The Role of Embryonic Stem Cell-expressed RAS (ERAS) in the Maintenance of Quiescent Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) were recently identified as liver-resident mesenchymal stem cells. HSCs are activated after liver injury and involved in pivotal processes, such as liver development, immunoregulation, regeneration, and also fibrogenesis. To date, several studies have reported candidate pathways that regulate the plasticity of HSCs during physiological and pathophysiological processes. Here we analyzed the expression changes and activity of the RAS family GTPases and thereby investigated the signaling networks of quiescent HSCs versus activated HSCs. For the first time, we report that embryonic stem cell-expressed RAS (ERAS) is specifically expressed in quiescent HSCs and down-regulated during HSC activation via promoter DNA methylation. Notably, in quiescent HSCs, the high level of ERAS protein correlates with the activation of AKT, STAT3, mTORC2, and HIPPO signaling pathways and inactivation of FOXO1 and YAP. Our data strongly indicate that in quiescent HSCs, ERAS targets AKT via two distinct pathways driven by PI3Kα/δ and mTORC2, whereas in activated HSCs, RAS signaling shifts to RAF-MEK-ERK. Thus, in contrast to the reported role of ERAS in tumor cells associated with cell proliferation, our findings indicate that ERAS is important to maintain quiescence in HSCs.

Bile acids and stellate cells.

Hepatic stellate cells are mainly known for their contribution to fibrogenesis in chronic liver diseases, but their identity and function in normal liver remain unclear. They were recently identified as liver-resident mesenchymal stem cells (MSCs), which can differentiate not only into adipocytes and osteocytes, but also into liver epithelial cells such as hepatocytes and bile duct cells as investigated in vitro and in vivo. During hepatic differentiation, stellate cells and other MSCs transiently develop into liver progenitor cells with epithelial characteristics before hepatocytes are established. Transplanted stellate cells from the liver and pancreas are able to contribute to liver regeneration in stem cell-based liver injury models and can also home into the bone marrow, which is in line with their classification as MSCs. There is experimental evidence that bile acids support liver regeneration and are able to activate signaling pathways in hepatic stellate cells. For this reason, it is important to analyze the influence of bile acids on developmental fate decisions of hepatic stellate cells and other MSC populations.

Hepatic stellate cells support hematopoiesis and are liver-resident mesenchymal stem cells.

BACKGROUND/AIMS: Hematopoiesis can occur in the liver, when the bone marrow fails to provide an adequate environment for hematopoietic stem cells. Hepatic stellate cells possess characteristics of stem/progenitor cells, but their contribution to hematopoiesis is not known thus far. METHODS: Isolated hepatic stellate cells from rats were characterized with respect to molecular markers of bone marrow mesenchymal stem cells (MSC) and treated with adipocyte or osteocyte differentiation media. Stellate cells of rats were further co-cultured with murine stem cell antigen-1(+) hematopoietic stem cells selected by magnetic cell sorting. The expression of murine hematopoietic stem cell markers was analyzed by mouse specific quantitative PCR during co-culture. Hepatic stellate cells from eGFP(+) rats were transplanted into lethally irradiated wild type rats. RESULTS: Desmin-expressing stellate cells were associated with hematopoietic sites in the fetal rat liver. Hepatic stellate cells expressed MSC markers and were able to differentiate into adipocytes and osteocytes in vitro. Stellate cells supported hematopoietic stem/progenitor cells during co-culture similar to bone marrow MSC, but failed to differentiate into blood cell lineages after transplantation. CONCLUSION: Hepatic stellate cells are liver-resident MSC and can fulfill typical functions of bone marrow MSC such as the differentiation into adipocytes or osteocytes and support of hematopoiesis.

Frequent promoter hypermethylation of Wnt pathway inhibitor genes in malignant astrocytic gliomas.

Aberrant activation of wingless (Wnt) signaling is involved in the pathogenesis of various cancers. Recent studies suggested a role of Wnt signaling in gliomas, the most common primary brain tumors. We investigated 70 gliomas of different malignancy grades for promoter hypermethylation in 8 genes encoding members of the secreted frizzled-related protein (SFRP1, SFRP2, SFRP4, SFRP5), dickkopf (DKK1, DKK3) and naked (NKD1, NKD2) families of Wnt pathway inhibitors. All tumors were additionally analyzed for mutations in exon 3 of the beta-catenin gene (CTNNB1). While none of the tumors carried CTNNB1 mutations, we found frequent promoter hypermethylation of Wnt pathway inhibitor genes, with at least one of these genes being hypermethylated in 6 of 16 diffuse astrocytomas (38%), 4 of 14 anaplastic astrocytomas (29%), 7 of 10 secondary glioblastomas (70%) and 23 of 30 primary glioblastomas (77%). Glioblastomas often demonstrated hypermethylation of 2 or more analyzed genes. Hypermethylation of SFRP1, SFRP2 and NKD2 each occurred in more than 40% of the primary glioblastomas, while DKK1 hypermethylation was found in 50% of secondary glioblastomas. Treatment of SFRP1-, SFRP5-, DKK1-, DKK3-, NKD1- and NKD2-hypermethylated U87-MG glioblastoma cells with 5-aza-2'-deoxycytidine and trichostatin A resulted in increased expression of each gene. Furthermore, SFRP1-hypermethylated gliomas showed significantly lower expression of the respective transcripts when compared with unmethylated tumors. Taken together, our results suggest an important role of epigenetic silencing of Wnt pathway inhibitor genes in astrocytic gliomas, in particular, in glioblastomas, with distinct patterns of hypermethylated genes distinguishing primary from secondary glioblastomas.

ECRG4 is a candidate tumor suppressor gene frequently hypermethylated in colorectal carcinoma and glioma.

BACKGROUND: Cancer cells display widespread changes in DNA methylation that may lead to genetic instability by global hypomethylation and aberrant silencing of tumor suppressor genes by focal hypermethylation. In turn, altered DNA methylation patterns have been used to identify putative tumor suppressor genes. METHODS: In a methylation screening approach, we identified ECRG4 as a differentially methylated gene. We analyzed different cancer cells for ECRG4 promoter methylation by COBRA and bisulfite sequencing. Gene expression analysis was carried out by semi-quantitative RT-PCR. The ECRG4 coding region was cloned and transfected into colorectal carcinoma cells. Cell growth was assessed by MTT and BrdU assays. ECRG4 localization was analyzed by fluorescence microscopy and Western blotting after transfection of an ECRG4-eGFP fusion gene. RESULTS: We found a high frequency of ECRG4 promoter methylation in various cancer cell lines. Remarkably, aberrant methylation of ECRG4 was also found in primary human tumor tissues, including samples from colorectal carcinoma and from malignant gliomas. ECRG4 hypermethylation associated strongly with transcriptional silencing and its expression could be re-activated in vitro by demethylating treatment with 5-aza-2'-deoxycytidine. Overexpression of ECRG4 in colorectal carcinoma cells led to a significant decrease in cell growth. In transfected cells, ECRG4 protein was detectable within the Golgi secretion machinery as well as in the culture medium. CONCLUSIONS: ECRG4 is silenced via promoter hypermethylation in different types of human cancer cells. Its gene product may act as inhibitor of cell proliferation in colorectal carcinoma cells and may play a role as extracellular signaling molecule.

Laminin-521 promotes quiescence in isolated stellate cells from rat liver.

The laminin α5 protein chain is an element of basement membranes and important to maintain stem cells. Hepatic stellate cells (HSC) are liver-resident mesenchymal stem cells, which reside in a quiescent state on a basement membrane-like structure in the space of Dissé. In the present study, laminin α5 chain was detected in the space of Dissé of normal rat liver. Since HSC are critical for liver regeneration and can contribute to fibrosis in chronic liver diseases, the effect of laminins on HSC maintenance was investigated. Therefore, isolated rat HSC were seeded on uncoated polystyrene (PS) or PS coated with either laminin-521 (PS/LN-521) or laminin-211 (PS/LN-211). PS/LN-521 improved HSC adhesion and better preserved their retinoid stores as well as quiescence- and stem cell-associated phenotype, whereas HSC on PS/LN-211 or PS developed into myofibroblasts-like cells. To improve the homogeneity as well as the presentation of laminin molecules on the culture surface to HSC, laminin-functionalized, gold-nanostructured glass surfaces were generated. This approach further enhanced the expression of quiescence-associated genes in HSC. In conclusion, the results indicate that LN-521 supports the quiescent state of HSC and laminin α5 can be regarded as an important element of their niche in the space of Dissé.

Transplanted Human Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Support Liver Regeneration in Gunn Rats.

Gunn rats bear a mutation within the uridine diphosphate glucuronosyltransferase-1a1 (Ugt1a1) gene resulting in high serum bilirubin levels as seen in Crigler-Najjar syndrome. In this study, the Gunn rat was used as an animal model for heritable liver dysfunction. Induced mesenchymal stem cells (iMSCs) derived from embryonic stem cells (H1) and induced pluripotent stem cells were transplanted into Gunn rats after partial hepatectomy. The iMSCs engrafted and survived in the liver for up to 2 months. The transplanted iMSCs differentiated into functional hepatocytes as evidenced by partially suppressed hyperbilirubinemia and expression of multiple human-specific hepatocyte markers such as albumin, hepatocyte nuclear factor 4α, UGT1A1, cytokeratin 18, bile salt export pump, multidrug resistance protein 2, Na/taurocholate-cotransporting polypeptide, and α-fetoprotein. These findings imply that transplanted human iMSCs can contribute to liver regeneration in vivo and thus represent a promising tool for the treatment of inherited liver diseases.

Combined Methylome and Transcriptome Analysis During Rat Hepatic Stellate Cell Activation.

Hepatic stellate cells (HSCs) are mesenchymal stem cells (MSCs) of the liver. They are unique among MSCs, since HSCs remain in a quiescent, retinoid-storing state in the normal liver but become activated after liver injury and contribute to tissue repair. The epigenetic mechanisms accompanying the transition of HSCs from a quiescent to an activated state are in the focus of the present study. We investigated the methylome and transcriptome during this process and observed profound changes. While the promoter methylation correlated negatively with gene expression, the gene-body methylation revealed no clear correlation. Most genes with altered expression were associated with cell differentiation. Among them, Wilms tumor 1 (Wt1) and Deltex4 (Dtx4) genes were identified as epigenetically regulated. Since HSCs were reported to derive from multipotent Wt1-positive cells and many differentially expressed genes were associated with cell differentiation during their activation, epigenetic alterations are presumably required to enable HSC development.

Epigenetic Changes during Hepatic Stellate Cell Activation.

BACKGROUND AND AIMS: Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC. METHODS AND RESULTS: The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism. CONCLUSIONS: In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.

Bile acids induce hepatic differentiation of mesenchymal stem cells.

Mesenchymal stem cells (MSC) have the potential to differentiate into multiple cell lineages and their therapeutic potential has become obvious. In the liver, MSC are represented by stellate cells which have the potential to differentiate into hepatocytes after stimulation with growth factors. Since bile acids can promote liver regeneration, their influence on liver-resident and bone marrow-derived MSC was investigated. Physiological concentrations of bile acids such as tauroursodeoxycholic acid were able to initiate hepatic differentiation of MSC via the farnesoid X receptor and transmembrane G-protein-coupled bile acid receptor 5 as investigated with knockout mice. Notch, hedgehog, transforming growth factor-ß/bone morphogenic protein family and non-canonical Wnt signalling were also essential for bile acid-mediated differentiation, whereas ß-catenin-dependent Wnt signalling was able to attenuate this process. Our findings reveal bile acid-mediated signalling as an alternative way to induce hepatic differentiaion of stem cells and highlight bile acids as important signalling molecules during liver regeneration.

Histone deacetylase inhibitors induce attenuation of Wnt signaling and TCF7L2 depletion in colorectal carcinoma cells.

Histone deacetylase inhibitors (HDIs) specifically affect cancer cells by inducing cell cycle arrest, activate apoptotic pathways and re-activate epigenetically silenced tumor suppressor genes, but their pleiotropic mode of action is not fully understood. Despite the clinical effects of HDIs in the treatment of hematological malignancies, their potency against solid tumors is still unclear. We investigated the effects and mechanisms of HDI action in colorectal carcinoma cell lines with an activated Wnt signaling pathway, which is implicated in different aspects of tumorigenesis, including cell proliferation, apoptosis, angiogenesis and metastasis. We assessed the effects of HDI treatment in colorectal carcinoma cell lines by measuring histone hyperacetylation, cell viability and expression of Wnt target genes. Upon treatment with HDIs of the hydroxamate class, we found attenuation of Wnt signaling with concomitant induction of apoptosis and colorectal cancer cell death. Strikingly, the effects of HDIs on Wnt signaling were independent of histone hyperacetylation, thus we investigated the role of non-histone target proteins of histone deacetylases (HDACs). The compounds TSA and SAHA induced a rapid proteasome-dependent depletion of the Wnt transcription factor TCF7L2, which may be mediated by inhibition of HDAC 6 and 10. Our findings provide a molecular rationale for the use of HDIs against colorectal carcinomas with activated Wnt signaling.

Hepatic stellate cells contribute to progenitor cells and liver regeneration.

Retinoid-storing hepatic stellate cells (HSCs) have recently been described as a liver-resident mesenchymal stem cell (MSC) population; however, it is not clear whether these cells contribute to liver regeneration or serve as a progenitor cell population with hepatobiliary characteristics. Here, we purified HSCs with retinoid-dependent fluorescence-activated cell sorting from eGFP-expressing rats and transplanted these GFP(+) HSCs into wild-type (WT) rats that had undergone partial hepatectomy in the presence of 2-acetylaminofluorene (2AAF) or retrorsine, both of which are injury models that favor stem cell-based liver repair. Transplanted HSCs contributed to liver regeneration in host animals by forming mesenchymal tissue, progenitor cells, hepatocytes, and cholangiocytes and elevated direct bilirubin levels in blood sera of GUNN rats, indicating recovery from the hepatic bilirubin-handling defect in these animals. Transplanted HSCs engrafted within the bone marrow (BM) of host animals, and HSC-derived cells were isolated from BM and successfully retransplanted into new hosts with injured liver. Cultured HSCs transiently adopted an expression profile similar to that of progenitor cells during differentiation into bile acid-synthesizing and -transporting hepatocytes, suggesting that stellate cells represent a source of liver progenitor cells. This concept connects seemingly contradictory studies that favor either progenitor cells or MSCs as important players in stem cell-based liver regeneration.

MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation.

Background Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the molecules and pathways signaling glioma cell invasion thus appear of major relevance for the development of targeted and individualized therapies. By miRNA expression profiling of microdissected human tumor biopsy specimens we identified miR-328 as one of the main miRNAs upregulated in invading glioma cells in vivo and further investigated its role in glioma pathogenesis. Methods We employed miRNA mimics and inhibitors to functionally characterize miR-328, 3' untranslated region luciferase assays, and T-cell factor/lymphoid enhancer factor reporter assays to pinpoint miR-328 targets and signaling pathways, and analyzed miR-328 expression in a large panel of gliomas. Results First, we corroborated the invasion-promoting role of miR-328 in A172 and TP365MG glioma cells. Secreted Frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signaling, was then pinpointed as a direct miR-328 target. SFRP1 expression is of prognostic relevance in gliomas with reduced expression, being associated with significantly lower overall patient survival in both the Repository of Molecular Brain Neoplasia Data (REMBRANDT) and The Cancer Genome Atlas. Of note, miR-328 regulated both SFRP1 protein expression levels and Wnt signaling pathway activity. Finally, in human glioma tissues miR-328 appeared to account for the downregulation of SFRP1 preferentially in lower-grade astrocytic gliomas and was inversely related to SFRP1 promoter hypermethylation. Conclusion Taken together, we report on a novel molecular miR-328-dependent mechanism that via SFRP1 inhibition and Wnt activation contributes to the infiltrative glioma phenotype at already early stages of glioma progression, with unfavorable prognostic implications for the final outcome of the disease.

Stellate cells from rat pancreas are stem cells and can contribute to liver regeneration.

The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC), which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries.