Differentiation-associated changes in the expression of chondroitin sulfate proteoglycan in induced U-937 cells

A monoblastic cell line U-937 (clone 4), was induced to differentiate along the monocytoid lineage by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), and vitamin D3 (VD3). By immunochemical and morphological criteria the cells were found to differentiate into macrophage-like cells in the presence of all three inducers. The expression of proteoglycans was investigated in control cultures and in cells differentiated in the presence of both TPA, RA, and VD3. The cells were labeled with [35S]sulfate and cell and medium-associated 35S-macromolecules were either solubilized in sodium dodecyl sulfate or subjected to proteolytic digestion. By use of chondroitinase ABC digestions and deaminative cleavage at pH 1.5 it was demonstrated that all cell cultures incorporated [35S]sulfate exclusively into chondroitin sulfate proteoglycan (CSPG). The expression of CSPG was found to decrease with differentiation to 60% in the presence of TPA, 67% in RA, and 40% in VD3 of control cultures on a cellular basis. The CSPG synthesized was consistently recovered from the medium fractions, whereas free glycosaminoglycan (GAG) chains were found in the cell fraction in all the cell cultures. GAG chains from both control and TPA-, RA-, and VD3-induced cultures were found to be exclusively of the chondroitin 4-sulfate type. However, the CSPGs from RA- and VD3-treated cells were found to differ in molecular size from those of control and TPA-induced cultures, as judged by Sepharose CL-6B gel chromatography. This difference in macromolecular properties following the induced differentiation of the monoblastic cells into macrophage-like cells was found to reside in expression of CSPGs (in the presence of RA and VD3) with smaller GAG chains. Control cells and TPA-induced cells synthesized CSPGs with GAG chains of approximate Mr of 30,000, contrasted by approximate Mr of 17,000 and 16,000 in RA- and VD3-induced cells, respectively. Accordingly, all three agents used in this study were found to induce differentiation of the U-937-4 cells and a decrease in the expression of CSPG, but only RA and VD3 were found to influence the structure of the proteoglycans synthesized.