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 (HStotal) biosynthesis and a 170-fold increase in anticoagulantly active HS (HSact) 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. HSact precursor pool measurements reveal that 30% of control F9 HStotal can be converted into HSact while only an additional 10% of RACT F9 HStotal can be transformed into HSact. Disaccharide analysis of metabolic labeled HS indicated that 32% 3-O-sulfate containing disaccharides, i.e. GlcA-anManR3S and GlcA-anManR3S6S, are present in HSact and 68% GlcA-anManR3S and GlcA-anManR3S6S are found in anticoagulantly inactive HS (HSinact). By using adenosine 3'-phosphate 5'-phosphosulfate and purified 3-O-sulfotransferase-1, 30% of 3-O-sulfation occurs in HSact and 70% of 3-O-sulfation occurs in HSinact. The similar ratio of 3-O-sulfate distribution in HSact versus HSinact suggests that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. Extensively 3-O-sulfated HSinact may play an important functional role under in vivo conditions within the murine placenta.