RB and RB2/p130 genes demonstrate both specific and overlapping functions during the early steps of in vitro neural differentiation of marrow stromal stem cells.
Marrow stromal stem cells (MSCs) are stem-like cells that are currently being tested for their potential use in cell therapy for a number of human diseases. MSCs can differentiate into both mesenchymal and nonmesenchymal lineages. In fact, in addition to bone, cartilage and fat, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes. RB and RB2/p130 genes are involved in the differentiation of several systems. For this reason, we evaluated the role of RB and RB2/p130 in the differentiation and apoptosis of MSCs under experimental conditions that allow for MSC differentiation toward the neuron-like phenotype. To this end, we ectopically expressed either RB or RB2/p130 and monitored proliferation, differentiation and apoptosis in rat primary MSC cultures induced to differentiate toward the neuron-like phenotype. Both RB and RB2/P130 decreased cell proliferation rate. In pRb-overexpressing cells, the arrest of cell growth was also observed in the presence of the HDAC-inhibitor TSA, suggesting that its antiproliferative activity does not rely upon the HDAC pathway, while the addition of TSA to pRb2/p130-overexpressing cells relieved growth inhibition. TUNEL reactions and studies on the expression of genes belonging to the Bcl-2 family showed that while RB protected differentiating MSCs from apoptosis, RB2/p130 induced an increase of apoptosis compared to controls. The effects of both RB and RB2/p130 on programmed cell death appeared to be HDAC- independent. Molecular analysis of neural differentiation markers and immunocytochemistry revealed that RB2/p130 contributes mainly to the induction of generic neural properties and RB triggers cholinergic differentiation. Moreover, the differentiation potentials of RB2/p130 and RB appear to rely, at least in part, on the activity of HDACs.
Department of Neurological Sciences, Second University of Naples, Naples, Italy.
F P
FP
Jori
M A B
MA
Melone
M A
MA
Napolitano
M
M
Cipollaro
A
A
Cascino
A
A
Giordano
U
U
Galderisi
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004268:DNA-Binding Proteins / Q000235:genetics; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000502:physiology; D006877:Hydroxamic Acids / Q000494:pharmacology; D007150:Immunohistochemistry; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050717:Retinoblastoma-Like Protein p130; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D009419:Nerve Tissue Proteins; D016900:Neurofilament Proteins; D011506:Proteins; C493839:Rbl2 protein, rat; D016160:Retinoblastoma Protein; D050717:Retinoblastoma-Like Protein p130; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C012589:trichostatin A; D000110:Acetylcholinesterase; D006655:Histone Deacetylases