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Finding ATF4/p75NTR/IL-8 signal pathway in endothelial-mesenchymal transition by safrole oxide.

Ge D, Jing Q, Zhao W, Yue H, Su L, Zhang S, Zhao J - PLoS ONE (2014)

Bottom Line: Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h.Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly.The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO.

View Article: PubMed Central - PubMed

Affiliation: Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan, China.

ABSTRACT
Targeting the endothelial-to-mesenchymal transition (EndoMT) may be a novel therapeutic strategy for cancer and various diseases induced by fibrosis. We aimed to identify a small chemical molecule as an inducer of EndoMT and find a new signal pathway by using the inducer. Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h. To understand the underlying molecular mechanism, we performed microarray, quantitative real-time PCR and western blot analysis to find key factors involved in SFO-induced EndoMT and demonstrated the involvement of the factors by RNAi. The expression of activating transcription factor 4 (ATF4), p75 neurotrophin receptor (p75NTR), and interleukin 8 (IL-8) was greatly increased in SFO-induced EndoMT. Knockdown of ATF4 inhibited the SFO-induced EndoMT completely, and knockdown of p75NTR or IL-8 partially inhibited the EndoMT, which suggests that all three factors were involved in the process. Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly. The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO. Our findings support potential novel targets for the therapeutics of cancer and fibrosis disease.

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ATF4, p75NTR and IL-8 participated in SFO-induced EndoMT in HUVECs.Cells were preincubated with 40 nM ATF4, IL-8 or 60 nM p75NTR siRNA or scramble siRNA for 48 h, then 50 µg/ml SFO for 3 h for qPCR and 6 h for western blot assay. (A and B) Western blot analysis of siRNA-mediated downregulation of ATF4 and p75NTR in HUVECs. Data below the western blot bands were used to quantify the density of the bands. (C) qPCR analysis of siRNA-mediated downregulation of IL-8. GAPDH was a normalization control. (D and E) Analysis of effect of ATF4, p75NTR and IL-8 siRNA on SFO-induced α-SMA in HUVECs by qPCR (D) and western blot (E). (F) qPCR analysis of siRNA effects on eNOS expression in HUVECs. Expression was normalized to that of GAPDH. (*p<0.05, **p<0.01, n = 3).
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pone-0099378-g005: ATF4, p75NTR and IL-8 participated in SFO-induced EndoMT in HUVECs.Cells were preincubated with 40 nM ATF4, IL-8 or 60 nM p75NTR siRNA or scramble siRNA for 48 h, then 50 µg/ml SFO for 3 h for qPCR and 6 h for western blot assay. (A and B) Western blot analysis of siRNA-mediated downregulation of ATF4 and p75NTR in HUVECs. Data below the western blot bands were used to quantify the density of the bands. (C) qPCR analysis of siRNA-mediated downregulation of IL-8. GAPDH was a normalization control. (D and E) Analysis of effect of ATF4, p75NTR and IL-8 siRNA on SFO-induced α-SMA in HUVECs by qPCR (D) and western blot (E). (F) qPCR analysis of siRNA effects on eNOS expression in HUVECs. Expression was normalized to that of GAPDH. (*p<0.05, **p<0.01, n = 3).

Mentions: To further confirm the role of ATF4, p75NTR and IL-8 in SFO-increased α-SMA expression, we transfected ATF4, p75NTR and IL-8 siRNA into HUVECs followed by treatment with SFO. Transfection of the selected siRNAs significantly inhibited the expression of the corresponding genes (Fig. 5A to C). Knockdown of ATF4, p75NTR and IL-8 significantly inhibited SFO-induced α-SMA in mRNA and protein levels (Fig. 5D and E). Furthermore, knockdown of ATF4, p75NTR and IL-8 rescued SFO-inhibited eNOS expression (Fig.5F).


Finding ATF4/p75NTR/IL-8 signal pathway in endothelial-mesenchymal transition by safrole oxide.

Ge D, Jing Q, Zhao W, Yue H, Su L, Zhang S, Zhao J - PLoS ONE (2014)

ATF4, p75NTR and IL-8 participated in SFO-induced EndoMT in HUVECs.Cells were preincubated with 40 nM ATF4, IL-8 or 60 nM p75NTR siRNA or scramble siRNA for 48 h, then 50 µg/ml SFO for 3 h for qPCR and 6 h for western blot assay. (A and B) Western blot analysis of siRNA-mediated downregulation of ATF4 and p75NTR in HUVECs. Data below the western blot bands were used to quantify the density of the bands. (C) qPCR analysis of siRNA-mediated downregulation of IL-8. GAPDH was a normalization control. (D and E) Analysis of effect of ATF4, p75NTR and IL-8 siRNA on SFO-induced α-SMA in HUVECs by qPCR (D) and western blot (E). (F) qPCR analysis of siRNA effects on eNOS expression in HUVECs. Expression was normalized to that of GAPDH. (*p<0.05, **p<0.01, n = 3).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048316&req=5

pone-0099378-g005: ATF4, p75NTR and IL-8 participated in SFO-induced EndoMT in HUVECs.Cells were preincubated with 40 nM ATF4, IL-8 or 60 nM p75NTR siRNA or scramble siRNA for 48 h, then 50 µg/ml SFO for 3 h for qPCR and 6 h for western blot assay. (A and B) Western blot analysis of siRNA-mediated downregulation of ATF4 and p75NTR in HUVECs. Data below the western blot bands were used to quantify the density of the bands. (C) qPCR analysis of siRNA-mediated downregulation of IL-8. GAPDH was a normalization control. (D and E) Analysis of effect of ATF4, p75NTR and IL-8 siRNA on SFO-induced α-SMA in HUVECs by qPCR (D) and western blot (E). (F) qPCR analysis of siRNA effects on eNOS expression in HUVECs. Expression was normalized to that of GAPDH. (*p<0.05, **p<0.01, n = 3).
Mentions: To further confirm the role of ATF4, p75NTR and IL-8 in SFO-increased α-SMA expression, we transfected ATF4, p75NTR and IL-8 siRNA into HUVECs followed by treatment with SFO. Transfection of the selected siRNAs significantly inhibited the expression of the corresponding genes (Fig. 5A to C). Knockdown of ATF4, p75NTR and IL-8 significantly inhibited SFO-induced α-SMA in mRNA and protein levels (Fig. 5D and E). Furthermore, knockdown of ATF4, p75NTR and IL-8 rescued SFO-inhibited eNOS expression (Fig.5F).

Bottom Line: Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h.Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly.The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO.

View Article: PubMed Central - PubMed

Affiliation: Shandong Provincial Key Laboratory of Animal Cells and Developmental Biology, School of Life Science, Shandong University, Jinan, China.

ABSTRACT
Targeting the endothelial-to-mesenchymal transition (EndoMT) may be a novel therapeutic strategy for cancer and various diseases induced by fibrosis. We aimed to identify a small chemical molecule as an inducer of EndoMT and find a new signal pathway by using the inducer. Safrole oxide (SFO), 50 µg/ml, could most effectively induce EndoMT within 12 h. To understand the underlying molecular mechanism, we performed microarray, quantitative real-time PCR and western blot analysis to find key factors involved in SFO-induced EndoMT and demonstrated the involvement of the factors by RNAi. The expression of activating transcription factor 4 (ATF4), p75 neurotrophin receptor (p75NTR), and interleukin 8 (IL-8) was greatly increased in SFO-induced EndoMT. Knockdown of ATF4 inhibited the SFO-induced EndoMT completely, and knockdown of p75NTR or IL-8 partially inhibited the EndoMT, which suggests that all three factors were involved in the process. Furthermore, knockdown of p75NTR inhibited the SFO-increased IL-8 expression and secretion, and knockdown of ATF4 inhibited SFO-increased p75NTR level significantly. The ATF4/p75NTR/IL-8 signal pathway may have an important role in EndoMT induced by SFO. Our findings support potential novel targets for the therapeutics of cancer and fibrosis disease.

Show MeSH
Related in: MedlinePlus