Syndecan-1 downregulates syndecan-4 expression by suppressing the ERK1/2 and p38 MAPK signaling pathways in cultured vascular endothelial cells

Abstract

Syndecan-1 and syndecan-4 are members of the syndecan family of transmembrane heparan sulfate proteoglycans. Vascular endothelial cells synthesize both species of proteoglycans and use them to regulate the blood coagulation-fibrinolytic system and their proliferation via their heparin-like activity and FGF-2 binding activity, respectively. However, little is known about the crosstalk between the expressions of the proteoglycan species. Previously, we reported that biglycan, a small leucine-rich dermatan sulfate proteoglycan, intensifies ALK5-Smad2/3 signaling by TGF-β₁ and downregulates syndecan-4 expression in vascular endothelial cells. In the present study, we investigated the crosstalk between the expressions of syndecan-1 and other proteoglycan species (syndecan-4, perlecan, glypican-1, and biglycan) in bovine aortic endothelial cells in a culture system. These data suggested that syndecan-1 downregulated syndecan-4 expression by suppressing the endogenous FGF-2-dependent ERK1/2 pathway and FGF-2-independent p38 MAPK pathway in the cells. Moreover, this crosstalk was a one-way communication from syndecan-1 to syndecan-4, suggesting that syndecan-4 compensated for the reduced activity in the regulation of vascular endothelial cell functions caused by the decreased expression of syndecan-1 under certain conditions.

Keywords: MAPK; Syndecan-1; Syndecan-4; Vascular endothelial cells.

Graphical abstract

Fig. 1

Effects of siRNA-mediated syndecan-1 or syndecan-4 knockdown on the levels of PG mRNAs in vascular endothelial cells. Bovine aortic endothelial cells were transfected with siCont, siSDC-1-1, siSDC-1-2, siSDC-4-1, or siSDC-4-2 for 8 h, and then incubated for 16 h in fresh medium. [A] Effects of siRNA-mediated syndecan-1 knockdown on the levels of other PG mRNAs. [B] Effects of siRNA-mediated syndecan-4 knockdown on the levels of other PG mRNAs. Values are the means ± SE of three independent experiments. Significantly different from the corresponding “siCont”, *P < 0.05; **P < 0.01.

Fig. 2

Effects of siRNA-mediated syndecan-1 knockdown on syndecan-1 mRNA and core protein expression, and the effects of siRNA-mediated syndecan-4 knockdown on syndecan-1 core protein expression in vascular endothelial cells. [A] Time course of the levels of syndecan-4 mRNA after syndecan-1 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h, and then incubated for 3, 6, 12, 18, and 24 h in fresh medium. Values are the means ± SE of three independent experiments. **Significantly different from the corresponding siCont, P < 0.01. [B] Expression of syndecan-4 core protein after syndecan-1 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h, and then incubated for 24 h in fresh medium. The blots present syndecan-1 and syndecan-4 expression (left panels) and the measured proportion of the intensity of syndecan-4 expression in the cell layer (right panel). [C] Expression of syndecan-1 core protein after syndecan-4 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-4-1 for 8 h, and then incubated for 24 h in fresh medium. The blots present syndecan-1 and syndecan-4 expression (left panels) and the measured proportion of the intensity of syndecan-1 expression in the cell layer (right panel).

Fig. 3

Activation of MAPKs (ERK1/2, p38 MAPK, and JNK) after siRNA-mediated syndecan-1 knockdown and the involvement of this activation in the elevation of syndecan-4 mRNA level in vascular endothelial cells. [A] Activation of MAPKs after siRNA-mediated syndecan-1 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h and incubated for 6, 12, and 24 h in fresh medium. The blots present MAPK expression (left panels) and the calculated ratio of the intensity of phosphorylated MAPK expression to that of total MAPK expression (right panels). The values presented in the bar graphs were calculated using data presented in the blots. [B,C] Involvement of ERK1/2 and p38 MAPK activation in the elevation of the syndecan-4 mRNA level by siRNA-mediated syndecan-1 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h; treated with [B] PD98059 and [C] SB203580 at 5 and 10 μM for 1 h after changing medium; and incubated for 23 h in fresh medium. Values are the means ± SE of three independent experiments. Significantly different from the corresponding siCont, **P < 0.01. ^##Significantly different from the corresponding “absence of inhibitor”, ^#P < 0.05; ^##P < 0.01.

Fig. 4

Involvement of FGFR and ALK5 in the elevation of the syndecan-4 mRNA level by siRNA-mediated syndecan-1 knockdown in vascular endothelial cells. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h; treated with [A] PD161570 and [C] LY364947 at 1, 5, and 10 μM for 1 h; and incubated for 23 h in fresh medium. Values are the means ± SE of three independent experiments. **Significantly different from the corresponding siCont, P < 0.01. ^##Significantly different from the corresponding “absence of inhibitor”, P < 0.01. [B] Involvement of FGFR in the elevation of ERK1/2 and p38 MAPK phosphorylation by siRNA-mediated syndecan-1 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h; treated with PD161570 at 1, 5, and 10 μM for 1 h; and incubated for 23 h in fresh medium. The blots present ERK1/2 and p38 MAPK expression (top panels) and the calculated ratio of the intensity of phosphorylated ERK1/2 (middle panels) and phosphorylated p38 MAPK (bottom panels) expressions to that of total ERK1/2 and p38 MAPK expressions, respectively. The values presented in the bar graphs were calculated using data presented in the blots. [D] Involvement of ALK5 in the elevation of p38 MAPK phosphorylation by siRNA-mediated syndecan-1 knockdown. Bovine aortic endothelial cells were transfected with siCont or siSDC-1-1 for 8 h; treated with LY364947 at 1, 5, and 10 μM for 1 h; and incubated for 23 h in fresh medium. The blots present p38 MAPK expression (top panels) and the calculated ratio of the intensity of phosphorylated p38 MAPK expression (bottom panels) to that of total p38 MAPK expression. The values presented in the bar graphs were calculated using data presented in the blots.