Transglutaminase 2 associated with PI3K and PTEN in a membrane-bound signalosome platform blunts cell death.

Atypically expressed transglutaminase 2 (TG2) has been identified as a poor prognostic factor in a variety of cancers. In this study, we evaluated the contribution of TG2 to the prolonged cell survival of differentiated acute promyelocytic leukaemia (APL) cells in response to the standard treatment with combined retinoic acid (ATRA) and arsenic trioxide (ATO). We report that one advantage of ATRA + ATO treatment compared to ATRA alone diminishes the amount of activated and non-activated CD11b/CD18 and CD11c/CD18 cell surface integrin receptors. These changes suppress ATRA-induced TG2 docking on the cytosolic part of CD18 ß2-integrin subunits and reduce cell survival. In addition, TG2 overexpresses and hyperactivates the phosphatidylinositol-3-kinase (PI3K), phospho-AKT S473, and phospho-mTOR S2481 signalling axis. mTORC2 acts as a functional switch between cell survival and death by promoting the full activation of AKT. We show that TG2 presumably triggers the formation of a signalosome platform, hyperactivates downstream mTORC2-AKT signalling, which in turn phosphorylates and inhibits the activity of FOXO3, a key pro-apoptotic transcription factor. In contrast, the absence of TG2 restores basic phospho-mTOR S2481, phospho-AKT S473, PI3K, and PTEN expression and activity, thereby sensitising APL cells to ATO-induced cell death. We conclude, that atypically expressed TG2 may serve as a hub, facilitating signal transduction via signalosome formation by the CD18 subunit with both PI3K hyperactivation and PTEN inactivation through the PI3K-PTEN cycle in ATRA-treated APL cells.

Zbtb46-dependent altered developmental program in embryonic stem cell-derived blood cell progenitors.

Zbtb46 is a recently identified dendritic cell (DC)-specific transcription factor with poorly defined biology. Although Zbtb46 is highly expressed in conventional DCs, evidence also points to its presence in erythroid progenitors and endothelial cells suggesting that this factor might influence the early hematopoietic development. Here, we probe the effect of this transcription factor in embryonic stem cell (ESC)-derived blood cell progenitors using chemically inducible mouse cell lines. Unexpectedly, forced expression of this protein elicited a broad repressive effect at the early stage of ESC differentiation. Ectopic expression of Zbtb46 interfered with the mesoderm formation and cell proliferation was also negatively impacted. More importantly, reduced number of CD11b+ myeloid blood cells were generated from ESC-derived Flk1+ mesoderm cells in the presence of Zbtb46. Consistent with this finding, our gene expression profiling revealed that numerous myeloid and immune response related genes, including Irf8, exhibited lower expression in the Zbtb46-primed cells. Despite these repressive effects, however, Zbtb46 overexpression was associated with enhanced formation of erythroid blood cell colonies and increased adult hemoglobin (Hbb-b1) expression at the early phase of ESC differentiation. Moreover, elevated percent of CD105 (Endoglin) positive cells were detected in the Zbtb46-primed samples. In summary, our results support that Zbtb46 suppresses the ESC-derived myeloid development and diverts mesoderm cells toward erythroid developmental pathway. Moreover, our transcriptomic data provide a resource for exploration of the Zbtb46 regulatory network in ESC-derived progenitors.

Cells Lacking PA200 Adapt to Mitochondrial Dysfunction by Enhancing Glycolysis via Distinct Opa1 Processing.

The conserved Blm10/PA200 proteins are proteasome activators. Previously, we identified PA200-enriched regions in the genome of SH-SY5Y neuroblastoma cells by chromatin immunoprecipitation (ChIP) and ChIP-seq analysis. We also found that selective mitochondrial inhibitors induced PA200 redistribution in the genome. Collectively, our data indicated that PA200 regulates cellular homeostasis at the transcriptional level. In the present study, our aim is to investigate the impact of stable PA200 depletion (shPA200) on the overall transcriptome of SH-SY5Y cells. RNA-seq data analysis reveals that the genetic ablation of PA200 leads to overall changes in the transcriptional landscape of SH-SY5Y neuroblastoma cells. PA200 activates and represses genes regulating metabolic processes, such as the glycolysis and mitochondrial function. Using metabolic assays in live cells, we showed that stable knockdown of PA200 does not change basal respiration. Spare respiratory capacity and proton leak however are slightly, yet significantly, reduced in PA200-deficient cells by 99.834% and 84.147%, respectively, compared to control. Glycolysis and glycolytic capacity show a 42.186% and 26.104% increase in shPA200 cells, respectively, compared to control. These data suggest a shift from oxidative phosphorylation to glycolysis especially when cells are exposed to oligomycin-induced stress. Furthermore, we observed a preserved long and compact tubular mitochondrial morphology after inhibition of ATP synthase by oligomycin, which might be associated with the glycolytic change of shPA200 cells. The present study also demonstrates that the proteolytic cleavage of Opa1 is affected, and that the level of OMA1 is significantly reduced in shPA200 cells upon oligomycin-induced mitochondrial insult. Together, these findings suggest a role for PA200 in the regulation of metabolic changes in response to selective inhibition of ATP synthase in an in vitro cellular model.

The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.

Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

The proteasome activator PA200 regulates expression of genes involved in cell survival upon selective mitochondrial inhibition in neuroblastoma cells.

The conserved Blm10/PA200 activators bind to the proteasome core and facilitate peptide and protein turnover. Blm10/PA200 proteins enhance proteasome peptidase activity and accelerate the degradation of unstructured proteasome substrates. Our knowledge about the exact role of PA200 in diseased cells, however, is still limited. Here, we show that stable knockdown of PA200 leads to a significantly elevated number of cells in S phase after treatment with the ATP synthase inhibitor, oligomycin. However, following exposure to the complex I inhibitor rotenone, more PA200-depleted cells were in sub-G1 and G2/M phases indicative of apoptosis. Chromatin immunoprecipitation (ChIP) and ChIP-seq data analysis of collected reads indicate PA200-enriched regions in the genome of SH-SY5Y. We found that PA200 protein peaks were in the vicinity of transcription start sites. Gene ontology annotation revealed that genes whose promoters were enriched upon anti-PA200 ChIP contribute to the regulation of crucial intracellular processes, including proliferation, protein modifications and metabolism. Selective mitochondrial inhibitors induced PA200 redistribution in the genome, leading to protein withdrawal from some gene promoters and binding to others. Collectively, the results support a model in which PA200 potentially regulates cellular homeostasis at the transcriptional level, in addition to its described role as an alternative activator of the proteasome.

Juvenile Huntington's Disease Skin Fibroblasts Respond with Elevated Parkin Level and Increased Proteasome Activity as a Potential Mechanism to Counterbalance the Pathological Consequences of Mutant Huntingtin Protein.

Huntington's disease (HD) is an inherited neurodegenerative disorder, caused by an abnormal polyglutamine (polyQ) expansion in the huntingtin protein (Htt). Mitochondrial dysfunction and impairment of the ubiquitin-proteasome system (UPS) are hallmarks of HD neurons. The extraneural manifestations of HD are still unclear. We investigated the crosstalk between mitochondria and proteolytic function in skin fibroblasts from juvenile HD patients. We found reduced mitosis, increased cell size, elevated ROS and increased mitochondrial membrane potential in juvenile HD fibroblasts, while cellular viability was maintained. Mitochondrial OXPHOS analysis did not reveal significant differences compared to control. However, the level of mitochondrial fusion and fission proteins was significantly lower and branching in the mitochondria network was reduced. We hypothesized that juvenile HD fibroblasts counterbalance cellular damage and mitochondrial network deficit with altered proteasome activity to promote cell survival. Our data reveal that juvenile HD fibroblasts exhibit higher proteasome activity, which was associated with elevated gene and protein expression of parkin. Moreover, we demonstrate elevated proteasomal degradation of the mitochondrial fusion protein Mfn1 in diseased cells compared to control cells. Our data suggest that juvenile HD fibroblasts respond to mutant polyQ expansion of Htt with enhanced proteasome activity and faster turnover of specific UPS substrates to protect cells.

RUNX3-Mediated Immune Cell Development and Maturation.

The transcription factor RUNX3 is a prominent regulator of multiple hematopoietic cell lineages. Gene loss of function studies demonstrated the unique and essential roles of this master regulator in differentiated lymphoid and myeloid cells. As a complementary approach, RUNX3 was upregulated in various leukocyte subsets to probe the instructive role of this 'multilineage'-specific transcription factor. In this report, we overview the immunomodulatory functions of RUNX3 within the hematopoietic compartment to gain insight into the consequences of Runx3 deletion or overexpression in committed immune cells. Genetic studies revealed the essential role of RUNX3 in Langerhans cell development. Moreover, this transcription factor is necessary for the differentiation and maintenance of the cytotoxic CD8+ T cells. In addition, T helper, natural killer, and B cells are also influenced by RUNX3. Importantly, the ectopic expression of Runx3 enhances the immunogenicity of cytotoxic T cells and pluripotent stem-cell-derived dendritic cells, suggesting that this protein can be applied in cell-based immunotherapies.

Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism.

Retinoid X receptor (RXR) regulates several key functions in myeloid cells, including inflammatory responses, phagocytosis, chemokine secretion, and proangiogenic activity. Its importance, however, in tumor-associated myeloid cells is unknown. In this study, we demonstrate that deletion of RXR in myeloid cells enhances lung metastasis formation while not affecting primary tumor growth. We show that RXR deficiency leads to transcriptomic changes in the lung myeloid compartment characterized by increased expression of prometastatic genes, including important determinants of premetastatic niche formation. Accordingly, RXR-deficient myeloid cells are more efficient in promoting cancer cell migration and invasion. Our results suggest that the repressive activity of RXR on prometastatic genes is mediated primarily through direct DNA binding of the receptor along with nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors and is largely unresponsive to ligand activation. In addition, we found that expression and transcriptional activity of RXRα is down-modulated in peripheral blood mononuclear cells of patients with lung cancer, particularly in advanced and metastatic disease. Overall, our results identify RXR as a regulator in the myeloid cell-assisted metastatic process and establish lipid-sensing nuclear receptors in the microenvironmental regulation of tumor progression.

Immunogenic Dendritic Cell Generation from Pluripotent Stem Cells by Ectopic Expression of Runx3.

Application of dendritic cells (DCs) to prime responses to tumor Ags provides a promising approach to immunotherapy. However, only a limited number of DCs can be manufactured from adult precursors. In contrast, pluripotent embryonic stem (ES) cells represent an inexhaustible source for DC production, although it remains a major challenge to steer directional differentiation because ES cell-derived cells are typically immature with impaired functional capacity. Consistent with this notion, we found that mouse ES cell-derived DCs (ES-DCs) represented less mature cells compared with bone marrow-derived DCs. This finding prompted us to compare the gene expression profile of the ES cell- and adult progenitor-derived, GM-CSF-instructed, nonconventional DC subsets. We quantified the mRNA level of 17 DC-specific transcription factors and observed that 3 transcriptional regulators (Irf4, Spi-B, and Runx3) showed lower expression in ES-DCs than in bone marrow-derived DCs. In light of this altered gene expression, we probed the effects of these transcription factors in developing mouse ES-DCs with an isogenic expression screen. Our analysis revealed that forced expression of Irf4 repressed ES-DC development, whereas, in contrast, Runx3 improved the ES-DC maturation capacity. Moreover, LPS-treated and Runx3-activated ES-DCs exhibited enhanced T cell activation and migratory potential. In summary, we found that ex vivo-generated ES-DCs had a compromised maturation ability and immunogenicity. However, ectopic expression of Runx3 enhances cytokine-driven ES-DC development and acts as an instructive tool for the generation of mature DCs with enhanced immunogenicity from pluripotent stem cells.

Improved transgene expression in doxycycline-inducible embryonic stem cells by repeated chemical selection or cell sorting.

Transgene-mediated programming is a preeminent strategy to direct cellular identity. To facilitate cell fate switching, lineage regulating genes must be efficiently and uniformly induced. However, gene expression is often heterogeneous in transgenic systems. Consistent with this notion, a non-uniform reporter gene expression was detected in our doxycycline (DOX)-regulated, murine embryonic stem (ES) cell clones. Interestingly, a significant fraction of cells within each clone failed to produce any reporter signals upon DOX treatment. We found that the majority of these non-responsive cells neither carry reporter transgene nor geneticin/G418 resistance. This observation suggested that our ES cell clones contained non-recombined cells that survived the G418 selection which was carried out during the establishment of these clones. We successfully eliminated most of these corrupted cells with repeated chemical (G418) selection, however, even after prolonged G418 treatments, a few cells remained non-responsive due to epigenetic silencing. We found that cell sorting has been the most efficient approach to select those cells which can uniformly and stably induce the integrated transgene in this ES cell based platform. Together, our data revealed that post-cloning chemical re-selection or cell sorting strongly facilitate the production of ES cell lines with a uniform transgene induction capacity.