is mediated by the EP2 and EP4 receptors. (A) iDCs and mDCs express the EP2 and EP4 receptors. On iDCs and mDCs, an RT-PCR for the PGE2 receptors EP1-EP4 was performed. Controls include β-actin (actin) and minus RT (–). A specific product is detected for EP2 and EP4 but not for EP1 and EP3 receptors. (B) EP2 and EP4 antagonists block PGE2-induced podosome dissolution. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE2 in the presence or absence of AH6809 (5 µM for 30 minutes) or AH23848 (50 µM for 30 minutes), or with AH6809 or AH23848 alone, and stained with anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was counted in seven images per condition per experiment and an average (with s.e.m.) of four experiments is shown. (C) EP2 and EP4 agonists and cAMP elevation mimic PGE2 in inducing podosome dissolution. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE2, butaprost (buta, 2 µM for 15 minutes), PGE1-OH (5 µg/ml for 30 minutes), sulprostone (sulp, 2 µM for 15 minutes), IBMX (10 µM for 15 minutes) or IBMX with forskolin (IBMX/fors, 10 µM for 15 minutes), and stained for vinculin and F-actin. The number of cells displaying podosomes was counted in seven images per condition per experiment and an average (with s.e.m.) of three experiments for the EP agonists (buta, PGE1-OH and sulp) and seven experiments for cAMP elevation (IBMX and fors) is shown. Asterisks indicate significant differences (P<0.05).

View larger version (64K): [in this window] [in a new window]   Fig. 4. Myosin II is enriched in podosomes and myosin II function is needed for PGE2-induced podosome dissolution. (A) Myosin IIA is the predominant isoform in DCs. Cell lysates from iDCs and N1E-115 cells (N115), as a positive control that expresses all isoforms, were analyzed for expression of myosin (myo) IIA, IIB and IIC. (B) Myosin IIA is enriched in the rings of podosomes, whereas myosin IIB is nearly undetectable. iDCs were plated on FN-coated coverslips and subsequently stained with an anti-myosin-IIA or an anti-myosin-IIB antibody (green) and phalloidin-Texas Red (to detect F-actin, red). Representative images are depicted. (C) Inhibition of myosin II function blocks PGE2-induced podosome dissolution. iDCs were plated on FN-coated coverslips and left untreated or were stimulated with PGE2 for 5 minutes in the presence or absence of blebbistatin (bleb, 10 µM for 30 minutes), or were treated with blebbistatin alone, and stained with anti-vinculin antibodies and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was counted in seven images per condition per experiment and an average (with s.e.m.) of three experiments is shown. (D) Inhibition of myosin II function blocks PGE2-induced myosin IIA redistribution. iDCs were plated on FN-coated coverslips and left untreated or were stimulated with PGE2 for 5 minutes in the presence or absence of blebbistatin (bleb, 10 µM for 30 minutes), or were treated with blebbistatin alone, and were stained with anti-myosin IIA antibodies and phalloidin-Texas Red (to detect F-actin). Scale bars: 20 µm (A), 10 µm (B). Asterisks indicate significant differences (P<0.05).

View larger version (25K): [in this window] [in a new window]   Fig. 5. HL-60 cells form podosomes upon PMA stimulation and respond to PGE2. (A,B) HL-60 cells form podosomes upon stimulation with PMA, which dissolve after stimulation with PGE2 or an EP4 agonist. HL-60 cells seeded on FN-coated coverslips were left untreated or were stimulated with PMA for 24 hours and then left untreated or were further stimulated with PGE2 for 5 minutes. Cells were then stained with an anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). (A) Representative images are depicted. (B) The number of cells displaying podosomes was counted in seven images per condition per experiment and an average (with s.e.m.) of three experiments is shown. (C) HL-60 cells show expression of EP2 and EP4, whereas EP1 and EP3 receptors are nearly undetectable, as determined by RT-PCR. Asterisks indicate significant differences (P<0.05).

View larger version (40K): [in this window] [in a new window]   Fig. 6. PGE2-receptor stimulation leads to activation of RhoA and inactivation of Rac1 and Cdc42. (A) Activation of the small GTPase RhoA in response to PGE2. GTP-RhoA levels were determined in lysates (pd, pull down; TL, total lysate) of HL-60 cells using a Rhotekin-GST pull-down assay. In cells treated with PGE2 for 5 minutes, Rho-GTP levels showed a 2.6±0.9 (s.d.)-fold increase relative to untreated cells (n=7). (B) Loss of Rac1 GTPase activity in response to PGE2. Rac1-GTP levels were determined in lysates of HL-60 cells using a biotinylated Pak1 peptide. In cells stimulated with PGE2, Rac1 activity was decreased 0.5±0.2 (s.d.)-fold relative to untreated cells (n=6). (C) Loss of Cdc42 GTPase activity in response to PGE2. Cdc42-GTP levels were determined in lysates of HL-60 cells using a biotinylated Pak1 peptide. In cells stimulated with PGE2, Cdc42 activity was decreased 0.7±0.2 (s.d.)-fold relative to untreated cells (n=6). (D,E) Activation of the small GTPase RhoA in response to PGE1-OH or 8-Bromo-cAMP. GTP-RhoA levels were determined in lysates of HL-60 cells using a Rhotekin-GST pull-down assay. (D) In cells treated for 5 minutes with an EP4 agonist (PGE1-OH), Rho-GTP levels showed a 1.6±0.5 (s.d.)-fold increase relative to untreated cells (n=8). (E) In cells treated for 5 minutes with 8-Bromo-cAMP (8Bromo, 200 µM), which elevates cAMP levels, Rho-GTP levels showed a 2.5±0.9 (s.d.)-fold increase relative to untreated cells (n=5). Asterisks indicate significant differences (P<0.05).

View larger version (54K): [in this window] [in a new window]   Fig. 7. Podosome loss induced by PGE2, EP agonists and elevated cAMP is mediated by Rho kinase. (A) Rho-kinase inhibition blocks PGE2- and EP-agonist-induced podosome dissolution. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with PGE2 (for 5 minutes), butaprost (buta, 2 µM for 15 minutes) or PGE1-OH (5 µg/ml for 30 minutes), in the presence or absence of Y27632 (20 µM for 1 hour), or were treated with Y27632 alone, and stained with anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was determined in seven images per condition per experiment and an average (with s.e.m.) of three experiments is shown. (B) IBMX and IBMX/forskolin-induced podosome dissolution is efficiently blocked by Y27632. iDCs seeded on FN-coated coverslips were left untreated or were stimulated with IBMX (10 µM for 5 minutes) or IBMX with forskolin (IBMX/fors, both 10 µM for 5 minutes) in the presence or absence of Y27632 (20 µM for 1 hour), or with Y27632 alone, and stained with anti-vinculin mAb and phalloidin-Texas Red (to detect F-actin). The number of cells displaying podosomes was counted in seven images per condition per experiment and an average (with s.e.m.) of three experiments is shown. Asterisks indicate significant differences (P<0.05). (C) Rho-kinase inhibition blocks podosome loss in response to PGE2, EP2/EP4 agonists or cAMP elevation. Representative images are depicted.

View larger version (31K): [in this window] [in a new window]   Fig. 8. Regulation of adhesion dynamics in dendritic cells by PGE2. PGE2 signalling, mediated by EP2 and EP4 receptors, leads to elevation of the level of cAMP, activation of RhoA and inactivation of Rac1. PGE2-mediated activation of RhoA promotes Rho-kinase activity and, subsequently, myosin-II-dependent contraction, leading to podosome dissolution and FA formation. By contrast, pathways leading to activation of Rac1 are known to promote podosome formation and induce cytoskeletal relaxation accompanied by a loss of FAs.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter