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Calcium (Ca2+) is a versatile second messenger that regulates a wide range of cellular functions. Although it is not established how a single second messenger coordinates diverse effects within a cell, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity. Ca2+ signaling patterns can vary in different regions of the cell and Ca2+ signals in nuclear and cytoplasmic compartments have been reported to occur independently. No general paradigm has been established yet to explain whether, how, or when Ca2+ signals are initiated within the nucleus or their function. Here we highlight that receptor tyrosine kinases rapidly translocate to the nucleus. Ca2+ signals that are induced by growth factors result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. This novel signaling mechanism may be responsible for growth factor effects on cell proliferation.
Intracellular Ca2+ can regulate cellular processes as distinct as cell death and proliferation (1). To achieve this versatility, there is increasing evidence that the spatial patterns of Ca2+ signals may determine their specificity (2). Ca2+ signals in nuclear and cytoplasmic compartments occur independently in several different cell types (3). However, the mechanisms and pathways that promote localized increases of free Ca2+ levels in the nucleus have not been entirely defined.
Recently, ligand-dependent translocation of receptor tyrosine kinases (RTKs) to the nucleus has been reported (4–7). RTKs can activate phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2), generating two intracellular products: inositol 1,4,5-trisphosphate (InsP3), a universal Ca2+-mobilizing second messenger, and diacylglycerol, an activator of protein kinase C (PKC) (8,9). It has also been reported that the interior of the nucleus has all the Ca2+ signaling machinery necessary to produce nuclear Ca2+ signaling (10–15). The translocation of RTK to the nucleus indicates a new mechanism by which RTK increases Ca2+ in the nucleus and a new paradigm to explain the mechanism and pathways that promote nuclear Ca2+ signaling. This review highlights the recent advances in this area.
PLC hydrolyzes PIP2 to generate InsP3 (16), and InsP3 then binds to the InsP3 receptor (InsP3R) to release Ca2+ from internal stores. It is well established that components necessary for InsP3-mediated Ca2+ signaling are present in the plasma membrane and the endoplasmic reticulum, and there is evidence that these components are also present in the nuclear envelope as well. These components include PIP kinase (PIPK) (17,18), which synthesizes PIP2, plus PLC (19) and the InsP3R (20–22). InsP3R is found on both the cytoplasmic and the intranuclear side of the nuclear membrane (11,23), and the nuclear envelope contains sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps for Ca2+ reuptake as well (24). The nucleus, therefore, is equipped to produce InsP3 and to release and take up free Ca2+, independent of cytosolic InsP3 or Ca2+. Although Ca2+ can spread passively from the cytosol into the nucleus under certain circumstances (25–27), intranuclear InsP3 can increase Ca2+ directly within the nucleus as well, both in isolated nuclei (12,20,28) and in nuclei within intact cells (23,29,30). Moreover, RTKs may selectively activate nuclear isoforms of PLC (18,31). However, until recently it was not known whether such receptors use this mechanism to increase Ca2+ in the nucleus. Two additional details about nuclear Ca2+ signaling have recently been established. First, the relative distribution of InsP3R isoforms in the nucleus and cytosol can differ among cell types (21). Because each InsP3R isoform has distinct sensitivities to InsP3 (32) and to Ca2+ (33,34), this differential distribution provides a mechanism by which the nucleus may be more sensitive than the cytosol to InsP3-mediated Ca2+ release in certain cell types (21). Second, InsP3-gated Ca2+ stores are found not only within the nuclear envelope, but also along a nucleoplasmic reticulum (23). PIPK and PIP2 are present in the interior of the nucleus (14), and insulin and hepatocyte growth factor (HGF) can induce InsP3 production in nuclei (6,7,35). These findings suggest that Ca2+ signaling machinery is present not only along the nuclear envelope but within the interior of the nucleus as well, which may provide an additional level of spatial control of nuclear Ca2+ signaling. In fact, Ca2+ signals induced by HGF and insulin begin in the nucleus (6,7); nuclear Ca2+ signals are initiated in both SKHep-1 cells and primary hepatocytes when PIP2 is hydrolyzed to form InsP3 (6,7). Moreover, both the HGF receptor (c-met) and insulin receptor translocate to the nucleus (Figure 1). Translocation of the HGF receptor to the nucleus depends upon the adaptor protein Gab1, that contains a nuclear localization sequence and importin-β1, and the formation of Ca2+ signals depends upon this translocation (6). Transport of proteins through the nuclear pore complex typically involves importins α/β and exportins. Specifically, importin-β binds to the classical lysine-rich nuclear localization signal in the cargo, and importin-β interacts with the importin-β/cargo complex to guide it through the nuclear pore (6). Together, these data indicate that RTKs can activate the calcium signaling machinery within the nucleus.
Nuclear Ca2+ signaling directly regulates cellular functions such as activation of kinases within the nucleus (23,36), protein transport across the nuclear envelope (11,37), and transcription of certain genes (38–40). For example, nuclear Ca2+ activates calmodulin kinase IV (36) and induces translocation of intranuclear but not cytosolic PKC (23). Gene transcription mediated by either the cAMP response element (CRE), CRE binding protein (CREB), or CREB binding protein (CBP) specifically depends upon increases in nuclear Ca2+, whereas gene transcription mediated by the serum response element instead is mediated by increases in cytoplasmic Ca2+ (38,39). Transcriptional activation of Elk-1 by epidermal growth factor also depends upon nuclear rather than cytosolic Ca2+ (40). Moreover, Ca2+ can bind to and directly regulate certain nuclear transcription factors (41), and can affect DNA structure as well (42). Nuclear Ca2+ can negatively regulate the activity of transcription factors as well (43). This was demonstrated by examining the relative effects of nuclear and cytosolic Ca2+ on the activity of the transcription enhancer factor TEF/TEAD. Chelation of nuclear but not cytosolic Ca2+ increased TEAD activity to twice that of controls, providing evidence that nuclear Ca2+ negatively regulates the activity of this transcription factor. Collectively, these findings show that nuclear Ca2+ regulates the expression of certain genes. Exogenous expression of the Ca2+ buffering protein parvalbumin has shown that intracellular Ca2+ regulates cell growth (44), but lack of effective experimental tools has made it difficult to demonstrate whether the effect of Ca2+ on cell growth is due to nuclear or cytosolic Ca2+ signals. Initial functional studies of nuclear Ca2+ on gene transcription relied on microinjection of Ca2+ chelators into either the nucleus or cytosol of individual cells (39), but it is impractical to use this labor-intensive approach to conduct biochemical, cell population, or in vivo studies. However, a newer approach has been developed in which cells are infected with adenovirus to deliver Ca2+ chelators such as parvalbumin that are targeted to be expressed in either the nucleus or cytosol (45). Nuclear Ca2+ stimulates cell proliferation rather than apoptosis and specifically permits cells to advance through early prophase (45). Furthermore, nuclear Ca2+ regulates cell proliferation in multiple cell lines and in vivo (45).
The current evidence suggests that nucleoplasmic Ca2+ regulates cell cycle progression. RTKs move to the nucleus to generate InsP3 and therefore Ca2+ signals within the nucleus, and this nuclear Ca2+ signaling is important for cell proliferation. Further work is needed to identify the mechanism by which RTKs move to the nucleus and how nucleoplasmic Ca2+ control the pathways involved in cell cycle progression.
Research supported by NIH (#DK57751, #DK34989, and #DK45710), and by CNPq, FAPEMIG, and Howard Hughes Medical Institute.
Presented at the IV Miguel R. Covian Symposium, Ribeirão Preto, SP, Brazil, May 23–25, 2008.