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

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posted on 21.02.2013 by Claude Lalou, Nathalie Scamuffa, Samia Mourah, Francois Plassa, Marie-Pierre Podgorniak, Nadem Soufir, Nicolas Dumaz, Fabien Calvo, Nicole Basset-Seguin, Abdel-Majid Khatib

(A) 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) Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for Furin, PACE4, PC5, PC7 as described in Materials and Methods. 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 (C) and proIGF-IR (D) 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. (E) 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. (F) 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<0.0001.