c-Myb–Dependent Cell Cycle Progression and Ca ²⁺ Storage in Cultured Vascular Smooth Muscle Cells

Abstract

Considerable controversy surrounds the role of the c-myb proto-oncogene in vascular smooth muscle cells (VSMCs). Previous investigations using antisense approaches have suggested a relationship between c-myb expression, cell cycle progression, and cytoplasmic Ca2+ concentration ([Ca2+]cyt). However, the ability of certain antisense oligonucleotides to bind and inactivate growth factors allows alternative explanations. To define more specifically the role of c-Myb in cultured VSMCs (SVE and A10 cell lines), we have generated stable cell clones expressing a dominant-negative c-Myb lacking critical elements of the DNA binding domain (delta5-SVE) and transiently transfected cell populations (GRE-MEn-SVE and GRE-MEn-A10) expressing a glucocorticoid-inducible chimeric protein that targets the Drosophila Engrailed repressor domain to c-Myb-responsive promoters. The delta5-SVE clones and GRE-MEn cell populations exhibit a 60% reduction in mean intracellular c-Myb activity, as measured by cotransfection assays with a c-Myb-responsive reporter, a 42% decrease in the mean S phase entry of growth-arrested (G[0]) cells after serum stimulation, and a 36% inhibition of mean cell proliferation over 4 days. These cells also display 28% (34-nmol/L) and 30% (42-nmol/L) reductions in mean [Ca2+]cyt at G(0) and at the G1/S interface, respectively, as well as significant reductions in the peak [Ca2+]cyt responses to thapsigargin (5 micromol/L) and caffeine (10 mmol/L). These latter reductions in operationally defined Ca2+ pools were observed both at different stages of the cell cycle and after transient induction of the dominant-interfering construct, suggesting that c-Myb regulates these releasable Ca2+ stores independent of its effects on cell cycle progression.

Tissue-selective expression of dominant-negative proteins for the regulation of vascular smooth muscle cell proliferation

“…Other studies utilizing c-myb cDNA lacking the DNAbinding domain as well as a myb-engrailed fusion demonstrated that in addition to its effect on the cell cycle, cmyb has a role in regulating intracellular stores of calcium. 34 For c-myc to induce apoptosis, however, it requires association with its dimerisation partner Max. 43 In addition, Myc-induced apoptosis is usually associated with c-myc overexpression, 44 although the reverse appears to hold for some B-lymphocyte lines.…”

“…33 Similar deletion or chimeric c-myb constructs have also been used to examine the role of c-myb in cell-cycle progression and the regulation of cytoplasmic Ca 2+ concentration in rat VSMC cell lines. 33,34 The dominant-negative chimeric myb-engrailed (MEnT, MybEn, MERT) and myc-engrailed (MycEn) constructs used in this study were designed on the basis that the activity of c-myb and c-myc proteins is dependent on the integrity of their trans-activation and DNA-binding domains. As described previously, 33 the dominantnegative chimeric myb-engrailed gene constructs (MybEn 35 Design of the myc-engrailed construct was slightly different as the functional activity of c-myc requires dimerisation with Max.…”

Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension

“…Although its etiology remains unclear, elevated levels of circulating mitogens, dysfunction or down-regulation of receptors and ion channels, upregulation of transporters, and heightened activity of elastases and glycoproteins have been implicated in IPAH (5,6,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Transient receptor potential (TRP) channel genes may encode subunits that form receptor-(ROC) and store-(SOC) operated Ca 2ϩ channels in many cell types, including PASMC and pulmonary artery endothelial cells (PAEC) (28,(30)(31)(32)(33)(34). Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21). High levels of [Ca 2ϩ ] cyt and sufficient levels of Ca 2ϩ in the SR are required for vascular smooth muscle cell proliferation (22,25,39).…”

“…Ca 2ϩ entry through ROC and SOC increases [Ca 2ϩ ] cyt , allowing for phosphorylation of signal transduction proteins and transcription factors (23,24,(35)(36)(37)(38), that are essential for the progression of the cell cycle (21). High levels of [Ca 2ϩ ] cyt and sufficient levels of Ca 2ϩ in the SR are required for vascular smooth muscle cell proliferation (22,25,39).…”

c-Myb-binding Sites Mediate G1/S-associated Repression of the Plasma Membrane Ca2+-ATPase-1 Promoter

“…We have shown that c-Myb activity regulates [Ca 2ϩ ] i in both VSMC and fibroblasts (9 -11). Experiments employing either wild-type or dominant negative forms of c-Myb, in which cell cycle-associated Ca 2ϩ homeostasis was monitored, implicated the plasma membrane Ca 2ϩ -ATPase (PMCA) family of Ca 2ϩ efflux pumps as critical mediators of c-Myb-dependent [Ca 2ϩ ] i (9,10).…”