Review Article | Open Access
Nikolai M. Soldatov, "1.2, Cell Proliferation, and New Target in Atherosclerosis", International Scholarly Research Notices, vol. 2013, Article ID 463527, 13 pages, 2013. https://doi.org/10.1155/2013/463527
1.2, Cell Proliferation, and New Target in Atherosclerosis
1.2 calcium channels are the principal proteins involved in electrical, mechanical, and/or signaling functions of the cell. 1.2 couples membrane depolarization to the transient increase in intracellular Ca2+ concentration that is a trigger for muscle contraction and CREB-dependent transcriptional activation. The CACNA1C gene coding for the 1.2 pore-forming subunit is subject to extensive alternative splicing. This review is the first attempt to follow the association between cell proliferation, 1.2 expression and splice variation, and atherosclerosis. Based on insights into the association between the atherosclerosis-induced molecular remodeling of 1.2, proliferation of vascular smooth muscle cells, and CREB-dependent transcriptional signaling, this review will give a perspective outlook for the use of the CACNA1C exon skipping as a new potential gene therapy approach to atherosclerosis.
It has been long known that Cav1.2 calcium channel blockers inhibit human brain tumor , pancreatic cancer [2, 3], breast cancers  and small cell lung cancer  because they inhibit cell proliferation and DNA synthesis. Correlation between the oncogenic transformation and expression of both Cav1.2 and Cav3 channels was demonstrated in spontaneously immortalized 3T3 fibroblasts [6, 7] suggesting that both types of calcium channels may play a role in cell proliferation. Indeed, studies showed that Cav3 (T-type) calcium channels regulate proliferation, for example, of BC3H1 cells , vascular smooth muscle (VSM) cells , and glioma, neuroblastoma, and neuroblastoma × glioma hybrid cells . Unlike the majority of cells, including the listed ones, normal human fibroblasts express only Cav1.2 . The pore-forming subunit of this channel was cloned from human fibroblasts and identified as a “short” (exon 1) isoform of the -coding gene CACNA1C . A variety of Cav1.2 calcium channel blockers, including dihydropyridines (DHPs) nifedipine and nicardipine, as well as diltiazem and verapamil inhibit cell proliferation and DNA synthesis in fibroblasts . Thus, human fibroblast is an excellent cell type to study the roles of Cav1.2 in proliferation not complicated by expression of other Cav genetic variants, cell transformation, and/or differentiation.
2. Ca1.2 and Proliferation of Normal Human Fibroblasts
Our earlier studies performed on normal human diploid fibroblasts have revealed a number of important features that point to the plasticity of the Cav1.2 expression in response to cell culture conditions, including cell-cell contact inhibition, presence of mitogens, and second messengers. The expression of Cav1.2 in the plasma membrane was measured using the DHP radioligand binding assay. DHPs bind to Cav1.2 with high affinity in equimolar ratio and are excellent probes for the expression of total (functional and dormant) Cav1.2 in the plasma membrane. The test system was based on the measurement of specific binding of 2,6-dimethyl-3-methoxycarbonyl-5-([2,3-3H2]-n-propoxycarbonyl)-4-(2′-difluoromethoxyphenyl)-1,4-dihydropyridine ([3H]PMD, NCBI PubChem CID 14267917)  to human embryonic diploid fibroblasts grown in Eagle’s medium supplemented with 10% serum. Under standard conditions of incubation (1 h at room temperature in Tris-buffered saline) [3H]PMD interacted with .9 nM with a single class of DHP receptors that were present at the maximum density of ≈1.2 pmol/106 cells in a sparse culture of fibroblasts (≈3–5 × 1,000/cm2) . The turnover rate of DHP receptors is approximately exponential with a half-life of ≈12 h, as it was estimated from the rate of loss of [3H]PMD binding sites in response to the net inhibition of protein synthesis by cycloheximide. With progression to confluent monolayers, the value for [3H]PMD binding did not change, but the value decreased ≈4 fold (Figure 1(a), compare bars 1 and 2), suggesting that expression of Cav1.2 is responsive to the arrest of fibroblasts proliferation by cell-cell contact inhibition.
The involvement of Cav1.2 in proliferation of human fibroblasts was further supported by the finding that concentration of DHP receptors, and, respectively, of Cav1.2 is strongly affected by mitogens and second messengers. Demonstrating a remarkable plasticity of the Cav1.2 expression in normal human fibroblasts (Figure 1(b)), serum deprivation induced a 2-fold increase in the density of DHP receptors that reached its maximum after 3-4 days of cultivation in the absence of serum (compare also bars 2 and 6 in Figure 1(a)). The elevation of the Cav1.2 expression was fully reversible and highly sensitive to serum and other mitogens. An addition of 10% serum reduced the density of DHP receptors to the initial level with almost the same time course (Figure 1(b)). Thus, inhibition of cell growth, proliferation, and DNA synthesis in fibroblasts by serum deprivation stimulates expression of Cav1.2 with a time course comparable with the DHP receptor turnover rate.
The response of fibroblasts to serum deprivation by boosting Cav1.2 expression is essentially identical to that caused by the inhibition of Cav1.2 by 1 μM diltiazem (Figure 1(a), compare bars 5 and 6), the calcium channel blocker that does not compete with DHPs for binding with the channel. In fact, diltiazem was present in the DHP binding assay medium throughout all experiments to enhance the affinity of the DHP probe to the channel receptor . Thus, it is reasonable to suggest that by boosting the Cav1.2 expression, the cell recruits more routes for the Ca2+ entry through the plasma membrane to overcome the lack of mitogens in serum deprivation or lack of conducting channels in the presence of diltiazem, both aimed at supporting cell proliferation until cell-cell contact inhibition terminates it and turns the cells into a quiescent state.
If this hypothesis is true, then the addition of DHP-insensitive routes for Ca2+ entry through the plasma membrane should eliminate the need in higher level of Cav1.2 expression. This observation exactly has been made when the serum-stimulated cells were supplemented with Ca2+ ionophore A23187 (Figure 1(a), bar 4). The A23187-induced Ca2+ entry dramatically reduced the cellular expression of Cav1.2. A similar effect was observed with 8Br-cAMP, the plasma membrane-permeable derivative of cAMP, showing that stimulation of the alternative cAMP-dependent cell signaling pathway may reduce the needs in Cav1.2 in proliferation of fibroblasts.
Stimulation of Cav1.2 expression in fibroblasts, arrested in the quiescent state by serum deprivation, strongly depends on cell proliferation. The measurement of [14C]thymidine incorporation as an assay for DNA synthesis in cells showed (Figure 2, dark bars) that individual mitogens stimulated cell proliferation in the order bFGF > insulin > EGF. In contrast, in that very order the same mitogens suppressed expression of Cav1.2 in fibroblasts as evidenced by the measurement of DHP receptor binding (open bars). Consistent with relatively low cell toxicity of dihydropyridines, blockade of Cav1.2 attenuated entry of cells into the S phase of cell cycle . Thus, the plasticity of Cav1.2 in fibroblast proliferation is associated with transient Cav1.2 expression responsive to both primary effectors and second messengers of cell proliferation.
3. Ca1.2 Variability
One of the most important features of Cav1.2 is its remarkable molecular diversity. The channel consists of the pore-forming protein, which binds calcium channel blockers. Three types of regulatory subunits, β, α2δ, and calmodulin (CaM), are constitutively tethered to . All these subunits are products of different genes that are located on different chromosomes. Cav1.2 shares the accessory subunits with other Cav1 and Cav2 channels. Moreover, , β, and α2δ are subject to individual alternative splicing that generates a large diversity of Cav1.2 channel complexes. The functional significance of the Cav1.2 splice variation is poorly understood not only because of the difficulties in identification of individual splice variants in the naturally occurring oligomeric proteins. The tendency of Cav1.2 to form large clusters in the plasma membrane [19–23] as well as homo- and heterooligomerization of β subunits  create additional major challenges for the investigation of Cav1.2 in regulation of cell proliferation, differentiation, and other functions—not to speak of an adequate elucidation of their roles in disease and search for new therapeutic approaches.
Molecular cloning showed that transcripts in human fibroblasts are composed of exons 1–50  with alternative splicing of exons 21/22 and 31/32 and constitutive splicing of exons 33 and 45 (Figure 3(a)) . However, variability associated with exons 1, 7, 8, 9, 34, 41, and 42 that were later found in human hippocampus, heart, and VSM cells [25–30] was not observed in fibroblasts.
It is not known which Cav1.2 splice variants are expressed in fibroblasts in response to serum deprivation, and whether they are structurally different from those present in proliferating cells. However, electrophysiological properties of the three of fibroblast splice variants (coexpressed with and α2δ-1) were compared in Xenopus oocyte expression system . Characteristics of the voltage-dependence and kinetics of inactivation of the barium current through the channel encoded by the CACNA1C transcript lacking exons 22, 31, 41A and 45 were found to be very similar to those recorded with (lacking exons 21, 31, 41A and 45) and (also lacking exon 33) (Figure 3(b)). However, voltage dependence of the DHP inhibition of the current was significantly different in the and channels: the IC50 values for the concentration dependence of the barium current inhibition by (+)isradipine, almost identical (6.2 and 7.3 nM, resp.) at −40 mV, were significantly different at −90 mV (680 and 79 nM for and , resp.). While such a difference in the pharmacological properties of the exon 21- and exon 22-splice variants is deemed unimportant in the case of fibroblasts, it may significantly contribute to the tissue specificity of this major class of calcium channel blockers in cardiac, vascular, and other responsive cells affected by cardiovascular diseases.
4. Ca1.2 in Atherosclerosis
Atherosclerosis is perhaps the single most deadly disease, leading to about 600,000 deaths annually in the USA, most of these due to the progression of the disease to heart attack or stroke . In spite of significant efforts, the molecular mechanisms of atherosclerosis are not currently well understood, and effective molecular targets for prevention and treatment are not elaborated. Atherosclerosis is an inflammatory process in medium and large size arteries that causes endothelial perturbation and local release of cytokines, as well as dedifferentiation, proliferation, and migration of VSM cells . Arterial VSM cells constitute the media of the artery and play a crucial role in its elasticity and contractility. Migration of VSM cells from the media to the intima of the arterial wall and proliferation of intimal smooth muscle cells are the major early events in the formation of atherosclerotic lesions. Recent advances in molecular genetics studies have revealed that genetic polymorphisms significantly influence susceptibility to atherosclerotic vascular diseases . However, none of the discovered susceptibility genes was directly implicated for proliferation and migration of VSM cells, one of the major pathophysiological responses to atherosclerosis at the cellular level.
The presence and activity of Cav1.2 calcium channels in VSM cells has been established both in patch clamp and molecular cloning experiments [29, 35–38]. Cav1.2 calcium channels play a major role in atherosclerosis because they are essential for Ca2+ signal transduction in VSM cells. Contraction of VSM cells is triggered by the Ca2+ current () through Cav1.2, and thus is affected by Ca2+ channel blockers. Since the 1990s, it is known that DHPs, particularly, the charged 2-aminoethoxymethyl DHP derivative amlodipine , exert a number of vasoprotective effects, including potent antiatherogenic action , inhibition of migration of VSM cells , and reduction of arterial intimal-medial thickening and plaque formation [42, 43]. Although it is tempting to link this activity in part to pleiotropic effects of calcium channel blockers , it is the inhibition of proliferation that essentially underlies it . The density of DHP receptors was found to depend on VSM cell proliferation . Association of Cav1.2 with mitogenesis in VSM cells is supported by the findings that DHPs reduced DNA synthesis stimulated by serum and PdGF [47–49], serotonin , EGF , and H2O2 (in mesangial cells) . Expression of Cav1.2 in VSM cells was shown to be cell cycle dependent, with the highest calcium current density in G1 phase . Whether these changes are reflected in the molecular repertoire of the Cav1.2 splice variants is the issue particularly important for the elucidation of new therapeutic targets in diseases leading to pathogenic proliferation of VSM cells, such as atherosclerosis.
Investigation of the alternative splicing in VSM cells [28, 54, 55] revealed the involvement of a number of CACNA1C exons generating impressive diversity of human vascular that includes possibly the VSM-specific splicing of exons 9/9a [35, 55] and exon 34 . To establish an association between the disease and CACNA1C splice variation at the level of cell, we have completed  the single-gene profiling of the molecular remodeling in VSM cells of an artery caused by atherosclerosis. The VSM cells were identified in frozen sections of six surgical biopsy samples of femoral and carotid arteries by immunostaining with an antibody against smooth muscle α-actin , used as a marker for VSM cells. The α-actin staining correlated with immunostaining by an anti- antibody in serial sections and was reduced in atherosclerotic regions (Figure 4) consistent with dedifferentiation of VSM cells [57, 58]. The reduced expression of at the protein level was corroborated by the quantitative RT-PCR data showing that the relative mRNA level in VSM cells (normalized to 18S RNA) was reduced fold (mean ± SEM) in the atherosclerotic region. Overall, the reduced expression of caused by the locally elaborated cytokines in the atherosclerotic regions of arteries resembles the reduced expression of DHP receptors observed in fibroblasts exposed to mitogens and/or second messengers after serum deprivation (Figure 1).
To find out whether the altered expression of in atherosclerotic VSM cells is accompanied by changes in the CACNA1C alternative splicing pattern, we isolated the immunohistochemically identified VSM cells by laser-capture microdissection from adjacent regions of arteries affected and not affected by atherosclerosis and identified the CACNA1C splice variants by RT-PCR. Our findings revealed an extended repertoire of the exon 21 splice isoforms in nonatherosclerotic VSM cells characterized by a complex splicing pattern of exons 9, 9A, 31–34, and 41A, including the electrophysiologically characterized (GenBank # z34811), (z34812), (AY830711), (AY830713), and (AY830712) splice isoforms. However, only the exon 22 isoform of was identified in atherosclerotic VSM cells. Thus, the switch of the CACNA1C alterative splicing from exon 21 to exon 22 (Figure 3(b)) is a molecular signature of the Cav1.2 remodeling of VSM cells to a pathophysiological proliferating state in atherosclerosis. The age, gender, ethnicity, drug exposure, and other co-morbid conditions did not appreciably affect this common pattern of the splice variation in VSM cells in response to atherosclerosis.
Careful electrophysiological analysis exhibited a number of differences in the properties of the “atherosclerotic” channel as compared to the isoforms in healthy VSM cells. The largest differences were found between the and channels (Figure 5). In response to step depolarization applied from the holding potential of −90 mV, both channels generate calcium currents () that inactivate with almost identical kinetics (Figure 5(a)). However, we found that through the channel recovers from inactivation significantly faster than that in (Figure 5(b)) and other isoforms present in healthy VSM cells. This finding suggests that alternative splicing in atherosclerosis may affect vascular tone as a result of the increase in the density in VSM cells. A hyperpolarization shift of the activation curve for the atherosclerotic channel variant, as compared to Cav1.2 in healthy VSM cells (Figure 5(c)), may also result in an increase of calcium entry in VSM cells. However, the overall 3-4-fold reduction in the expression of Cav1.2 in atherosclerotic VSM cells may scale down some of the observed electrophysiological changes.
5. CACNA1C Exon 22 as a New Therapeutic Target in Atherosclerosis
Direct DNA sequencing of the crude PCR amplification products indicated that the switch to the exon 22 isoform of vascular was almost complete in atherosclerosis because no distortion of the nucleotide peaks in the region of exon 21/22 was seen when compared to the exon 20 invariant region (Figure 6(a)). Thus, Cav1.2 underwent almost quantitative exon 21/22 remodeling in VSM cells of diseased artery regions.
Although the cellular mechanisms leading to the CACNA1C exon 21/22 switch may be very complex, the association with VSM cell proliferation is obvious. Indeed, a similar exon switch was observed in primary human aortic cells in culture after the quiescent nonproliferating cells, containing predominantly exon 21 splice variants, were exposed to serum (Figure 6(b)). Unlike exon 21, exon 22 contains the AvrII restriction site that allows for the assessment of its presence in PCR amplification product of CACNA1C transcripts isolated from the cells. The AvrII-sensitive exon 22 isoform of the transcript was not detected in the quiescent nonproliferating aortic cells (Figure 6(b), lane 2). However, when 5% serum was added to the medium with nonconfluent aortic cells, DNA biosynthesis was activated, while the level of the transcript decreased ≈3 fold, and the presence of the AvrII-sensitive exon 22 isoforms of was easily detected (Figure 6(b), lane 4). The isoform remodeling simulated in aortic primary cells in vitro was not complete as compared to VSM cells in atherosclerotic regions of artery occluded with heavy plaque burden, which were selected for the molecular profiling. However, the cell culture results demonstrate that in a different experimental system, there is an obvious association between proliferation of VSM cells, downregulation of the CACNA1C expression, and synthesis of the exon 22 isoform.
Recent strategies targeting VSM cells to treat cardiovascular diseases suggest indiscriminate disruption of Cav1.2 [59–61]. Is it possible to correct the described CACNA1C splice defects induced by atherosclerosis without affecting the transcripts of the gene lacking the "pathogenic" exon 22? Correction of defective genes responsible for disease development is achieved by gene therapy. Usually it requires an insertion into the genome of a normal gene in place of a defective one causing disease. Such technique, however, is poorly controlled. Currently, one of the most promising, cutting-edge therapeutic approaches to correct defects associated with disease-induced expression of abnormal splice variants is antisense-mediated exon skipping. It is based on the use of antisense oligonucleotides targeting specific exons to be removed. The adenovirus-directed exon 22 skipping-induced inhibition of VSM cell proliferation (and, respectively, migration) can be used to rescue VSM cells from remodeling in atherosclerosis. The exon 22-skipping will not alter the open reading frame of the transcript because alternative exons 21 and 22 are of the equal size (60 nt). The modified nonspliceosomal snRNA U7 gene along with its natural promoter and 3′ elements, exon 22-antisense sequence and supplemented with Sm ribonucleoprotein-binding sequence, may be incorporated into the adenovirus vector for high efficiency transfer . The cytokine receptors (e.g., PdGF-β receptor) based recognition targeting of viral liposomes or nanoparticles may be especially advantageous in connection with selective gene transfer to VSM cells affected by atherosclerosis, while reducing the probability of the transfection of other cells.
6. Ca1.2 and CREB-Dependent Transcriptional Activation
How Cav1.2 activity is translated into a proliferation-effected modality is another important question to be asked. Cav1.2 calcium channels generate a transient rise in cytosolic Ca2+-concentration activated by membrane depolarization. Cellular responses associated with the rise of [Ca2+]i range from sarcomeric contraction to cell growth and proliferation. Cytoplasmic domains of Cav1.2 have evolved a fairly intricate CaM-dependent signaling mechanism that provides for the negative feedback inhibition of the calcium current, known as Ca2+ dependent inactivation (CDI), which is mediated by different determinants of [25, 63]. Such a mechanism of CDI, resulting in acceleration of inactivation in response to the rise of intracellular Ca2+, was first identified in cardiac Cav1.2 . Similar experiments performed on the recombinant Cav1.2 also showed that the replacement of extracellular Ca2+ by Ba2+ eliminates CDI, and the channel inactivates by a slower voltage-dependent mechanism . The very fact that two distantly located determinants, one in the pore region responsible for slow inactivation, and the CaM-binding one in the proximal locus of the C-tail, are independently crucial for CDI indicates that not only their specific molecular structure but also their mutual folding and/or interaction are essential. Experimental evidence show that this interaction reacts dynamically to membrane voltage supporting state-dependent transitions of the channel between resting, open, and inactivated conformations, which are essential for CREB-dependent transcriptional activation [66, 67].
CREB is a transcription factor of general importance in a large variety of cells. CREB phosphorylation promotes the activation of genes and is regulated by protein kinases under control of the major second messengers, cAMP and/or Ca2+. Indeed, CREB functions as a “molecular determinant of VSM cells fate .” CREB content depends on proliferation of VSM cells both in situ and in culture. Serum deprivation increased CREB content in VSM cells, while exposure to PdGF decreased it. Consistent with this observation, an overexpression of the constitutively active CREB in VSM cells arrested cell cycle progression.
To investigate the association of Cav1.2 with CREB transcriptional activation at the molecular level, we combined patch clamp with fluorescent resonance energy transfer (FRET) microscopy in the live cell. In voltage clamped cells, FRET provided optical measurements under state-dependent conditions showing that the shorter N-terminal tail of (e.g., ) does not rearrange vis-à-vis the plasma membrane in response to voltage gating . In sharp contrast, the C-tail shows voltage-dependent conformational rearrangements, which are much larger in size than that, for example, in the potassium Kv2.1 channel . Measurements of and corrected FRET between the enhanced yellow (EYFP) and cyan fluorescent proteins (ECFP), genetically attached, respectively, to the N- and C-termini of showed no significant effect on voltage-dependence and kinetics of the channel current. However, there was a substantial increase in FRET signal accompanying inactivation of the channel that was fully reversible upon its transition into the resting state in response to hyperpolarization . The plasma-membrane anchoring of the C-tail by the fusion of the pleckstrin homology domain (PH) eliminated CDI but not . Do the voltage-gated conformational rearrangements of the C-tail, and CDI, play a role in Ca2+ signal transduction that is utilized in Ca2+-induced activation or CREB-dependent transcription? To answer this question, we used the test system based on the measurement of interaction between KID domain of CREB and KIX domain of coactivator CREB-binding protein (CBP, Figure 7(A), inset) under voltage-clamp conditions by monitoring FRET between EYFP-KID and ECFP-KIX . Ca2+-dependent phosphorylation of KID stimulates its binding to KIX, bringing EYFP and ECFP close enough to observe the interaction by FRET. In perforated whole clamped cell, where the integrity of the cytoplasmic content is intact (Figure 7A)), no activation of CREB transcription (Figure 7B), panel (a)) and rearrangement of the C-tail (panel (b)) was observed when the C-tail was anchored to the plasma membrane. CREB transcriptional activity remained low in spite of a large sustained inward (panel (d)) and the corresponding increase in [Ca2+]i detected by the fluorescence of Ca2+ indicator Fura4 (panel (c)). Release of the C-tail by the activation of PIP2 hydrolysis upon activation of M1AchR (Figure 7C)) at −90 mV caused significant elevation of [Ca2+]i that also was not utilized by the cell for CREB transcriptional activation (Figure 7C), panel (a)) until a depolarizing pulse to +20-mV was applied and the released C-tail was permitted to rearrange (Figure 7D)). This experiment provides compelling evidence that neither large inward nor the subsequent rise in [Ca2+]i lead to CREB transcription activation, unless the conformational rearrangement of the subunit C-tail provides the precise targeting of the Ca2+ signal transduction (Figure 7D), scheme) .
There is general agreement that CaM binds to LA and IQ domains of the C-tail, and acts as a sensor that conveys CDI (for review, see ). The affinity of CaM for both domains depends on [Ca2+]i. Our data indicate that CDI and Ca2+-signal transduction depend on the voltage-gated mobility of the C-tail. It is therefore reasonable to suggest that the LA-domain is a Ca2+-sensitive apo-CaM-mediated lock for the mechanism of slow voltage-dependent inactivation of the channel . Apo-CaM associated with LA is able to cross-link it to another, still unidentified apo-CaM binding site in the polypeptide bundle underlying the pore. As the result of this specific localization, apo-CaM/LA “lock” is hidden from the cytoplasmic Ca2+ so that, for example, the intracellular Ca2+ released from the intracellular stores or Ca2+ caging compounds does not accelerate significantly the inactivation of Cav1.2 . Thus, apo-CaM associated with LA binds predominantly Ca2+ ions permeating through the pore. A Ca2+-dependent transfer of CaM from LA to the IQ-motif opens the “lock” and initiates a large rearrangement of the C-terminal tail. This in turn facilitates inactivation of the channel. The Ca2+/CaM complex with the IQ-motif is then transferred by the mobile C-tail to a downstream target of the Ca2+-signaling cascade (such as CaMKII ), where Ca2+ is released as an activating stimulus, while CaM switches back to LA and returns the C-tail to the resting position available for the next cycle of Ca2+-signal transduction.
Activation of CREB-dependent transcription by the L-type is mediated through multiple cell signaling pathways. Using FRET probes of CREB activity and 2D wavelet transform analysis, we applied principles of quantitative biology to detail the mechanism of Ca2+-activated CREB-dependent transcription within localized regions (microdomains) of the nucleus. We reached this goal by applying continuous wavelet analysis in two dimensions with a 2D wavelet as a deconvolution algorithm for FRET microscopy image analysis . Continuous wavelet analysis is a mathematical technique that allows us to analyze a signal over several different frequencies across the entire signal [73, 74]. It is especially useful for finding heterogeneity in a signal because it can easily find where the pattern (i.e., frequency) of a signal changes. In these experiments, we, for the first time, obtained evidence of CREB signaling microdomains within the nucleus that respond differentially to stimulation and cAMP (Figure 8(A)). Results of the study revealed that CREB-dependent transcriptional signaling occurs in discrete signaling microdomains underlying the architecture of nuclear signaling. Continuous activation of CREB-dependent transcriptional signaling by cAMP and Ca2+ resulted in a gradual increase of the number of microdomains. Four different categories of cAMP and Ca2+-induced CREB signaling microdomains were characterized in COS1 cells expressing recombinant Cav1.2 with the “atherosclerotic” splice variant (Figure 8). In up to 65% of the microdomains, transcription was activated in additive manner by cAMP and Ca2+. Approximately 15% of signaling domains were activated only by and 5% of domains were activated only by cAMP. Finally, 15% of the domains were transient, and activated by both cAMP and (Figure 8(B)) . A similar spatiotemporal organization of CREB-dependent signaling was observed in spontaneously beating neonatal rat cardiomyocytes. Although COS1 cells that were used in our experiments shown in Figures 7 and 8 are naturally deprived of Cav1.2 , they inherited the ability to replicate the Cav1.2-dependent activation of CREB signaling with the exogenous recombinant channel. Thus, this experimental approach fits the task, which, in my opinion, is the most important unresolved issue for the coupling of Cav1.2 to CREB signaling: does the splice variation of affect the spatiotemporal organization of CREB-dependent signaling in a way that may affect cell proliferation and other crucial function?
Association of Cav1.2 with regulation of transcription, cell proliferation, and its pathophysiology, as in the case of atherosclerosis, requires detailed investigation of the roles of the naturally occurring splice variants. It will limit the traditionally intuitive approach to Cav1.2 in physiology and help to define new principle approaches to the treatment of various Cav1.2 channelopathy-related dysfunctions, above all cardiovascular diseases. Humgenex Inc. provides consulting and logistic support on a broad range of issues reported in this review.
|ECFP:||Enhanced cyan fluorescent protein|
|EYFP:||Enhanced yellow fluorescent protein|
|FALI:||Fluorophore-assisted light inactivation|
|FRET:||Fluorescent resonance energy transfer|
|PH:||Pleckstrin homology domain|
|VSM:||Vascular smooth muscle.|
- Y. S. Lee, M. M. Sayeed, and R. D. Wurster, “Inhibition of cell growth and intracellular Ca2+ mobilization in human brain tumor cells by Ca2+ channel antagonists,” Molecular and Chemical Neuropathology, vol. 22, no. 2, pp. 81–95, 1994.
- K. Sato, J. Ishizuka, C. W. Cooper et al., “Inhibitory effect of calcium channel blockers on growth of pancreatic cancer cells,” Pancreas, vol. 9, no. 2, pp. 193–202, 1994.
- V. Bertrand, M. J. Bastie, N. Vaysse, and L. Pradayrol, “Inhibition of gastrin-induced proliferation of AR4-2J cells by calcium channel antagonists,” International Journal of Cancer, vol. 56, no. 3, pp. 427–432, 1994.
- J. M. Taylor and R. U. Simpson, “Inhibition of cancer cell growth by calcium channel antagonists in the athymic mouse,” Cancer Research, vol. 52, no. 9, pp. 2413–2418, 1992.
- M. G. Cattaneo, M. Gullo, and L. M. Vicentini, “C2+ and Ca2+ channel antagonists in the control of human small cell lung carcinoma cell proliferation,” European Journal of Pharmacology, vol. 247, no. 3, pp. 325–331, 1993.
- C. Chen, M. J. Corbley, T. M. Roberts, and P. Hess, “Voltage-sensitive calcium channels in normal and transformed 3T3 fibroblasts,” Science, vol. 239, no. 4843, pp. 1024–1026, 1988.
- P. C. Dartsch, M. Ritter, M. Gschwentner, H. J. Lang, and F. Lang, “Effects of calcium channel blockers on NIH 3T3 fibroblasts expressing the Ha-ras oncogene,” European Journal of Cell Biology, vol. 67, no. 4, pp. 372–378, 1995.
- M. Biel, R. Hullin, S. Freundner et al., “Tissue-specific expression of high-voltage-activated dihydropyridine-sensitive L-type calcium channels,” European Journal of Biochemistry, vol. 200, no. 1, pp. 81–88, 1991.
- D. M. Rodman, K. Reese, J. Harral et al., “Low-voltage-activated (T-type) calcium channels control proliferation of human pulmonary artery myocytes,” Circulation Research, vol. 96, no. 8, pp. 864–872, 2005.
- A. Panner, L. L. Cribbs, G. M. Zainelli, T. C. Origitano, S. Singh, and R. D. Wurster, “Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells,” Cell Calcium, vol. 37, no. 2, pp. 105–119, 2005.
- C. Chen and P. Hess, “Calcium channels in mouse 3T3 and human fibroblasts,” Biophysical Journal, vol. 51, p. 226a, 1987.
- N. M. Soldatov, “Molecular diversity of L-type Ca2+ channel transcripts in human fibroblasts,” Proceedings of the National Academy of Sciences of the United States of America, vol. 89, no. 10, pp. 4628–4632, 1992.
- A. Fujii, H. Matsumoto, S. Nakao, H. Teshigawara, and Y. Akimoto, “Effect of calcium-channel blockers on cell proliferation, DNA synthesis and collagen synthesis of cultured gingival fibroblasts derived from human nifedipine responders and non-responders,” Archives of Oral Biology, vol. 39, no. 2, pp. 99–104, 1994.
- N. M. Soldatov, “Genomic structure of human L-type Ca2+ channel,” Genomics, vol. 22, no. 1, pp. 77–87, 1994.
- V. P. Shevchenko, N. F. Myasoedov, N. M. Soldatov, G. J. Duburs, V. V. Kastron, and I. P. Skrastins, “Synthesis and evaluation of biological activity of two novel tritium-labeled derivatives of riodipine,” Journal of Labelled Compounds and Radiopharmaceuticals, vol. 27, no. 6, pp. 721–730, 1989.
- S. M. Dudkin, S. N. Gnedoj, N. N. Chernyuk, and N. M. Soldatov, “1,4-dihydropyridine receptor associated with Ca2+ channels in human embryonic fibroblasts,” FEBS Letters, vol. 233, no. 2, pp. 352–354, 1988.
- A. DePover, M. A. Matlib, S. W. Lee et al., “Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem,” Biochemical and Biophysical Research Communications, vol. 108, no. 1, pp. 110–117, 1982.
- N. M. Soldatov, S. N. Gnedoj, N. N. Chernyuk, I. A. Britanova, and S. M. Dudkin, “Mitogenesis and 1,4-dihydropyridine receptor associated with Ca2+-channels in human embryonic fibroblasts,” Journal of Membrane Biology, vol. 5, pp. 1161–1167, 1988 (Russian).
- D. Lipscombe, D. V. Madison, M. Poenie, H. Reuter, R. Y. Tsien, and R. W. Tsien, “Spatial distribution of calcium channels and cytosolic calcium transients in growth cones and cell bodies of sympathetic neurons,” Proceedings of the National Academy of Sciences of the United States of America, vol. 85, no. 7, pp. 2398–2402, 1988.
- G. S. Harms, L. Cognet, P. H. M. Lommerse et al., “Single-molecule imaging of L-type CA2+ channels in live cells,” Biophysical Journal, vol. 81, no. 5, pp. 2639–2646, 2001.
- V. di Biase, G. J. Obermair, Z. Szabo et al., “Stable membrane expression of postsynaptic 1.2 calcium channel clusters is independent of interactions with AKAP79/150 and PDZ proteins,” Journal of Neuroscience, vol. 28, no. 51, pp. 13845–13855, 2008.
- M. F. Navedo, E. P. Cheng, C. Yuan et al., “Increased coupled gating of L-type Ca2+ channels during hypertension and timothy syndrome,” Circulation Research, vol. 106, no. 4, pp. 748–756, 2010.
- E. Kobrinsky, P. Abrahimi, S. Q. Duong et al., “Effect of β subunits on structural organization of 1.2 calcium channels,” PLoS One, vol. 4, no. 5, Article ID e5587, 2009.
- Q. Z. Lao, E. Kobrinsky, Z. Liu, and N. M. Soldatov, “Oligomerization of β subunits is an essential correlate of Ca2+ channel activity,” The FASEB Journal, vol. 24, no. 12, pp. 5013–5023, 2010.
- E. Kobrinsky, S. Tiwari, V. A. Maltsev et al., “Differential role of the α1C subunit tails in regulation of the 1.2 channel by membrane potential, β subunits, and Ca2+ ions,” Journal of Biological Chemistry, vol. 280, no. 13, pp. 12474–12485, 2005.
- S. Tiwari, Y. Zhang, J. Heller, D. R. Abernethy, and N. M. Soldatov, “Artherosclerosis-related molecular alteration of the human 1.2 calcium channel α1C subunit,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 45, pp. 17024–17029, 2006.
- I. Splawski, K. W. Timothy, L. M. Sharpe et al., “1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism,” Cell, vol. 119, no. 1, pp. 19–31, 2004.
- Z. Z. Tang, M. C. Liang, S. Lu et al., “Transcript scanning reveals novel and extensive splice variations in human L-type voltage-gated calcium channel, 1.2 α1 subunit,” Journal of Biological Chemistry, vol. 279, no. 43, pp. 44335–44343, 2004.
- P. Liao, T. F. Yong, M. C. Liang, D. T. Yue, and T. W. Soong, “Splicing for alternative structures of 1.2 Ca2+ channels in cardiac and smooth muscles,” Cardiovascular Research, vol. 68, no. 2, pp. 197–203, 2005.
- Y. Blumenstein, N. Kanevsky, G. Sahar, R. Barzilai, T. Ivanina, and N. Dascal, “A novel long N-terminal isoform of human L-type Ca2+ channel is up-regulated by protein kinase C,” Journal of Biological Chemistry, vol. 277, no. 5, pp. 3419–3423, 2002.
- N. M. Soldatov, A. Bouron, and H. Reuter, “Different voltage-dependent inhibition by dihydropyridines of human Ca2+ channel splice variants,” Journal of Biological Chemistry, vol. 270, no. 18, pp. 10540–10543, 1995.
- D. P. Faxon, M. A. Creager, S. C. Smith et al., “Atherosclerotic vascular disease conference: executive summary: atherosclerotic vascular disease conference proceeding for healthcare professionals from a special writing group of the American Heart Association,” Circulation, vol. 109, no. 21, pp. 2595–2604, 2004.
- R. Ross, “Atherosclerosis—an inflammatory disease,” The New England Journal of Medicine, vol. 340, no. 2, pp. 115–126, 1999.
- H. Roy, S. Bhardwaj, and S. Yla-Herttuala, “Molecular genetics of atherosclerosis,” Human Genetics, vol. 125, no. 5-6, pp. 467–491, 2009.
- M. Biel, P. Ruth, E. Bosse et al., “Primary structure and functional expression of a high voltage activated calcium channel from rabbit lung,” FEBS Letters, vol. 269, no. 2, pp. 409–412, 1990.
- X. Cheng, J. Liu, M. Asuncion-Chin et al., “A novel 1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits,” Journal of Biological Chemistry, vol. 282, no. 40, pp. 29211–29221, 2007.
- W. J. Koch, P. T. Ellinor, and A. Schwartz, “cDNA cloning of a dihydropyridine-sensitive calcium channel from rat aorta: evidence for the existence of alternatively spliced forms,” Journal of Biological Chemistry, vol. 265, no. 29, pp. 17786–17791, 1990.
- S. Richard, D. Neveu, G. Carnac, P. Bodin, P. Travo, and J. Nargeot, “Differential expression of voltage-gated Ca2+-currents in cultivated aortic myocytes,” Biochimica et Biophysica Acta, vol. 1160, no. 1, pp. 95–104, 1992.
- A. Schwartz, “Calcium antagonists: review and perspective on mechanism of action,” American Journal of Cardiology, vol. 64, no. 17, pp. 3I–9I, 1989.
- R. P. Mason, “Mechanisms of plaque stabilization for the dihydropyridine calcium channel blocker amlodipine: review of the evidence,” Atherosclerosis, vol. 165, no. 2, pp. 191–199, 2002.
- A. Ruiz-Torres, R. Lozano, J. Melón, and R. Carraro, “L-calcium channel blockade induced by diltiazem inhibits proliferation, migration and F-actin membrane rearrangements in human vascular smooth muscle cells stimulated with insulin and IGF-1,” International Journal of Clinical Pharmacology and Therapeutics, vol. 41, no. 9, pp. 386–391, 2003.
- H. Koshiyama, S. Tanaka, and J. Minamikawa, “Effect of calcium channel blocker amlodipine on the intimal-medial thickness of carotid arterial wall in type 2 diabetes,” Journal of Cardiovascular Pharmacology, vol. 33, no. 6, pp. 894–896, 1999.
- T. Yamashita, S. Kawashima, M. Ozaki et al., “A calcium channel blocker, benidipine, inhibits intimal thickening in the carotid artery of mice by increasing nitric oxide production,” Journal of Hypertension, vol. 19, no. 3, pp. 451–458, 2001.
- R. P. Mason, P. Marche, and T. H. Hintze, “Novel vascular biology of third-generation L-type calcium channel antagonists: ancillary actions of amlodipine,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 23, no. 12, pp. 2155–2163, 2003.
- O. Stepien, J. Gogusev, D. L. Zhu et al., “Amlodipine inhibition of serum-, thrombin-, or fibroblast growth factor- induced vascular smooth-muscle cell proliferation,” Journal of Cardiovascular Pharmacology, vol. 31, no. 5, pp. 786–793, 1998.
- V. Ruiz-Velasco, M. B. Mayer, E. W. Inscho, and L. J. Hymel, “Modulation of dihydropyridine receptors in vascular smooth muscle cells by membrane potential and cell proliferation,” European Journal of Pharmacology, vol. 268, no. 3, pp. 311–318, 1994.
- J. Nilsson, M. Sjölund, L. Palmberg, A. M. von Euler, B. Jonzon, and J. Thyberg, “The calcium antagonist nifedipine inhibits arterial smooth muscle cell proliferation,” Atherosclerosis, vol. 58, no. 1–3, pp. 109–122, 1985.
- J. Thyberg and L. Palmberg, “The calcium antagonist nisoldipine and the calmodulin antagonist W-7 synergistically inhibit initiation of DNA synthesis in cultured arterial smooth muscle cells,” Biology of the Cell, vol. 60, no. 2, pp. 125–132, 1987.
- E. Munro, M. Patel, P. Chan et al., “Effect of calcium channel blockers on the growth of human vascular smooth muscle cells derived from saphenous vein and vascular graft stenoses,” Journal of Cardiovascular Pharmacology, vol. 23, no. 5, pp. 779–784, 1994.
- T. A. Kent, A. Jazayeri, and J. M. Simard, “Calcium channels and nifedipine inhibition of serotonin-induced [3H]thymidine incorporation in cultured cerebral smooth muscle cells,” Journal of Cerebral Blood Flow and Metabolism, vol. 12, no. 1, pp. 139–146, 1992.
- A. Agrotis, P. J. Little, J. Saltis, and A. Bobik, “Dihydropyridine Ca2+ channel antagonists inhibit the salvage pathway for DNA synthesis in human vascular smooth muscle cells,” European Journal of Pharmacology, vol. 244, no. 3, pp. 269–275, 1993.
- I. Duque, M. R. Puyol, P. Ruiz, M. Gonzalez-Rubio, M. L. D. Marques, and D. R. Puyol, “Calcium channel blockers inhibit hydrogen peroxide-induced proliferation of cultured rat mesangial cells,” Journal of Pharmacology and Experimental Therapeutics, vol. 267, no. 2, pp. 612–616, 1993.
- T. Kuga, S. Kobayashi, Y. Hirakawa, H. Kanaide, and A. Takeshita, “Cell cycle-dependent expression of L- and T-type Ca2+ currents in rat aortic smooth muscle cells in primary culture,” Circulation Research, vol. 79, no. 1, pp. 14–19, 1996.
- E. M. Graf, M. Bock, J. F. Heubach et al., “Tissue distribution of a human 1.2 α1 subunit splice variant with a 75 bp insertion,” Cell Calcium, vol. 38, no. 1, pp. 11–21, 2005.
- P. Liao, D. Yu, S. Lu et al., “Smooth muscle-selective alternatively spliced exon generates functional variation in 1.2 calcium channels,” Journal of Biological Chemistry, vol. 279, no. 48, pp. 50329–50335, 2004.
- O. Skalli, P. Ropraz, and A. Trzeciak, “A monoclonal antibody against α-smooth muscle actin: a new probe for smooth muscle differentiation,” Journal of Cell Biology, vol. 103, no. 6, pp. 2787–2796, 1986.
- T. M. Doherty, K. Asotra, L. A. Fitzpatrick et al., “Calcification in atherosclerosis: bone biology and chronic inflammation at the arterial crossroads,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 20, pp. 11201–11206, 2003.
- G. K. Owens, “Regulation of differentiation of vascular smooth muscle cells,” Physiological Reviews, vol. 75, no. 3, pp. 487–517, 1995.
- J. P. Bannister, A. Adebiyi, G. Zhao et al., “Smooth muscle cell α2δ-1 subunits are essential for vasoregulation by 1.2 channels,” Circulation Research, vol. 105, no. 10, pp. 948–955, 2009.
- S. Sonkusare, M. Fraer, J. D. Marsh, and N. J. Rusch, “Disrupting calcium channel expression to lower blood pressure: new targeting of a well-known channel,” Molecular Interventions, vol. 6, no. 6, pp. 304–310, 2006.
- S. Télémaque, S. Sonkusare, T. Grain et al., “Design of mutant β2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells,” Journal of Pharmacology and Experimental Therapeutics, vol. 325, no. 1, pp. 37–46, 2008.
- A. Goyenvalle, A. Vulin, F. Fougerousse et al., “Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping,” Science, vol. 306, no. 5702, pp. 1796–1799, 2004.
- D. B. Halling, P. Aracena-Parks, and S. L. Hamilton, “Regulation of voltage-gated Ca2+ channels by calmodulin,” Science's STKE, vol. 2005, no. 315, p. re15, 2005.
- K. S. Lee, E. Marban, and R. W. Tsien, “Inactivation of calcium channels in mammalian heart cells: joint dependence on membrane potential and intracellular calcium,” Journal of Physiology, vol. 364, pp. 395–411, 1985.
- C. Shi and N. M. Soldatov, “Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel,” Journal of Biological Chemistry, vol. 277, no. 9, pp. 6813–6821, 2002.
- E. Kobrinsky, E. Schwartz, D. R. Abernethy, and N. M. Soldatov, “Voltage-gated mobility of the Ca2+ channel cytoplasmic tails and its regulatory role,” Journal of Biological Chemistry, vol. 278, no. 7, pp. 5021–5028, 2003.
- N. M. Soldatov, “Ca2+ channel moving tail: link between Ca2+-induced inactivation and Ca2+ signal transduction,” Trends in Pharmacological Sciences, vol. 24, no. 4, pp. 167–171, 2003.
- D. J. Klemm, P. A. Watson, M. G. Frid et al., “cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration,” Journal of Biological Chemistry, vol. 276, no. 49, pp. 46132–46141, 2001.
- E. Kobrinsky, L. Stevens, Y. Kazmi, D. Wray, and N. M. Soldatov, “Molecular rearrangements of the 2.1 potassium channel termini associated with voltage gating,” Journal of Biological Chemistry, vol. 281, no. 28, pp. 19233–19240, 2006.
- B. M. Mayr, G. Canettieri, and M. R. Montminy, “Distinct effects of cAMP and mitogenic signals on CREB-binding protein recruitment impart specificity to target gene activation via CREB,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 19, pp. 10936–10941, 2001.
- M. Morad and N. Soldatov, “Calcium channel inactivation: possible role in signal transduction and Ca2+ signaling,” Cell Calcium, vol. 38, no. 3-4, pp. 223–231, 2005.
- D. G. Wheeler, C. F. Barrett, R. D. Groth, P. Safa, and R. W. Tsien, “CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation-transcription coupling,” Journal of Cell Biology, vol. 183, no. 5, pp. 849–863, 2008.
- D. E. Mager, E. Kobrinsky, A. Masoudieh, A. Maltsev, D. R. Abernethy, and N. M. Soldatov, “Analysis of functional signaling domains from fluorescence imaging and the two-dimensional continuous wavelet transform,” Biophysical Journal, vol. 93, no. 8, pp. 2900–2910, 2007.
- E. Kobrinsky, D. E. Mager, S. A. Bentil, S. I. Murata, D. R. Abernethy, and N. M. Soldatov, “Identification of plasma membrane macro- and microdomains from wavelet analysis of FRET microscopy,” Biophysical Journal, vol. 88, no. 5, pp. 3625–3634, 2005.
- E. Kobrinsky, S. Q. Duong, A. Sheydina, and N. M. Soldatov, “Microdomain organization and frequency-dependence of CREB-dependent transcriptional signaling in heart cells,” The FASEB Journal, vol. 25, no. 5, pp. 1544–1555, 2011.
- A. Ravindran, Q. Z. Lao, J. B. Harry, P. Abrahimi, E. Kobrinsky, and N. M. Soldatov, “Calmodulin-dependent gating of 1.2 calcium channels in the absence of β subunits,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 23, pp. 8154–8159, 2008.
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