The epithelial to mensenchymal transition program regulates various aspects of embryonic development and tissue homeostasis, but aberrant activation of this pathway in cancer contributes to tumor ...progression and metastasis. TGF-b potently induces an epithelial to mensenchymal transition in cancers of epithelial origin by inducing transcriptional changes mediated by several key transcription factors. Here, we identify the developmental transcription factor SOX4 as a transcriptional target of TGF-b in immortalized human mammary epithelial cells. SOX4 expression and activity are rapidly induced in the early stages of the TGF-b-induced epithelial to mensenchymal transition. We demonstrate that conditional activation of Sox4 is sufficient to induce the expression of N-cadherin and additional mesenchymal markers including vimentin and fibronectin, but fails to induce complete EMT as no changes are observed in the expression of E-cadherin and b-catenin. Moreover, shRNA-mediated knockdown of SOX4 significantly delays TGF-b-induced mRNA and protein expression of mesenchymal markers. Taken together, these data suggest that TGF-b-mediated increased expression of SOX4 is required for the induction of a mesenchymal phenotype during EMT in human mammary epithelial cells.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Maintenance of Foxp3 protein expression in regulatory T cells (Treg) is crucial for a balanced immune response. We have previously demonstrated that Foxp3 protein stability can be regulated through ...acetylation, however the specific mechanisms underlying this observation remain unclear. Here we demonstrate that SIRT1 a member of the lysine deacetylase Sirtuin (SIRT) family, but not the related SIRTs 2-7, co-localize with Foxp3 in the nucleus. Ectopic expression of SIRT1, but not SIRTs 2-7 results in decreased Foxp3 acetylation, while conversely inhibition of endogenous SIRT activity increased Foxp3 acetylation. We show that SIRT1 inhibition decreases Foxp3 poly-ubiquitination, thereby increasing Foxp3 protein levels. Co-transfection of SIRT1 with Foxp3 results in increased Foxp3 proteasomal degradation, while SIRT inhibition increases FOXP3 transcriptional activity in human Treg. Taken together, these data support a central role for SIRT1 in the regulation of Foxp3 protein levels and thereby in regulation of Treg suppressive capacity. Pharmacological modulation of SIRT1 activity in Treg may therefore provide a novel therapeutic strategy for controlling immune responses.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The evolutionarily conserved Forkhead box O (FOXO) family of transcription factors regulates multiple transcriptional targets involved in various cellular processes, including proliferation, stress ...resistance, apoptosis, and metabolism. Target gene regulation appears to be controlled in a cell-type-specific manner due to association of FOXO isoforms with specific cofactors. Many of the cellular processes modulated by FOXO are themselves deregulated in tumorigenesis, and deletion of Foxo genes has demonstrated that these transcription factors function as tumor suppressors. Our understanding of the regulation of FOXO activity, and defining specific transcriptional targets, may provide clues to the molecular mechanisms controlling cell fate decisions. In this review we describe the functional consequences of FOXO activation based on our current knowledge of transcriptional targets.
The underlying molecular mechanisms for many autoimmune diseases are poorly understood. Juvenile idiopathic arthritis (JIA) is an exceptionally well-suited model for studying autoimmune diseases due ...to its early onset and the possibility to analyze cells derived from the site of inflammation. Epigenetic profiling, utilizing primary JIA patient-derived cells, can contribute to the understanding of autoimmune diseases. With H3K27ac chromatin immunoprecipitation, we identified a disease-specific, inflammation-associated, typical enhancer and super-enhancer signature in JIA patient synovial-fluid-derived CD4+ memory/effector T cells. RNA sequencing of autoinflammatory site-derived patient T cells revealed that BET inhibition, utilizing JQ1, inhibited immune-related super-enhancers and preferentially reduced disease-associated gene expression, including cytokine-related processes. Altogether, these results demonstrate the potential use of enhancer profiling to identify disease mediators and provide evidence for BET inhibition as a possible therapeutic approach for the treatment of autoimmune diseases.
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•Identification of a disease-specific, inflammation-associated, enhancer signature•Inflammatory-site-derived cells are epigenetically different from peripheral cells•Enrichment of disease-related SNPs in disease-associated super-enhancers•BET inhibition of JIA patient T cells preferentially reduced JIA gene expression
By defining the active super-enhancer profile of autoimmune disease patients, Peeters et al. identify a disease-specific, inflammation-associated super-enhancer signature. In addition, inhibition of super-enhancer activity, using a BET inhibitor, in autoimmune disease patient-derived cells preferentially reduced disease-associated gene expression. These findings suggest a role for enhancers and super-enhancers in autoimmune diseases and demonstrates the potential use of BET inhibitors for the treatment of such diseases.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lymphocytes have evolved to react rapidly and robustly to changes in their local environment by using transient adaptations and by regulating their terminal differentiation programmes. Forkhead box ...transcription factors (FTFs) can direct leukocyte-specific responses, and their functional diversification promotes a high degree of context-dependent specification. Many, often antagonistic, FTFs have overlapping expression patterns and can thereby compete for binding to the same chromosomal target sequences. Multiple molecular mechanisms also connect extracellular signals to the expression and functionality of specific FTFs and, in this way, fine-tune their activity. Through these diverse mechanisms, FTFs can function as context-dependent rheostats responding to diverse environmental stimuli. Focusing on the various mechanisms by which their functional activity is modulated, as well as on their mechanisms of action, we discuss how specific FTFs control lymphocyte function, allowing for the establishment and maintenance of immune homeostasis.
Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally ...associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The PI(3)K-PKB-FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO ...transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K-PKB-FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Osteoarthritis (OA) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. Recently, allogeneic human mesenchymal stromal/stem cells (MSC) entered ...clinical trials as a novel therapy for OA. Increasing evidence suggests that therapeutic efficacy of MSC depends on paracrine signalling. Here we investigated the role of extracellular vesicles (EVs) secreted by human bone marrow derived MSC (BMMSC) in human OA cartilage repair.
To test the effect of BMMSC-EVs on OA cartilage inflammation, TNF-alpha-stimulated OA chondrocyte monolayer cultures were treated with BMMSC-EVs and pro-inflammatory gene expression was measured by qRT-PCR after 48 h. To assess the impact of BMMSC-EVs on cartilage regeneration, BMMSC-EVs were added to the regeneration cultures of human OA chondrocytes, which were analyzed after 4 weeks for glycosaminoglycan content by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-O) and type II collagen (immunostaining).
We show that BMMSC-EVs inhibit the adverse effects of inflammatory mediators on cartilage homeostasis. When co-cultured with OA chondrocytes, BMMSC-EVs abrogated the TNF-alpha-mediated upregulation of COX2 and pro-inflammatory interleukins and inhibited TNF-alpha-induced collagenase activity. BMMSC-EVs also promoted cartilage regeneration
. Addition of BMMSC-EVs to cultures of chondrocytes isolated from OA patients stimulated production of proteoglycans and type II collagen by these cells.
Our data demonstrate that BMMSC-EVs can be important mediators of cartilage repair and hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis.
Here we have explored whether inhibition of autophagy can be used as a treatment strategy for acute myeloid leukemia (AML). Steady-state autophagy was measured in leukemic cell lines and primary ...human CD34
AML cells with a large variability in basal autophagy between AMLs observed. The autophagy flux was higher in AMLs classified as poor risk, which are frequently associated with TP53 mutations (TP53
), compared with favorable- and intermediate-risk AMLs. In addition, the higher flux was associated with a higher expression level of several autophagy genes, but was not affected by alterations in p53 expression by knocking down p53 or overexpression of wild-type p53 or p53
. AML CD34
cells were more sensitive to the autophagy inhibitor hydroxychloroquine (HCQ) than normal bone marrow CD34
cells. Similar, inhibition of autophagy by knockdown of ATG5 or ATG7 triggered apoptosis, which coincided with increased expression of p53. In contrast to wild-type p53 AML (TP53
), HCQ treatment did not trigger a BAX and PUMA-dependent apoptotic response in AMLs harboring TP53
. To further characterize autophagy in the leukemic stem cell-enriched cell fraction AML CD34
cells were separated into ROS
and ROS
subfractions. The immature AML CD34
-enriched ROS
cells maintained higher basal autophagy and showed reduced survival upon HCQ treatment compared with ROS
cells. Finally, knockdown of ATG5 inhibits in vivo maintenance of AML CD34
cells in NSG mice. These results indicate that targeting autophagy might provide new therapeutic options for treatment of AML since it affects the immature AML subfraction.
Autophagy is a highly regulated catabolic process that involves sequestration and lysosomal degradation of cytosolic components such as damaged organelles and misfolded proteins. While autophagy can ...be considered to be a general cellular housekeeping process, it has become clear that it may also play cell type‐dependent functional roles. In this study, we analyzed the functional importance of autophagy in human hematopoietic stem/progenitor cells (HSPCs), and how this is regulated during differentiation. Western blot‐based analysis of LC3‐II and p62 levels, as well as flow cytometry‐based autophagic vesicle quantification, demonstrated that umbilical cord blood‐derived CD34+/CD38− immature hematopoietic progenitors show a higher autophagic flux than CD34+/CD38+ progenitors and more differentiated myeloid and erythroid cells. This high autophagic flux was critical for maintaining stem and progenitor function since knockdown of autophagy genes ATG5 or ATG7 resulted in reduced HSPC frequencies in vitro as well as in vivo. The reduction in HSPCs was not due to impaired differentiation, but at least in part due to reduced cell cycle progression and increased apoptosis. This is accompanied by increased expression of p53, proapoptotic genes BAX and PUMA, and the cell cycle inhibitor p21, as well as increased levels of cleaved caspase‐3 and reactive oxygen species. Taken together, our data demonstrate that autophagy is an important regulatory mechanism for human HSCs and their progeny, reducing cellular stress and promoting survival. Stem Cells 2016;34:1651–1663
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