We hypothesized that cAMP response element-binding protein (CREB) could function as a molecular determinant of smooth muscle cell fate. In arterial sections from the systemic and pulmonary circulation, CREB content was high in proliferation-resistant medial subpopulations of smooth muscle cells and low in proliferation-prone regions. In vessels from neonatal calves exposed to chronic hypoxia, CREB content was depleted and smooth muscle cell (SMC) proliferation was accelerated. Induction of quiescence by serum deprivation in culture led to increased CREB content. Highly proliferative SMC in culture were observed to have low CREB content. Exposure to proliferative stimuli such as hypoxia or platelet-derived growth factor decreased SMC CREB content. Assessment of CREB gene transcription by nuclear run-on analysis and transcription from a CREB promoter-luciferase construct indicate that CREB levels in SMC are in part controlled at the level of transcription. Overexpression of wild type or constitutively active CREB in primary cultures of SMC arrested cell cycle progression. Additionally, expression of constitutively active CREB decreased both proliferation and chemokinesis. Consistent with these functional properties, active CREB decreased the expression of multiple cell cycle regulatory genes, as well as genes encoding growth factors, growth factor receptors, and cytokines. Our data suggest a unique mode of cellular phenotype determination at the level of the nuclear content of CREB.The vessel wall, once seen as simply a mechanical conduit for blood flow, is a complex organ whose dysfunction is responsible for the leading cause of death in the United States, cardiovascular disease. The lumen of blood vessels is lined with endothelial cells, which communicate with the underlying medial cell layer. For many years, the arterial media was considered to be made-up of a homogeneous population of smooth muscle cells (SMC) 1 arising from a common lineage. Thus the diverse activities of contraction, proliferation, migration, and extracellular matrix production were thought to reflect SMC response to either normal or pathological stimuli. Vascular remodeling is the compensatory response of the vasculature to stress or injury. Under pathological circumstances (hypoxia, mechanical injury, hyperlipidemia, and oxidative stress), SMC in the intimal and medial compartments of the arterial wall become proliferative, migratory, and produce excess matrix proteins. This switch in SMC phenotype is termed phenotypic modulation and is considered to play a key pathogenic role in atherosclerosis and pulmonary hypertension. While the stimuli for SMC activation are well known, the molecular events, which permit activation of SMC, remain poorly understood.Cyclic nucleotides (cAMP and cGMP) promote SMC quiescence in vitro and in vivo. ␤-Adrenergic stimulation of cAMP signaling is important for SMC quiescence and contractile function under normal conditions. It is believed that cAMP acts as a gate to prevent SMC mitogenic response to growth fac...

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect.Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB.In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf 3 MEK pathway and has an impact on physiologically relevant genes in these cells.Insulin binding to its cell surface receptor results in alterations in the expression of many genes for cellular growth, differentiation, and proliferation. Specific insulin responsive elements have been identified in the promoters of several genes (1-4). Peroxisome proliferator-activated receptors and other steroid hormone receptors have been implicated in regulating gene transcription through these insulin responsive elements (2, 4, 5). In other genes, insulin responsive sites have been mapped to regions containing cAMP responsive elements (CREs) 1 and serum responsive elements (6 -9). Consistent with these findings, many insulin-regulated genes are also regulated by extracellular stimuli that modulate intracellular cAMP levels (2, 10 -17) (Table I). Previously, we demonstrated that phosphorylation of CREB was stimulated by insulin in primary rat adipocytes and HIRc cells (18,19). This observation posed an important question regarding the impact of insulin-mediated CREB phosphorylation on CREB transactivation and the post-receptor pathways activated by insulin responsible for this effect. Cyclic AMP regulates the transcription of target genes primarily through the phosphorylation of the cAMP-response element binding protein (CREB) by protein kinase A (PKA) on serine 133 (of CREB-341 or serine 119 of CREB-327) of the CREB molecule (20 -23). We demonstrated an analogous response to insulin and identified that this increase in phosphorylation was at least in part due to a decrease in nuclear protein phosphatase-2A activity. These experiments were the first to show a transient increase in CREB phosphorylation and regulation of a nuclear, serine/threonine-specific protein phosphatase in response ...

Adipogenesis is the process by which mature, insulin-responsive adipocytes are generated from undifferentiated preadipocytes and mesenchymal progenitor cells (1). This process is crucial to the normal development of adipose tissue and its expansion in response to excess dietary energy intake. Alternatively, most lipodystrophic syndromes are characterized by a suppression of adipogenesis and an increase in adipocyte death.Cells destined to the adipose lineage arise late in development from multipotential stem cells of mesodermal origin (1-4). The commitment of the multipotent stem cells to the adipocyte lineage is a poorly understood process. However, once committed to the adipocyte lineage, nonproliferating preadipocytes become responsive to external stimuli that induce their differentiation to mature adipocytes. These stimuli include insulin-like growth factor-1 or insulin (which appears to work through the insulin-like growth factor-1 receptor), glucocorticoids, and agents that elevate intracellular cAMP levels (1).Exposure of these cells to adipogenic inducers initiates a temporally orchestrated cascade of gene expression events that characterize adipogenic differentiation. These agents initially induce a period of mitotic expansion during which expression of CCAAT/enhancer binding proteins (C/EBPs) 2 ␤ and ␦ is increased, whereas expression of factors like Pref-1, necdin, and Wnt10b are diminished. Following mitotic expansion, differentiation begins, during which peroxisome-proliferator-activated receptor ␥ (PPAR␥) and C/EBP ␣ are up-regulated. These transcription factors regulate the expression of many of the factors that characterize the mature adipocyte phenotype like GLUT4 (5), adiponectin, aP2 (6), and perilipin (7).We previously reported that the activity of the transcription factor CREB was stimulated by cAMP mimetics and insulin in both preadipocytes (8), suggesting that CREB might play a role in adipogenic conversion. Subsequent experiments demonstrated that ectopic expression of constitutively active forms of CREB could induce adipogenesis of 3T3-L1 cells (9, 10) and prevent their apoptotic death in response to insulin and/or serum deprivation and tumor necrosis factor ␣ (11). Alternately, ectopic expression of dominant negative forms of CREB blocked adipogenic conversion and stimulated apoptosis of mature adipocytes. Recent studies using these techniques as well as CREB-specific antisense and siRNA confirm these results and indicate that CREB may promote adipogenesis by

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In the present study, we observed superstimulated levels of cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter in cells infected with wild-type adenovirus expressing 12 S and 13 S E1a proteins, or in cells expressing 13 S E1a alone. cAMP-stimulated transcription was inhibited in cells expressing only 12 S E1a, but slightly elevated in cells expressing E1a proteins with mutations in conserved regions 1 or 2, leading us to conclude that the superstimulation was mediated by conserved region 3 of 13 S E1a. E1a failed to enhance cAMP-stimulated transcription from promoters containing mutations that abolish binding by cAMP response element binding protein (CREB) or CCAAT/enhancer binding proteins (C/EBPs). This result was supported by experiments in which expression of dominant-negative CREB and/or C/EBP proteins repressed E1a- and cAMP-stimulated transcription from the PEPCK gene promoter. In reconstitution experiments using a Gal4-responsive promoter, E1a enhanced cAMP-stimulated transcription when chimaeric Gal4-CREB and Gal4-C/EBPalpha were co-expressed. Phosphorylation of CREB on serine-133 was stimulated in cells treated with dibutyryl cAMP, whereas phosphorylation of C/EBPalpha was increased by E1a expression. Our data support a model in which cAMP agonists increase CREB activity and stimulate PEPCK gene transcription, a process that is enhanced by E1a through the phosphorylation of C/EBPalpha.