Thyroid Hormone Receptor Regulates Most Genes Independently of fibroblast Growth Factor 21 in Liver
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A ZHANG and others Thyroid hormone/FGF21 224:3 289–301 Research interactions Thyroid hormone receptor regulates most genes independently of fibroblast growth factor 21 in liver Aijun Zhang1, Douglas H Sieglaff1, Jean Philippe York1, Ji Ho Suh1, Stephen D Ayers1,*, Glenn E Winnier1, Alexei Kharitonenkov2, Christopher Pin3,4, Pumin Zhang5, Paul Webb1 and Xuefeng Xia1,6 1Houston Methodist Research Institute, Genomic Medicine Program, 6670 Bertner Ave, Houston, Texas 77030, USA 2College of Arts and Sciences, Chemistry Department, Indiana University Bloomington, Bloomington, Indiana, USA, 3Departments of Paediatrics, Oncology, and Physiology and Pharmacology, University of Western Ontario, Correspondence London, Ontario, Canada should be addressed 4Children’s Health Research Institute, London, Ontario, Canada to X Xia or P Webb 5Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, USA Emails 6The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China [email protected] or *(S D Ayers is now at Personalis Inc., Department of Genomics, Menlo Park, CA, USA) [email protected] Abstract Thyroid hormone (TH) acts through specific receptors (TRs), which are conditional transcription Key Words factors, to induce fibroblast growth factor 21 (FGF21), a peptide hormone that is usually " nuclear receptor induced by fasting and that influences lipid and carbohydrate metabolism via local hepatic and " FGF systemic endocrine effects. While TH and FGF21 display overlapping actions when administered, " gene expression " Journal of Endocrinology including reductions in serum lipids, according to the current models these hormones act metabolism independently in vivo. In this study,we examined mechanisms of regulation of FGF21 expression by TH and tested the possibility that FGF21 is required for induction of hepatic TH-responsive genes. We confirm that active TH (triiodothyronine (T3)) and the TRb-selective thyromimetic GC1 increase FGF21 transcript and peptide levels in mouse liver and that this effect requires TRb. T3 also induces FGF21 in cultured hepatocytes and this effect involves direct actions of TRb1, which binds a TRE within intron 2 of FGF21. Gene expression profiles of WTand Fgf21-knockout mice are very similar, indicating that FGF21 is dispensable for the majority of hepatic T3 gene responses. A small subset of genes displays diminished T3 response in the absence of FGF21. However, most of these are not obviously directly involved in T3-dependent hepatic metabolic processes. Consistent with these results, T3-dependent effects on serum cholesterol are K K maintained in the Fgf21 / background and we observe no effect of the Fgf21-knockout background on serum triglycerides and glucose. Our findings indicate that T3 regulates the genes involved in classical hepatic metabolic responses independently of FGF21. Journal of Endocrinology (2015) 224, 289–301 Introduction Fibroblast growth factor 21 (FGF21) is one of three expressed in the liver, pancreas, and white and brown endocrine FGFs that influence metabolic responses via a adipose tissue (Johnson et al. 2009, Fon Tacer et al. 2010, combination of local and systemic actions. FGF21 is Kliewer & Mangelsdorf 2010). Hepatic Fgf21 expression is http://joe.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-14-0440 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 06:06:38PM via free access Research A ZHANG and others Thyroid hormone/FGF21 224:3 290 interactions induced by the nuclear hormone receptor peroxisome the expression of selected T3-regulated genes in liver and proliferator-activated receptor a (PPARa) under fasting brown adipose tissue, or overall energy expenditure. conditions, and secreted FGF21 peptide exerts autocrine/ Conversely, FGF21 elicited similar alterations in serum lipids paracrine effects on liver, including regulation of genes and gene expression in hypothyroid and euthyroid mice. involved in lipogenesis, lipolysis, and fatty acid oxidation, In this study, we set out to understand the mechanisms as well as additional endocrine effects on other tissues and downstream effects of TH-dependent changes in hepatic (Badman et al.2007). Administration of FGF21 to over- Fgf21 expression. We find that both T3 and the TRb-selective weight and diabetic mice results in decreases in the levels of thyromimetic GC1 act through TRb to directly induce plasma triglycerides and glucose, and increases in both FGF21 expression and that the Fgf21 locus harbors a insulin sensitivity and thermogenesis associated with functional TRE within intron 2. This finding added another decreased body weight (Coskun et al.2008, Berglund et al. layer of complexity to previous studies of T3 regulation of 2009, Xu et al.2009). Conversely, mice that lack FGF21 FGF21 expression, which indicated that TRb works primarily exhibit late-onset obesity. Thus, there is intense interest in through PPARa bound to the FGF21 promoter (Adams et al. FGF21 as a therapeutic for metabolic disorders, weight loss, 2010). While patterns of hepatic T3 responses are similar in and diabetes. Potentially adverse effects of FGF21 have also WT and Fgf21-knockout mice, similar to previous studies come to light. FGF21 is involved in integration of of a subset of hepatic genes involved in lipid metabolism metabolism and bone turnover, raising the possibility that (Domouzoglou & Maratos-Flier 2011, Domouzoglou et al. FGF21 could promote bone loss, and elevated FGF21 levels 2014), we also find that optimal response of a small subset are associated with abnormal lipid profiles in coronary heart of hepatic TH-target genes requires Fgf21. These genes, disease, raising concerns about cardiovascular events however, are not obviously related to metabolic regulation (Iglesias et al.2012, Wei et al.2012). Moreover, FGF21 levels and we find no changes in serum metabolic parameters become elevated in obesity and nonalcoholic fatty liver in Fgf21-knockout mice. Thus, our data support the idea disease, indicating that serum FGF21 levels do not always that hepatic effects of TH on gene regulation and metab- correlate with an improved metabolic profile. Given these olism are largely independent of Fgf21. concerns, it is important to understand how to manage local production of FGF21 in desired target tissues and the consequences of changes in FGF21 concentrations. Materials and method Thyroid hormones (THs) act as master regulators of Journal of Endocrinology Mice metabolism and there is evidence for interdependency of TH and FGF21 signaling pathways. TH receptors (TRa and All animal experiments were approved by the Animal TRb) are members of the nuclear hormone receptor family Care and Use Committee of Houston Methodist Hospital K K that regulate target genes in response to the main active form Research Institute. Mouse strains C57BL/6, ApoE / , K/K K/K of TH, triiodothyronine (T3). TRs usually work by binding Ldlr , and TRb were purchased from The Jackson DNA-response elements (TREs) in the proximal promoters of Laboratory (Bar Harbor, ME, USA) at 9 weeks of age. K K target genes as heterodimers with retinoid X receptors(RXRs) Fgf21 / mice on a C57/BL6 background (Dutchak et al. but can also regulate target genes via protein–protein 2012) were received from The University of Texas South- interactions with heterologous transcription factors (Baxter western Medical Center (Dallas, TX, USA). For short-term & Webb 2009, Cheng et al. 2010, Lin et al. 2013). T3 induces ligand treatments (1 and 3 days) T3, GC1, or vehicle FGF21 in mouse liver and, in this study, TRb selectively (control) were administered to mice (nZ5) by oral gavage enhances FGF21 expression and promoter activity via at a dose of 1 mg/kg body weight, after which, mice were interactions with PPARa bound to PPARa-response elements, killed and dissected for tissue harvesting. For long-term but does not require PPARa to induce other T3-regulated treatments (2 weeks), GC1 was admixed into diet at a genes (Adams et al.2010). In addition, FGF21 administration dose of 0.8 mg/kg body weight, after which the mice were to mice reduces serum levels of the parental form of TH, killed for collection of blood and tissue samples. thyroxine (T4), and T3, implying mutual regulatory depen- dency of these signaling systems (Domouzoglou et al.2014). Quantitative RT-PCR In spite of these potential relationships, key actions of FGF21 and TH appear largely independent (Domouzoglou et al. Total RNA was isolated from livers or cultured cells using 2014). Recent assessments of administration of TH to Fgf21- the RNAeasy Kit (Qiagen) and reverse transcribed to cDNA knockout mice have revealed no differences in serum lipids, using the ABI Reverse Transcription Kit according to the http://joe.endocrinology-journals.org Ñ 2015 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-14-0440 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 06:06:38PM via free access Research A ZHANG and others Thyroid hormone/FGF21 224:3 291 interactions manufacturer’s instructions. Gene transcript expression FBS-DMEM media for 24 h before treatment. The cells were was measured by quantitative RT-PCR (qPCR) (Roche pretreated with CHX at 10 mg/ml for 30 min, followed by LightCycler 480 II), in 20 ml reactions that included 10 nM T3 incubation for 6 h, after which RNA was collected cDNA (10 ng of initial RNA input), 900 nM of each primer, using the RNAeasy Kit (Qiagen). For RNA interference assays, PCR-grade water, and 10 mlof2! TaqMan Master Mix HepG2-TRb cells were plated in 10% resin-stripped FBS- (Applied Biosystems). The transcript expression levels DMEM media and grown to 50% confluency. The cells were were normalized to peptidylprolyl isomerase A (PPIA) transfected with PPARa ON-TARGET plus SMART pool or expression levels. Independent samples were prepared negative control siRNA (Dharmacon, Waltham, MA, USA) from five to six different mice per genotype or three at a 50 nM final concentration, and 48 h later treated with separate cell culture seedings and analyzed in technical 10 nM T3 or bezofibrate for 6 h, after which RNA was coll- triplicates.