Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: Role of the BMP-4 gene

Abstract

Previous studies showed that exposure of C3H10T1͞2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1͞2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. 5-azacytidine ͉ adipogenesis ͉ determination ͉ mesenchymal stem cell ͉ preadipocyte

Wnt signaling and adipocyte lineage commitment

“…Moreover, inhibition of the action of this endogenous expression of BMP-4 with noggin, a naturally occurring BMP-4 antagonist, prevents A33 cells from differentiating into adipocytes when treated with differentiation inducers. 13 This result is remarkable given that A33 cells had not previously been exposed to exogenous BMP-4. Thus, BMP-4 is involved at an early stage of adipocyte development.…”

“…When 10T1/2 stem cells are exposed to BMP-4 during proliferation and growth arrest, subsequent treatment with adipocyte differentiation inducers results in adipocyte differentiation. 2,13,27 Without prior exposure to BMP-4, however, the differentiation inducers are without effect on 10T1/2 stem cells. Further, if 10T1/2 cells are exposed to BMP-4 in cell culture and then subcutaneously implanted into athymic mice, the BMP-4 treated cells differentiate into adipocytes producing fat pads.…”

“…One such preadipocyte line, the A33 line, was previously characterized. 2,13,14 Exposure of 10T1/2 cells to the DNA methyltransferase inhibitor 5-azacytidine is thought to cause a small fraction of these cells to commit to the myocyte or adipocyte lineages by activating the expression of one or a few commitment genes that were previously repressed due to methylation at CpG dinucleotides. 15 Indeed, the first transcription factor shown to be involved in myocyte determination, i.e., MyoD, was identified in a genetic screen with 10T1/2 cells and myoblasts derived from 10T1/2 cells following exposure to 5-azacytidine.…”

A Lox/CHOP‐10 crosstalk governs osteogenic and adipogenic cell fate by MSCs

“…Based on our previous studies that knockdown of Lox disrupts the BMP4‐induced adipocyte lineage commitment from MSCs10 and a theoretical inverse relationship exists between osteogenic and adipogenic lineage commitment and differentiation of MSCs,6 we hypothesized that knockdown of Lox would enhance BMP4‐induced osteogenesis of MSCs. To explore this, C3H10T1/2 cells, a well‐characterized mesenchymal stem cells20 and faithful model of MSCs to study adipocytic and osteogenic commitment and differentiation both in vitro and in vivo,8, 9, 21 was used. As expected, two key osteogenic transcription factors, Runx2 and Osterix, were up‐regulated by BMP4, which were further enhanced by Lox knockdown at the committed stage (day 0) (Figure 1A‐C).…”

A1 adenosine receptors play an essential role in protecting the embryo against hypoxia

“…One reverse transcription reaction, containing 1 g of RNA, was used for each 96-well PCR array. The hypoxia signaling pathway PCR Array (SuperArray) was used to analyze gene expression in isolated hearts (46). Each focused array contained five housekeeping genes to control for template concentration.…”