MicroRNAs Function as Cis- and Trans- Acting Modulators of Clock Gene Expression in SCN and Peripheral Circadian Oscillators

The circadian system in mammals is arranged as a hierarchical network of oscillators, with the master pacemaker of circadian rhythms located in the suprachiasmatic nuclei (SCN) of the hypothalamus and peripheral oscillators in most other organ and tissue systems of the body. The molecular machinery responsible for generating circadian rhythms is composed of interlocked transcriptional-translational feedback loops with the gene Brain Muscle Arnt-like 1 (Bmal1) functioning as a core positive regulator. Using the mouse, Mus musculus as a model system, we studied the post-transcriptional mechanisms regulating Bmal1 expression in the SCN pacemaker and in peripheral oscillators. Target prediction algorithms were used to identify microRNAs (miRNAs) predicted to target Bmal1. We profiled the temporal expression of miR-142-3p in the mouse SCN in vivo and in an immortalized SCN cell line and observed robust circadian rhythms in its expression in the SCN. Following luciferase-reporter and site-directed mutagenesis analyses, we identified miR-142-3p as a bona-fide post-transcriptional repressor of Bmal1. The temporal expression of potential Bmal1-targeting miRNAs was also examined in the circulation in mouse serum. In mice housed in a light-dark cycle, diurnal oscillations were observed in serum levels of miR-152 and miR-494, but not miR-142-3p expression. Luciferase reporter studies indicated that miR-494, both independently and synergistically with miR-142-3p, repressed the Bmal1 3′ UTR. Overexpression of these miRNAs disrupted ensemble circadian rhythms of PER2::LUCIFERASE activity in cultured fibroblasts. Overexpression of the miRNAs also increased their extracellular levels and their intracellular accumulation in recipient cultures exposed to conditioned medium. Furthermore, inhibition of exocytosis and endocytosis affected ensemble circadian rhythms in cultured fibroblasts. The results thus implicate miR-142-3p and miR-494 in the regulation of Bmal1 expression in the SCN and peripheral oscillators and suggest that miRNAs may function as both, intracellular and extracellular (cis- and trans- acting) signals, modulating the core clock mechanism in the SCN and in fine-tuning the synchronization of circadian rhythmicity between cell-autonomous oscillators in the periphery

PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells

Reduction of circulating PCSK9 and LDL-C levels by liver-specific knockdown of HNF1α in normolipidemic mice

The transcription factors hepatic nuclear factor (HNF)1α and HNF1β can bind to the HNF1 site on the proprotein convertase subtilisin/kexin type 9 (PCSK9) promoter to activate transcription in HepG2 cells. However, it is unknown whether one or both HNF1 factors are obligatory for transactivating hepatic PCSK9 gene expression in vivo. We developed shRNA adenoviral constructs (Ad-shHNF1α and Ad-shHNF1β) to examine the effects of knockdown of HNF1α or HNF1β on PCSK9 expression and its consequent impact on LDL receptor (LDLR) protein levels in cultured hepatic cells and liver tissue. We demonstrated that infection with Ad-shHNF1α, but not Ad-shHNF1β, markedly reduced PCSK9 mRNA expression in HepG2 cells with a concomitant increase in LDLR protein abundance. Injecting Ad-shHNF1α in mice fed a normal diet significantly (α 50%) reduced liver mRNA expression and serum concentration of PCSK9 with a concomitant increase (α 1.9-fold) in hepatic LDLR protein abundance. Furthermore, we observed a modest but significant reduction in circulating LDL cholesterol after knockdown of HNF1α in these normolipidemic mice. Consistent with the observation that knockdown of HNF1β did not affect PCSK9 mRNA or protein expression in cultured hepatic cells, Ad-shHNF1β infection in mice resulted in no change in the hepatic mRNA expression or serum content of PCSK9. Altogether, our study demonstrates that HNF1α, but not HNF1β, is the primary positive regulator of PCSK9 transcription in mouse liver