Here we outline a role for a member of the highly conserved groucho transcription factor family as a facilitator of nuclear receptor action during cell differentiation. TLE3 is a direct target for regulation by PPARγ and functions in a feed-forward loop with PPARγ to promote differentiation. Mechanistically, TLE3 functions as a coregulator of both PPARγ and Wnt signaling, driving the formation of active and repressive transcriptional complexes on the promoters of adipocyte genes. The dual ability of TLE3 to function as a coactivator for PPARγ and a corepressor for TCF provides an elegant mechanism for the integration of pro- and anti-adipogenic signals during adipocyte development.
The function of TLE proteins in adipocyte development has not previously been investigated. Our discovery of TLE3 as a coactivator for PPARγ-dependent transcription was unexpected, as groucho and mammalian TLEs have been primarily studied for their roles as corepressors (Chen and Courey, 2000
). TLE proteins lack a DNA-binding domain, and therefore their ability to regulate transcription is believed to be dependent on interaction with other proteins. Several transcription factors are known to interact with TLEs, including PAX, Hes, Engrailed, and TCFs (Buscarlet and Stifani, 2007
). TLEs are recruited to silence gene expression in various contexts through direct interactions with histones and histone modifying enzymes (Chen et al., 1999
; Sekiya and Zaret, 2007
). Our demonstration that TLE3 expression acts to positively reinforce PPARγ action has uncovered a previously unrecognized mode of action for this transcriptional cofactor.
Previous work has identified several coactivators that interact with PPARγ (Cho et al., 2009
; Ge et al., 2002
; Gelman et al., 1999
; Grontved et al.
; Louet et al., 2006
; Qi et al., 2003
; Takahashi et al., 2002
). Unlike TLE3, however, the levels of these factors are not regulated during differentiation. They are likely required for the efficient action of PPARγ and other transcription factors, but are not utilized as developmental switches per se. PGC-1α, a coregulator whose expression is highly regulated by physiological stimuli, is critical for the in brown adipocyte thermogenic program, but is not believed to play a central role in white adipose differentiation (Puigserver et al., 1998
). The high expression of TLE3 in WAT, relative to BAT, leads us to speculate that TLE3 may function as a white adipocyte counterpart to PGC-1α in brown adipocytes. Transcriptional profiling supports this, as the brown adipocyte markers, PRDM16, Cidea, Elovl3, Ucp1, and PGC-1a were not upregulated in 10T1/2 cells expressing TLE3.
TLE3 mRNA and protein expression accumulate during preadipocyte differentiation and in response to PPARγ activation. Thus, coactivation of PPARγ by TLE3 may serve as a feed-forward mechanism to enhance differentiation. Expression of TLE3 at levels present in differentiated adipocytes promotes preadipocyte differentiation, and this effect is highly dependent on PPARγ expression. Indeed, co-expression of PPARγ and TLE3 has a synergistic effect on the expression of a number of terminal adipocyte genes. Moreover, we found a high degree of overlap between the transcriptional programs regulated by TLE3 and PPARγ, indicating that TLE3 exerts a preferential effect on the PPARγ signaling pathway in this cell type. Further studies will be needed to determine whether there may be additional transcription factors other than PPARγ involved in TLE3 signaling in preadipocytes. Mechanistic studies indicate that TLE3 is recruited along with PPARγ to PPREs in adipocyte target genes in a differentiation-dependent manner. PPARγ and TLE3 can be localized to common transcription complexes by immunoprecipitation and biochemical purification, although the two proteins do not appear to interact directly.
The Wnt signaling pathway is important for the maintenance and proliferation of preadipocytes (Ross et al., 2000
). Differentiation is accompanied by the suppression of Wnt signaling and the concurrent activation of PPARγ (Ross et al., 2002
). Surprisingly, the mechanisms underlying this switch are poorly understood. In particular, it is unclear how the Wnt pathway is shut off. We propose that induction of TLE3 expression is a component of a developmental switch that silences Wnt signaling and allows adipogenesis to proceed. TCF4 and β-catenin are present on the aP2 promoter in preadipocytes, and this correlates with the suppression of transcription. In the course of differentiation, endogenous TLE3 is recruited to differentiation-dependent adipocyte promoters, where it can interact directly with TCF4 and compete for the binding of β-catenin. Forced expression of TLE3 in preadipocytes displaces β-catenin and relieves repression of differentiation-dependent genes. Interestingly, a similar mode of TCF action has recently been proposed to operate in the context of skin differentiation (Nguyen et al., 2006
). TCF3 was found to actively repress epidermal and sebaceous gland differentiation in the stem cell compartment through repression of lipid metabolism genes such as PPARγ and CD36.
There is precedence for dual function transcriptional cofactors. It is becoming increasingly clear that the strict labels of “coactivator” and “corepressor” may not accurately reflect the complex interactions of some of these nuclear proteins. For example, in addition to coactivating TCF/Lef1,β-catenin can also act as a repressor (Blauwkamp et al., 2008). The nuclear receptor cofactor RIP140 has also been reported to perform both coactivator and corepressor functions (Debevec et al., 2007
; Subramaniam et al., 1999
). Furthermore, the functional roles of transcriptional coregulators may be context-specific and vary with the transcriptional machinery present in a particular cell. One possibility is that TLE3 is directing chromatin remodeling and generating a chromatin structure that facilitates PPARγ-dependent transcription.
The physiological importance of TLE3 for the adipocyte program is illustrated by the demonstration that suppression of TLE3 expression compromises preadipocyte differentiation and PPARγ target gene expression. In addition, we showed that expression of TLE3 from the adipose-selective aP2 promoter in mice mimics the effect of synthetic PPARγ agonist administration. aP2-TLE3 transgenic mice challenged with a high-fat diet were partially protected against insulin resistance. Clamp studies showed that the improvement in glucose handling in aP2-TLE3 mice was largely attributable to improved hepatic insulin sensitivity. This result is in line with previously reported effects of low-dose TZD treatment. Submaximal doses of pioglitazone have been shown to increase the glucose infusion rate and suppress HGP in the absence of major effects on IS-GDR (Kubota et al., 2006
Improvements in hepatic insulin sensitivity may reflect redistribution of triglycerides away from liver and into adipose tissue. We found that aP2-TLE3 mice have increased adipose tissue, reduced liver mass, and reduced hepatic lipid accumulation on high-fat diet. A similar finding was reported in ob/ob animals expressing an adiponectin transgene (Kim et al., 2007
). Furthermore, adiponectin has been shown to act directly on the liver to suppress gluconeogenesis (Combs et al., 2001
). Therefore, our demonstration that aP2-TLE3 mice have higher plasma adiponectin levels provides a plausible mechanistic explanation for the beneficial effects of TLE3 on systemic glucose metabolism. Interestingly, despite having reduced food intake, leptin levels were not elevated in aP2-TLE3 mice, suggestive of a change in leptin sensitivity. Additional studies will be needed to explore this issue. Since TLE3 and aP2 are also expressed in macrophages, it will be interesting to address the function of TLE3 in this cell type and its potential contribution to the phenotype of the aP2-TLE3 mice. Finally, given the lethality of global TLE3-deficiency, future in vivo
loss of function studies will necessitate the generation of tissue-selective conditional deletions of TLE3.