Melatonin Signal Transduction Pathways Require E-Box-Mediated Transcription of Per1 and Per2 to Reset the SCN Clock at Dusk - PubMed (original) (raw)

Melatonin Signal Transduction Pathways Require E-Box-Mediated Transcription of Per1 and Per2 to Reset the SCN Clock at Dusk

Patty C Kandalepas et al. PLoS One. 2016.

Abstract

Melatonin is released from the pineal gland into the circulatory system at night in the absence of light, acting as "hormone of darkness" to the brain and body. Melatonin also can regulate circadian phasing of the suprachiasmatic nucleus (SCN). During the day-to-night transition, melatonin exposure advances intrinsic SCN neural activity rhythms via the melatonin type-2 (MT2) receptor and downstream activation of protein kinase C (PKC). The effects of melatonin on SCN phasing have not been linked to daily changes in the expression of core genes that constitute the molecular framework of the circadian clock. Using real-time RT-PCR, we found that melatonin induces an increase in the expression of two clock genes, Period 1 (Per1) and Period 2 (Per2). This effect occurs at CT 10, when melatonin advances SCN phase, but not at CT 6, when it does not. Using anti-sense oligodeoxynucleotides (α ODNs) to Per 1 and Per 2, as well as to E-box enhancer sequences in the promoters of these genes, we show that their specific induction is necessary for the phase-altering effects of melatonin on SCN neural activity rhythms in the rat. These effects of melatonin on Per1 and Per2 were mediated by PKC. This is unlike day-active non-photic signals that reset the SCN clock by non-PCK signal transduction mechanisms and by decreasing Per1 expression. Rather, this finding extends roles for Per1 and Per2, which are critical to photic phase-resetting, to a nonphotic zeitgeber, melatonin, and suggest that the regulation of these clock gene transcripts is required for clock resetting by diverse regulatory cues.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. At CT 10, melatonin induces of Per1 and Per2 transcription by 120 min.

A) qPCR amplification products migrate at the predicted size and are distinguishable on an 8% polyacrylamide gel stained with ethidium bromide (Per1 = 113 bp, Per2 = 90 bp, BMAL1 = 79 bp). B) Melatonin has no significant effect on the expression levels of Per1, Per2, or Bmal1 mRNA 30 min following the initiation of treatment (p ≥ 0.05, Student’s T Test). C) Melatonin treatment significantly increases Per1 and Per2, but not Bmal1, transcripts, at 120 min. Data are shown as percent change of relative mRNA levels compared to control ± SEM, n = 3-4/condition. ***p ≤ 0.001 (Per1), *p ≤ 0.05 (Per2), p ≥ 0.05 (Bmal1), Student’s T-test.

Fig 2

Fig 2. At CT 6, melatonin does not change the levels of Per1 and Per2 transcripts, although Bmal1 is reduced at 120 min.

Melatonin applied at CT 6 has no significant effect on the expression levels of Per 1, Per2, or Bmal1 mRNA after 30 min (A). After 120 min (B), only Bmal1 mRNA significantly decreases following initiation of melatonin treatment at CT 6. Data are shown as percent change of relative mRNA levels compared to control ± SEM, n = 3–9 /condition, p ≥ 0.05 (Per 1, Per 2), *p ≤ 0.05 (Bmal1), Student’s T-test.

Fig 3

Fig 3. The PKC inhibitor, chelerythrine chloride, blocks the increase of Per1 and Per2 mRNA induced by melatonin applied at CT 10.

Pre-treatment with 0.25 mM of the PKC inhibitor, chelerythrine chloride, blocks the melatonin-induced increase in Per1 (A) and Per2 (B) transcripts after 120 min. Data are shown as percent change of relative mRNA levels compared to control ± SEM, n = 3/condition (** p ≤ 0.01, *p ≤ 0.05, 1-way ANOVA, Tukey’s post-hoc analysis). Controls were exposed to sham treatment lacking MEL. MEL = melatonin. CC = chelerythrine chloride.

Fig 4

Fig 4. Per1 and Per2 αODN attenuate the expression of corresponding transcripts in the SCN.

2-h incubation of SCN slices with αODN results in a 45% decrease in Per1 transcripts (A) and a 60% decrease in Per2 transcripts (B) 4 h after initiation of treatment with the corresponding αODN. No change in GAPDH mRNA was evident following either treatment, which was used as a normalization control.

Fig 5

Fig 5. Per1 is required for melatonin to alter the phase of SCN neuronal activity rhythms at CT 10.

A) The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in controls. The dotted line indicates the mean time-of-peak for untreated slices. Long, vertical boxes represent subjective night, CT 12–14. B) At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (n = 3). Arrow = time of melatonin treatment. C) Per1 αODN application from CT 8–10 has no significant effect on the time-of-peak electrical activity (n = 3). Small box = duration of ODN exposure. D) The MEL-induced phase advance is completely blocked by Per1 αODN (n = 3). E) Per1 missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (n = 3). F) Per1 missense ODN does not block the MEL-induced phase advance at CT 10. G) Summary of the effects of Per1 ODN on MEL-induced phase advances at CT 10. **indicates statistically significant difference compared to controls (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s post-hoc analysis.

Fig 6

Fig 6. Per2 is required for melatonin to phase-shift SCN neuronal activity rhythms at CT 10.

A) The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in control SCN (n = 3). B) At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (n = 3). C) Per2 αODN application from CT 8–10 has no effect on the mean time-of-peak electrical activity (n = 3). Small box = duration of ODN exposure. D) The MEL-induced phase advance is blocked completely by pre-incubation from CT 8–10 with Per2 αODN (n = 3). E) Pre-incubation with Per2 missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (n = 3). F) Pre-incubation with Per2 missense ODN has no effect on the MEL-induced phase advance at CT 10 (n = 3). G) Summary of the effects of Per2 αODN pre-incubations on MEL-induced phase advances at CT 10. **indicates statistically significant differences (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s post-hoc analysis. Symbols as in Fig 5.

Fig 7

Fig 7. E-box decoy blocks binding at E-box sites in SCN 2.2 cells.

Electromobility shift assay of an E-box probe incubated with nuclear extracts of SCN 2.2 cells transfected with 1 μM E-box decoy or missense ODN. Media lane indicates non-transfected control. Arrow = retarded mobility of the E-box probe. This DNA-protein interaction is absent in SCN 2.2 cells transfected with the E-box decoy up to 24 h (n = 3).

Fig 8

Fig 8. Melatonin-induced increases in Per1 and Per2 mRNAs are blocked by E-box decoy ODN.

Pre-treatment of SCN slices with E-box decoy ODN (1 μM), blocks the melatonin-induced increase in Per1 (A) and Per2 (B) transcripts after 120 min. qPCR data are shown as percent change of relative mRNA levels compared to control ± SEM, n = 3/condition (** p ≤ 0.01, *p ≤ 0.05, 1-way ANOVA, Tukey’s post-hoc analysis). MEL = melatonin.

Fig 9

Fig 9. E-box promoter motif is required for melatonin to shift SCN neuronal activity rhythms at CT 10.

A) The spontaneous electrical activity rhythm in SCN brain slices peaks at CT 6.38 ± 0.13 in controls (n = 3). The dotted line indicates the mean time-of-peak for untreated slices. Large, vertical boxes represent subjective night, CT 12–14. B) At CT 10, MEL (1 nM, 10 min) advances the electrical activity rhythm by 3.6 h ± 0.10 (n = 3). Arrow = time of melatonin treatment. C) E-box decoy ODN has no significant effect on the time-of-peak electrical activity (n = 3). Small box = duration of ODN exposure. D) The MEL-induced phase advance is blocked by the E-box decoy ODN (n = 3). E) Missense ODN has no effect on the MEL-induced advance in time-of-peak electrical activity (n = 3). F) Missense ODN does not block the MEL-induced phase advance at CT 10 (n = 3). G) Summary of the effects of ODN on MEL-induced phase advances at CT 10. **indicates statistically significant difference compared to controls (p ≤ 0.001) as determined by 1-way ANOVA with Tukey’s post- hoc analysis.

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