Lactogenic differentiation of HC11 cells is not accompanied by downregulation of AP-2 transcription factor genes

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

The AP-2 family of transcription factors

The AP-2 family of transcription factors consists of five different proteins in humans and mice: AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ε. 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-2α(-/- )animals have impairments in neural-crest-derived facial structures. AP-2β is indispensable for kidney development and AP-2γ is necessary for the formation of trophectoderm cells shortly after implantation; AP-2α and AP-2γ 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

AP-2α regulates migration of GN-11 neurons via a specific genetic programme involving the Axl receptor tyrosine kinase

<p>Abstract</p> <p>Background</p> <p>Neuronal migration is a crucial process that allows neurons to reach their correct target location to allow the nervous system to function properly. AP-2α 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.</p> <p>Results</p> <p>We down-modulated AP-2α 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 (<it>Axl</it>) gene. We prove that <it>Axl </it>is able to coordinate migration per se and by ChIP and promoter analysis we observe that its transcription is directly driven by AP-2α via the binding to one or more functional AP-2α binding sites present in its regulatory region. Analysis of migration in AP-2α null mouse embryo fibroblasts also reveals an essential role for AP-2α in cell movement via the activation of a distinct genetic programme.</p> <p>Conclusion</p> <p>We show that AP-2α plays an essential role in cell movement via the activation of cell-specific genetic programmes. Moreover, we demonstrate that the AP-2α regulated gene <it>Axl </it>is an essential player in GN-11 neuron migration.</p

Роль компетентностного подхода при управлении профессиональными рисками

В статье рассмотрены соответствия между опасностью, вредом здоровью, профессиональным риском и компетентностью работников, которые могут послужить основой для системы выявления и управления профессиональными рисками, обусловленными человеческим фактором.In this article compliances between danger, harm to health, occupational risk and competence of employees are considered. They can serve as a basis for the system for the detection and management of occupational risks caused by human factors

BMP Inhibition in Seminomas Initiates Acquisition of Pluripotency via NODAL Signaling Resulting in Reprogramming to an Embryonal Carcinoma

Type II germ cell cancers (GCC) can be subdivided into seminomas and non-seminomas. Seminomas are similar to carcinoma in situ (CIS) cells, the common precursor of type II GCCs, with regard to epigenetics and expression, while embryonal carcinomas (EC) are totipotent and differentiate into teratomas, yolk-sac tumors and choriocarcinomas. GCCs can present as seminomas with a non-seminoma component, raising the question if a CIS gives rise to seminomas and ECs at the same time or whether seminomas can be repro- grammed to ECs. In this study, we utilized the seminoma cell line TCam-2 that acquires an EC-like status after xenografting into the murine flank as a model for a seminoma to EC tran- sition and screened for factors initiating and driving this process. Analysis of expression and DNA methylation dynamics during transition of TCam-2 revealed that many pluripotency- and reprogramming-associated genes were upregulated while seminoma-markers were downregulated. Changes in expression level of 53 genes inversely correlated to changes in DNA methylation. Interestingly, after xenotransplantation 6 genes ( GDF3 , NODAL , DNMT3B , DPPA3 , GAL , AK3L1 ) were rapidly induced, followed by demethylation of their genomic loci, suggesting that these 6 genes are poised for expression driving the repro- gramming. We demonstrate that inhibition of BMP signaling is the initial event in reprogram- ming, resulting in activation of the pluripotency-associated genes and NODAL signaling. We propose that reprogramming of seminomas to ECs is a multi-step process. Initially, the microenvironment causes inhibition of BMP signaling, leading to induction of NODAL sig- naling. During a maturation phase, a fast acting NODAL loop stimulates its own activity and temporarily inhibits BMP signaling. During the stabilization phase, a slow acting NODAL loop, involving WNTs re-establishes BMP signaling and the pluripotency circuitry. In parallel, DNMT3B-driven de novo methylation silences seminoma-associated genes and epigenetically fixes the EC state

A signaling cascade including ARID1A, GADD45B and DUSP1 induces apoptosis and affects the cell cycle of germ cell cancers after romidepsin treatment

In Western countries, the incidence of testicular germ cell cancers (GCC) is steadily rising over the last decades. Mostly, men between 20 and 40 years of age are affected. In general, patients suffering from GCCs are treated by orchiectomy and radio- or chemotherapy. Due to resistance mechanisms, intolerance to the therapy or denial of chemo- / radiotherapy by the patients, GCCs are still a lethal threat, highlighting the need for alternative treatment strategies. In this study, we revealed that germ cell cancer cell lines are highly sensitive to the histone deacetylase inhibitor romidepsin in vitro and in vivo, highlighting romidepsin as a potential therapeutic option for GCC patients. Romidepsin-mediated inhibition of histone deacetylases led to disturbances of the chromatin landscape. This resulted in locus-specific histone-hyper- or hypoacetylation. We found that hypoacetylation at the ARID1A promotor caused repression of the SWI/SNF-complex member ARID1A. In consequence, this resulted in upregulation of the stress-sensors and apoptosis-regulators GADD45B, DUSP1 and CDKN1A. RNAi-driven knock down of ARID1A mimicked in parts the effects of romidepsin, while CRISPR/Cas9-mediated deletion of GADD45B attenuated the romidepsin-provoked induction of apoptosis and cell cycle alterations. We propose a signaling cascade involving ARID1A, GADD45B and DUSP1 as mediators of the romidepsin effects in GCC cells

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O$_{2}$-dependent shuttle of oxidizable fatty acid substrates. MB’s impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively