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Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1.

Koyama Y, Kotani M, Sawamura T, Kuribayashi M, Konishi R, Michinaga S - J Neuroinflammation (2013)

Bottom Line: The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ET(B) antagonist.ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1.The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca(2+) chelation and staurosporine.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-Kita, Tonda-bayashi, Osaka, 584-8540, Japan. koyamay@osaka-ohtani.ac.jp

ABSTRACT

Background: Chemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes.

Methods: Astrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA.

Results: Treatment of cultured astrocytes with ET-1 and Ala(1,3,11,15)-ET-1, an ET(B) agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala(1,3,11,15)-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ET(B) antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca(2+) chelation and staurosporine.

Conclusion: These findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains.

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Effect of ET-1 on chemokine mRNA expression in cultured ratastrocytes. (A) Serum-starved astrocytes were treatedwith 100 nM ET-1 for the times indicated. The expression of CCL2, CXCL1,CCL5, CXCL12 and CX3CL1 mRNA was normalized to G3PDH and expressed asthe % of 0 hour. Data are the mean ± SEM of 6 to 16 experiments.*P <0.05 and **P <0.01 versus 0hour by one-way ANOVA followed by Dunnett’s test. (B)Astrocytes were treated with the indicated concentrations of ET-1 forone (CCL2 and CXCL1) or six (CX3CL1) hours. Data are the mean ± SEMof five to eight experiments. *P <0.05 and **P<0.01 versus none by one-way ANOVA followed byDunnett’s test. ANOVA, analysis of variance; ET-1, endothelin-1;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error ofthe mean.
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Figure 1: Effect of ET-1 on chemokine mRNA expression in cultured ratastrocytes. (A) Serum-starved astrocytes were treatedwith 100 nM ET-1 for the times indicated. The expression of CCL2, CXCL1,CCL5, CXCL12 and CX3CL1 mRNA was normalized to G3PDH and expressed asthe % of 0 hour. Data are the mean ± SEM of 6 to 16 experiments.*P <0.05 and **P <0.01 versus 0hour by one-way ANOVA followed by Dunnett’s test. (B)Astrocytes were treated with the indicated concentrations of ET-1 forone (CCL2 and CXCL1) or six (CX3CL1) hours. Data are the mean ± SEMof five to eight experiments. *P <0.05 and **P<0.01 versus none by one-way ANOVA followed byDunnett’s test. ANOVA, analysis of variance; ET-1, endothelin-1;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error ofthe mean.

Mentions: Treatment of cultured astrocytes with 100 nM ET-1 increased mRNA levels of CCL2and CXCL1, where the maximum increase was observed in one hour(Figure 1A). In contrast, CX3CL1 mRNA decreasedfollowing ET-1 exposure to approximately 40% of levels observed in non-treatedcells in six hours. ET-1 did not affect mRNA levels of CCL5 and CXCL12. Theeffect of ET-1 on CCL2 and CXCL1 mRNA levels was dose-dependent and asignificant increase was observed at 10 nM (Figure 1B). The ET-induced decrease in astrocytic CX3CL1 mRNA was significantat 10 nM. Treatment with 100 nM Ala1,3,11,15-ET-1, a selectiveETB agonist, also increased CCL2 and CXCL1 mRNA levels incultured astrocytes, while it decreased CX3CL1 mRNA (Figure 2). Increases in CCL2 and CXCL1 mRNA by ET-1 were inhibited by 1μM BQ788, an ETB antagonist (Figure 3). BQ788 also inhibited the ET-induced decrease in CX3CL1 mRNA.FR139317 (1 μM), an ETA antagonist, did not inhibit the effectsof ET-1 on astrocytic CCL2, CXCL1 and CX3CL1 mRNA levels. The effects of ET-1 onchemokine release from cultured astrocytes were examined. Treatment with 100 nMET-1 for 1.5 to 3 hours increased the release of CCL2 and CXCL1 protein in theculture medium, while release of CX3CL1 protein into the culture mediumdecreased in the presence of ET-1 (Figure 4).


Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1.

Koyama Y, Kotani M, Sawamura T, Kuribayashi M, Konishi R, Michinaga S - J Neuroinflammation (2013)

Effect of ET-1 on chemokine mRNA expression in cultured ratastrocytes. (A) Serum-starved astrocytes were treatedwith 100 nM ET-1 for the times indicated. The expression of CCL2, CXCL1,CCL5, CXCL12 and CX3CL1 mRNA was normalized to G3PDH and expressed asthe % of 0 hour. Data are the mean ± SEM of 6 to 16 experiments.*P <0.05 and **P <0.01 versus 0hour by one-way ANOVA followed by Dunnett’s test. (B)Astrocytes were treated with the indicated concentrations of ET-1 forone (CCL2 and CXCL1) or six (CX3CL1) hours. Data are the mean ± SEMof five to eight experiments. *P <0.05 and **P<0.01 versus none by one-way ANOVA followed byDunnett’s test. ANOVA, analysis of variance; ET-1, endothelin-1;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error ofthe mean.
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Related In: Results  -  Collection

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Figure 1: Effect of ET-1 on chemokine mRNA expression in cultured ratastrocytes. (A) Serum-starved astrocytes were treatedwith 100 nM ET-1 for the times indicated. The expression of CCL2, CXCL1,CCL5, CXCL12 and CX3CL1 mRNA was normalized to G3PDH and expressed asthe % of 0 hour. Data are the mean ± SEM of 6 to 16 experiments.*P <0.05 and **P <0.01 versus 0hour by one-way ANOVA followed by Dunnett’s test. (B)Astrocytes were treated with the indicated concentrations of ET-1 forone (CCL2 and CXCL1) or six (CX3CL1) hours. Data are the mean ± SEMof five to eight experiments. *P <0.05 and **P<0.01 versus none by one-way ANOVA followed byDunnett’s test. ANOVA, analysis of variance; ET-1, endothelin-1;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error ofthe mean.
Mentions: Treatment of cultured astrocytes with 100 nM ET-1 increased mRNA levels of CCL2and CXCL1, where the maximum increase was observed in one hour(Figure 1A). In contrast, CX3CL1 mRNA decreasedfollowing ET-1 exposure to approximately 40% of levels observed in non-treatedcells in six hours. ET-1 did not affect mRNA levels of CCL5 and CXCL12. Theeffect of ET-1 on CCL2 and CXCL1 mRNA levels was dose-dependent and asignificant increase was observed at 10 nM (Figure 1B). The ET-induced decrease in astrocytic CX3CL1 mRNA was significantat 10 nM. Treatment with 100 nM Ala1,3,11,15-ET-1, a selectiveETB agonist, also increased CCL2 and CXCL1 mRNA levels incultured astrocytes, while it decreased CX3CL1 mRNA (Figure 2). Increases in CCL2 and CXCL1 mRNA by ET-1 were inhibited by 1μM BQ788, an ETB antagonist (Figure 3). BQ788 also inhibited the ET-induced decrease in CX3CL1 mRNA.FR139317 (1 μM), an ETA antagonist, did not inhibit the effectsof ET-1 on astrocytic CCL2, CXCL1 and CX3CL1 mRNA levels. The effects of ET-1 onchemokine release from cultured astrocytes were examined. Treatment with 100 nMET-1 for 1.5 to 3 hours increased the release of CCL2 and CXCL1 protein in theculture medium, while release of CX3CL1 protein into the culture mediumdecreased in the presence of ET-1 (Figure 4).

Bottom Line: The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ET(B) antagonist.ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1.The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca(2+) chelation and staurosporine.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Pharmacology, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-Kita, Tonda-bayashi, Osaka, 584-8540, Japan. koyamay@osaka-ohtani.ac.jp

ABSTRACT

Background: Chemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes.

Methods: Astrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA.

Results: Treatment of cultured astrocytes with ET-1 and Ala(1,3,11,15)-ET-1, an ET(B) agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala(1,3,11,15)-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ET(B) antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca(2+) chelation and staurosporine.

Conclusion: These findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains.

Show MeSH
Related in: MedlinePlus