Lumican is a
small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) with anti-
tumor activity. We recently demonstrated that
lumican inhibits the migration of
melanoma cells and identified
beta1 integrin as mediator of this effect [M.F. D'Onofrio, S. Brézillon, T. Baranek, C. Perreau, P.J. Roughley, F.X. Maquart, Y. Wegrowski, Identification of
beta1 integrin as mediator of
melanoma cell adhesion to
lumican, Biochem. Biophys. Res. Commun. 365 (2008) 266-272]. The aim of the present work was to study
beta1 integrin, focal adhesion complexes, actin distribution and expression in the presence of
lumican substratum in comparison to
type I collagen or
fibronectin substrata in A375 human
melanoma cells. The
protein distribution was investigated by immunocytochemistry and confocal microscopy. In parallel, their expression was evaluated by Western immunoblotting and Real-time Reverse Transcription-PCR analyses. The interaction of
melanoma cells with the
lumican substratum resulted in heterogeneous distribution of
beta1 integrin on cell membrane after 24h of seeding. Concomitantly, a reorganization of actin stress fibers and a significant decrease in
vinculin immunostaining at focal adhesion complexes were observed. No alteration of the expression was detected at
protein and
mRNA levels. However, a cytosolic accumulation of
vinculin focal adhesion
protein was observed on
lumican substratum by confocal microscopy. Moreover,
vinculin expression was significantly increased in cytosolic fractions in comparison to cells seeded on
type I collagen or
fibronectin substrata. Our results suggest that
lumican induces an alteration of the link between actin filaments and
beta1 integrin, characterized by a cytosolic accumulation of
vinculin focal adhesion
protein, which could lead to a destabilization of focal adhesion complexes. In addition,
focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) was significantly decreased. Therefore, the cytoskeleton remodeling and the decreased pFAK phosphorylation induced by
lumican in
melanoma cells might explain, at least in part, the anti-invasive effect of this SLRP.