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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 ectopic overexpression of Cav1 on the levels of Cav1, SOCE, and cell migration after wounding. (Aa) structure of expression vector. (Ab) representative Cav1 and TRPC1 immunoblots in two different clones (C1 and C2) of stable Cav1‐transfected cells (IEC‐Cav1). IEC‐6 cells were transfected with the Cav1 expression vector or control empty vector (Null), and clones resistant to the selection medium containing 0.6 mg/mL G418 were isolated and screened for Cav1 and TRPC1 expression. (Ac) changes in the levels of Cav1 and TRPC1 in the complex IPed by anti‐Cav1 Ab in cells described in (Ab). Levels of TRPC1 and Cav1 were measured using western blot analysis. (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L CPA in the absence (0Ca2+) or presence of extracellular Ca2+ in cells described in (Ab). (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 cells transfected with the Null. (D) summarized data showing cell migration 6 h after wounding in cells described in (Ab). Values are means ± SEM from six dishes. *P < 0.05 compared with cells transfected with the Null.
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fig06: Effect of ectopic overexpression of Cav1 on the levels of Cav1, SOCE, and cell migration after wounding. (Aa) structure of expression vector. (Ab) representative Cav1 and TRPC1 immunoblots in two different clones (C1 and C2) of stable Cav1‐transfected cells (IEC‐Cav1). IEC‐6 cells were transfected with the Cav1 expression vector or control empty vector (Null), and clones resistant to the selection medium containing 0.6 mg/mL G418 were isolated and screened for Cav1 and TRPC1 expression. (Ac) changes in the levels of Cav1 and TRPC1 in the complex IPed by anti‐Cav1 Ab in cells described in (Ab). Levels of TRPC1 and Cav1 were measured using western blot analysis. (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L CPA in the absence (0Ca2+) or presence of extracellular Ca2+ in cells described in (Ab). (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 cells transfected with the Null. (D) summarized data showing cell migration 6 h after wounding in cells described in (Ab). Values are means ± SEM from six dishes. *P < 0.05 compared with cells transfected with the Null.

Mentions: To further define the role of Cav1 in Ca2+ influx in IECs, stable Cav1‐transfected IEC‐6 cells (IEC‐Cav1) were developed in this study. The expression vector encoding the full‐length cDNA of the human Cav1 under the control of the CMV promoter was constructed as shown in Figure 6Aa. Two clones that were resistant to the selection medium containing 0.6 mg/mL G418 were characterized by examining the levels of Cav1 protein. Levels of Cav1 protein in stable IEC‐Cav1 cells were approximately five‐fold of the levels in IEC‐6 cells transfected with the control vector containing no Cav1 cDNA (Null) (relative protein levels from 0.55 ± 0.07 in Null to 2.41 ± 0.3 in IEC‐Cav1‐C1; P < 0.05). On the other hand, there were no differences in the levels of TRPC1 between IEC‐Cav1 and Null cells. To determine if increased levels of Cav1 enhanced Cav1/TRPC1 complex, whole‐cell lysates were immunoprecipitated with the specific anti‐Cav1 antibody. As shown in Figure 6Ac, levels of Cav1/TRPC1 complexes were higher in IEC‐Cav1 cells than those observed in Null cells. Consistently, IEC‐Cav1 cells exhibited an increase in SOCE and cell migration after wounding. Levels of resting [Ca2+]cyt and SOCE in IEC‐Cav1 cells were increased by ~60% compared with Null cells (Fig. 6B and C), and the number of cells migrating over the wounded edge in IEC‐Cav1 cells were increased by ~55%. Increased migration in IEC‐Cav1 cells is not simply due to clonal variation, since identical results were observed when two independently transfected clones (IEC‐Cav1‐C1, IEC‐Cav1‐C2) were analyzed. These results indicate that ectopic overexpression of the Cav1 gene activates TRPC1‐mediated Ca2+ signaling and promotes epithelial restitution after wounding.


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 ectopic overexpression of Cav1 on the levels of Cav1, SOCE, and cell migration after wounding. (Aa) structure of expression vector. (Ab) representative Cav1 and TRPC1 immunoblots in two different clones (C1 and C2) of stable Cav1‐transfected cells (IEC‐Cav1). IEC‐6 cells were transfected with the Cav1 expression vector or control empty vector (Null), and clones resistant to the selection medium containing 0.6 mg/mL G418 were isolated and screened for Cav1 and TRPC1 expression. (Ac) changes in the levels of Cav1 and TRPC1 in the complex IPed by anti‐Cav1 Ab in cells described in (Ab). Levels of TRPC1 and Cav1 were measured using western blot analysis. (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L CPA in the absence (0Ca2+) or presence of extracellular Ca2+ in cells described in (Ab). (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 cells transfected with the Null. (D) summarized data showing cell migration 6 h after wounding in cells described in (Ab). Values are means ± SEM from six dishes. *P < 0.05 compared with cells transfected with the Null.
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fig06: Effect of ectopic overexpression of Cav1 on the levels of Cav1, SOCE, and cell migration after wounding. (Aa) structure of expression vector. (Ab) representative Cav1 and TRPC1 immunoblots in two different clones (C1 and C2) of stable Cav1‐transfected cells (IEC‐Cav1). IEC‐6 cells were transfected with the Cav1 expression vector or control empty vector (Null), and clones resistant to the selection medium containing 0.6 mg/mL G418 were isolated and screened for Cav1 and TRPC1 expression. (Ac) changes in the levels of Cav1 and TRPC1 in the complex IPed by anti‐Cav1 Ab in cells described in (Ab). Levels of TRPC1 and Cav1 were measured using western blot analysis. (B) representative records showing the time course of [Ca2+]cyt changes after exposure to 10 μmol/L CPA in the absence (0Ca2+) or presence of extracellular Ca2+ in cells described in (Ab). (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 cells transfected with the Null. (D) summarized data showing cell migration 6 h after wounding in cells described in (Ab). Values are means ± SEM from six dishes. *P < 0.05 compared with cells transfected with the Null.
Mentions: To further define the role of Cav1 in Ca2+ influx in IECs, stable Cav1‐transfected IEC‐6 cells (IEC‐Cav1) were developed in this study. The expression vector encoding the full‐length cDNA of the human Cav1 under the control of the CMV promoter was constructed as shown in Figure 6Aa. Two clones that were resistant to the selection medium containing 0.6 mg/mL G418 were characterized by examining the levels of Cav1 protein. Levels of Cav1 protein in stable IEC‐Cav1 cells were approximately five‐fold of the levels in IEC‐6 cells transfected with the control vector containing no Cav1 cDNA (Null) (relative protein levels from 0.55 ± 0.07 in Null to 2.41 ± 0.3 in IEC‐Cav1‐C1; P < 0.05). On the other hand, there were no differences in the levels of TRPC1 between IEC‐Cav1 and Null cells. To determine if increased levels of Cav1 enhanced Cav1/TRPC1 complex, whole‐cell lysates were immunoprecipitated with the specific anti‐Cav1 antibody. As shown in Figure 6Ac, levels of Cav1/TRPC1 complexes were higher in IEC‐Cav1 cells than those observed in Null cells. Consistently, IEC‐Cav1 cells exhibited an increase in SOCE and cell migration after wounding. Levels of resting [Ca2+]cyt and SOCE in IEC‐Cav1 cells were increased by ~60% compared with Null cells (Fig. 6B and C), and the number of cells migrating over the wounded edge in IEC‐Cav1 cells were increased by ~55%. Increased migration in IEC‐Cav1 cells is not simply due to clonal variation, since identical results were observed when two independently transfected clones (IEC‐Cav1‐C1, IEC‐Cav1‐C2) were analyzed. These results indicate that ectopic overexpression of the Cav1 gene activates TRPC1‐mediated Ca2+ signaling and promotes epithelial restitution after wounding.

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