The retinoic acid and cAMP-dependent up-regulation of 3-O- sulfotransferase-1 leads to a dramatic augmentation of anticoagulantly active heparan sulfate biosynthesis in F9 embryonal carcinoma cells

Abstract

Retinoic acid (RA) and dibutyryl cAMP plus theophilline (CT) trigger F9 cells to differentiate into parietal endoderm. The differentiation induces a 9-fold increase in total heparan sulfate (HS(total)) biosynthesis and a 170- fold increase in anticoagulantly active HS (HS(act)) biosynthesis. Measurement of 3-O-sulfotransferase-1 mRNA and enzymatic activity demonstrated an increase of over 100-fold whereas determination of N-, 2-O, and 6-O-sulfotransferase enzymatic activities showed elevations of 2-, 3.5-, and 3.7-fold, respectively. HS(act) precursor pool measurements reveal that 30% of control F9 HS(total) can be converted into HS(act) while only an additional 10% of RACT F9 HS(total) can be transformed into HS(act). Disaccharide analysis of metabolic labeled HS indicated that 32% 3-O-sulfate containing disaccharides, i.e. GlcA-anMan(R)3S and GlcA-anMan(R)3S6S, are present in HS(act) and 68% GlcA-anMan(R)3S and GlcA-anMan(R)3S6S are found in anticoagulantly inactive HS (HS(inact)). By using adenosine 3'-phosphate 5'- phosphosulfate and purified 3-O-sulfotransferase-1, 30% of 3-O-sulfation occurs in HS(act) and 70% of 3-O-sulfation occurs in HS(inact). The similar ratio of 3-O-sulfate distribution in HS(act) versus HS(inact) suggests that HS(act) production in the F9 system is determined by the abundance of 3-O- sulfotransferase-1 as well as the size of the HS(act) precursor pool. Extensively 3-O-sulfated HS(inact) may play an important functional role under in vivo conditions within the murine placenta.