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A Drosophila ABC transporter regulates lifespan.

Huang H, Lu-Bo Y, Haddad GG - PLoS Genet. (2014)

Bottom Line: MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes.By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition.We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics (Division of Respiratory Medicine), University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.

No MeSH data available.


Related in: MedlinePlus

dMRP4 is required for oxidative stress resistance.(A) Molecular analysis of dMRP4 mutants. The solid bar represents the genomic region of dMRP4. The bent arrow indicates the transcription start site of dMRP4 gene. The open triangles show the insertion positions of EP3655 and EP3177. Open boxes below the solid bar represent exons of dMRP4 transcript and filled boxes indicate the encoding protein sequences. The span of both deletions was determined by sequencing the corresponding regions with specific primers. The arrows were primers for dMRP4-related semi-quantitative RT-PCR and arrowheads for qt-PCR experiments. The deleted sequences were described in Materials and Methods. (B) Expression of dMRP4 mRNA in two mutant alleles. Semi-quantitative RT-PCR was used to determine the levels of dMRP4 mRNA expression. dMRP4 mRNA was under-detectable in dMRPM1/M1 (M1) or dMRPM2/M2 (M2). Actin5C served as an internal standard. (C) qt-PCR analysis of dMRP4 mRNA in two dMRP4 alleles. (D) Effects of paraquat-induced oxidative stress on dMRP4 mutant flies (n = 180 for each group). (E) Effects of hydrogen peroxide-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). (F) Effects of hyperoxia-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). Error bars represent S.E.
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pgen-1004844-g002: dMRP4 is required for oxidative stress resistance.(A) Molecular analysis of dMRP4 mutants. The solid bar represents the genomic region of dMRP4. The bent arrow indicates the transcription start site of dMRP4 gene. The open triangles show the insertion positions of EP3655 and EP3177. Open boxes below the solid bar represent exons of dMRP4 transcript and filled boxes indicate the encoding protein sequences. The span of both deletions was determined by sequencing the corresponding regions with specific primers. The arrows were primers for dMRP4-related semi-quantitative RT-PCR and arrowheads for qt-PCR experiments. The deleted sequences were described in Materials and Methods. (B) Expression of dMRP4 mRNA in two mutant alleles. Semi-quantitative RT-PCR was used to determine the levels of dMRP4 mRNA expression. dMRP4 mRNA was under-detectable in dMRPM1/M1 (M1) or dMRPM2/M2 (M2). Actin5C served as an internal standard. (C) qt-PCR analysis of dMRP4 mRNA in two dMRP4 alleles. (D) Effects of paraquat-induced oxidative stress on dMRP4 mutant flies (n = 180 for each group). (E) Effects of hydrogen peroxide-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). (F) Effects of hyperoxia-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). Error bars represent S.E.

Mentions: To test whether dMRP4 indeed might play a role in oxidative stress resistance, we generated two mutations by excision of two independent EP elements near the dMRP4 gene (Fig. 2A). Analysis of the dMRP4 expression by RT-PCR indicated that dMRP4 RNA was undetectable in these mutants (Fig. 2B). However, the more sensitive assay with qt-PCR revealed about 8% dMRP4 mRNA retaining in both homozygous mutations (Fig. 2C). Currently it is not clear if this transcript residual was resulted from splice forms of the predicted full length mRNA or from an alternative transcription start site of the remaining dMRP4 transcript after the truncation. Nevertheless, these results indicate that the two dMRP4 alleles represent strong loss-of-function mutations. In addition, flies homozygous for both mutations were viable and fertile, suggesting that dMRP4 may not be an essential gene for development. However, it cannot be ruled out that the remaining residual in these mutations might still retain some vital function during development.


A Drosophila ABC transporter regulates lifespan.

Huang H, Lu-Bo Y, Haddad GG - PLoS Genet. (2014)

dMRP4 is required for oxidative stress resistance.(A) Molecular analysis of dMRP4 mutants. The solid bar represents the genomic region of dMRP4. The bent arrow indicates the transcription start site of dMRP4 gene. The open triangles show the insertion positions of EP3655 and EP3177. Open boxes below the solid bar represent exons of dMRP4 transcript and filled boxes indicate the encoding protein sequences. The span of both deletions was determined by sequencing the corresponding regions with specific primers. The arrows were primers for dMRP4-related semi-quantitative RT-PCR and arrowheads for qt-PCR experiments. The deleted sequences were described in Materials and Methods. (B) Expression of dMRP4 mRNA in two mutant alleles. Semi-quantitative RT-PCR was used to determine the levels of dMRP4 mRNA expression. dMRP4 mRNA was under-detectable in dMRPM1/M1 (M1) or dMRPM2/M2 (M2). Actin5C served as an internal standard. (C) qt-PCR analysis of dMRP4 mRNA in two dMRP4 alleles. (D) Effects of paraquat-induced oxidative stress on dMRP4 mutant flies (n = 180 for each group). (E) Effects of hydrogen peroxide-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). (F) Effects of hyperoxia-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). Error bars represent S.E.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256198&req=5

pgen-1004844-g002: dMRP4 is required for oxidative stress resistance.(A) Molecular analysis of dMRP4 mutants. The solid bar represents the genomic region of dMRP4. The bent arrow indicates the transcription start site of dMRP4 gene. The open triangles show the insertion positions of EP3655 and EP3177. Open boxes below the solid bar represent exons of dMRP4 transcript and filled boxes indicate the encoding protein sequences. The span of both deletions was determined by sequencing the corresponding regions with specific primers. The arrows were primers for dMRP4-related semi-quantitative RT-PCR and arrowheads for qt-PCR experiments. The deleted sequences were described in Materials and Methods. (B) Expression of dMRP4 mRNA in two mutant alleles. Semi-quantitative RT-PCR was used to determine the levels of dMRP4 mRNA expression. dMRP4 mRNA was under-detectable in dMRPM1/M1 (M1) or dMRPM2/M2 (M2). Actin5C served as an internal standard. (C) qt-PCR analysis of dMRP4 mRNA in two dMRP4 alleles. (D) Effects of paraquat-induced oxidative stress on dMRP4 mutant flies (n = 180 for each group). (E) Effects of hydrogen peroxide-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). (F) Effects of hyperoxia-induced oxidative stress on dMRP4 mutant flies (n = 200 for each group). Error bars represent S.E.
Mentions: To test whether dMRP4 indeed might play a role in oxidative stress resistance, we generated two mutations by excision of two independent EP elements near the dMRP4 gene (Fig. 2A). Analysis of the dMRP4 expression by RT-PCR indicated that dMRP4 RNA was undetectable in these mutants (Fig. 2B). However, the more sensitive assay with qt-PCR revealed about 8% dMRP4 mRNA retaining in both homozygous mutations (Fig. 2C). Currently it is not clear if this transcript residual was resulted from splice forms of the predicted full length mRNA or from an alternative transcription start site of the remaining dMRP4 transcript after the truncation. Nevertheless, these results indicate that the two dMRP4 alleles represent strong loss-of-function mutations. In addition, flies homozygous for both mutations were viable and fertile, suggesting that dMRP4 may not be an essential gene for development. However, it cannot be ruled out that the remaining residual in these mutations might still retain some vital function during development.

Bottom Line: MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes.By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition.We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics (Division of Respiratory Medicine), University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling.

No MeSH data available.


Related in: MedlinePlus