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First published online 31 January 2006
doi: 10.1242/jcs.02787

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A signalling cascade involving PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in response to phorbol ester

¹ Institut Européen de Chimie-Biologie, 2 rue Robert Escarpit, 33600 Pessac, France
² INSERM Unité 441 / Université Victor Segalen Bordeaux 2, Avenue du Haut-Lévêque, 33600 Pessac, France

View larger version (84K): [in a new window]   Fig. 1. Podosome formation in HUVECs and PAE cells. (A) V12Cdc42 induced podosomes in HUVECs. HUVECs were transfected with GFP-V12Cdc42 or GFP-V14RhoA. After 24 hours, F-actin was labelled with Alexa-Fluor-633-phalloidin (red) and vinculin with anti-vinculin and secondary Alexa Fluor 568 antibodies (green). For better visualisation, the colours of the images were modified in Photoshop. Only cells expressing the GFP-tagged constructs are shown. Bar, 10 µm. (B) v-Src expression induces podosomes in HUVECs and PAE cells. HUVECs and PAE cells were transfected with v-Src-encoding plasmid. On the next day, cells were fixed and stained for actin (red) and vinculin (green). Bar, 10 µm. (C) PMA induced actin remodelling in endothelial cells. HUVECs and PAE cells were treated with PMA for either 1 or 3 hours, fixed and stained with rhodamine-phalloidin to visualise F-actin. Arrows indicate individual podosomes and rosettes. Bars, 10 µm.

View larger version (68K): [in a new window]   Fig. 2. (A) PMA induced a time-dependent remodelling of actin in HUVECs. Cells were treated with PMA for over a 6-hour period and stained with rhodamine-phalloidin to visualise F-actin. Upon addition of PMA, stress fibres rapidly disappeared and were found greatly reduced at 30 minutes when F-actin dots became detectable. Podosomes remained visible up to 3 hours and then progressively disappeared. At 6 hours, podosomes were not detectable and stress fibres had returned. Bar, 10 µm. (B) PMA-induced individual podosomes and rosettes. HUVECs were treated with PMA for 1 hour. F-actin was labelled with rhodamine-phalloidin. High-magnification images show the presence of individual podosomes and clusters of podosomes defined as rosettes. Bar, 10 µm. (C) Quantitation of the experiment described in A. Cells showing either individual podosomes or rosettes were counted after treatment with PMA; data are presented as percentage of the total population. Error bars represent the mean ± s.d. of three independent experiments where approximately 200 cells per coverslip were counted.

View larger version (43K): [in a new window]   Fig. 3. PMA translocates PKCs to the plasma membrane. HUVEC protein extracts were made at indicated times after PMA addition and then fractionated. Membrane and cytosol fractions were analysed by western blotting with either anti-PKC, anti-Ser657PKC, anti-PKC or anti-Ser643PKC antibodies.

View larger version (41K): [in a new window]   Fig. 4. PMA-induced podosomes depend on PKC and PKC activation. (A) Both the isotype-restricted and the isotype-selective inhibitor prevented PMA-induced podosomes in HUVECs. Cells were pretreated with either 50 nM Gö6976 or 3 µM rottlerin for 1 hour and then activated with PMA for 1 hour in the presence of the inhibitor. Cells were labelled for F-actin. Bar, 10 µm. (B) Quantitation of the experiment described in A. Cells showing podosome-like structures were counted after 1 hour treatment with PMA. Error bars represents the mean ± s.d. of three independent experiments. (C) siRNA designed against PKC and PKC downregulated their respective target protein. HUVECs were transfected with PKC or PKC siRNA and protein extracts were analysed by western blot. (D) Knockdown of PKC or PKC expression inhibited PMA-induced podosome formation. HUVECs were transfected with siRNA designed against PKC or PKC. Cells showing podosomes after 1 hour of PMA treatment were counted and data are presented as percentage of the total population. Error bars represent the mean ± s.d. of three independent experiments.

View larger version (87K): [in a new window]   Fig. 5. Active PKC induces podosomes in HUVECs. (A) Cells were transfected with plasmids encoding GFP-PKCWT or GFP-PKCWT and F-actin was labelled with rhodamine-phalloidin. Bars, 10 µm. (B) The same experiment as in A with GFP-PKCA25E or GFP-PKCA147E constructs. Bars, 10 µm.

View larger version (59K): [in a new window]   Fig. 6. PMA-induced podosomes depend on RhoGTPases in HUVECs. (A) V12Cdc42-induced podosomes are insensitive to PKC inhibitors. HUVECs were transfected with a GFP-V12Cdc42 construct, and treated with Gö6976 and rottlerin. F-actin was labelled with rhodamine-phalloidin. Arrows indicate cells expressing the GFP-tagged constructs (visualised by the green GFP signal). The percentage of cells transfected with V12Cdc42 that show podosomes is indicated at the top of each image. Bars, 10 µm. (B) siRNA designed against RhoA, Rac1 or Cdc42 downregulated their respective target protein. HUVECs were transfected with the indicated siRNA. After 48 hours, protein extracts were analysed by western blot. (C) Knockdown of Cdc42 or RhoA expression inhibited PMA-induced podosome formation. HUVECs were transfected with indicated siRNA and analysed by immunofluorescence. Quantitation of the experiment was done by counting the number of cells showing podosomes after transfection and is presented as percentage of the control response obtained with cells transfected with control siRNA. Error bars represent the mean ± s.d. of two independent experiments.

View larger version (53K): [in a new window]   Fig. 7. PMA-induced podosomes depend on Src kinase in HUVECs. (A) PP2 inhibits PMA- but not V12Cdc42-induced podosomes. HUVECs were pretreated or not with 10 µM PP2 for 1 hour and then activated or not by PMA for 1 hour in the presence of the inhibitor. Alternatively, HUVECs were transfected with the GFP-V12Cdc42 construct and treated with PP2. Cells were labelled for F-actin. Bars, 10 µm. (B) Cdc42 is required for v-Src-induced podosome formation in PAE cells. PAE cells were co-transfected with v-Src-encoding plasmid together with GFP or GFP-N17Cdc42 constructs. GFP-positive cells showing podosomes were counted. Error bars represent the mean ± s.d. of three independent experiments. (C) v-Src activates Cdc42. PAE cells were transfected using a GFP-encoding (control) or a v-Src-encoding plasmid. Active GTPases were affinity-precipitated with GST-RBD-rhotekin or GST-CRIB-PAK and analysed by western blot using relevant antibodies. v-Src expression was visualised by western blot with anti-avian-Src antibodies. For each point, a fraction of the lysate was run to monitor the amount of GTPase before precipitation. (D) The graph shows quantitation of bands from two independent experiments; error bars represent the mean ± s.d.

View larger version (45K): [in a new window]   Fig. 8. Proteolytic activity associated with HUVEC podosomes. (A) ECM degradation is associated with v-Src- and PMA-induced podosomes. HUVECs were seeded on FITC-gelatin-coated coverslips. The cells were either transfected with v-Src-encoding or V12Cdc42-encoding plasmids or treated with PMA for 1 hour. Cells were fixed and stained for F-actin. Areas of degraded gelatin that overlap (arrows) or not (filled arrowheads) with podosomes are shown. Podosomes that do not colocalise with matrix degradation areas are also shown (opened arrowheads). Cell contours have been digitally rendered in the micrographs. (B) ECM degradation is inhibited by MMP inhibitors. The experiment described in A for PMA-treated cells was in the presence of 10 µM of GM6001 or 10 µM TSRI265. `Black holes' representative of gelatin degradation were quantified and are presented as percentage of the control response obtained with untreated cells. Error bars represent the mean ± s.d. of three independent experiments.

View larger version (47K): [in a new window]   Fig. 9. (A) MMP-2 is activated upon PMA treatment. Cells lysates and supernatants from HUVECs treated with PMA for indicated times were analysed by gelatin zymography. The active form of MMP-2 (62 kDa) became apparent, revealing the processing of pro-MMP-2. One representative experiment out of four is shown. (B) Localisation of MMP-2 and MT1-MMP to podosomes. HUVECs were treated with PMA for 1 hour, fixed and stained with anti-MMP-2 or anti-MT1-MMP antibodies and rhodamine-phalloidin to visualise MMP-2 or MT1-MMP (green) and F-actin (red), respectively. Bar, 10 µm.

View larger version (41K): [in a new window]   Fig. 10. MT1-MMP is essential for matrix degradation. (A) Inhibition of MT1-MMP expression by siRNA MT1-MMP was visualised by western blot. (B) siRNA MT1-MMP blocked MMP2 activation. Cells lysates from HUVECls treated with PMA in the presence or absence of siRNA MT1-MMP, were analysed by gelatin zymography. (C) MT1-MMP-expression knock-down increases the number of cells with podosomes. HUVECs were treated with PMA in the presence or absence of siRNA MT1-MMP. Cells showing podosomes were counted and the data are presented as percentage of the total population. Error bars represent the mean ± s.d. of two independent experiments.

View larger version (16K): [in a new window]   Fig. 11. Schematic representation of the positioning of PKCs, Src and Cdc42 in the signalling cascade leading to podosome assembly in HUVECs in response to PMA. Asterisks indicate podosome inducers in endothelial cells. PKC is involved in PMA-induced podosome formation in HUVECs but its exact role and position in the cascade remains to be elucidated.

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