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In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation.

Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re-ChIP, and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor-β1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.

Cyclooxygenase-2 (COX-2), with its main antifibrotic metabolite PGE2, is regarded as an antifibrotic gene. Repressed COX-2 expression and deficient PGE2 have been shown to contribute to the activation of lung fibroblasts and excessive deposition of collagen in pulmonary fibrosis. We have previously demonstrated that COX-2 expression in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) is epigenetically silenced and can be restored by epigenetic inhibitors. This study aimed to investigate whether COX-2 downregulation induced by the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in normal lung fibroblasts could be prevented by epigenetic inhibitors. We found that COX-2 protein expression and PGE2 production were markedly reduced by TGF-β1 and this was prevented by the pan-histone deacetylase inhibitor suberanilohydroxamic acid (SAHA) and to a lesser extent by the DNA demethylating agent Decitabine (DAC), but not by the G9a histone methyltransferase (HMT) inhibitor BIX01294 or the EZH2 HMT inhibitor 3-deazaneplanocin A (DZNep). However, chromatin immunoprecipitation assay revealed that the effect of SAHA was unlikely mediated by histone modifications. Instead 3′-untranslated region (3′-UTR) luciferase reporter assay indicated the involvement of post-transcriptional mechanisms. This was supported by the downregulation by SAHA of the 3′-UTR mRNA binding protein TIA-1 (T-cell intracellular antigen-1), a negative regulator of COX-2 translation. Furthermore, TIA-1 knockdown by siRNA mimicked the effect of SAHA on COX-2 expression. These findings suggest SAHA can prevent TGF-β1-induced COX-2 repression in lung fibroblasts post-transcriptionally through a novel TIA-1-dependent mechanism and provide new insights into the mechanisms underlying its potential antifibrotic activity. Abbreviations Unlabelled Table SAHA suberanilohydroxamic acid TGF-β1 transforming growth factor-β1 COX-2 cyclooxygenase-2 TIA-1 T-cell intracellular antigen-1 PGE2 prostaglandin E2 IPF idiopathic pulmonary fibrosis DAC Decitabine HMT histone methyltransferase EZH2 enhancer of zeste homolog 2 DZNep 3-deazaneplanocin A 3′-UTR 3′-untranslated region α-SMA α-smooth muscle actin ECM extracellular matrix COL1 collagen 1 DNMT DNA methyltransferase HAT histone acetyltransferase HDAC histone deacetylase H3K9me3 histone H3 lysine 9 trimethylation ARE AUUUA-rich element HuR human antigen R ELAV1 ELAV-like RNA binding protein 1 TTP Tristetraprolin CUGBP2 CUG triplet repeat, RNA binding protein 2 F-NL fibroblast from non-fibrotic lung FCS fetal calf serum, Highlights • The HDAC inhibitor SAHA upregulates the expression of the antifibrotic gene COX-2 post-transcriptionally. • The mechanism relies on the downregulation of TIA-1, a negative regulator of COX-2 translation. • SAHA has a therapeutic potential by preventing COX-2 repression induced by TGF-β1 in human lung fibroblasts.

Dynamic culture protocols have recently emerged as part of (bone) tissue engineering strategies due to their ability to represent a more physiological cell environment in vitro. Here, we described how a perfusion flow induced by a simple bioreactor system improves proliferation and osteogenic commitment of human bone marrow stromal cells. L88/5 cells were cultured in poly(methyl methacrylate) custom-milled communicating well plates, in the presence of an osteogenic cocktail containing 1α,25-dihydroxyvitamin D3, L-ascorbic acid 2-phosphate, andβ-glycerophosphate. The dynamic cell culture was maintained under perfusion flow stimulation at 1 mL/min for up to 4 days and compared with a static control condition. A cell viability assay showed that the proliferation associated with the dynamic cell culture was 20% higher vs. the static condition. A significantly higher upregulation of the osteogenic markers runt-related transcription factor 2 (RUNX2), collagen type I (COL1A1), osteocalcin (BGLAP), alkaline phosphatase (ALPL), and osteopontin (SPP1) was detected when the perfusion flow stimulation was administered to the cells treated with the osteogenic cocktail. An in silico analysis showed that in the dynamic cell culture condition (i) the shear stress in the proximity of the cell layer approximates 10-3 Pa, (ii) the nutrient and the waste product concentration is more homogeneously distributed than in the static counterpart, and (iii) perfusion flow was associated with higher nutrient consumption. In summary, increased cell proliferation and enhanced early phenotype commitment indicate that dynamic cell culture conditions, delivered via bioreactor systems, produce an enhanced in vitro environment for both basic and translational research in tissue engineering and regenerative medicine.

COPD is a common chronic inflammatory lung disease with cigarette smoke (CS) as the major cause. The receptor for advanced glycation end products (RAGE) and its ligands are critically involved in inflammation. Inhaled corticosteroids are poorly effective in improving the symptoms of COPD. This underlines the need for new anti-inflammatory therapies. Cordycepin is a purine nucleoside antimetabolite. Simvastatin is a lipid-lowering agent. But the effect of Cordycepin and Simvastatin on CSE-induced inflammatory gene expression in human bronchial epithelial cells has not been explored. Human BEAS-2B bronchial epithelial cells were treated with Cordycepin + CSE (2.5%) and Simvastatin + CSE (2.5%). IL-8 andIL-6 production was measured by ELISA. RAGE and RAGE ligand HMBG-1 expression was analysed by Western blot. We found that CSE-induced IL-8 production was inhibited by Cordycepin and Simvastatin but IL-6 production was enhanced by Cordycepin and not affected by Simvastatin. CSE and both drugs had no effect on basal RAGE and HMGB-1 expression. Since IL-6 contribute to the pathogenesis of COPD by modulating pulmonary function but not necessarily inflammation and neutrophil-driven airway inflammation (by IL-8 and other chemokines) is critically involved in the pathogenesis of COPD, our findings suggest cigarette smoke, by inducing inflammatory responses from human bronchial epithelial cells, may play an important role in airway inflammation in COPD and that Cordycepin and Simvastatin, by reducing IL-8 production (unlikely via RAGE inhibition), may have therapeutic potential in COPD.

Background Bivalent chromatin refers to overlapping regions containing activating histone H3 Lys4 trimethylation (H3K4me3) and inactivating H3K27me3 marks. Existence of such bivalent marks on the same nucleosome has only recently been suggested. Previous genome-wide efforts to characterize bivalent chromatin have focused primarily on individual marks to define overlapping zones of bivalency rather than mapping positions of truly bivalent mononucleosomes. Results Here, we developed an efficacious sequential ChIP technique for examining global positioning of individual bivalent nucleosomes. Using next generation sequencing approaches we show that although individual H3K4me3 and H3K27me3 marks overlap in broad zones, bivalent nucleosomes are focally enriched in the vicinity of the transcription start site (TSS). These seem to occupy the H2A.Z nucleosome positions previously described as salt-labile nucleosomes, and are correlated with low gene expression. Although the enrichment profiles of bivalent nucleosomes show a clear dependency on CpG island content, they demonstrate a stark anti-correlation with methylation status. Conclusions We show that regional overlap of H3K4me3 and H3K27me3 chromatin tend to be upstream to the TSS, while bivalent nucleosomes with both marks are mainly promoter proximal near the TSS of CpG island-containing genes with poised/low expression. We discuss the implications of the focal enrichment of bivalent nucleosomes around the TSS on the poised chromatin state of promoters in stem cells. Electronic supplementary material The online version of this article (doi:10.1186/s12920-016-0221-6) contains supplementary material, which is available to authorized users.

Lung cancer is the leading cause of cancer mortality worldwide and despite efforts made to improve clinical results, continuing poor survival rates indicate that novel therapeutic approaches are needed. Valproic acid (VPA), a short-chain branched fatty acid used mainly for the treatment of epilepsy and bipolar disorder, has been shown to inhibit class I histone deacetylases (HDAC-I), a group of enzymes involved in chromatin remodeling and which are thought to play a role in tumor development. Although evidence of VPA's therapeutic efficacy has also been observed in patients with solid tumors, the very high concentration required to induce antitumor activity limits its clinical usefulness. We used a panel of NSCLC cell lines to evaluate the activity and mechanisms of action of organosulfur valproic acid derivatives, a promising new class of compounds designed to improve the safety and efficacy of the valproic acid molecule and created by coupling it with a hydrogen sulfide (H(2) S)-releasing moiety. Our results highlighted the increased cytotoxic activity of the novel organosulfur derivatives, ACS33 and ACS2, with respect to VPA, starting from low concentrations. In particular, ACS2 exhibited important pro-apoptotic activity triggered by the mitochondrial pathway and also showed anti-invasion potential. Furthermore, our in vitro results identified a highly effective combination schedule of ACS2 and cisplatin capable of inducing a synergistic interaction even when the two drugs were used at low concentrations, which could prove a valid alternative to traditional chemotherapeutic regimens used for advanced lung cancer. Further studies are needed to confirm these preliminary findings.

Introduction and objectives: Idiopathic Pulmonary Fibrosis (IFP) is a fatal interstitial lung disease with unknown aetiology. Lung myofibroblasts (activated fibrobalsts) are the major effector cells in the pathogenesis of IPF. Transforming growth factor-β (TGF-β1) is a potent activator of fibroblasts. Lack of effective treatment options necessitates novel therapeutic approaches. Epigenetic drugs, by inhibiting chromatin modifying enzymes involved in gene expression control, represent promising agents capable of modulating the cellular phenotype. We previously demonstrated that the cyclooxygenase-2 (COX-2) gene is epigenetically silenced in lung fibroblasts from IPF patients (F-IPF)1 and epigenetic inhibitors and restore COX-2 expression. However, whether epigenetic inhibitors can alter fibroblast phenotype remains unknown. This study aimed to investigate the effect of four different epigenetic enzyme inhibitors on fibroblast phenotype change in IPF. Methods: F-IPF and fibroblasts from non-fibrotic lung (F-NL) treated with TGF-β1 were cultured to test the effects of the epigenetic inhibitors BIX01294 (BIX, G9a histone methyltransferase inhibitor), 3-deazaneplanocin A (DZNep, EZH2 histone methyltransferase inhibitor), SAHA (histone deacetylases inhibitor) and Decitabine (DAC, DNA demethylating agent), in comparison with the COX-2 products prostaglandin E2 (PGE2). The expression of COX-2 and myofibroblast markers collagen 1 (COL1) and α-smooth muscle actin (α-SMA) was assessed. The COX-2 DNA promoter methylation level was analysed by bisulfite sequencing. Results: TGF-β1 induced a myofibroblast phenotype in F-NL characterised by COL1 and α-SMA upregulation and COX-2 downregulation, similar to F-IPF. PGE2 and SAHA were able to maintain/restore COX-2 expression in TGF-β1-induced myofibroblasts and F-IPF. DAC demonstrated similar effect in TGF-β1 treated F-NL only. SAHA also reduced COL1 and α-SMA expression. But DZNep and BIX showed no effect. No differences in the COX-2 promoter methylation was detected between F-NL and F-IPF. Conclusions: Among the epigenetic inhibitors tested, SAHA shows a promising antifibrotic effect by inhibiting fibroblast activation and the underlying molecular mechanisms are currently under investigation.

Introduction and Objectives: Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive interstitial lung disease that is refractory to current treatment options. Transforming growth factor (TGF)-β1 is a key pro-fibrotic cytokine that plays a crucial role in IPF pathogenesis. Our group previously demonstrated distinct epigenetic modifications involved in repression of the antifibrotic gene cyclooxygenase-2 (COX-2) in fibroblasts from IPF (F-IPF) lungs compared with fibroblasts from non-fibrotic lungs (F-NL). Epigenetic drugs capable of inhibiting DNA and histone modifications may, therefore, represent a putative novel therapy. The aim of this study was to investigate the ability of 4 epigenetic inhibitors to regulate TGF-β-driven fibrosis in ex vivo mouse lung. Methods: A precision-cut lung slice (PCLS) model of fibrosis was established using the previously described [1] CC10-tTS-rtTA-TGFβ1 transgenic (tgTGF-β1) mouse. The model was first assessed by investigating PCLS overexpression of TGF-β1 in response to stimulation of the transgene by doxycycline treatment. Gene expression of COX-2 and fibrotic markers including collagen were assessed after 4 days of treatment. The anti-fibrotic potential of 4 epigenetic inhibitors; BIX01294 (BIX, inhibitor of G9a histone methyltransferase), 3-deazaneplanocin A (DZNep, inhibitor of EZH2 histone methyltransferase), SAHA (inhibitor of histone deacetylases, HDACs) and Decitabine (DAC, DNA demethylating agent) was investigated. Viability of PCLS was assessed by MTT and Prestoblue® viability assay. Results: Treatment of PCLS from tgTGF-β1 mice with doxycycline induced a concentration-dependent increase in global TGF-β1, pro-fibrotic markers including collagen and pro-inflammatory COX-2, which was comparable to recombinant TGF-β1 treatment. Treatment with three of the epigenetic inhibitors BIX01294, DZNep and DAC did not reduce the pro-fibrotic response following doxycycline treatment. However SAHA demonstrated a significant suppressive effect on COX-2 and collagen expression, while not directly affecting TGF-β1 transgene expression. Conclusions: The data suggests that SAHA has the potential to reduce fibrosis in a TGF-β1 driven model of pulmonary fibrosis. Further work is currently underway to assess the anti-fibrotic potential of this drug in tgTGF-β1 animals.