Circadian expression of clock genes in human oral mucosa and skin: association with specific cell-cycle phases

Abstract

We studied the relative RNA expression of clock genes throughout one 24-hour period in biopsies obtained from the oral mucosa and skin from eight healthy diurnally active male study participants. We found that the human clock genes hClock, hTim, hPer1, hCry1, and hBmal1 are expressed in oral mucosa and skin, with a circadian profile consistent with that found in the suprachiasmatic nuclei and the peripheral tissues of rodents. hPer1, hCry1, and hBmal1 have a rhythmic expression, peaking early in the morning, in late afternoon, and at night, respectively, whereas hClock and hTim are not rhythmic. This is the first human study to show a circadian profile of expression for all five clock genes as documented in rodents, suggesting their functional importance in man. In concurrent oral mucosa biopsies, thymidylate synthase enzyme activity, a marker for DNA synthesis, had a circadian variation with peak activity in early afternoon, coinciding with the timing of S phase in our previous study on cell-cycle timing in human oral mucosa. The major peak in hPer1 expression occurs at the same time of day as the peak in G(1) phase in oral mucosa, suggesting a possible link between the circadian clock and the mammalian cell cycle.

Figure 1.

Melatonin and cortisol rhythms during the 24-hour biopsy period. Normalized data for the serum melatonin and cortisol in eight study participants during the 24-hour biopsy period are shown. Time point means (normalized value) and SE are depicted along the 24-hour time scale. The best fitting 24-hour single component cosine curve is shown. The time for the acrophase (95% confidence limits), P value from cosinor analysis, and time-effect by analysis of variance are shown.

Figure 2.

Examples of the results for duplex PCR for gene expression in human oral mucosa. Primers for hClock were co-amplified with β-actin (A). After showing that there was no significant variation throughout 24 hours in the expression of hClock versus β-actin, hPer1 (B), hCry1 (C), hBmal1 (D), hTim (E), and TS (F) were co-amplified with hClock. A to F show the relative expression of all four clock genes throughout 24 hours, a control lane (ddH₂O), and a marker lane.

Figure 3.

The timing of hClock and hTim expression in oral mucosa and skin. Normalized data for the circadian expression of hClock, and hTim in human oral mucosa and skin of eight study participants. Time point means (normalized values) and SE are depicted along the 24-hour time scale.

Figure 4.

The timing of hPer1, hBmal1, and hCry1 expression in oral mucosa and skin. Normalized data for the circadian expression of hPer1, hCry1, and hBmal1 in human oral mucosa and skin of eight study participants. Time point means (normalized values) and SE are depicted along the 24-hour time scale. For hBmal1 and hCry1, a 24-hour single-component cosine curve is shown along with the predicted acrophase (95% confidence limits) and P values from cosinor analysis and time-effect by analysis of variance. For hPer1, a 12/24 hours composite cosinor curve is shown along with the predicted acrophases for each peak (95% confidence limits) and P values for each rhythm component from cosinor analysis.

Figure 5.

Circadian TS activity and TS mRNA expression in oral mucosa. Normalized data for the circadian variation in TS enzyme activity and TS mRNA expression in human oral mucous in eight study participants. Time point means (normalized value) and SE are depicted along the 24-hour time scale. The best fitting 24-hour single-component cosine curve is shown. The time for the acrophase (95% confidence limits), P value from cosinor analysis, and time-effect by analysis of variance are shown.

Figure 6.

Association of circadian clock gene expression with the timing of cell-cycle phases. A: The high points for the best-fitting cosine for p53, cyclin-E, cyclin-A, and cyclin-B1 are depicted along the 24-hour time scale based on our previous study on human oral mucosa. The 95% confidence limits for each variable are shown. The cell-cycle phase, for which each protein is a marker, is shown in parentheses on the y axis and on the graph. B: The acrophases for the major (filled circle) and minor (open circle) peaks of expression for hPer1 (B1) and for the single peak in expression of hBmal1 (B2) in oral mucosa are depicted along the 24-hour time scale. In B3, the acrophase for the peak activity of TS (S phase) in oral mucosa is depicted along the 24-hour time scale. The 95% confidence limits for each variable are shown. C: The acrophases for the major (filled circle) and minor (open circle) peaks of expression for hPer1 (C1) and for the single peak in expression of hBmal1 (C2) in skin are depicted along the 24-hour time scale. The 95% confidence limits for each variable are shown. D: The acrophases for S phase and M phase in human skin based on pooled data from 12 studies looking at M phase and 14 studies looking at S phase throughout 24 hours are depicted along a 24-hour time scale. The 95% confidence limits for each variable are shown.