RE-1 silencing transcription factor (REST): a regulator of neuronal development and neuronal/endocrine function.

RE-1 silencing transcription factor (REST) is a transcriptional repressor that has been proposed to function as a master negative regulator of neurogenesis, as REST target genes encode neuronal receptors, ion channels, neuropeptides and synaptic proteins. During neuronal differentiation, REST expression levels are reduced, allowing expression of selected REST target genes. The analysis of neural stem/progenitor cells that are either devoid of REST or overexpress REST revealed that REST is not the master regulator that is solely responsible for the acquisition of the neuronal fate. Rather, REST provides a regulatory hub that coordinately regulates multiple tiers of neuronal development in vitro. In addition, REST may play an important role for maintaining the integrity of adult neurons. REST confers oxidative stress resistance and is essential for maintaining neuronal viability. Furthermore, the concentration of REST has been reported to influence the pathogenic outcome by neuronal diseases, including stroke, epilepsy and Alzheimer's disease. Experiments performed with PC12 pheochromocytoma cells indicate that REST may function as a key regulator of the neurosecretory phenotype. Moreover, transgenic mice overexpressing REST in pancreatic ß-cells showed impaired insulin secretion leading to significantly reduced plasma insulin levels. Based on the fact that REST plays a prominent role in controlling stimulus-induced secretion in endocrine cells, we propose that REST may also be important for neurotransmitter release via regulation of genes that encode important proteins of the exocytotic machinery.

Chromatin structure and expression of the AMPA receptor subunit Glur2 in human glioma cells: major regulatory role of REST and Sp1.

It has been suggested that reduced glutamate receptor expression protects glioma cells from glutamate toxicity. GluR2 is the critical subunit of the GluR2 subtype of AMPA glutamate receptors as this subunit determines the Ca(2+) permeability of the receptor. The gene encoding the GluR2 subtype of AMPA receptors has been described as a target gene for the transcription repressor REST. However, we recently showed that the GluR2 gene is not regulated by REST in several neuronal and neuroendocrine cell lines, due to a repressive chromatin environment. Here, we show that the GluR2 gene has an open chromatin configuration in human glioma cells. Overexpression of REST reduced GluR2 mRNA levels while shRNA-mediated depletion of REST or expression of a REST mutant, that contained a transcriptional activation domain, enhanced GluR2 expression. Incubation with trichostatin A (TSA), a histone deacetylase inhibitor, induced acetylation of histone 4 of the GluR2 locus in glioma cells, leading to an upregulation of GluR2 expression. Together, these data suggest that REST is responsible for the reduced expression of GluR2 in glioma cells. The transcription factor Sp1 additionally binds under physiological conditions to the GluR2 gene in human glioma cells and expression of a dominant-negative mutant of Sp1 reduced expression of GluR2. Thus, the regulation via Sp1 represents a further control point for GluR2 expression in glioma cells. Together, we show that the GluR2 gene is embedded into an open chromatin configuration in glioma cells and expression of GluR2 is controlled by REST and Sp1.

rAAV Vectors as Safe and Efficient Tools for the Stable Delivery of Genes to Primary Human Chondrosarcoma Cells In Vitro and In Situ.

Treatment of chondrosarcoma remains a major challenge in orthopaedic oncology. Gene transfer strategies based on recombinant adenoassociated viral (rAAV) vectors may provide powerful tools to develop new, efficient therapeutic options against these tumors. In the present study, we tested the hypothesis that rAAV is adapted for a stable and safe delivery of foreign sequences in human chondrosarcoma tissue by transducing primary human chondrosarcoma cells in vitro and in situ with different reporter genes (E. coli lacZ, firefly luc, Discosoma sp. RFP). The effects of rAAV administration upon cell survival and metabolic activities were also evaluated to monitor possibly detrimental effects of the gene transfer method. Remarkably, we provide evidence that efficient and prolonged expression of transgene sequences via rAAV can be safely achieved in all the systems investigated, demonstrating the potential of the approach of direct application of therapeutic gene vectors as a means to treat chondrosarcoma.

Regulation of cellular proliferation, differentiation and cell death by activated Raf.

The protein kinases Raf-1, A-Raf and B-Raf connect receptor stimulation with intracellular signaling pathways and function as a central intermediate in many signaling pathways. Gain-of-function experiments shed light on the pleiotropic biological activities of these enzymes. Expression experiments involving constitutively active Raf revealed the essential functions of Raf in controlling proliferation, differentiation and cell death in a cell-type specific manner.

Chromatin structure and expression of synapsin I and synaptophysin in retinal precursor cells.

Synapsin I and synaptophysin are major proteins of small synaptic vesicles. In neurons the transcriptional repressor REST is a major regulator of synapsin I and synaptophysin gene transcription. Gene regulation by REST is influenced by the configuration of the chromatin and cell type specific variations have been observed. Here, we have investigated the regulation of the synapsin I and synaptophysin genes in R28 retinal precursor cells. Chromatin immunoprecipitation experiments revealed that both genes are embedded in open chromatin in R28 retinal precursor cells. In contrast, in fibroblasts the synapsin I and synaptophysin genes are found in nucleosomes that carried an epigenetic marker that is linked to a condensed form of chromatin and gene silencing. Synapsin I and synaptophysin gene expression in retinal precursor cells was enhanced following inhibition of histone deacetylase activity, indicating that these genes are regulated via histone acetylation/deacetylation in R28 cells. Inhibition of histone deacetylase activity did not induce synapsin I and synaptophysin expression in fibroblasts, indicating that these genes are silenced in this cell type in a histone acetylation/deacetylation-independent manner. Moreover, elevated levels of synapsin I and synaptophysin mRNA were found in retinal precursor cells that expressed a mutant of REST that activated gene transcription. In contrast, the ribeye gene, encoding a major structural protein of synaptic ribbons, was neither regulated by histone acetylation/deacetylation nor by the REST mutant in retinal precursor cells. These data reveal that the synapsin I and synaptophysin genes are bona fide target genes for REST in R28 retinal precursor cells.

SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells.

INTRODUCTION: Transplantation of genetically modified human bone marrow-derived mesenchymal stem cells (hMSCs) with an accurate potential for chondrogenic differentiation may be a powerful means to enhance the healing of articular cartilage lesions in patients. Here, we evaluated the benefits of delivering SOX9 (a key regulator of chondrocyte differentiation and cartilage formation) via safe, maintained, replication-defective recombinant adeno-associated virus (rAAV) vector on the capability of hMSCs to commit to an adequate chondrocyte phenotype compared with other mesenchymal lineages. METHODS: The rAAV-FLAG-hSOX9 vector was provided to both undifferentiated and lineage-induced MSCs freshly isolated from patients to determine the effects of the candidate construct on the viability, biosynthetic activities, and ability of the cells to enter chondrogenic, osteogenic, and adipogenic differentiation programs compared with control treatments (rAAV-lacZ or absence of vector administration). RESULTS: Marked, prolonged expression of the transcription factor was noted in undifferentiated and chondrogenically differentiated cells transduced with rAAV-FLAG-hSOX9, leading to increased synthesis of major extracellular matrix components compared with control treatments, but without effect on proliferative activities. Chondrogenic differentiation (SOX9, type II collagen, proteoglycan expression) was successfully achieved in all types of cells but strongly enhanced when the SOX9 vector was provided. Remarkably, rAAV-FLAG-hSOX9 delivery reduced the levels of markers of hypertrophy, terminal and osteogenic/adipogenic differentiation in hMSCs (type I and type X collagen, alkaline phosphatase (ALP), matrix metalloproteinase 13 (MMP13), and osteopontin (OP) with diminished expression of the osteoblast-related transcription factor runt-related transcription factor 2 (RUNX2); lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma 2 (PPARG2)), as well as their ability to undergo proper osteo-/adipogenic differentiation. These effects were accompanied with decreased levels of ß-catenin (a mediator of the Wnt signaling pathway for osteoblast lineage differentiation) and enhanced parathyroid hormone-related protein (PTHrP) expression (an inhibitor of hypertrophic maturation, calcification, and bone formation) via SOX9 treatment.

Human mesenchymal stem cells overexpressing therapeutic genes: from basic science to clinical applications for articular cartilage repair.

Adult articular cartilage has a limited capacity for self repair. Reproduction of a native structure and functional integrity in damaged cartilage remains a major problem in orthopaedic surgery. Strategies based on the implantation of genetically modified cells to sites of injury may provide workable options to treat articular cartilage lesions like those resulting from acute trauma or associated with the progression of osteoarthritis. Mesenchymal stem cells have remarkable properties that make them an attractive source of cells to treat cartilage disorders due to their self-renewal capability, stemness maintenance, and chondrogenic differentiation potential. For these reasons, such progenitor cells might be further modified by gene transfer protocols to reinforce their potency and consequently, to enhance the healing processes in damaged tissue following transplantation in sites of cartilage injury. Here, we propose an overview of the current approaches employed for cell- and gene-based treatment of articular cartilage disorders using mesenchymal stem cells.

Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2.

The genetic manipulation of bone marrow-derived mesenchymal stem cells (MSCs) is an attractive approach to produce therapeutic platforms for settings that aim at restoring articular cartilage defects. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated overexpression of human fibroblast growth factor 2 (hFGF-2), a mitogenic factor also known to influence MSC differentiation, upon the proliferative and chondrogenic activities of human MSCs (hMSCs) in a three-dimensional environment that supports chondrogenesis in vitro. Prolonged, significant FGF-2 synthesis was noted in rAAV-hFGF-2-transduced monolayer and aggregate cultures of hMSCs, leading to enhanced, dose-dependent cell proliferation compared with control treatments (rAAV-lacZ transduction and absence of vector administration). Chondrogenic differentiation (proteoglycans, type-II collagen, and SOX9 expression) was successfully achieved in all types of aggregates, without significant difference between conditions. Most remarkably, application of rAAV-hFGF-2 reduced the expression of type-I and type-X collagen, possibly due to increased levels of matrix metalloproteinase-13, a key matrix-degrading enzyme. FGF-2 overexpression also decreased mineralization and the expression of osteogenic markers such as alkaline phosphatase, with diminished levels of RUNX-2, a transcription factor for osteoblast-related genes. Altogether, the present findings show the ability of rAAV-mediated FGF-2 gene transfer to expand hMSCs with an advantageous differentiation potential for future, indirect therapeutic approaches that aim at treating articular cartilage defects in vivo.

Transcription of genes encoding synaptic vesicle proteins in human neural stem cells: chromatin accessibility, histone methylation pattern, and the essential role of rest.

Human HNSC.100 neural stem cells up-regulate expression of GFAP following withdrawal of mitogens. Activation of the ERK signaling pathway prevented the up-regulation of GFAP expression. Incubation of cells with retinoic acid in the absence of mitogens enhanced basal neuronal differentiation that was accompanied by an up-regulation of neuronal gene expression and a down-regulation of GFAP and nestin expression. Retinoic acid treatment changed the histone code of neuronal genes encoding synapsin I, synaptophysin, and synaptotagmins II, IV, and VII from a transcriptionally inactive (methylation of lysine residue 9 of histone 3) to a transcriptionally active state (methylation of lysine residue 4 of histone 3). In contrast, the chromatin structure of the GFAP gene is transformed from a transcriptionally active state in unstimulated neural stem cells to a transcriptionally inactive state in retinoic acid-stimulated cells. Additionally, retinoic acid treatment reduced the binding of histone deacetylase-1 and REST to neuronal genes. The inhibition of histone deacetylase activity induced expression of genes encoding synaptic vesicle proteins in unstimulated neural stem cells. Similarly, neuronal gene transcription was enhanced following expression of a mutant of REST that contained a transcriptional activation domain. These data indicate that in undifferentiated human neural stem cells, neuronal genes encoding synaptic vesicle proteins are accessible for the REST mutant and are sensitive to enhanced histone acetylation.