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1.
Melatonin and Vitamin D Orchestrate Adipose Derived Stem Cell Fate by Modulating Epigenetic Regulatory Genes.
Santaniello, S, Cruciani, S, Basoli, V, Balzano, F, Bellu, E, Garroni, G, Ginesu, GC, Cossu, ML, Facchin, F, Delitala, AP, et al
International journal of medical sciences. 2018;(14):1631-1639
Abstract
Melatonin, that regulates many physiological processes including circadian rhythms, is a molecule able to promote osteoblasts maturation in vitro and to prevent bone loss in vivo, while regulating also adipocytes metabolism. In this regard, we have previously shown that melatonin in combination with vitamin D, is able to counteract the appearance of an adipogenic phenotype in adipose derived stem cells (ADSCs), cultured in an adipogenic favoring condition. In the present study, we aimed at evaluating the specific phenotype elicited by melatonin and vitamin D based medium, considering also the involvement of epigenetic regulating genes. ADSCs were cultured in a specific adipogenic conditioned media, in the presence of melatonin alone or with vitamin D. The expression of specific osteogenic related genes was evaluated at different time points, together with the histone deacetylases epigenetic regulators, HDAC1 and Sirtuins (SIRT) 1 and 2. Our results show that melatonin and vitamin D are able to modulate ADSCs commitment towards osteogenic phenotype through the upregulation of HDAC1, SIRT 1 and 2, unfolding an epigenetic regulation in stem cell differentiation and opening novel strategies for future therapeutic balancing of stem cell fate toward adipogenic or osteogenic phenotype.
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Butyrate and docosahexaenoic acid interact in alterations of specific lipid classes in differentiating colon cancer cells.
Tylichová, Z, Slavík, J, Ciganek, M, Ovesná, P, Krčmář, P, Straková, N, Machala, M, Kozubík, A, Hofmanová, J, Vondráček, J
Journal of cellular biochemistry. 2018;(6):4664-4679
Abstract
Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.
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3.
ZNF521 Represses Osteoblastic Differentiation in Human Adipose-Derived Stem Cells.
Chiarella, E, Aloisio, A, Scicchitano, S, Lucchino, V, Montalcini, Y, Galasso, O, Greco, M, Gasparini, G, Mesuraca, M, Bond, HM, et al
International journal of molecular sciences. 2018;(12)
Abstract
Human adipose-derived stem cells (hADSCs) are multipotent mesenchymal cells that can differentiate into adipocytes, chondrocytes, and osteocytes. During osteoblastogenesis, the osteoprogenitor cells differentiate into mature osteoblasts and synthesize bone matrix components. Zinc finger protein 521 (ZNF521/Zfp521) is a transcription co-factor implicated in the regulation of hematopoietic, neural, and mesenchymal stem cells, where it has been shown to inhibit adipogenic differentiation. The present study is aimed at determining the effects of ZNF521 on the osteoblastic differentiation of hADSCs to clarify whether it can influence their osteogenic commitment. The enforced expression or silencing of ZNF521 in hADSCs was achieved by lentiviral vector transduction. Cells were cultured in a commercial osteogenic medium for up to 20 days. The ZNF521 enforced expression significantly reduced osteoblast development as assessed by the morphological and molecular criteria, resulting in reduced levels of collagen I, alkaline phosphatase, osterix, osteopontin, and calcium deposits. Conversely, ZNF521 silencing, in response to osteoblastic stimuli, induced a significant increase in early molecular markers of osteogenesis and, at later stages, a remarkable enhancement of matrix mineralization. Together with our previous findings, these results show that ZNF521 inhibits both adipocytic and osteoblastic maturation in hADSCs and suggest that its expression may contribute to maintaining the immature properties of hADSCs.
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4.
Cellular differentiation, bioactive and mechanical properties of experimental light-curing pulp protection materials.
Sauro, S, Babbar, A, Gharibi, B, Feitosa, VP, Carvalho, RM, Azevedo Rodrigues, LK, Banerjee, A, Watson, T
Dental materials : official publication of the Academy of Dental Materials. 2018;(6):868-878
Abstract
OBJECTIVE Materials for pulp protection should have therapeutic properties in order to stimulate remineralization and pulp reparative processes. The aim of this study was to evaluate the mechanical properties, biocompatibility, cell differentiation and bioactivity of experimental light-curable resin-based materials containing bioactive micro-fillers. METHODS Four calcium-phosphosilicate micro-fillers were prepared and incorporated into a resin blend: 1) Bioglass 45S5 (BAG); 2) zinc-doped bioglass (BAG-Zn); 3) βTCP-modified calcium silicate (β-CS); 4) zinc-doped β-CS (β-CS-Zn). These experimental resins were tested for flexural strength (FS) and fracture toughness (FT) after 24h and 30-day storage in simulated body fluid (SBF). Cytotoxicity was evaluated using MTT assay, while bioactivity was evaluated using mineralization and gene expression assays (Runx-2 & ALP). RESULTS The lowest FS and FT at 24h was attained with β-CS resin, while all the other tested materials exhibited a decrease in FS after prolonged storage in SBF. β-CS-Zn maintained a stable FT after 30-day SBF aging. Incorporation of bioactive micro-fillers had no negative effect on the biocompatibility of the experimental materials tested in this study. The inclusion of zinc-doped fillers significantly increased the cellular remineralization potential and expression of the osteogenic genes Runx2 and ALP (p<0.05). SIGNIFICANCE The innovative materials tested in this study, in particular those containing β-CS-Zn and BAG-Zn may promote cell differentiation and mineralization. Thus, these materials might represent suitable therapeutic pulp protection materials for minimally invasive and atraumatic restorative treatments.
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5.
Targeting Histone Deacetylase Activity to Arrest Cell Growth and Promote Neural Differentiation in Ewing Sarcoma.
Souza, BK, da Costa Lopez, PL, Menegotto, PR, Vieira, IA, Kersting, N, Abujamra, AL, Brunetto, AT, Brunetto, AL, Gregianin, L, de Farias, CB, et al
Molecular neurobiology. 2018;(9):7242-7258
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Abstract
There is an urgent need for advances in the treatment of Ewing sarcoma (EWS), an aggressive childhood tumor with possible neuroectodermal origin. Inhibition of histone deacetylases (HDAC) can revert aberrant epigenetic states and reduce growth in different experimental cancer types. Here, we investigated whether the potent HDAC inhibitor, sodium butyrate (NaB), has the ability to reprogram EWS cells towards a more differentiated state and affect their growth and survival. Exposure of two EWS cell lines to NaB resulted in rapid and potent inhibition of HDAC activity (1 h, IC50 1.5 mM) and a significant arrest of cell cycle progression (72 h, IC50 0.68-0.76 mM), marked by G0/G1 accumulation. Delayed cell proliferation and reduced colony formation ability were observed in EWS cells after long-term culture. NaB-induced effects included suppression of cell proliferation accompanied by reduced transcriptional expression of the EWS-FLI1 fusion oncogene, decreased expression of key survival and pluripotency-associated genes, and re-expression of the differentiation neuronal marker βIII-tubulin. Finally, NaB reduced c-MYC levels and impaired survival in putative EWS cancer stem cells. Our findings support the use of HDAC inhibition as a strategy to impair cell growth and survival and to reprogram EWS tumors towards differentiation. These results are consistent with our previous studies indicating that HDis can inhibit the growth and modulate differentiation of cells from other types of childhood pediatric tumors possibly originating from neural stem cells.
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Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells.
Chlebowska-Tuz, J, Sokolowska, O, Gaj, P, Lazniewski, M, Firczuk, M, Borowiec, K, Sas-Nowosielska, H, Bajor, M, Malinowska, A, Muchowicz, A, et al
Haematologica. 2018;(11):1843-1852
Abstract
A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.
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Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy.
Schick, R, Mekies, LN, Shemer, Y, Eisen, B, Hallas, T, Ben Jehuda, R, Ben-Ari, M, Szantai, A, Willi, L, Shulman, R, et al
PloS one. 2018;(10):e0205719
Abstract
AIMS: Dilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome-decreased ventricular function. Titin (TTN)-a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS). METHODS AND RESULTS iPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to β-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells. CONCLUSIONS These findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.
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Beneficial Effects of Concentrated Growth Factors and Resveratrol on Human Osteoblasts In Vitro Treated with Bisphosphonates.
Borsani, E, Bonazza, V, Buffoli, B, Nocini, PF, Albanese, M, Zotti, F, Inchingolo, F, Rezzani, R, Rodella, LF
BioMed research international. 2018;:4597321
Abstract
Bisphosphonates are primary pharmacological agents against osteoclast-mediated bone loss and widely used in the clinical practice for prevention and treatment of a variety of skeletal conditions, such as low bone density and osteogenesis imperfecta, and pathologies, such as osteoporosis, malignancies metastatic to bone, Paget disease of bone, multiple myeloma, and hypercalcemia of malignancy. However, long-term bisphosphonate treatment is associated with pathologic conditions including osteonecrosis of the jaw, named BRONJ, which impaired bone regeneration process. Clinical management of BRONJ is controversy and one recent approach is the use of platelet concentrates, such as Concentrated Growth Factors, alone or together with biomaterials or antioxidants molecules, such as resveratrol. The aim of the present study was to investigate the in vitro effects of Concentrated Growth Factors and/or resveratrol on the proliferation and differentiation of human osteoblasts, treated or not with bisphosphonates. Human osteoblasts were stimulated for 3 days in complete medium and for 21 days in mineralization medium. At the end of the experimental period, the in vitro effect on osteoblast proliferation and differentiation was evaluated using different techniques such as MTT, ELISA for the quantification/detection of osteoprotegerin and bone morphogenetic protein-2, immunohistochemistry for sirtuin 1 and collagen type I, and the Alizarin Red S staining for the rate of mineralization. Results obtained showed that Concentrated Growth Factors and/or resveratrol significantly increased osteoblast proliferation and differentiation and that the cotreatment with Concentrated Growth Factors and resveratrol had a protective role on osteoblasts treated with bisphosphonates. In conclusion, these data suggest that this approach could be promised in the clinical management of BRONJ.
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GPR182 is a novel marker for sinusoidal endothelial differentiation with distinct GPCR signaling activity in vitro.
Schmid, CD, Schledzewski, K, Mogler, C, Waldburger, N, Kalna, V, Marx, A, Randi, AM, Géraud, C, Goerdt, S, Koch, PS
Biochemical and biophysical research communications. 2018;(1):32-38
Abstract
Endothelial cells (EC) along the vascular tree exhibit organ-specific angiodiversity. Compared to most other ECs, liver sinusoidal endothelial cells (LSEC) that constitute the organ-specific microvasculature of the liver are morphologically and functionally unique. Previously, we showed that transcription factor Gata4 acts as a master regulator controlling LSEC differentiation. Upon analysis of the molecular signature of LSEC, we identified GPR182 as a potential LSEC-specific orphan G-protein coupled receptor (GPCR). Here, we demonstrate that GPR182 is expressed by LSEC and by EC with sinusoidal differentiation in spleen, lymph node and bone marrow in healthy human tissues. In a tissue microarray analysis of human hepatocellular carcinoma (HCC) samples, endothelial GPR182 expression was significantly reduced in tumor samples compared to peritumoral liver tissue samples (p = 0.0105). Loss of endothelial GPR182 expression was also seen in fibrotic and cirrhotic liver tissues. In vitro, GPR182 differentially regulated canonical GPCR signaling pathways as shown using reporter luciferase assays in HEK293T cells. Whereas ERK and RhoA signaling were inhibited, CREB and Calcium signaling were activated by ectopic GPR182 overexpression. Our data demonstrate that GPR182 is an endothelial subtype-specific marker for human sinusoidal EC of the liver, spleen, lymph node and bone marrow. In addition, we provide evidence for GPR182-dependent downstream signaling via ERK and SRF pathways that may be involved in regulating endothelial subtype-specific sinusoidal differentiation and sinusoidal functions such as permeability.
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10.
Downregulation of p53 drives autophagy during human trophoblast differentiation.
Gauster, M, Maninger, S, Siwetz, M, Deutsch, A, El-Heliebi, A, Kolb-Lenz, D, Hiden, U, Desoye, G, Herse, F, Prokesch, A
Cellular and molecular life sciences : CMLS. 2018;(10):1839-1855
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Abstract
The placental barrier is crucial for the supply of nutrients and oxygen to the developing fetus and is maintained by differentiation and fusion of mononucleated cytotrophoblasts into the syncytiotrophoblast, a process only partially understood. Here transcriptome and pathway analyses during differentiation and fusion of cultured trophoblasts yielded p53 signaling as negative upstream regulator and indicated an upregulation of autophagy-related genes. We further showed p53 mRNA and protein levels decreased during trophoblast differentiation. Reciprocally, autophagic flux increased and cytoplasmic LC3B-GFP puncta became more abundant, indicating enhanced autophagic activity. In line, in human first trimester placenta p53 protein mainly localized to the cytotrophoblast, while autophagy marker LC3B as well as late autophagic compartments were predominantly detectable in the syncytiotrophoblast. Importantly, ectopic overexpression of p53 reduced levels of LC3B-II, supporting a negative regulatory role on autophagy in differentiating trophoblasts. This was also shown in primary trophoblasts and human first trimester placental explants, where pharmacological stabilization of p53 decreased LC3B-II levels. In summary our data suggest that differentiation-dependent downregulation of p53 is a prerequisite for activating autophagy in the syncytiotrophoblast.