Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo

Side population (SP) cells, which can be identified by their ability to exclude Hoechst 33342 dye, are one of the candidates for somatic stem cells. Although bone marrow SP cells are known to be long-term repopulating hematopoietic stem cells, there is little information about the characteristics of cardiac SP cells (CSPs). When cultured CSPs from neonatal rat hearts were treated with oxytocin or trichostatin A, some CSPs expressed cardiac-specific genes and proteins and showed spontaneous beating. When green fluorescent protein–positive CSPs were intravenously infused into adult rats, many more (∼12-fold) CSPs were migrated and homed in injured heart than in normal heart. CSPs in injured heart differentiated into cardiomyocytes, endothelial cells, or smooth muscle cells (4.4%, 6.7%, and 29% of total CSP-derived cells, respectively). These results suggest that CSPs are intrinsic cardiac stem cells and involved in the regeneration of diseased hearts

An Id-like molecule, HHM, is a synexpression group-restricted regulator of TGF-β signalling

Transforming growth factor (TGF)-β induces various cellular responses principally through Smad-dependent transcriptional regulation. Activated Smad complexes cooperate with transcription factors in regulating a group of target genes. The target genes controlled by the same Smad-cofactor complexes are denoted a synexpression group. We found that an Id-like helix-loop-helix protein, human homologue of Maid (HHM), is a synexpression group-restricted regulator of TGF-β signalling. HHM suppressed TGF-β-induced growth inhibition and cell migration but not epithelial–mesenchymal transition. In addition, HHM inhibited TGF-β-induced expression of plasminogen activator inhibitor-type 1 (PAI-1), PDGF-B, and p21WAF, but not Snail. We identified a basic-helix-loop-helix protein, Olig1, as one of the Smad-binding transcription factors affected by HHM. Olig1 interacted with Smad2/3 in response to TGF-β stimulation, and was involved in transcriptional activation of PAI-1 and PDGF-B. HHM, but not Id proteins, inhibited TGF-β signalling-dependent association of Olig1 with Smad2/3 through physical interaction with Olig1. HHM thus appears to regulate a subset of TGF-β target genes including the Olig1-Smad synexpression group. HHM is the first example of a cellular response-selective regulator of TGF-β signalling with clearly determined mechanisms

Deoxycytidine kinase inactivation enhances gemcitabine resistance and sensitizes mitochondrial metabolism interference in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment

The H3K27 demethylase, Utx, regulates adipogenesis in a differentiation stage-dependent manner.

Understanding the molecular mechanisms that drive adipogenesis is important in developing new treatments for obesity and diabetes. Epigenetic regulations determine the capacity of adipogenesis. In this study, we examined the role of a histone H3 lysine 27 demethylase, the ubiquitously transcribed tetratricopeptide repeat protein on the X chromosome (Utx), in the differentiation of mouse embryonic stem cells (mESCs) to adipocytes. Using gene trapping, we examined Utx-deficient male mESCs to determine whether loss of Utx would enhance or inhibit the differentiation of mESCs to adipocytes. Utx-deficient mESCs showed diminished potential to differentiate to adipocytes compared to that of controls. In contrast, Utx-deficient preadipocytes showed enhanced differentiation to adipocytes. Microarray analyses indicated that the β-catenin/c-Myc signaling pathway was differentially regulated in Utx-deficient cells during adipocyte differentiation. Therefore, our data suggest that Utx governs adipogenesis by regulating c-Myc in a differentiation stage-specific manner and that targeting the Utx signaling pathway could be beneficial for the treatment of obesity, diabetes, and congenital utx-deficiency disorders

PRRX1 induced by BMP signaling decreases tumorigenesis by epigenetically regulating glioma-initiating cell properties via DNA methyltransferase 3A

Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs

Effects of Utx deficiency on gene expression in mESCs and adipocytes.

<p>(A) Heat map visualizations of the 79 genes that are differentially expressed in <i>Utx</i>-deficient and control mESCs and adipocytes. Two clones were used for each group. Data for the heat maps are normalized using average linkage clustering on entities and represent median-centered log-transformed values. Red and blue correspond to high and low expression, respectively, compared with the experiment-wide median. (B) mRNA levels of <i>Mgp</i> and <i>Dcn</i> normalized to <i>β-actin</i>. The experiments were performed independently with three clones, and the results are expressed as mean ± SE (n = 3); *p<0.05. (C) GSEA enrichment score curves described with two sets of adipocytes from the Molecular Signatures Database (MSigDB). The graph at the bottom of each panel represents the ranked, ordered, non-redundant list of genes. Vertical black lines indicate the position of genes from the studied gene set in the ordered, non-redundant data set. The green curve corresponds to the enrichment score (ES) curve, which is the running sum of the weighted enrichment score obtained with the GSEA software. NES, normalized enrichment score: FDR, false discovery rate; NOM, nominal. (D, E) mRNA levels of <i>c-Myc</i> normalized to <i>β-actin</i> in (D) mESC and adipocytes, (E) mESCs, EBs, and RA-induced differentiation. The experiments were performed independently with three clones, and the results are expressed as mean ± SE. (n = 3) *p<0.05. (F) mRNA levels of <i>aP2</i> normalized to <i>Gapdh</i> in <i>Utx</i>-deficient or control mESCs and adipocytes in the presence of 10058-F4. The representative result is shown as mean ± SE. (n = 3) *p<0.05.</p

<i>Utx</i>-deficient mESCs showed diminished differentiation to adipocytes.

<p>(A) Oil-red O staining of adipocytes. Representative results from three independent mESC clones are shown. (B-F), mRNA levels of <i>aP2</i>, <i>Pparg2</i>, <i>C/EBP alpha</i>, <i>C/EBP beta</i>, <i>C/EBP delta</i> (B)<i>; Nanog</i>, <i>Sox2</i> (C); <i>Pou5f1</i>, <i>Brachyury</i> (D); <i>Prdm16</i>, <i>Ucp1</i>, <i>Pgc1 alpha</i> (E); <i>Utx</i> (F) normalized to <i>β-actin</i> in mESCs and adipocytes. The experiments were performed with three independent clones, and the results are expressed as mean ± SE (n = 3); *p<0.05.</p

Construction and characterization of <i>Utx</i>-deficient mESCs.

<p>(A) Conditional gene targeting of the murine <i>Utx</i> locus. (B) Immunoblotting for Utx and β-actin in <i>Utx</i>-deficient and control mESCs. A representative result from three independent mESC clones is shown. Band densities of Utx and β-actin were quantified by ImageQuant TL ver. 8.1 and normalized to β-actin. The data are presented relative to the signal of control mESCs. (C) Immunoblotting for H3, H3K4me3, H3K9me3, H3K27me3, and H3K36me3 in mESCs. A representative result from three independent mESC clones is shown. Band density of histones was quantified by ImageQuant TL ver. 8.1 (GE healthcare life sciences, Pittsburgh, PA, USA), and data are shown by the relative value of histone methylations normalized to H3.</p