This report describes an analysis of circadian rhythms of mRNA expression in the liver of adult male rats. Animals were sacrificed at nine times during a 12 hour light period and nine corresponding times during a 12 hour dark period. Liver RNAs from each of the 54 animals were applied to individual Affymetrix GeneChips (RAE230A). Analysis yielded 265 probe sets with a 24 hour frequency. Because of the richness of this dataset, we were able to apply QT clustering and identified 5 groups with maxima at different times during the cycle. Two peaked during the light period, two during the dark period, and one very close to the light to dark transition. Approximately two-thirds of the probe sets reach maximum expression during the active (dark) period. The chip in several cases contained more than one probe set for the same gene. In 14 out of 15 instances, all probe sets for the same gene sorted to the same cluster. The single exception was Pvrl2 which sorted to both Clusters 1 and 2. The correlation coefficient of the probe set in Cluster 1 (1375216_at) was 0.84 while the correlation coefficient of the probe set in Cluster 2 (1370345_at) was also 0.84. Both have amongst the lowest correlations with the centroids of their respective clusters.
Regulation of the central clock involves a number of other transcription factors that may be expressed in peripheral tissues. The array used here contained probe sets for PER1, PER2 and, PER3. However, only PER2 showed significant expression and circadian oscillation while PER1 and PER3 had very low signals. Similarly, probe sets for CRY2 and Bhlhb3 also had very low signals. A very low signal can be due to either the lack of expression of the gene or inadequacy of the probe set to measure the signal. In contrast, the chip contained probe sets for Bhlhb2, NFIL3A, Clock, RORC and RORB, and these signals were reasonably strong but without oscillation. It has been reported that at least in some tissues, Clock is expressed at tonic levels and that cycling is due to the rhymicity of its heterodimeric partner BMAL (Reddy et al., 2005
). Of the remaining transcription factors that have been implicated in regulation of circadian changes in gene expression only PER2, BMAL DBP, Nr1d1, and Nr1d2 showed a pattern of circadian oscillation. PER2 was in Cluster 5 during the dark period while BMAL was in Cluster 1 during the light period. DBP, Nr1d1 and Nr1d2 are all in Cluster 3.
The fact that BMAL1, PER2, DBP, Nr1d1 and Nr1d2 all begin to oscillate in response to acute MPL dosing suggest that they are all glucocorticoid sensitive either directly or indirectly. The observation that following chronic dosing an initial oscillation of BMAL occurs followed by what appears to be continuous up-regulation while PER2, DBP, Nr1d1 and Nr1d2 shows oscillation followed by what appears to be continuous down-regulation is potentially informative. However, the apparent continuous up- or down-regulation of the genes may actually be an artifact of sampling times. Just as reasonable an interpretation of the results is that all five genes continue to oscillate throughout the infusion period with BMAL being out of phase with PER2, DBP, Nr1d1 and Nr1d2.
Because synthetic glucocorticoids are a widely used class of drugs, we compared circadian regulated gene expression with those directly regulated by corticosteroids. These datasets together allowed us to address two basic but related questions. The first is: do all genes that respond to corticosteroids have circadian rhythms? The second is: do all genes with circadian rhythms respond to corticosteroid? The answer to both questions is no. Seventy-seven of the genes identified were both circadian and MPL responsive. The fact that all genes that respond to MPL are not circadian and that all genes with circadian rhythms do not respond to MPL suggests that there exist some diversity in mediating mechanisms. This result is consistent with previously described observations comparing our acute and chronic profiles (Almon et al., 2007
If an animal is diurnal, changes in mRNA expression near the end of the dark period begins to prepare the animal for the activity and feeding time. Similarly, changes during the end of the light period prepare the diurnal animal for inactivity and rest. Rats are nocturnal and cycling of gene expression in peripheral tissues like the liver is reversed relative to humans who are essentially diurnal. We explored the results with a focus of potential chronotherapeutic insight.
We identified several genes transcripts that are closely associated with hypocholesterolemic drug strategies. Among these are transcripts for HMG-CoA reductase, the statin target, Sqle, involved in cholesterol synthesis, and Cyp7a1 which is the fibrate target in conversion of cholesterol to bile acids. These transcripts are in Cluster 4 which reaches a maximum 4 hr into the animal’s dark/active period. The current practice of having patients take statins before they go to bed (Staels, 2006
) would seem inappropriate since available data indicates that in humans, HMG-CoA reductase has a maximum expression at about 10 AM (Harwood et al., 1987
; Stacpoole et al., 1987
). In those experiments the investigators were directly measuring enzymatic activity in serially drawn mononuclear leukocyte. The assumption was that activity in mononuclear leukocyte mirrors activity in the liver. In contrast, whole body cholesterol biosynthesis has been reported to peak between midnight and 3 AM (Parker et al., 1982
). In those experiments, the investigators used plasma mevalonic acid as a biomarker for whole body cholesterol biosynthesis. Mevalonic acid is the direct product of HMG-CoA reductase. In addition, urinary mevalonic acid has become an indicator of the effectiveness of statin drug treatment (Hiramatsu et al., 1998
). However, our data is consistent with the data of Harwood et al. indicating the enzyme reaches its peak during the animal’s active feeding period which in the case of rats would be during the dark period as opposed to the light period in humans. The presence of squalene epoxidase in Cluster 4 further reinforces the validity of these observations. What is confusing is why plasma and urine mevalonic acid peak during the inactive period in humans. Most of the mevalonic acid synthesized in the liver is used for cholesterol and then bile acid biosynthesis. However, the preponderance of mevalonic acid in circulation is metabolized by the kidney with the primary products being squaline and lanosterol (Raskin and Siperstein, 1974
). The reason that the timing of the use of statins is important may have less to do with efficacy in lowering cholesterol and more to do with the toxic side effect associated with destabilization of muscle membranes and the development of rhabdomyolysis.
Of the transcripts with circadian rhythms, 35 were for proteins related to cell cycle and apoptosis. In contrast to the cholesterol synthesis related genes, the genes in this functional group are distributed in all five clusters. A relationship between circadian rhythms and cell cycle is well established and our data simply confirms and elaborates on the observations of others. However, the exploitation of these observations in cancer chemotherapy is not straightforward. In cancer therapeutics the important consideration is outcomes, which is based on the balance between toxic and therapeutic effects. If all dividing cells are entrained the same way to the circadian rhythm, then attaining an optimum balance is more complicated. However, some evidence is available that at least in some cases cancer cells have altered organization of the cell cycle relative to the circadian rhythm (Canaple et al., 2003
; Garcia-Saenz et al., 2006
). To the degree that this is true then knowledge of the circadian expression of drug targets in normal cells may provide a basis for reducing toxicity. Adding to the complexity are the observations that endogenous circadian rhythms are often disrupted in cancer patients.
Of the 265 circadian transcripts, 65 are associated with translation and protein processing. Out of these, only 8 reach a maximum during the light/inactive period. Cluster 3, which reaches a maximum shortly after the transition, contains only one transcript, FKBP5. FKBP5 is associated with glucocorticoid signaling (Binder et al., 2004
) and reaches a maximum expression very close to the maximum of the corticosterone circadian rhythm. Clusters 4 and 5 contain the remaining 55 transcripts. Prominent among these are 20 chaperone related proteins. Both inhibiting and enhancing chaperone activities are evolving drug strategies. An important set of drugs in this category are geldanamycin derivatives which inhibit Hsp90 causing the degradation of proteins involved in a large variety of cellular processes from cell cycle and apoptosis to angiogenesis. Because misfolded proteins are associated with several diseases there are a variety of approaches being developed to enhance the activity of Hsp90 and other chaperonins (Powers and Workman, 2006
). Two particularly interesting areas are chaperone-mediated enzyme enhancement and gene therapy. What is also clear is that protein synthesis related expression occurs primarily in Cluster 5. The fact that six proteins that are part of the 60S ribosomal subunit are co-expressed in Cluster 5 tends to validate our results. Proteins that work together are expressed together.
With the burgeoning development of antisense oligonucleotides it is probable that more transcripts will become drug targets. Timing will be an important aspect in the use of antisense technology when applied to transcripts with circadian rhythms.