Dataset Information

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

ABSTRACT: Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell cycle regulation. Our data provide evidence that the inactivation of RB1 or RB2/P130 in uncommitted bone marrow stromal cells (BMSC) facilitates the first steps of adipogenesis. In cultures with silenced RB1 or RB2/P130, we observed an increase of clones with adipogenic potential and a higher percentage of cells accumulating lipid droplets. Nevertheless, the absence of RB1 or RB2/P130 impaired the terminal adipocyte differentiation and gave rise to dysregulated adipose cells, with alteration in lipid uptake and release. For the first time, we evidenced that RB2/P130 plays a role in bone marrow adipogenesis. Our data suggest that while the inactivation of retinoblastoma proteins may delay the onset of last cell division and allow more BMSC to be committed to adipocyte, it did not allow a permanent cell cycle exit, which is a prerequisite for adipocyte terminal maturation.

SUBMITTER: Capasso S

PROVIDER: S-EPMC3956544 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

Capasso Stefania S Alessio Nicola N Di Bernardo Giovanni G Cipollaro Marilena M Melone Mariarosa Ab MA Peluso Gianfranco G Giordano Antonio A Galderisi Umberto U

Cell cycle (Georgetown, Tex.) 20131126 3

Bone marrow adipose tissue (BMAT) is different from fat found elsewhere in the body, and only recently have some of its functions been investigated. BMAT may regulate bone marrow stem cell niche and plays a role in energy storage and thermogenesis. BMAT may be involved also in obesity and osteoporosis onset. Given the paramount functions of BMAT, we decided to better clarify the human bone marrow adipogenesis by analyzing the role of the retinoblastoma gene family, which are key players in cell ...[more]

PMID: 24281253

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Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Project description:BackgroundBone marrow-derived mesenchymal stromal cells (MSCs) are multipotent cells with a high constitutive level of autophagy and low expression of CD99. Under certain conditions, MSCs may develop tumorigenic properties. However, these transformation-induced conditions are largely unknown. Recently, we have identified an association between Hsp70, a main participant in cellular stress response and tumorigenesis, and CD99. Preliminary observations had revealed upregulation of both proteins in stressed long-term cultured MSCs. And so we hypothesized that CD99 is implicated in stress-induced mechanisms of cellular transformation in MSCs. Hence, we investigated the effects of prolonged stress on MSCs and the role of CD99 and autophagy in their survival.MethodsHuman telomerase reverse transcriptase (hTERT) overexpressing immortalized MSCs and primary bone marrow stromal cells were used to investigate the influence of long-term serum deprivation and hypoxia on growth and differentiation of MSCs. Cell proliferation and apoptosis were evaluated using flow cytometry, differentiation capabilities of MSCs were assessed by immunohistochemical staining followed by microscopic examination. CD99, Hsp70 expression were analyzed using flow cytometry, western blotting, and reverse transcriptase polymerase chain reaction. Autophagy was explored with specific inhibitors using cell morphology examination and western blotting.ResultsChronic stress factors are able to change the morphology of MSCs and to inhibit spontaneous differentiation into adipocyte lineage. Furthermore, CD99 elevation and downregulation of p53 and p21 accompanied defective autophagy, which is usually associated with tumor formation. We found that inhibition of autophagy by chloroquine promoted cell detachment and modulated CD99 expression level whereas incorporation of CD99 recombinant protein into the cells suppressed autophagy.ConclusionsObtained results provide a model for chronic stress-induced transformation of MSCs via CD99 and may therefore be highly relevant to mesenchymal tumorigenesis.

Dataset Information

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

Publications

Silencing of RB1 and RB2/P130 during adipogenesis of bone marrow stromal cells results in dysregulated differentiation.

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