Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activation

Transcription is controlled by promoter-selective transcriptional factors (TFs), which bind to cis-regulatory enhancers elements, termed hormone response elements (HREs), in a specific subset of genes. Regulation by these factors involves either the recruitment of coactivators or corepressors and direct interaction with the basal transcriptional machinery (1). Hormone-activated nuclear receptors (NRs) are well characterized transcriptional factors (2) that bind to the promoters of their target genes and recruit primary and secondary coactivator proteins which possess many enzymatic activities required for gene expression (1,3,4). In the present study, using single-cell high-resolution fluorescent microscopy and high throughput microscopy (HTM) coupled to computational imaging analysis, we investigated transcriptional regulation controlled by the estrogen receptor alpha (ERalpha), in terms of large scale chromatin remodeling and interaction with the associated coactivator SRC-3 (Steroid Receptor Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a steroid-dependent transcriptional factor (16) that belongs to the NRs superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2), regulates transcription of distinct target genes involved in development, puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the nucleus and undergoes a rapid (within minutes) intranuclear redistribution following the addition of either agonist or antagonist (17,18,19). We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated reporter gene array (PRL-array) containing multicopy hormone response-binding elements for ERalpha that are derived from the physiological enhancer/promoter region of the prolactin gene. Following GFP-ER transfection of PRL-HeLa cells, we were able to observe in situ ligand dependent (i) recruitment to the array of the receptor and associated coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout of the reporter gene. Addition of E2 causes a visible opening (decondensation) of the PRL-array, colocalization of RNA Polymerase II, and transcriptional readout of the reporter gene, detected by mRNA FISH. On the contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI), a dramatic condensation of the PRL-array was observed, displacement of RNA Polymerase II, and complete decreasing in the transcriptional FISH signal. All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family member and a known oncogenic protein (4,34). SRC-3 is regulated by a variety of posttranslational modifications, including methylation, phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These events have been shown to be important for its interaction with other coactivator proteins and NRs and for its oncogenic potential (37,39). A number of extracellular signaling molecules, like steroid hormones, growth factors and cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by a wide range of kinases, including extracellular-regulated kinase 1 and 2 (ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs) (41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein, whose cellular localization is regulated by phosphorylation and interaction with ERalpha. Using a combination of high throughput and fluorescence microscopy, we show that both chemical inhibition (with U0126) and siRNA downregulation of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by EGF signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known partecipants in the regulation of SRC-3 activity (39). Accordingly, the cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further supports these results. In the presence of ERalpha, U0126 also dramatically reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell lysates; localization of SRC-3 at the ER-targeted prolactin promoter array (PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of estrogen receptor. While a wealth of studies have revealed the molecular functions of NRs and coregulators, there is a paucity of data on how these functions are spatiotemporally organized in the cellular context. Technically and conceptually, our findings have a new impact upon evaluating gene transcriptional control and mechanisms of action of gene regulators.

Studio degli effetti di citochine a basso dosaggio (LOW DOSE) su cellule di origine animale (3T3-L1) ed umana (hASC)

Nella sindrome metabolica l’insulino-resistenza e l’obesità rappresentano i fattori chiave nello sviluppo di tale patologia, ma il principale player risulta un’infiammazione cronica di basso grado (Chronic Low Grade Inflammation) a carico del tessuto adiposo. Lo scopo di questo progetto di ricerca è quindi stato quello di testare citochine a basso dosaggio come possibile trattamento dell’infiammazione cronica. Le citochine utilizzate (GUNA®-Interleukin 4 (IL-4), GUNA®-Interleukin 10 (IL-10), GUNA®-Melatonin, GUNA®-Melatonin+GUNA®-IL-4.) sono state fornite dall’azienda GUNA S.p.a. Poiché l’infiammazione cronica a basso grado inizia in seguito ad un aumento eccessivo del tessuto adiposo, inizialmente si è valutato l’effetto su una linea di preadipociti murini (3T3-L1). Questa prima parte dello studio ha messo in evidenza come le citochine a basso dosaggio non modificano la vitalità cellulare, anche se agiscono sull’espressione e la localizzazione di vimentina e E-caderina. Inoltre IL-4 e IL-10 sembrano avere una parziale attività inibitoria, non significativa, sull’adipogenesi ad eccezione dell’espressione dell’adiponectina che appare significativamente aumentata. In ultimo i trattamenti con IL-4 e IL-10 hanno mostrato una diminuzione del contenuto di ROS e una ridotta attività antiinfiammatoria dovuta alla diminuzione di IL-6 secreto. Un’altra popolazione cellulare principale nel tessuto adiposo è rappresentata dalle ASC (Adipose Stem Cell). Per tale motivo si è proseguito valutando l’effetto che le citochine low-dose su questo citotipo, evidenziando che il trattamento con le citochine non risulta essere tossico, anche se sembrerebbe rallentare la crescita cellulare, e determina un’inibizione del processo adipogenico. Inoltre il trattamento con IL-10 sembra stimolare le ASC a produrre fattori che inducono una maggiore vasculogenesi e le induce a produrre fattori chemiotattici che determinano una maggiore capacità di rigenerazione tissutale da parte di MSC da derma. Infine, il trattamento con IL-4 e IL-10 stimola probabilmente una minore produzione di citochine pro-infiammatorie che inducono in maniera significativa una minore mobilità di cellule MSC.

Gene expression and genome-wide association studies analysis to select heavy pigs for protected designation of origin cured meats production

The high quality of protected designation of origin (PDO) dry-cured pork products depends largely on the chemical and physical parameters of the fresh meat and their variation during the production process of the final product. The discovery of the mechanisms that regulate the variability of these parameters was aided by the reference genome of swine adjuvant to genetic analysis methods. This thesis can contribute to the discovery of genetic mechanisms that regulate the variability of some quality parameters of fresh meat for PDO dry-cured pork production. The first study is of gene expression and showed that between low and high glycolytic potential (GP) samples of Semimembranosus muscle of Italian Large White (ILW) pigs in early postmortem, the differentially expressed genes were all but one over expressed in low GP. These were involved in ATP biosynthesis processes, calcium homeostasis, and lipid metabolism including the potential master regulator gene Peroxisome Proliferator-Activated Receptor Alpha (PPARA). The second is a study in commercial hybrid pigs to evaluate correlations between carcass and fresh ham traits, including carcass and fresh ham lean meat percentages, the former, a potential predictor of the latter. In addition, a genome-wide association study allowed the identification of chromosome-wide associations with phenotypic traits for 19 SNPs, and genome-wide associations for 14 SNPs for ferrochelatase activity. The latter could be a determinant for color variation in nitrite-free dry-cured ham. The third study showed gene expression differences in the Longissimus thoracis muscle of ILW pigs by feeding diets with extruded linseed (source of polyunsaturated fatty acids) and vitamin E and selenium (diet three) or natural (diet four) antioxidants. The diet three promoted a more rapid and massive immune system response possibly determined by improvement in muscle tissue function, while the diet four promoted oxidative stability and increased the anti-inflammatory potential of muscle tissue.