Cytokines released from human adipose tissue-derived stem cells by bFGF stimulation: Effects of IL-8 and CXCL-1 on wound healing.

Objectives: Adipose-derived stem cells (ADSCs) are widely used in wound care because they release a variety of cytokines. However, the molecular mechanism of paracrine action remains unclear. It has been reported that basic fibroblast growth factor (bFGF) enhances the therapeutic potential of ADSCs. In this study, we searched for cytokines whose release from ADSCs is enhanced by bFGF stimulation. Results: Quantitative RT-PCR and ELISA analyses revealed that bFGF upregulates CXCL-1 and IL-8 mRNA synthesis and secretion from ADSCs. Both cytokines showed the ability to promote important processes for wound healing, including tube formation of vascular and lymphatic endothelial cells and cell migration of fibroblasts in vitro. Conclusions: These results suggest that bFGF stimulation increases the secretion of CXCL-1 and IL-8 from ADSCs and that these cytokines may promote angiogenesis, lymphangiogenesis, and cell migration, leading to enhanced efficiency of wound healing.

Differential metabolic secretion between muscular dystrophy mouse-derived spindle cell sarcomas and rhabdomyosarcomas drives tumor type development.

The dystrophin gene (Dmd) is recognized for its significance in Duchenne muscular dystrophy (DMD), a lethal and progressive skeletal muscle disease. Some patients with DMD and model mice with muscular dystrophy (mdx) spontaneously develop various types of tumors, among which rhabdomyosarcoma (RMS) is the most prominent. By contrast, spindle cell sarcoma (SCS) has rarely been reported in patients or mdx mice. In this study, we aimed to use metabolomics to better understand the rarity of SCS development in mdx mice. Gas chromatography-mass spectrometry was used to compare the metabolic profiles of spontaneously developed SCS and RMS tumors from mdx mice, and metabolite supplementation assays and silencing experiments were used to assess the effects of metabolic differences in SCS tumor-derived cells. The levels of 75 metabolites exhibited differences between RMS and SCS, 25 of which were significantly altered. Further characterization revealed downregulation of nonessential amino acids, including alanine, in SCS tumors. Alanine supplementation enhanced the growth, epithelial mesenchymal transition, and invasion of SCS cells. Reduction of intracellular alanine via knockdown of the alanine transporter Slc1a5 reduced the growth of SCS cells. Lower metabolite secretion and reduced proliferation of SCS tumors may explain the lower detection rate of SCS in mdx mice. Targeting of alanine depletion pathways may have potential as a novel treatment strategy.NEW & NOTEWORTHY To the best of our knowledge, SCS has rarely been identified in patients with DMD or mdx mice. We observed that RMS and SCS tumors that spontaneously developed from mdx mice with the same Dmd genetic background exhibited differences in metabolic secretion. We proposed that, in addition to dystrophin deficiency, the levels of secreted metabolites may play a role in the determination of tumor-type development in a Dmd-deficient background.

MEK inhibitors increase the mortality rate in mice with LPS-induced inflammation through IL-12-NO signaling.

Lipopolysaccharide (LPS) is an endotoxin that can cause an acute inflammatory response. Nitric oxide (NO) is one of the most important innate immune system components and is synthesized by inducible NOS (iNOS) in macrophages in response to stimulation with LPS. LPS activates the RAS-RAF-mitogen-activated protein kinase/ERK kinase (MEK)-extracellular-signal-regulated kinase (ERK) signaling cascade in macrophages. The purpose of this study was to examine how the combination of LPS and MEK inhibitors, which have been used as anticancer agents in recent years, affects inflammation. We showed that MEK inhibitors enhanced iNOS expression and NO production in LPS-stimulated mouse bone marrow-derived macrophages. A MEK inhibitor increased the mortality rate in mice with LPS-induced inflammation. The expression of the cytokine interleukin-12 (IL-12) in macrophages was enhanced by the MEK inhibitor, as shown by a cytokine array and ELISA. IL-12 enhanced iNOS expression and NO production in response to LPS. We also showed that tumor necrosis factor (TNF-α) was secreted by macrophage after stimulation with LPS and that TNF-α and IL-12 synergistically induced iNOS expression and NO production. An anti-IL-12 neutralizing antibody prevented NO production and mortality in an LPS-induced inflammation mouse model in the presence of a MEK inhibitor. These results suggest that the MEK inhibitor increases the mortality rate in mice with LPS-induced inflammation through IL-12-NO signaling.

Long-term glucocorticoid treatment increases CD204 expression by activating the MAPK pathway and enhances modified LDL uptake in murine macrophages.

Atherosclerotic plaques develop from the accumulation of macrophage-derived foam cells via the uptake of modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Although glucocorticoids are suspected to exacerbate atherosclerosis, the precise mechanisms have not been fully elucidated. We investigated the effects of long-term treatment (2 weeks) with both a natural glucocorticoid (hydrocortisone, HC, 1 µM) and a synthetic glucocorticoid (dexamethasone, Dex, 100 nM) on murine bone marrow-derived macrophages using flow cytometry and western blotting. Treatment with HC and Dex enhanced CD204 expression but not CD36 expression and acetylated LDL (Ac-LDL) uptake. Treatment with HC and Dex also induced the phosphorylation of extracellular signal-regulated kinase (ERK). The Dex-induced enhancement in CD204 expression and Ac-LDL uptake were suppressed by an inhibitor of the mitogen-activated protein kinase (MAPK)/ERK kinase. These results suggest that glucocorticoids activate the MAPK/ERK pathway, which enhances CD204 expression and results in increased uptake of Ac-LDL in macrophages. The MAPK/ERK pathway in macrophages might be a key target to prevent atherosclerosis that is worsened by glucocorticoids.

The stem cell transcription factor ZFP296 transforms NIH3T3 cells and promotes anchorage-independent growth of cancer cells.

Cancer cells and embryonic stem (ES) cells share several biological properties, suggesting that some genes expressed in ES cells may play an important role in cancer cell growth. In this study, we investigated the possible role of zinc finger protein 296 (ZFP296), a transcription factor expressed in ES cells, in cancer development. First, we found that overexpression of Zfp296 in NIH3T3 mouse fibroblasts induced two phenomena indicative of cell transformation: enhanced proliferation under low-serum conditions and anchorage-independent growth. We also found that Zfp296 expression was upregulated in the tumor area of a mouse model of colon carcinogenesis. In addition, the expression levels of ZFP296 in various human cell lines were generally low in normal cells and relatively high in cancer cells. Finally, using a soft agar assay, we found that overexpression of ZFP296 promoted the anchorage-independent growth of cancer cells, while its knockdown had the opposite effect. Overall, these results suggest a possible role of the ES-specific transcription factor ZFP296 in cancer.

Ionizing radiation induces cutaneous lipid remolding and skin adipocytes confer protection against radiation-induced skin injury.

BACKGROUND: Radiation-induced skin injury is a serious concern during radiotherapy and radiation accidents. Skin fat represents the dominant architectural component of the human skin. However, the interplay between skin fat and the progression of radiation-induced skin injury remains largely unexplored. OBJECTIVE: This study aims to elucidate the interplay between skin fat and the progression of radiation-induced skin injury. METHODS: SD rats were irradiated with an electron beam. mRNA profiles were determined by RNA-Seq. The skin lipid mass was monitored by magnetic resonance imaging (MRI) and lipid profiles were measured by liquid chromatography-mass spectrometry (LC-MS). Human mature adipocytes isolated from dermal and subcutaneous white adipose tissues (WATs) were co-cultured with human keratinocytes (HaCaT) and skin fibroblasts (WS1) in the transwell culture system. Cell migration ability was measured by migration assay. RESULTS: Radiation modulated cutaneous lipid metabolism by downregulating multiple pathways. Moreover, radiation decreased skin fat mass with altered lipid metabolite profiles. The rats fed with a high-fat diet showed resistance to radiogenic skin injury compared with that with a control diet, indicating that skin lipid plays a radioprotective role. Mature adipocytes promoted the migration but not the proliferation of co-cultured skin keratinocytes and fibroblasts. Palmitic acid, the most abundant fatty acid in skin tissues, facilitated the migration of WS1 cells. Moreover, fatty acid-binding protein 4 (FABP4) could be incorporated into skin cells and promote DNA damage repair in irradiated skin fibroblasts. CONCLUSION: Radiation induces cutaneous lipid remolding, and skin adipocytes confer a protective role against radiation-induced skin injury.

The Embryonic Stem Cell-Specific Transcription Factor ZFP57 Promotes Liver Metastasis of Colorectal Cancer.

BACKGROUND: The embryonic stem cell-specific transcription factor, ZFP57, has been shown to play an important role in tumor formation. In this study, we examined if ZFP57 is involved in colorectal cancer metastasis. MATERIALS AND METHODS: First, we used colorectal cancer cell lines to perform in vivo metastatic experiments with nude mice. Next, we carried out immunohistochemical analysis of clinical specimens of colorectal cancers. RESULTS: In liver metastatic experiments using human colorectal cancer HT29 and HCT116 cells, liver polymetastases occurred at high frequency in ZFP57-overexpressing HT29 and HCT116 cells, whereas both control cells only resulted in oligometastases. Next, we analyzed ZFP57 expression using clinical specimens. Liver metastasis-positive cases were more frequently associated with ZFP57 overexpression than negative cases in primary lesions of colorectal cancer, and the overexpression was particularly remarkable in tumor invasive lesions. Furthermore, ZFP57 overexpression was significantly correlated not only with liver metastasis but also with lymph node metastasis. In addition, the expression level of ZFP57 was significantly correlated with that of the metastasis-related gene NANOG. We also found that ZFP57 overexpression reduced the progression-free survival rate of patients with colorectal cancer. CONCLUSIONS: This study demonstrated that ZFP57 plays an important role in the hematogenous metastasis of colorectal cancer, suggesting that it could be used as a novel treatment target.

Possible Role of NADPH Oxidase 4 in Angiotensin II-Induced Muscle Wasting in Mice.

Background: Muscle wasting is a debilitating phenotype associated with chronic heart failure (CHF). We have previously demonstrated that angiotensin II (AII) directly induces muscle wasting in mice through the activation of NADPH oxidase (Nox). In this study, we tested the hypothesis that deficiency of NADPH oxidase 4 (Nox4), a major source of oxidative stress, ameliorates AII-induced muscle wasting through the regulation of redox balance. Methods and Results: Nox4 knockout (KO) and wild-type (WT) mice were used. At baseline, there were no differences in physical characteristics between the WT and KO mice. Saline (vehicle, V) or AII was infused via osmotic minipumps for 4 weeks, after which, the WT + AII mice showed significant increases in Nox activity and NOX4 protein compared with the WT + V mice, as well as decreases in body weight, gastrocnemius muscle weight, and myocyte cross-sectional area. These changes were significantly attenuated in the KO + AII mice (27 ± 1 vs. 31 ± 1 g, 385 ± 3 vs. 438 ± 13 mg, and 1,330 ± 30 vs. 2281 ± 150 µm2, respectively, all P < 0.05). The expression levels of phospho-Akt decreased, whereas those of muscle RING Finger-1 (MuRF-1) and MAFbx/atrogin-1 significantly increased in the WT + AII mice compared with the WT + V mice. Furthermore, nuclear factor erythroid-derived 2-like 2 (Nrf2) and the expression levels of Nrf2-regulated genes significantly decreased in the WT + AII mice compared with the WT + V mice. These changes were significantly attenuated in the KO + AII mice (P < 0.05). Conclusion: Nox4 deficiency attenuated AII-induced muscle wasting, partially through the regulation of Nrf2. The Nox4-Nrf2 axis may play an important role in the development of AII-induced muscle wasting.

Cortisol overproduction results from DNA methylation of CYP11B1 in hypercortisolemia.

Adrenocortical hormone excess, due to primary aldosteronism (PA) or hypercortisolemia, causes hypertension and cardiovascular complications. In PA, hypomethylation of aldosterone synthase (CYP11B2) is associated with aldosterone overproduction. However, in hypercortisolemia, the role of DNA methylation of 11ß-hydroxylase (CYP11B1), which catalyzes cortisol biosynthesis and is highly homologous to CYP11B2, is unclear. The aims of our study were to determine whether the CYP11B1 expression was regulated through DNA methylation in hypercortisolemia with cortisol-producing adenoma (CPA), and to investigate a possible relationship between DNA methylation and somatic mutations identified in CPA. Methylation analysis showed that the CYP11B1 promoter was significantly less methylated in CPA than in adjacent unaffected adrenal tissue and white blood cells. Furthermore, in CPA with somatic mutations in either the catalytic subunit of protein kinase A (PRKACA) or the guanine nucleotide-binding protein subunit alpha (GNAS) gene, the CYP11B1 promoter was significantly hypomethylated. In addition, DNA methylation reduced CYP11B1 promoter activity using a reporter assay. Our study results suggest that DNA methylation at the CYP11B1 promoter plays a role in the regulation of CYP11B1 expression and cortisol production in CPA, and that somatic mutations associated with CPA reduce DNA methylation at the CYP11B1 promoter.

Esrrb directly binds to Gata6 promoter and regulates its expression with Dax1 and Ncoa3.

Estrogen-related receptor beta (Esrrb) is expressed in embryonic stem (ES) cells and is involved in self-renewal ability and pluripotency. Previously, we found that Dax1 is associated with Esrrb and represses its transcriptional activity. Further, the disruption of the Dax1-Esrrb interaction increases the expression of the extra-embryonic endoderm marker Gata6 in ES cells. Here, we investigated the influences of Esrrb and Dax1 on Gata6 expression. Esrrb overexpression in ES cells induced endogenous Gata6 mRNA and Gata6 promoter activity. In addition, the Gata6 promoter was found to contain the Esrrb recognition motifs ERRE1 and ERRE2, and the latter was the responsive element of Esrrb. Associations between ERRE2 and Esrrb were then confirmed by biotin DNA pulldown and chromatin immunoprecipitation assays. Subsequently, we showed that Esrrb activity at the Gata6 promoter was repressed by Dax1, and although Dax1 did not bind to ERRE2, it was associated with Esrrb, which directly binds to ERRE2. In addition, the transcriptional activity of Esrrb was enhanced by nuclear receptor co-activator 3 (Ncoa3), which has recently been shown to be a binding partner of Esrrb. Finally, we showed that Dax1 was associated with Ncoa3 and repressed its transcriptional activity. Taken together, the present study indicates that the Gata6 promoter is activated by Esrrb in association with Ncoa3, and Dax1 inhibited activities of Esrrb and Ncoa3, resulting maintenance of the undifferentiated status of ES cells.

ETS-related transcription factors ETV4 and ETV5 are involved in proliferation and induction of differentiation-associated genes in embryonic stem (ES) cells.

The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5 (Etv4/5 double knock-out (dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5 dKO ES cells. The artificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells.

Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells.

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, have a characteristic cell cycle with truncated G1 and G2 phases. Recent findings that suppression of Oct3/4 expression results in a reduced proliferation rate of ES cells suggest the involvement of Oct3/4 in the regulation of ES cell growth, although the underlying molecular mechanism remains unclear. In the present study, we identified E2F3a as a direct target gene of Oct3/4 in ES cells. Oct3/4 directly bound to the promoter region of the E2F3a gene and positively regulated expression of E2F3a in mouse ES cells. Suppression of E2F3a activity by E2F6 overexpression led to the reduced proliferation in ES cells, which was relieved by co-expression of E2F3a. Furthermore, cell growth retardation caused by loss of Oct3/4 was rescued by E2F3a expression. These results suggest that Oct3/4 upregulates E2F3a expression to promote ES cell growth.

Nanog positively regulates Zfp57 expression in mouse embryonic stem cells.

To maintain the self-renewal of embryonic stem (ES) cells, several core transcription factors, including Oct3/4, STAT3, and Nanog, regulate the expression of their target genes. Zinc finger protein 57 (Zfp57) is specifically expressed in self-renewing ES cells and its expression level is reduced upon ES cell differentiation, suggesting that expression of this transcription factor is regulated by core transcription factors. In the present study, we investigated whether Zfp57 expression is regulated by Nanog. Nanog overexpression resulted in the upregulation of Zfp57. On the other hand, knockdown of Nanog reduced the expression level of Zfp57. In addition, we identified the Nanog-responsive region in the promoter of the Zfp57 gene. These results suggest that Nanog is an upstream regulator of Zfp57. Moreover, Nanog overexpression promoted the growth of ES cells in soft agar and this was suppressed by Zfp57 knockdown, suggesting that the Nanog/Zfp57 pathway plays a central role in anchorage-independent growth of ES cells. Interestingly, NANOG overexpression also led to the upregulation of ZFP57 in two human tumor cell lines. Taken together, our results suggest that Nanog positively regulates Zfp57 expression in multiple types of cells.

Zfp296 is a novel Klf4-interacting protein and functions as a negative regulator.

Pluripotency and self-renewing ability of embryonic stem (ES) cells are regulated by several transcription factors, including Oct3/4, Sox2, Kruppel-like factor 4 (Klf4), and c-Myc. These transcription factors reprogram somatic cells into induced pluripotent stem (iPS) cells. Zinc finger protein (Zfp) 296 has been reported to enhance iPS cell formation. Here we found that Zfp296 interacts with Klf4. A maltose-binding protein pull-down assay demonstrated that Klf4 binds to the Zfp296 158-483 amino acid region, and that Zfp296 binds to the Klf4 DNA-binding domain (DBD). A quantitative reverse transcription-polymerase chain reaction analysis revealed that expression of Zfp296 and Klf4 decreased during differentiation of E14 and ZHBTc4 ES cells. We also found that green fluorescent protein-labeled Zfp296 and Klf4 were localized to the nucleus. Because Zfp296 bound to the Klf4 DBD, we next examined the influence of Zfp296 on Klf4 DNA-binding activity. A biotin DNA pull-down assay showed that Klf4 binds to the Lefty1 promoter region, and that binding activity was sustained even in the presence of Zfp296. In contrast, a reporter assay showed that the Lefty1 promoter was activated by Klf4, and that the enhanced activity was repressed by Zfp296. These findings suggest that Zfp296 is a functional regulator of Klf4 in ES cells.

Dax1 associates with Esrrb and regulates its function in embryonic stem cells.

Self-renewal capacity and pluripotency, which are controlled by the Oct3/4-centered transcriptional regulatory network, are major characteristics of embryonic stem (ES) cells. Nuclear hormone receptor Dax1 is one of the crucial factors in the network. Here, we identified an orphan nuclear receptor, Esrrb (estrogen-related receptor beta), as a Dax1-interacting protein. Interaction of Dax1 and Esrrb was mediated through LXXLL motifs of Dax1 and the activation- and ligand-binding domains of Esrrb. Furthermore, Esrrb enhanced the promoter activity of the Dax1 gene via direct binding to Esrrb-binding site 1 (ERRE1, where "ERRE" represents "Esrrb-responsive element") of the promoter. Expression of Dax1 was suppressed followed by Oct3/4 repression; however, overexpression of Esrrb maintained expression of Dax1 even in the absence of Oct3/4, indicating that Dax1 is a direct downstream target of Esrrb and that Esrrb can regulate Dax1 expression in an Oct3/4-independent manner. We also found that the transcriptional activity of Esrrb was repressed by Dax1. Furthermore, we revealed that Oct3/4, Dax1, and Esrrb have a competitive inhibition capacity for each complex. These data, together with previous findings, suggest that Dax1 functions as a negative regulator of Esrrb and Oct3/4, and these molecules form a regulatory loop for controlling the pluripotency and self-renewal capacity of ES cells.

RYBP-PRC1 complexes mediate H2A ubiquitylation at polycomb target sites independently of PRC2 and H3K27me3.

Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.

WITHDRAWN: Involvement of Gli proteins in undifferentiated state maintenance and proliferation of embryonic stem cells.

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Eed/Sox2 regulatory loop controls ES cell self-renewal through histone methylation and acetylation.

Transcription factors and epigenetic modulators are involved in the maintenance of self-renewal in embryonic stem (ES) cells. Here, we demonstrate the existence of a regulatory loop in ES cells between Sox2, an indispensable transcription factor for self-renewal, and embryonic ectoderm development (Eed), an epigenetic modulator regulating histone methylation. We found that Sox2 and Eed positively regulate each other's expression. Interestingly, Sox2 overexpression suppressed the induction of differentiation-associated genes in Eed-deficient ES cells without restoring histone methylation. This Sox2-mediated suppression was prevented by knockdown of the histone acetyltransferase (HAT), Tip60 or Elp3, and Sox2 stimulated expression of these HATs. Furthermore, forced expression of either HAT resulted in repression of differentiation-associated genes in Eed-deficient cells. These results suggest that Sox2 overcame the phenotype of Eed-deficient ES cells by promoting histone acetylation. We also found that knockout of Eed and knockdown of these HATs synergistically enhanced the upregulation of differentiation-associated genes in ES cells. Taken together, our results suggest that the Eed/Sox2 regulatory loop contributes to the maintenance of self-renewal in ES cells by controlling histone methylation and acetylation.

ß-catenin enhances Oct-4 activity and reinforces pluripotency through a TCF-independent mechanism.

Understanding the mechanisms regulating pluripotency in embryonic and induced pluripotent stem cells is required to ensure their safe use in clinical applications. Glycogen synthase kinase-3 (GSK-3) has emerged as an important regulator of pluripotency, based primarily on studies with small-molecule GSK-3 inhibitors. Here, we use mouse embryonic stem cells (ESCs) lacking GSK-3 to demonstrate that a single GSK-3 substrate, ß-catenin, controls the ability of ESCs to exit the pluripotent state and to differentiate into neurectoderm. Unexpectedly, the effects of ß-catenin on pluripotency do not appear to be dependent on TCF-mediated signaling, based on experiments utilizing a ß-catenin C-terminal truncation mutant or highly efficient dominant-negative TCF strategies. Alternatively, we find that stabilized ß-catenin forms a complex with and enhances the activity of Oct-4, a core component of the transcriptional network regulating pluripotency. Collectively, our data suggest previously underappreciated, divergent TCF-dependent and TCF-independent roles for ß-catenin in ESCs.