Phospholipase C-gamma1 is required for the activation of store-operated Ca2+ channels in liver cells - PubMed (original) (raw)

Phospholipase C-gamma1 is required for the activation of store-operated Ca2+ channels in liver cells

Tom Litjens et al. Biochem J. 2007.

Abstract

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

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Figures

Figure 1

Figure 1. Effect of the PLC inhibitor U73122 on the ISOC in H4IIE cells

(A) Pre-incubation of H4IIE cells with 2 μM of U73122 for 2–5 min inhibited activation of ISOC by intracellular perfusion with 20 μM IP3 (closed symbols, _n_=6). For comparison, activation of ISOC by IP3 is shown in untreated cells (open symbols, _n_=4).(B) Application of U73122 to the bath after the ISOC had fully developed had no effect on its amplitude. Each point represents the amplitude of ISOC at −118 mV taken from voltage ramps from −138 to 102 mV, applied every 2 s.

Figure 2

Figure 2. Knockdown of PLC-γ1 in H4IIE cells by a specific siRNA

(A) Immunofluorescence microscopy of PLC-γ1 (green) in cells transfected for 96 h with negative control siRNA (first row) and PLC-γ1-01 siRNA (second row). Second column shows staining of the cells nuclei with DAPI (blue), and third column shows merged images. Results are representative of four individual experiments. Bar=20 μm. (B) Reduction of the PLC-γ1 protein as determined by Western blotting. Expression of GAPDH served as a loading control. Molecular mass markers are indicated on the left-hand side of the Figure. Results are representative of four individual experiments.

Figure 3

Figure 3. Immunofluorescence microscopy analysis of siRNA transfection efficiency

H4IIE cells were co-transfected with 38 nM unlabelled PLC-γ1-01 siRNA and 38 nM AlexaFluor-546-labelled negative control siRNA. At 96 h after transfection the cells were fixed, immunostained with anti-PLC-γ1 antibody, followed by incubation with FITC-conjugated secondary antibody and then the nuclei were counterstained with DAPI (blue). H4IIE cells co-transfected with PLC-γ1-01 siRNA (unlabelled) and AlexaFluor-546-labelled negative control siRNA (red) show reduced PLC-γ1 expression (green) compared with neighbouring non-transfected cells. Bar=20 μm.

Figure 4

Figure 4. Effect of PLC-γ1 knockdown on ISOC in H4IIE cells

(A) Development of ISOC in response to intracellular IP3 in H4IIE cells treated with either control siRNA or PLC-γ1-01 siRNA for 72 and 96 h. (B) Scatter plot of the data presented in panel (A) and the data obtained on non-transfected cells and cells treated with transfection reagent only at 90 s after establishing whole cell. The P values are relative to the Control (cells transfected with control siRNA). (C) Activation of ISOC by intracellular application of thapsigargin in H4IIE cells treated with either control siRNA or PLC-γ1-01 siRNA for 96 h.

Figure 5

Figure 5. Effect of PLC-γ1 knockdown on thapsigargin-induced Ca2+ entry in H4IIE cells

(A) Reduction of thapsigargin-induced Ca2+ entry in H4IIE cells treated with PLC-γ1-01 siRNA (open symbols) compared with cells treated with control siRNA (closed symbols), at 96 h post-transfection. The results are shown as means±S.E.M. for 15–20 cells; _n_=4. (B) Reduction of the initial rate and the peak concentration of thapsigargin-induced Ca2+ entry by PLC-γ1 knockdown in four separate experiments (96 h post-transfection). The results are shown as means±S.E.M. for four independent experiments.

Figure 6

Figure 6. Dependence of the ISOC in H4IIE cells on PIP2

Development of the ISOC in H4IIE cells transfected with control siRNA for 72–96 h and treated with 10 μM PIP2 for either 20 min or overnight. Each point represents the amplitude of ISOC at −118 mV taken from voltage ramps from −138 to 102 mV, applied every 2 s.

Figure 7

Figure 7. Knockdown of STIM1 in H4IIE cells by specific siRNA

Immunofluorescence microscopy of STIM1 (green) in cells transfected for 96 h with negative control siRNA (first row) and STIM1-01 siRNA (second row). Second column shows staining of the cells nuclei with DAPI (blue) and the third column shows the merged images. Results are representative of three individual experiments. Bar=20 μm.

Figure 8

Figure 8. Effect of STIM1 knockdown on ISOC in H4IIE cells

Development of ISOC in response to intracellular IP3 in H4IIE cells treated with either control siRNA or STIM1-01 siRNA for 48 and 72 h. Each point represents the amplitude of ISOC at −118 mV taken from voltage ramps from −138 to 102 mV, applied every 2 s.

Figure 9

Figure 9. Immunoprecipitation of STIM1 and PLC-γ1

(A) Cell lysates (100 μg) or lysis buffer only (LB) were immunoprecipitated (IP) with anti-STIM1 antibody (+) [or PBS as a negative control (−)] and the immunoprecipitates were analysed by immunoblotting (IB) with anti-PLCγ1 antibody. H4IIE cells were either untreated (−TG) or treated (+TG) with 1 μM thapsigargin and cell lysates were prepared using Ca2+-supplemented (5 mM free Ca2+) lysis buffer. Positive controls included non-immunoprecipitated lysates (10 μg) from cells untreated (C1) or treated (C2) with 1 μM thapsigargin. No STIM1-immunoprecipitated 150 kDa PLC-γ1 band was observed in either the untreated or thapsigargin treated cell lysates. Molecular mass markers (M) are indicated to the right-hand side of the Figure. The results are representative of two independent experiments. (B) Cell lysates (100 μg) or lysis buffer only (LB) were immunoprecipitated (IP) with anti-PLC-γ1 antibody (+) [or PBS (−)] and the immunoprecipitates were analysed by immunoblotting (IB) with anti-STIM1 antibody. No PLCγ1-immunoprecipitated 85 kDa STIM1 band was observed in either the untreated or thapsigargin-treated cell lysates. The results are representative of two independent experiments. The band at approx. 75 kDa present in the IP(+) samples, including the lysis buffer only sample, is most probably non-denatured anti-PLCγ1 immunoprecipitating antibody. The faint band at approx. 95 kDa present in both IP(−) and IP(+) samples, including the lysis buffer only samples, is most probably an artefact due to the Protein A/G Plus agarose beads.

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