Depletion of cAMP-response element-binding protein/ATF1 inhibits adipogenic conversion of 3T3-L1 cells ectopically expressing CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBP beta, or PPAR gamma 2.

The differentiation of preadipocytes to adipocytes is orchestrated by the expression of the "master adipogenic regulators," CCAAT/enhancer-binding protein (C/EBP) beta, peroxisome proliferator-activated receptor gamma (PPARgamma), and C/EBP alpha. In addition, activation of the cAMP-response element-binding protein (CREB) is necessary and sufficient to promote adipogenic conversion and prevent apoptosis of mature adipocytes. In this report we used small interfering RNA to deplete CREB and the closely related factor ATF1 to explore the ability of the master adipogenic regulators to promote adipogenesis in the absence of CREB and probe the function of CREB in late stages of adipogenesis. Loss of CREB/ATF1 blocked adipogenic conversion of 3T3-L1 cells in culture or 3T3-F442A cells implanted into athymic mice. Loss of CREB/ATF1 prevented the expression of PPARgamma, C/EBP alpha, and adiponectin and inhibited the loss of Pref-1. Loss of CREB/ATF1 inhibited adipogenic conversion even in cells ectopically expressing C/EBP alpha, C/EBP beta, or PPARgamma2 individually. CREB/ATF1 depletion did not attenuate lipid accumulation in cells expressing both PPARgamma2 and C/EBP alpha, but adiponectin expression was severely diminished. Conversely ectopic expression of constitutively active CREB overcame the blockade of adipogenesis due to depletion of C/EBP beta but not due to loss of PPARgamma2 or C/EBP alpha. Depletion of CREB/ATF1 did not suppress the expression of C/EBP beta as we had previously observed using dominant negative forms of CREB. Finally results are presented showing that CREB promotes PPARgamma2 gene transcription. The results indicate that CREB and ATF1 play a central role in adipogenesis because expression of individual master adipogenic regulators is unable to compensate for their loss. The data also indicate that CREB not only functions during the initiation of adipogenic conversion but also at later stages.

Platelet-derived growth factor BB induces nuclear export and proteasomal degradation of CREB via phosphatidylinositol 3-kinase/Akt signaling in pulmonary artery smooth muscle cells.

Cyclic AMP response element binding protein (CREB) content is diminished in smooth muscle cells (SMCs) in remodeled pulmonary arteries from animals with pulmonary hypertension and in the SMC layers of atherogenic systemic arteries and cardiomyocytes from hypertensive individuals. Loss of CREB can be induced in cultured SMCs by chronic exposure to hypoxia or platelet-derived growth factor BB (PDGF-BB). Here we investigated the signaling pathways and mechanisms by which PDGF elicits depletion of SMC CREB. Chronic PDGF treatment increased CREB ubiquitination in SMCs, while treatment of SMCs with the proteasome inhibitor lactacystin prevented decreases in CREB content. The nuclear export inhibitor leptomycin B also prevented depletion of SMC CREB alone or in combination with lactacystin. Subsequent studies showed that PDGF activated extracellular signal-regulated kinase, Jun N-terminal protein kinase, and phosphatidylinositol 3 (PI3)-kinase pathways in SMCs. Inhibition of these pathways blocked SMC proliferation in response to PDGF, but only inhibition of PI3-kinase or its effector, Akt, blocked PDGF-induced CREB loss. Finally, chimeric proteins containing enhanced cyan fluorescent protein linked to wild-type CREB or CREB molecules with mutations in several recognized phosphorylation sites were introduced into SMCs. PDGF treatment reduced the levels of each of these chimeric proteins except for one containing mutations in adjacent serine residues (serines 103 and 107), suggesting that CREB loss was dependent on CREB phosphorylation at these sites. We conclude that PDGF stimulates nuclear export and proteasomal degradation of CREB in SMCs via PI3-kinase/Akt signaling. These results indicate that in addition to direct phosphorylation, proteolysis and intracellular localization are key mechanisms regulating CREB content and activity in SMCs.