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Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF.

Monville C, Fages C, Feyens AM, D'Hondt V, Guillet C, Vernallis A, Gascan H, Peschanski M - BMC Cell Biol. (2002)

Bottom Line: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression.The only effective proteins were IFNgamma and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF).These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U421/IM3, Créteil, France. monvillec@cf.ac.uk

ABSTRACT

Background: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures.

Results: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNgamma and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines (but not IFNgamma) stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNgamma produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF (which is internalised with its receptor) produced an overexpression of P-gp in CNTF-deficient astrocytes.

Conclusion: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

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Effect of cytokines on CNTF-intracellular content astrocytes. Among all the molecules tested, only LIF and CT-1 induced a CNTF-modulation in long term astrocyte culture. Addition of rhLIF during 24 h elicited a significant increase of P-gp expression both at 10 and 30 ng/ml (+56.12 % ± 25, factorial ANOVA significant at 95%, t-test p < 0.05* and +33.8 % ± 8, factorial ANOVA significant at 95%, t-test p < 0.01** respectively). The same pattern of regulation was observed with CT-1 (+44.5 % ± 12, factorial ANOVA significant at 95%, t-test p < 0.01** and +15.1 % ± 5, factorial ANOVA significant at 95%, t-test p < 0.05*).
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Figure 7: Effect of cytokines on CNTF-intracellular content astrocytes. Among all the molecules tested, only LIF and CT-1 induced a CNTF-modulation in long term astrocyte culture. Addition of rhLIF during 24 h elicited a significant increase of P-gp expression both at 10 and 30 ng/ml (+56.12 % ± 25, factorial ANOVA significant at 95%, t-test p < 0.05* and +33.8 % ± 8, factorial ANOVA significant at 95%, t-test p < 0.01** respectively). The same pattern of regulation was observed with CT-1 (+44.5 % ± 12, factorial ANOVA significant at 95%, t-test p < 0.01** and +15.1 % ± 5, factorial ANOVA significant at 95%, t-test p < 0.05*).

Mentions: The progressive maturation of P-gp during development and the regulation of its expression by members of the IL-6 family of cytokines are two features that P-gp shares with CNTF in astrocytes (see discussion section). Moreover, like P-gp, CNTF regulation is very specific, since, among different molecules that normally trigger astrogliosis, only those belonging to the IL-6 family provoked an increase of CNTF (Fig. 7). We, therefore, explored the possibility of a functional link between the two molecules by analysing whether the presence of endogenous CNTF in astrocytes was required for the observed modulation of P-gp expression by IL-6 type cytokines and IFN-γ. This was tested by analysing the effects of the cytokines in astrocytes taken from CNTF knockout mice. In these cultures, the modulation of P-gp by LIF, CT-1 or IL-6 (even in presence of its specific α receptor subunit) was completely lost (Fig. 4). In contrast, addition of rrCNTF (250 ng/ml) produced a significant increase of P-gp intracellular level. In the CNTF -/- astrocytes, addition of IFN-γ to the medium triggered an increase of P-gp cellular level comparable to that observed in wild-type astrocytes, underlining its direct effect on P-gp modulation (Fig. 6b).


Astroglial expression of the P-glycoprotein is controlled by intracellular CNTF.

Monville C, Fages C, Feyens AM, D'Hondt V, Guillet C, Vernallis A, Gascan H, Peschanski M - BMC Cell Biol. (2002)

Effect of cytokines on CNTF-intracellular content astrocytes. Among all the molecules tested, only LIF and CT-1 induced a CNTF-modulation in long term astrocyte culture. Addition of rhLIF during 24 h elicited a significant increase of P-gp expression both at 10 and 30 ng/ml (+56.12 % ± 25, factorial ANOVA significant at 95%, t-test p < 0.05* and +33.8 % ± 8, factorial ANOVA significant at 95%, t-test p < 0.01** respectively). The same pattern of regulation was observed with CT-1 (+44.5 % ± 12, factorial ANOVA significant at 95%, t-test p < 0.01** and +15.1 % ± 5, factorial ANOVA significant at 95%, t-test p < 0.05*).
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Related In: Results  -  Collection

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Figure 7: Effect of cytokines on CNTF-intracellular content astrocytes. Among all the molecules tested, only LIF and CT-1 induced a CNTF-modulation in long term astrocyte culture. Addition of rhLIF during 24 h elicited a significant increase of P-gp expression both at 10 and 30 ng/ml (+56.12 % ± 25, factorial ANOVA significant at 95%, t-test p < 0.05* and +33.8 % ± 8, factorial ANOVA significant at 95%, t-test p < 0.01** respectively). The same pattern of regulation was observed with CT-1 (+44.5 % ± 12, factorial ANOVA significant at 95%, t-test p < 0.01** and +15.1 % ± 5, factorial ANOVA significant at 95%, t-test p < 0.05*).
Mentions: The progressive maturation of P-gp during development and the regulation of its expression by members of the IL-6 family of cytokines are two features that P-gp shares with CNTF in astrocytes (see discussion section). Moreover, like P-gp, CNTF regulation is very specific, since, among different molecules that normally trigger astrogliosis, only those belonging to the IL-6 family provoked an increase of CNTF (Fig. 7). We, therefore, explored the possibility of a functional link between the two molecules by analysing whether the presence of endogenous CNTF in astrocytes was required for the observed modulation of P-gp expression by IL-6 type cytokines and IFN-γ. This was tested by analysing the effects of the cytokines in astrocytes taken from CNTF knockout mice. In these cultures, the modulation of P-gp by LIF, CT-1 or IL-6 (even in presence of its specific α receptor subunit) was completely lost (Fig. 4). In contrast, addition of rrCNTF (250 ng/ml) produced a significant increase of P-gp intracellular level. In the CNTF -/- astrocytes, addition of IFN-γ to the medium triggered an increase of P-gp cellular level comparable to that observed in wild-type astrocytes, underlining its direct effect on P-gp modulation (Fig. 6b).

Bottom Line: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression.The only effective proteins were IFNgamma and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF).These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U421/IM3, Créteil, France. monvillec@cf.ac.uk

ABSTRACT

Background: The P-glycoprotein (P-gp), an ATP binding cassette transmembrane transporter, is expressed by astrocytes in the adult brain, and is positively modulated during astrogliosis. In a search for factors involved in this modulation, P-gp overexpression was studied in long-term in vitro astroglial cultures.

Results: Surprisingly, most factors that are known to induce astroglial activation in astroglial cultures failed to increase P-gp expression. The only effective proteins were IFNgamma and those belonging to the IL-6 family of cytokines (IL-6, LIF, CT-1 and CNTF). As well as P-gp expression, the IL-6 type cytokines (but not IFNgamma) stimulated the expression of endogenous CNTF in astrocytes. In order to see whether an increased intracellular level of CNTF was necessary for induction of P-gp overexpression by IL-6 type cytokines, by the same cytokines analysis was carried out on astrocytes obtained from CNTF knockout mice. In these conditions, IFNgamma produced increased P-gp expression, but no overexpression of P-gp was observed with either IL-6, LIF or CT-1, pointing to a role of CNTF in the intracellular signalling pathway leading to P-gp overexpression. In agreement with this suggestion, application of exogenous CNTF (which is internalised with its receptor) produced an overexpression of P-gp in CNTF-deficient astrocytes.

Conclusion: These results reveal two different pathways regulating P-gp expression and activity in reactive astrocytes, one of which depends upon the intracellular concentration of CNTF. This regulation of P-gp may be one of the long searched for physiological roles of CNTF.

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