Lumican affects actin cytoskeletal organization in human melanoma A375 cells.
OBJECTIVE
Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line.


METHODS
Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms.


RESULTS
A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican.


CONCLUSIONS
Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.
Radwanska
Agata
A
Department of Cell Pathology, Faculty of Biotechnology, University of Wroclaw, ul. Przybyszewskiego 63/77, 51-148 Wroclaw, Poland. agatar@ibmb.uni.wroc.pl
Baczynska
Dagmara
D
Nowak
Dorota
D
Brézillon
Stéphane
S
Popow
Agnieszka
A
Maquart
François-Xavier
FX
Wegrowski
Yanusz
Y
Malicka-Blaszkiewicz
Maria
M
D000199:Actins / Q000187:drug effects*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D011508:Chondroitin Sulfate Proteoglycans / Q000494:pharmacology*; D003094:Collagen / Q000737:chemistry; D003599:Cytoskeleton / Q000187:drug effects* / Q000648:ultrastructure*; D003600:Cytosol / Q000187:drug effects / Q000648:ultrastructure; D005353:Fibronectins / Q000737:chemistry; D006801:Humans; D007536:Isomerism; D007632:Keratan Sulfate / Q000494:pharmacology*; D000071234:Lumican; D008545:Melanoma / Q000648:ultrastructure*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019311:Staurosporine / Q000494:pharmacology; D013778:Tetrazolium Salts; D013844:Thiazoles
D000199:Actins; D011508:Chondroitin Sulfate Proteoglycans; D005353:Fibronectins; C000604306:LUM protein, human; D000071234:Lumican; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D003094:Collagen; D007632:Keratan Sulfate; C022616:thiazolyl blue; D019311:Staurosporine