Epidermal growth factor (EGF) triggers nuclear calcium signaling through the intranuclear phospholipase Cδ-4 (PLCδ4).

Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.

Nuclear inositol 1,4,5-trisphosphate receptors regulate local Ca2+ transients and modulate cAMP response element binding protein phosphorylation.

Several lines of evidence indicate that increases in nuclear Ca(2+) have specific biological effects that differ from those of cytosolic Ca(2+), suggesting that they occur independently. The mechanisms involved in controlling nuclear Ca(2+) signaling are both controversial and still poorly understood. Using hypotonic shock combined with mechanical disruption, we obtained and characterized a fraction of purified nuclei from cultured rat skeletal myotubes. Both immunoblot studies and radiolabeled inositol 1,4,5-trisphosphate [IP(3)] binding revealed an important concentration of IP(3) receptors in the nuclear fraction. Immunofluorescence and immunoelectron microscopy studies localized type-1 and type-3 IP(3) receptors in the nucleus with type-1 receptors preferentially localized in the inner nuclear membrane. Type-2 IP(3) receptor was confined to the sarcoplasmic reticulum. Isolated nuclei responded to IP(3) with rapid and transient Ca(2+) concentration elevations, which were inhibited by known blockers of IP(3) signals. Similar results were obtained with isolated nuclei from the 1B5 cell line, which does not express ryanodine receptors but releases nuclear Ca(2+) in an IP(3)-dependent manner. Nuclear Ca(2+) increases triggered by IP(3) evoked phosphorylation of cAMP response element binding protein with kinetics compatible with sequential activation. These results support the idea that Ca(2+) signals, mediated by nuclear IP(3) receptors in muscle cells, are part of a distinct Ca(2+) release component that originates in the nucleus and probably participates in gene regulation mediated by cAMP response element binding protein.