Human adipose tissue-derived mesenchymal stem cells (hASCs) are useful for regeneration of inflamed or injured tissues. To identify secreted hASC proteins during inflammation, hASCs were exposed to ...tumor necrosis factor-α (TNF-α) and conditioned media derived from hASCs were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified 187 individual proteins as secreted proteins (secretome) in hASC-conditioned media; 118 proteins were secreted at higher levels upon TNF-α treatment. The TNF-α-induced secretome included a variety of cytokines and chemokines such as interleukin-6 (IL-6), IL-8, chemokine (C-X-C motif) ligand 6, and monocyte chemotactic protein-1 (MCP-1). TNF-α also increased expression of various proteases including cathepsin L, matrix metalloproteases and protease inhibitors, and induced secretion of long pentraxin 3, a key inflammatory mediator implicated in innate immunity. TNF-α-conditioned media stimulated migration of human monocytes, which play a key role in inflammatory responses. This migration was abrogated by pretreatment with neutralizing anti-IL-6, anti-IL-8, and anti-MCP-1 antibodies, suggesting that IL-6, IL-8, and MCP-1 are involved in migration of monocytes. Taken together, these results suggest that TNF-α-induced secretome may play a pivotal role in inflammatory responses and that shotgun proteomic analysis will be useful for elucidation of the paracrine functions of mesenchymal stem cells.
Glucose homeostasis is maintained by the orchestration of peripheral glucose utilization and hepatic glucose production, mainly by insulin. In this study, we found by utilizing a combined parallel ...chromatography mass profiling approach that lysophosphatidylcholine (LPC) regulates glucose levels. LPC was found to stimulate glucose uptake in 3T3-L1 adipocytes dose- and time-dependently, and this activity was found to be sensitive to variations in acyl chain lengths and to polar head group types in LPC. Treatment with LPC resulted in a significant increase in the level of GLUT4 at the plasma membranes of 3T3-L1 adipocytes. Moreover, LPC did not affect IRS-1 and AKT2 phosphorylations, and LPC-induced glucose uptake was not influenced by pretreatment with the PI 3-kinase inhibitor LY294002. However, glucose uptake stimulation by LPC was abrogated both by rottlerin (a protein kinase Cδ inhibitor) and by the adenoviral expression of dominant negative protein kinase Cδ. In line with its determined cellular functions, LPC was found to lower blood glucose levels in normal mice. Furthermore, LPC improved blood glucose levels in mouse models of type 1 and 2 diabetes. These results suggest that an understanding of the mode of action of LPC may provide a new perspective of glucose homeostasis.
5' AMP-activated protein kinase (AMPK) is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase ...cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-3-(3-nitro-phenyl)-thioureido-ethyl}-amide (Xn) and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-3-(3-cyano-phenyl)-thioureido-ethyl}-amide (Xc) elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4). Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
2,3,7,8-Tetrachlorodibenzo- p -dioxin (TCDD) is a widespread environmental pollutant with many toxic effects, including endocrine disruption, reproductive
dysfunction, immunotoxicity, liver damage, ...and cancer. These are mediated by TCDD binding to and activating the aryl hydrocarbon
receptor (AhR), a basic helix-loop-helix transcription factor. In this regard, targeting the AhR using novel small molecule
inhibitors is an attractive strategy for the development of potential preventive agents. In this study, by screening a chemical
library composed of approximately 10,000 compounds, we identified a novel compound, 2-methyl-2 H -pyrazole-3-carboxylic acid (2-methyl-4- o -tolylazo-phenyl)-amide (CH-223191), that potently inhibits TCDD-induced AhR-dependent transcription. In addition, CH-223191
blocked the binding of TCDD to AhR and inhibited TCDD-mediated nuclear translocation and DNA binding of AhR. These inhibitory
effects of CH-223191 prevented the expression of cytochrome P450 enzymes, target genes of the AhR. Unlike many known antagonists
of AhR, CH-223191 did not have detectable AhR agonist-like activity or estrogenic potency, suggesting that CH-223191 is a
specific antagonist of AhR. It is noteworthy that CH-223191 potently prevented TCDD-elicited cytochrome P450 induction, liver
toxicity, and wasting syndrome in mice. Taken together, these results demonstrate that this novel compound, CH-223191, may
be a useful agent for the study of AhR-mediated signal transduction and the prevention of TCDD-associated pathology.
Transforming growth factor-β1 (TGF-β1) induces the differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle cells. Lipid rafts are cholesterol-rich ...microdomains in cell membranes that reportedly play a key role in receptor-mediated signal transduction and cellular responses. In order to clarify whether lipid rafts are involved in TGF-β1-induced differentiation of hASCs into smooth muscle cells, we analyzed the lipid raft proteome of hASCs.
Pretreatment of hASCs with the lipid raft disruptor methyl-β-cyclodextrin abrogated TGF-β1-induced expression of α-smooth muscle actin, a smooth muscle cell marker, suggesting a pivotal role of lipid rafts in TGF-β1-induced differentiation of hASCs to smooth muscle cells. Sucrose density gradient centrifugation along with a shotgun proteomic strategy using liquid chromatography-tandem mass spectrometry identified 1002 individual proteins as the lipid raft proteome, and 242 of these were induced by TGF-β1 treatment. ADAM12, a disintegrin and metalloproteases family member, was identified as the most highly up-regulated protein in response to TGF-β1 treatment. TGF-β1 treatment of hASCs stimulated the production of both ADAM12 protein and mRNA. Silencing of endogenous ADAM12 expression using lentiviral small hairpin RNA or small interfering RNA abrogated the TGF-β1-induced differentiation of hASCs into smooth muscle cells.
These results suggest a pivotal role for lipid raft-associated ADAM12 in the TGF-β1-induced differentiation of hASCs into smooth muscle cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High‐grade gliomas are one of the most common brain tumors and notorious for poor prognosis due to their malignant nature. Gliomas have an extensive area of hypoxia, which is critical for glioma ...progression by inducing aggressiveness and activating the angiogenesis process in the tumor microenvironment. To resolve the factors responsible for the highly malignant nature of gliomas, we comprehensively profiled the U373MG glioma cell secretome—exosome and soluble fraction under hypoxic and normoxic conditions. A total of 239 proteins were identified from the exosome and soluble fractions. Vascular endothelial growth factor, stanniocalcin 1 (STC1) and stanniocalcin 2, and insulin‐like growth factor binding protein 3 and 6, enriched in the soluble fraction, and lysyl oxidase homolog 2 enriched in the exosomal fraction were identified as upregulated proteins by hypoxia based on a label‐free quantitative analysis. STCs and insulin‐like growth factor binding proteins, which were identified as secretory proteins under hypoxic conditions, were highly correlated with glioma grade in human patients by microarray analysis. An in vitro scratch wound assay revealed that STC1 and 2 have important functions in the induction of cell migration in a hypoxia‐dependent manner, suggesting that they are hypoxia‐dependent migration factors.
There are limitations to current colorectal cancer (CRC)-specific diagnostic methods and therapies. Tumorigenesis proceeds because of interaction between cancer cells and various surrounding cells; ...discovering new molecular mediators through studies of the CRC secretome is a promising approach for the development of CRC diagnostics and therapies.
A comparative secretomic analysis was performed using primary and metastatic human isogenic CRC cells. Proliferation was determined by MTT and thymidine incorporation assay, migration was determined by wound-healing assay (ELISA). The level of palmitoleoyl-protein carboxylesterase (NOTUM) in plasma from patients with CRC was determined by enzyme-linked immunosorbent assay.
NOTUM expression was increased in metastatic cells. Proliferation was suppressed by inhibiting expression of NOTUM. Knockdown of NOTUM genes inhibited proliferation as well as migration, with possible involvement of p38 and c-JUN N-terminal kinase in this process. The result was verified in patients with CRC.
NOTUM may be a new candidate for diagnostics and therapy of CRC.
Oct4 has been implicated in regulation of pluripotency in embryonic stem cells (ESCs) and reprogramming of somatic cells into induced pluripotent stem cells. However, the molecular mechanisms ...involved in Oct4‐dependent regulation of pluripotency and reprogramming have not been clear. To gain insight into the mechanism of regulation of Oct4‐mediated self‐renewal of ESCs and reprogramming of somatic cells, we attempted to identify Oct4‐binding proteins using affinity purification and mass spectrometry. We identified Reptin, a key component of ATP‐dependent chromatin remodeling complexes, as an Oct4‐binding protein. Depletion of endogenous Reptin using lentiviral short hairpin RNA (shRNA) led to a decrease in the number and size of alkaline phosphatase‐positive colonies of mouse ESCs. In addition, shRNA‐mediated silencing of Reptin resulted in decreased expression of pluripotency‐specific marker genes, including Oct4, Sox2, Nanog, and SSEA‐1. Results of the Oct4 reporter assay showed synergism between Oct4 and Reptin, and depletion of endogenous Reptin abolished Oct4 transcriptional activity. Results of a chromatin immunoprecipitation assay showed the overlapping interaction of Reptin and Oct4 to CR4 in the Oct4 enhancer in ESCs. Knockdown of Reptin using shRNA suppressed the reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells, whereas overexpression of Reptin resulted in enhanced efficiency of induced pluripotent stem cell generation. These results strongly suggest that Reptin plays a key role in maintaining the pluripotency of ESCs and in establishing the pluripotency during reprogramming of somatic cells by regulation of Oct4‐mediated gene regulation. Stem Cells 2014;32:3126–3136
Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in the signal transduction of various cellular responses.
However, although it is undeniably important that modulators of PLC ...activity be identified, no direct PLC activity modulator
has been identified until now. In this study, by screening more than 10,000 different compounds in human neutrophils, we identified
a compound that strongly enhances superoxide-generating activity, which is well known to be PLC-dependent. The active compound
2,4,6-trimethyl- N -( meta -3-trifluoromethyl-phenyl)-benzenesulfonamide ( m -3M3FBS) stimulated a transient intracellular calcium concentration (Ca 2+ i ) increase in neutrophils. Moreover, m -3M3FBS stimulated the formation of inositol phosphates in U937 cells, indicating that it stimulates PLC activity. The compound
showed no cell-type specificity in terms of Ca 2+ i increase in the various cell lines including leukocytes, fibroblasts, and neuronal cells. We also ruled out the possible
involvement of heterotrimeric G proteins in m -3M3FBSâstimulated signaling by confirming the following: 1) pertussis toxin does not inhibit m -3M3FBSâinduced Ca 2+ i increase; 2) m -3M3FBS does not stimulate cyclic AMP generation; and 3) the inhibition of G q by the regulator of G protein-signaling 2 does not affect the m -3M3FBSâinduced Ca 2+ i increase. We also observed that m -3M3FBS stimulated PLC activity in vitro. The purified isoforms of PLC that were tested (i.e., β2, β3, γ1, γ2, and δ1) were
activated by m -3M3FBS and showed no isoform specificity. Taken together, these results demonstrate that m -3M3FBS modulates neutrophil functions by directly activating PLC. Because m -3M3FBS is the first compound known to directly activate PLC, it should prove useful in the study of the basic molecular mechanisms
of PLC activation and PLC-mediated cell signaling.
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