The nuclear receptor Nr2f6 as new molecular player for thermogenesis in skeletal muscle cells

Abstract

Transcription factors (TFs) are proteins that bind to specific DNA sequences in order to direct which genes should be turned on or off to control cellular metabolism. The regulatory elements found at many promoters and enhancers afford the cells the opportunity to promptly regulate the expression of genes set to affect a specific cellular response. In physiological context, dysfunction in this TF- mediated signaling is directly associated with metabolic diseases such as obesity, diabetes and cancer. Thus, the seek for transcription factors and their respective regulatory sequences will provide scientists many opportunities to originally study gene regulation in different physiological processes including those affected by metabolic diseases, when mitochondrial function is markedly impaired. Given the enormous number of TFs and potential regulatory sequences, we firstly analyzed large datasets including RNAseq and ChIPseq at National Center for Biotechnology Information (NCBI). The analysis originally revealed a much lower expression of the transcription factor Nr2f6 in skeletal muscle and adipocytes of mice submitted to cold or physical exercise, two well-known conditions to induce the oxidative phynotype in the peripheral tissues. Next, Nr2f6 was knocked-down in C2C12 cells and the RNAseq technique carried out to see what pathways could be affected. The terms of genes associated with metabolic process, cellular differentiation, muscle contraction, and Ca2+ transport were markedly upregulated underscoring the role of Nr2f6 on the cellular metabolism. Of particular interest, studies have demonstrated that skeletal muslce increases heat production by uncoupling of Serca ATP hydrolysis from Ca2+ transport, clearly suggesting a role of Nr2f6 in the muscle thermogenic process. Consistent with the thermogenesis activity in skeletal muscle, knockdown of Nr2f6 markedly induced the AMPK phosphorylation, an effect that was reflected in increased insulin response. In addition, genes associated with thermogenic process in skeletal muscle including Slc25a4 (ATP/ADP carrier) and ATP1A1 (ATPase Na+/k+ transporting) and ryanodine receptor (Ryr1) were also upregulated in Nr2f6 knockdown cells. We next sought to Nr2f6 responsive elements at DNA regulatory sequence of genes associated with Ca2+ transport and thermogenesis. We found Nr2f6 responsive elements at DNA regulatory sequences of Ca2+ transport genes including Sln, ATP2a1 (Serca 1A), ATP2a2 (Serca 2A), and Ryr1. Likewise, Nr2f6 responsive element was also found at DNA regulatory sequence of PPARGC1±, an important player of thermogenesis process in brown adipocytes and skeletal muscle cells. Take together this finding suggest that the nuclear receptor, Nr2f6, might be an important regulator of mitochondrial function and thermogenic process in skeletal muscle. (AU)