Nuclear hormone receptors (NRs) are ligand-dependent DNA binding proteins that translate nutritional and physiological signals into gene regulation. The significance of NRs in human health and disease is underscored by the availability of drugs that targets NRs for treating several diseases. In this context, a subgroup of NR family has been proposed to regulate metabolism in a cell/tissue specific manner. The Rev-erb subgroup of NRs consists of two isoforms Rev-erb and Rev-erb. These two receptors have been shown to regulate different aspects of human physiology such as metabolism, inflammation, and circadian rhythm. Many NRs are expressed in skeletal muscle, a major mass peripheral tissue that accounts for ~40% of the total body weight and energy expenditure. This lean tissue is a major site for lipid and glucose homeostasis. Skeletal muscle express and secrete cytokines which perform autocrine and paracrine function with other tissues such as adipose. Accordingly, skeletal muscle plays important role in blood lipid profile, insulin sensitivity and progression of diseases such as type 2 diabetes and obesity. In addition, many studies have shown that NRs in skeletal muscle regulate glucose, lipid and energy homeostasis. Therefore, understanding the function of NRs in skeletal muscle provides a platform for potential new therapeutic treatments for metabolic disease. Rev-erb is expressed in skeletal muscle; however the function of this receptor in skeletal muscle metabolism has not been examined. Nevertheless, considering the importance of Rev-erb subfamily in metabolism, circadian control and the role of skeletal muscle in lipid homeostasis, the function of Rev-erb in skeletal muscle metabolism needs to be further investigated. We tested the hypothesis that Rev-erb (directly and/or indirectly) regulated the genetic programs that control lipid homeostasis in skeletal muscle. Initially, we exogenously expressed a truncated version of Rev-erb without the ligand-binding domain (Rev-erb) in vitro (in the C2C12 skeletal muscle cell culture system); and in vivo (in mouse tibialis anterior muscle. Moreover, we also attenuated Rev-erb expression in skeletal muscle cells using siRNAs targeting N-terminus and hinge regions of Rev-erb. We performed candidate based expression profiling utilizing quantitative RT-PCR analysis on the Taqman Low Density Array (TLDA) platform to identify putative downstream primary and/or secondary targets of Rev-erb action in skeletal muscle cells in the context of metabolism and muscle growth. Exogenous expression of Rev-erb in skeletal muscle cells in vitro decreased the expression of several genes involved in fatty acid/lipid absorption (including Cd36, and Fabp3 and 4). Interestingly, the mRNA encoding the master lipogenic regulator, SREBP-1c was also increased after ectopic Rev-erb expression. Moreover, we observed significant induction in the mRNAs encoding interleukin-6 and IKB that are involved in the regulation of the inflammatory cascade. Finally, we also observed the marked repression of myostatin mRNA, an important protein implicated in negative regulation of muscle hypertrophy/hyperplasia and a positive regulator of body fat accumulation. In summary, our in vitro study suggested that Rev-erb regulates genes involved in metabolism, inflammation and muscle growth. Quantitative PCR analysis that utilised the Taqman Low Density Array (TLDA) platform revealed Rev-erb siRNA expression down-regulated (in a subtle but significant manner) several genes involved in lipid/glucose homeostasis and the TGF- signalling pathway. Interestingly, genes that are involved in the myostatin and TGF- signalling pathway such as Activin A receptor type 2a (ACVR2A), Smad specific E3 ubiquitin protein ligase 1 (Smurf1), and TGF- receptor 2 (TGFBR2) were identified potential (direct and/or indirect) target of Rev-erb action in skeletal muscle cells. Moreover, genes such as Citrate Synthase (CS), V-akt murine thymoma viral oncogene homolog 2 (Akt2), Peroxisome proliferator- activated receptor- coactivator (PGC)-1 (PGC1) were also significantly modulated by Rev-erb in these analyses. The expression of two mRNAs encoding a) SREBP1c and b) IKB increased by ectopic Rev-erb expression was examined in more detail. These were selected because Rev-erb has been presumed to function as a transcriptional silencer. Secondly, we had demonstrated that in vivo expression of Rev-erb (after injection and electroporation of mouse tibialis anterior muscle) increased SREBP-1c expression, and Rev-erb siRNA studies suggested that this orphan NR was necessary for optimal SREBP-1c mRNA expression. Consequently, we tested the hypothesis that Rev-erb encodes the potential to function as a transcriptional activator in skeletal muscle. To test this hypothesis, we examined whether the SREBP1c and IKB promoters were trans-activated by co-transfected Rev-erb in skeletal muscle cells. We initially tested whether Rev-erb regulates the SREBP1c promoter. Transfection experiments showed Rev-erb expression trans-activated this promoter. This observation was in contrast to previous promoter studies showing that Rev-erb is a potent repressor of gene transcription. Therefore, we subsequently performed an experiment in which we simultaneously used the Rev-erb promoter (previously characterized to be repressed by Rev-erb) and SREBP1c promoter to examine the effect of Rev-erb expression. This experiment showed that Rev-erb repressed the activity of Rev-erb promoter, and in parallel trans-activated the SREBP1c promoter. Bioinformatics analysis identified two regions covering putative Rev-erb response elements RERE1 (-1342 to -1158) and RERE2 (-525 to -401) in the SREBP1c promoter. Chromatin immuno-precipitation assays demonstrated that Rev-erb is selectively recruited to RERE2 between nucleotide positions –525 to –401 in the promoter. Unidirectional deletion analysis of the SREBP1c promoter coupled with the analysis of mutants in the LXR response elements (of the SREBP-1c promoter) confirmed that Rev-erb mediated trans-activation of SREBP1c promoter does not function through LXR response elements. Interestingly, treatment of skeletal muscle cells with Hemin, a molecule recently proposed to function as a ligand for Rev-erbs, increased SREBP1c mRNA expression. In summary these data show that Rev-erb is a novel positive regulator of SREBP1c mRNA expression in skeletal muscle. We subsequently cloned the previously characterised human IKB promoter region spanning the potential ROR and Rev-erb binding site. Transfection experiments showed that in accordance to previously published observation, ROR trans-activated the IKB promoter. However, both Rev-erb and Rev-erb when co-transfected with the IKB promoter had minimal effects on the activity of this promoter. Studies have shown that Rev-erb functions as a competitor for ROR and block ROR mediated trans-activation of its target gene expression. Interestingly, our co-transfection experiments showed that both Rev-erb and Rev-erb blocks ROR-mediated trans-activation of IKB promoter. Together, this data suggests that Rev-erb-mediated regulation of IKB transcription in skeletal muscle cells could occur through indirect mechanisms. In conclusion, our studies have shown Rev-erb directly and indirectly regulates the expression of genes involved in metabolism, inflammation and muscle growth suggesting that Rev-erb in skeletal muscle has the potential to be exploited in a therapeutic manner.
| Identifer | oai:union.ndltd.org:ADTP/283961 |
| Creators | Sathiya Ramakrishnan |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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