Study shows that lithium regulates circadian gene expression through a nuclear hormone receptor
By Melissa Lee Phillips | February 17, 2006
Lithium treatment leads to synchronized circadian oscillations in human cells through the orphan nuclear receptor Rev-erba, according to a study in this week's Science. This molecular pathway may underlie lithium's effects on the circadian clock in people with bipolar disorder, a condition associated with aberrant circadian rhythms, according to the authors.
It was already established that Rev-erba is part of the circadian clock and that lithium acts on a protein involved in circadian rhythm, glycogen synthase kinase 3b (GSK3b), said Eric Herzog of Washington University in St. Louis, who was not involved in the research. The researchers "link those two stories," Herzog noted, demonstrating "a pretty convincing specific interaction between lithium, GSK3, and circadian genes."
In the classic molecular circadian loop in mammals, the transcription factors BMAL1 and CLOCK activate the clock genes Per and Cry, whose proteins feed back into the nucleus to inhibit their own transcription. Several years ago, biologists discovered another, parallel negative feedback loop involving the nuclear receptor Rev-erba. Specifically, the BMAL1-CLOCK heterodimer also activates Rev-erba transcription, and Rev-erba protein feeds back and represses the transcription of Bmal1.
Led by first author Lei Yin and senior author Mitchell Lazar, the University of Pennsylvania researchers noticed that Rev-erba contains several potential sites for phosphorylation of GSK3b, Lazar told The Scientist. Previous work had shown that mutations in the Drosophila homolog of GSK3b lengthen the flies' circadian period. "We realized that GSK3b was a wonderful candidate" to explain how external signals might influence the Rev-erba feedback loop, Lazar said.
He and his colleagues found that inhibiting GSK3b with siRNA in human embryonic kidney cells led to a near complete loss of Rev-erba protein, suggesting that GSK3b is necessary for Rev-erba stabilization. They also found that treating cultures with high concentrations of serum - known to synchronize circadian oscillations - inhibits GSK3b activity by phosphorylating it. Again, Rev-erba protein expression dropped, and Bmal1 transcription was activated, which begins the 24-hour circadian gene expression cycle, Lazar said.
The authors next treated these cells with lithium, since lithium is known to inhibit GSK3b. They found that lithium treatment also reduced Rev-erba protein levels and induced Bmal1 transcription.
In addition, the researchers found that serum shock cannot initiate circadian oscillations in cells if Rev-erba is replaced with a mutant form that is resistant to degradation. "If Rev-erb can't be degraded, the clock never starts," Lazar said. Something in serum is responsible for Rev-erba degradation, Lazar said, but "we're still trying to tease out exactly what hormonal cue in the serum is causing GSK3 to be phosphorylated."
The researchers also discovered that lithium treatment fails to induce Bmal1 expression in the absence of wild-type Rev-erba, suggesting that lithium initiates synchronized circadian oscillations by degrading Rev-erba.
Despite the compelling evidence, Herzog cautioned that Lazar's group still cannot say that this pathway is in fact relevant to circadian rhythms or bipolar disorder in vivo.
In addition, in most experiments, the researchers use concentrations of lithium that are "about twenty times higher than lithium's usual therapeutic doses," Husseini Manji of the National Institutes of Mental Health, not a co-author, told The Scientist. The authors did show that a clinically relevant concentration of lithium will also reduce Rev-erba protein and induce Bmal1 transcription, but their conclusions would have been stronger if every experiment had used lower lithium concentrations, Manji said. Still, the work suggests that Rev-erba degradation may be a potential target for developing novel bipolar or circadian disorder treatments, he added.
Melissa Lee Phillips
Links within this article
L. Yin et al., "Nuclear receptor Rev-erba is a critical lithium-sensitive component of the circadian clock," Science, February 17, 2006.
M. Abe et al., "Lithium lengthens the circadian period of individual suprachiasmatic nucleus neurons," Neuroreport, September 28, 2000.
A. N. Schweitzer, "Bipolar understanding," The Scientist, March 29, 2004.
H. A. Mansour et al., "Circadian genes and bipolar disorder," Annals of Medicine, May 2005
S. M. Reppert et al., "Molecular analysis of mammalian circadian rhythms," Annual Review of Physiology, 2001.
K. Y. Kreeger, "Collecting clues to the mammalian clock," The Scientist, April 15, 2002.
N. Preitner et al., "The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator," Cell, July 26, 2002.
S. Martinek et al., "A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock," Cell, June 15, 2001.
A. Balsalobre et al., "A serum shock induces circadian gene expression in mammalian tissue culture cells," Cell, June 12, 1998.
K. Kreeger, "High-throughput technology tackles circadian rhythms," The Scientist, November 17, 2003.
P. S. Klein et al., "A molecular mechanism for the effect of lithium on development," PNAS, August 6, 1996.