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Epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality.

Crambert G, Ernandez T, Lamouroux C, Roth I, Dizin E, Martin PY, Féraille E, Hasler U - Physiol Rep (2014)

Bottom Line: Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress.Both hyperosmolality and chemical induction of ER stress decreased ENaC expression in vitro.ENaC depletion by either stimulus was abolished by transcriptional inhibition and by the chemical chaperone 4-phenylbutyric acid and was partly abrogated by either PERK or ATF6 silencing.

View Article: PubMed Central - PubMed

Affiliation: UPMC/INSERM/Paris Descartes U1138 CNRS ERL 8228, Equipe 3 Métabolisme et Physiologie Rénale, Centre de Recherche des Cordeliers, Paris, France.

No MeSH data available.


Related in: MedlinePlus

PERK and ATF6 activities are increased by NaCl. (A) Western blot analysis of phosphorylated PERK in mCCDcl1 cells challenged or not (Ctl) with NaCl (500 mOsmol/kg), thapsigargin (Tg, 1 μmol/L) or tunicamycin (Tun, 3 μmol/L) for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (B) PCR analysis of XBP‐1 mRNA splicing in lysates of cells challenged or not (Ctl) with Tg or Tun for 6 h or with NaCl for 30 min to 6 h. Bands >200 bp were consistently observed in samples from cells challenged with either chemical compound and may be experimental artifacts. A representative gel from three similar experiments is shown. (C) Western blot analysis of nuclear ATF4, XBP‐1 and ATF6 in cells challenged with NaCl for 1 or 3 h or Tg or Tun for 3 h. Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots of cytoplasmic and nuclear extracts are shown. α‐tubulin was used as a control of nuclear extract purity. (D) mRNA abundance of ATF6 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 μmol/L) for 3 h (left panel) or in mCCDcl1 cells challenged or not (Ctl) with Tg or Tun for 6 h (left panel) or with NaCl or urea (500 mOsmol/kg) for 1–24 h (right panel). Data is represented as fold difference over non‐stimulated CCD/cells and is expressed as the mean ± SEM of data from at least six animals or 4 experiments (for mCCDcl1 cells). (E) Confocal z‐stacks of ATF4, XBP‐1 and ATF6 in cells challenged or not (Ctl) with NaCl, Tg or Tun for 3 h. (F) Confocal single‐frame images and z‐stacks of ATF6 (green) and GM130 (red), a cis‐Golgi matrix protein, in cells challenged or not (Ctl) with NaCl for 30 min or 3 h. ATF6 accumulated in the Golgi after 30 min of challenge, as suggested by increased yellow staining, and in the nucleus (stained with Hoechst 33342, blue, shown in z‐stacks) after 3 h of challenge. Inserts depict enlarged images of regions depicted by white squares. For (E) and (F), representative images of three similar experiments are shown. Bar, 10 μmol/L.
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fig05: PERK and ATF6 activities are increased by NaCl. (A) Western blot analysis of phosphorylated PERK in mCCDcl1 cells challenged or not (Ctl) with NaCl (500 mOsmol/kg), thapsigargin (Tg, 1 μmol/L) or tunicamycin (Tun, 3 μmol/L) for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (B) PCR analysis of XBP‐1 mRNA splicing in lysates of cells challenged or not (Ctl) with Tg or Tun for 6 h or with NaCl for 30 min to 6 h. Bands >200 bp were consistently observed in samples from cells challenged with either chemical compound and may be experimental artifacts. A representative gel from three similar experiments is shown. (C) Western blot analysis of nuclear ATF4, XBP‐1 and ATF6 in cells challenged with NaCl for 1 or 3 h or Tg or Tun for 3 h. Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots of cytoplasmic and nuclear extracts are shown. α‐tubulin was used as a control of nuclear extract purity. (D) mRNA abundance of ATF6 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 μmol/L) for 3 h (left panel) or in mCCDcl1 cells challenged or not (Ctl) with Tg or Tun for 6 h (left panel) or with NaCl or urea (500 mOsmol/kg) for 1–24 h (right panel). Data is represented as fold difference over non‐stimulated CCD/cells and is expressed as the mean ± SEM of data from at least six animals or 4 experiments (for mCCDcl1 cells). (E) Confocal z‐stacks of ATF4, XBP‐1 and ATF6 in cells challenged or not (Ctl) with NaCl, Tg or Tun for 3 h. (F) Confocal single‐frame images and z‐stacks of ATF6 (green) and GM130 (red), a cis‐Golgi matrix protein, in cells challenged or not (Ctl) with NaCl for 30 min or 3 h. ATF6 accumulated in the Golgi after 30 min of challenge, as suggested by increased yellow staining, and in the nucleus (stained with Hoechst 33342, blue, shown in z‐stacks) after 3 h of challenge. Inserts depict enlarged images of regions depicted by white squares. For (E) and (F), representative images of three similar experiments are shown. Bar, 10 μmol/L.

Mentions: The mammalian UPR involves three canonical ER‐resident transmembrane proteins: RNA‐dependent protein kinase‐like ER kinase (PERK), inositol‐requiring ER‐to‐nucleus signal kinase 1 (IRE1α) and activating transcription factor 6 (ATF6). PERK phosphorylation, and consequent activation, was transiently increased by NaCl, albeit at lower levels than by either Tg or Tun, as would be expected (Fig. 5A). X box‐binding protein 1 (XBP1) mRNA splicing, used as an indicator of IRE1 activity, was induced by Tg and Tun but not NaCl (Fig. 5B). Analysis of nuclear ATF4, XBP‐1 and ATF6 expression, by Western blot analysis of nuclear extracts (Fig. 5C) and immunofluorescence (Fig. 5E) revealed that NaCl increased nuclear localization of XBP‐1 and ATF6 but not ATF4. ATF6 activation by NaCl was examined further. Tg and Tun increased, and NaCl transiently increased, ATF6 mRNA (Fig. 5D), reflecting increased ATF6 protein abundance (Fig. 5C). ATF6 mRNA levels were also significantly increased in microdissected CD challenged with either NaCl or Tg (Fig. 5D). We further investigated the ATF6 arm of the UPR by examining its intracellular distribution following NaCl challenge (Fig. 5F). ER stress increases translocation of an inactive precursor of ATF6 from the ER to the Golgi, where it is cleaved by Golgi‐resident proteases. The cytosolic fragment is an active transcription factor that translocates to the nucleus. Under isotonic conditions, low levels of ATF6 were observed in the nucleus and Golgi, as revealed by its co‐localization with Hoechst 33342, a nuclear stain, and GM130, a cis‐Golgi matrix protein (Fig. 5F). We have previously observed strong alterations of Golgi morphology upon NaCl challenge, characterized by the appearance of symmetrical Golgi structures that circumvent the centrosome (Nunes et al. 2013). These structural alterations were accompanied by an accumulation of ATF6 in the Golgi after 30 min of NaCl challenge (Fig. 5F), indicating increased translocation of pre‐existing ATF6 to the Golgi. Golgi morphology recovered after 3 h of NaCl challenge, at which time ATF6 accumulated in both the Golgi and nucleus (Fig. 5F), presumably as a consequence of de novo ATF6 protein synthesis (Fig. 5C and D). ATF6 signal specificity was verified using another anti‐ATF6 antibody (Sigma‐Aldrich) and the signal obtained by either antibody was abated by siRNA against ATF6 (not shown). Together, these observations indicate that while Tg and Tun strongly activate PERK, IRE1α and ATF6, NaCl activates PERK and ATF6.


Epithelial sodium channel abundance is decreased by an unfolded protein response induced by hyperosmolality.

Crambert G, Ernandez T, Lamouroux C, Roth I, Dizin E, Martin PY, Féraille E, Hasler U - Physiol Rep (2014)

PERK and ATF6 activities are increased by NaCl. (A) Western blot analysis of phosphorylated PERK in mCCDcl1 cells challenged or not (Ctl) with NaCl (500 mOsmol/kg), thapsigargin (Tg, 1 μmol/L) or tunicamycin (Tun, 3 μmol/L) for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (B) PCR analysis of XBP‐1 mRNA splicing in lysates of cells challenged or not (Ctl) with Tg or Tun for 6 h or with NaCl for 30 min to 6 h. Bands >200 bp were consistently observed in samples from cells challenged with either chemical compound and may be experimental artifacts. A representative gel from three similar experiments is shown. (C) Western blot analysis of nuclear ATF4, XBP‐1 and ATF6 in cells challenged with NaCl for 1 or 3 h or Tg or Tun for 3 h. Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots of cytoplasmic and nuclear extracts are shown. α‐tubulin was used as a control of nuclear extract purity. (D) mRNA abundance of ATF6 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 μmol/L) for 3 h (left panel) or in mCCDcl1 cells challenged or not (Ctl) with Tg or Tun for 6 h (left panel) or with NaCl or urea (500 mOsmol/kg) for 1–24 h (right panel). Data is represented as fold difference over non‐stimulated CCD/cells and is expressed as the mean ± SEM of data from at least six animals or 4 experiments (for mCCDcl1 cells). (E) Confocal z‐stacks of ATF4, XBP‐1 and ATF6 in cells challenged or not (Ctl) with NaCl, Tg or Tun for 3 h. (F) Confocal single‐frame images and z‐stacks of ATF6 (green) and GM130 (red), a cis‐Golgi matrix protein, in cells challenged or not (Ctl) with NaCl for 30 min or 3 h. ATF6 accumulated in the Golgi after 30 min of challenge, as suggested by increased yellow staining, and in the nucleus (stained with Hoechst 33342, blue, shown in z‐stacks) after 3 h of challenge. Inserts depict enlarged images of regions depicted by white squares. For (E) and (F), representative images of three similar experiments are shown. Bar, 10 μmol/L.
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fig05: PERK and ATF6 activities are increased by NaCl. (A) Western blot analysis of phosphorylated PERK in mCCDcl1 cells challenged or not (Ctl) with NaCl (500 mOsmol/kg), thapsigargin (Tg, 1 μmol/L) or tunicamycin (Tun, 3 μmol/L) for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (B) PCR analysis of XBP‐1 mRNA splicing in lysates of cells challenged or not (Ctl) with Tg or Tun for 6 h or with NaCl for 30 min to 6 h. Bands >200 bp were consistently observed in samples from cells challenged with either chemical compound and may be experimental artifacts. A representative gel from three similar experiments is shown. (C) Western blot analysis of nuclear ATF4, XBP‐1 and ATF6 in cells challenged with NaCl for 1 or 3 h or Tg or Tun for 3 h. Data is represented as fold difference over non‐stimulated cells (Ctl) and is expressed as the mean ± SEM of three independent experiments. Representative Western blots of cytoplasmic and nuclear extracts are shown. α‐tubulin was used as a control of nuclear extract purity. (D) mRNA abundance of ATF6 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 μmol/L) for 3 h (left panel) or in mCCDcl1 cells challenged or not (Ctl) with Tg or Tun for 6 h (left panel) or with NaCl or urea (500 mOsmol/kg) for 1–24 h (right panel). Data is represented as fold difference over non‐stimulated CCD/cells and is expressed as the mean ± SEM of data from at least six animals or 4 experiments (for mCCDcl1 cells). (E) Confocal z‐stacks of ATF4, XBP‐1 and ATF6 in cells challenged or not (Ctl) with NaCl, Tg or Tun for 3 h. (F) Confocal single‐frame images and z‐stacks of ATF6 (green) and GM130 (red), a cis‐Golgi matrix protein, in cells challenged or not (Ctl) with NaCl for 30 min or 3 h. ATF6 accumulated in the Golgi after 30 min of challenge, as suggested by increased yellow staining, and in the nucleus (stained with Hoechst 33342, blue, shown in z‐stacks) after 3 h of challenge. Inserts depict enlarged images of regions depicted by white squares. For (E) and (F), representative images of three similar experiments are shown. Bar, 10 μmol/L.
Mentions: The mammalian UPR involves three canonical ER‐resident transmembrane proteins: RNA‐dependent protein kinase‐like ER kinase (PERK), inositol‐requiring ER‐to‐nucleus signal kinase 1 (IRE1α) and activating transcription factor 6 (ATF6). PERK phosphorylation, and consequent activation, was transiently increased by NaCl, albeit at lower levels than by either Tg or Tun, as would be expected (Fig. 5A). X box‐binding protein 1 (XBP1) mRNA splicing, used as an indicator of IRE1 activity, was induced by Tg and Tun but not NaCl (Fig. 5B). Analysis of nuclear ATF4, XBP‐1 and ATF6 expression, by Western blot analysis of nuclear extracts (Fig. 5C) and immunofluorescence (Fig. 5E) revealed that NaCl increased nuclear localization of XBP‐1 and ATF6 but not ATF4. ATF6 activation by NaCl was examined further. Tg and Tun increased, and NaCl transiently increased, ATF6 mRNA (Fig. 5D), reflecting increased ATF6 protein abundance (Fig. 5C). ATF6 mRNA levels were also significantly increased in microdissected CD challenged with either NaCl or Tg (Fig. 5D). We further investigated the ATF6 arm of the UPR by examining its intracellular distribution following NaCl challenge (Fig. 5F). ER stress increases translocation of an inactive precursor of ATF6 from the ER to the Golgi, where it is cleaved by Golgi‐resident proteases. The cytosolic fragment is an active transcription factor that translocates to the nucleus. Under isotonic conditions, low levels of ATF6 were observed in the nucleus and Golgi, as revealed by its co‐localization with Hoechst 33342, a nuclear stain, and GM130, a cis‐Golgi matrix protein (Fig. 5F). We have previously observed strong alterations of Golgi morphology upon NaCl challenge, characterized by the appearance of symmetrical Golgi structures that circumvent the centrosome (Nunes et al. 2013). These structural alterations were accompanied by an accumulation of ATF6 in the Golgi after 30 min of NaCl challenge (Fig. 5F), indicating increased translocation of pre‐existing ATF6 to the Golgi. Golgi morphology recovered after 3 h of NaCl challenge, at which time ATF6 accumulated in both the Golgi and nucleus (Fig. 5F), presumably as a consequence of de novo ATF6 protein synthesis (Fig. 5C and D). ATF6 signal specificity was verified using another anti‐ATF6 antibody (Sigma‐Aldrich) and the signal obtained by either antibody was abated by siRNA against ATF6 (not shown). Together, these observations indicate that while Tg and Tun strongly activate PERK, IRE1α and ATF6, NaCl activates PERK and ATF6.

Bottom Line: Hyperosmotic stress profoundly challenges cellular homeostasis and induces endoplasmic reticulum (ER) stress.Both hyperosmolality and chemical induction of ER stress decreased ENaC expression in vitro.ENaC depletion by either stimulus was abolished by transcriptional inhibition and by the chemical chaperone 4-phenylbutyric acid and was partly abrogated by either PERK or ATF6 silencing.

View Article: PubMed Central - PubMed

Affiliation: UPMC/INSERM/Paris Descartes U1138 CNRS ERL 8228, Equipe 3 Métabolisme et Physiologie Rénale, Centre de Recherche des Cordeliers, Paris, France.

No MeSH data available.


Related in: MedlinePlus