Wnt5A activates the calpain-mediated cleavage of filamin A.
We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.
O'Connell
Michael P
MP
Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA.
Fiori
Jennifer L
JL
Baugher
Katherine M
KM
Indig
Fred E
FE
French
Amanda D
AD
Camilli
Tura C
TC
Frank
Brittany P
BP
Earley
Rachel
R
Hoek
Keith S
KS
Hasskamp
Joanne H
JH
Elias
E George
EG
Taub
Dennis D
DD
Bernier
Michel
M
Weeraratna
Ashani T
AT
D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D002614:Chelating Agents / Q000494:pharmacology; D003285:Contractile Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000502:physiology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D064448:Filamins; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008545:Melanoma / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D008840:Microfilament Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D000071818:Wnt-5a Protein
D002614:Chelating Agents; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; C078717:ROR2 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors; D002154:Calpain; C484806:CAPN1 protein, human