PCAF Promotes Adipocyte Differentiation Through The Activation Of FOXO1

Objective:The accumulation of adipose tissue is the result of the increasing of the number of fat cells as well as the size of individual cells.The number of fat cells is decided by the differentiation of pluripotent stem cells to preadipocytes, while the volume of individual cells is related to the preadipocyte-to-adipocyte differentiation and the triglyceride accumulation.PCAF(P300/CBP-associated factor) is an obesity related gene that showed significant associations with the body weight in our previous studies. As a histone acetyltransferase, PCAF promotes chromatin activation catalyting the acetylation of lysine residues. But the role of PCAF in preadipocyte differentiation is still not clear. This research is to investigate the regulation of PCAF on adipocyte differentiations and its potential molecular mechanism. Methords:In this research, we used the 3T3-L1 cell as a model system to investigate preadipocyte differeniations. Real-time PCR and Western Blot were performed to observe PCAF expressions during the differentiation process.Cells were transfected by recombinant lentivirus vectors that carrying PCAF(overexpression) or shRNA(knockdown). Stable cell lines of high and low expression of PCAF were obtained after puromycin and flow cytometry selection. Cell counting and Oil-Red-O staining were used to evaluate proliferations and differentiations of the impact of 3T3-L1 cells. PCR array and Ch IP-on-chip were employed to analyze downstream genes and pathways of PCAF. Western Blot and ChIP qPCR were used to validate potential target genes of PCAF. Each experiment was repeated three times or more, Statistical analyses were done by SPSS18.0. Measurements from 2 groups were compared by independent sample t test(mean ± standard deviation). Results:1. The expression of PCAF during adipocytes differentiationReal-time PCR was used to detect PCAF expressions at day0, day2, day4, day6, and day8 of adipocytes differentiation. RNA samples were extracted from 3T3-L1 cells. Results showed that the PCAF expression is at the highest on day2, then decreased gradually. We further refined the time point, we found that PCAF expressions increased at 10 min, 1h, 6h, and day2 when compared with day0, while the PCAF expression slightly decreased on day1 when compared with day0.2. The construction of stable cell lines with PCAF up and down-regulationsLentiviral vectors with the PCAF ORF and shRNA sequences were transfected into 3T3-L1 cells, creating RNA interfering(3T3-sh B1), over-expression(3T3-PCAF), and control cell lines(3T3-control). Real-time PCR showed that the PCAF expression was down-regulated by 73%(P<0.05) in 3T3-sh1 cell line than in the 3T3-control cell line, while the PCAF expression was up-regulated 3.8 folds(P<0.05) in the 3T3-PCAF cell line than the control cell line.3. The effects of PCAF expressions on adipocytes proliferation and differentiation.3T3-shB1, 3T3-PCAF, and 3T3-control cell lines were cultured for 7 days. Growth curves showed no significant difference of cell proliferation among the three cell lines. However, the 3T3-sh B1 cell line showed significantly less differentiated adipocytes at day 8 comparing with the 3T3-PCAF and the control cell lines using Oil-Red O staining.4. The molecular mechanism of PCAF regulating adipocyte differentiations(1) PCR array showed that expressions of FOXO1, CEBPD, and RUNX1T1 were positively correlated with the expression of PCAF.When PCAF was up-regulated or down-regulated, the three genes were up or down-regulated in accord with PCAF.(2) Ch IP-on-chip experiments were performed on undiffereniated 3T3-L1 cells(day0) and differentiated cells(day2). PATHWAY analyses showed that PCAF regulated 2249 genes in 73 pathways. Furthermore, the promotor of the FOXO1 gene was regulated by PCAF; PI3 K, PDPK1, AKT were also regulated by PCAF. The PATHWAY analysis shows that the insulin signaling pathway(KEGG: mmu04910) was among one of the targeted pathways that regulated by PCAF(FDR=0.0439);(3) ChIP-qPCR was used to detect the PCAF binding on the promoters of insulin signaling pathway genes PI3 K, PDPK1, AKT, and FOXO1 during adipocyte differeniations. It showed that the binding of PCAF with PI3 K and AKT promoters was not significant different at different time points. The binding of PCAF with the PDPK1 promotor were strong at 1h and day1, while were weaker at 10 min and day2; the binding of PCAF with the FOXO1 promoter were weak at 10 min, 1h and day1, while were stronger at day2. Real-time PCR was used to detect the expression of PDPK1, the results showed that expressions of PDPK1 were up-regulated at 1h and day1. Western blot showed that there was no correaltion between AKT and PCAF, however, phosphorylated of AKT at 1h and day1 were up-regulated by PDPK1. The phosphorylated FOXO1 was up-regulated by p-AKT.Conclusions:1. This research showed that PCAF was expressed in both undifferentiated and differentiated 3T3-L1 cells, and the PCAF expression increased in differentiated cells.2. There was no significant effect on cell proliferation when 3T3-L1 cells were transfected by 3T3-sh1, 3T3-PCAF, and 3T3-control lentiviral vectors. However, the 3T3-sh1 cell line showed significantly less differentiated adipocytes than the 3T3-PCAF and the 3T3-control cell lines. These results suggested that PCAF involved in the regulation of adipocyte differentiation.3. PCAF regulated pre-adipocytes differentiations via the PI3 K / PDPK1 / AKT / FOXO1 pathway. In the early stage of differentiation, PCAF regulated the expression of PDPK1, therefore changed the phosphorylation statuses of AKT, and followed by the regulating of the FOXO1 phosphorylation. In the middle stage of adipocyte differentiation, PCAF directly up-regulated the FOXO1 expression, therefore promoted the cells into the terminal differentiation stage.