Episodes of Prolactin Gene Expression in GH3 Cells Are Dependent on Selective Promoter Binding of Multiple Circadian Elements

Mar 11, 2010Endocrinology

Prolactin gene activity in GH3 cells depends on specific daily rhythm signals binding to its control regions

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Abstract

Pulses of prolactin mRNA expression are significantly linked to the binding of circadian factors at the E-box133 site.

Prolactin gene expression in mammotropes occurs in pulses, with mechanisms not fully understood.
The E-box133 region in the rat prolactin promoter interacts with circadian factors CLOCK and BMAL1, which is necessary for pulse activity.
In serum-shocked GH(3) cells, the expression of bmal1, cry1, per1, and per3 mRNA temporally aligns with prolactin mRNA pulses.
Circadian factors bind differentially to two E-box sites in the promoter, indicating distinct functional roles.
Down-regulating any circadian factor that binds to E-box133 significantly reduces prolactin mRNA levels during pulse periods.

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Prolactin (PRL) gene expression in mammotropes occurs in pulses, but the mechanism(s) underlying this dynamic process remains obscure. Recent findings from our laboratory of an E-box in the rat PRL promoter (E-box133) that can interact with the circadian factors, circadian locomoter output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, and was necessary for pulse activity raised the intriguing possibility that the circadian system may be central to this oscillatory process. In this study, we used serum-shocked GH(3) cells, established previously to synchronize PRL pulses between cells in culture, to reveal that pulses of PRL mRNA are linked temporally to the expression of bmal1, cry1, per1, and per3 mRNA in these cells. Moreover, we found that each of these circadian factors binds to the rat PRL promoter by chromatin immunoprecipitation analysis. Using EMSA analysis, we observed that two sites present in the proximal promoter region, E-box133 and E-box10, bind circadian factors differentially (E-box133 interacted with BMAL1, cryptochrome-1, period (PER)-1, and PER3 but not PER2 and E-box10 bound BMAL1, cryptochrome-1, PER2, PER3 but not PER1). More importantly, down-regulation of any factor binding E-box133 significantly reduced PRL mRNA levels during pulse periods. Our results demonstrate clearly that certain circadian elements binding to the E-box133 site are required for episodes of PRL mRNA expression in serum-shocked GH(3) cultures. Moreover, our findings of binding-related differences between functionally distinct E-boxes demonstrate not only that E-boxes can bind different components but suggest that the number and type of circadian elements that bind to an E-box is central in dictating its function.

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Episodes of Prolactin Gene Expression in GH3 Cells Are Dependent on Selective Promoter Binding of Multiple Circadian Elements

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