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  • Open access
  • 37 Reads
Modulation of retinoic acid receptor signaling pathway via all trans retinoic acid in Merkel cell carcinoma cells
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The biological activity of retinoic acid or all-trans retinoic acid (ATRA) is mediated by retinoic receptors, which are ligand-dependent transcription factors that activate genes crucial for cell differentiation. Dysregulations of retinoic receptor pathway lead to carcinogenesis. A strong in vitro/in vivo antitumor activity of ATRA by modulating the retinoic pathway has been proved in carcinoma of different histotypes. However, the effect of this molecule in Merkel cell carcinoma (MCC), a rare but aggressive skin neoplasm of viral origin in 80% of cases, is unknown. Herein, we investigated the antineoplastic effect of ATRA in Merkel cell polyomavirus (MCPyV)-positive/-negative MCC cells and in human fibroblasts, as control. The antineoplastic effect of ATRA was evaluated at day 3 of treatment by testing MCC cell proliferation, migration and clonogenicity. Apoptosis, cell death/cycle were evaluated via Annexin-V/propidium iodide (P.I.) and TALI assays, respectively. Apoptotic and retinoic pathways were evaluated by RT2 Profiler PCR mRNA array, that allows the analysis of pro/anti-apoptotic and retinoic pathway genes (84+84 genes), and by western blot (WB) analysis. ATRA treatment led to a strong reduction in MCC cell proliferation, migration and clonogenicity, while inducing cell cycle arrest and promoting apoptosis/death in MCC cells, with a more pronounced effect in MCPyV-positive cells. A significant overexpression of various pro-apoptotic markers in ATRA-treated MCC cells compared to untreated cells was determined by gene expression array and WB analyses. No phenotypic and molecular effects were identified in ATRA-treated fibroblast control cells. Upon ATRA treatments in MCC cells, numerous retinoic signaling genes, such as BMP2, FOXA1, MAFB, RBP4, OLIG2, UCP1 were found to be differentially expressed compared to untreated cells. Our in vitro data indicate that ATRA is effective in reducing MCC cell growth, while presenting strong pro-apoptotic effects and favoring cell cycle arrest/death via retinoic receptor pathway regulation.

  • Open access
  • 46 Reads
Androgen-receptor in skeletal muscle biopsies and cells

Aging is characterized by the loss of skeletal muscle mass, likely caused by a decrease in sex steroid hormone levels. Androgens increase, indeed, the muscle size through the androgen receptor (AR), which activates both genomic and non-genomic pathways to trigger various biological responses. Non-genomic androgen effects occur through the upstream interaction of AR with effectors or scaffolds, including the Src tyrosine kinase or filamin A. As a consequence, activation of several downstream effectors (paxillin, FAK, MAPK, Akt) follows. Such events induce cell proliferation and survival, motility and invasion as well as metabolic changes.

The activation status of signalling hubs linking the AR non-genomic circuit with cytoskeleton organization has been analyzed by Western blot of lysate proteins from human skeletal muscle biopsies (obtained by young or old females) and C2C12 skeletal muscle cells. Phosphorylation of both Ser-2152 filamin A and Tyr-118 paxillin is stronger in biopsies from old females, as compared with those observed in young females. Conversely, AR is weakly expressed in samples from old females, as compared with young females. Consistent with these findings, C2C12 cells express abundant amounts of AR that seems involved in the androgen-triggered rapid activation of several signalling effectors (e.g. MAPK, Akt, Src, FAK).

Taken together, our findings suggest that derangement of androgen/AR axis occurs in skeletal muscle of old females, thus allowing to excessive metabolic functions and loss of skeletal muscle. Further investigation in cultured cells and mouse models might help us in targeting the skeletal muscle AR axis with new compounds, such as new selective androgen receptor modulators or small drugs specifically interfering in the non genomic androgen actions, to restore the muscle functions and improve the clinical outcome of age-related frailty and sarcopenia.

  • Open access
  • 31 Reads
The nerve growth factor signaling in gender-related cancers.

The nerve growth factor (NGF) was initially identified as a promoter of neuronal survival and differentiation. As such, it captured for a long time the interest of neurobiologists. Nowadays, NGF is considered as a multifaceted molecule with pleiotropic effects in quite divergent cell types, including hormone-dependent cancer cells. Many tumors exhibit derangements of nerve growth factor and its receptors, including the tropomyosin receptor kinase A (TrkA). This receptor is frequently expressed in triple-negative breast cancers (TNBC) as well as prostate cancers (PC), although its role in pathogenesis and aggressiveness of these diseases is still under investigation.

We now report that treatment of TNBC as well as PC-derived cells with NGF triggers proliferation and survival of these cells. Simultaneously, NGF fosters cell motility and induces invasiveness in these cells by acting on the release of metalloproteases-9 (MMP-9). Somatic knockdown of TrkA or its pharmacologic inhibition by the specific inhibitor, GW441756 impair these effects. A strong reduction in TNBC or PC-derived spheroid size is observed upon GW441756 treatment.

The relevance of our studies is based on the novelty that further exploration of NGF pathway derangements in gender-related cancers will likely offer to envision innovative targets and treatment opportunities in clinical management of TNBC as well as PC patients.

  • Open access
  • 32 Reads
Effects of Electrical Stimulation on Proteins Related to Signal Transduction, c-Src and Focal Adhesion Kinase in Fibroblasts.

In the field of acupuncture and moxibustion, electrical stimulation of the skin and muscles is known to increase blood flow and metabolism locally and maintain the body in a sustained healthy state. However, little is known about the changes in cellular morphology in response to the electrical stimuli or how the localization of specific proteins is regulated by such stimulation. To gain greater understanding in this respect, the present study examined the effects of electrical stimulation on the cytoskeletal system of cultured fibroblasts. Cultured fibroblastic cells were subjected to periodic electrical stimulation for 0 (unstimulated control), 2, 5, and 20 hours. After approximately 2 hours, the stress fibers and focal adhesions in the cells had become enlarged, the stress fibers exhibiting an increase in thickness, while the cells had a contracted appearance. During 20 hours of periodic stimulation, both the stress fibers and focal adhesions gradually became larger and thicker. After the electrical stimulation, the cells exhibited increased staining of focal adhesions with anti-phosphotyrosine antibody (PY-20). They also exhibited increased staining of tyrosine-phosphorylated focal adhesion kinase (FAK) (pY397) and tyrosine-phosphorylated c-Src (pY418), indicating that the electrical stimulation had affected proteins related to signal transduction. ELISA analysis showed that 20 hours of electrical stimulation gradually increased the activity of tyrosine-phosphorylated c-Src until it was approximately tripled, whereas 5 hours of electrical stimulation approximately doubled the activity of tyrosine-phosphorylated FAK, this being the maximum reached. These results strongly suggest that electrical stimulation induces changes in the activation of c-Src and FAK signaling-related proteins and affects the formation of the cytoskeletal system.

  • Open access
  • 37 Reads
New insights on signaling pathways deregulated in LAP1-deficient cells: a proteomics study

Mutations in genes encoding nuclear envelope (NE) proteins, despite being rare, represent a major threat to cell homeostasis by compromising nuclear integrity and function as well as nucleocytoplasmic communication. In the last decade, several diseases have been associated to mutations in the TOR1AIP1 gene that codes for lamina-associated polypeptide 1 (LAP1), a NE protein ubiquitously expressed in human tissues. Although this is suggestive of an important physiological role of LAP1, it remains unclear which cellular activities are regulated by this protein. To address this, we investigated the molecular repercussions of its deficiency in patient-derived skin fibroblasts carrying a pathological LAP1 mutation (p.E482A), previously reported in a case of severe dystonia, cerebellar atrophy and cardiomyopathy. Using liquid chromatography with tandem mass spectrometry (LC–MS/MS), a quantitative proteome analysis was performed to identify up-/downregulated proteins in LAP1 E482A fibroblasts relative to age-matched control fibroblasts. A subsequent functional characterization of the LC–MS/MS-identified differentially expressed proteins using bioinformatics tools unraveled various signaling pathways/biological processes potentially deregulated in LAP1 E482A fibroblasts, such as DNA repair, neurodevelopment and myogenesis, among others. This work sheds light on dysfunctional molecular mechanisms in LAP1-deficient cells, which will contribute to a better understanding of LAP1’s physiological relevance for the maintenance of cell homeostasis and, hopefully, allow to uncover potential therapeutic targets for LAP1-associated pathologies.

  • Open access
  • 20 Reads
Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?
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Pemphigus vulgaris, a blistering skin and/or mucosa disease, is caused by autoantibodies against the desmosomal cadherins, mainly Dsg-3. Binding of IgG type antibodies to the neonatal Fc receptor (FcRn) results in antibody recycling and increases the plasma half-life of all IgGs, including pathogenic autoantibodies, contributing to disease phenotype. Recently, it has been shown that blocking FcRn can lead to a rapid decrease of pathogenic IgG and an improvement of various autoimmune diseases, including pemphigus, myasthenia gravis, and immune thrombocytopenia. Efgartigimod is an engineered Fc fragment that inhibits FcRn activity and may be useful for the treatment of IgG-mediated autoimmune diseases.

To study the pathogenic action of anti-desmoglein antibodies in vitro, mouse monoclonal anti-Dsg-3 antibodies, especially AK23, have been developed that mimic the pathogenic effect of patient sera in cultured keratinocytes. However, mouse IgG binds poorly to human FcRn. Therefore, to study the potential function of FcRn in pemphigus in human keratinocytes, we have here used chimeric AK23 anti-Dsg-3 antibodies that contain human Fc domains.

We show that these antibodies induce changes in Dsg-3 localization and result in acantholysis in a monolayer dissociation assay in hTert keratinocytes. Surprisingly, the effects on keratinocyte adhesion can be inhibited by blocking IgG binding to FcRn with efgartigimod. These data suggest that in keratinocytes, FcRn may play a further role in the pathogenesis of pemphigus, beyond its known contribution to IgG recycling

  • Open access
  • 24 Reads
Delay of Brain Development and Maturation in Pups Undergoing to Maternal Hyperhomocysteinemia
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The effect of the toxic amino acid homocysteine on both: mother and embryo causes disruption of placental blood flow and can lead to disturbances of the brain formation in offspring. The molecular and cellular mechanisms of these effects are poorly studied. The effects of maternal hyperhomocysteinemia (mHHC) on the neuronal migration, neural tissue maturation and expression of some neuronal genes were analyzed. mHHC was induced in female rats by per os administration of 0.15% aqueous methionine solution from the 4th day of pregnancy until delivery. Ultrastructure of both cortical and hippocampus tissue in mHHC pups demonstrated the developmental delay, accompanied by a retardation in motor development and body weight. The absence of synaptic glomeruli in hippocampus tissue of P20 mHHC pups suggested more essential delay in development compared to the cortical tissue. Neuronal cells labeled on E14 or E18 showed decreased number and disturbed positioning, indicating mHHC disrupted both generation and radial migration of the neuroblasts into the cortical plate. In E14 mHHC fetus brain there were decreased expression of the Kdr gene (an angiogenesis system component) and increased content of SEMA3E and the MMP-2 activity level. On E20 the level of Bdnf gene expression was increased in mHHC group. The increase in proBDNF/mBDNF ratio might affect neuronal cell viability, positioning and maturation in mHHC pups. The decrease in the level of procaspase-8 with Caspase-3 activation in the brain of E20 mHHC fetuses may indicate the development of apoptosis. It can be concluded that mHHC disturbs the mechanisms of early brain development leading to the delay in brain tissue maturation in neocortex and hippocampus of pups during the first month of their postnatal ontogenesis.

  • Open access
  • 46 Reads
Pioneering organelle structural biology: Golgi apparatus dysfunction and cascades of fatal pathways in cancer

The Golgi apparatus (GA) dysfunctions in Parkinson’s Disease (PD), neurodevelopmental disorders (NDDs), cancer, and organelle structural biology (OSB) can provide insights into therpeutic targets, gene therapy, and drug design. Primary defects and fragmentation within the GA are implicated in a wide range of neurodegenerative diseases. GA defects typically result in mislocation of proteins, accumulation of undegraded proteins, and impaired glycosylation of proteins. Inhibition of vesicular trafficking by α-synuclein (aSyn) may affect the dopamine-producing neu- rons and neuromodulators. GA regulates apoptosis during pathological mechanisms of neurological diseases and could provide new avenues in treatments through translation research. PD patients bearing the hereditary E46K disease mutation manifest the clinical picture of parkinsonism. How do we provide high resolution nanoimages of the GA during disease to capture dysfunction? Could we visualize the aSyn traffic jam between vesicles in the organelles ER and GA? OSB is emerging as a field as more technology advances and is more accessible. Structural studies of the GA will advance the field of neurological disease forward with an in depth understanding of dysfunction, fragmentation, and defects. Discoveries of the GA in PD, NDDs, and cancer would break new ground and provide translational medicine data of these diseases. Future research could be visualizing high angle annular dark field-STEM (HAADF-STEM) tomograms, cryogenic electron tomography (cryo-ET), multiplex correlative light and electron microscopy (cryo-CLEM), nanobody-assisted tissue immunostaining for volumetric EM (NATIVE) and using soft X-ray tomography (SXT) and computational reconstruction of the GA.

  • Open access
  • 45 Reads
Roles of PPARα in Liver Health and Diseases
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Nuclear receptors (NRs) are ligand-dependent transcription factors. Their activation modulates the expression of genes controlling vital processes including development, metabolism and reproduction. Among them, the peroxisome proliferator-activated receptors (PPARs) are activated by fatty acids and their derivatives. Multiples roles of the PPARα isotype in liver will be discussed. In the mouse, PPARα controls genes required for lipid catabolism already before birth. We identified an endocrine developmental axis in which fetal glucocorticoid receptor primes the activity of PPARα in anticipation of the sudden shift to milk as postnatal nutrient source from which energy can be efficiently extracted. PPARα plays a pivotal role in the management of energy stores during fasting by orchestrating the genomic and metabolic responses required for homeostasis under this energy stress condition. Among the many regulated pathways, a major one is the biosynthesis of ketone bodies. PPARα is also required together with the carbohydrate-sensitive transcription factor carbohydrate-responsive element-binding protein (ChREBP) to balance the fibroblast growth factor 21 (FGF21) glucose response. Recently, we reported that hepatocyte PPARα activity is involved in the cross-talk between adipose tissues and the liver during fat mobilization. Ketone body and FGF21 production, two PPARα-dependent responses, is impaired upon fasting in a genetically-induced absence of adipose triglyceride lipase (ATGL) in adipocytes. Interestingly, liver gene expression analyses unveiled a set of fasting-induced genes sensitive to both ATGL deletion in adipocytes and PPARα deletion in hepatocytes. The PPARα-dependent responses in the liver also affect brown adipose tissue (BAT) activity. Liver PPARα is protective against NAFLD as shown by hepatocyte-specific PPARα deficiency in different models of steatosis and during ageing. In diet-induced mouse models of NAFLD, PPARα emerged as a sexually dimorphic transcription factor. Further in vivo experiments demonstrated that hepatocyte PPARα also determines a sex-specific response to fasting thereby identifying PPARα as a potential sexually dimorphic drug target. Similarly, liver molecular signatures in humans also showed sexually dimorphic gene expression profiles with a sex-specific co-expression network for PPARα. In conclusion, the multifaceted roles of PPARα offer an attractive field for the future development of ligands with numerous potential clinical applications.

  • Open access
  • 34 Reads
Blood flow diverts extracellular vesicles from endothelial degradative compartments to promote angiogenesis

Extracellular vesicles released by tumors (tEVs) disseminate via circulatory networks and promote microenvironmental changes in distant organs favoring metastatic seeding. Despite their abundance in the bloodstream, how hemodynamics affect the function of circulating tEVs remains unsolved. We experimentally tuned flow profiles in vitro (microfluidics) and in vivo (zebrafish) and demonstrated that efficient uptake of tEVs occurs in endothelial cells subjected to capillary-like hemodynamics. Such flow profiles partially reroute internalized tEVs towards non-acidic and non-degradative Rab14-positive endosomes, at the expense of lysosomes, suggesting that endothelial mechanosensing diverts tEVs from degradation. Subsequently, tEVs promote the expression of pro-angiogenic transcription factors in flow-stimulated endothelial cells and favor vessel sprouting in zebrafish. Altogether, we demonstrate that capillary-like flow profiles potentiate the pro-tumoral function of circulating tEVs by promoting their uptake and rerouting their trafficking. We propose that tEVs contribute to premetastatic niche formation by exploiting endothelial mechanosensing in specific vascular regions with permissive hemodynamics.