a b s t r a c tThe phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.

It has been proposed that robust rhythmic gene expression requires clock-controlled elements (CCEs). Transcription of Per1 was reported to be regulated by the E-box and D-box in conventional reporter assays. However, such experiments are inconclusive in terms of how the CCEs and their combinations determine the phase of the Per1 gene. Whereas the phase of Per2 oscillation was found to be the most delayed among the three Period genes, the phase-delaying regions of the Per2 promoter remain to be determined. We therefore investigated the regulatory mechanism of circadian Per1 and Per2 transcription using an in vitro rhythm oscillation-monitoring system. We found that the copy number of the E-box might play an important role in determining the phase of Per1 oscillation. Based on real-time bioluminescence assays with various promoter constructs, we provide evidence that the non-canonical E-box is involved in the phase delay of Per2 oscillation. Transfection experiments confirmed that the non-canonical E-box could be activated by CLOCK/BMAL1. We also show that the D-box in the third conserved segment of the Per2 promoter generated high amplitude. Our experiments demonstrate that the copy number and various combinations of functional CCEs ultimately led to different circadian phases and amplitudes.

Proinsulin C-peptide causes multiple molecular and physiological effects, and improves renal and neuronal dysfunction in patients with diabetes. However, whether C-peptide controls the inhibitor kappaB (IkappaB)/NF-kappaB-dependent transcription of genes, including inflammatory genes is unknown. Here we showed that 1 nM C-peptide increased the expression of cyclooxygenase-2 (COX-2) mRNA and its protein in Swiss 3T3 fibroblasts. Consistently, C-peptide enhanced COX-2 gene promoter-activity, which was inhibited by GF109203X and Go6976, specific PKC inhibitors, and BAY11-7082, a specific nuclear factor-kappaB (NF-kappaB) inhibitor, accompanied by increased phosphorylation and degradation of IkappaB. These results suggest that C-peptide stimulates the transcription of inflammatory genes via activation of a PKC/IkappaB/NF-kappaB signaling pathway.