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AP-2 family transcription factors are essential for development and morphogenesis of diverse tissues and organs, but their precise roles in specification of neural crest stem cell (NCSC)-derived cell types have not been determined. Among three members known to be expressed in the NCSC (i.e. AP-2alpha, AP-2beta, and AP-2gamma), we found that only AP-2beta is predominantly expressed in the sympathetic ganglia of developing mouse embryos, supporting its role in sympathetic development. Indeed, AP-2beta null mice expressed significantly reduced levels of both noradrenaline (NA) and NA-synthesizing dopamine beta-hydroxylase in the peripheral nervous system. Strikingly, we also found that NA neuron development was significantly compromised in the locus coeruleus as well. Pharmacological treatment with an NA intermediate during pregnancy significantly rescues the neonatal lethality of AP-2beta(-/-) mice, indicating that NA deficiency is one of the main causes for lethality found in AP-2beta(-/-) mice. We also showed that forced expression of AP-2beta, but not other AP-2 factors, in NCSC favors their differentiation into NA neurons. In summary, we propose that AP-2beta plays critical and distinctive roles in the NA phenotype specification in both the peripheral and central nervous system during development.

We have previously demonstrated that fibronectin (Fn) stimulates the proliferation of non-small cell lung carcinoma (NSCLC) cell growth through the induction of cyclooxygenase-2 (COX-2) and prostaglandin E2 secretion. Here, we demonstrate that NSCLC cells express mRNA and protein for the prostaglandin E2 receptor EP4 and that Fn enhances its stimulatory effect by inducing the expression of EP4, but not of EP1, EP2, and EP3 receptor subtypes. The effect of Fn on EP4 was inhibited by an antibody against alpha5beta1 integrin and by inhibitors of phosphoinositide 3-kinase (wortmannin) and extracellular signal-regulated kinase (PD98095), but not by inhibitors of protein kinase C (calphostin C), of protein kinase A (H-89), or of mammalian target of rapamycin (rapamycin). A COX-2 small interfering RNA was also inhibitory. Fn significantly increased AP-2 binding activity in the promoter of the EP4 gene, and AP-2 antisense oligonucleotides blocked Fn-induced EP4 expression. Using full-length and mutated EP4 promoter constructs, we found that Fn stimulation of EP4 gene expression was inhibited when one AP-2 site (-1000 bp) was mutated. Fn induced nuclear AP-2alpha protein expression through multiple signaling pathways. Our results indicate that Fn-induced NSCLC cell proliferation is mediated through EP4. Furthermore, they show that Fn induces EP4 expression through the activation of alpha5beta1-dependent signals that include induction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathways as well as expression of COX-2. These events lead to activation of the transcription factor AP-2alpha, which interacts with specific regions in the EP4 gene promoter, leading to transcription of the EP4 gene.

We previously reported the association between the activating enhancer-binding protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with the disease-susceptible allele of AP-2beta showed stronger expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta leads to lipid accumulation by enhancing glucose transport and inducing insulin resistance in 3T3-L1 adipocytes. In this study we demonstrated that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the expression and secretion of adiponectin and increased those of interleukin-6 (IL-6). Interestingly, the effects of AP-2beta on the expressions of adiponectin and IL-6 and the mechanisms by which AP-2beta modulated their expressions were different. We found that the promoter activity of adiponectin gene was inhibited by AP-2beta overexpression and enhanced by knockdown of endogenous AP-2beta, whereas IL-6 was unaffected. Electrophoretic mobility shift assays revealed the existence of putative responsive elements for AP-2beta and NF-YA in human and mouse adiponectin promoter regions, and mutation of this AP-2beta binding site abolished the inhibitory effect of AP-2beta. Furthermore, chromatin immunoprecipitation assays demonstrated that AP-2beta and NF-YA competitively bind to the same region of the adiponectin promoter. Our results clearly demonstrated that AP-2beta directly inhibits adiponectin gene expression by displacing NF-YA and binding to its promoter. We conclude that AP-2beta might modulate the expression of adiponectin by directly inhibiting its transcriptional activity.

Activator protein 2alpha (AP-2alpha) induces cytotoxicity by inducing cell cycle arrest and apoptosis. In this study we investigated the mechanism of apoptosis induction by AP-2alpha. We found that AP-2alpha induced apoptosis efficiently in cells treated with benzyloxycar-bonyl-IETD-fluoromethyl ketone or FADD-silenced cells but failed to do so in benzyloxycarbonyl-LEHD-fluoromethyl ketone-treated or apoptosis protease activation factor-1 (Apaf1)-silenced cells, suggesting the central role of mitochondria in AP-2alpha-induced apoptosis. In good correlation, cells overexpressing AP-2alpha showed a reduction in mitochondrial membrane potential (Deltapsi(m)), cytochrome c and Smac/DIABLO release into cytosol, and Bax translocation into mitochondria. We found that the pro-apoptotic protein Bax is important for AP-2alpha-induced apoptosis as adenovirus AP2 failed to induce apoptosis in HCT116 Bax(-/-) cells. However, we found the IAP (inhibitor of apoptosis) inhibitor Smac/DIABLO may have a limited role in AP-2alpha-induced apoptosis as we found the IAP member Survivin down-regulated by AP-2alpha. Although the total Bax level remains unaltered, we found a time-dependent increase in the activated form of Bax in adenovirus AP2-infected cells. In addition, we show that AP-2alpha transcriptionally represses Bcl-2 by binding to its promoter both in vitro and in vivo and that this is essential for AP-2alpha-induced apoptosis as ectopic expression of Bcl-2 efficiently inhibited apoptosis induced by AP-2alpha. Furthermore, we show that chemotherapy-induced endogenous AP-2alpha down-regulates Bcl-2 and induces apoptosis in an AP-2alpha-dependent manner. Moreover, we demonstrate that inhibition of okadaic acid or staurosporine-sensitive pathways in AP-2alpha overexpressing breast cancer cells resulted in AP-2alpha-dependent apoptosis induction. These results suggest that AP-2alpha induces apoptosis by down-regulating Bcl-2 and utilizing a bax/cytochrome c/Apaf1/caspase 9-dependent mitochondrial pathway.