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.