Exposure to Endocrine Disruptors and Nuclear Receptors Gene Expression in Infertile and Fertile Men from Italian Areas with Different Environmental Features

Internal levels of selected endocrine disruptors (EDs) (i.e., perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), di-2-ethylhexyl-phthalate (DEHP), mono-(2-ethylhexyl)-phthalate (MEHP), and bisphenol A (BPA)) were analyzed in blood/serum of infertile and fertile men from metropolitan, urban and rural Italian areas. PFOS and PFOA levels were also evaluated in seminal plasma. In peripheral blood mononuclear cells (PBMCs) of same subjects, gene expression levels of a panel of nuclear receptors (NRs), namely estrogen receptor α (ERα) estrogen receptor β (ERβ), androgen receptor (AR), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptor γ (PPARγ) and pregnane X receptor (PXR) were also assessed. Infertile men from the metropolitan area had significantly higher levels of BPA and gene expression of all NRs, except PPARγ, compared to subjects from other areas. Subjects from urban areas had significantly higher levels of MEHP, whereas subjects from rural area had higher levels of PFOA in both blood and seminal plasma. Interestingly, ERα, ERβ, AR, PXR and AhR expression is directly correlated with BPA and inversely correlated with PFOA serum levels. Our study indicates the relevance of the living environment when investigating the exposure to specific EDs. Moreover, the NRs panel in PBMCs demonstrated to be a potential biomarker of effect to assess the EDs impact on reproductive health

PO-099 Targeting the mitogen activated protein kinase ERK5 in human melanoma

Introduction Melanoma is the most aggressive skin cancer with a poor prognosis in advanced stages. Available treatments for melanoma are unsatisfactory, because rapidly lead to an acquired resistance in the majority of cases. Therefore, there is urgent need to identify novel possible targets involved in melanoma growth. ERK5/BMK1 is a member of the Mitogen-Activated Protein Kinases (MAPK) family and regulates cell functions critical for tumour development. Indeed, several studies reported a direct involvement of ERK5 in several types of cancer including prostate and breast cancer and hepatocellular carcinoma. However, no data have been reported about a possible role of ERK5 in melanoma. Material and methods Cell lines and patient-derived primary melanoma cells (wild type B-RAF: SSM2c and M26c; BRAFV600E: A375, SK-Mel-5, SK-Mel-28, 501-Mel, expressing; NRASQ61R: SK-Mel-2; MeWo) have been used for in vitro and in vivo experiments. HEK293T cells were used for protein overexpression. ERK5 inhibition was achieved using ERK5 and MEK5 inhibitors or lentiviral vectors encoding shRNA specific for ERK5. BRAF inhibition was achieved using Vemurafenib, a BRAFV600E inhibitor. Results and discussions In silico data analysis indicated that components of the ERK5 pathway are upregulated in up to 47% melanoma patients. Accordingly, we found that ERK5 is consistently expressed and active in commercial and patients derived melanoma cell lines. On that basis, we investigated the role of ERK5 in melanoma cell growth. In vitro , pharmacological or genetic inhibition of ERK5 decreased the number of viable cells in several melanoma cell lines. Moreover, xenografts performed using LV-shERK5-transduced A375 or SSM2c cells showed a reduced tumour growth when compared to those transduced with control LV-shC. We also found that oncogenic BRAF positively regulates expression, phosphorylation and nuclear localization of exogenous and endogenous ERK5. Accordingly, combined pharmacological inhibition of BRAFV600E and MEK5 is required to decrease nuclear ERK5, that is critical for the regulation of cell proliferation. Furthermore, the combination of MEK5 or ERK5 inhibitors with vemurafenib is more effective than single treatments in reducing 2D colony formation and growth of BRAFV600E melanoma cells and xenografts. Conclusion Our results identify ERK5 as a critical regulator of melanoma growth in vitro and in vivo , and point toward the possibility of targeting ERK5, alone or in combination with BRAF-MEK1/2 inhibitors, for the treatment of melanoma

Different proteolipid protein mutants exhibit unique metabolic defects

PMD (Pelizaeus–Merzbacher disease), a CNS (central nervous system) disease characterized by shortened lifespan and severe neural dysfunction, is caused by mutations of the PLP1 (X-linked myelin proteolipid protein) gene. The majority of human PLP1 mutations are caused by duplications; almost all others are caused by missense mutations. The cellular events leading to the phenotype are unknown. The same mutations in non-humans make them ideal models to study the mechanisms that cause neurological sequelae. In the present study we show that mice with Plp1 duplications (Plp1tg) have major mitochondrial deficits with a 50% reduction in ATP, a drastically reduced mitochondrial membrane potential and increased numbers of mitochondria. In contrast, the jp (jimpy) mouse with a Plp1 missense mutation exhibits normal mitochondrial function. We show that PLP in the Plp1tg mice and in Plp1-transfected cells is targeted to mitochondria. PLP has motifs permissive for insertion into mitochondria and deletions near its N-terminus prevent its co-localization to mitochondria. These novel data show that Plp1 missense mutations and duplications of the native Plp1 gene initiate uniquely different cellular responses

The multiple roles of myelin protein genes during the development of the oligodendrocyte

It has become clear that the products of several of the earliest identified myelin protein genes perform functions that extend beyond the myelin sheath. Interestingly, these myelin proteins, which comprise proteolipid protein, 2′,3′-cyclic nucleotide 3′-phosphodiesterase and the classic and golli MBPs (myelin basic proteins), play important roles during different stages of oligodendroglial development. These non-myelin-related functions are varied and include roles in the regulation of process outgrowth, migration, RNA transport, oligodendrocyte survival and ion channel modulation. However, despite the wide variety of cellular functions performed by the different myelin genes, the route by which they achieve these many functions seems to converge upon a common mechanism involving Ca2+ regulation, cytoskeletal rearrangements and signal transduction. In the present review, the newly emerging functions of these myelin proteins will be described, and these will then be discussed in the context of their contribution to oligodendroglial development