TR4 transcriptional activity is negatively regulated by β-catenin
Previous reports showed that Wnt/β-catenin signaling suppresses the differentiation of 3T3-L1 preadipocytes and that β-catenin levels decrease as adipogenesis progresses [25]. To determine the correlation between TR4 and β-catenin during adipogenesis, we compared their relative protein levels during the differentiation of 3T3-L1 preadipocytes using immunoblotting. Consistent with previous reports, β-catenin was highly expressed in 3T3-L1 preadipocytes and then gradually decreased as adipocyte differentiation progressed (Fig. 1a). In contrast, TR4 levels were relatively constant and the levels of aP2, an adipogenic marker, were dramatically increased during this period. To further examine the possible role of β-catenin in the regulation of TR4 activity, we next generated pools of stable 3T3-L1 preadipocytes constitutively expressing β-catenin (3T3-L1-β-catenin), and determined the effect of β-catenin overexpression on the expression of TR4 and TR4 target genes. The protein levels of the TR4 target genes, Fatp1, and Cd36, on day 5 of differentiation, were dramatically reduced by 56% and 67%, respectively, due to β-catenin overexpression (Fig. 1b). Next, we performed a reporter gene assay to determine whether β-catenin suppresses TR4 transcriptional activity. As expected, TR4 dramatically induced the activity of the luciferase reporter gene fused to the mouse FATP1 promoter (FATP1pro-Luc) in both HEK293T and NIH-3T3 cells (Fig. 1c). However, when a β-catenin expression plasmid was co-transfected with TR4, TR4 transcriptional activation of FATP1pro-Luc was reduced by ~ 53% and 60%. In addition, β-catenin also suppressed TR4-mediated induction of luciferase activity from the cDR1-Luc construct, which contains three copies of a consensus DR1 sequence, an ideal TR4RE. β-catenin is known to regulate the expression of target genes through interaction with transcription factors such as TCF4 and NF-κB [26, 27]. To determine whether β-catenin inhibits TR4 activity via a physical interaction with TR4, we performed a GST pull-down assay. As shown in Fig. 1d, while 35S-labeled β-catenin interacted with GST-RXRα (a positive control), it was not able to interact with either the full-length GST-TR4 fusion protein (GST-TR4-FL) or the different deletion mutants of TR4 fused to GST (GST-TR4-N [aa 1–125], GST-TR4-∆C [aa 1–348], and GST-TR4-LBD [aa 224–615]).
β-catenin inhibits TR4 target gene expression through induction of Slug expression
Wnt/β-catenin signaling plays its role, in part, via induction of various transcription factors. Accumulating evidence shows that Snail and Slug can be induced by β-catenin and mediate the effects of β-catenin during a variety of cellular events, including epithelial-mesenchymal transition and cell proliferation [16, 28]. Thus, β-catenin may exert its inhibitory effect against TR4 function and adipogenesis of 3T3-L1 preadipocytes by inducing these genes. To test this hypothesis, we first determined whether β-catenin could induce Snail and Slug expression in 3T3-L1 preadipocytes. As shown in Fig. 2a, on day 5 of differentiation, the mRNA levels of Snail and Slug in 3T3-L1-β-catenin adipocytes were 1.8- and 2.4-fold higher, respectively, compared to control adipocytes (3T3-L1-pCI) along with decreased expression of TR4 target genes, Fatp1 (58%) and Pc (79%). We next determined the effects of Snail and Slug on TR4 transcriptional activity in HEK293T cells using a reporter assay. As expected, TR4 strongly increased the promoter activity of FATP1 (FATP1pro-Luc) and PC (PCpro-Luc) (Fig. 2b). However, TR4-mediated transcriptional activation of these reporter genes was dramatically repressed when a Snail- or Slug-expressing plasmid was co-transfected with TR4 expression plasmid into HEK293T cells. Interestingly, we found E-box sites that are located close to the TR4REs in both the FATP1 and PC promoters. Thus, to determine whether the inhibitory effects of these Snail family genes on TR4 transcriptional activity is due to the presence of E-boxes, Snail family binding sites, in FATP1 and PC promoters, we generated two different reporter genes containing the three TR4REs from each promoter without E-box. The results of the reporter gene assay showed that Slug, but not Snail, suppressed TR4-mediated transactivation of FATP1-DR1-Luc and PC-DR1-Luc (Fig. 2b).
Previous studies have reported that unlike the flexible binding capacity of Snail to various E box sequences, Slug binds to the E box only in the presence of “GG” or “CC” in the E box (CANNTG) [29]. Interestingly, sequence analysis revealed that all the E boxes in the FATP1 and PC promoters do not have “CAGGTG” or “CACCTG” sequences [1, 2]. Previous studies and our data suggest that Slug inhibits TR4 transactivation in an E-box-independent manner. Therefore, we decided to determine how Slug negatively regulates TR4 activity. We first examined the expression profile of Slug during adipocyte differentiation using immunoblotting. Although, Slug was highly expressed in 3T3-L1 preadipocytes, its expression was dramatically decreased at day 2 and almost disappeared after 6 days of differentiation (Fig. 2c), indicating that Slug and β-catenin levels decreased together during adipocyte differentiation. We next determined whether Slug knockdown could abolish β-catenin-induced suppression of TR4 transcriptional activity using a reporter gene assay. TR4 induced FATP1 promoter activity in NIH-3T3 cells (Fig. 2d). Moreover, Slug knockdown with a Slug-specific microRNA (SlugmiR) significantly enhanced TR4-mediated induction of FATP1 promoter activity (approximately 2.5-fold) compared to the cells transfected with control microRNA. As expected, when β-catenin was cotransfected with TR4, TR4 transactivation was reduced by approximately 75%. However, β-catenin-mediated inhibition of TR4 activity was completely abolished by the addition of SlugmiR. RT-qPCR analysis revealed that, on day 8 of differentiation, the mRNA levels of both Fatp1 and Pc in 3T3-L1 adipocytes overexpressing TR4 (3T3-L1-TR4) were upregulated compared with control adipocytes (3T3-L1-C) (Fig. 2e). In contrast, overexpression of β-catenin strongly suppressed TR4-mediated induction of Fatp1 and Pc expression in 3T3-L1-TR4 adipocytes. However, when SlugmiR was cotransfected with β-catenin, suppressive effect of β-catenin was strongly inhibited. These results showed that Slug mediates the inhibitory effect of β-catenin on transcriptional activity.
Slug suppresses lipid accumulation in 3T3-L1 adipocytes
To further determine the role of Slug in β-catenin-induced suppression of TR4 function and adipogenesis, we next generated pools of 3T3-L1 cells stably overexpressing Slug (3T3-L1-Slug) and analyzed the effect of Slug on adipogenesis in 3T3-L1 preadipocytes using Oil Red O staining. As shown in Fig. 3a, lipid accumulation was significantly inhibited in day 7 3T3-L1-Slug adipocytes compared to the levels in control adipocytes stably transfected with an empty vector (3T3-L1-C). Furthermore, in day 9 3T3-L1-Slug adipocytes, the mRNA levels of two TR4 target genes Fatp1 and Pc were 40% and 70% lower, respectively, than those in control adipocytes (3T3-L1-C) (Fig. 3b). In contrast, silencing of Slug in 3T3-L1 preadipocytes, by constitutively expressing SlugmiR (3T3-L1-SlugmiR) enhanced lipid accumulation and mRNA levels of lipogenic genes (Fatp1 and Pc) in 3T3-L1 adipocytes on day 7 and day 9, respectively, as compared to those of control 3T3-L1 adipocytes (Fig. 3c and d). As expected, TR4 mRNA levels in 3T3-L1 adipocytes were not affected by Slug knockdown. These results suggest that Slug strongly inhibits adipogenesis of 3T3-L1 preadipocytes.
Slug inhibits TR4 transcriptional activity via physical interaction with TR4
To address the molecular mechanism by which Slug suppresses TR4 function in lipogenesis, we tested whether Slug physically interacts with TR4. First, we performed a GST pull-down assay using various deletion mutants of TR4 fused to GST. Only GST-TR4-FL and GST-TR4-ΔC were able to interact with Slug (Fig. 4a). Next, we performed a mammalian two-hybrid assay to determine which region of Slug is required for its interaction with TR4. When VP16-TR4 was co-transfected with either GAL4-Slug-ΔDBD (aa 1–130) or GAL4-Slug-ΔSNAG (aa 33–269), but not with GAL4-Slug-ΔDBD (aa 131–269), strong pG5-Luc activity was observed in HEK293T cells (Fig. 4b). In addition, GAL4-Slug-2 (aa 33–130), which contains a region of Slug present in both GAL4-Slug-ΔDBD and GAL4-Slug-ΔSNAG, also showed a strong interaction with VP16-TR4. Furthermore, Slug-2 fused to GST (GST-Slug-2), but not GST alone, interacted with TR4, indicating that the Slug-2 domain is responsible for Slug binding to TR4 (Fig. 4c). To further confirm whether the Slug-2 region is sufficient to repress TR4 transactivation, we performed a reporter gene assay in HEK293T cells using cDR1-Luc. As expected, TR4 significantly induced cDR1-Luc activity, and this TR4 transactivation was dramatically repressed not only by full-length Slug, but also by Slug-2 (Fig. 4d). In contrast, the SNAG region (Slug-SNAG) did not affect TR4 transcriptional activity.
Slug suppresses TR4-enhanced lipid accumulation in 3T3-L1 adipocytes by interrupting TR4 homodimerization
Given that the inhibitory role of Slug against TR4 activity is mediated through its physical interaction with TR4, Slug may inhibit TR4 transcriptional activity by preventing TR4 from binding to its cognate response elements. To prove this hypothesis, we performed a gel shift assay using TR4RE sequences located in the TR4 target gene promoters (FATP1-DR1 and PC-DR1) as probes. As shown in Fig. 5a, TR4, but not Slug, formed complexes with both DR1 sequences, indicating that Slug-mediated inhibition of TR4 activity is not due to competition with TR4 for binding to TR4REs. However, these TR4-DNA complexes were dramatically reduced in the presence of Slug. Next, we prepared chromatin samples from 3T3-L1-C and 3T3-L1-Slug cells, and then, performed a ChIP assay using an anti-TR4 antibody. PCR analysis showed that recruitment of TR4 to the FATP1 promoter region (− 554 bp to − 341 bp) containing FATP1-TR4RE and an E box, but not to the downstream region (− 143 bp to + 84 bp) lacking a TR4RE and an E box, was observed in chromatin samples prepared from both control and Slug-overexpressing cells. However, this PCR amplification was markedly lower in Slug-overexpressing cells than in control cells (Fig. 5b). Because TR4 usually binds to TR4REs as a homodimer and Slug inhibits TR4 binding to TR4REs, we tested whether Slug affects the formation of TR4-TR4 homodimer. As shown in Fig. 5c, 35S-labeled Slug could be pulled down by both GST-TR4-ΔC and GST-TR4-FL in the absence of TR4. However, when 35S-Slug was incubated with GST-TR4-ΔC or GST-TR4-FL in the presence of non-radiolabeled TR4, no interaction between Slug and either GST-TR4-ΔC or GST-TR4-FL was observed. Furthermore, non-radiolabeled Slug or Slug-2 also inhibited the binding between GST-TR4-FL and 35S-labeled TR4, indicating that Slug suppresses TR4 binding to TR4REs through inhibition of TR4 homodimerization (Fig. 5d). We next determined whether Slug can suppress TR4-mediated induction of target genes in 3T3-L1 adipocytes stably overexpressing TR4 (3T3-L1-TR4) using RT-qPCR analysis. On day 8 of differentiation, mRNA levels of both Fatp1 and Pc in 3T3-L1-TR4 adipocytes were upregulated by 4.8-fold, as compared with those in control adipocytes (Fig. 5e). However, when Slug was introduced into 3T3-L1-TR4 adipocytes, the mRNA levels of these genes were significantly reduced with no change in TR4 mRNA levels (Fig. 5e). In contrast, when Slug was silenced in day 6 3T3-L1-TR4 adipocytes by transfection of SlugmiR, mRNA levels of Fatp1 and Pc were slightly increased as compared with those in 3T3-L1-TR4 adipocytes. We next determined whether Slug affects TR4-mediated lipid accumulation in 3T3-L1 adipocytes. As expected, TR4 enhanced lipid accumulation in day 8 3T3-L1 adipocytes by 44%, as compared with that in control adipocytes (Fig. 5f). When Slug was overexpressed in day 8 3T3-L1-TR4 adipocytes, TR4-induced lipid accumulation was reduced by 39%. However, Slug knockdown by SlugmiR did not significantly affect TR4-mediated lipid accumulation in day 8 3T3-L1-TR4 adipocytes. Together, these results show that Slug exhibits inhibitory effect on adipogenesis, at least in part, via inhibition of TR4 activity.