Elsevier

Metabolism

Volume 109, August 2020, 154280
Metabolism

Basic Science
GATA3 induces the upregulation of UCP-1 by directly binding to PGC-1α during adipose tissue browning

https://doi.org/10.1016/j.metabol.2020.154280Get rights and content

Highlights

  • GATA3 expression was induced upon cold exposure in mice.

  • Ectopic expression of GATA3 induced thermogenic program, whereas GATA3 knockdown decreased thermogenic activation.

  • GATA3 induced increased UCP-1 expression by binding to its promoter region with PGC-1α.

  • GATA3 induced the in vivo activation of the thermogenic program.

  • GATA3 may represent a promising target for the prevention and treatment of obesity by regulating thermogenic capacity.

Abstract

Objective

Obesity is recognized as the cause of multiple metabolic diseases and is rapidly increasing worldwide. As obesity is due to an imbalance in energy homeostasis, the promotion of energy consumption through browning of white adipose tissue (WAT) has emerged as a promising therapeutic strategy to counter the obesity epidemic. However, the molecular mechanisms of the browning process are not well understood. In this study, we investigated the effects of the GATA family of transcription factors on the browning process.

Methods

We used qPCR to analyze the expression of GATA family members during WAT browning. In order to investigate the function of GATA3 in the browning process, we used the lentivirus system for the ectopic expression and knockdown of GATA3. Western blot and real-time qPCR analyses revealed the regulation of thermogenic genes upon ectopic expression and knockdown of GATA3. Luciferase reporter assays, co-immunoprecipitation, and chromatin immunoprecipitation were performed to demonstrate that GATA3 interacts with proliferator-activated receptor-γ co-activator-1α (PGC-1α) to regulate the promoter activity of uncoupling protein-1 (UCP-1). Enhanced energy expenditure by GATA3 was confirmed using oxygen consumption assays, and the mitochondrial content was assessed using MitoTracker. Furthermore, we examined the in vivo effects of lentiviral GATA3 overexpression and knockdown in inguinal adipose tissue of mice.

Results

Gata3 expression levels were significantly elevated in the inguinal adipose tissue of mice exposed to cold conditions. Ectopic expression of GATA3 enhanced the expression of UCP-1 and thermogenic genes upon treatment with norepinephrine whereas GATA3 knockdown had the opposite effect. Luciferase reporter assays using the UCP-1 promoter region showed that UCP-1 expression was increased in a dose-dependent manner by GATA3 regardless of norepinephrine treatment. GATA3 was found to directly bind to the promoter region of UCP-1. Furthermore, our results indicated that GATA3 interacts with the transcriptional coactivator PGC-1α to increase the expression of UCP-1. Taken together, we demonstrate that GATA3 has an important role in enhancing energy expenditure by increasing the expression of thermogenic genes both in vitro and in vivo.

Conclusion

GATA3 may represent a promising target for the prevention and treatment of obesity by regulating thermogenic capacity.

Introduction

Obesity has been shown to be intimately associated with serious metabolic diseases such as type II diabetes, hypertension, and various types of cancer. Nevertheless, obesity is rapidly increasing worldwide. Fundamentally, obesity is caused by an energy imbalance, in which energy intake exceeds energy expenditure [1,2]. Therefore, as one strategy for addressing obesity, brown and beige adipocytes have emerged as promising targets [3]. Indeed, both brown and beige adipocytes can consume chemical energy and dissipate it in the form of heat in a primarily uncoupling protein-1 (UCP-1)-dependent manner [[4], [5], [6]]. In particular, beige adipocytes, which have only recently been discovered, may prove to be an important breakthrough for overcoming obesity. One of the remarkable characteristics of beige adipocytes is that their development is highly inducible by various external stimuli, including cold exposure, catecholamine treatment, and exercise [[7], [8], [9]]. In addition, it has been shown that human beige adipocytes can be trans-differentiated (termed “browning”) from subcutaneous white adipose tissue (scWAT) [[10], [11], [12]]. Therefore, increasing energy expenditure by inducing and/or promoting the browning process is considered an efficient way to prevent and/or treat obesity. However, a deeper molecular understanding of the browning process is essential.

Although the differentiation of white adipocytes and brown adipocytes has been extensively studied, relatively little is known regarding the molecular mechanisms of the browning process. It is known that cold exposure induces the release of catecholamines from terminal neurons, which act as agonists of adrenergic receptors on certain adipocytes within the scWAT. This binding to β-adrenoreceptors then activates the G protein-coupled receptor pathway, which increases intracellular cAMP concentrations and induces protein kinase A (PKA). The activated PKA then induces the phosphorylation of cAMP response element-binding protein (CREB) and p38 [13]. Subsequently, p38 MAPK phosphorylates peroxisome proliferator-activated receptor-γ (PPARγ) co-activator-1α (PGC-1α) and transcription factor activating transcription factor 2, which directly promotes pgc-1α gene transcription [14,15]. The phosphorylated PGC-1α also serves as a co-activator for PPARγ, inducing the transcription of downstream thermogenic genes such as ucp-1 [16]. Elevated UCP-1 then allows protons to leak across the mitochondrial inner membrane, resulting in increased oxygen consumption along with heat production. Although these signaling pathways are known, detailed mechanisms of the overall browning process are not well understood. In particular, with the exception of IRF4, little is known regarding how PGC-1α co-activates the thermogenic gene expression program [17].

The GATA family of transcription factors consists of six members (GATA1–6) characterized by their ability to bind to “GATA” DNA sequences. The GATA family is known to be intimately involved in a number of key physiological and pathological processes [18]. Indeed, the importance of GATA factors is evidenced by the embryonic lethality of most single GATA knockout-mice [19]. Among the GATA members, GATA3 is a transcription factor that contains the zinc-finger DNA-binding domain Cys-X2-C-X17-Cys-X2-Cys, which binds to the consensus 5′-(A/T)GATA(A/G)-3′ motif. In addition, GATA3 controls the expression of a wide range of biologically and clinically important genes [20,21]. In particular, GATA3 suppresses key adipogenic regulators, such as PPARγ2, by binding to their promoters [[19], [20], [21]]. Genetic variations in human GATA3 were shown to be associated with type 2 diabetes raising the possibility that GATA3 may be linked to metabolic disease in humans [22]. Recently, Bjȍrkqvist et al. reported that GATA3 is significantly upregulated in the scWAT from R6/2 mice, which is an animal model of Huntington's disease (HD) [23]. The R6/2 HD mouse model exhibits adipose tissue abnormalities in combination with weight loss and increased energy expenditure due to enhanced browning activity. The authors suggested that increased GATA3, which inhibits adipocyte differentiation, may be involved in the browning process [[24], [25], [26]]. However, little is known regarding the role of GATA3 in the browning process. In this study, we identified GATA3 as a novel transcriptional partner of PGC-1α in the promotion of the thermogenic program during browning. Enhanced expressions of UCP-1 and relevant thermogenic genes by GATA3, with a subsequent increase in oxygen consumption, were observed in both cell lines and an in vivo mouse model. Our findings suggest that GATA3 may mitigate obesity by activating thermogenesis and improving energy expenditure via the upregulation of UCP-1 expression through its interaction with PGC-1α.

Section snippets

Animal experiments

All use of animals was approved by and in accordance with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) animal care and use committee guidelines. Mice were maintained at a constant temperature (23 °C) and allowed free access to food and water, along with a standard chow diet unless otherwise stated. Nine-week-old C57BL/6 mice were exposed to cold conditions in order to induce browning. Male mice were housed in a cold chamber maintained at 6 °C for up to 1 week. For

GATA3 expression was induced upon cold exposure in mice

To identify the role of the GATA family of transcription factors in WAT browning, we first established the appropriate conditions to induce browning by exposing mice to cold temperatures, followed by qPCR analysis of ingWAT, which is a representative subcutaneous WAT (scWAT). As shown in Fig. 1A, the expressions of various thermogenic genes, including UCP-1 and PGC-1α, were upregulated, indicating that browning was induced under our experimental conditions. We then examined the expression

Discussion

With growing interest to develop effective anti-obesity drugs, recent research has focused on the promotion of energy expenditure through the activation of BAT and the conversion of white adipose adipocytes into brown-like adipocytes (beige adipocytes) [[38], [39], [40], [41]]. Indeed, brown and beige adipocytes activate the thermogenic program in response to external stimuli in order to release energy in the form of heat. UCP-1 plays a pivotal role in this thermogenic program, and the

Conclusion

In summary, this study demonstrated that GATA3 enhanced energy expenditure by regulation of thermogenesis. The GATA3 binds directly to the promoter region of UCP-1 and regulates gene expression by interaction with PGC-1α. This interaction of PGC-1α and GATA3 was essential for the improved thermogenic program and increasing the mitochondrial content. In conclusion, our results reveal a novel role for GATA3 in WAT browning suggesting that GATA3 may be a valuable target for the treatment of

CRediT authorship contribution statement

Min Jeong Son: Conceptualization, Formal analysis, Investigation, Writing - original draft. Kyoung-Jin Oh: Conceptualization, Formal analysis, Investigation. Anna Park: Conceptualization, Formal analysis, Investigation. Min-Gi Kwon: Investigation. Jae Myoung Suh: Writing - original draft, Writing - review & editing. Il-Chul Kim: Writing - review & editing. Seyun Kim: Writing - original draft, Writing - review & editing. Sang Chul Lee: Conceptualization, Formal analysis, Writing - review &

Acknowledgments

We thank Drs. Baek-Soo Han, Seung-Wook Chi, Jeong-Ki Min, and Eun Woo Lee for their helpful advice. The authors would like to thank Enago (www.enago.co.kr) for English language review. This work was supported by grants from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and the Research Program (grants 2017M3A9C4065954, 2015M3A9D7029882, 2016M3A9B690287123, 2017R1E1A1A01074745, 2018R1A2A3075389 and 2019R1A2C1006035) through the National Research Foundation of Korea.

Declaration of competing interest

The authors have no conflicts of interest and declare no competing financial interests.

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