Cancer du sein et micro-environnement tumoral: rôle de la protéase ...

pro-cath-D was analyzed in HMF fibroblasts in which endogenous LRP1 expressionhad or had not been silenced. As previously observed (23), labeled 52-kDa proenzymewas transformed into a 48-kDa intermediate, and a 34-kDa mature enzymeafter binding and internalization into cells (Fig. 4B, panel a). Moreover, as expected,mannose-6-phosphate, that neutralizes the cath-D primary receptor (e.g. M6Preceptors) (23), strongly reduced pro-cath-D internalization (Fig. 4B, panel a,compare lanes 1 and 3). Interestingly, in the presence of M6P, siRNA-mediatedinhibition of LRP1 expression (Fig. 4B, panel b) led to a significant reduction in theuptake of pro-cath-D independent of the M6P receptors (Fig. 4B, panel a, comparelanes 3 and 4; panel c for quantification). These findings strongly suggest that LRP1is a cath-D alternative endocytosis receptor. To support these results, additionalexperiments were performed using LRP1-/- MEF fibroblasts transfected or not with fulllengthLRP1. LRP1-dependent cath-D endocytosis was also observed in LRP1-/-fibroblasts transfected with LRP1 (MEF-B41) (Fig. 4C, panels a-b). Altogether, thesefindings indicate that pro-cath-D is partially endocytosed by LRP1 in fibroblasts. We,therefore, propose that LRP1 is an internalization pathway of pro-cath-D alternativeto the M6P receptors.LRP1 is the receptor that mediates the cath-D-induced stimulation of fibroblastoutgrowthWe previously observed that pro-cath-D secreted by cancer cells mediates fibroblastoutgrowth in a paracrine manner (15). As we identified LRP1 as the novel receptorfor cath-D on fibroblasts, we further checked whether paracrine cath-D would inducefibroblast outgrowth via its interaction with LRP1. To find out whether cath-D requiresLRP1 expression to trigger fibroblast outgrowth, we performed 3D culture assays12