Limits...
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

Levels of Cav1 and its interaction with TRPC1 in different lines of IECs. (A) representative immunoblot of Cav1 in IEC‐6 cells, differentiated IEC‐Cdx2L1 cells, and IECs stably overexpressing TRPC1 (IEC‐TRPC1). Levels of total Cav1 were examined by western blot analysis, and actin immunoblotting was performed as an internal control for equal loading (upper panel). Quantitative analysis of western immunoblots by densitometry that were corrected for actin loading from cells described above. Values are means ± SEM; P < 0.05 compared with parental IEC‐6 cells (lower panel). (B) levels of Cav1 and TRPC1 in the complex IP by the anti‐Cav1 or anti‐TRPC1 Ab in cells described in (A). Three separate experiments were performed that showed similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4255804&req=5

fig04: Levels of Cav1 and its interaction with TRPC1 in different lines of IECs. (A) representative immunoblot of Cav1 in IEC‐6 cells, differentiated IEC‐Cdx2L1 cells, and IECs stably overexpressing TRPC1 (IEC‐TRPC1). Levels of total Cav1 were examined by western blot analysis, and actin immunoblotting was performed as an internal control for equal loading (upper panel). Quantitative analysis of western immunoblots by densitometry that were corrected for actin loading from cells described above. Values are means ± SEM; P < 0.05 compared with parental IEC‐6 cells (lower panel). (B) levels of Cav1 and TRPC1 in the complex IP by the anti‐Cav1 or anti‐TRPC1 Ab in cells described in (A). Three separate experiments were performed that showed similar results.

Mentions: Our previous studies have shown that TRPC1 functions as SOC in IECs and mediates Ca2+ influx after store depletion (Rao et al. 2006, 2010). To further determine the possibility that Cav1 regulates Ca2+ influx through its interaction with TRPC1, basal levels of Cav1 and its interaction with TRPC1 were examined in three lines of IECs including IEC‐6, differentiated IEC‐Cdx2L1, and stable TRPC1‐transfected cells (IEC‐TRPC1). As shown in Figure 4A, differentiated IEC‐Cdx2L1 and IEC‐TRPC1 cells expressed higher levels of Cav1 compared to that observed in IEC‐6 cells. Both IEC‐Cdx2L1 and IEC‐TRPC1 cells exhibited four‐fold increase of IEC‐6 cell in the level of Cav1 protein, which was associated with increased Ca2+ influx after store depletion as reported previously (Rao et al. 2006, 2010). To determine whether Cav1 forms Cav1/TRPC1 complexes in different IECs, whole‐cell lysates were immunoprecipitated (IP) with either anti‐Cav1 or anti‐TRPC1 antibody, and then these precipitates were examined by western blot analysis using specific antibody against TRPC1 or Cav1. As shown in Figure 4B, IP of Cav1 or TRPC1 resulted in co‐IP of TRPC1 and Cav1 in all three lines of IECs, although the levels of TRPC1 protein in IEC‐TRPC1 cells were higher than those observed in IEC‐6 and IEC‐Cdx2L1 cells. We also used IgG as a negative control in IP assays and found that incubation with IgG at the same condition did not pull down either Cav1 or TRPC1 (data not shown). These results indicate that Cav1 physically interacts with TRPC1 in various IECs.


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)

Levels of Cav1 and its interaction with TRPC1 in different lines of IECs. (A) representative immunoblot of Cav1 in IEC‐6 cells, differentiated IEC‐Cdx2L1 cells, and IECs stably overexpressing TRPC1 (IEC‐TRPC1). Levels of total Cav1 were examined by western blot analysis, and actin immunoblotting was performed as an internal control for equal loading (upper panel). Quantitative analysis of western immunoblots by densitometry that were corrected for actin loading from cells described above. Values are means ± SEM; P < 0.05 compared with parental IEC‐6 cells (lower panel). (B) levels of Cav1 and TRPC1 in the complex IP by the anti‐Cav1 or anti‐TRPC1 Ab in cells described in (A). Three separate experiments were performed that showed similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255804&req=5

fig04: Levels of Cav1 and its interaction with TRPC1 in different lines of IECs. (A) representative immunoblot of Cav1 in IEC‐6 cells, differentiated IEC‐Cdx2L1 cells, and IECs stably overexpressing TRPC1 (IEC‐TRPC1). Levels of total Cav1 were examined by western blot analysis, and actin immunoblotting was performed as an internal control for equal loading (upper panel). Quantitative analysis of western immunoblots by densitometry that were corrected for actin loading from cells described above. Values are means ± SEM; P < 0.05 compared with parental IEC‐6 cells (lower panel). (B) levels of Cav1 and TRPC1 in the complex IP by the anti‐Cav1 or anti‐TRPC1 Ab in cells described in (A). Three separate experiments were performed that showed similar results.
Mentions: Our previous studies have shown that TRPC1 functions as SOC in IECs and mediates Ca2+ influx after store depletion (Rao et al. 2006, 2010). To further determine the possibility that Cav1 regulates Ca2+ influx through its interaction with TRPC1, basal levels of Cav1 and its interaction with TRPC1 were examined in three lines of IECs including IEC‐6, differentiated IEC‐Cdx2L1, and stable TRPC1‐transfected cells (IEC‐TRPC1). As shown in Figure 4A, differentiated IEC‐Cdx2L1 and IEC‐TRPC1 cells expressed higher levels of Cav1 compared to that observed in IEC‐6 cells. Both IEC‐Cdx2L1 and IEC‐TRPC1 cells exhibited four‐fold increase of IEC‐6 cell in the level of Cav1 protein, which was associated with increased Ca2+ influx after store depletion as reported previously (Rao et al. 2006, 2010). To determine whether Cav1 forms Cav1/TRPC1 complexes in different IECs, whole‐cell lysates were immunoprecipitated (IP) with either anti‐Cav1 or anti‐TRPC1 antibody, and then these precipitates were examined by western blot analysis using specific antibody against TRPC1 or Cav1. As shown in Figure 4B, IP of Cav1 or TRPC1 resulted in co‐IP of TRPC1 and Cav1 in all three lines of IECs, although the levels of TRPC1 protein in IEC‐TRPC1 cells were higher than those observed in IEC‐6 and IEC‐Cdx2L1 cells. We also used IgG as a negative control in IP assays and found that incubation with IgG at the same condition did not pull down either Cav1 or TRPC1 (data not shown). These results indicate that Cav1 physically interacts with TRPC1 in various IECs.

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