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Transforming growth factor-beta1 (TGF-beta1) is crucially involved in the fibrotic events characterizing interstitial lung diseases (ILDs), as well as in the airway remodeling process typical of asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal and fibrotic human lung fibroblasts (HLFs), the effects of TGF-beta1 on mitogen-activated protein kinase (MAPK) phosphorylation, cell proliferation, and production of interleukins 6 (IL-6) and 11 (IL-11), in the presence or absence of a pretreatment with budesonide (BUD). MAPK phosphorylation was detected by Western blotting, cell viability and proliferation were evaluated using Trypan blue staining and [(3)H]-thymidine incorporation assay, respectively, and the release of IL-6 and IL-11 into cell culture supernatants was assessed by ELISA. TGF-beta1 (10 ng/ml) significantly stimulated MAPK phosphorylation (P < 0.01), and also enhanced cell proliferation as well as the secretion of both IL-6 and IL-11, which reached the highest increases at the 72nd h of cell exposure to this growth factor. All such effects were prevented by BUD (10(-8) M) and, with the exception of IL-6 release, also by a mixture of MAPK inhibitors. Therefore, our findings suggest that the fibrotic action exerted by TGF-beta1 in the lung is mediated at least in part by MAPK activation and by an increased synthesis of the profibrogenic cytokines IL-6 and IL-11; all these effects appear to be prevented by corticosteroids via inhibition of MAPK phosphorylation.

The redox state of the cell is involved in the regulation of many physiological functions as well as in the pathogenesis of several diseases, and is strictly dependent on the amount of iron in its catalytically active state. Alterations of iron homeostasis determine increased steady-state concentrations of Reactive Oxygen Species (ROS) that cause lipid peroxidation, DNA damage and altered protein folding. Ferritin keeps the intracellular iron in a non-toxic and readily available form and consequently plays a central role in iron and redox homeostasis. The protein is composed by 24 subunits of the H- and L-type, coded by two different genes, with structural and functional differences. The aim of this study was to shed light on the role of the single H ferritin subunit (FHC) in keeping the native correct protein three-dimensional structure. To this, we performed Raman spectroscopy on protein extracts from K562 cells subjected to FHC silencing. The results show a significant increase in the percentage of disordered structures content at a level comparable to that induced by H2O2 treatment in control cells. ROS inhibitor and iron chelator were able to revert protein misfolding. This integrated approach, involving Raman spectroscopy and targeted-gene silencing, indicates that an imbalance of the heavy-to-light chain ratio in the ferritin composition is able to induce severe but still reversible modifications in protein folding and uncovers new potential pathogenetic mechanisms associated to intracellular iron perturbation.

Background Tuberculin skin test based on in vivo intradermal inoculation of purified protein derivative from Mycobacterium bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). Methods Proteomic analysis was performed on different bPPD preparations from M. bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC–MS/MS analysis. Mass spectrometry analysis was performed on a Q-exactive hybrid quadrupole-Orbitrap mass spectrometer coupled online to an Easy nano-LC1000 system. Results Three hundred and fifty-six proteins were identified and quantified by at least 2 peptides (99% confidence per peptide). One hundred and ninety-eight proteins, which had not been previously described, were detected; furthermore, the proteomic profile shared 80 proteins with previous proteomes from bPPDs from the United Kingdom and Brazil and 139 protein components from bPPD from Korea. Locus name of M. bovis (Mb) with orthologs from M. tuberculosis H37Rv, comparative gene and protein length, molecular mass, functional categories, gene name and function of each protein were reported. Ninety-two T cell mycobacterial antigens responsible for delayed-type hypersensitivity were detected, fifty-two of which were not previously reported in any bPPD proteome. Data are available via ProteomeXchange with identifier PXD005920. Conclusions This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. Indeed, many antigens contained within bPPD are currently responsible for the cross-reactivity resulting in false-positive results as they are shared between non-tuberculous and tuberculous mycobacteria. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1172-1) contains supplementary material, which is available to authorized users.

The EGFR (epidermal growth factor receptor) a member of the family of transmembrane protein kinase receptors known as the erbB family shows a significant correlation with the presence of metastases and poorly differentiated oral cancer. Aim of the present work is to define the key-role of EGFR in oral cancer prognosis. We have analyzed the EGFR expression on 149 cases of oral squamous cell cancers (OSCC) and we have found that it was poorly expressed in normal oral epithelium, but its expression was significantly increased in OSCCs. Moreover, we have recorded that both pEGFR-Tyr 845 and pEGFR-Tyr 1068 were mainly distributed in high histological grading and in advanced stages. Western blotting has confirmed the total absence of EGFR phosphorylation in normal oral epithelium and the higher level of protein phosphorylation in representative cases of OSCCs. The EGF-R amplification was found by fluorescence in situ hybridization (FISH) in 14% of OSCC; interestingly, EGF-R amplification was mainly observed in OSCC with higher histological grading (G2 and G3) and advanced stage (pT4) sub-groups. Kaplan-Meyer survival analysis suggested that patients with positive pEGFR-Tyr 845 tumors had a worse prognosis and were bad responders to chemotherapy. These results confirm the central role of EGF-R activation status as a prognostic biomarker in OSCC The EGFR (epidermal growth factor receptor) a member of the family of transmembrane protein kinase receptors known as the erbB family shows a significant correlation with the presence of metastases and poorly differentiated oral cancer. Aim of the present work is to define the key-role of EGFR in oral cancer prognosis. We have analyzed the EGFR expression on 149 cases of oral squamous cell cancers (OSCC) and we have found that it was poorly expressed in normal oral epithelium, but its expression was significantly increased in OSCCs. Moreover, we have recorded that both pEGFR-Tyr 845 and pEGFR-Tyr 1068 were mainly distributed in high histological grading and in advanced stages. Western blotting has confirmed the total absence of EGFR phosphorylation in normal oral epithelium and the higher level of protein phosphorylation in representative cases of OSCCs. The EGF-R amplification was found by fluorescence in situ hybridization (FISH) in 14% of OSCC; interestingly, EGF-R amplification was mainly observed in OSCC with higher histological grading (G2 and G3) and advanced stage (pT4) sub-groups. Kaplan-Meyer survival analysis suggested that patients with positive pEGFR-Tyr 845 tumors had a worse prognosis and were bad responders to chemotherapy. These results confirm the central role of EGF-R activation status as a prognostic biomarker in OSCC. © 2012 Landes Bioscience.

Objectives The aim of this study is to evaluate the potential impact of mutations in the promoter region of the L ferritin gene on its transcriptional activity. Design and methods To search for the presence of mutations in the promoter of the L gene, we amplified by PCR a DNA region of about 385 n.t. in 100 healthy subjects from Southern Italy. Results A subject carrying a C to A transition in position − 216 was identified. This transition causes an increased transcriptional activity in vitro. This finding was substantiated by Real Time Quantitative PCR, which showed increased levels of L ferritin mRNA. Conclusions A previously unidentified mutation in the promoter region of the L ferritin gene was detected in an individual. Interestingly, this subject is affected by bilateral cataract, a disease that has been correlated, in a subset of patients, with high levels of circulating ferritin. We hypothesize that changes in the expression of the L ferritin might be linked, at least to a certain extent, to the pathogenesis of this rare eye disease.

In our body, cells are continuously exposed to physical forces that can regulate different cell functions such as cell proliferation, differentiation and death. In this work, we employed two different strategies to mechanically stress cancer cells. The cancer and healthy cell populations were treated either with mechanical stress delivered by a micropump (fabricated by deep X-ray nanolithography) or by ultrasound wave stimuli. A specific down-regulation of Major Histocompatibility Complex (MHC) class I molecules expression on cancer cell membrane compared to different kinds of healthy cells (fibroblasts, macrophages, dendritic and lymphocyte cells) was observed, stimulating the cells with forces in the range of nano-newton, and pressures between 1 and 10 bar (1 bar5100.000 Pascal), depending on the devices used. Moreover, Raman spectroscopy analysis, after mechanical treatment, in the range between 700–1800 cm-1, indicated a relative concentration variation of MHC class I. PCA analysis was also performed to distinguish control and stressed cells within different cell lines. These mechanical induced phenotypic changes increase the tumor immunogenicity, as revealed by the related increased susceptibility to Natural Killer (NK) cells cytotoxic recognition.

Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells. In this study we sought to define the repertoire of genes whose expression might be affected by FHC during the hemin-induced differentiation of the erythromyeloid cell line K562. To this aim, gene expression profiling was performed in four different sets of cells: i) wild type K562; ii) sh-RNA FHC-silenced K562; iii) hemin-treated wild-type K562; and iv) hemin-treated FHC-silenced K562. Statistical analysis of the gene expression data, performed by two-factor ANOVA, identified three distinct classes of transcripts: a) Class 1, including 657 mRNAs whose expression is modified exclusively during hemin-induced differentiation of K562 cells, independently from the FHC relative amounts; b) Class 2, containing a set of 70 mRNAs which are consistently modified by hemin and FHC-silencing; and c) Class 3, including 128 transcripts modified by FHC-silencing but not by hemin. Our data indicate that FHC may function as a modulator of gene expression during erythroid differentiation and add new findings to the knowledge of the complex gene network modulated during erythroid differentiation.

Brugada Syndrome (BS) is a polygenic inherited cardiac disease characterized by life-threatening arrhythmias and high incidence of sudden death. In this study, two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (LC-MS/MS) was used to investigate specific changes in the plasma proteome of BS patients and family members sharing the same gene mutation (SCN5AQ1118X), with the aim to identify novel disease biomarkers. Our data demonstrate that the levels of several proteins were significantly altered in BS patients compared with controls. In particular, apolipoprotein E, prothrombin, vitronectin, complement-factor H, vitamin-D-binding protein, voltage-dependent anion-selective channel protein 3 and clusterin were considerably increased in plasma sample of BS patients, whereas alpha-1-antitrypsin, fibrinogen and angiotensinogen were considerably decreased; moreover, post-translational modifications of antithrombin-III were detected in all affected individuals. On the light of these results, we hypothesize that these proteins might be considered as potential markers for the identification of disease status in BS.

Cancer is currently considered as the end point of numerous genomic and epigenomic mutations and as the result of the interaction of transformed cells within the stromal microenvironment. The present work focuses on breast cancer, one of the most common malignancies affecting the female population in industrialized countries. In this study, we perform a proteomic analysis of bioptic samples from human breast cancer, namely, interstitial fluids and primary cells, normal vs disease tissues, using tandem mass tags (TmT) quantitative mass spectrometry combined with the MudPIT technique. To the best of our knowledge, this work, with over 1700 proteins identified, represents the most comprehensive characterization of the breast cancer interstitial fluid proteome to date. Network analysis was used to identify functionally active networks in the breast cancer associated samples. From the list of differentially expressed genes, we have retrieved the associated functional interaction networks. Many different signaling pathways were found activated, strongly linked to invasion, metastasis development, proliferation, and with a significant cross-talking rate. This pilot study presents evidence that the proposed quantitative proteomic approach can be applied to discriminate between normal and tumoral samples and for the discovery of yet unknown carcinogenesis mechanisms and therapeutic strategies.

BackgroundCalpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle.Methods and findingsHere we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca(2+)-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting.ConclusionThe role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role. The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53–NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis. These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation.

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ETA or ETB selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ETA antagonist BQ-123, but not by the specific ETB antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ETA receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation. © 2005 Wiley-Liss, Inc.

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes which play a key role in signal transduction mediated by cytokines, growth factors, neurotransmitters and various types of environmental stresses. In the airways, these extracellular stimuli elicit complex inflammatory and structural changes leading to the typical features of asthma including T cell activation, eosinophil and mast cell infiltration, as well as bronchial hyperresponsiveness and airway remodelling. Because MAPKs represent an important point of convergence for several different signalling pathways, they affect multiple aspects of normal airway function and also significantly contribute to asthma pathophysiology. Therefore, this review focuses on the crucial involvement of MAPKs in asthma pathogenesis, thus also discussing their emerging role as molecular targets for anti-asthma drugs.

Airway epithelial cells play a central role in the inflammatory, apoptotic, and remodeling processes associated with asthma. Within this context, a key function is exerted by transforming growth factor-beta (TGF-beta), whose biological effects are mediated at least in part by mitogen-activated protein kinases (MAPKs). The aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the effects of TGF-beta (10 ng/ml) on both MAPK activation and apoptosis, in the presence or absence of a pretreatment with budesonide (10-8 M). MAPK activation was detected by Western blotting, using anti-phospho-MAPK monoclonal antibodies, which specifically recognize the phosphorylated, active forms of these enzymes. Apoptosis was assayed by caspase-3 activation and fluorescence microscopy, using annexin-V (An-V) and propidium iodide (PI) as markers of cell death. Our results show that TGF-beta induced a marked ( reverse similar 9-fold) increase in p38 MAPK phosphorylation, and also dramatically enhanced cell death, which was completely prevented by specific MAPK inhibitors. Both MAPK activation and apoptosis were effectively inhibited by budesonide (BUD), thereby suggesting that the powerful antiapoptotic action of inhaled glucocorticoids may be very important for their protective role against epithelial injury, which represents a key pathogenic event in asthma.

Background — Reactive oxygen species play a critical role in inducing apoptosis. The small GTPase p21 Ras and the ERK1/2 MAPK have been proposed as key regulators of the signaling cascade triggered by oxidative stress (H 2 O 2 ). Harvey-Ras (Ha-Ras) and Kirsten-Ras (Ki-Ras) isoforms are so far functionally indistinguishable, because they activate the same downstream effectors, including ERK1/2. Moreover, ERK1/2 signaling has been involved in both protection and induction of apoptosis. Methods and Results — Human umbilical vein endothelial cells (HUVECs) were subjected to H 2 O 2 , and apoptosis was detected by fluorescence-activated cell sorting analysis, fluorescence microscopy, and caspase-3 activation. Transfection of Ha- Ras and Ki- Ras genes in HUVECs was performed to evaluate the response to H 2 O 2 . We have found that, whereas Ha- Ras decreases tolerance to oxidative stress, Ki- Ras has a potent antiapoptotic activity. Both effects are mediated by ERK1/2. Tolerance to H 2 O 2 is encoded by a unique stretch of lysines at the COOH terminus of the Ki-Ras, lacking in Ha-Ras, and it is relatively independent of the farnesylated anchor. Inhibition of p21 Ras signaling by farnesylation inhibitors increased the resistance to apoptosis in Ha-Ras–expressing cells. Conclusions — These findings explain the opposite effects of ERK1/2 stimulation on apoptosis found in different cell types and suggest that local activation of ERK1/2 signaling may account for the opposing response to oxidative stress by Ha-Ras or Ki-Ras–expressing cells. Modulation of cell reactivity to oxidative stress by p21 Ras points to the specific and predictive effects of Ras inhibitors in vivo as potential therapeutic drugs in disorders produced by increase of reactive oxygen species inside the cells.

AIM—To investigate in vivo the intermediate cytoskeletal filaments desmin and vimentin in myocardial tissues from patients with dilated cardiomyopathy, and to determine whether alterations in these proteins are associated with impaired contractility. METHODS—Endomyocardial biopsies were performed in 12 patients with dilated cardiomyopathy and in 12 controls (six women with breast cancer before anthracycline chemotherapy and six male donors for heart transplantation). Biopsy specimens were analysed by light microscopy and immunochemistry (desmin, vimentin). Myocyte contractile protein function was evaluated by the actin-myosin in vitro motility assay. Left ventricular ejection fraction was assessed by echocardiography and radionuclide ventriculography. RESULTS—Patients with dilated cardiomyopathy had a greater cardiomyocyte diameter than controls (p

Friedreich Ataxia (FA) is a neurodegenerative disorder characterised by progressive gait disturbance, dysarthria, dysmetria and other coordination disorders. The genetic defect is represented by an expansion of GAA repeats in the frataxin gene (FRDA or X25). Hypertrophic cardiomyopathy is a common finding in FA, and it is widely recognised as specific for the diagnosis of disease status. In this study, we report the co-existence, in a 5-year old boy with FA, of a double mutation in two distinct genes [X25 (A allele: 850 triplets; B allele: 1000 triplets), and cardiac troponin T (TNNT2) (287G>A)]. TNNT2 gene mutations have been previously identified in individuals with a familial form of hypertrophic cardiomyopathy (FHC), an autosomal dominant inherited disease characterised by unexplained cardiac hypertrophy and high incidence of sudden death. Although we cannot rule out the impact of each gene defect on cardiac morphology, it is of interest that these two mechanisms may be acting in a synergistic fashion to produce the extreme degree of cardiac hypertrophy detected in the child. This is, to our knowledge, the first description of a double gene defect in individuals with FA and FHC.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1–2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

The pathogenic mechanisms underlying cardiovascular diseases involve significant alterations in myocardial gene and protein expression. Proteomics analysis can define new protein and peptide changes associated with cardiac pathology, including myocardial infarction. The aim of the present study was to analyze serum proteome of patients with ST-Elevation myocardial infarction (STEMI). Serum samples were collected from STEMI patients (age 65.0+/-10.3) at 5.3+/-2.7 hours after the onset of typical chest pain and before initiating standard therapy. Ten age- and sex-matched donors were used as controls. The samples were albumin- and IgG-depleted. Isotope-coded affinity tag method was employed to label cysteine residues and liquid chromatography-Tandem Mass Spectrometry analysis was performed to measure the labeled proteins. Our proteomic approach identified increased levels of vitamin D-binding protein precursor (VDB) in the serum of STEMI patients when compared to control donors. Western blot analysis confirmed the increase in VDB protein in STEMI patients. Moreover, fresh thrombotic plaques, obtained during primary angioplasty, showed high expression of VDB protein. Mechanistically, VDB protein reduces platelet aggregation and prolongs coagulation time ex vivo.