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Caveolin-1 enhances rapid mucosal restitution by activating TRPC1-mediated Ca2+ signaling.

Rathor N, Chung HK, Wang SR, Wang JY, Turner DJ, Rao JN - Physiol Rep (2014)

Bottom Line: Cav1 silencing in stable TRPC1-transfected cells by transfection with siCav1 reduced SOCE without effect on the level of resting [Ca(2+)]cyt.Inhibition of Cav1 expression by siCav1 and subsequent decrease in Ca(2+) influx repressed epithelial restitution, as indicated by a decrease in cell migration over the wounded area, whereas stable ectopic overexpression of Cav1 increased Cav1/TRPC1 complex, induced SOCE, and enhanced cell migration after wounding.These results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1-mediated Ca(2+) signaling and rapid mucosal restitution after injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland, USA Baltimore VA Medical Center, Baltimore, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus

Effect of Cav1 silencing on Cav1/TRPC1 complex, SOCE, and cell migration in stable IEC‐TRPC1 cells. (Aa) representative Cav1 and TRPC1 immunoblots. After cells were transfected with either siRNA targeting the Cav1 mRNA coding region (siCav1) or control siRNA (C‐siRNA) for 24 and 48 h, whole‐cell lysates were harvested for western blot analysis to monitor the expression of Cav1, TRPC1 and loading control actin. (Ab) changes in the levels of Cav1/TRPC1 protein in the complex IP by anti‐TRPC1 antibody in cells described in (Aa). (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L cyclopiazonic acid (CPA) in the absence (0Ca2+) or presence of extracellular Ca2+ in parent IEC‐6 cells and IEC‐TRPC1 cells transfected with C‐siRNA or siCav1 for 48 h. (C) summarized data showing resting [Ca2+]cyt (left) and the amplitude of CPA‐induced Ca2+ influx (right) from cells described in (B). Values are means ± SEM; n = 25. *P < 0.05 compared with parent IEC‐6 cells; +P < 0.05 compared with cells transfected with C‐siRNA. (D) images of cell migration after wounding: (a) 0 h after wounding; (b) 6 h after wounding in parent IEC‐6 cells; (c) 6 h after wounding in IEC‐TRPC1 cells transfected with C‐siRNA; and (d) 6 h after wounding in IEC‐TRPC1 cells transfected with siCav1 for 48 h. (E) summarized data showing rates of cell migration after wounding in cells described in (D). Data were expressed as means ± SEM from six dishes. *P < 0.05 compared with parent IEC‐6 cells;+P < 0.05 compared with cells transfected with C‐siRNA.
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fig05: Effect of Cav1 silencing on Cav1/TRPC1 complex, SOCE, and cell migration in stable IEC‐TRPC1 cells. (Aa) representative Cav1 and TRPC1 immunoblots. After cells were transfected with either siRNA targeting the Cav1 mRNA coding region (siCav1) or control siRNA (C‐siRNA) for 24 and 48 h, whole‐cell lysates were harvested for western blot analysis to monitor the expression of Cav1, TRPC1 and loading control actin. (Ab) changes in the levels of Cav1/TRPC1 protein in the complex IP by anti‐TRPC1 antibody in cells described in (Aa). (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L cyclopiazonic acid (CPA) in the absence (0Ca2+) or presence of extracellular Ca2+ in parent IEC‐6 cells and IEC‐TRPC1 cells transfected with C‐siRNA or siCav1 for 48 h. (C) summarized data showing resting [Ca2+]cyt (left) and the amplitude of CPA‐induced Ca2+ influx (right) from cells described in (B). Values are means ± SEM; n = 25. *P < 0.05 compared with parent IEC‐6 cells; +P < 0.05 compared with cells transfected with C‐siRNA. (D) images of cell migration after wounding: (a) 0 h after wounding; (b) 6 h after wounding in parent IEC‐6 cells; (c) 6 h after wounding in IEC‐TRPC1 cells transfected with C‐siRNA; and (d) 6 h after wounding in IEC‐TRPC1 cells transfected with siCav1 for 48 h. (E) summarized data showing rates of cell migration after wounding in cells described in (D). Data were expressed as means ± SEM from six dishes. *P < 0.05 compared with parent IEC‐6 cells;+P < 0.05 compared with cells transfected with C‐siRNA.

Mentions: Our previous study shows that ectopic TRPC1 overexpression increases SOCE and stimulates IEC migration after wounding (Rao et al. 2006). In this study, we further determined if Cav1 is necessary for TRPC1‐mediated Ca2+ influx during restitution after wounding. siRNA targeting Cav1 mRNA (siCav1) was used to specifically block endogenous Cav1 in stable IEC‐TRPC1 cells. Initially, we determined the transfection efficiency of the siRNA nucleotides and demonstrated that more than 95% of IEC‐TRPC1 cells were positive when they were transfected with a fluorescent FITC‐conjugated C‐siRNA for 48 h (data not shown). As shown in Figure 5Aa, transfection with siCav1 for 48 h decreased Cav1 protein levels by ~90%, but it did not affect TRPC1 content (relative protein levels from 1.4 ± 0.17 in C‐siRNA to 0.21 ± 0.018 in 48 h siCav1; P < 0.05). Transfection with control siRNA (C‐siRNA) at the same concentrations showed no significant effect on Cav1 level. Cav1 silencing by siCav1 reduced Cav1/TRPC1 complexes as measured by IP assays (Fig. 5Ab). Although Cav1 silencing had no effect on the levels of resting [Ca2+]cyt, it significantly inhibited cyclopiazonic acid (CPA)‐induced Ca2+ influx in stable IEC‐TRPC1 cells (Fig. 5B and C). The level of Ca2+ influx after store depletion was decreased by ~70% in Cav1‐silenced cells compared with those observed in either parent IEC‐6 or cells transfected with C‐siRNA (from 1129 ± 96 nmol/L in C‐siRNA to 452 ± 47 nmol/L in 48 h siCav1; n= 25, P < 0.05). Cav1 silencing also impaired epithelial restitution after wounding in stable IEC‐TRPC1 cells (Fig. 5D and E). The number of cells migrating over the denuded area 6 h after wounding was decreased by ~40% in Cav1‐silenced cells. We also examined the effect of Cav1 silencing on Ca2+ influx and epithelial restitution in other lines of IECs and demonstrated that decreased levels of Cav1 by transfection with siCav1 also inhibited SOCE and repressed cell migration after wounding in differentiated IEC‐Cdx2L1 cells (data not shown). In addition, neither siCav1 nor C‐siRNA affected cell viability as measured by Trypan blue staining (data not shown). These findings strongly indicate that Cav1 is crucial for stimulation of cell migration after wounding by activating TRPC1‐mediated Ca2+ signaling.


Caveolin-1 enhances rapid mucosal restitution by activating TRPC1-mediated Ca2+ signaling.

Rathor N, Chung HK, Wang SR, Wang JY, Turner DJ, Rao JN - Physiol Rep (2014)

Effect of Cav1 silencing on Cav1/TRPC1 complex, SOCE, and cell migration in stable IEC‐TRPC1 cells. (Aa) representative Cav1 and TRPC1 immunoblots. After cells were transfected with either siRNA targeting the Cav1 mRNA coding region (siCav1) or control siRNA (C‐siRNA) for 24 and 48 h, whole‐cell lysates were harvested for western blot analysis to monitor the expression of Cav1, TRPC1 and loading control actin. (Ab) changes in the levels of Cav1/TRPC1 protein in the complex IP by anti‐TRPC1 antibody in cells described in (Aa). (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L cyclopiazonic acid (CPA) in the absence (0Ca2+) or presence of extracellular Ca2+ in parent IEC‐6 cells and IEC‐TRPC1 cells transfected with C‐siRNA or siCav1 for 48 h. (C) summarized data showing resting [Ca2+]cyt (left) and the amplitude of CPA‐induced Ca2+ influx (right) from cells described in (B). Values are means ± SEM; n = 25. *P < 0.05 compared with parent IEC‐6 cells; +P < 0.05 compared with cells transfected with C‐siRNA. (D) images of cell migration after wounding: (a) 0 h after wounding; (b) 6 h after wounding in parent IEC‐6 cells; (c) 6 h after wounding in IEC‐TRPC1 cells transfected with C‐siRNA; and (d) 6 h after wounding in IEC‐TRPC1 cells transfected with siCav1 for 48 h. (E) summarized data showing rates of cell migration after wounding in cells described in (D). Data were expressed as means ± SEM from six dishes. *P < 0.05 compared with parent IEC‐6 cells;+P < 0.05 compared with cells transfected with C‐siRNA.
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fig05: Effect of Cav1 silencing on Cav1/TRPC1 complex, SOCE, and cell migration in stable IEC‐TRPC1 cells. (Aa) representative Cav1 and TRPC1 immunoblots. After cells were transfected with either siRNA targeting the Cav1 mRNA coding region (siCav1) or control siRNA (C‐siRNA) for 24 and 48 h, whole‐cell lysates were harvested for western blot analysis to monitor the expression of Cav1, TRPC1 and loading control actin. (Ab) changes in the levels of Cav1/TRPC1 protein in the complex IP by anti‐TRPC1 antibody in cells described in (Aa). (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L cyclopiazonic acid (CPA) in the absence (0Ca2+) or presence of extracellular Ca2+ in parent IEC‐6 cells and IEC‐TRPC1 cells transfected with C‐siRNA or siCav1 for 48 h. (C) summarized data showing resting [Ca2+]cyt (left) and the amplitude of CPA‐induced Ca2+ influx (right) from cells described in (B). Values are means ± SEM; n = 25. *P < 0.05 compared with parent IEC‐6 cells; +P < 0.05 compared with cells transfected with C‐siRNA. (D) images of cell migration after wounding: (a) 0 h after wounding; (b) 6 h after wounding in parent IEC‐6 cells; (c) 6 h after wounding in IEC‐TRPC1 cells transfected with C‐siRNA; and (d) 6 h after wounding in IEC‐TRPC1 cells transfected with siCav1 for 48 h. (E) summarized data showing rates of cell migration after wounding in cells described in (D). Data were expressed as means ± SEM from six dishes. *P < 0.05 compared with parent IEC‐6 cells;+P < 0.05 compared with cells transfected with C‐siRNA.
Mentions: Our previous study shows that ectopic TRPC1 overexpression increases SOCE and stimulates IEC migration after wounding (Rao et al. 2006). In this study, we further determined if Cav1 is necessary for TRPC1‐mediated Ca2+ influx during restitution after wounding. siRNA targeting Cav1 mRNA (siCav1) was used to specifically block endogenous Cav1 in stable IEC‐TRPC1 cells. Initially, we determined the transfection efficiency of the siRNA nucleotides and demonstrated that more than 95% of IEC‐TRPC1 cells were positive when they were transfected with a fluorescent FITC‐conjugated C‐siRNA for 48 h (data not shown). As shown in Figure 5Aa, transfection with siCav1 for 48 h decreased Cav1 protein levels by ~90%, but it did not affect TRPC1 content (relative protein levels from 1.4 ± 0.17 in C‐siRNA to 0.21 ± 0.018 in 48 h siCav1; P < 0.05). Transfection with control siRNA (C‐siRNA) at the same concentrations showed no significant effect on Cav1 level. Cav1 silencing by siCav1 reduced Cav1/TRPC1 complexes as measured by IP assays (Fig. 5Ab). Although Cav1 silencing had no effect on the levels of resting [Ca2+]cyt, it significantly inhibited cyclopiazonic acid (CPA)‐induced Ca2+ influx in stable IEC‐TRPC1 cells (Fig. 5B and C). The level of Ca2+ influx after store depletion was decreased by ~70% in Cav1‐silenced cells compared with those observed in either parent IEC‐6 or cells transfected with C‐siRNA (from 1129 ± 96 nmol/L in C‐siRNA to 452 ± 47 nmol/L in 48 h siCav1; n= 25, P < 0.05). Cav1 silencing also impaired epithelial restitution after wounding in stable IEC‐TRPC1 cells (Fig. 5D and E). The number of cells migrating over the denuded area 6 h after wounding was decreased by ~40% in Cav1‐silenced cells. We also examined the effect of Cav1 silencing on Ca2+ influx and epithelial restitution in other lines of IECs and demonstrated that decreased levels of Cav1 by transfection with siCav1 also inhibited SOCE and repressed cell migration after wounding in differentiated IEC‐Cdx2L1 cells (data not shown). In addition, neither siCav1 nor C‐siRNA affected cell viability as measured by Trypan blue staining (data not shown). These findings strongly indicate that Cav1 is crucial for stimulation of cell migration after wounding by activating TRPC1‐mediated Ca2+ signaling.

Bottom Line: Cav1 silencing in stable TRPC1-transfected cells by transfection with siCav1 reduced SOCE without effect on the level of resting [Ca(2+)]cyt.Inhibition of Cav1 expression by siCav1 and subsequent decrease in Ca(2+) influx repressed epithelial restitution, as indicated by a decrease in cell migration over the wounded area, whereas stable ectopic overexpression of Cav1 increased Cav1/TRPC1 complex, induced SOCE, and enhanced cell migration after wounding.These results indicate that Cav1 physically interacts with and activates TRPC1, thus stimulating TRPC1-mediated Ca(2+) signaling and rapid mucosal restitution after injury.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Cell Biology Group, University of Maryland School of Medicine, Baltimore, Maryland, USA Baltimore VA Medical Center, Baltimore, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus