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Pin1 enhances TF expression and activity
tiated our study by assessing the effect of Pin1 on TF gene expression. TF gene expression was measured in cultured ECs and SMCs after Pin1 gain and loss-of-function. Pin1 overexpression significantly increased TF mRNA levels in human SMCs and TNF-α-activated ECs, while knock- down of Pin1 by siRNA or Pin1 isomerase activity inhibi- tion with Juglone28 resulted in significantly decreased TF mRNA expression in ECs (Figure 1A and B). Additionally, pharmacological inhibition of Pin1 isomerase activity with Juglone significantly decreased PMA-induced activation of
the TF promoter in HEK293T cells (Figure 1C).
The promoter of the TF gene contains binding motifs for NF-κB, AP-1, Sp1, and Egr-1, which are important tran- scription factors for both basal expression and stimulus- specific induction of TF expression.14,15 Consistent with our gene expression data, Pin1 overexpression markedly enhanced the activity of the wild-type TF promoter reporter construct in both HEK293T cells and SMCs (Figure 1D-F). However, this activation of the TF promoter by Pin1 was significantly diminished when treating
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Figure 1. Pin1 enhances Tissue Factor (TF) mRNA expression and TF promoter activation by NF-κB and AP-1. (A and B) Expression of the TF gene (F3) in human umbilical vein endothelial cells (HUVEC) and smooth muscle cells (SMC) after Pin1 overexpression, Pin1 knockdown (siPin1), or treatment with the Pin1 inhibitor Juglone. HUVECs were treated with TNF-α for 6 hours to induce TF gene expression. (C) Activity of the wild-type TF gene promoter luciferase reporter construct in PMA-stimulated HEK293T treated with Pin1 inhibitor Juglone or vehicle (DMSO) control. Inset shows schematic representation of the TF gene promoter luciferase reporter construct. (D) Activity of the wild-type TF gene promoter luciferase reporter construct after Pin1 overexpression in PMA-stimulated HEK293T and either vehicle (DMSO), NF-κB signaling inhibitor (BAY-117085), or AP-1 signaling inhibitor (SP600125) treatment. (E and F) Activity of luciferase reporter constructs con- taining either the wild-type TF gene promoter or the TF gene promoter in which the response element for NF-κB (NF-κB RE) or AP-1 (AP-1 RE) was mutated in PMA- stimulated HEK293T (E) or TNF-α-stimulated SMCs (F) transiently over-expressing Pin1. (G and H) Activity of the wild-type TF gene promoter luciferase reporter con- struct after transient overexpression of Pin1 or Pin1 mutants containing a disrupted WW-domain (Pin1:W34A) or lacking isomerase activity (Pin1:K63A) in HEK293T (G) or SMCs (H) left untreated or stimulated with PMA. Data are shown as mean±Standard Error of Mean. (A-C) P-values were calculated for comparisons between Pin1 overexpression, Pin1 knockdown (siPin1), or Juglone treatment versus control transduced (Ctrl) or vehicle-treated groups using the two-tailed Student’s t-test. (D-H) P-values were calculated using one-way ANOVA as indicated. *P<0.05, **P<0.01, ***P<0.001. AU: arbitrary units; mut: mutated.
haematologica | 2018; 103(6)
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