Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS.
From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied.
Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.
Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1tau mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1tau overexpression in O. tauri.
Overexpression of the CK1tau in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau. Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins.
The identical phenotype for overexpression of wild-type CK1 and CK1tau is in line with the absence of critical targets for rodent CK1tau in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau. These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism.
Casein Kinase 1; Circadian clock; Minimal model; Ostreococcus tauri; Quantitative mass spectrometry; Phospho-proteomics; Bioinformatics
ADF/cofilin proteins are key modulators of actin dynamics in metastasis and invasion of cancer cells. Here we focused on the roles of ADF and cofilin-1 individually in the development of polarized migration of rat mammary adenocarcinoma (MTLn3) cells, which express nearly equal amounts of each protein. Small interference RNA (siRNA) technology was used to knockdown (KD) the expression of ADF and cofilin-1 independently.
Either ADF KD or cofilin KD caused cell elongation, a reduction in cell area, a decreased ability to form invadopodia, and a decreased percentage of polarized cells after 180 s of epidermal growth factor stimulation. Moreover, ADF KD or cofilin KD increased the rate of cell migration and the time of lamellipodia protrusion but through different mechanisms: lamellipodia protrude more frequently in ADF KD cells and are more persistent in cofilin KD cells. ADF KD cells showed a significant increase in F-actin aggregates, whereas cofilin KD cells showed a significant increase in prominent F-actin bundles and increased cell adhesion. Focal adhesion area and cell adhesion in cofilin KD cells were returned to control levels by expressing exogenous cofilin but not ADF. Return to control rates of cell migration in ADF KD cells was achieved by expression of exogenous ADF but not cofilin, whereas in cofilin KD cells, expression of cofilin efficiently rescued control migration rates.
Although ADF and cofilin have many redundant functions, each of these isoforms has functional differences that affect F-actin structures, cell adhesion and lamellipodial dynamics, all of which are important determinants of cell migration.
ADF; Cofilin; Metastasis; Invadopodia; Adhesion; Lamellipodia
A few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.
Introduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24+ and CD44+ populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-β, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays, a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.
Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks.
Physiological Wnt/β-catenin signaling; Nasopharyngeal carcinoma; Self-renewal network; Chromosome 3 transfer; Stemness transition; Tumor suppressor genes; Cancer stem cell markers
Fallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane.
We have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples.
This 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.
Fallopian tube secretory epithelial cells; Gene expression microarray; Three-dimensional in vitro models; Tissue microenvironment; Ovarian cancer
Epithelial tissues depend on intercellular homodimerization of E-cadherin and loss of E-cadherin is central to the epithelial to mesenchymal transition seen in multiple human diseases. Signaling pathways regulate E-cadherin function and cellular distribution via phosphorylation of the cytoplasmic region by kinases such as casein kinases but the protein phosphatases involved have not been identified.
This study shows protein Ser/Thr phosphatase-6 catalytic subunit (PP6c) is expressed in epithelial tissue and its mRNA and protein are robustly up-regulated in epithelial cell lines at high vs. low density. PP6c accumulates at adherens junctions, not tight junctions, co-immunoprecipitates with E-cadherin-catenin complexes without a canonical SAPS subunit, and associates directly with the E-cadherin cytoplasmic tail. Inducible shRNA knockdown of PP6c dispersed E-cadherin from the cell surface and this response was reversed by chemical inhibition of casein kinase-1 and prevented by alanine substitution of Ser846 in murine E-cadherin.
PP6c associates with E-cadherin in adherens junctions and is required to oppose casein kinase-1 to maintain cell surface localization of E-cadherin. There is feedback signaling to enhance PP6c transcription and boost protein levels in high density epithelial cells.
Catenin; Casein kinase; SAPS; Occludin; Caco-2; ARPE-19
Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes.
The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α.
UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.
Adipogenesis; ATRA; Brown adipocyte; UCP1; White adipocyte
Ischemic preconditioning has been proposed to involve changes in mitochondrial H+ and K+ fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K+ channels (MitoKATP). The objectives of the present study were to explore how increased H+ and K+ fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT).
Isolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H+ and K+ conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K+- and H+-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K+ and H+ conductance.
It is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H+ or K+ conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.
Ischemic preconditioning; Mitochondrial permeability transition; Potassium channels; Respiratory uncoupling; Reactive oxygen species
Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.
In this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation.
Cellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.
Protein kinase C; Myoblast differentiation; Myoblast fusion; Insulin receptor substrate
Angiogenesis is the main therapeutic mechanism of cell therapy for cardiovascular diseases, but diabetes is reported to reduce the function and number of progenitor cells. Therefore, we studied the effect of streptozotocin-induced diabetes on the bone marrow-mesenchymal stem cell (MSC) function, and examined whether diabetes-impaired MSC could be rescued by pretreatment with oxytocin.
MSCs were isolated and cultured from diabetic (DM) or non-diabetic (non-DM) rat, and proliferation rate was compared. DM-MSC was pretreated with oxytocin and compared with non-DM-MSC. Angiogenic capacity was estimated by tube formation and Matrigel plug assay, and therapeutic efficacy was studied in rat myocardial infarction (MI) model.
The proliferation and angiogenic activity of DM-MSC were severely impaired but significantly improved by pretreatment with oxytocin. Krüppel-like factor 2 (KLF2), a critical angiogenic factor, was dramatically reduced in DM-MSC and significantly restored by oxytocin. In the Matrigel plug assay, vessel formation of DM-BMSCs was attenuated but was recovered by oxytocin. In rat MI model, DM-MSC injection did not ameliorate cardiac injury, whereas oxytocin-pretreated DM-MSC improved cardiac function and reduced fibrosis.
Our results show that diabetes influenced MSC by reducing angiogenic capacity and therapeutic potential. We demonstrate the striking effect of oxytocin on stem cell dysfunction and suggest the use of oxytocin as a priming reagent in autologous stem cell therapy.
Diabetes; Angiogenesis; Stem cells; Oxytocin; Krüppel-like factor 2
Previous research indicates that the membrane ruffles and leading edge of lamellipodia of myogenic cells contain presumptive fusion sites. A micrometer-sized lipid raft (microraft) is organized at the presumptive fusion site of mouse myogenic cells in a cell-contact independent way and serves as a platform tethering adhesion proteins that are relevant to cell fusion. However, the mechanisms underlying recruitment of adhesion proteins to lipid rafts and microraft organization remain unknown.
Here we show that small G-protein Rac1 was required for microraft organization and subsequent cell fusion. However, Rac1 activity was unnecessary for recruitment of M-cadherin to lipid rafts. We found that p120 catenin (p120) binds to M-cadherin exclusively in lipid rafts of differentiating myogenic cells. The Src kinase inhibitor SU6656 prevented p120 binding to M-cadherin and their recruitment to lipid rafts, then suppressed microraft organization, membrane ruffling, and myogenic cell fusion. Suppression of membrane ruffling in SU6656-treated cells was partially restored by pretreatment with the protein tyrosine phosphatase inhibitor vanadate. The present analyses using an antibody to tyrosine phosphorylated p120 suggest that Src family kinases play a role in binding of p120 to M-cadherin and the recruitment of M-cadherin to lipid rafts through phosphorylation of putative substrates other than p120.
The present study showed that the procedure establishing fusion-competent sites consists of two sequential events: recruitment of adhesion complexes to lipid rafts and organization of microrafts. The recruitment of M-cadherin to lipid rafts depended on interaction with p120 catenin, whereas the organization of microrafts was controlled by a small G protein, Rac1.
Myogenesis; Cell fusion; M-cadherin; p120 catenin; Rac1; Tyrosine phosphorylation
Tissue microenvironments comprise different extracellular matrix (ECM) proteins that regulate cellular responsiveness to growth factors. In vitro culture of adherent cells on ECM-coated substrata is commonly used to study microenvironmental influence on specific cell signaling responses. Phosphorylation-specific flow cytometry can be utilized to quantify intracellular phosphorylation-dependent signaling events in single cells. However this approach necessitates trypsinization of adherent cells to accommodate flow cytometric analysis. Trypsin is a potent activator of cell signaling and can obscure signal transduction events induced by other factors.
To address this we developed a cold trypsin-phosphorylation-specific flow cytometry protocol, where adherent cells are prepared for flow cytometric analysis on ice (~0°C), a temperature where trypsin retains activity but where intracellular kinases are inactive. We show that this straightforward approach can be used to quantify intracellular pERK levels in single adherent primary human vascular smooth muscle cells grown on different ECM.
Exploiting the limited temperature dependence of trypsin facilitated development of a generally applicable phosphorylation-specific flow cytometry method for analysis of adherent cell types including primary patient derived cells. We demonstrate the utility of cold trypsin-phosphorylation-specific flow cytometry analysis of cell signaling to measure microenvironmental influence in single adherent cells.
Flow cytometry; Phosphorylation; Cell signaling; Extracellular matrix
Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions.
In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes.
By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides.
Endolysosome; Antigen processing; Proteases; Cathepsins; Protein degradation; Primary cells; Mass spectrometry; T cell epitope production; MHC; HIV
Obesity is associated with a higher risk of developing cancer and co-morbidities that are part of the metabolic syndrome. Adipose tissue is recognized as an endocrine organ, as it affects a number of physiological functions, and contains adipose tissue-derived stem cells (ASCs). ASCs can differentiate into cells of multiple lineages, and as such are applicable to tissue engineering and regenerative medicine. Yet the question of whether ASC functionality is affected by the donor’s body mass index (BMI) still exists.
ASCs were isolated from patients having different BMIs (BMI-ASCs), within the ranges of 18.5-32.8. It was hypothesized that overweight BMI-ASCs would be more compromised in early adipogenic and osteogenic potential, and ability to form colonies in vitro. BMI was inversely correlated with ASC proliferation and colony forming potential as assessed by CyQUANT proliferation assay (fluorescence- based measurement of cellular DNA content), and colony forming assays. BMI was positively correlated with early time point (day 7) but not later time point (day 15) intracytoplasmic lipid accumulation as assessed by Oil-Red-O staining. Alizarin red staining and RT-PCR for alkaline phosphatase demonstrated that elevated BMI resulted in compromised ASC mineralization of extracellular matrix and decreased alkaline phosphatase mRNA expression.
These data demonstrate that elevated BMI resulted in reduced ASC proliferation, and potentially compromised osteogenic capacity in vitro; thus BMI is an important criterion to consider in selecting ASC donors for clinical applications.
Adipose stromal/stem cells (ASCs); Body mass index (BMI); Osteogenesis; Proliferation; Colony formation; Cell size
The transcription factor E2F4 controls proliferation of normal and cancerous intestinal epithelial cells. E2F4 localization in normal human intestinal epithelial cells (HIEC) is cell cycle-dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, the intracellular signaling mechanisms regulating such E2F4 localization remain unknown.
Treatment of quiescent HIEC with serum induced ERK1/2 activation, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition while inhibition of MEK/ERK signaling by U0126 prevented these events. Stimulation of HIEC with epidermal growth factor (EGF) also led to the activation of ERK1/2 but, in contrast to serum or lysophosphatidic acid (LPA), EGF failed to induce E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition. Furthermore, Akt and GSK3β phosphorylation levels were markedly enhanced in serum- or LPA-stimulated HIEC but not by EGF. Importantly, E2F4 phosphorylation, E2F4 nuclear translocation and G1/S phase transition were all observed in response to EGF when GSK3 activity was concomitantly inhibited by SB216763. Finally, E2F4 was found to be overexpressed, phosphorylated and nuclear localized in epithelial cells from human colorectal adenomas exhibiting mutations in APC and KRAS or BRAF genes, known to deregulate GSK3/β-catenin and MEK/ERK signaling, respectively.
The present results indicate that MEK/ERK activation and GSK3 inhibition are both required for E2F4 phosphorylation as well as its nuclear translocation and S phase entry in HIEC. This finding suggests that dysregulated E2F4 nuclear localization may be an instigating event leading to hyperproliferation and hence, of tumor initiation and promotion in the colon and rectum.
E2F; Intestinal epithelium; Proliferation; Colorectal cancer; Cell cycle; GSK3; ERK; EGF
Apoptosis is a form of programmed cell death that is regulated by the Bcl-2 family and caspase family of proteins. The caspase cascade responsible for executing cell death following cytochrome c release is well described; however the distinct roles of caspases-9, -3 and -7 during this process are not completely defined.
Here we demonstrate several unique functions for each of these caspases during cell death. Specific inhibition of caspase-9 allows for efficient release of cytochrome c, but blocks changes in mitochondrial morphology and ROS production. We show that caspase-9 can cleave Bid into tBid at amino acid 59 and that this cleavage of Bid is required for ROS production following serum withdrawal. We also demonstrate that caspase-3-deficient MEFs are less sensitive to intrinsic cell death stimulation, yet have higher ROS production. In contrast, caspase-7-deficient MEFs are not resistance to intrinsic cell death, but remain attached to the ECM.
Taken together, these data suggest that caspase-9 is required for mitochondrial morphological changes and ROS production by cleaving and activating Bid into tBid. After activation by caspase-9, caspase-3 inhibits ROS production and is required for efficient execution of apoptosis, while effector caspase-7 is required for apoptotic cell detachment.
Caspase; Bid; ROS; Intrinsic apoptosis; Mitochondria; Cell detachment
In the progression towards diabetes, glucolipotoxicity is one of the main causes of pancreatic beta cell pathology. The aim of this study was to examine the in vitro effects of chronic glucolipotoxic conditions on cellular responses in pancreatic islets, including glucose and fat metabolism, Calcium mobilization, insulin secretion and insulin content.
Exposure of islets to chronic glucolipotoxic conditions decreased glucose stimulated insulin secretion in vitro. Reduced protein levels of Glut2/slc2a2, and decreased glucokinase and pyruvate carboxylase mRNA levels indicated a significant lowering in glucose sensing. Concomitantly, both fatty acid uptake and triglyceride accumulation increased significantly while fatty acid oxidation decreased. This general suppression in glucose metabolism correlated well with a decrease in mitochondrial number and activity, reduction in cellular ATP content and dampening of the TCA cycle. Further, we also observed a decrease in IP3 levels and lower Calcium mobilization in response to glucose. Importantly, chronic glucolipotoxic conditions in vitro decreased insulin gene expression, insulin content, insulin granule docking (to the plasma membrane) and insulin secretion.
Our results present an integrated view of the effects of chronic glucolipotoxic conditions on known and novel signaling events, in vitro, that results in reduced glucose responsiveness and insulin secretion.
Type 2 diabetes; Rat islets; Glucolipotoxicity; Glucose metabolism; Insulin content; Insulin secretion
While the essential role of 3D nuclear architecture on nuclear functions has been demonstrated for various cell types, information available for neutrophils, essential components of the immune system, remains limited. In this study, we analysed the spatial arrangements of telomeres which play a central role in cell fate. Our studies were carried out in swine, which is an excellent model organism for both biomedical research and agronomic applications. We isolated bacterial artificial chromosome (BAC)-containing subtelomeric p and q sequences specific to each porcine chromosome. This allowed us to study the behaviour of p and q telomeres of homologous chromosomes for seven pairs chosen for their difference in length and morphology. This was performed using 3D-FISH on structurally preserved neutrophils, and confocal microscopy. Resting and lipopolysaccharide (LPS)-activated states were investigated to ascertain whether a response to a pathogen aggression modifies this organization.
The positions of the p and q telomeres relative to the nuclear outer border were determined in the two states. All p telomeres changed their position significantly during the activation process, although the effect was less pronounced for the q telomeres. The patterns of telomeric associations between homologs and their frequencies were analysed for 7 pairs of chromosomes. This analysis revealed that the distribution of pp, qq and pq associations differs significantly among the 7 chromosomes. This distribution does not fit with the theoretical distribution for each chromosome, suggesting that preferential associations occur between subtelomeres.
The percentage of nuclei harbouring at least one telomeric association between homologs varies significantly among the chromosomes, the smallest metacentric chromosome SSC12, which is also the richest in gene-density, harbouring the highest value. The distribution of types of telomeric associations is highly dependent on the chromosomes and is not affected by the activation process. The frequencies of telomeric associations are also highly dependent on the type of association and the type of chromosome. Overall, the LPS-activation process induces only minor changes in these patterns of associations. When telomeric associations occur, the associations of p and q arms from the same chromosome are the most frequent, suggesting that “chromosome bending” occurs in neutrophils as previously observed in gametes.
Neutrophils; Telomere Associations; LPS-activation; 3D-FISH; Nuclear Architecture
Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized.
Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, β or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α.
We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity.
Autophagy; Drosophila; HIF-1α/sima; Hypoxia; p62/Ref2P; Proteasome
Renal podocytes form the main filtration barrier possessing a unique phenotype maintained by proteins including podocalyxin and nephrin, the expression of which is suppressed in pathological conditions. We used an in vitro model of human glomerular epithelial cells (HGEC) to investigate the role of high glucose in dysregulating the podocytic epithelial phenotype and determined the time needed for this change to occur.
In our in vitro podocyte system changes indicating podocyte dedifferentiation in the prolonged presence of high glucose included loss of podocalyxin, nephrin and CD10/CALLA concomitant with upregulation of mesenchymal vimentin. Our study demonstrates for the first time that podocyte-specific markers undergo changes of expression at different time intervals, since glucose-mediated podocalyxin downregulation is a progressive process that precedes downregulation of nephrin expression. Finally we demonstrate that high glucose permanently impaired WT1 binding to the podocalyxin gene promoter region but did not affect WT1 binding on the nephrin gene promoter region.
The presence of high glucose induced a phenotypic conversion of podocytes resembling partial dedifferentiation. Our study demonstrates that dysregulation of the normal podocytic phenotype is an event differentially affecting the expression of function-specific podocytic markers, exhibiting downregulation of the epithelial marker CD10/CALLA and PC first, followed by stably downregulated nephrin. Furthermore, it is herein suggested that WT1 may not be directly involved with upregulation of previously reduced PC and nephrin expression.
Podocalyxin; Nephrin; Dedifferentiation; Podocytes; WT1
Autophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg8 protein plays an important role in the yeast autophagy process. In humans, six Atg8 homologs, including MAP1LC3A, MAP1LC3B, MAP1LC3C (refer to LC3A, LC3B, and LC3C hereafter), GABARAP, GABARAPL1, and GABARAPL2 have been reported. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes.
In this study, we found 3 new alternative splicing isoforms in LC3B, GABARAP, and GABARAPL1, (designated as LC3B-a, GABARAP-a and GABARAPL1-a, respectively). None of them can go through the PE-conjugation process and be located to autophagosomes. Interestingly, compared with LC3B, LC3B-a has a single amino acid (Arg68) deletion due to the NAGNAG alternative splicing in intron 3. Through structural simulations, we found that the C-terminal tail of LC3B-a is less mobile than that of LC3B, thus affecting its C-terminal cleavage by human ATG4 family proteins. Furthermore, we found that Arg68 is an essential residue facilitating the interaction between human Atg8 family proteins and ATG4B by forming a salt bridge with Asp171 of ATG4B. Depletion of this salt bridge reduces autophagosomes formation and autophagic flux under both normal and nutrition starvation conditions.
These results suggest Arg68 is an essential residue for the C-terminal cleavage of Atg8 family proteins during the autophagy process.
Atg8; MAP1LC3B; Autophagy; Alternative splicing
H19 is a paternally imprinted gene that has been shown to be highly expressed in the trophoblast tissue. Results from previous studies have initiated a debate as to whether noncoding RNA H19 acts as a tumor suppressor or as a tumor promotor in trophoblast tissue. In the present study, we developed lentiviral vectors expressing H19-specific small interfering RNA (siRNA) to specifically block the expression of H19 in the human choriocarcinoma cell line JAR. Using this approach, we investigated the impact of the H19 gene on the proliferation, invasion and apoptosis of JAR cells. Moreover, we examined the effect of H19 knockdown on the expression of insulin-like growth factor 2 (IGF2), hairy and enhancer of split homologue-1 (HES-1) and dual-specific phosphatase 5 (DUSP5) genes.
H19 knockdown inhibited apoptosis and proliferation of JAR cells, but had no significant impact on cell invasion. In addition, H19 knockdown resulted in significant upregulation of HES-1 and DUSP5 expression, but not IGF2 expression in JAR cells.
The finding that H19 downregulation could simultaneously inhibit proliferation and apoptosis of JAR cells highlights a putative dual function for H19 in choriocarcinoma and may explain the debate on whether H19 acts as a tumor suppressor or a tumor promotor in trophoblast tissue. Furthermore, upregulation of HES-1 and DUSP5 may mediate H19 downregulation-induced suppression of proliferation and apoptosis of JAR cells.
H19; JAR cells; Choriocarcinoma; HES-1; DUSP5; IGF2
The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines.
Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments.
Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.
Colorectal cancer; Cancer-stroma interaction; Proteoglycans; Syndecan-2; Fibronectin
We have recently characterized two distinct populations of Satellite Cells (SCs) that differ in proliferation, regenerative potential, and mitochondrial coupling efficiency and classified these in Low Proliferative Clones (LPC) and High Proliferative Clones (HPC). Herewith, we have investigated their cell metabolism and individuated features that remark an intrinsic difference in basal physiology but that are retrievable also at the initial phases of their cloning.
Indeed, LPC and HPC can be distinguished for mitochondrial membrane potential (ΔΨm) just after isolation from the fiber. This is matched by mitochondrial redox state measured via NAD+/NADH analysis and alternative respiratory CO2 production in cloned cells. All these parameters are accountable for metabolic differences reflected indeed by alternative expression of the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3). Also Ca2+ handling by mitochondria is different together with the sensitivity to apoptosis triggered via this pathway. Finally, according to the above, we were able to determine which one among the clones represents the suitable stem cell.
These experimental observations report novel physiological features in the cell biology of SCs and refer to an intrinsic heterogeneity within which their stemness may reside.
Satellite cells; Clones; Metabolism; CO2 production; Apoptosis
The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance.
MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform.
Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes.
Circulating; Microparticles; Exosomes; Microvesicles; Disease; Diagnostics; Therapy