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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

In vivo LNA ASO-induced downregulation ofAnks3 expression in mouse kidney.(A) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (B-D) Anks3 renal decreased expression does not affect expression of Anks6. Abundance of Anks3 transcripts (B) and protein (C), and Anks6 transcripts (D) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to Actb gene expression level. (C) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.
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pone.0136781.g005: In vivo LNA ASO-induced downregulation ofAnks3 expression in mouse kidney.(A) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (B-D) Anks3 renal decreased expression does not affect expression of Anks6. Abundance of Anks3 transcripts (B) and protein (C), and Anks6 transcripts (D) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to Actb gene expression level. (C) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.

Mentions: In rodents, only 20% of nephrons are present at birth and the nephrogenesis occurs from E11 to postnatal day 7 (PN7) [16]. To study the in vivo function of ANKS3 in the kidney, we tested the biological consequences of Anks3 downregulated expression induced by Locked Nucleic Acid modified Antisense Oligonucleotides (LNA ASO) in newborn mice. Confocal microscopy analysis revealed the presence of LNA ASO in the kidney of both 22- and 30-days old mice (i.e. 4 and 12 days after the last injection) and the co-localisation of ANKS3 and ANKS3 ASO in the same tubules (Fig 5A). Four days after the final injection of LNA ASOs, renal abundance of Anks3 transcripts was significantly reduced by over 70% in ANKS3 ASO mice when compared to control mice treated with either saline or a scrambled oligonucleotide (SCR ASO) (Fig 5B). Even 12 days after the final injection of LNA ASOs, renal Anks3 mRNA levels remained significantly lower in ANKS3 ASO mice than in saline- and SCR ASO-treated mice (-22%). Reduced amount of ANKS3 transcripts in ANKS3 ASO mice was paralleled by significant reduction in abundance of ANKS3 protein when compared to mice treated with saline or SCR ASO (up to -20%; P<0.02), which confirms that significant inhibition of ANKS3 expression in vivo was achieved and was specific to LNA ASO treatment (Fig 5C).


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)

In vivo LNA ASO-induced downregulation ofAnks3 expression in mouse kidney.(A) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (B-D) Anks3 renal decreased expression does not affect expression of Anks6. Abundance of Anks3 transcripts (B) and protein (C), and Anks6 transcripts (D) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to Actb gene expression level. (C) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.
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pone.0136781.g005: In vivo LNA ASO-induced downregulation ofAnks3 expression in mouse kidney.(A) Presence of ANKS3 in kidneys treated with saline (CTR) and scrambled AntiSense Oligonucleotides (SCR ASO) 12 days post injection and with ANKS3 Locked Nucleic Acid modified AntiSense Oligonucleotides (ANKS3 LNA ASOs). Representative photomicrographs of kidney sections from 5 mice four and twelve days after the last injection of LNA ASOs (D4 post inj., D12 post inj.). Kidneys were stained with ANKS3 (revealed by Alexa Fluor 488 in green) and the LNA ASOs were localised with the Alexa Fluor 647 marker in red. (B-D) Anks3 renal decreased expression does not affect expression of Anks6. Abundance of Anks3 transcripts (B) and protein (C), and Anks6 transcripts (D) were evaluated in mouse kidneys four days (D4 post inj., n = 3) and twelve days (D12 post inj., n = 5) after the final injection. cDNA quantification was performed in duplicate and normalized to Actb gene expression level. (C) Western blots (upper panel) and quantitative protein analysis (lower panel) performed with extracts from kidneys of 5 mice treated with saline (Ctr), scramble ASO (SCR ASO) or ANKS3 ASO four and twelve days after the last injection showed that expression of ANKS3 normalized to that of β-actin is significantly decreased in mice treated with ANKS3 ASO. Data are mean ± SEM. Non-parametric Mann-Whitney U test was used to assess differences between ANKS3 ASO and SCR ASO treated mice.**P<0.01; *P<0.05 significantly different to control mice treated with saline (Ctr) or SCR ASO.
Mentions: In rodents, only 20% of nephrons are present at birth and the nephrogenesis occurs from E11 to postnatal day 7 (PN7) [16]. To study the in vivo function of ANKS3 in the kidney, we tested the biological consequences of Anks3 downregulated expression induced by Locked Nucleic Acid modified Antisense Oligonucleotides (LNA ASO) in newborn mice. Confocal microscopy analysis revealed the presence of LNA ASO in the kidney of both 22- and 30-days old mice (i.e. 4 and 12 days after the last injection) and the co-localisation of ANKS3 and ANKS3 ASO in the same tubules (Fig 5A). Four days after the final injection of LNA ASOs, renal abundance of Anks3 transcripts was significantly reduced by over 70% in ANKS3 ASO mice when compared to control mice treated with either saline or a scrambled oligonucleotide (SCR ASO) (Fig 5B). Even 12 days after the final injection of LNA ASOs, renal Anks3 mRNA levels remained significantly lower in ANKS3 ASO mice than in saline- and SCR ASO-treated mice (-22%). Reduced amount of ANKS3 transcripts in ANKS3 ASO mice was paralleled by significant reduction in abundance of ANKS3 protein when compared to mice treated with saline or SCR ASO (up to -20%; P<0.02), which confirms that significant inhibition of ANKS3 expression in vivo was achieved and was specific to LNA ASO treatment (Fig 5C).

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