PTHrP in differentiating human mesenchymal stem cells: transcript isoform expression, promoter methylation, and protein accumulation.
Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5' and 3' ends and the existence of three different transcriptional promoters (P1, P2 and P3), two of which (P2 and P3) contain CpG islands. It is known that the expression of PTHrP isoforms may be differentially regulated in a developmental stage- and tissue-specific manner. To search for novel molecular markers of stemness/differentiation, here we have examined isoform expression in fat-derived mesenchymal stem cells both maintained in stem conditions and induced toward adipo- and osteogenesis. In addition, the expression of the splicing isoforms derived from P2 and P3 promoters was correlated to the state of methylation of the latter. Moreover, we also performed a quantitative evaluation of intracellular and secreted PTHrP protein product in undifferentiated stem cells and in parallel cultures at various differentiation stages. The data obtained indicate that from the stemness condition to that of osteo- and adipo-genic differentiated cells, the expression of isoforms becomes increasingly selective, thereby being a potential gene signature for the monitoring of cell stem or committed/differentiating state and that the switching-off of PTHrP isoform expression is mostly promoter methylation-dependent. Moreover, PTHrP intracellular retention is down-regulated in osteo-differentiating cells whereas the secretion of the protein in the extracellular medium is up-regulated with respect to stem cells, thereby suggesting that these variations of the intracellular and extracellular levels of PTHrP could potentially be enclosed in the list of the available protein signature of osteogenic differentiation.
['Longo|Alessandra|A|', 'Librizzi|Mariangela|M|', 'Naselli|Flores|F|', 'Caradonna|Fabio|F|', 'Tobiasch|Edda|E|', 'Luparello|Claudio|C|']
D017667:Adipocytes D017398:Alternative Splicing D002454:Cell Differentiation D002478:Cells, Cultured D018899:CpG Islands D019175:DNA Methylation D005091:Exons D005786:Gene Expression Regulation D006801:Humans D007438:Introns D059630:Mesenchymal Stem Cells D010011:Osteocytes D044162:Parathyroid Hormone-Related Protein D011401:Promoter Regions, Genetic D020033:Protein Isoforms D014158:Transcription, Genetic
PTHrP Mesenchymal stem cells Mesenchymal stem cells Promoter methylation Osteogenesis 3,3′,5,5′-tetramethylbenzidine 5-bromo-4-chloro-3′-indolyphosphate Adipogenesis BCIP BMP D-PBS Dulbecco's phosphate buffered saline EDTA FCS Gene expression MICE MSC MSRE NBT PCR PMSF SDS SM TBS-T TMB Tris-buffered saline-Tween 20 bone morphogenetic protein dNTPs deoxynucleotide triphosphates ethylenediaminetetraacetic acid fetal calf serum mesenchymal stem cells methylation-sensitive restriction endonuclease microtiter immunocytochemical ELISA assay nitro-blue tetrazolium parathyroid hormone-related protein phenylmethylsulfonyl fluoride polymerase chain reaction s.e.m. semi-quantitative multiplex sodium dodecyl sulfate standard error of the mean