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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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Dose-dependent inhibition of ET-induced changes in CCL2, CXCL1 andCX3CL1 mRNA levels by signal transduction inhibitors.Serum-starved astrocytes were treated with 100 nM ET-1 for one (CCL2 andCXCL1) or six (CX3CL1) hours. Different concentrations of signaltransduction inhibitors (SN50, mithramycin, SB203580, SP600125 andstaurosporine) were included in the medium 30 minutes before treatmentwith ET-1. The expression of CCL2, CXCL1 and CX3CL1 mRNA was normalizedto G3PDH and expressed as the % of no treatment cultures. Data are themean ± SEM of 6 to 12 experiments. *P <0.05,**P <0.01 versus no treatment,#P <0.05, ##P <0.01versus ET-1 with no inhibitor by one-way ANOVA followed byFisher’s PLSD test. ANOVA, analysis of variance; ET, endothelin;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PLSD, protected leastsignificant difference; SEM, standard error of the mean.
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Figure 6: Dose-dependent inhibition of ET-induced changes in CCL2, CXCL1 andCX3CL1 mRNA levels by signal transduction inhibitors.Serum-starved astrocytes were treated with 100 nM ET-1 for one (CCL2 andCXCL1) or six (CX3CL1) hours. Different concentrations of signaltransduction inhibitors (SN50, mithramycin, SB203580, SP600125 andstaurosporine) were included in the medium 30 minutes before treatmentwith ET-1. The expression of CCL2, CXCL1 and CX3CL1 mRNA was normalizedto G3PDH and expressed as the % of no treatment cultures. Data are themean ± SEM of 6 to 12 experiments. *P <0.05,**P <0.01 versus no treatment,#P <0.05, ##P <0.01versus ET-1 with no inhibitor by one-way ANOVA followed byFisher’s PLSD test. ANOVA, analysis of variance; ET, endothelin;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PLSD, protected leastsignificant difference; SEM, standard error of the mean.

Mentions: ETB receptors belong to Gq-protein coupled receptors. Activation ofastrocytic ETB receptors induces an increase in cytosolicCa2+ and activation of protein kinase C (PKC) andmitogen-activated protein (MAP) kinases [25,33-35]. Ca2+ chelation (a combination of 0.5 mM ethylene glycoltetraacetic acid (EGTA) and 30 μM 1,2-bis(2-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester. (BAPTA/AM)) and PKC inhibition(staurosporine, 10 nM) did not affect ET-induced CCL2 and CXCL1 mRNA expression(Table 2). On the other hand, the decrease inCX3CL1 expression was inhibited by Ca2+ chelation and staurosporine.The inhibition by staurosporine was BAPTA/AM dose-dependent, where a significanteffect was obtained at 10 nM (Figure 6). SB203580 (ap38 inhibitor) and SP600125 (a JNK inhibitor) inhibited the effect of ET-1 onCCL2 and CXCL1 expression in a dose-dependent manner, but PD98059 (an ERKinhibitor, 50 μM) had no effect (Table 2 andFigure 6). The ET-induced decrease in CX3CL1expression was not affected by these MAP kinase inhibitors (Table 2). Pyrrolidine dithiocarbamate (PDTC, 100 μM) and SN50(10 μM), which inhibits the transcriptional activities of nuclearfactor-kappaB (NFκB), reduced ET-induced CCL2 and CXCL1 expressions, whilethese inhibitors did not alter the effects of ET-1 on CX3CL1 expression(Table 2 and Figure 6). Mithramycin (500 nM), an inhibitor of transcription factor SP1,diminished ET-induced CCL2 and CXCL1 expression, but had no effect on thedecrease of CX3CL1 expression (Table 2 andFigure 6). At the highest concentrations used,these signal transduction inhibitors did not largely affect basal expressions ofastrocytic CCL2, CXCL1 and CX3CL1 mRNAs [see Additional file 1].


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)

Dose-dependent inhibition of ET-induced changes in CCL2, CXCL1 andCX3CL1 mRNA levels by signal transduction inhibitors.Serum-starved astrocytes were treated with 100 nM ET-1 for one (CCL2 andCXCL1) or six (CX3CL1) hours. Different concentrations of signaltransduction inhibitors (SN50, mithramycin, SB203580, SP600125 andstaurosporine) were included in the medium 30 minutes before treatmentwith ET-1. The expression of CCL2, CXCL1 and CX3CL1 mRNA was normalizedto G3PDH and expressed as the % of no treatment cultures. Data are themean ± SEM of 6 to 12 experiments. *P <0.05,**P <0.01 versus no treatment,#P <0.05, ##P <0.01versus ET-1 with no inhibitor by one-way ANOVA followed byFisher’s PLSD test. ANOVA, analysis of variance; ET, endothelin;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PLSD, protected leastsignificant difference; SEM, standard error of the mean.
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Figure 6: Dose-dependent inhibition of ET-induced changes in CCL2, CXCL1 andCX3CL1 mRNA levels by signal transduction inhibitors.Serum-starved astrocytes were treated with 100 nM ET-1 for one (CCL2 andCXCL1) or six (CX3CL1) hours. Different concentrations of signaltransduction inhibitors (SN50, mithramycin, SB203580, SP600125 andstaurosporine) were included in the medium 30 minutes before treatmentwith ET-1. The expression of CCL2, CXCL1 and CX3CL1 mRNA was normalizedto G3PDH and expressed as the % of no treatment cultures. Data are themean ± SEM of 6 to 12 experiments. *P <0.05,**P <0.01 versus no treatment,#P <0.05, ##P <0.01versus ET-1 with no inhibitor by one-way ANOVA followed byFisher’s PLSD test. ANOVA, analysis of variance; ET, endothelin;G3PDH, glyceraldehyde-3-phosphate dehydrogenase; PLSD, protected leastsignificant difference; SEM, standard error of the mean.
Mentions: ETB receptors belong to Gq-protein coupled receptors. Activation ofastrocytic ETB receptors induces an increase in cytosolicCa2+ and activation of protein kinase C (PKC) andmitogen-activated protein (MAP) kinases [25,33-35]. Ca2+ chelation (a combination of 0.5 mM ethylene glycoltetraacetic acid (EGTA) and 30 μM 1,2-bis(2-aminophenoxy)ethaneN,N,N',N'-tetraacetic acid acetoxymethyl ester. (BAPTA/AM)) and PKC inhibition(staurosporine, 10 nM) did not affect ET-induced CCL2 and CXCL1 mRNA expression(Table 2). On the other hand, the decrease inCX3CL1 expression was inhibited by Ca2+ chelation and staurosporine.The inhibition by staurosporine was BAPTA/AM dose-dependent, where a significanteffect was obtained at 10 nM (Figure 6). SB203580 (ap38 inhibitor) and SP600125 (a JNK inhibitor) inhibited the effect of ET-1 onCCL2 and CXCL1 expression in a dose-dependent manner, but PD98059 (an ERKinhibitor, 50 μM) had no effect (Table 2 andFigure 6). The ET-induced decrease in CX3CL1expression was not affected by these MAP kinase inhibitors (Table 2). Pyrrolidine dithiocarbamate (PDTC, 100 μM) and SN50(10 μM), which inhibits the transcriptional activities of nuclearfactor-kappaB (NFκB), reduced ET-induced CCL2 and CXCL1 expressions, whilethese inhibitors did not alter the effects of ET-1 on CX3CL1 expression(Table 2 and Figure 6). Mithramycin (500 nM), an inhibitor of transcription factor SP1,diminished ET-induced CCL2 and CXCL1 expression, but had no effect on thedecrease of CX3CL1 expression (Table 2 andFigure 6). At the highest concentrations used,these signal transduction inhibitors did not largely affect basal expressions ofastrocytic CCL2, CXCL1 and CX3CL1 mRNAs [see Additional file 1].

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