Effect of <FONT FACE=Symbol>b</font>-D-xylosides bearing different aglycones on the synthesis of proteoglycans during the cell cycle of endothelial cells in culture

EFFECT OF b-D-XYLOSIDES BEARING DIFFERENT AGLYCONES ON THE SYNTHESIS OF PROTEOGLYCANS DURING THE CELL CYCLE OF ENDOTHELIAL CELLS IN CULTURE

MOREIRA C.R., LOPES C.C., PORCIONATTO M.A., ESKO J.D., DIETRICH C.P. AND NADER H.B.¹

Disciplina de Biologia Molecular, UNIFESP, São Paulo, SP.

¹ University of California San Diego, LaJolla, CA, USA.

PMA, an activator of PKC, stimulated the synthesis of heparan sulfate proteoglycan secreted to the medium of endothelial cells mainly during G₁ phase of the cell cycle (Porcionatto et al. 1998. J Cell Biochem 70: 563-572). This result led us to investigate the effect of b-D-xylosides (that specifically stimulate the synthesis of proteoglycans) at different phases of the cell cycle. Glycosaminoglycan (GAG) biosynthesis initiates through the transfer of b-D-xylose from UDP-xylose to specific serine residues of the protein core. GAG biosynthesis can also occur on exogenous b-D-xylosides, but unlike endogenous xylosylated core proteins, b-D-xylosides preferentially stimulate chondroitin sulfate or dermatan sulfate synthesis and only weakly heparan sulfate or heparin synthesis (Schwartz et al. 1974. Proc Natl Acad Sci 71: 40-51; Dietrich et al. 1982. Biochim Biophys Acta 717: 478-485). Recently, it has been shown that the composition of glycosaminoglycans synthesized on b-D-xylose depends on the structure of the aglycone. Fritz & coworkers (1994. J Biol Chem 269: 300-307) studying different aglycones linked to b-D-xyloside have shown that 2-naphtol-b-D-xyloside (NX) and cis/trans-decahydro-2-naphtol-b-D-xyloside (DX) were able to prime the synthesis of heparan sulfate chains in CHO cells. In the present study we compare the effect of NX, DX, p-nitro- and o-nitrophenyl-b-D-xylosides as GAG acceptors in an endothelial cell line. It was observed stimulation of the synthesis of GAGs in a dose dependent manner. When cells were treated with 50mM NX for 15 hours there was a 6-fold increase in the amount of heparan sulfate (HS) and a 19-fold increase in the amount of chondroitin sulfate (CS) secreted to the medium, reaching a plateau with 100mM (9-and 26-fold, respectively). On the other hand, no differences in the amounts of HS from the cell extract were observed between control and treated cultures, whereas for CS a 9-fold increase (50mM) and 12-fold increase (100mM) were observed. The results obtained for the two different isomers of nitrophenyl-b-D-xylosides show that the compounds displace different dose response curves. The optimum concentration for enhancement of the GAG synthesis was 500mM for p-nitrophenyl and 100mM for the o-nitrophenyl derivative. For the p-nitrophenyl there was a 7-fold increase in the amount of HS and a 26-fold increase in the amount CS secreted to the medium. The amount of CS present in the cell was increased 4 fold. For the o-nitrophenyl there was a 20-fold increase in the amount of HS and a 100-fold increase in the amount CS secreted to the medium. The amount of CS present in the cell was increased 6 fold. On the other hand, no differences in the amounts of HS from the cell extract were observed between control and treated cultures with both isomers. The CS synthesized in the presence of the xylosides shows a different electrophoretic migration (more dermatan sulfate-like) when compared to the ones from control cultures. To study the effect of these compounds on the cell cycle, the cells were submitted to 24 hours of starvation and then stimulated to proliferate with the addition of fetal calf serum (FCS) in the absence or in the presence of b-D-xylosides in different periods of time. NX, DX and p-nitrophenyl did not alter the cell cycle whereas the mitogenic effect of FCS on the cells was abolished after treatment with o-nitrophenyl (75mM). These results indicate that hidrophobicity of the aglycones linked to the b-D-xylosides is not the only factor involved in priming of the GAGs. Thus the o-nitrophenyl (less hidrophobic than NX and DX) is the best acceptor for HS synthesis and curiously was the only compound capable of interfering with the cell cycle, blocking the cell entrance in the S phase.

— ( September 14, 1999 ) .

* Supported by: FAPESP and CNPq.

Publication Dates

• Publication in this collection
  07 Aug 2000
• Date of issue
  Mar 2000