Ca²⁺ Control of Transcription

Ca²⁺-Mediated Phosphorylation of CREB

The Ca²⁺/cAMP response element binding protein CREB is a transcription factor.¹¹ CREB transcriptional activation occurs through its binding to the CREB-binding protein, CBP, a co-activator protein that links to many factors of the general transcriptional machinery. Phosphorylation of CREB at Ser-133 increases its affinity for CBP, and it can be observed with phospho-specific antibodies. CREB can be phosphorylated not only at SER-133 but also at S-142. Phosphorylation is mediated by a variety of kinases, with the cAMP/PKA pathway often being the principal modulator.¹² In the case of Ca²⁺-dependent activation, CREB phosphorylation is thought to result from the following signaling cascade:¹³ Ca²⁺ binds to calmodulin, Ca²⁺/CaM enters the nucleus and activates calmodulin-dependent kinase IV (CaMKIV), with Ca²⁺/CaMKIV phosphorylating CREB. In addition, Ca²⁺/CaM activates CaMKIV kinases that substantially enhance Ca²⁺/CaMKIV activity. Further, Ca²⁺/CaMKIV can regulate CREB binding protein by direct phosphorylation.¹³ Whether and to what extent these cascades apply to VSMC have not yet been clarified.

Ca²⁺ Controls Transcription Beyond CREB

Ca²⁺/CaM modulate transcriptional activation via signaling cascades in addition to the CREB cascade. For example, Ca²⁺/CaM activates the phosphatase calcineurin (CaN) that activates NFAT.¹⁴ Dephosphorylated NFAT can enter the nucleus, where it binds to and activates CBP in synergy with MEF2. Diverse transcription-dependent forms of neuronal plasticity suggest that the Ca²⁺ concentration in the cytosol, [Ca²⁺]_c, and in the nuclear matrix, [Ca²⁺]_nu, can modulate transcription differentially.¹⁵ For example, [Ca²⁺]_nu is known to activate the retinoic acid orphan receptor via Ca²⁺/CaMKIV. [Ca²⁺]_nu activates members of the MEF2 family of transcription factors via Ca²⁺/CaMKIV phosphorylation. Independently of CaM, elevated [Ca²⁺]_nu can activate p38, which in turn phosphorylates and activates MEF2.¹³ Increments in [Ca²⁺]_nu and Ca²⁺/CaM have been reported to inhibit transcription when they activate Cabin-1, which competes with CBP for binding to MEF2. Because Cabin-1 associates with histone deacetylases 1 and 2, its dissociation from MEF2 may replace a repressing complex with an activating complex. Thus, Ca²⁺/CaMKIV could enhance MEF2 transcription in an indirect way.¹⁶

The list of cascades involved in the Ca²⁺ modulation of transcription could be extended. It has been previously reported that elevated Ca²⁺ modulates transcription not through a single pathway, but via multiple cascades. Extrapolation of the present knowledge from neuronal to vascular tissue requires a re-analysis of the existence and function of these components. The use of transgenic mice will likely further our understanding of which one of the cascades described is important for VSMC. Future experiments should extend global measurements of [Ca²⁺]_c through Ca²⁺ imaging to provide information on whether the Ca²⁺ concentration increases close to the sarcolemma, either in the central parts of the cytosol or within the nuclear matrix.

Elevated [Ca²⁺]_c Results From Multiple Ca²⁺ Fluxes

The cellular distribution of Ca²⁺ ions is tightly controlled. In VSMC, [Ca²⁺]_c increases as consequences of passive Ca²⁺ influx through the sarcolemma (Ca²⁺ channels and nonselective cation channels) and Ca²⁺ release from the sarcoplasmic reticulum SR (ryanodine and IP₃ receptors). [Ca²⁺]_c decreases because of ATPases that transport Ca²⁺ ions into the extracellular space (PMCA) and into the SR Ca²⁺ store (SERCA). In addition, [Ca²⁺]_c decreases when Ca²⁺ ions bind to the mobile and fixed buffers in the cytosol and translocate into cell compartments such as the mitochondria (Ca²⁺ uniporter in the inner mitochondrial membrane) or the nucleus (pathways are controversial at present).

Ca²⁺ influx through dihydropyridine-sensitive L-type Ca²⁺ channels generates the Ca²⁺ current I_Ca-L. In VSMC, I_Ca-L has been thoroughly studied. The results help to explain how I_Ca-L increases [Ca²⁺]_c and activates contraction, and how drugs relax VSMC (eg, dihydropyridines blocking Ca²⁺ channels; K⁺ channel openers gating Ca²⁺ channels). Whereas L-type Ca²⁺ channels are voltage-gated, a small (+10 mV) change in membrane potential has been shown to increase [Ca²⁺]_c by 75 nM.¹⁷ The influence of voltage-gated I_Ca-L is often demonstrated by measuring increments in [Ca²⁺]_c or in contractile force by superfusing solutions with elevated [KCl]. The specific contribution of I_Ca-L is shown by blocking the events with dihydropyridines. In this way, the Burlington group^3,4 has elaborated on the impact of membrane depolarisation and Ca²⁺ influx through L-type Ca²⁺ channels for transcriptional activation by correlating increments in [Ca²⁺]_c, CREB-phosphorylation, and c-fos expression. They also defined the components of the cascade by means of “specific” inhibitors: dihydropyridines for I_Ca-L, calmidazolium for CaM, and KN-93 for inhibition of calmodulin-dependent kinases.

The article by Pulver et al⁶ asks whether transcriptional activation can be activated by additional Ca²⁺ influx pathways. The authors demonstrate that SOCE can both increase [Ca²⁺]_c and activate CREB phosphorylation and c-fos expression, similar to I_Ca-L. In cultured VSMC, SOCE seems to be the only vehicle for significant Ca²⁺ influx, as can be expected from a preparation that rapidly loses the L-type Ca²⁺ channels when it dedifferentiates in tissue culture. However, results from cultured VSMC may be misleading. For example, studies in rat aortic VSMC indicated that loading the cells with BAPTA did not prevent but rather stimulated the KCl-induced or stretch-induced CREB phosphorylation.¹⁸ It must then be recognized that Pulver et al repeated their experiments in VSMC of intact arteries. In doing so, the authors demonstrated that VSMC of intact arteries responded to thapsigargin with increments in [Ca²⁺]_c and CREB phosphorylation, and that the efficacy of thapsigargin was at least as high as the efficacy of membrane depolarisation.

Does SOCE Increase [Ca²⁺]_c at Physiological Conditions?

The work by Thrastup et al shows activation of SOCE by treating VSMC with thapsigargin, an inhibitor of the SR Ca²⁺-ATPase SERCA.¹⁹ Instead of using electrophysiology, the authors define Ca²⁺ influx via SOCE by measuring thapsigarin-induced increments in [Ca²⁺]_c with a fluorescent Ca²⁺ chameleon indicator. Involvement of SOCE is demonstrated by blocking the [Ca²⁺]_c increment, which is accomplished by lowering the extracellular Ca²⁺ concentration or by blocking the SOCE with Ni²⁺ or specific drugs. Although the Ca²⁺ flux is not directly measured, these experiments are state-of-the-art. The results demonstrate without doubt the importance of SOCE for the [Ca²⁺]_c balance of VSMC when SERCA activity is inhibited or blocked.

Although these results are easy to accept in context with membrane physiology, it becomes problematic to apply these results to transcriptional control in vivo, where SERCA is active. Sun et al compared the increments in [Ca²⁺]_c caused by thapsigargin with those caused by administration of serum, an intervention known to stimulate IP₃-mediated Ca²⁺ release.²⁰ Serum transiently increased [Ca²⁺]_c, and the comparison between increments in [Ca²⁺]_c caused by to serum and thapsigargin suggests that [Ca²⁺]_c can be reliably measured with chameleon Ca²⁺ indicator. The comparison, however, does not address the point of whether the “physiological” serum-induced increment in [Ca²⁺]_c can activate CREB phosphorylation at all or to an extent similar to post-thapsigargin application.

These unanswered questions should not exclude SOCE as a potentially important player in the Ca²⁺ control of transcription. Typically, SOCE caused by IP₃-induced SR Ca²⁺ release can be measured only if SERCA was inhibited (thapsigargin), or if Ca²⁺ was heavily buffered by millimolar concentrations of EGTA or BAPTA.^8,9 Recent work on white blood cells (RBL-1 cells) demonstrates that IP₃-induced Ca²⁺ transients can activate SOCE at physiological conditions, providing that the mitochondria take-up Ca²⁺ from SOCE.⁹ In other words, in the absence of exogenous buffers and without competition from noninhibited SERCA, the Ca²⁺ ions released from SR are rapidly transferred into mitochondria. Mitochondria may be suitable for just such a job, because they colocalize with the domains of Ca²⁺ entry via SOCE and with the SERCA in the ER.²¹ In this way, mitochondria can reduce the rate and amount of refilling SR with Ca²⁺ and can therefore reduce the inactivation of SOCE. These results suggest that SOCE can significantly contribute to the physiological Ca²⁺ balance of leukocytes.⁹ Whether similar arguments apply to SOCE in VSMC has yet to be analyzed.

Ca²⁺ Control of Transcription in VSMC: Where Do We Stay?

Neuroscience suggests that the Ca²⁺ control of transcription can adapt cell function to a wide variety of demands, from synaptic plasticity to cell metabolism. The diversity of the effects is attributed to the distribution of the Ca²⁺ signals over time and space. In neurons, the time domain is the interval between the groups of action potentials.²² The space domain depends on the place where the Ca²⁺-permeable neuronal synapses localize. With regard to long-term potentiation, Ca²⁺ influx through L-type Ca²⁺ channels has a higher efficacy than Ca²⁺ influx via NMDA receptors.²³ The results suggest that the colocalization of the Ca²⁺-permeable membrane channel with submembranous proteins could be a key factor. The information changes, for example, after Ca²⁺ ions have bound to diffusible proteins (such as CaM)—it can slowly spread over longer distances than can the Ca²⁺ ions, whose diffusion is fast but limited in space by Ca²⁺ binding. The effects of increments in cytosolic [Ca²⁺]_c have been separated from those of elevated nuclear [Ca²⁺]_nu, ie, these 2 events seem to modulate different Ca²⁺-dependent cascades.²⁴ Understanding how [Ca²⁺]_c and [Ca²⁺]_nu operate together is only the beginning.

In comparison with neuroscience, vascular biology analyzes the “Ca²⁺ control of transcription” with fewer groups and over a shorter time period. The article by Pulver et al⁶ is an important step in furthering our understanding. However, it is also an example that much work is still needed to provide information on specific signaling cascades, to differentiate effects caused by cytosolic from those caused by nuclear Ca²⁺, and to find out if the state of mitochondrial respiration modulates the efficacy by which SOCE can contribute to increments in [Ca²⁺]_c or in transcription. Expanding on the work of the Burlington group will surely further our understanding of VSMC, their plasticity between differentiated contractile phenotype, and the proliferating phenotype involved in the development of vascular pathologies. Any new developments will likely profit from the neurosciences, because any new studies will initially apply similar questions, models, and methods as those already used in the neurosciences field. Pulver et al⁶ have introduced the idea that transcription may be activated when the Ca²⁺ stores are emptied. Whether this hypothesis holds true when the store is emptied by more physiological, agonist-induced SR Ca²⁺ release remains to be tested. Regardless, ideas and results presented by Pulver et al will certainly stimulate the community of cardiovascular biologists to further test and study the Ca²⁺ hypothesis in future experiments.