Low FasL levels promote proliferation of human bone marrow-derived mesenchymal stem cells, higher levels inhibit their differentiation into adipocytes.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that can differentiate into several cell types. Bone marrow (BM)-MSCs mainly differentiate into osteoblasts or adipocytes. MSC interactions with their microenvironment directly affect their self-renewal/differentiation program. Here, we show for the first time that Fas ligand (FasL), a well-explored proapoptotic cytokine, can promote proliferation of BM-derived MSCs in vitro and inhibits their differentiation into adipocytes. BM-MSCs treated with a low FasL dose (0.5 ng/ml) proliferated more rapidly than untreated cells without undergoing spontaneous differentiation or apoptosis, whereas higher doses (25 ng/ml) induced significant though not massive BM-MSC death, with surviving cells maintaining a stem cell phenotype. At the molecular level, 0.5 ng/ml FasL induced ERK1/2 phosphorylation and survivin upregulation, whereas 25 ng/ml FasL induced caspase activation. Importantly, 25 ng/ml FasL reversibly prevented BM-MSC differentiation into adipocytes by modulating peroxisome proliferator-activated receptor gamma (PPARγ) and FABP4/aP2 expression induced by adipogenic medium. All such effects were inhibited by anti-Fas neutralizing antibody. The in vitro data regarding adipogenesis were confirmed using Fas(lpr) mutant mice, where higher PPARγ and FABP4/aP2 mRNA and protein levels were documented in whole tibia. These data show for the first time that the FasL/Fas system can have a role in BM-MSC biology via regulation of both proliferation and adipogenesis, and may have clinical relevance because circulating Fas/FasL levels decline with age and several age-related conditions, including osteoporosis, are characterized by adipocyte accumulation in BM.

Dexamethasone affects Fas- and serum deprivation-induced cell death of human osteoblastic cells through survivin regulation.

Glucocorticoid-induced bone loss is the most prevalent form of secondary osteoporosis. Such loss could be due to the alteration of osteoclast and osteoblast lifespan through regulated apoptosis. The current study investigated the effect of dexamethasone on Fas- and starvation-induced apoptosis of mature osteoblasts and their precursors. Using the human osteoblastic hFOB1.19 and the MG63 osteosarcoma cell lines, we found that sub-lethal doses of dexamethasone act on pre-osteoblasts but not on mature cells by increasing their susceptibility to apoptosis. Apoptosis occurs in a caspase-dependent manner as both DNA fragmentation and mitochondrial transmembrane potential dissipation (ΔΨm) are inhibited by the pan-caspase inhibitor zVAD. The increased susceptibility of osteoblast precursors to apoptosis could be due to dexamethasonemediated down-regulation of survivin expression. Dexamethasone can up-regulate survivin, and to a lesser extent Bcl-2, in mature cells but not in pre-osteoblasts. In addition, it can induce FLIP over-expression in osteosarcoma cells. All these effects are inhibited by the glucocorticoid antagonist RU486, indicating that dexamethasone action is specific and, furthermore, that it depends on glucocorticoid receptor. Finally, we have found that survivin and Bcl-2 are essential for pre- and mature osteoblast survival as their silencing is sufficient to induce spontaneous apoptosis in both cell types. In conclusion, our data outline a new molecular mechanism of glucocorticoid-mediated bone loss due to the enhanced apoptosis of precursors compared to mature osteoblasts. Furthermore, the data suggest a mechanism of dexamethasone-induced resistance of osteosarcoma cells to Fas- and stress-induced apoptosis.

Loss of pericentromeric DNA methylation pattern in human glioblastoma is associated with altered DNA methyltransferases expression and involves the stem cell compartment.

Cancer is generally characterized by loss of CG dinucleotides methylation resulting in a global hypomethylation and the consequent genomic instability. The major contribution to the general decreased methylation levels seems to be due to demethylation of heterochromatin repetitive DNA sequences. In human immunodeficiency, centromeric instability and facial anomalies syndrome, demethylation of pericentromeric satellite 2 DNA sequences has been correlated to functional mutations of the de novo DNA methyltransferase 3b (DNMT3b), but the mechanism responsible for the hypomethylated status in tumors is poorly known. Here, we report that human glioblastoma is affected by strong hypomethylation of satellite 2 pericentromeric sequences that involves the stem cell compartment. Concomitantly with the integrity of the DNMTs coding sequences, we report aberrations in DNA methyltrasferases expression showing upregulation of the DNA methyltransferase 1 (DNMT1) and downregulation of the de novo DNA methyltransferase 3a (DNMT3a). Moreover, we show that DNMT3a is the major de novo methyltransferase expressed in normal neural progenitor cells (NPCs) and its forced re-expression is sufficient to partially recover the methylation levels of satellite 2 repeats in glioblastoma cell lines. Thus, we speculate that DNMT3a decreased expression may be involved in the early post-natal inheritance of an epigenetically altered NPC population that could be responsible for glioblastoma development later in adult life.