Novel polysome messages and changes in translational activity appear after induction of adipogenesis in 3T3-L1 cells

Abstract

Background: Control of translation allows for rapid adaptation of the cell to stimuli, rather than the slower transcriptional control. We presume that translational control is an essential process in the control of adipogenesis, especially in the first hours after hormonal stimulation. 3T3-L1 preadipocytes were cultured to confluency and adipogenesis was induced by standard protocols using a hormonal cocktail. Cells were harvested before and 6 hours after hormonal induction. mRNAs attached to ribosomes (polysomal mRNAs) were separated from unbound mRNAs by velocity sedimentation. Pools of polysomal and unbound mRNA fractions were analyzed by microarray analysis. Changes in relative abundance in unbound and polysomal mRNA pools were calculated to detect putative changes in translational activity. Changes of expression levels of selected genes were verified by qPCR and Western blotting.

Results: We identified 43 genes that shifted towards the polysomal fraction (up-regulated) and 2 genes that shifted towards free mRNA fraction (down-regulated). Interestingly, we found Ghrelin to be down-regulated. Up-regulated genes comprise factors that are nucleic acid binding (eIF4B, HSF1, IRF6, MYC, POLR2a, RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa, TSC22d3), form part of ribosomes (RPL18, RPL27a, RPL6, RPL7a, RPS18, RPSa), act on the regulation of translation (eIF4B) or transcription (HSF1, IRF6, MYC, TSC22d3). Others act as chaperones (BAG3, HSPA8, HSP90ab1) or in other metabolic or signals transducing processes.

Conclusions: We conclude that a moderate reorganisation of the functionality of the ribosomal machinery and translational activity are very important steps for growth and gene expression control in the initial phase of adipogenesis.

Figure 1

Heatmap for spike-normalized microarray expression data. Columns 1 to 4 show log2 expression data for polysomal (p) and non-polysomal (np) fractions at two time points (0 h and 6 h after hormonal induction). Column 5 shows log2 fold change M ((p6-np6) - (p0-np0)). Only genes with fdr <0.05 were selected. Genes were identified as up-regulated (Up; highest expression in polysomal fractions at time point 6 h after hormonal induction) with M-value > 2, p6 > p0, p6 > np6, np6 < np0, p0 < np0. Genes were identified as down regulated (Down) with M-value < -2, p6 < p0 and as neither up nor down regulated (Equal) with equivalence test parameter 'epsilon = 0,2'. High expression levels are shown in dark grey, low expression levels in white.

Figure 2

Western blot results. 3T3-L1 cells were differentiated with insulin, dexamethasone and IBMX. Protein was isolated from whole cell extracts 0 h (T0) and 6 h (T6) after hormonal induction. 30 μg of each sample was subjected to Western blot analysis for eIF4B, IMPDH2, RPL27a and UBE2k/HIP2 expression. Changes in protein expression were quantified by densitometry and normalized with appropriate expression data of βActin. The values represent the average of three independent experiments, and the asterisk denotes a p-value < 0.05. eIF4B and RPL27a protein expression is 1.4 fold higher (p-value < 0.05) at T6 than time point T0. IMPDH2 and UBE2k/HIP2 show no significant differences in protein expression between T0 and T6.

Figure 3

Ribosomal and extraribosomal functions of the ribosomal proteins up-regulated in this study. Microarray results of polysomal fractions from 3T3-L1 cell lysate (6 h after hormonal induction) show that that ribosomal proteins (RP) are prominent among the up-regulated genes. For some RPs, extraribosomal functions have been demonstrated. In the nucleus, RPSa binds to DNA by histone binding, in the cytoplasm it is associated with the 40S small ribosomal subunit and at the cell surface it acts as a receptor for various components [34]. RPL6 over-expression promotes G1 to S phase transition of gastric cancer cells and promotes cell growth [28]. RPL7a interacts with the human thyroid hormone receptor and inhibits transactivation. Hyperthyroidism favours osteosarcoma cell growth and down-regulation of RPL7a might enhance sensitivity to TR and disrupt growth control [61]. RPL18 was shown to inhibit autophosphorylation of the double-stranded RNA-activated protein kinase (PKR) and PKR mediated phosphorylation of the translation initiation factor eIF2α. Over-expression of RPL18 reduced eIF2α phosphorylation and stimulated translation of a reporter gene in vivo [31]. A polymorphism in the promoter region of the RPL27a gene was associated with meat marbling in Japanese Black beef cattle [32]. These known extraribosomal functions might be important in early adipogenesis. Additionally an enhanced amount of RPs promotes an increase in translation process of adipocyte specific genes. In the beginning of translational process, the 43S ribosomal subunit scans mRNAs for start codons. Strong secondary structures inhibit processing of the complex on the mRNAs. eIF4B increases the helicases activity of the complex and allows translation mRNAs with strong secondary structures in the 5´UTR.

Figure 4

Schematic overview of the pathway controlling translational changes in adipogenesis. The PI3K/AKT/mTORC1 pathway, which is stimulated by insulin, leads to activation of eIF4B, which changes preferences in translation activity [38]. Regulation of C/EBPα could possibly be explained by up-regulation of eIF4b activity, as members of the C/EBP family are regulated at the translational level (dashed line). Additionally an increase in translation of adipogenesis genes mediated by eIF4B is thinkable (dashed line). c-MYC over-expression in cycling cells has been reported to block exit from the cell cycle, accelerate cell division, and increase cell size (reviewed in [45]). When c-MYC levels are high, 3T3-L1 adipoblasts are locked in a proliferation-competent state and normal differentiation can not be activated. Persisting high levels of c-MYC can inhibit the expression of genes that promote adipogenesis namely C/EBPα and PPARγ2 and therefore prevent terminal differentiation of preadipocytes to mature adipocytes [46,47]. c-MYC is an important regulator of ribogenesis, as it activates Pol I, Pol II and Pol III [49]. As a supplement in media, Ghrelin promotes the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARγ and C/EBPα [52]. Ghrelin mRNA over-expressing 3T3-L1 cells, on the other hand, demonstrated significantly attenuated differentiation of preadipocytes into adipocytes [53]. Down-regulation of Ghrelin levels in the early phase of adipogenesis fits the known facts indicating a role of decreased endogenous Ghrelin levels in promoting adipogenesis).