Abstract
Brain and muscle ARNT-like protein-1 (BMAL-1) is an important component of the cellular circadian clock. Proteins such as epidermal (EGF) or nerve growth factor (NGF) affect the cellular clock via extracellular signal-regulated kinases-1/2 (ERK-1/2) in NIH3T3 or neuronal stem cells, but no such data are available for the insulin-like growth factor-1 (IGF-1). The hypothalamus expresses receptors for all three growth factors, acts as a central circadian pacemaker, and releases hormones in a circadian fashion. However, little is known about growth factor-induced modulation of clock gene activity in hypothalamic cells. Here, we investigated effects of IGF-1, EGF, or NGF on the Bmal-1 promoter in two hypothalamic cell lines. We found that only IGF-1 but not EGF or NGF enhanced activity of the Bmal-1 promoter. Inhibition of ERK-1/2 activity did not affect IGF-1-induced Bmal-1 promoter activation and all three growth factors similarly phosphorylated ERK-1/2, questioning a role for ERK-1/2 in controlling BMAL-1 promoter activity. Of note, only IGF-1 induced sustained phosphorylation of glycogen synthase kinase-3β (GSK-3β). Moreover, the GSK-3β inhibitor lithium or siRNA-mediated GSK-3β knockdown diminished the effects of IGF-1 on the Bmal-1 promoter. When IGF-1 was used in the context of temperature cycles entraining hypothalamic clock gene expression to a 24-h rhythm, it shifted the phase of Bmal-1 promoter activity, indicating that IGF-1 functions as a zeitgeber for cellular hypothalamic circadian clocks. Our results reveal that IGF-1 regulates clock gene expression and that GSK-3β but not ERK-1/2 is required for the IGF-1-mediated regulation of the Bmal-1 promoter in hypothalamic cells.
Highlights
Brain and muscle ARNT-like protein-1 (BMAL-1) is an important component of the cellular circadian clock
Our results reveal that insulin-like growth factor-1 (IGF-1) regulates clock gene expression and that GSK-3 but not extracellular signal–regulated kinases-1/2 (ERK-1/2) is required for the IGF-1– mediated regulation of the Bmal-1 promoter in hypothalamic cells
Our data suggest that IGF-1– promoted circadian clock gene expression in hypothalamic cells is unique among the three tested growth factors and distinct from what is known in other cell models, because it does not correlate with the cAMP-response elements (Cre) promoter activation, c-FOS induction, or ERK-1/2 phosphorylation
Summary
Hypothalamic mHypoA-2/10 cells were used to investigate the effects of growth factors on Bmal-1 promoter activity (38 – 40). EGF and NGF failed again to activate this clock gene reporter at any time and in either cell line, suggesting that both growth factors do not affect clock gene expression in hypothalamic cells at all. Our data suggest that IGF-1– promoted circadian clock gene expression in hypothalamic cells is unique among the three tested growth factors and distinct from what is known in other cell models, because it does not correlate with the Cre promoter activation, c-FOS induction, or ERK-1/2 phosphorylation. IGF-1 and EGF induced reporter activation in these cells (Fig. 4C) in a PD-184352-dependent manner (Fig. 4D) It appears that IGF-1–induced circadian gene expression in hypothalamic cells is unique among growth factors and cell types. Temperature-induced Bmal-1 promoter activation was significantly inhibited, indicating the following sequence of temperature-controlled transcription factor activation: PER-2, REV-ERB␣, and BMAL-1 (Fig. 9C)
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