BMP4 Induces The Commitment Of Pluripotent Mesenchymal C3H10T1/2 Cells To The Brown Adipocyte Lineage

Objectives:There are 2 different types of fat present in mammals:white adipose tissue, the primary site of energy storage, and brown adipose tissue, which is specialized in energy expenditure. Factors that specify the developmental fate and function of brown fat are poorly understood. Bone morphogenic proteins (BMPs) play an important role in adipogenesis. Previous studies showed that while BMP2 and BMP4 are capable of triggering commitment of stem cells to the white adipocyte lineage, BMP7 and BMP8B trigger commitment of progenitor cells to a brown adipocyte lineage and activates brown adipogenesis. However, the role of BMP4 in the development of brown adipocytes is unclear. This study aimed to investigate the effects of BMP4 on the commitment of brown adipocyte lineage in pluripotent mesencnymal C3H10T1/2 cells.Research Design and Methods:To evaluate the physiological roles of BMP4 on the commitment of brown adipocyte lineage, C3H10T1/2 pluripotent cells were pretreated with BMP4 and BMP7, followed by 3 different adipogenic induction cocktails. Measure the expression of brown-fat specific markers by western-blot and realtime PCR; define the mitochondrial density and function by Mito Tracker staining and transmission electron microscopy; study the cellular mechanisms by Stealth TM RNAi.Results:Both BMP4 and BMP7 unexpectedly activated a full program of brown adipogenesis, including induction of the brown-fat-defining marker uncoupling protein 1 (UCP1), and induction of mitochondrial biogenesis and oxygen consumption. Morever, BMP4 induce UCP1 expression in a dose-responsive manner in C3H10T1/2 cells. Implantation of BMP4-pretreated C3H10T1/2 cells into nude mice resulted in the development of adipose tissue depots containing UCP1-positive brown adipocytes. To further elucidate mechanisms of BMP4 in commitment of brown adipocyte, we investigated the expression of early regulators of brown fat fate PRDM16 (PR-domain-containing 16) during brown adipocyte lineage commitment of BMP4-pretreated cells. We found PRDM16 was markedly up regulated in both protein and mRNA levels, when C3H10T1/2 cells were treated with BMP4. To figure out whether PRDM16 was required for the brown adipocyte lineage commitment process induced by BMP4, PRDM16 expression was disrupted with specific Stealth TM RNAi in proliferating C3H10T1/2 stem sells. It was found that PRDM16 RNAi blocked commitment/differentiation into brown adipocytes. Furthermore, we identified the brown adipocyte lineage commitment of C3H10T1/2 stem cells induced by BMP4 mainly through two downstream signal pathways, p38 MAPK and Smad. We blocked them with specific Stealth TM RNAi to define the regulation of BMP signal pathway to PRDM16. As we thought, PRDM16 could be remarkablely repressed by Smad4 RNAi. These findings show that BMP4/PRDM16 signaling is required for BMP4-induced brown adipocyte lineage commitment of C3H10T1/2 stem cells.Conclusions:From the above, we demonstrate that BMP4 up regulates PRDM16 expresssion via Smads pathwaty during brown adipocyte lineage commitment.