Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-beta in rat clonal dental pulp cells

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.