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15-Deoxy-Delta-Prostaglandin J(2) Upregulates the Expression of LPS-Induced IL-8/CXCL8 mRNA in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats.

Kim JH, Kim HS - Immune Netw (2009)

Bottom Line: As a result of these properties, we examined the effect of 15d-PGJ(2) on the LPS-induced IL-8/CXCL8 mRNA expression in VSMCs from SHR.However, inhibition of the p38 signaling pathway augmented the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 mRNA.Our results indicate that the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 expression in SHR VSMCs is mediated through the PPARgamma and ERK pathway, and may be related to NAD(P)H oxidase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, College of Medicine, and Aging-associated Vascular Disease Research Center, Yeungnam University, Daegu, Korea.

ABSTRACT

Background: 15d-PGJ(2) has been known to act as an anti-inflammatory agent and has anti-hypertensive effects. As a result of these properties, we examined the effect of 15d-PGJ(2) on the LPS-induced IL-8/CXCL8 mRNA expression in VSMCs from SHR.

Methods: Effect and action mechanism of 15d-PGJ(2) on the expression of LPS-induced IL-8/CXCL8 mRNA in VSMCs from SHR and WKY were examined by using real-time polymerase chain reaction, electrophoretic mobility shift assay for NF-kappaB avtivity, Western blotting analysis for ERK and p38 phosphorylation and flow cytometry for NAD(P)H oxidase activity.

Results: 15d-PGJ(2) decreased the expression of LPS-induced IL-8/CXCL8 mRNA in WKY VSMCs, but increased the expression of LPS-induced IL-8/CXCL8 mRNA in SHR VSMCs. The upregulatory effect of 15d-PGJ(2) in SHR VSMCs was mediated through PPARgamma, and dependent on NF-kappaB activation and ERK phosphorylation. However, inhibition of the p38 signaling pathway augmented the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 mRNA. A NAD(P)H oxidase inhibitor inhibited the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs, and an increase in NAD(P)H oxidase activity was detected in SHR VSMCs treated with 15d-PGJ(2)/LPS.

Conclusion: Our results indicate that the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 expression in SHR VSMCs is mediated through the PPARgamma and ERK pathway, and may be related to NAD(P)H oxidase activity. However, p38 inactivation may also play an important role in 15d-PGJ(2)/LPS-induced IL-8/CXCL8 expression in SHR VSMCs.

No MeSH data available.


Related in: MedlinePlus

Blocking of p38 phosphorylation increased the expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs. (A) VSMCs were untreated (NT) or pretreated with PD169316 (p38 inhibitor, 10 µM) for 30 min, and then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Real-time PCR was performed after total mRNAs were isolated. Bars represent means±SD from three independent experiments. *p<0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the phospho-p38 antibody. Data shown are representative of four independent experiments. (C) VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with p38 siRNA oligomers (20 nmol/l). VSMCs were then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Bars represent means±SEM from three independent experiments.
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Figure 5: Blocking of p38 phosphorylation increased the expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs. (A) VSMCs were untreated (NT) or pretreated with PD169316 (p38 inhibitor, 10 µM) for 30 min, and then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Real-time PCR was performed after total mRNAs were isolated. Bars represent means±SD from three independent experiments. *p<0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the phospho-p38 antibody. Data shown are representative of four independent experiments. (C) VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with p38 siRNA oligomers (20 nmol/l). VSMCs were then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Bars represent means±SEM from three independent experiments.

Mentions: Next we investigated whether the MAPK signaling pathways are involved in the upregulatory effect of 15d-PGJ2 in SHR VSMCs. After VSMCs were untreated (NT) or pretreated with PD98059 (ERK inhibitor, 10 µM, 4A), or PD169316 (p38 inhibitor, 10 µM, 5A) for 30 min, cells were untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Real-time PCR was then performed on these treated cells. In addition, these results were further confirmed by investigating the phosphorylation of MAP kinases in VSMCs that had been treated with 15d-PGJ2/LPS. The expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA was decreased by the ERK inhibitor PD98059. And the expression of LPS alone-induced IL-8/CXCL8 mRNA was also decreased by PD98059 (Fig. 4A). However, although ERK phosphorylation in cells treated 15d-PGJ2 alone was not detected, a remarkable increase in ERK phosphorylation in VSMCs that were treated with 15d-PGJ2/LPS relative to VSMCs that were treated with LPS alone was also detected (Fig. 4B). PD169316 increased the IL-8/CXCL8 mRNA expression in VSMCs stimulated with 15d-PGJ2/LPS, rather than inhibiting IL-8/CXCL8 expression (Fig. 5A). Moreover, 15d-PGJ2 decreased LPS-induced p38 phosphorylation (Fig. 5B). More specifically, blocking p38 phosphorylation caused an increase in 15d-PGJ2/LPS-induced IL-8/CXCL8 expression. To confirm this result, real-time PCR using p38-directed small interfering RNA (siRNA) was performed. In this experiment we found that while LPS inhibited the expression of IL-8/CXCL8 mRNA, 15d-PGJ2 induced IL-8/CXCL8 expression and 15d-PGJ2/LPS increased IL-8/CXCL8 mRNA expression in p38 siRNA transfected SHR VSMCs (Fig. 5C).


15-Deoxy-Delta-Prostaglandin J(2) Upregulates the Expression of LPS-Induced IL-8/CXCL8 mRNA in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats.

Kim JH, Kim HS - Immune Netw (2009)

Blocking of p38 phosphorylation increased the expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs. (A) VSMCs were untreated (NT) or pretreated with PD169316 (p38 inhibitor, 10 µM) for 30 min, and then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Real-time PCR was performed after total mRNAs were isolated. Bars represent means±SD from three independent experiments. *p<0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the phospho-p38 antibody. Data shown are representative of four independent experiments. (C) VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with p38 siRNA oligomers (20 nmol/l). VSMCs were then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Bars represent means±SEM from three independent experiments.
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Related In: Results  -  Collection

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Figure 5: Blocking of p38 phosphorylation increased the expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs. (A) VSMCs were untreated (NT) or pretreated with PD169316 (p38 inhibitor, 10 µM) for 30 min, and then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Real-time PCR was performed after total mRNAs were isolated. Bars represent means±SD from three independent experiments. *p<0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) Cell lysates were separated on 10% SDS-polyacrylamide gels and then immunoblotted with the phospho-p38 antibody. Data shown are representative of four independent experiments. (C) VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with p38 siRNA oligomers (20 nmol/l). VSMCs were then untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Bars represent means±SEM from three independent experiments.
Mentions: Next we investigated whether the MAPK signaling pathways are involved in the upregulatory effect of 15d-PGJ2 in SHR VSMCs. After VSMCs were untreated (NT) or pretreated with PD98059 (ERK inhibitor, 10 µM, 4A), or PD169316 (p38 inhibitor, 10 µM, 5A) for 30 min, cells were untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ2 (10 µM) for 4 h. Real-time PCR was then performed on these treated cells. In addition, these results were further confirmed by investigating the phosphorylation of MAP kinases in VSMCs that had been treated with 15d-PGJ2/LPS. The expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA was decreased by the ERK inhibitor PD98059. And the expression of LPS alone-induced IL-8/CXCL8 mRNA was also decreased by PD98059 (Fig. 4A). However, although ERK phosphorylation in cells treated 15d-PGJ2 alone was not detected, a remarkable increase in ERK phosphorylation in VSMCs that were treated with 15d-PGJ2/LPS relative to VSMCs that were treated with LPS alone was also detected (Fig. 4B). PD169316 increased the IL-8/CXCL8 mRNA expression in VSMCs stimulated with 15d-PGJ2/LPS, rather than inhibiting IL-8/CXCL8 expression (Fig. 5A). Moreover, 15d-PGJ2 decreased LPS-induced p38 phosphorylation (Fig. 5B). More specifically, blocking p38 phosphorylation caused an increase in 15d-PGJ2/LPS-induced IL-8/CXCL8 expression. To confirm this result, real-time PCR using p38-directed small interfering RNA (siRNA) was performed. In this experiment we found that while LPS inhibited the expression of IL-8/CXCL8 mRNA, 15d-PGJ2 induced IL-8/CXCL8 expression and 15d-PGJ2/LPS increased IL-8/CXCL8 mRNA expression in p38 siRNA transfected SHR VSMCs (Fig. 5C).

Bottom Line: As a result of these properties, we examined the effect of 15d-PGJ(2) on the LPS-induced IL-8/CXCL8 mRNA expression in VSMCs from SHR.However, inhibition of the p38 signaling pathway augmented the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 mRNA.Our results indicate that the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 expression in SHR VSMCs is mediated through the PPARgamma and ERK pathway, and may be related to NAD(P)H oxidase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, College of Medicine, and Aging-associated Vascular Disease Research Center, Yeungnam University, Daegu, Korea.

ABSTRACT

Background: 15d-PGJ(2) has been known to act as an anti-inflammatory agent and has anti-hypertensive effects. As a result of these properties, we examined the effect of 15d-PGJ(2) on the LPS-induced IL-8/CXCL8 mRNA expression in VSMCs from SHR.

Methods: Effect and action mechanism of 15d-PGJ(2) on the expression of LPS-induced IL-8/CXCL8 mRNA in VSMCs from SHR and WKY were examined by using real-time polymerase chain reaction, electrophoretic mobility shift assay for NF-kappaB avtivity, Western blotting analysis for ERK and p38 phosphorylation and flow cytometry for NAD(P)H oxidase activity.

Results: 15d-PGJ(2) decreased the expression of LPS-induced IL-8/CXCL8 mRNA in WKY VSMCs, but increased the expression of LPS-induced IL-8/CXCL8 mRNA in SHR VSMCs. The upregulatory effect of 15d-PGJ(2) in SHR VSMCs was mediated through PPARgamma, and dependent on NF-kappaB activation and ERK phosphorylation. However, inhibition of the p38 signaling pathway augmented the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 mRNA. A NAD(P)H oxidase inhibitor inhibited the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs, and an increase in NAD(P)H oxidase activity was detected in SHR VSMCs treated with 15d-PGJ(2)/LPS.

Conclusion: Our results indicate that the upregulatory effect of 15d-PGJ(2) on LPS-induced IL-8/CXCL8 expression in SHR VSMCs is mediated through the PPARgamma and ERK pathway, and may be related to NAD(P)H oxidase activity. However, p38 inactivation may also play an important role in 15d-PGJ(2)/LPS-induced IL-8/CXCL8 expression in SHR VSMCs.

No MeSH data available.


Related in: MedlinePlus