Uncaging Reveals Calcium’s Role in Cell Cycle
Progression
Calcium ions are known to play a role in many aspects of cell function, but gaps exist
in the understanding of calcium’s role in the cell cycle. Research led by Professor Mike
White and Dr. Violaine Sée at the University of Liverpool used targeted flash photolysis
to elucidate intracellular calcium’s role in signaling the cell to progress through the cell
cycle.
D-type cyclins such as CD1 are known to play an important role
in cell cycle progression. Its level varies during cell cycle, and the
amount of CD1 protein is directly dependent on the rate of CD1
gene transcription. CD1 gene transcription is regulated by multiple
transcription factors, including nuclear factor kappa B (NF-κΒ), which
can control cell growth and differentiation through transcriptional
regulation of CD1.
Even though NF-κΒ appears to play a role in cell cycle progression,
the intracellular events leading to this transcription factor’s activation
upon growth factor stimulation are unknown. Stimulation of non
dividing cells with growth factors or mitogens causes a transient rise
in intracellular free calcium concentration. Intracellular calcium has
been shown to play a role in the G1/S and G2/M phases of the cell
cycle, but the University of Liverpool researchers wanted to find out
more about its role in the G0–G1 transition.
Using targeted photolysis to uncage a calcium chelator and a calcium
donor in living cells allowed them to observe how the cells responded
to precisely-timed absence of (normally-present) calcium signals and
increases in calcium levels. This allowed them to understand the role
that calcium plays in CD1 gene transcription and subsequent cell
proliferation.
To study the long-term role of the serum-induced intracellular calcium
increase, the researchers needed a noninvasive method to eliminate this
transient increase. They used the caged calcium scavenger Diazo-2. Its
normally low affinity to calcium increases 30-fold when illuminated
at 360 nm. To eliminate the serum-induced calcium peak, they applied
the illumination to Diazo-2-loaded cells just after serum induction.
The Diazo-2 photolysis inhibited the serum-induced calcium peak
without effecting subsequent intracellular calcium levels.
The researchers used the MicroPoint system to uncage Diazo-2 in
a cell of interest by exposing it to a train of 4 s flashes of 360 nm
light over 10 s. They performed simultaneous imaging and photo-
stimulation of the specimen, and could therefore follow the targeted
cells and normal cells with time-lapse confocal microscopy. In this
way they directly monitored the effects of uncaging on intracellular
signaling and cell fate.
As Violaine Sée explains, they did not detect NF-κΒ translocation
into the nucleus or NF-κΒ-dependent gene transcription from the NF-
κΒ consensus and CD1 promoters in the targeted cells. These facts
suggested that the serum-induced calcium peak appears to signal the
down-stream events.
To further study the relationship between the transient calcium increase
and later NF-κΒ activation, they performed targeted photolysis of the
caged calcium donor NP-EGTA. Activated NP-EGTA causes a 22%
transient increase in intracellular calcium. This increase is of the same
magnitude as the serum-stimulated increase, but it did not induce
detectable p65 translocation to the nucleus.
The result of suppressing and replicating serum-induced calcium
peaks using caged molecules suggested that NF-κΒ translocation and
downstream regulation of gene transcription depends on a transient
increase of intracellular calcium, but other serum-dependent signals
are required to activate NF-κΒ. These results, combined with other
experiments, led the researchers to propose that the G0–G1 transition
depends on mitogenic kinase activity as well as calcium signaling and
that NF-kB activation has a critical role in the transduction of these
mitogenic signals to the nucleus.
“The work showed that calcium has a lead role in cell cycle. Calcium
ions have been implicated in many aspects of cell function, but its
role in the cell cycle was not well described,” said Violaine Sée. “We
found that calcium can initiate the cell cycle through activation of the
mitogen-activated protein kinase–NF-κΒ pathway.”
Acknowledgement:
Appreciation is gratefully extended to Professor Mike
White, Dr. Violaine Sée and Dr. Dave Spiller, University of Liverpool
Research Paper:
Violaine Sée, Nina K.M. Rajala, David G. Spiller, and Michael R.H. White,
Calcium-dependent regulation of the cell cycle via a novel MAPK–NF-κΒ
pathway in Swiss 3T3 cells, The Journal of Cell Biology, Volume 166, Number
5, August 30, 2004 661–672.