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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, FĆ©raille 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 MĆ©tabolisme et Physiologie RĆ©nale, Centre de Recherche des Cordeliers, Paris, France.

No MeSH data available.


Related in: MedlinePlus

ER stress is transiently induced by NaCl. (A) mRNA abundance of ERā€stress responsive genes in mCCDcl1 cells challenged or not (Ctl) with thapsigargin (Tg, 1 Ī¼mol/L) or tunicamycin (Tun, 3 Ī¼mol/L) for 6 h. (Bā€“E) mRNA abundance of ERā€stress responsive genes in mCCDcl1 (B and D) and mpkCCDcl4 (B) cells challenged or not with NaCl (500 mOsmol/kg, B and C) or urea (500 mOsmol/kg, D and E) for 1ā€“24 h. Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of four independent experiments. (F) mRNA abundance of ATF3 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 Ī¼mol/L) for 3 h. Data is represented as fold difference over nonā€stimulated CCD and is expressed as the mean Ā± SEM of data from at least six animals. (G) Western blot analysis of Grp78 protein in mCCDcl1 cells challenged or not (Ctl) with NaCl, Tg or Tun for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (H) Confocal maximum projections of mCCDcl1 cells loaded with ERā€Tracker Red challenged or not (0 min) with NaCl or Tg for 2 or 25 min. Shown are representative images of three similar experiments. Arrows depict the transient appearance of spherical clusters by NaCl. Bar, 10 Ī¼mol/L.
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fig04: ER stress is transiently induced by NaCl. (A) mRNA abundance of ERā€stress responsive genes in mCCDcl1 cells challenged or not (Ctl) with thapsigargin (Tg, 1 Ī¼mol/L) or tunicamycin (Tun, 3 Ī¼mol/L) for 6 h. (Bā€“E) mRNA abundance of ERā€stress responsive genes in mCCDcl1 (B and D) and mpkCCDcl4 (B) cells challenged or not with NaCl (500 mOsmol/kg, B and C) or urea (500 mOsmol/kg, D and E) for 1ā€“24 h. Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of four independent experiments. (F) mRNA abundance of ATF3 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 Ī¼mol/L) for 3 h. Data is represented as fold difference over nonā€stimulated CCD and is expressed as the mean Ā± SEM of data from at least six animals. (G) Western blot analysis of Grp78 protein in mCCDcl1 cells challenged or not (Ctl) with NaCl, Tg or Tun for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (H) Confocal maximum projections of mCCDcl1 cells loaded with ERā€Tracker Red challenged or not (0 min) with NaCl or Tg for 2 or 25 min. Shown are representative images of three similar experiments. Arrows depict the transient appearance of spherical clusters by NaCl. Bar, 10 Ī¼mol/L.

Mentions: We next examined whether hyperosmolality can induce ER stress and UPR signaling in vitro. ER stress activates the UPR, which in turn increases expression of genes that help restore ER homeostasis. We began our analysis of ER stress by hyperosmotic stress by examining the induction of genes that are typically upregulated by the UPR. We used Tg and Tun as control stimuli of ER stress. As expected, strong induction of ER stress by either Tg or Tun in mCCDcl1 cells dramatically increased UPR target gene expression (Fig. 4A). Some, but not all, of these genes were significantly upregulated by either NaCl (500 mOsmol/kg, Fig. 4B and C) or urea (500 mOsmol/kg, Fig. 4D and E) in both mCCDcl1 and mpkCCDcl4 cells. Of all genes tested, ATF3 mRNA upregulation by NaCl was strongest. Consistent with previous studies (Tian and Cohen 2002; Cai and Brooks 2011), this gene was also strongly upregulated in response to urea in both cell lines (Fig. 4D and E) and was also upregulated by either NaCl or Tg in microdissected CD (Fig. 4F). We continued our investigation of ER stress induction by hyperosmolality by examining Grp78 protein abundance (Fig. 4G). Similar to Grp78 mRNA, NaCl transiently increased Grp78 protein abundance. Induction peaked shortly following challenge, reaching levels similar to those achieved by either Tg or Tun (Fig. 4G). NaCl also altered ER morphology, as revealed by spherical clusters that immediately, but transiently, appeared upon NaCl but not Tg challenge (Fig. 4H). Possibly, an increase of intracellular osmolality upon NaCl challenge may affect ER volume, which could be connected with ER stress. Together, these data indicate that hyperosmolality transiently induces ER stress, albeit at levels lower than that achieved by chemical inducers.


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, FĆ©raille E, Hasler U - Physiol Rep (2014)

ER stress is transiently induced by NaCl. (A) mRNA abundance of ERā€stress responsive genes in mCCDcl1 cells challenged or not (Ctl) with thapsigargin (Tg, 1 Ī¼mol/L) or tunicamycin (Tun, 3 Ī¼mol/L) for 6 h. (Bā€“E) mRNA abundance of ERā€stress responsive genes in mCCDcl1 (B and D) and mpkCCDcl4 (B) cells challenged or not with NaCl (500 mOsmol/kg, B and C) or urea (500 mOsmol/kg, D and E) for 1ā€“24 h. Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of four independent experiments. (F) mRNA abundance of ATF3 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 Ī¼mol/L) for 3 h. Data is represented as fold difference over nonā€stimulated CCD and is expressed as the mean Ā± SEM of data from at least six animals. (G) Western blot analysis of Grp78 protein in mCCDcl1 cells challenged or not (Ctl) with NaCl, Tg or Tun for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (H) Confocal maximum projections of mCCDcl1 cells loaded with ERā€Tracker Red challenged or not (0 min) with NaCl or Tg for 2 or 25 min. Shown are representative images of three similar experiments. Arrows depict the transient appearance of spherical clusters by NaCl. Bar, 10 Ī¼mol/L.
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fig04: ER stress is transiently induced by NaCl. (A) mRNA abundance of ERā€stress responsive genes in mCCDcl1 cells challenged or not (Ctl) with thapsigargin (Tg, 1 Ī¼mol/L) or tunicamycin (Tun, 3 Ī¼mol/L) for 6 h. (Bā€“E) mRNA abundance of ERā€stress responsive genes in mCCDcl1 (B and D) and mpkCCDcl4 (B) cells challenged or not with NaCl (500 mOsmol/kg, B and C) or urea (500 mOsmol/kg, D and E) for 1ā€“24 h. Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of four independent experiments. (F) mRNA abundance of ATF3 in microdissected CCD challenged or not (Ctl) with NaCl (500 mOsmol/kg) or Tg (1 Ī¼mol/L) for 3 h. Data is represented as fold difference over nonā€stimulated CCD and is expressed as the mean Ā± SEM of data from at least six animals. (G) Western blot analysis of Grp78 protein in mCCDcl1 cells challenged or not (Ctl) with NaCl, Tg or Tun for 6 h (left panel) or NaCl for 10 min to 24 h (right panel). Data is represented as fold difference over nonā€stimulated cells and is expressed as the mean Ā± SEM of three independent experiments. Representative Western blots are shown. GAPDH was used as a loading control. (H) Confocal maximum projections of mCCDcl1 cells loaded with ERā€Tracker Red challenged or not (0 min) with NaCl or Tg for 2 or 25 min. Shown are representative images of three similar experiments. Arrows depict the transient appearance of spherical clusters by NaCl. Bar, 10 Ī¼mol/L.
Mentions: We next examined whether hyperosmolality can induce ER stress and UPR signaling in vitro. ER stress activates the UPR, which in turn increases expression of genes that help restore ER homeostasis. We began our analysis of ER stress by hyperosmotic stress by examining the induction of genes that are typically upregulated by the UPR. We used Tg and Tun as control stimuli of ER stress. As expected, strong induction of ER stress by either Tg or Tun in mCCDcl1 cells dramatically increased UPR target gene expression (Fig. 4A). Some, but not all, of these genes were significantly upregulated by either NaCl (500 mOsmol/kg, Fig. 4B and C) or urea (500 mOsmol/kg, Fig. 4D and E) in both mCCDcl1 and mpkCCDcl4 cells. Of all genes tested, ATF3 mRNA upregulation by NaCl was strongest. Consistent with previous studies (Tian and Cohen 2002; Cai and Brooks 2011), this gene was also strongly upregulated in response to urea in both cell lines (Fig. 4D and E) and was also upregulated by either NaCl or Tg in microdissected CD (Fig. 4F). We continued our investigation of ER stress induction by hyperosmolality by examining Grp78 protein abundance (Fig. 4G). Similar to Grp78 mRNA, NaCl transiently increased Grp78 protein abundance. Induction peaked shortly following challenge, reaching levels similar to those achieved by either Tg or Tun (Fig. 4G). NaCl also altered ER morphology, as revealed by spherical clusters that immediately, but transiently, appeared upon NaCl but not Tg challenge (Fig. 4H). Possibly, an increase of intracellular osmolality upon NaCl challenge may affect ER volume, which could be connected with ER stress. Together, these data indicate that hyperosmolality transiently induces ER stress, albeit at levels lower than that achieved by chemical inducers.

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 MĆ©tabolisme et Physiologie RĆ©nale, Centre de Recherche des Cordeliers, Paris, France.

No MeSH data available.


Related in: MedlinePlus