Calcium Regulation of Gene Expression: Isn't That Spatial?
Authors: David D. Ginty
Journal: Neuron (1997)

Elevation of intracellular calcium ion concentrations can influence a wide variety of biochemical processes. In the nervous system, excitatory neurotransmission can markedly increase the concentration of free calcium ions within neurons, and this second messenger activates expression of genes critical for long-term neuronal adaptive responses, including the maintenance phases of LTP and LTD. Thus, calcium-sensitive genes may be important substrates of complex neurobiological phenomena underlying learning and memory. This review focuses on the mechanisms of calcium activation of gene expression. In particular, I highlight recent exciting results which demonstrate that spatially distinct pools of calcium can differentially influence the nature of the genetic response (9Hardingham G.E. Chawla S. Johnson C.M. Bading H. Nature. 1997; 385: 260-265Crossref PubMed Scopus (626) Google Scholar). Bursts of neuronal activity rapidly trigger activation of transcription of a class of genes known as immediate early genes (IEGs). At least 50 distinct IEGs can be induced by synaptic activity, and it is likely that many IEGs remain to be identified. Several IEGs, including the prototypical IEG the c-fos proto-oncogene, encode transcription factors that regulate expression of a second class of genes termed late response genes. Late response genes encode proteins that affect many aspects of neuron function, and include growth factors, neurotransmitter synthesizing enzymes, synaptic vesicle proteins, ion channels, and structural proteins. In addition, many IEGs themselves encode proteins, such as an inducible form of cyclooxygenase (19Yamagata K. Andreasson K.I. Kaufman W.E. Barnes C.A. Nathans D. Worley P.F. Neuron. 1993; 11: 372-386Abstract Full Text PDF Scopus (1056) Google Scholar), that may directly influence the physiological state of a neuron. Importantly, the characterization of calcium regulation of transcription of c-fos has provided a structural framework useful for understanding how this second messenger ion governs early nuclear events that affect neuronal plasticity. There at least two distinct DNA sequence motifs within the upstream regulatory region of c-fos that are critical for calcium activation of c-fos transcription, the CRE (cyclic AMP response element) and the SRE (serum response element). The CRE is constitutively occupied by members of the CREB/ATF family of transcription factors. CREB is the prototypical member of this family of basic/leucine zipper-containing transcription factors (reviewed in12Meyer T.E. Habener J.F. Endocrine Rev. 1993; 14: 269-290PubMed Google Scholar). Distinct family members can bind to the CRE as homodimers, or as heterodimers with other family members. In contrast, the SRE is occupied by a ternary complex that consists of a serum response factor (SRF) dimer, and a single member of the p62TCF family of Ets domain-containing transcription factors. The importance of these sequence motifs for calcium-mediated expression of c-fos is reflected by the observation that the integrity of all of the major cis-regulatory elements are required for proper calcium-sensitive activation of c-fos transcription in transgenic animals (Robertson et al., 1996). Moreover, either the CRE or the SRE alone is sufficient to confer calcium induction of an otherwise non-responsive reporter gene in transfected cells (1Bading H. Ginty D.D. Greenberg M.E. Science. 1993; 260: 181-186Crossref PubMed Scopus (941) Google Scholar, 13Misra R.P. Bonni A. Miranti C.K. Rivera V.M. Sheng M. Greenberg M.E. J. Biol. Chem. 1994; 269: 25483-25493Abstract Full Text PDF PubMed Google Scholar). Since most IEGs contain one or more CREs and/or SREs within their transcriptional regulatory regions, it is very likely that CREB, SRF, and p62TCF are critical mediators of the general nuclear response to glutamate and other neurotransmitters that activate gene expression through calcium-dependent signaling pathways. Seconds to minutes after synaptic activation and calcium entry into a neuron, CREB becomes newly phosphorylated on its transcriptional regulatory residue, serine 133 (Ser133) (8Ginty D.D. Kornhauser J.M. Thompson M.A. Bading H. Mayo K.E. Takahashi J.S. Greenberg M.E. Science. 1993; 260: 238-241Crossref PubMed Scopus (721) Google Scholar). Since mutation of Ser133 to a non-phosphorylatable alanine residue abolishes its ability to confer calcium activation of c-fos transcription, this phosphorylation event is believed to be a critical trigger that initiates c-fos transcription. This idea has naturally led to two important questions: what are the enzymes responsible for phosphorylation and dephosphorylation of CREB on Ser133, and how does this phosphorylation event influence gene transcription? Multiple calcium-regulated CREB kinases exist. CaMKII was initially thought to be an important calcium-regulated CREB kinase, but recent evidence points towards other enzymes as the key mediators of CREB Ser133 phosphorylation. For example, although nuclear isoforms of CaMKII exist within discrete neuronal populations, CaMKII is generally not found in the nucleus and, therefore, it is unlikely that it catalyzes phosphorylation of CREB in most neurons. Furthermore, CaMKII phosphorylates CREB on Ser142 as well as on Ser133. Phosphorylation of Ser142 inhibits CREB-mediated gene expression (reviewed by7Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1213) Google Scholar). Since calcium is believed to be responsible for activating CREB-mediated transcription in most neurons, CaMKII may not be the calcium-regulated CREB Ser133 kinase. Rather, calcium-sensitive phosphorylation of CREB Ser133 is likely to be catalyzed by any of a number of other enzymes. CaMKIV is one excellent candidate because it is localized to the nucleus, it phosphorylates CREB on Ser133, but not on Ser142, and because application of antisense CaMKIV oligonucleotides to cultured hippocampal neurons can block calcium-mediated phosphorylation of CREB on Ser133 (4Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar). However, CaMKIV has a relatively restricted expression pattern and, therefore, it may not catalyze calcium-dependent phosphorylation of CREB on Ser133 in all neurons. Other candidate calcium-regulated CREB kinases include p70S6K, pp90RSK family members, and cAMP-dependent protein kinase (PKA). Each of these enzymes can efficiently phosphorylate CREB on Ser133 in vitro, and each may be activated, albeit indirectly, by neurotransmitters that trigger an increase in intracellular levels of calcium. Moreover, upon their activation, each of these enzymes is localized, at least in part, within the nucleus. Thus, multiple calcium-regulated CREB kinases exist. It is likely that the type of neuron, the mode of calcium entry into the neuron, the magnitude of rise in intracellular calcium, the distance between the synapse and the nucleus, and the pool of activator calcium (nuclear versus cytoplasmic) are all factors that influence the mechanism by which calcium triggers phosphorylation of CREB on Ser133. In addition to calcium-regulated protein kinases, the phosphorylation state of CREB on Ser133 is modulated by protein phosphatases. Two serine threonine phosphatases, PP-1 and PP-2B, are the best candidate Ser133-phosphorylated CREB phosphatases. PP-1 is likely to catalyze this dephosphorylation event in hippocampal neurons (4Bito H. Deisseroth K. Tsien R.W. Cell. 1996; 87: 1203-1214Abstract Full Text Full Text PDF PubMed Scopus (953) Google Scholar). In addition, PP-1 activity may be sensitive to synaptic activity (e.g.,14Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (874) Google Scholar), providing an additional regulatory mechanism whereby calcium modulates the phosphorylation state of CREB Ser133, and CREB-mediated transcription. Upon its phosphorylation on Ser133, CREB becomes bound by a transcriptional co-activator protein, CBP, or its close relative p300. CBP and p300 are large molecules that contain multiple domains that mediate a variety of protein-protein interactions. In addition to directly binding to Ser133-phosphorylated CREB, CBP contacts general transcription initiation factors which likely influences assembly of the pre-initiation complex (PIC). Exciting new studies have shown that CBP itself has an intrinsic enzymatic activity; CBP is a histone acetyl transferase (HAT) (16Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell, 953–959. 1996Google Scholar, 3Bannister A.J. Koukarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1499) Google Scholar). Moreover, CBP binds to another protein, P/CAF (p300/CBP-associated protein), that also has HAT activity (20Yang X.-J. Ogryzko V.V. Nishikawa J.-I. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1289) Google Scholar). HATs catalyze acetylation of lysine residues located within amino terminal tails of histones. These positively charged histone tails mediate histone-DNA interactions which form the structural basis of the nucleosome, and acetylation neutralizes this positive charge. Therefore, acetylation of histones loosens histone-DNA interactions which is likely to facilitate the association between regulatory transactivator proteins and nucleosomal DNA. Thus, through mediating protein-protein interactions and via its intrinsic HAT activity, CBP may promote assembly of the PIC at the promoter of IEGs. This model (Figure 1) may explain how phosphorylation of CREB Ser133, through recruitment of the co-activators CBP or p300 to the promoter, influences initiation of IEG transcription. While CREB is an important calcium responsive transcription factor, internal deletions within the c-fos gene that abolish CREB binding do not preclude calcium activation of c-fos transcription. This finding, together with the observation that SRE-driven reporter genes are calcium-responsive, indicates that the SRE is also a calcium response element. Like CREB, SRF and members of the p62TCF family of transcription factors that bind to the SRE are important mediators of calcium-sensitive c-fos transcription. SRE-mediated transcription that requires both SRF and a p62TCF family member is termed TCF-dependent transcription, while that which requires SRF, but not a p62TCF family member, is termed TCF-independent transcription. An increase in intracellular calcium can activate both TCF-dependent, and TCF-independent transcription. How does calcium influence TCF-dependent and TCF-independent transcription of c-fos and other IEGs? While little is known about how the SRE mediates calcium-sensitive transcription, clues to the answer come, in part, from studies of growth factor regulation of c-fos transcription. Many polypeptide growth factors, including NGF and EGF, induce TCF-dependent transcription. These growth factors activate the Ras/MAPK signal transduction cascade, which includes the GTP binding protein Ras, and the protein kinases Raf, MEK, MAPK and pp90RSK. Upon activation, MAPK translocates from the cytoplasm to the nucleus where it phosphorylates the p62TCF family member ELK-1 on several serine and threonine residues. MAPK phosphorylation of ELK-1 is critical for growth factor sensitive, TCF-dependent transcription (10Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1994; 73: 395-406Abstract Full Text PDF Scopus (328) Google Scholar). It is unclear how phosphorylation of ELK-1 influences gene expression, but CBP and p300 may contribute to TCF-dependent transcription (11Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar). Like growth factors, neurotransmitters that increase intracellular levels of calcium activate the Ras/MAPK pathway (2Bading H. Greenberg M.E. Science. 1991; 253: 912-914Crossref PubMed Scopus (406) Google Scholar). Thus, MAPK may also be an important mediator of calcium-sensitive, TCF-dependent transcription. In support of this idea, recent experiments employing MKP-1, an enzyme that dephosphorylates and inactivates ERKs and other MAPKs including p38MAPK and SAPK, demonstrated that MAPKs are responsible for mediating calcium-sensitive, TCF-dependent transcription in cultured cortical neurons (18Xia Z. Dudek H. Miranti C.K. Greenberg M.E. J. Neurosci. 1996; 16: 5425-5436Crossref PubMed Google Scholar). However, the mechanism by which this occurs is unclear because it has not yet been demonstrated that any p62TCF family member is a substrate for protein kinases activated by calcium signals in neurons. In addition to TCF-dependent transcription, the SRE can mediate calcium-sensitive, TCF-independent transcription. The mechanism by which calcium regulates TCF-independent transcription is unknown, although it does appear to be SRF-dependent. Further studies should emphasize the mechanism by which SRF and p62TCFs mediate calcium-sensitive transcription. Calcium may enter a neuron via several portals including voltage-sensitive calcium channels (VSCC), such as the L-type calcium channel, and ionotropic neurotransmitter receptors, such as the NMDA subtype of glutamate receptors. Alternatively, calcium may be released from its internal storage depot, the endoplasmic reticulum. Interestingly, depending on its route of entry into the neuron, calcium may influence distinct transcriptional responses in neurons. For example, calcium influx via VSCCs is more effective than calcium influx through NMDA receptors in triggering activation of expression of the BDNF gene in cortical neurons (reviewed in7Ghosh A. Greenberg M.E. Science. 1995; 268: 239-247Crossref PubMed Scopus (1213) Google Scholar). Moreover, calcium influx via VSCCs is more effective than calcium influx via NMDA receptors in activating CRE-mediated transcription of c-fos in hippocampal neurons (1Bading H. Ginty D.D. Greenberg M.E. Science. 1993; 260: 181-186Crossref PubMed Scopus (941) Google Scholar). Yet calcium entry via either VSCCs or NMDA receptors is sufficient to trigger SRE-mediated transcription of c-fos. How can the same second messenger ion differentially regulate CRE-mediated and SRE-mediated transcription within the same neuron? Are the levels of calcium attained different depending on its mode of entry, or does the route of calcium entry influence distinct biochemical processes? One potential explanation is that the CRE and the SRE can discriminate between spatially distinct pools of calcium. This possibility was addressed recently in experiments in which calcium within the nucleus of AtT20 cells, a pituitary cell line, was buffered to prevent its accumulation following activation of VSCCs (9Hardingham G.E. Chawla S. Johnson C.M. Bading H. Nature. 1997; 385: 260-265Crossref PubMed Scopus (626) Google Scholar). The buffer, BAPTA coupled to a 70kDa dextran molecule, was microinjected into the nucleus, and it did not leak from the nucleus to the cytoplasm. Therefore, the buffer largely prevented accumulation of nuclear calcium ions, but it did not affect accumulation of cytoplasmic calcium following activation of VSCCs. Under these conditions, SRE-mediated gene expression was normal following calcium entry via VSCCs. In dramatic contrast, CREB-mediated transcription was inhibited. Therefore, an increase in cytoplasmic calcium is not sufficient to promote CREB-mediated transcription; an increase in nuclear calcium is critical for CREB-mediated gene expression. Whether an increase in cytoplasmic calcium is sufficient to promote TCF-dependent transcription, TCF-independent transcription, or both remains to be determined. Since calcium-sensitive, TCF-dependent transcription is mediated by a member of the MAPK family of protein kinases, and since MAPK can translocate from the cytosol to the nucleus, it is reasonable to suspect that an increase in calcium levels exclusively within the cytoplasm may be sufficient to trigger MAPK-mediated, TCF-dependent transcription. Thus, spatially distinct pools of calcium do influence gene expression in unique ways. An increase in nuclear calcium concentration is critical for CREB-mediated transcription, and since phosphorylation of CREB on Ser133 influences CREB-mediated transcription, one may predict that a rise in nuclear calcium levels is also critical for phosphorylation of CREB Ser133. However, in hippocampal neurons, an increase in calcium concentration within the cytoplasm at the synapse, but not within the nucleus, appears to be necessary to promote phosphorylation of CREB on Ser133 (6Deisseroth K. Bito H. Tsien R.W. Neuron. 1996; 16: 89-101Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar). At first glance, these two observations may seem contradictory. One might immediately dismiss the significance of this apparent discrepancy by suggesting that calcium signaling mechanisms are profoundly different between these two cell types. For example, AtT20 cells are non-neuronal cells that do not express NMDA receptors, and calcium entry via NMDA receptors into hippocampal neurons is necessary for activity-dependent phosphorylation of CREB on Ser133. However, other explanations exist. For example, phosphorylation of CREB Ser133, while critical for CREB-mediated gene expression, may not be sufficient to promote IEG transcription. This idea is best exemplified by the observation that many extracellular stimuli induce robust phosphorylation of CREB on Ser133, yet only certain of these stimuli can induce CREB-mediated transcription in the absence of other transactivators. Thus, other critical regulatory event(s) are likely to accompany phosphorylation of CREB Ser133 during calcium activation of CREB-mediated transcription. Other events may include additional modifications of CREB, modifications of CBP or P/CAF, or associations between CBP and other regulatory proteins (e.g.,15Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Therefore, an increase in cytoplasmic calcium concentration in hippocampal neurons may be necessary or perhaps even sufficient to trigger phosphorylation of CREB Ser133, but it may not be sufficient to promote CREB-mediated transcription. A current major challenge is to identify and characterize regulatory events, in addition to phosphorylation of Ser133, that influence CREB-mediated IEG expression. One exciting prospect of the recent work on calcium signaling to the nucleus is that spatially distinct calcium signals may influence the activities of transcription factor complexes in unique ways. Thus, synaptic input that influences nuclear and cytoplasmic calcium levels differently may regulate distinct complex programs of gene expression, and therefore, distinct long-term neuronal changes. In other words, two excitatory synapses that differ only in their proximity to the cell body of the post-synaptic neuron may have profoundly different influence over the nature of the nuclear response. For example, a synapse in a distal dendritic process may have strong influence over SRE-mediated gene expression, and one in which the synapse is more proximal to the cell body may have control over SRE- as well as CRE-containing genes (Figure 2). Since some IEGs contain multiple SRE-like elements, they may be more sensitive to changes in cytoplasmic levels of calcium. Other IEGs, including somatostatin, proenkephalin and other neuropeptide genes, contain one or more CREs, but no SRE. As such, expression of these genes may be influenced by synaptic activity that affects nuclear calcium levels, but not by synaptic activity that increases calcium levels only within distal processes. This idea may explain the observation that LTP-inducing stimuli more efficiently triggers activation of Zif/268, which contains four putative SREs and only one or two CREs, than c-fos, which contains only one SRE and three functional CREs (5Cole A.J. Saffen D.W. Baraban J.M. Worley P.F. Nature. 1989; 340: 474-475Crossref PubMed Scopus (880) Google Scholar). Perhaps LTP-inducing stimuli more efficiently increase calcium concentration within distal spines and cytoplasm than in the nucleus. Thus, while much emphasis has been placed on CREB and its role in activity-dependent gene transcription, it is likely that SRF and p62TCF family members are also critical for activity- dependent expression of IEGs in neurons, and for mediating activity-dependent long-term neuronal adaptive responses. Future experiments should clarify the mechanisms by which the calcium signal is propagated from the synapse to the nucleus to trigger TCF-dependent and TCF-independent transcription, and the roles of SRF, Elk-1, Sap-1a and other p62TCFs in activity-dependent neuronal plasticity. Thus, it is possible that expression of entire complex genetic programs that control neuronal adaptive responses may be influenced by spatial aspects of calcium signaling, and the nature of excitatory synaptic input. While this idea remains to be experimentally verified, it seems clear that IEGs have the capacity to distinguish between different forms of calcium signaling, at least in part, because they can respond to spatially distinct pools of calcium.