BACKGROUND
Activator protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2alpha and AP-2gamma is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.
METHODS
We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.
RESULTS
We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed Cell Death and Cell Adhesion and Migration as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. Ctgf, Nrp1, Tnfaip3, Gsta3) and AP-2 and other main apoptosis players and to create a unique network.
CONCLUSIONS
Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.
A causative role of the membrane-bound tyrosine kinase ErbB-2 in breast tumorigenesis has been well established. MMTV/neu transgenic mice which overexpress ErbB-2 consistently develop mammary carcinomas with a high incidence. In human breast cancer, ErbB-2 is overexpressed in 25-30 of all cases and is representing a clinical marker of a poor prognosis. Besides to gene amplification, ErbB-2 overexpression has been attributed to transcription factors of the AP-2 family which were shown to control the ErbB-2 gene promoter in cell culture studies. Particularly AP-2alpha and gamma are often coexpressed in ErbB-2-positive breast carcinomas. However, LTRgamma transgenic mice which overexpress AP-2gamma in their mammary epithelium display only a very weak upregulation of the erbB-2 gene and do not develop mammary carcinoma. These findings therefore raise the possibility of functional cooperativity between both genes in breast cancer. To experimentally address the impact of AP-2gammaon ErbB-2-induced breast carcinogenesis we crossed MMTV/neu transgenic mice with LTRgamma transgenic mice and monitored tumor development in bitransgenic female progeny. AP-2gamma overexpression negatively influenced tumor incidence, as reflected by a reduced tumor number and a prolonged tumor latency. Histological analysis revealed three major types of tumors corresponding to different stages of tumor progression. Interestingly, an increased proportion of advanced stage carcinomas was observed in bitransgenic mice. Moreover, the AP-2gamma transgene differentially affected proliferation rates between the different progression stages: proliferation was enhanced at early stages but reduced in advanced stages in comparison to control tumors. Therefore, AP-2gamma while reducing the incidence of mammary tumors is promoting tumor progression.
The development and growth of the skull is controlled by cranial sutures, which serve as growth centers for osteogenesis by providing a pool of osteoprogenitors. These osteoprogenitors undergo intramembranous ossification by direct differentiation into osteoblasts, which synthesize the components of the extracellular bone matrix. A dysregulation of osteoblast differentiation can lead to premature fusion of sutures, resulting in an abnormal skull shape, a disease called craniosynostosis. Although several genes could be linked to craniosynostosis, the mechanisms regulating cranial suture development remain largely elusive. We have established transgenic mice conditionally expressing an autoactivated platelet-derived growth factor receptor alpha (PDGFRalpha) in neural crest cells (NCCs) and their derivatives. In these mice, premature fusion of NCC-derived sutures occurred at early postnatal stages. In vivo and in vitro experiments demonstrated enhanced proliferation of osteoprogenitors and accelerated ossification of osteoblasts. Furthermore, in osteoblasts expressing the autoactivated receptor, we detected an upregulation of the phospholipase C-gamma (PLC-gamma) pathway. Treatment of differentiating osteoblasts with a PLC-gamma-specific inhibitor prevented the mineralization of synthesized bone matrix. Thus, we show for the first time that PDGFRalpha signaling stimulates osteogenesis of NCC-derived osteoblasts by activating the PLC-gamma pathway, suggesting an involvement of this pathway in the etiology of human craniosynostosis.
Murine neural crest stem cells (NCSCs) are a multipotent transient population of stem cells. After being formed during early embryogenesis as a consequence of neurulation at the apical neural fold, the cells rapidly disperse throughout the embryo, migrating along specific pathways and differentiating into a wide variety of cell types. In vitro the multipotency is lost rapidly, making it difficult to study differentiation potential as well as cell fate decisions. Using a transgenic mouse line, allowing for spatio-temporal control of the transforming c-myc oncogene, we derived a cell line (JoMa1), which expressed NCSC markers in a transgene-activity dependent manner. JoMa1 cells express early NCSC markers and can be instructed to differentiate into neurons, glia, smooth muscle cells, melanocytes, and also chondrocytes. A cell-line, clonally derived from JoMa1 culture, termed JoMa1.3 showed identical behavior and was studied in more detail. This system therefore represents a powerful tool to study NCSC biology and signaling pathways. We observed that when proliferative and differentiation stimuli were given, enhanced cell death could be detected, suggesting that the two signals are incompatible in the cellular context. However, the cells regain their differentiation potential after inactivation of c-MycER(T). In summary, we have established a system, which allows for the biochemical analysis of the molecular pathways governing NCSC biology. In addition, we should be able to obtain NCSC lines from crossing the c-MycER(T) mice with mice harboring mutations affecting neural crest development enabling further insight into genetic pathways controlling neural crest differentiation.
The AP-2 family of transcription factors consists of five different proteins in humans and mice: AP-2alpha, AP-2beta, AP-2gamma, AP-2delta and AP-2epsilon. Frogs and fish have known orthologs of some but not all of these proteins, and homologs of the family are also found in protochordates, insects and nematodes. The proteins have a characteristic helix-span-helix motif at the carboxyl terminus, which, together with a central basic region, mediates dimerization and DNA binding. The amino terminus contains the transactivation domain. AP-2 proteins are first expressed in primitive ectoderm of invertebrates and vertebrates; in vertebrates, they are also expressed in the emerging neural-crest cells, and AP-2alpha-/- animals have impairments in neural-crest-derived facial structures. AP-2beta is indispensable for kidney development and AP-2gamma is necessary for the formation of trophectoderm cells shortly after implantation; AP-2alpha and AP-2gamma levels are elevated in human mammary carcinoma and seminoma. The general functions of the family appear to be the cell-type-specific stimulation of proliferation and the suppression of terminal differentiation during embryonic development.
Transcription factor AP-2gamma, a novel marker of gonocytes and seminomatous germ cell tumors
(2005)
BACKGROUND
During pregnancy the mammary epithelium undergoes a complex developmental process which culminates in the generation of the milk-secreting epithelium. Secretory epithelial cells display lactogenic differentiation which is characterized by the expression of milk protein genes, such as beta-casein or whey acidic protein (WAP). Transcription factors AP-2alpha and AP-2gamma are downregulated during lactation, and their overexpression in transgenic mice impaired the secretory differentiation of the mammary epithelium, resulting in lactation failure. To explore whether the downregulation of AP-2alpha and AP-2gamma is of functional significance for lactogenic differentiation, we analyzed the expression of the AP-2 family members during the lactogenic differentiation of HC11 mammary epithelial cells in vitro. Differentiation of HC11 cells was induced following established protocols by applying the lactogenic hormones prolactin, dexamethasone and insulin.
FINDINGS
HC11 cells express all AP-2 family members except AP-2delta. Using RT-PCR we could not detect a downregulation of any of these genes during the lactogenic differentiation of HC11 cells in vitro. This finding was confirmed for AP-2alpha and AP-2gamma using Northern analysis. Differentiating HC11 cells displayed lower expression levels of milk protein genes than mammary glands of mid-pregnant or lactating mice.
CONCLUSION
The extent of lactogenic differentiation of HC11 cells in vitro is limited compared to mammary epithelium undergoing secretory differentiation in vivo. Downregulation of AP-2 transcription factor genes is not required for lactogenic differentiation of HC11 cells but may functionally be involved in aspects of lactogenic differentiation in vivo that are not reflected by the HC11 system.
BACKGROUND
Neuronal migration is a crucial process that allows neurons to reach their correct target location to allow the nervous system to function properly. AP-2alpha is a transcription factor essential for neural crest cell migration and its mutation results in apoptosis within this cell population, as demonstrated by genetic models.
RESULTS
We down-modulated AP-2alpha expression in GN-11 neurons by RNA interference and observe reduced neuron migration following the activation of a specific genetic programme including the Adhesion Related Kinase (Axl) gene. We prove that Axl is able to coordinate migration per se and by ChIP and promoter analysis we observe that its transcription is directly driven by AP-2alpha via the binding to one or more functional AP-2alpha binding sites present in its regulatory region. Analysis of migration in AP-2alpha null mouse embryo fibroblasts also reveals an essential role for AP-2alpha in cell movement via the activation of a distinct genetic programme.
CONCLUSION
We show that AP-2alpha plays an essential role in cell movement via the activation of cell-specific genetic programmes. Moreover, we demonstrate that the AP-2alpha regulated gene Axl is an essential player in GN-11 neuron migration.
