Up-regulation of transient receptor potential canonical 1 (TRPC1) following sarco(endo)plasmic reticulum Ca2+ ATPase 2 gene silencing promotes cell survival: a potential role for TRPC1 in Darier's disease

Abstract

The mechanism(s) involved in regulation of store operated calcium entry in Darier's disease (DD) is not known. We investigated the distribution and function of transient receptor potential canonical (TRPC) in epidermal skin cells. DD patients demonstrated up-regulation of TRPC1, but not TRPC3, in the squamous layers. Ca2+ influx was significantly higher in keratinocytes obtained from DD patients and showed enhanced proliferation compared with normal keratinocytes. Similar up-regulation of TRPC1 was also detected in epidermal layers of SERCA2+/- mice. HaCaT cells expressed TRPC1 in the plasma membrane. Expression of sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA)2 small interfering RNA (siRNA) in HaCaT cells increased TRPC1 levels and thapsigargin-stimulated Ca2+ influx, which was blocked by store-operated calcium entry inhibitors. Thapsigargin-stimulated intracellular Ca2+ release was decreased in DD cells. DD keratinocytes exhibited increased cell survival upon thapsigargin treatment. Alternatively, overexpression of TRPC1 or SERCA2-siRNA in HaCaT cells demonstrated resistance to thapsigargin-induced apoptosis. These effects were dependent on external Ca2+ and activation of nuclear factor-kappaB. Isotretinoin reduced Ca2+ entry in HaCaT cells and decreased survival of HaCaT and DD keratinocytes. These findings put forward a novel consequence of compromised SERCA2 function in DD wherein up-regulation of TRPC1 augments cell proliferation and restrict apoptosis. We suggest that the anti-apoptotic effect of TRPC1 could potentially contribute to abnormal keratosis in DD.

Figure 1.

TRPC1 and TRPC3 expression in control and DD patients. Immunofluorescence in skin sections (10 μm) obtained from control (A and C) or DD patient (B and D), using anti-TRPC1 (A and B), anti-SERCA2, and anti TRPC3 (C and D) at 1:100 dilutions. Fluorescently labeled secondary antibodies were used to label TRPC1, TRPC3, and SERCA2 proteins. Images were taken using 63× objective. (E) Bar graph indicating immunofluorescence values of SERCA2, TRPC1, and TRPC3 in control and DD patients. (F) Western blot using crude membranes prepared from control and DD patients and probed using TRPC1, SERCA2, and actin antibodies. (G) Images (H&E staining and DIC) from control and DD patients.

Figure 2.

SERCA2^+/− mice exhibit increased TRPC1 expression. (A) Immunofluorescence in skin sections (10 μm) obtained from SERCA2^+/+ or SERCA2^+/− mice using anti-TRPC1 and anti-SERCA2 antibodies. Fluorescently labeled secondary antibodies were used to label TRPC1 and SERCA2 proteins. Images were taken using 63× objective. (B) Western blots performed on samples obtained from SERCA2^+/+ and SERCA2^+/− mice and probed using SERCA2 and β-actin antibodies.

Figure 3.

Expression of TRPC1 and TRPC3 in HaCaT cells. (A) RT-PCR on HaCaT cells using trp1- and trp3-specific primers (top) or GAPDH primers (middle) as described in Bollimuntha et al. (2005) and Kobayashi et al. (2003), bottom, is control PCR using plasmid DNA. (B) Western blot on crude membranes prepared from HaCaT cells and probed using TRPC1 or TRPC3 antibodies. Control samples were obtained from HSG cells expressing endogenous TRPC1 and 293 cells expressing endogenous TRPC3. (C) Localization of endogenous TRPC1 and TRPC3 in HaCaT and SERCA2-siRNA–expressing cells using TRPC1 and TRPC3 antibodies followed by rhodamine-conjugated secondary antibodies. Control indicates staining without TRPC1 or TRPC3 antibodies but treated with rhodamine-labeled secondary antibody.

Figure 4.

Ca²⁺ influx in HaCaT cells and DD keratinocytes. (A) Tg-stimulated fluorescence trace in HaCaT cells transiently expressing adenovirus encoding control or SERCA2-siRNA (MOI of 10 plaque-forming units/cell) in a Ca²⁺ containing SES media. (B) Traces of intracellular release (mean traces from >50 cells each). (C) Fluorescence traces of control or cells expressing SERCA2-siRNA or TRPC1 upon carbachol stimulation. (D) Ca²⁺ influx values measured as area under the curve; number of cells imaged also is labeled. (E) Traces (mean value of 50–60 cells) in cells pretreated with 1 mM lanthanum for 15 min or 1 μM SOCE blocker BTP2 for 60 min. (F) Tg-stimulated fluorescence trace in keratinocytes isolated from normal and DD patients. (G) Western blots using SERCA2, TRPC1, and actin antibodies and representative of three to five individual experiments (quantification added to the blot). Asterisk (*) indicates mean values that are significantly different (p < 0.05).

Figure 5.

Overexpression of TRPC1 restricts Tg-mediated apoptosis. Marker for apoptosis (YO-PRO-1) was used to stain control and TRPC1-overexpressing cells as described in Bollimuntha et al. (2005). Tg (1 μM) was treated for 0 or 30 min (A). Fluorescence images of cells stained with YO-PRO-1 were taken using 10× or 40× objective, and green cells were counted along with total number of cells. (B) Fluorescence images of Annexin-V–positive cells taken immediately using 10× or 40× objectives, and green cells were counted along with total number of cells. (C) Represents mean bar graph from >1000 cells in each group. (D) Represents mean bar graph from >800 cells in each group. (E) Western blot on crude membranes and whole extracts prepared from HaCaT cells or TRPC1-overexpressing cells and probed using TRPC1, Bcl-xL, caspase-8, caspase-9, Apaf-1, and GAPDH antibodies. Asterisk (*) indicates values significantly different from its counterpart (p < 0.05).

Figure 6.

Overexpression of SERCA2-siRNA or TRPC1 increases cell proliferation. (A) MTT assays performed on control, TRPC1-, TRPC3-, and SERCA2-siRNA–overexpressing cells treated with 1 μM Tg for 30 min in the absence (left) or presence of 100 μM isotretinoin, 12 h (right) with or without Ca²⁺. (B) Cell survival (MTT assay) on control, TRPC1-, and SERCA2-siRNA–overexpressing cells in the presence of isotretinoin. (C) Fluorescence images on HaCaT cells overexpressing HA-TRPC1 or endogenous SERCA2, or cells expressing SERC2 siRNA. TRPC1 was stained using HA antibody (1:500), whereas SERCA2 was stained using SERCA2 antibody (1:1000). Proteins were detected using FITC-labeled specific secondary antibodies. (D) Cell survival in control and TRPC1-overexpressing HaCaT cell treated with SOCE inhibitor BTP2 (1 μM) and 1 mM La³⁺ for 12 h. (E) Cell proliferation upon 100 μM carbachol stimulation for 12 h. (F) Cell survival of keratinocytes isolated from normal and DD patients, respectively, in presence of 1 μM Tg or 100 μM isotretinoin for 12 h. Asterisk (*) indicates values significantly different from its counterpart (p < 0.05).

Figure 7.

SERCA2-siRNA or TRPC1-mediated protection depends on NF-κB stimulation. (A) Fluorescence images on control and TRPC1-expressing cells treated with 1 μM Tg for 30 min and stained using p65 antibody and 4,6-diamidino-2-phenylindole (DAPI) (to counterstain nucleus). Average data are shown as bar graph. Asterisk (*) indicates values significantly different from its counterpart (p < 0.05). (B) TNF-α (50 ng/ml; 4 h) or Tg (1 μM; 4 h) stimulated activation of NF-κB luciferase reporter construct in control and SERC2-siRNA–expressing HaCaT cells, in the absence or presence of 100 nM NF-κB inhibitor. (C) Cell survival (MTT assay) on control, TRPC1, and SERCA2-siRNA–overexpressing cells in the absence or presence of 100 nM NF-κB inhibitor for 12 h and extracellular Ca²⁺, respectively. (D) Western blots on control and NF-κB inhibitor (100 nM; 12 h)-treated HaCaT cells using TRPC1, SERCA2, and β-tubulin antibodies.

Figure 8.

Proposed model for the role of TRPC1 in Darier's disease: We propose that in Darier's disease keratinocytes, the nonfunctional SERCA fails to maintain the adequate ER calcium pool (⊝, compromised function). This decreased SERCA activity in turn up-regulates the store-operated plasma membrane calcium channel TRPC1, in an undefined mechanism dependent on NF-κB. Overexpressed TRPC1 channels augment store-operated calcium influx. The incoming calcium is pumped out via the plasma membrane calcium ATPase (PMCA) and also used for the cells physiological need. An optimal level of this calcium brings about proper growth and proliferation (⊕, positive effect) by activating NF-κB pathway. This confers resistance to apoptosis by regulating the expression of proproliferative/antiapoptotic genes. However, if there is an overload this calcium might be detrimental, leading to stress-induced apoptosis or uncontrolled proliferation accounting for cancerous state (?). It is also proposed that isotretinoin, by yet another undefined mechanism (?) regulates the calcium influx via TRPC1 and blocks the calcium overload, thereby inhibiting hyperproliferation and probably cancer.