PTHrP in differentiating human mesenchymal stem cells: Transcript isoform expression, promoter methylation, and protein accumulation

Abstract

Human PTHrP gene displays a complex organization with nine exons producing diverse mRNA variants due to alternative splicing at 5 0 and 3 0 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. Ó 2013 Published by Elsevier Masson SAS.

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16INK4a gene in human colorectal carcinoma (Caco-2) cells

“…Co-amplification of IL-4 internal region was concurrently carried out as a control (998 bp product; forward: 5 0 -CCCCAAGTGACT GACAATCTGG-3 0 and reverse: 5 0 -GTGAGAGTATTTGG TTTTTCAGAAAT-3 0 ). The MSREM-PCR products were resolved as described [27].…”

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

“…Methylation Sensitive Restriction Endonuclease-PCR (MSRE-PCR) (Longo et al, 2013) was performed in order to determine the methylation status of the sites CpG rich, present in the 5′ flanking region (− 849 bp and − 457 bp to the start site, see Fig. 1) of the CYP2E1 gene.…”

Type V collagen counteracts osteo-differentiation of human mesenchymal stem cells

“…The media were carefully changed once a week. After four weeks, osteogenic differentiation was visualized by staining of extracellular calcium deposits with Alizarin Red S [3]. …”

“…LW28, LW36 and LW40 MSC cultures, isolated from adipose tissue samples obtained by liposuction of three female patients, respectively 28, 36 and 40 years old, were differentiated at passage 5 towards osteoblast lineage, and the differentiation checked as already shown by Longo et al [3].…”

“…The cDNAs were synthesized using SuperScript II reverse transcriptase (Invitrogen) as reported in Ref. [3].…”