Retinoic acid-enhanced invasion through reconstituted basement membrane by human SK-N-SH neuroblastoma cells involves membrane-associated tissue-type plasminogen activator

Al-trans retinoic acid (RA) enhanced human, S-type, SK-N-SH neuroblastoma cell invasion of reconstituted basement membrane in vitro but did not induce terminal differentiation of this cell line. In contrast to basal invasion, which was urokinase (uPA)- and plasmin-dependent, RA-enhanced invasion was dependent on tissue-type plasminogen activator (t-PA) and plasmin activity. Neither basal nor RA-enhanced invasion involved TIMP-2 inhibitable metalloproteinases. Enhanced invasion was associated with the induction of t-PA expression, increased expression of the putative t-PA receptor amphoterin, increased association of t-PA with cell membranes and increased net membrane-associated PA activity. Enhanced invasion was not associated with significant changes in the expression of uPA or its membrane receptor UPAR; plasminogen activator inhibitors PAI-1 and PAI-2; metalloproteinases MMP-1, MMP-2, MMP-3, MMP-9 and membrane type MMP1; or tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. RA stimulated the association of t-PA with the external cell membrane surface, which could be inhibited by heparin sulphate but not by mannose sugars or chelators of divalent cations, consistent with a role for amphoterin. Our data indicate that RA can promote the malignant behavior of S-type neuroblastoma cells refractory to RA-mediated terminal differentiation by enhancing their basement membrane invasive capacity. We suggest that this results from the action of a novel, RA-regulated mechanism involving stimulation of t-PA expression and its association with the cell membrane leading to increased PA-dependent matrix degradation.