The role of Akt on Arsenic trioxide suppression of 3T3-L1 preadipocyte differentiation

Tang

International Journal of Molecular Medicine

et al. 2014

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), α1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)γ2 and C/EBPα. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis.

Section: Discussionmentioning

confidence: 54%

Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation

Tang

International Journal of Molecular Medicine

et al. 2014

J. Biochem. Mol. Toxicol.

“…Certainly, such a differentiation-inhibiting action of TCDD is well known (see Introduction) and, therefore, it is likely that it significantly contributes to the observation reported here. On the other hand, the differentiation-inhibiting action of TCDD appears to differ greatly from that induced by other chemicals such as arsenic trioxide [28], octanoate [29], p38 MAPK inhibitors [30], TGF␤ [31], and endrin [32], with respect to changes in DNA-binding activities of nuclear transcription factors, expression of several adipocyte markers as well as the persistence of their effects as assessed at the later stages of adipocyte differentiation. For example, endrin-induced inhibition of differentiation of 3T3-L1 is not accompanied with the significant increase of C/EBP␤, particularly its LIP content [32], unlike the case of TCDD [4] as assessed at several time points during differentiation including day 8 [32], despite the fact that both agents cause severe inhibition of adipocyte differentiation.…”

Section: Discussionmentioning

confidence: 90%

TCDD suppresses insulin-responsive glucose transporter (GLUT-4) gene expression through C/EBP nuclear transcription factors in 3T3-L1 adipocytes

Liu

Matsumura

2006

TCDD is known to reduce significantly the level of the functionally active form of glucose transporter type 4 (GLUT4) in vivo in adipose tissue and muscles. To study the mechanistic basis of this phenomenon, we conducted transient transfection and DNA deletion analysis in 3T3-L1 cells using chloramphenicol acetyltransferase (CAT) reporter plasmids containing the GLUT4 promoter joined to the bacterial CAT. It was found that in transfected control samples, CAT activity was significantly higher in cells transfected with p469CAT and p273CAT than those with p78CAT, indicating that the region between -78 and -273 contained elements that play major roles in transactivation of this gene. Treatment with TCDD decreased CAT activity with p469CAT and p273CAT, but not with p78CAT, indicating the same region to contain the element(s) affected by TCDD. A gel-shift (EMSA) analysis result indicated that TCDD shows the profound effect only on the nuclear proteins binding to the [(32)P]-labeled probe containing C/EBP response element equivalent of the -265 to -242 stretch of the GLUT4 promoter. The results of supershift analysis showed that TCDD caused a decrease in the tier of C/EBPalpha and an increase in that of C/EBPbeta among the proteins bound to this C/EBP response element. We studied the effect of TCDD in cells overexpressing either C/EBPalpha, C/EBPbeta, or C/EBPdelta through transient transfection of p273CAT or p469CAT. The results clearly showed that the effect of TCDD to suppress the CAT activity of p273 or p469 disappeared in those cells overexpressing C/EBPalpha or C/EBPbeta. These results implicate the C/EBP proteins to be the main mediator of suppressive action of TCDD on GLUT4 gene expression in 3T3-L1 cells.

“…In ATO-treated MSCs, p21 was increased significantly whereas p53 was not changed, indicating that p21 may be important in the MSC senescence induced by ATO. Wang et al [15] reported that ATO inhibited 3T3-L1 preadipocyte differentiation through C/EBPa and PPARg. Similarly, our study also showed that ATO inhibited MSC adipogenic differentiation but promoted its osteogenic differentiation.…”

Section: Discussionmentioning

confidence: 99%

Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

Cheng¹,

Lin²,

Zhang³

et al. 2011

ABBS

Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells. Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However, reports about the effects of ATO on MSCs are limited. In this study, we found that 1 mM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/ enhancer-binding protein alpha C/EBPa and peroxisome proliferator-activated receptor gamma PPARg might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.