Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney

Abstract

Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1–3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.

A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention

“…We (30) have also previously shown that Per1 and Cry2 modulate opposing actions on Per1 target gene expression in the kidney and liver and that Per1 heterozygous mice have an approximate 50% reduction in Per1 protein expression in these tissues. Therefore, to determine if Per1 regulates aldosterone levels, plasma aldosterone was assessed in WT and Per1 heterozygous mice by ELISA at noon (middle of the inactive phase) and midnight (middle of the active phase) under normal dietary conditions.…”

“…We (30) have recently shown that Per1 and Cry2 mediate opposing actions on Per1 target gene expression in the liver and kidney and that mice with reduced levels of Per1 have increased levels of Cry2. To test the effect of reduced Per1 on Cry2 adrenal gland cells and tissue, Cry2 mRNA levels were determined by quantitative PCR.…”

“…Per1 may not act as a canonical repressor, however, as increasing evidence suggests that Per1 may activate gene expression through an unknown mechanism, and this may occur in a tissue-and gene-specific manner (8,16,18,31,36). However, our recent work (30) demonstrated that this possible mechanism could be through repression of the circadian repressor Cry2. In this study, reduced expression of Per1 in vivo was associated with increased Cry2 expression and changes in Per1 target gene expression.…”

“…Per1 must be phosphorylated by CK1-␦/ε to enter the nucleus (20). We (30,31) have previously shown that pharmacological inhibition of CK1-␦/ε using PF-670462 recapitulated the effects of Per1 knockdown, including increased levels of Cry2, decreased ␣-ENaC mRNA and protein expression, and, importantly, decreased ENaC channel activity. PF-670462 has been previously shown to have an IC 50 for CK1-␦ of 15 nM and 7.7 nM for CK1-ε (5).…”

“…Casein kinase (CK)1-␦/ε inhibitor experiments were performed as previously described (30,31). For dose and time course, NCI-H295R cells were treated with either 0.1, 1, 10, or 100 M of the CK1-␦/ε inhibitor PF-670462 or vehicle (water) for 24, 48, or 72 h. For nuclear entry experiments, NCI-H295R cells were treated with 0.1 M PF-670462 or vehicle for 24 h.…”