Furin, PACE4, PC5 and PC7 expression and activity in human primary melanoma cells.

<p>(<b>A</b>) Expression of the indicated PCs was analyzed in M10 cells using specific primers for the PCs found in the secretory pathway (Furin, PACE4, PC5, PC7) and reverse transcription-PCR analysis assay. Note that all these PCs are expressed in the M10 cells. (<b>B</b>) Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for Furin, PACE4, PC5, PC7 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. During PCR, the transcription of β2-microglobulin that was evaluated in each sample was used as endogenous control. Results shown in the bar graph are expressed as ratio of PCs mRNA transcripts (M10)/(M10/PDX) deduced from values derived from M10 and M10/PDX cells mRNA analysis. Data are shown as means ± S.E of three experiments performed in duplicate. Real-time PCR analysis revealed that expression of α1-PDX in M10 cells did not affect significantly the expression levels of these PCs in M10 cells. Processing of proPDGF-A (<b>C</b>) and proIGF-IR (<b>D</b>) analyzed by Western blotting revealed that expression of α1-PDX in M10 cells (M10/PDX) completely inhibited the processing of pro-IGF-1R and proPDGF-A. (<b>E</b>) PCs activity in M10 cells and M10/PDX cells was assessed by evaluating the cell extracts for their ability to digest the universal PCs substrate, the fluorogenic peptide pERTKR-MCA at the indicated time points. Expression of α1-PDX in M10 cells reduced their PCs activity. (<b>F</b>) Results shown in the bar graph represent the PCs activity at 2 hours of incubation of the indicated tumor cells. Results are representative of three experiments and data are mean ± S.E performed in triplicate. ***p<i><</i>0.0001.</p

Schematic representation of the effects of PCs inhibition on migration and invasion of human melanoma cells with altered <i>CDKN2A</i>, <i>p53</i> and <i>Ras</i> genes.

<p>The <i>CDKN2A</i> locus codes for the tumor suppressors p16 and ARF that regulate cell cycle progression, DNA repair and cell invasion via activating pRb, and the p53 pathway via inhibiting the activity of MDM2, respectively. Mutations or deletions of the <i>CDKN2A</i> gene result in altered MMPs/TIMPs and/or uPA/uPAR/PAI-1 expression and activity, which in turn lead to tumor cell invasion. The <i>Ras</i> gene is important for regulating the ERK activity, and hence expression of MMPs and uPA/uPAR. An activating mutation in the Ras gene can result in uncontrolled activity of MMPs and the urokinase system, which can lead to increased melanoma invasion. Blockade of PCs activity can reduce the expression and/or generation of active MMPs, uPA and uPAR leading to reduced tumor cells invasiveness. The asterisks indicate altered genes.</p

Effect of PCs inhibition on uPA, uPAR and PAI-1 expression.

<p>(<b>A–C</b>), Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for indicated genes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. Results are shown in the bar graph and are expressed as the mRNA transcript ratios. Data are shown as means ± S.E of three experiments performed in duplicate. Student's <i>t</i> test was used for statistical analysis. *, <i>P</i><0.05 and ***, <i>P</i><0.001. (<b>D</b>), Tumor cells were incubated at 37°C in serum-free media. After 24 hours, media were collected and analyzed for the presence of uPAR using ELISA kit. Results shown are representative of 3 experiments. Data are mean ± SEM (<i>n = </i>6 per group). Student's <i>t</i> test was used for statistical analysis ***, <i>P</i><0.001</p

Expression of PC5 during fin regeneration.

<p>(A) Total RNA was isolated from fins (15-20 fins per time point) and analyzed by real-time PCR using specific primers for zebrafish PC5 or β-actin. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to control (0 dpa). Results are shown as means ± S.E. of three experiments performed in triplicate. (B) Immunofluorescence analysis revealed that PC5 is expressed in all area of the regenerating fin and low signal was observed on the vessels (red signal, 25× objective).</p

Uprocessed ProVEGF-C inhibits fin regeneration.

<p>(A) 24 h prior amputation of caudal fins (6 per group), empty vector (Control) or vector containing wild-type (wt) or mutant (mut) proVEGF-C constructs were injected in the fins and animals were allowed to regenerate at 28.5°C after fins amputation for 5 days. The microinjection of vector containing wild-type proVEGF-C had no effect on normal regeneration. In contrast, injection of the mutant proVEGF-C resulted in severe inhibition of fin regeneration. Results are representative of tree experiments. The corresponding percentages of regenerated area were deduced from the ratio of 100 x (fin surface regenerated in VEGF-Cwt)/(fin surface regenerated in Control) and 100 x (fin surface regenerated in VEGF-Cmut/fin surface regenerated in Control). (B) Total RNA derived from uncut fins was subjected to real-time PCR analysis using specific primers for the VEGF-C receptors R2, R3 or β-actin. During PCR, the transcription of β-actin that was evaluated in each sample was used as endogenous control. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to R2 transcript assigned to 100%. Results are shown as means ± S.E. of three experiments performed in duplicate.</p

Processing of zebrafish proVEGF-C.

<p>(A) Processing of proVEGF-C was analyzed by Western blotting in zebrafish ZF4 cells transiently cotransfected with pSecTagB (Myc tag) vector containing the zebrafish proVEGF-C cDNA and the empty pIRES2-EGFP vector (Control) or pSecTagB expressing proVEGF-C and pIRES2-EGFP containing the PCs inhibitors; ppFurin or α1-PDX. Results of band intensities are shown in the bar graph that were deduced from the ratio of VEGF-C/(pro-VEGF-C + VEGF-C). Results are representative of three experiments. (B) The processing of proVEGF-C was analyzed by Western blotting using anti-Myc antibody on media derived from PCs-deficient LoVo cells transiently cotransfected with either pIRES2-EGFP vector alone and pSecTagB (Myc tag) containing the zebrafish VEGF-C cDNA (Control) or pIRES2-EGFP vector containing Furin, PACE4, PC5 or PC7 cDNA. The corresponding percentages of band intensities were deduced from the ratio of VEGF-C/(proVEGF-C+VEGF-C). Results are representative of three experiments.</p

Effect of proVEGF-C processing on ZF4 cells proliferation.

<p>(A) ZF4 cells were serum deprived overnight and then treated for 24 h with media derived from LoVo cells transiently cotransfected with empty vectors (Control) or empty vector and vector containing proVEGF-C construct or vector expressing proVEGF-C cDNA and vector expressing Furin cDNA. Cell proliferation was assessed using Cell Titer96 non-radioactive cell proliferation assay. Results are shown as means ± S.E. of three experiments performed in triplicate. (B) Total RNA derived from ZF4 cells was subjected to real-time PCR analysis using specific primers for the zebrafish VEGF-C receptors R2, R3 or β-actin. During PCR, the transcription of β-actin that was evaluated in each sample was used as endogenous control. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to R2 transcript assigned to 100%. Results are shown as means ± S.E. of three experiments performed in duplicate.</p

PCs expression and activity in ZF4 cells.

<p>(A) Following total RNA extraction from 10⁴ x ZF4 cells, real-time PCR analysis was performed using specific primers for Furin, PC5 or β-actin zebrafish as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011438#s2" target="_blank">Material and Methods</a>. During PCR, the transcription of β-actin that was evaluated in each sample was used as endogenous control. Results are shown in the bar graph and are expressed as the percentage of the indicated transcripts relative to Furin transcript (100%). Data are shown as means ± S.E of three experiments performed in duplicate. (B) PCs activity in ZF4 cells was assessed by evaluating the cells protein extract ability to digest the universal PCs substrate, the fluorogenic peptide pERTKR-MCA at the indicated time periods. Digestion of pERTKR-MCA by recombinant Furin (2 unit/µl) is given for comparison. As can be seen, the PCs inhibitor peptidyl chloromethyl ketones (CMK) (10 µM) reduced dramatically the PCs activity in ZF4 cells and the activity of recombinant Furin. Results are representative of two experiments performed in triplicate and data are mean ± S.E. *p<0.005; **p<0.0001.</p