Nuclear localization and new isoforms detection give new insights on Hsp10 functions in normal and cigarette smoke-stressed lung cells

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular dis- tribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is a potent stressor for the respiratory system, but its effects on the expression, function, and cellular locali- zation of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and charac- terized the effects of non-lethal doses of CS extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial cells (16HBE). We applied various in vitro methods: IHC, subcellular fractionation analyses (SFA), western blotting (WB), ICC, transmission electron microscopy (TEM) immunogold, chromati protein extracts (CPE), as well as 2D-gel based proteomics analyses. Bioinformatics was used to gather structural in silico data. IHC showed that Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers. ICC, SFA, and WB showed that 16HBE and HFL-1 cells featured nuclear Hsp10, before and after CSE exposure; TEM immunogold further confirmed this observation. Proteomics data showed that CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts. Bioinformatics analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals. Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein as suggested by CPE/gel retardation experiments. Data reported here indicate that in human cells of the respiratory mucosa there are at least three different intracellular locales for Hsp10: mitochondrial, nuclear, and cyto- solic. Further experiments are en route for the definition of the mechanisms underlying the transfer of Hsp10 to the nucleus and other cellular/extracellular compartments. This work was supported by grants from University of Palermo (FFR 2012) to GLR

Cigarette Smoke Condensate Exposure Changes RNA Content of Extracellular Vesicles Released from Small Airway Epithelial Cells

Exposure to environmental tobacco smoke (ETS) is a known risk factor for the development of chronic lung diseases, cancer, and the exacerbation of viral infections. Extracellular vesicles (EVs) have been identified as novel mediators of cell–cell communication through the release of biological content. Few studies have investigated the composition/function of EVs derived from human airway epithelial cells (AECs) exposed to cigarette smoke condensate (CSC), as surrogates for ETS. Using novel high-throughput technologies, we identified a diverse range of small noncoding RNAs (sncRNAs), including microRNA (miRNAs), Piwi-interacting RNA (piRNAs), and transfer RNA (tRNAs) in EVs from control and CSC-treated SAE cells. CSC treatment resulted in significant changes in the EV content of miRNAs. A total of 289 miRNAs were identified, with five being significantly upregulated and three downregulated in CSC EVs. A total of 62 piRNAs were also detected in our EV preparations, with five significantly downregulated and two upregulated in CSC EVs. We used TargetScan and Gene Ontology (GO) analysis to predict the biological targets of hsa-miR-3913-5p, the most represented miRNA in CSC EVs. Understanding fingerprint molecules in EVs will increase our knowledge of the relationship between ETS exposure and lung disease, and might identify potential molecular targets for future treatments

Role of Hydrogen Sulfide in NRF2- and Sirtuin-Dependent Maintenance of Cellular Redox Balance

Hydrogen sulfide (H2S) has arisen as a critical gasotransmitter signaling molecule modulating cellular biological events related to health and diseases in heart, brain, liver, vascular systems and immune response. Three enzymes mediate the endogenous production of H2S: cystathione β-synthase (CBS), cystathione γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). CBS and CSE localizations are organ-specific. 3-MST is a mitochondrial and cytosolic enzyme. The generation of H2S is firmly regulated by these enzymes under normal physiological conditions. Recent studies have highlighted the role of H2S in cellular redox homeostasis, as it displays significant antioxidant properties. H2S exerts antioxidant effects through several mechanisms, such as quenching reactive oxygen species (ROS) and reactive nitrogen species (RNS), by modulating cellular levels of glutathione (GSH) and thioredoxin (Trx-1) or increasing expression of antioxidant enzymes (AOE), by activating the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2). H2S also influences the activity of the histone deacetylase protein family of sirtuins, which plays an important role in inhibiting oxidative stress in cardiomyocytes and during the aging process by modulating AOE gene expression. This review focuses on the role of H2S in NRF2 and sirtuin signaling pathways as they are related to cellular redox homeostasis

Transformation of primary human hepatocytes in hepatocellular carcinoma.

The present study investigated primary human hepatocyte cultures obtained from liver specimens of patients affected by cirrhosis and HCC, their proliferation and transformation. The degree of invasiveness of cells acquiring neoplastic characteristics was studied with a transwell migration assay. With the present study, we are the first to have identified and describe the existence of human hepatocytes near the cancerous lesion that can transform in HCC in vitro

Umbilical cord revisited: from Wharton’s jelly myofibroblasts to mesenchymal stem cells

The umbilical cord (UC) is an essential part of the placenta, contributing to foetal development by ensuring the blood flow between mother and foetus. The UC is formed within the first weeks of gestation by the enclosure of the vessels (one vein and two arteries) into a bulk of mucous connective tissue, named Wharton’s jelly (WJ) and lined by the umbilical epithelium. Since their first identification, cells populating WJ were described as unusual fibroblasts (or myofibroblasts). Recent literature data further highlighted the functional interconnection between UC and the resident cells. The UC represents a reservoir of progenitor populations which are collectively grouped into MSCs (mesenchymal stem cells). Such cells have been sourced from each component of the cord, namely the subamnion layer, the WJ, the perivascular region, and the vessels. These cells mainly show adherence to the phenotype of adult MSCs (as bone marrow-derived ones) and can differentiate towards mature cell types belonging to all the three germ layers. In addition, cells from human UC are derived from an immunoprivileged organ, namely the placenta: in fact, its development and function depend on the elusion of the maternal immune response towards the semi-allogeneic embryo. This is reflected in the expression of immunomodulatory molecules by UC-derived MSCs. The present paper describes UC structural features and the cell types which can be derived, with a focus on their phenotype and the novel results which boosted the use of UC-derived cells for regenerative medicine applications