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Melatonin differentially modulates the expression and function of the hMT1 and hMT2 melatonin receptors upon
Monica I Masana1, Paula A Witt-Enderby, Margarita L Dubocovich
1Department of Molecular Pharmacology and Biological Chemistry (S215), Northwestern University Medical School, 303 East Chicago Ave., IL 60611, USA.
Abstract:
Melatonin is synthesized and released following a circadian rhythm and reaches its highest blood levels during the night. It relays signals of darkness to target tissues involved in regulating circadian and seasonal rhythms. Here, we report the expression of human melatonin receptors type 1 and 2 (hMT(1) and hMT(2), respectively) in Chinese hamster ovary (CHO) cells following exposure to melatonin treatments mimicking the amplitude (400 pM) and duration (8 hr) of the nightly melatonin peak and upon withdrawal. Exposure of CHO-MT(1) cells to melatonin (400 pM) for 0.5, 1, 2, 4, and 8 hr significantly increased specific 2-[125I]iodomelatonin (500 pM) binding to hMT(1) melatonin receptors upon 16-hr withdrawal. However, the same treatment did not affect the expression of hMT(2) melatonin receptors. The increase in specific 2-[125I]iodomelatonin (500 pM) binding (162+/-29%, N=3, P<0.05) 16 hr after melatonin withdrawal was parallel to increases in hMT(1) melatonin receptor mRNA (231+/-33%, N=4, P<0.05). This effect was due to an increase in the total number of hMT(1) receptors [B(max) 833+/-97 fmol/mg protein (N=3), control; 1449+/-41 fmol/mg protein (N=3), treated], with no change in binding affinity. The melatonin-mediated increase in MT(1) melatonin receptor expression upon withdrawal was not mediated through either a direct effect of the hormone in the promoter's vector or in the rate of mRNA degradation. In conclusion, melatonin differentially regulates the expression of its own receptors, which may have important implications in the transduction of dark signals in vivo.
Insights
Melatonin treatment increases human melatonin receptor type 1 (hMT1) expression in CHO cells after withdrawal. This upregulation of hMT1 receptors is linked to increased mRNA levels, suggesting a role in signaling darkness.
Area of Science:
- Endocrinology
- Molecular Biology
- Chronobiology
Background:
- Melatonin, a hormone synthesized in a circadian rhythm, signals darkness to regulate biological rhythms.
- Human melatonin receptors, hMT1 and hMT2, are key mediators of melatonin's effects.
Purpose of the Study:
- To investigate the effect of melatonin exposure and withdrawal on the expression of human melatonin receptors (hMT1 and hMT2) in Chinese hamster ovary (CHO) cells.
- To determine the mechanism underlying melatonin's regulation of its own receptors.
Main Methods:
- CHO cells expressing hMT1 or hMT2 receptors were treated with melatonin mimicking physiological nighttime levels.
- Specific 2-[125I]iodomelatonin binding assays were performed after melatonin withdrawal.
- Quantitative analysis of hMT1 receptor mRNA levels and receptor density (Bmax) was conducted.
Main Results:
- Melatonin treatment followed by 16-hour withdrawal significantly increased specific 2-[125I]iodomelatonin binding to hMT1 receptors in CHO-MT(1) cells.
- This increase in binding correlated with elevated hMT1 receptor mRNA levels and an increased receptor number (Bmax), without altering binding affinity.
- No significant changes were observed in hMT2 receptor expression or binding.
Conclusions:
- Melatonin differentially regulates its receptor expression, specifically upregulating hMT1 receptors upon withdrawal after a period of stimulation.
- The observed increase in hMT1 receptor expression is mediated at the transcriptional level and not by direct hormonal effects on the promoter or mRNA degradation.
- These findings suggest a novel feedback mechanism for melatonin signaling, potentially crucial for transducing dark signals in vivo.