Transforming Growth Factor-β1 Modulates the Expression of Syndecan-4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Abstract

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor-β₁ (TGF-β₁ ) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine-rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF-β₁ first upregulates and then downregulates the expression of syndecan-4, a transmembrane heparan sulfate proteoglycan, via the TGF-β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF-β₁ . Involvement of the downstream signaling pathways of ALK5-the Smad and MAPK pathways-in syndecan-4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3-p38 MAPK pathway mediates the early upregulation of syndecan-4 by TGF-β₁ , whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF-β₁ . J. Cell. Biochem. 118: 2009-2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Keywords: ENDOTHELIAL CELL; PROTEOGLYCAN; SYNDECAN-4; TGF-β.

Figure 1

Effects of TGF‐β₁ on the expression of syndecan‐4 mRNA in vascular endothelial cells. Bovine aortic endothelial cells were treated with 1 and 5 ng/mL TGF‐β₁ at 37°C for 6 or 24 h ([A] and [B], respectively). Values are the mean ± S.E. of four samples. Significantly different from the corresponding control, **P < 0.01.

Figure 2

Effects of TGF‐β₁ on the activation of ERK1/2, JNK, p38 MAPK, and Smad2/3 in vascular endothelial cells. Bovine aortic endothelial cells were treated with 1 and 5 ng/mL TGF‐β₁ at 37°C for 1, 2, 4, 6, 8, or 12 h. [A] Expression of P‐ERK1/2, ERK1/2, P‐JNK, JNK, P‐p38 MAPK, p38 MAPK, P‐Smad2/3, and Smad2/3 proteins. Open and filled arrowheads indicate the position of Smad2 and Smad3, respectively. Arrows indicate the positions of P‐ERK1/2, ERK1/2, P‐JNK, and JNK. [B] The ratio of the intensity of P‐Smad2/3 to that of Smad2/3 in [A].

Figure 3

Effects of the MAPK pathway inhibitors PD98059, LY364947, and SB203580 on the expression of syndecan‐4 in vascular endothelial cells. Bovine aortic endothelial cells were pretreated with [A] the MEK1 inhibitor PD98059 at 20 μM, [B] JNK inhibitor SP600125 at 10 μM, or [C] p38 MAPK inhibitor SB203580 at 10 μM at 37°C for 1 h and then treated with 5 ng/mL TGF‐β₁ for 6 or 24 h. Values are the mean ± S.E. of four samples. *P < 0.05, **P < 0.01 versus control; ⁻P < 0.01 versus MAPK inhibitor without TGF‐β₁; ⁺⁺ P < 0.01 versus TGF‐β₁. [D] The expression of syndecan‐4 core protein. Arrowhead indicates the position of syndecan‐4. Bovine aortic endothelial cells were pretreated with 10 μM SB203580 at 37°C for 1 h and then treated with 5 ng/mL TGF‐β₁ for 6 h.

Figure 4

Effects of siRNA‐mediated knockdown of Smad2 or Smad3 on the expression of syndecan‐4 in vascular endothelial cells. Bovine aortic endothelial cells were transfected with siSmad2 or siSmad3 at 37°C for 24 h and then treated with 5 ng/mL TGF‐β₁ for 6 or 24 h. The expression of mRNAs for [A] Smad2, [B] Smad3, [C and D] PAI‐1, and [E and F] syndecan‐4. Values are the means ± S.E. of four samples. *P < 0.05, **P < 0.01 versus siControl; ⁻ P < 0.01 versus siSmad2 or siSmad3 without TGF‐β₁; ⁺⁺ P < 0.01 versus TGF‐β₁. [G] The expression of syndecan‐4 core protein. Arrowhead indicates the position of syndecan‐4. Bovine aortic endothelial cells were transfected with siControl, siSmad2, or siSmad3 at 37°C for 24 h and then treated with 5 ng/mL TGF‐β₁ for 24 h.

Figure 5

Effects of siRNA‐mediated knockdown of Smad2 or Smad3 on the activation of p38 MAPK in vascular endothelial cells. Bovine aortic endothelial cells were transfected with siSmad2 or siSmad3 at 37°C for 24 h and then treated with 5 ng/mL TGF‐β₁ for 4, 8, or 12 h. [A] Expression of P‐p38 MAPK, p38 MAPK, Smad2/3, and GAPDH proteins. Open and filled arrowheads indicate the position of Smad2 and Smad3, respectively. [B] The ratio of the intensity of P‐p38 MAPK to that of p38 MAPK in [A].