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ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Delestré L, Bakey Z, Prado C, Hoffmann S, Bihoreau MT, Lelongt B, Gauguier D - PLoS ONE (2015)

Bottom Line: Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction.Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation.Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universities, University Pierre and Marie Curie, University Paris Descartes, Sorbonne Paris Cité, INSERM, UMR_S1138, Cordeliers Research Centre, Paris, France.

ABSTRACT
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

No MeSH data available.


Related in: MedlinePlus

Induction of apoptosis and proliferation in in vivo Anks3 knock-down mice.(A) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(B, C) Abundance of caspase 9 (B) and caspase 3 (C) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (D) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (E) p53 mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to Gusb gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.
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pone.0136781.g007: Induction of apoptosis and proliferation in in vivo Anks3 knock-down mice.(A) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(B, C) Abundance of caspase 9 (B) and caspase 3 (C) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (D) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (E) p53 mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to Gusb gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.

Mentions: Owing to the role of many cilia proteins in cell cycle progression and proliferation and because increased apoptosis and proliferation capacity are critical early cellular events in cyst formation and PKD development in humans and mice, we investigated the role of Anks3 on these mechanisms in mouse kidneys. The proportion of apoptotic cells was significantly higher in ANKS3 ASO treated mice twelve days after LNA ASO injection when compared to SCR ASO controls (p<0.05) (Fig 7A). Increased apoptosis in ANKS3 ASO treated mice was associated with higher renal protein abundance of the initiator caspase (caspase 9) (Fig 7B), whereas expression of the effector caspase (caspase 3) was not affected (Fig 7C). Also, the percentage of proliferating cell nuclear antigen (PCNA) positive cells was markedly increased in the kidney of ANKS3 ASO treated mice four days after the last injection, but this effect was not statistically significant (p = 0.56) (Fig 7D). In addition, Anks3 downregulated expression was paralleled by reduced renal expression of p53 (p = 0.013 at day 4 post LNA ASO treatment), an actor of the cell cycle arrest (Fig 7E). However, the statistical significance of this difference appeared to be due to elevated expression of p53 in SCR ASO mice.


ANKS3 Co-Localises with ANKS6 in Mouse Renal Cilia and Is Associated with Vasopressin Signaling and Apoptosis In Vivo in Mice.

Delestré L, Bakey Z, Prado C, Hoffmann S, Bihoreau MT, Lelongt B, Gauguier D - PLoS ONE (2015)

Induction of apoptosis and proliferation in in vivo Anks3 knock-down mice.(A) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(B, C) Abundance of caspase 9 (B) and caspase 3 (C) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (D) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (E) p53 mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to Gusb gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.
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Related In: Results  -  Collection

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pone.0136781.g007: Induction of apoptosis and proliferation in in vivo Anks3 knock-down mice.(A) Immunofluorescence TUNEL was used to evaluate the percentage of apoptotic cells in mouse kidneys four days (D4 post inj.) and twelve days (D12 post inj.) after the final injection of scrambled (SCR) or ANKS3 AntiSense Oligonucleotides (ASO). Apoptosis was significantly increased in SCR ASO kidneys and in ANKS3ASO kidneys 12 days after the last injection.(B, C) Abundance of caspase 9 (B) and caspase 3 (C) proteins was evaluated by Western blot twelve days after final Locked Nucleic Acid modified (LNA) ASO injection (n = 5). Protein quantification was tested in duplicate and normalized to the level of ß-actin protein. (D) Percentage of PCNA positive cells in kidneys from mice that received SCR and ANKS3 ASO, 4 and 12 days after the last injection. (E) p53 mRNA expression was evaluated by quantitative RT-PCR four (n = 3) and twelve days (n = 5) post treatment in mice injected with LNA ASO, saline (CTR) and SCR ASO. Data are means ± SEM. Quantification of each cDNA was performed in duplicate and normalized to Gusb gene expression. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and control mice.*P<0.05 significantly different to control mice.
Mentions: Owing to the role of many cilia proteins in cell cycle progression and proliferation and because increased apoptosis and proliferation capacity are critical early cellular events in cyst formation and PKD development in humans and mice, we investigated the role of Anks3 on these mechanisms in mouse kidneys. The proportion of apoptotic cells was significantly higher in ANKS3 ASO treated mice twelve days after LNA ASO injection when compared to SCR ASO controls (p<0.05) (Fig 7A). Increased apoptosis in ANKS3 ASO treated mice was associated with higher renal protein abundance of the initiator caspase (caspase 9) (Fig 7B), whereas expression of the effector caspase (caspase 3) was not affected (Fig 7C). Also, the percentage of proliferating cell nuclear antigen (PCNA) positive cells was markedly increased in the kidney of ANKS3 ASO treated mice four days after the last injection, but this effect was not statistically significant (p = 0.56) (Fig 7D). In addition, Anks3 downregulated expression was paralleled by reduced renal expression of p53 (p = 0.013 at day 4 post LNA ASO treatment), an actor of the cell cycle arrest (Fig 7E). However, the statistical significance of this difference appeared to be due to elevated expression of p53 in SCR ASO mice.

Bottom Line: Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction.Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation.Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universities, University Pierre and Marie Curie, University Paris Descartes, Sorbonne Paris Cité, INSERM, UMR_S1138, Cordeliers Research Centre, Paris, France.

ABSTRACT
Mutations in Ankyrin repeat and sterile alpha motif domain containing 6 (ANKS6) play a causative role in renal cyst formation in the PKD/Mhm(cy/+) rat model of polycystic kidney disease and in nephronophthisis in humans. A network of protein partners of ANKS6 is emerging and their functional characterization provides important clues to understand the role of ANKS6 in renal biology and in mechanisms involved in the formation of renal cysts. Following experimental confirmation of interaction between ANKS6and ANKS3 using a Yeast two hybrid system, we demonstrated that binding between the two proteins occurs through their sterile alpha motif (SAM) and that the amino acid 823 in rat ANSK6 is key for this interaction. We further showed their interaction by co-immunoprecipitation and showed in vivo in mice that ANKS3 is present in renal cilia. Downregulated expression of Anks3 in vivo in mice by Locked Nucleic Acid (LNA) modified antisense oligonucleotides was associated with increased transcription of vasopressin-induced genes, suggesting changes in renal water permeability, and altered transcription of genes encoding proteins involved in cilium structure, apoptosis and cell proliferation. These data provide experimental evidence of ANKS3-ANKS6 direct interaction through their SAM domain and co-localisation in mouse renal cilia, and shed light on molecular mechanisms indirectly mediated by ANKS6 in the mouse kidney, that may be affected by altered ANKS3-ANKS6 interaction. Our results contribute to improved knowledge of the structure and function of the network of proteins interacting with ANKS6, which may represent therapeutic targets in cystic diseases.

No MeSH data available.


Related in: MedlinePlus