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Copper(II) Bis(diethyldithiocarbamate) Induces the Expression of Syndecan-4, a Transmembrane Heparan Sulfate Proteoglycan, via p38 MAPK Activation in Vascular Endothelial Cells

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Copper(II) Bis(diethyldithiocarbamate) Induces the Expression of Syndecan-4, a Transmembrane Heparan Sulfate Proteoglycan, via p38 MAPK Activation in Vascular Endothelial Cells

Takato Hara et al. Int J Mol Sci.

Abstract

Proteoglycans synthesized by vascular endothelial cells are important for regulating cell function and the blood coagulation-fibrinolytic system. Since we recently reported that copper(II) bis(diethyldithiocarbamate) (Cu(edtc)₂) modulates the expression of some molecules involving the antioxidant and blood coagulation systems, we hypothesized that Cu(edtc)₂ may regulate the expression of proteoglycans and examined this hypothesis using a bovine aortic endothelial cell culture system. The experiments showed that Cu(edtc)₂ induced the expression of syndecan-4, a transmembrane heparan sulfate proteoglycan, in a dose- and time-dependent manner. This induction required the whole structure of Cu(edtc)₂-the specific combination of intramolecular copper and a diethyldithiocarbamate structure-as the ligand. Additionally, the syndecan-4 induction by Cu(edtc)₂ depended on the activation of p38 mitogen-activated protein kinase (MAPK) but not the Smad2/3, NF-E2-related factor2 (Nrf2), or epidermal growth factor receptor (EGFR) pathways. p38 MAPK may be a key molecule for inducing the expression of syndecan-4 in vascular endothelial cells.

Keywords: bioorganometallics; metal coordination complexes; organocopper compound; proteoglycan; syndecan-4; vascular endothelial cell.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Induction of syndecan-4 mRNA by Cu(edtc)2 treatment in vascular endothelial cells. (A) The structure of Cu(edtc)2. The expression of syndecan-4 mRNA; the cells were incubated at 37 °C (B) for 8 h in the presence of Cu(edtc)2 (0.5, 1, 2, 3, and 5 µM) or (C) for 2, 4, 8, 12, and 24 h in the presence of Cu(edtc)2 (3 µM). Syndecan-4 mRNA levels were then determined by real-time reverse transcription polymerase chain reaction (RT-PCR). Values are means ± S.E. of four replicates. ** p < 0.01 vs. the corresponding control.
Figure 2
Figure 2
Effects of Cu(edtc)2 on the expression of perlecan, biglycan, syndecan-1, syndecan-2, and syndecan-3 mRNA in vascular endothelial cells. The cells were incubated at 37 °C for 8 h in the presence of Cu(edtc)2 (0.5, 1, 2, 3, and 5 µM). Proteoglycan mRNA levels were then determined by RT-PCR. Values are means ± S.E. of four replicates. ** p < 0.01 vs. control.
Figure 3
Figure 3
Comparison of the syndecan-4 mRNA induction by different structural components of Cu(edtc)2. (A) Structure of Cu(edtc)2 and Na(edtc); (B) expression of syndecan-4 mRNA. The cells were incubated at 37 °C for 8 h in the presence of Cu(edtc)2 (3 µM), CuSO4 (3 µM), or Na(edtc) (6 µM). Syndecan-4 mRNA levels were then determined by RT-PCR. Values are means ± S.E. of four replicates. ** p < 0.01 vs. the corresponding control. ## p < 0.01 vs. CuSO4. ++ p < 0.01 vs. Na(edtc).
Figure 4
Figure 4
Role of copper and EDTC in the Cu(edtc)2 molecule in syndecan-4 mRNA induction in vascular endothelial cells. (A) Structure of Cu(edtc)2, Zn(edtc)2, Ni(edtc)2, and Fe(edtc)3; (B) expression of syndecan-4 mRNA. The cells were incubated at 37 °C for 8 h in the presence of Cu(edtc)2, Zn(edtc)2, Ni(edtc)2, or Fe(edtc)3 (3 µM each), then syndecan-4 mRNA levels were measured by RT-PCR; (C) structure of Cu(edtc)2, and copper complexes 15; (D) expression of syndecan-4 mRNA. The cells were incubated at 37 °C for 8 h in the presence of copper complexes (3 µM each), then syndecan-4 mRNA levels were measured by RT-PCR. Values are means ± S.E. of four replicates. * p < 0.05; ** p < 0.01 vs. control.
Figure 5
Figure 5
Effects of Cu(edtc)2 on the expression of NF-E2-related factor2 (Nrf2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and Smad2/3 in vascular endothelial cells. The cells were incubated at 37 °C for 0.5, 1, 2, 4, and 6 h in the presence of Cu(edtc)2 (3 µM). (A) Cell lysates were then subjected to western blotting; (BD) the ratio of the intensity of Nrf2, phosphorylated p38 MAPK (P-p38 MAPK), and phosphorylated Smad2/3 (P-Smad2/3) in (A) to those of β-Actin, p38 MAPK, and Smad2/3, respectively; values are means of two replicates from two independent experiments (BD).
Figure 6
Figure 6
A p38 MAPK inhibitor SB203580 suppresses the syndecan-4 induction by Cu(edtc)2 in vascular endothelial cells. The expression of syndecan-4 (A) mRNA and (B) core protein; values are means ± S.E. of four replicates. ** p < 0.01 vs. control; ## p < 0.01 vs. Cu(edtc)2. The position of syndecan-4 core protein is indicated by the arrowhead; (C) the intensity of the blots of cell layer in (B); the cells were treated with Cu(edtc)2 (3 µM) for 8 h after pretreatment with of SB203580 (10 µM) for 3 h. Values are means of two replicates from two independent experiments (C).
Figure 7
Figure 7
Nrf2 does not contribute to the activation of p38 MAPK and induction of syndecan-4 by Cu(edtc)2 in vascular endothelial cells; (A) effect of Nrf2 on the activation of p38 MAPK by Cu(edtc)2; (B,C) the ratio of the intensity of Nrf2 and P-p38 MAPK in (A) to those of β-Actin and p38 MAPK, respectively; (D) expression of syndecan-4 mRNA. siRNA-transfected cells were treated with Cu(edtc)2 (3 µM) for 8 h. Values are means of two replicates from two independent experiments (B,C); ** p < 0.01 vs. control; ## p < 0.01.
Figure 8
Figure 8
Epidermal growth factor receptor (EGFR) activation does not contribute to the Cu(edtc)2-mediated activation of p38 MAPK and induction of syndecan-4 in vascular endothelial cells. (A) Activation of EGFR by Cu(edtc)2; the cells were incubated at 37 °C for 0.5, 1, 2, 3, and 4 h in the presence of Cu(edtc)2 (3 µM), then lysates were subjected to Western blotting. The ratio of the intensity of (B) phosphorylated EGFR (P-EGFR)(Y1068) and (C) P-EGFR(Y992) in (A) to that of EGFR; (D) EGFR-dependent p38 MAPK activation. The cells were treated with Cu(edtc)2 (3 µM) for 4 h after pretreatment with an EGFR inhibitor PD153035 (5, 10, and 20 µM) for 1 h. Lysates were then subjected to immunoblotting. (E) The ratio of the intensity of P-p38 MAPK to that of p38 MAPK in (D); (F) expression of syndecan-4 mRNA. The cells were treated with Cu(edtc)2 (3 µM) for 8 h after pretreatment with of PD153035 (5, 10, and 20 µM) for 1 h, then syndecan-4 mRNA levels were determined by RT-PCR. Values are means of two replicates from two independent experiments (B,C,E).

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