A proteomic analysis reveals that Snail regulates the expression of the nuclear orphan receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and interleukin 17 (IL-17) to inhibit adipocyte differentiation

Abstract

Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.

Fig. 1.

Study of protein alterations in nuclear extracts of Snail1 transfected 3T3-L1 cells. A, Verification of Snail1 overexpression in 3T3-L1 and mMSC cells by Western blot analysis. B, 3T3-L1/Snail1 and 3T3-L1/Mock cells were analyzed at indicated cell confluence. The abundance of Snail1 and S100-A4 was quantified by Western blot. Tubulin was used as loading control. According to the results, cell confluence was set up at 100% for 48h for the rest of the experiments. C, Schematic representation of proteomics experiments with 3T3-L1 cells. For metabolic labeling, 3T3-L1/Snail1 or control cells were grown and maintained in light and heavy-labeled DMEM medium supplemented with 10% dialyzed FBS. D, The quality of the subcellular fractionation was assessed by Western blot before mass spectrometry. Cytoplasm and nuclear fractions were assayed using Lamin B and Rho GDI as nuclear and cytoplasmic protein controls, respectively. E, Proteins identified and quantified in 3T3-L1 forward and reverse SILAC experiments. In total, we identified 3920 proteins with 2800 overlapping proteins, whereas we quantified 3483 proteins with 2251 common proteins in both experiments. F, The enrichment of nuclear fraction with cellular component analyses was evaluated using DAVID Database. In total, 458 out of 2251 quantified proteins in 3T3-L1 were previously observed in nucleus.

Fig. 2.

Significant altered networks and pathways induced by Snail1. A, Distribution of protein ratios versus protein abundance in Snail1 and control cells by SILAC analysis. Down-regulated and up-regulated proteins are indicated in black, red, and blue, respectively. Unaltered proteins by Snail are represented in green. B, IPA database was used to identify the most significant altered pathways and biological functions caused by Snail1 overexpression in 3T3-L1 cells. C, IPA database predicted down-regulation of PPARγ and up-regulation of C/EBPβ mediators that are critical for the inhibition of final differentiation to adipocytes in 3T3-L1 cells.

Fig. 3.

Validation of Snail1-deregulated proteins in 3T3-L1 and mMSC cells by PCR and Western blot. A, cDNA synthesized from total RNA from 3T3-L1/Snail1 and control cells was subjected to semi-quantitative RT-PCR. B, Quantitative PCR analysis of Trip4, Nr2f6, OsmR, Prrx1, and CBX6 transcription factors in 3T3-L1/Snail1 and mMSC/Snail1 cells in comparison to control. Data represent the median and S.D. of three independent experiments. C, Nuclear protein extracts of 3T3-L1/Snail1, mMSC/Snail1, and control cells were subjected to Western blot using specific antibodies against the indicated proteins. Lamin B was used as nuclear and loading control. D, 3T3-L1/Snail1 and control cells were transfected with the amplified promoters cloned in the pGL3 plasmid. E-cadherin promoter was used as control. Data represents the mean of Firefly luciferase ± S.E. of three independent experiments performed on triplicate. **: p < 0.005; ***: p < 0.001 compared with control cells. E, To confirm the regulation of these transcription factors by Snail1, we performed ChIP assay using a specific anti-Snail1 antibody. PTEN was used as positive control of Snail1 regulation. An irrelevant IgG was used as negative control. Data represent the median/mean ± S.D. of the results. F, Western blot analysis of the reversion of the expression of the indicated transcription factors by knockdown of Snail1. Lamin B was used as loading control.

Fig. 4.

Adipocyte differentiation of 3T3-L1 cells is inhibited by silencing of Nr2f6 transcription factor. A, 3T3-L1 preadipocytes were transfected with Trip4, Nr2f6, Prrx1, CBX6, and control siRNA. On day 10, after treatment with adipogenic mixture, cells were stained with Oil Red O. Photographs of differentiation were acquired with Olympus CK40 microscope equipped with an Olympus DP12 camera at x40 magnification. B, Oil Red O stained cells were dissolved in isopropanol and staining was quantified by absorbance at 500 nm. Data represents the mean ± S.E. of three independent experiments performed on duplicate. *: p < 0.01; **: p < 0.005; ***: p < 0.001 compared with control cells. C, Western blot analysis of Nr2f6 knockdown. D, Western blot analysis of the expression levels of Nr2f6, and Snail1. RhoGDI was used as loading control. E, cDNA synthesized from total RNA from 3T3-L1/Snail1 and mock cells as control were subjected to qPCR analysis to amplify PPARγ, c/EBPα/β, Nr2f6, and Snail1. Murine ribosomal RNA 18S was used as control.

Fig. 5.

Enhanced expression of Nr2f6 reverts the effects of Snail1 and induced a fast adipocyte differentiation of 3T3-L1 cells. A, 3T3-L1/Mock and 3T3-L1/Snail cells were transfected with pcDNA3.1/Nr2f6 or empty vector and analyzed by Western blot. B, cDNA from transfected cells was subjected to qPCR analysis for PPARγ, C/EBPα/β, Nr2f6, and Snail1. Murine ribosomal RNA 18S was used as control. C, Representative images of f 3T3-L1 cells transfected with pcDNA3.1/Nr2f6 or empty vector were recorded at indicated times with an Olympus CK40 microscope equipped with an Olympus DP12 camera at ×40 magnification. D, Transfectants cells were stained with Oil Red O after 7 days of adypocite differentiation. Oil Red O stained cells were dissolved in isopropanol and staining was quantified by absorbance at 500 nm. Data represents the mean ± S.E. of two independent experiments performed on triplicate. *: p < 0.01; **: p < 0.005; ***: p < 0.001 compared with control cells. E, 3T3-L1 proliferation was determined by MTT assays after 48 h of culture. Optical density was significantly decreased by Nr2f6 overexpression (***: p < 0.001, compared with 3T3-L1/Mock cells).

Fig. 6.

Expression of IL-17 in 3T3-L1 and mMSC cells overexpressing Snail1 and IL-17 effect on adipogenesis. A, Murine cytokine antibody microarrays were incubated with the indicated conditioned medium. In both 3T3-L1 and mMSC cells, Snail1 promoted the expression of IL-17 and TNFα among other cytokines. Bar graph was calculated for each cytokine with the mean and S.E. of replicated spots in the array. B, IL-17 expression is promoted through the inhibition of Nr2f6 transcription factor by Snail1. IL-17 expression was quantified by ELISA in the conditioned medium of Snail1stably transfected 3T3-L1, mock, and mMSC cell lines. C, We quantified IL-17 expression in Nr2f6-silenced and ectopically-expressing cells. Nr2f6 expression induces variations in IL-17 expression levels. D, Adipocyte differentiation was performed in the presence or not of anti-IL17A antibody (500 ng/ml). Cells were stained with Oil Red O. Oil Red O stained cells were dissolved in isopropanol and staining was quantified by absorbance at 500 nm. Data represents the mean ± S.E. of two independent experiments performed on triplicate. *: p < 0.01; **: p < 0.005; ***: p < 0.001 compared with control cells. Anti-IL-17 antibody was able to reverse the effects of Snail1 overexpression on adipocyte differentiation. Images were taken as above.

Fig. 7.

Snail1 role in adipocyte differentiation. A model of the action of Snail in adipocyte differentiation. Snail1 overexpression down-regulates Nr2f6 expression, which in turn increases expression of IL-17. IL-17 up-regulates C/EBPβ and down-regulates C/EBPα and PPARγ. In addition, Snail1 down-regulates Trip4, another candidate mediator, which also interacts with PPARγ. Nr2f2 represents a central node between Nr2f6 and Trip4. These interactions will require further investigation.