c-Myb–Dependent Inositol 1,4,5-Trisphosphate Receptor Type-1 Expression in Vascular Smooth Muscle Cells

【摘要】  Objective— The IP3 receptor-1 (IP3R1) mediates Ca 2+ signals critical to vascular smooth muscle cell (VSMC) proliferation. The cell cycle–associated transcription factor c-Myb increases Ca 2+ at the G 1 /S transition. Here we show the mechanism through which c-Myb regulates expression of IP3R1.

Methods & Results— Ribonuclease protection confirmed transcriptional start (TS), and qRT-PCR revealed a 6-fold increase in IP3R1 mRNA as immortalized VSMC progress from G 0 to G 1 /S. A c-Myb neutralizing antibody decreased IP3R1 mRNA expression 3-fold, and abolished the 3.4-fold increase in IP3R1 protein observed at G 1 /S. Primary aortic VSMCs in culture and proliferating carotid VSMCs in vivo showed similar regulation of IP3R1 mRNA and protein. Sequence analysis of a 3.1-Kb mouse IP3R1 promoter revealed 17 putative c-Myb binding sites. Reporter assays demonstrated a 2-fold increase in promoter activity in G 1 /S- versus G 0 -synchronized VSMCs, which was abolished by functional c-Myb knockdown or deletion of promoter sequences upstream and downstream of TS. Point mutations in Myb sites-13 or -15 significantly blunted G 1 /S-specific promoter induction in both immortalized and primary VSMCs. Gel shift and ChIP confirmed binding of c-Myb to sites-13 and -15 in G 1 /S stage VSMCs.

Conclusion— c-Myb regulates cell cycle–associated IP3R1 transcription in VSMCs via specific highly conserved Myb-binding sites in the IP3R1 promoter.

The IP3R1 promoter contains several putative binding sites for the c-Myb transcription factor, two of which are shown by gel shift and ChIP to bind c-Myb. Point mutations in these abolished promoter activity in immortalized and primary VSMCs. These data provide a mechanism for cell cycle– and c-Myb–responsive IP3R1 expression.

【关键词】  inositol trisphosphate receptor cmyb cell cycle VSMC

Introduction

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca 2+ channels located primarily in the ER membrane, 1 which may closely appose the nucleus, mitochondria, or plasma membrane store-operated Ca 2+ channels to stimulate capacitative Ca 2+ entry (CCE). 2,3 IP3Rs are encoded by 3 separate genes, giving rise to 3 subtypes (IP3R1-3). Additional heterogeneity is created by alternative splicing and homo- or heterotetramerization. Almost all tissues express all 3 IP3R subtypes to various extents, 1 but IP3R1 predominates in adult vascular smooth muscle cells (VSMCs). 4

Proliferation of VSMCs underlies both atherogenesis and restenosis after angioplasty. 5 In mammalian cells, changes in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) regulate several steps in cell cycle progression such as G 0 -to-G 1 phase, G 1 -to-S phase, and G 2 -to-M phase transitions, 6 and modulate activity of cyclinE/cdk2 in particular in VSMCs 7. Cell cycle entry and proliferation can be induced by growth factors and agonists which bring about sustained increases in [Ca 2+ ] i attributable to a combination of increased IP3 binding to IP3 receptors, enhanced activity of store-operated Ca 2+ channels, and decreased Ca 2+ efflux via plasma membrane Ca 2+ -ATPase (PMCA) and sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) Ca 2+ pumps. 8,9 Knockdown of some of these Ca 2+ transporters has confirmed their importance in proliferation. For example, PMCA1 knockdown inhibits cell proliferation in a breast cancer cell line. 10 Similarly, in the rat VSMC cell line A7r5, knockdown of IP3R1, but not IP3R3, abolished IP3 induced Ca 2+ release (IICR) as well as CCE and arrested proliferation. 11

Other lines of evidence further support a critical role for IP3 and IP3R1 in cell cycle progression. IICR causes M phase–arrested oocytes to reenter cell cycle after fertilization. 12 In sea urchin embryos, IICR initiated nuclear envelope breakdown, metaphase-anaphase transition, and cytokinesis. 13 In mammalian cells, an anti-IP3R1 antibody blocked IICR and cell cycle after fertilization. 14 Thapsigargin-induced depletion of IP3-sensitive Ca 2+ stores completely inhibited cyclin-A expression, cdk2 activation, DNA synthesis, and G 1 /S transitions in human fibroblasts, 15 and proliferation was reduced in mouse fibroblasts stably expressing truncated IP3R1 mutants. 16 IP3R1 knockdown in T cells blocked [Ca 2+ ] i elevation as well as interleukin (IL)-2 induction, implicating IP3R1 in T cell proliferation. 17 Similarly, B cell proliferation also involves IICR. 18 Finally, when normally quiescent rat aortic VSMCs were incubated in serum-containing medium, their proliferation was accompanied by a marked increase in IP3R1 but not IP3R2 mRNA levels. 19

c-Myb is a transcription factor expressed in diverse cell types including VSMCs. 20 c-Myb is strongly associated with cell proliferation and G 1 /S transitions 21 and functions via binding to a consensus DNA sequence (TAAC T/G T/G) in the promoters of Myb-dependent genes. 22 c-Myb is known to regulate the promoters of key cell cycle genes such as c-myc (5 Myb binding sites 23 ), cyclin A1, and cdk1 (2 sites each 24,25 ), proliferation-enabling enzymes such as DNA topoisomerase II and methionine adenosyltransferase-2A (1 site each 26,27 ), a G protein–coupled receptor (adenosine receptor 2B; 33 sites 28 ) involved in endothelial cell proliferation, and Ca 2+ transporters such as PMCA1 (2 sites 29 ) known to be involved in VSMC proliferation. We and others have previously shown that c-Myb–dependent transcription has a significant role in regulating [Ca 2+ ] i at the G 1 /S transition of VSMCs 9,29,30. We have shown that c-Myb represses both PMCA1 and PMCA4 expression at the G 1 /S interface of VSMCs and that this decrease in expression of Ca 2+ -efflux pumps enables the increased [Ca 2+ ] i required for S-phase entry and cell proliferation. 9,29–31 Given our initial finding herein of increased IP3R1 mRNA expression levels at the G 1 /S transition of VSMCs, and that c-Myb is capable of both activating 23,25,26 and repressing transcription, 29,32 we hypothesized that cell cycle–regulated expression of the IP3R1 gene may also be mediated by c-Myb–dependent transcription.

In examining the IP3R1 promoter sequences from human, chimpanzee, mouse, rat, cow, and dog genomes, we discovered the presence of multiple putative c-Myb binding sites at conserved positions in all 6 species. We found IP3R1 mRNA expression levels in mouse VSMCs to be responsive to positive and negative regulation of c-Myb expression, and that these 2 proteins undergo coincident upregulation in proliferating mouse VSMCs in vivo. With a 3.1-Kb mouse IP3R1 promoter fragment, reporter assays using deletion and point mutants of this promoter, c-Myb modulating constructs, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays, we present compelling evidence of c-Myb–dependent activation of IP3R1 transcription during G 1 to S cell cycle progression in both primary and immortalized VSMCs.

Methods

Animal Protocols

All experiments were conducted with 12-week-old C57Bl/6J mice and in accordance with approved institutional operating protocols.

VSMC Isolation and Cell Lines

Isolation of primary mouse aortic VSMCs and derivation of our clonal cell line "MOVAS" has been described 31 (#CRL-2797, ATCC, Manassas, Fla). Early Northern blot experiments employed the Syrian hamster ductus deferens SMC cell line (DDT-MF2; ATCC, Manassas, Fla).

Carotid Artery Injury

Carotid artery injury or sham surgery was performed as previously described. 33

Functional Myb Knockdown

For details regarding the anti-c-Myb antibody construct (pNuMybsFv) 34 see supplemental Methods, available online at http://atvb.ahajournals.org.

Immunostaining

For details regarding reporter constructs, antibodies, and staining protocols used see supplemental Methods. EGFP, Texas red, and Alexa Fluor images were captured using the Olympus IX/81 Fluoview confocal microscope (Olympus America). Cell boundaries were delineated from 5 or more observation fields on each cover slip (n 15 cells from 2 separate experiments) in vitro and on each of 3 randomly selected sections per animal (n 15 cells), and 3 animals per group (sham versus injured) in vivo. Cell fluorescent intensity was quantified at 15 planes in 15 µm wide z-stacks using Olympus FV1000 software.

Calcium Imaging

See supplemental Methods for details.

Ribonuclease Protection Assays

Total RNA was isolated (Nucleospin II, Takara Clontech) from G 0 and G 1 /S stage MOVAS as described. 31 A 370 bp IP3R1-specific probe (–179 to +191) was PCR cloned (see supplemental Table I for all primer sequences). RPA was carried out (Kit III; Ambion) and protected bands were aligned with the sequencing ladders generated from the reverse primer used to clone the probe (Sequenase kit; Amersham).

Quantitative RT-PCR

Total RNA was extracted from G 0 - and G 1 /S-synchronized MOVAS either nontransfected or electroporated (Protocol A-033: 10 6 cells, 5 µg DNA/cuvette; VSMC kit; Amaxa) with pNuMybsFv and a c-Myb responsive promoter-luciferase reporter (mim1-luc; J. Lipsick, Stanford University). Total RNA was also extracted from G 0 - and G 1 /S-synchronized primary VSMCs isolated from C57Bl6 mouse aortas, which were either nontransfected or electroporated with mim1-luc and various IP3R1 promoter-luciferase constructs. DNase-treated RNA samples were each spiked with 0.1 pg of in vitro transcribed EGFP RNA as a control for efficiency of cDNA synthesis and used in quantitative PCR (SYBR GREEN; Applied Biosystems; supplemental Table I) for IP3R1, luciferase, EGFP, and β-2 microglobulin, a housekeeping gene used as an internal control. 35 IP3R1 and luciferase mRNA copy numbers were normalized to EGFP and β-2 microglobulin mRNA copy numbers and finally to their respective mean G 0 mRNA levels.

Cloning, Deletion and Point Mutations of the Mouse IP3R1 Promoter

See supplemental Methods for full details.

Promoter Luciferase Assay

MOVAS cells were transfected (Lipofectamine; Invitrogen) with either a promoterless luciferase vector (pGL3-Basic; Promega, Madison, WI) or the IP3R1 promoter-driven luciferase vector with or without c-Myb-modulating cDNA constructs expressing either a dominant negative c-Myb, 30 the anti–c-Myb antibody, 34 or wild-type mouse c-Myb. 36 See supplemental Methods for further details.

Electrophoretic Mobility Shift Assays

G 1 /S stage MOVAS cells were used to make nuclear protein extracts as described. 29 EMSA was carried out 32 using 32 P-end labeled, gel purified double stranded oligos (supplemental Table I). Double stranded oligos used correspond to the native (C57Bl/6J) and point mutated Myb-13 and -15 c-Myb binding sites in the mouse IP3R1 promoter (GenBank Accession # AY857958; supplemental Figure III).

Chromatin Immunoprecipitation

G 1 /S stage MOVAS cells were used for ChIP assays as described (Upstate) using a ChIP-certified anti–c-Myb rabbit polyclonal antibody (Santa Cruz Biotechnology) and G-Protein Sepharose (GE/Healthcare). Precleared chromatin pulled down with normal rabbit IgG served as a negative control. The immunoprecipitated DNA fragments were purified (MinElute; Qiagen), diluted 6-fold, and used for quantitative PCR (SYBR GREEN; Applied Biosystems) with primers specific for Myb-13, Myb-15, and the CArG element upstream of transcriptional start in the mouse Myosin Heavy Chain (MHC) promoter (representative of promoters active in VSMCs but not regulated by c-Myb). ChIP primer sequences are shown in supplemental Table I. Real time PCR absolute quantification involved standard curves obtained by using cloned amplicons representing IP3R1 promoter?s Myb site-13 and –15 and the mouse MHC promoter?s CArG element.

Statistics

Luciferase assays, immunostaining quantification, Ca 2+ microfluorimetry, and real time RT-PCR data are shown as means±SEM and represent results from at least 3 separate experiments (n 3). Student t test was used for statistical comparisons. Statistical significance was defined as P 0.05.

Results

c-Myb Modulates Cell Cycle–Regulated Expression of IP3R1

Having previously shown cell cycle–regulated [Ca 2+ ]i in VSMCs, 30 we first examined whether IP3R-responsive Ca 2+ stores play a role in this process. Ratiometric imaging in cell cycle–synchronized MOVAS showed that [Ca 2+ ]i increased 2-fold as cells progressed from G 0 to G 1 /S (66.8±9.0 versus 132.8±9.1 nmol/L; P <0.01; supplemental Figure I), whereas the magnitude of UTP-stimulated (ie, IP3-mediated) Ca 2+ release was higher at G 0 than at G 1 /S ( stimulated-basal: 84.7±6.7 versus 29.9±4.6 nmol/L; P <0.01; supplemental Figure I). These data point to a G 1 /S-associated depletion of IP3R-dependent Ca 2+ stores.

We next examined cell cycle–associated expression levels of IP3R1. Northern blots in synchronized DDT-MF2 SMC showed that IP3R1 mRNA levels increased 2-fold as cells moved from G 0 to G 1 /S (data not shown). Quantitative RT-PCR in synchronized MOVAS cell populations showed a 6-fold increase in steady state IP3R1 mRNA levels from G 0 to G 1 /S (1.00±0.39 versus 6.74±0.72; n=3; P =0.002; Figure 1 ). Electroporation of MOVAS cells with a GFP-encoding vector (pmaxGFP; ESBE) revealed a transfection efficiency of 70%. Quantitative RT-PCR in MOVAS electroporated with pNuMybsFv (encoding an anti–c-Myb neutralizing antibody), showed a 3-fold decrease in G 1 /S stage IP3R1 mRNA levels compared with vector only controls (n 5; P =0.003; Figure 1 ).

Figure 1. IP3R1 mRNA expression during cell cycle progression. qRT-PCR showed 4- to 6-fold increases in IP3R1 mRNA levels during G 0 to G 1 /S progression of nontransfected or empty vector–transfected MOVAS (n=3 each; * P <0.01), but a 3-fold reduction in G 1 /S IP3R1 levels following anti-Myb antibody expression (n 5; ** P =0.003). Bars represent mean IP3R1 mRNA copy number normalized to EGFP, β-2-microglobulin, and G 0 IP3R1 mRNA levels.

Image analysis of MOVAS cells immunostained for IP3R1 demonstrated that IP3R1 protein levels increased 3.4±0.3-fold as cells progressed from G 0 to G 1 /S (n=20 cells; P <0.001; Figure 2a and 2 b). As previously observed in rat 30, c-Myb protein levels in MOVAS increased 72±7% at G 1 /S versus G 0 (n=15 cells each; P <0.001). In G 1 /S-synchronized MOVAS transfected to express either EGFP alone, or EGFP and pNuMybsFv, the IP3R1-specific immunostaining revealed 52±3% less IP3R1 protein in anti–Myb-expressing cells (n=16 cells each; P <0.001; Figure 2c through 2 f). To ascertain whether IP3R1 expression levels were regulated in proliferating VSMCs in vivo, we examined injured mouse carotid arteries (supplemental Figure II). At day 9 after surgery, proliferating medial VSMCs exhibited a 77±9% increase in IP3R1 immunostaining in injured versus uninjured arteries (n=3 animals per group, P <0.001). Consistent with previous studies, and coincident with increased IP3R1 expression, adjacent sections revealed a 27±5% increase in Myb immunostaining of VSMCs (n=3; P <0.01). Of note, c-Myb expression observed in some cells lacking SM -actin staining may reflect endothelial or inflammatory cell expression of c-Myb, or VSMCs in which SM -actin was downregulated.

Figure 2. IP3R1 protein expression during cell cycle progression. Confocal microscopy showed increased Texas Red–stained IP3R1 protein (red) in nontransfected G 1 /S- versus G 0 -synchronized MOVAS (b vs a), and decreased IP3R1 expression in G 1 /S-stage MOVAS cotransfected with EGFP+anti-Myb antibody (pNuMybsFv; e, f) vs EGFP alone (c, d). Quantitative imaging (see Results) was performed only in EGFP-expressing MOVAS (arrows) examined with filters for EGFP (c, e) and Texas Red (d, f).

Taken together, the above data revealed cell cycle–associated regulation of UTP-defined IP3R1 activity and c-Myb-responsive expression levels of IP3R1 mRNA and protein in vitro, with coincident upregulation of c-Myb and IP3R1 protein expression in proliferating VSMCs in vivo.

Conserved Structure of the IP3R1 Promoter

A 3.1-Kb mouse IP3R1 promoter fragment was cloned, sequenced (GenBank Acc. #AY857958), and found to contain noncoding exon-1 (257 bp) and part of intron 1 to 2 (357 of 849 bp) at its 3' end (supplemental Figure III). MatInspector (Genomatix Suite) analysis of transcription factor sites showed the presence of 17 putative c-Myb binding sites in the IP3R1 promoter, with overlapping Myb sites -16 and -17 on opposing DNA strands (supplemental Figure III and supplemental Table II).

Mikoshiba et al previously reported a transcriptional start for IP3R1 at #2524 in mouse neuronal cells (GenBank Acc. #D78171). 37 We carried out 2 independent ribonuclease protection assays on RNA from G 0 - and G 1 /S-synchronized MOVAS. Identical sized RNase protected bands were observed in RNA samples from G 0 - and G 1 /S-synchronized MOVAS, revealing transcriptional start (+1) in VSMCs at position #2526 (supplemental Figure IV).

A 1.0-Kb mouse IP3R1 promoter sequence (–500 to +500) was used to search for IP3R1 promoter sequences from rat, human, chimpanzee, dog, and cow on ENSEMBL Genome Browser. Promoter sequences corresponding to –2526 to +614 were downloaded from each of these species and scanned for putative Myb binding sites using MatInspector. 38 Comparisons revealed that all 6 mammalian species have the transcriptional start sequence CAGTAA (experimentally confirmed in mouse), a TATA sequence 29 to 31 bp upstream, and multiple putative c-Myb–binding sites upstream and downstream of transcriptional start. Notably, only Myb site-13 from the mouse IP3R1 promoter was conserved in all mammalian IP3R1 promoters analyzed ( Figure 3 ).

Figure 3. IP3R1 gene promoter structure. Features of the IP3R1 promoter in 6 mammalian species. Transcriptional start (bent arrow, confirmed in mouse), TATA box (rectangle), and putative c-Myb binding sites (vertical lines) are shown, with conserved sites highlighted (thick vertical lines) and assigned numbers established in mouse.

Activation of the IP3R1 Promoter Requires Multiple Intact c-Myb-Binding Sites

When the mim-1- luc construct (containing 3 copies of the mim-1 Myb binding site upstream of a minimal promoter) was transfected into MOVAS ( Figure 4 A), luciferase activity doubled as cells progressed from G 0 to G 1 /S (1.0±0.2 versus 1.8±0.1; P =0.02). The anti–c-Myb construct blunted this rise in promoter activity (1.8±0.1 versus 0.63±0.1; P <0.001), whereas overexpression of c-Myb resulted in increased reporter activity at G 0 (1.0±0.2 versus 2.0±0.3; P <0.01). These results show that endogenous c-Myb activity increases during G 0 to G 1 /S progression and that the anti-c-Myb antibody effectively inhibits c-Myb responsive promoter activity.

Figure 4. IP3R1 promoter-reporter activity is cell cycle–regulated and c-Myb–responsive. A, Expression of the anti-Myb antibody can inhibit luciferase-defined promoter activity of c-Myb–responsive mim-1- luc (mim-1: G 0 versus G 1 /S, * P =0.02; G 1 /S: mim-1 versus mim-1+anti-Myb, ** P <0.001; G 0 : mim-1 versus mim-1+ c-Myb, *** P <0.01). B, IP3R1 promoter-luciferase reporter assays show significant differences when the reporter is cotransfected with a dominant negative c-Myb mutant, anti-Myb cDNA, or wild-type c-Myb cDNA. (* P <0.05 vs IP3R promoter alone at G 0; ** P <0.05 vs IP3R promoter alone at both G 0 and G 1 /S). C, Luciferase reporter assays for IP3R1 point mutants show significant differences between wild-type and point mutant IP3R1 promoters at G 1 /S (* P <0.05 vs wild-type G 0; ** P <0.05 vs wild-type G 1 /S).

MOVAS cells were next transfected with wild-type 3.1-Kb IP3R1 promoter-luciferase reporter and Myb-modulating constructs ( Figure 4 B). Wild-type IP3R1 promoter activity increased 2.20±0.15-fold as cells moved from G 0 to G 1 /S ( P <0.001). Transient expression of a dominant negative Myb (G 1 /S=0.37±0.04; P <0.05) or a Myb-neutralizing antibody (G 1 /S=0.28±0.04; P <0.05) depressed IP3R1 promoter function at both G 0 and G 1 /S. Overexpression of wild-type c-Myb led to a 2.50±0.46-fold increase in IP3R1 promoter activity at G 0, as compared with promoter activity at G 0 in the absence of c-Myb overexpression ( P <0.05). However, c-Myb overexpression did not cause more than normal activation of the promoter at G 1 /S, possibly indicating limiting amounts of a cooperating transcription factor or coactivator at G 1 /S. Together, these data show cell cycle–regulated and c-Myb responsive activity of the wild-type IP3R1 promoter.

Deleting various regions of the 3.1-Kb IP3R1 promoter and testing luciferase reporter activity suggested that transcriptional start was flanked by two important regulatory regions: an upstream region from –2500 to –2000 and a downstream region from +100 to +600, both containing putative c-Myb binding sites (supplemental Figure V, sites 1 to 6 upstream and sites 13 to 17 downstream of transcriptional start). When Myb sites 1 to 6, or 1 to-11, were deleted, G 1 /S-specific IP3R1 promoter activity decreased to 0.57±0.32 and 0.75±0.15 ( P <0.05) of G 0 levels (G 0 =1.00), respectively, compared with 2.20±0.15 ( P <0.05) for the wild-type promoter. Deletion of Myb sites 15 to 17, or 13-to 17, decreased G 1 /S-specific IP3R1 promoter activity to 0.12±0.05 ( P <0.05) and 0.22±0.11 ( P <0.05), respectively, pointing to a more significant contribution. Promoter deletants harboring only Myb sites 12 to 14, or 12 alone, exhibited reduced promoter activity (G 1 /S=0.32±0.07 and 0.37±0.02, respectively; P <0.05 in both cases), suggesting again the importance of both upstream and downstream sequences.

Point mutations in Myb sites 13 and 15 significantly reduced G 1 /S-specific transactivation of the IP3R1 promoter (G 1 /S=0.8±0.16 and 0.46±0.12, respectively; P <0.05 in both cases; Figure 4 C). However, some Myb sensitivity still remained in these point mutants, as their activity could be further repressed by the Myb-neutralizing antibody. Point mutations in Myb sites 1, 6, and 16/17 had no significant effect on promoter activity (data not shown).

When primary mouse aortic VSMCs were electroporated with a control vector (mim-1- luc; supplemental Figure VI), qRT-PCR analysis showed that IP3R1 mRNA levels doubled as cells moved from G 0 to G 1 /S (1.0±0.1 versus 2.4±0.6; P =0.04). When electroporated with the wild-type 3.1 Kb IP3R1 promoter reporter, luciferase mRNA levels rose 5-fold as cells moved from G 0 to G 1 /S (1.0±0.1 versus 5.4±0.1; P =0.013). However, qRT-PCR in primary VSMCs transfected with point mutated Myb-13- or Myb-15-IP3R1 promoter-reporters revealed a significant drop in promoter activity (G 1 /S: wild-type=5.4±0.1 versus Myb-13 mutant=1.1±0.2, P =0.014; and Myb-15 mutant=0.9±0.2, P =0.013). Together, these data show that the mouse IP3R1 promoter undergoes cell cycle–associated c-Myb–dependent transactivation in both immortalized and primary VSMCs.

Specific c-Myb–Binding Sites in the IP3R1 Promoter

Electrophoretic mobility shift assays (EMSA) with nuclear protein extracts from G 1 /S-synchronized MOVAS cells and radio-labeled double-stranded oligodeoxynucleotides (ds oligos) corresponding to Myb sites 13 and 15 demonstrated that a protein-DNA complex was formed in a dose-dependent fashion with increasing amounts of MOVAS cell protein. These protein-DNA complexes could be competed out with 150-fold excess unlabeled wild-type (nonmutated) ds oligos identical to Myb site 13 or 15, but not with 150-fold excess unlabeled point-mutated ds oligos for Myb site 13 or 15 ( Figure 5 ).

Figure 5. EMSA-defined c-Myb binding sites. Electrophoretic mobility shift assays using IP3R1 Myb site-13 or -15 ds oligos show Myb-specific DNA-protein complex formation with G 1 /S stage MOVAS nuclear protein extracts. Competitive binding reactions used 150-fold excess of the indicated ds oligo.

Chromatin immunoprecipitation carried out with an anti–c-Myb antibody ( Figure 6 ) and primers specific for Myb sites 13 and 15 showed that c-Myb is bound to Myb sites 13 and 15 at the G 1 /S stage in MOVAS cells. Promoters which are normally transcriptionally active in VSMCs but are not regulated by c-Myb (such as the mouse myosin heavy chain promoter?s CArG element) were not present in chromatin pulled down with the anti–c-Myb antibody.

Figure 6. ChIP-defined c-Myb binding sites. Chromatin immunoprecipitation assays with G 1 /S stage MOVAS and quantitative PCR show c-Myb bound to Myb site-13 and -15 in the IP3R1 promoter. In addition, chromatin pulled down with anti-Myb antibody did not contain promoters active in VSMCs but not regulated by c-Myb (Myosin Heavy Chain promoter?s CArG element).

Discussion

Previous work has suggested that Ca 2+ signals mediating cell cycle transitions are generated by Ca 2+ released from intracellular Ca 2+ stores. 39–41 Indeed, IP3R expression is maintained while L-type Ca 2+ channel and RyR expression is lost when quiescent VSMCs undergo a phenotypic switch to the proliferating/synthetic state. 42,43 Moreover, Wilkerson et al 44 have recently used a cerebral artery organ culture to show that an IP3R-mediated increase in Ca 2+ wave frequency is critically required for mouse VSMC proliferation, whereas Wang et al demonstrated that IP3R1 but not IP3R3 knockdown severely inhibits rat VSMC proliferation. 11

Based on the above, our demonstration of c-Myb–dependent IP3R1 promoter transactivation is consistent with that of other c-Myb–responsive genes critically involved in cell proliferation. 23, 25–27, 37 Most recently, Nakata et al have shown that c-Myb transactivates the human cyclin B1 promoter by binding to the highly conserved +22 c-Myb binding site also found in rat, mouse, and 4 other mammalian species. 45

We have previously shown that repression of the promoter for a Ca 2+ efflux pump (PMCA1) contributes to the generation of the late G 1 -associated Ca 2+ signal 9,30 and also that this transcriptional repression is brought about by the binding of c-Myb to 2 specific Myb recognition elements in the PMCA1 promoter. 29 Evidence presented here brings a novel addition to the list of c-Myb target genes involved in Ca 2+ -mediated regulation of the cell cycle. Here we show that c-Myb can bring about the transcriptional activation of the IP3R1 promoter at the G 1 /S transition by binding to at least 2 specific c-Myb recognition elements, one of which is highly conserved in the IP3R1 promoters of human, chimpanzee, mouse, rat, dog, and cow.

The findings we have detailed include (1) cell cycle-regulated induction of IP3R1 in immortalized and primary VSMCs in vitro and proliferation-associated induction of IP3R1 in injured carotid arteries; (2) downregulation of IP3R1 promoter activity by a dominant negative c-Myb or an anti–c-Myb antibody; (3) multiple putative c-Myb binding sites upstream and downstream of IP3R1 transcriptional start; (4) promoter deletant data supporting regulatory function in both upstream and downstream regions; (5) confirmation via point mutations that Myb-binding sites 13 and 15 play a major role in transactivation in both immortalized and primary VSMCs; and (6) evidence that c-Myb can bind to oligos encoding Myb sites 13 and 15 as well as to these sites in G 1 /S-stage native chromatin. Considering the role of c-Myb in repressing the PMCA1 promoter 29 and activating the IP3R1 promoter (this report), we believe that c-Myb acts to coordinately regulate the promoters of various Ca 2+ transporter genes and influences the generation of Ca 2+ transients which mediate progression through the VSMC cell cycle.

Acknowledgments

Sources of Funding

This study was supported by operating grants to M.H. from the Canadian Institutes of Health Research (CIHR; MOP-14648) and the Heart & Stroke Foundation of Ontario (HSFO; NA-4389). M.H. was a Clinician-Scientist of the CIHR, and is recipient of a Career Investigator Award of the HSFO (CI5503).

Disclosures

None.

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作者单位：Division of Cell and Molecular Biology (T.A., A.M.S., M.A.M., M.H.), Toronto General Hospital Research Institute; Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research (S.H., M.H.) and the Departments of Medicine (M.H.) and Physiology (S.G., S.H., M.H.), University of Toro