EGF enhances Ca2 mobilization and capacitative Ca2 entry in mouse mammary epithelial cells

Abstract

Effects of epidermal growth factor (EGF) on the intracellular Ca(2+) ([Ca(2+)](i)) responses to nucleotides, Ca(2+) release from thapsigargin-sensitive stores and capacitative Ca(2+) entry were investigated in cultured mouse mammary epithelial cells. EGF treatment induced proliferation of mammary epithelial cells. We checked for mitotic activity by immunocytochemistry with an anti-PCNA (proliferating cell nuclear antigen) antibody, which stains nuclei of the cells in S-phase of cell cycle. EGF treatment apparently increased the number of PCNA-stained cells compared to those treated with differentiating hormones (insulin, prolactin and cortisol) or without any hormone. Application of EGF did not induce any acute [Ca(2+)](i) response. EGF treatment for 1-2 days in culture, however, enhanced [Ca(2+)](i) responses including [Ca(2+)](i) increase by ATP, UTP and other nucelotides, Ca(2+) release from thapsigargin-sensitive stores, as well as capacitative Ca(2+) entry. Genistein, a tyrosine kinase inhibitor, prevented EGF-induced cell proliferation and the [Ca(2+) ](i) responses in a dose-dependent manner. These results indicate that EGF treatment enhances Ca(2+) mobilization and capacitative Ca(2+) entry, well correlated with cellular proliferation in mammary epithelial cells.

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

“…Fluo‐4 fluorescence change (F/F 0 ) was used to measure the changes in [Ca 2+ ] i . Calibration for converting the fluorescence change to intracellular Ca 2+ concentration was achieved according to the cell‐free calibration method, described previously (Ichikawa et al, 2000). Experiments were performed at room temperature (about 25°C).…”

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

“…Fluo‐4 fluorescence change (F/F 0 ) was used to measure the changes in [Ca 2+ ] i . Calibration for converting the fluorescence change to intracellular Ca 2+ concentration was achieved according to the cell‐free calibration method, described previously (Ichikawa et al, 2000). Experiments were performed at room temperature (about 25°C).…”

Coupling of P2Y receptors to G proteins and other signaling pathways

“…Activation of receptor tyrosine kinases by growth factors has been shown to resensitize P2Y receptors in many different cell types by PKC‐dependent P2Y receptor phosphorylation 87,88. In retinal glial cells, P2Y receptors are desensitized by ATP and resensitized by growth factors (e.g., EGF, PDGF, NGF), via activation of PI3K and protein phosphatases, whereas resensitization is not dependent on the activity of PKC, Src kinases, or ERK1/2 89…”

Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression

“…Moreover, in MCF-7 cells, ATP acting extracellularly through the purinergic receptor, P2Y 2 , raised intracellular Ca 2ϩ levels and increased proliferation (49). In mouse mammary epithelial cells, the enhancement of Ca 2ϩ mobilization and capacitative Ca 2ϩ entry by EGF induced the proliferating stage (21).…”

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

“…Fluo‐4 fluorescence change (F/F 0 ) was used to measure the changes in [Ca 2+ ] i . Calibration for converting the fluorescence change to intracellular Ca 2+ concentration was achieved according to the cell‐free calibration method, described previously (Ichikawa et al, 2000). Experiments were performed at room temperature (about 25°C).…”

Coupling of P2Y receptors to G proteins and other signaling pathways

“…Activation of receptor tyrosine kinases by growth factors has been shown to resensitize P2Y receptors in many different cell types by PKC‐dependent P2Y receptor phosphorylation 87,88. In retinal glial cells, P2Y receptors are desensitized by ATP and resensitized by growth factors (e.g., EGF, PDGF, NGF), via activation of PI3K and protein phosphatases, whereas resensitization is not dependent on the activity of PKC, Src kinases, or ERK1/2 89…”

Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression

“…Moreover, in MCF-7 cells, ATP acting extracellularly through the purinergic receptor, P2Y 2 , raised intracellular Ca 2ϩ levels and increased proliferation (49). In mouse mammary epithelial cells, the enhancement of Ca 2ϩ mobilization and capacitative Ca 2ϩ entry by EGF induced the proliferating stage (21).…”

Cell density‐dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells

“…Fluo‐4 fluorescence change (F/F 0 ) was used to measure the changes in [Ca 2+ ] i . Calibration for converting the fluorescence change to intracellular Ca 2+ concentration was achieved according to the cell‐free calibration method, described previously (Ichikawa et al, 2000). Experiments were performed at room temperature (about 25°C).…”

Coupling of P2Y receptors to G proteins and other signaling pathways

“…Activation of receptor tyrosine kinases by growth factors has been shown to resensitize P2Y receptors in many different cell types by PKC‐dependent P2Y receptor phosphorylation 87,88. In retinal glial cells, P2Y receptors are desensitized by ATP and resensitized by growth factors (e.g., EGF, PDGF, NGF), via activation of PI3K and protein phosphatases, whereas resensitization is not dependent on the activity of PKC, Src kinases, or ERK1/2 89…”

Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression

“…Moreover, in MCF-7 cells, ATP acting extracellularly through the purinergic receptor, P2Y 2 , raised intracellular Ca 2ϩ levels and increased proliferation (49). In mouse mammary epithelial cells, the enhancement of Ca 2ϩ mobilization and capacitative Ca 2ϩ entry by EGF induced the proliferating stage (21).…”