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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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MDR1mRNA and P-gp expression in astroglial cultures. a/ RT-PCR showing MDR1mRNA. b/ Western blotting using rabbit anti-P-gp polyclonal antibody demonstrating the presence of P-gp as early as 2 div. Between 2 div and 5 div, a band is observed at about 120 kDa (lower black arrow); a doublet appears at 6 div at about 150–170 kDa (black arrows); the band corresponding to the mature form of P-gp, is expected at 170 kDa (grey arrow), and becomes increased after 15 div.
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Figure 1: MDR1mRNA and P-gp expression in astroglial cultures. a/ RT-PCR showing MDR1mRNA. b/ Western blotting using rabbit anti-P-gp polyclonal antibody demonstrating the presence of P-gp as early as 2 div. Between 2 div and 5 div, a band is observed at about 120 kDa (lower black arrow); a doublet appears at 6 div at about 150–170 kDa (black arrows); the band corresponding to the mature form of P-gp, is expected at 170 kDa (grey arrow), and becomes increased after 15 div.

Mentions: Astrocytes in culture expressed P-glycoprotein as early as 24 h after plating. The RT-PCR performed on a fragment of the P-gp transcript yielded a 167-bp product (Fig. 1a), present in all astrocyte samples and in the MES-SA/MX2 cells used as a positive control. At the protein level, the results varied over time: until the 4th day in vitro (div), the antibody raised against P-gp detected a band at about 120 kDa; from the 6th div a doublet was detected at about 160–170 kDa; finally, at 15 div this doublet became predominant and its expression increased (Fig. 1b).


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)

MDR1mRNA and P-gp expression in astroglial cultures. a/ RT-PCR showing MDR1mRNA. b/ Western blotting using rabbit anti-P-gp polyclonal antibody demonstrating the presence of P-gp as early as 2 div. Between 2 div and 5 div, a band is observed at about 120 kDa (lower black arrow); a doublet appears at 6 div at about 150–170 kDa (black arrows); the band corresponding to the mature form of P-gp, is expected at 170 kDa (grey arrow), and becomes increased after 15 div.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117802&req=5

Figure 1: MDR1mRNA and P-gp expression in astroglial cultures. a/ RT-PCR showing MDR1mRNA. b/ Western blotting using rabbit anti-P-gp polyclonal antibody demonstrating the presence of P-gp as early as 2 div. Between 2 div and 5 div, a band is observed at about 120 kDa (lower black arrow); a doublet appears at 6 div at about 150–170 kDa (black arrows); the band corresponding to the mature form of P-gp, is expected at 170 kDa (grey arrow), and becomes increased after 15 div.
Mentions: Astrocytes in culture expressed P-glycoprotein as early as 24 h after plating. The RT-PCR performed on a fragment of the P-gp transcript yielded a 167-bp product (Fig. 1a), present in all astrocyte samples and in the MES-SA/MX2 cells used as a positive control. At the protein level, the results varied over time: until the 4th day in vitro (div), the antibody raised against P-gp detected a band at about 120 kDa; from the 6th div a doublet was detected at about 160–170 kDa; finally, at 15 div this doublet became predominant and its expression increased (Fig. 1b).

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.

Show MeSH