EDITORIAL FOCUS
Cell checkpoints and enterocyte differentiation: a
recipe for sequential stages Focus on "Caco-2
intestinal cell differentiation is associated with
G1 arrest and suppression of CDK2
and CDK4"
John M.
Carethers
Department of Medicine, University of California, San Diego, La
Jolla, California 92093-0688
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ARTICLE |
THE COMPLICATED PROCESS of maturation and
differentiation of cells has been difficult to unravel, and the
absorptive intestinal epithelial cell (also known as the enterocyte) is
no exception. Enterocytes proliferate from anchored stem cells in the
crypts of Lieberkühn by mitosis and migrate up the crypt while
simultaneously maturing, expressing enzymes for luminal and
intracellular digestion and absorption. Enterocytes later become
senescent near the villus tips and are subsequently sloughed into the
lumen of the intestine. This process is completed in 3 to 7 days in
humans. The billions of cells that comprise our absorptive interface
are rapidly turned over to resupply critical proteins by cell renewal
that are important for effective nutrient assimilation.
Curiously, the process of proliferation, differentiation, and
programmed senescence in the enterocyte is not well understood. A
complex mix of cell-cell and cell-matrix interactions likely play a
role, modulated by growth factors, luminal factors, and perhaps
hormones. The physiological presence of pancreatic enzymes and bile can
also hasten the life span of the enterocyte. Indeed, after the advent
of adverse conditions or injury, the enormous proliferative potential
of the small intestine is awakened, summoning replacement enterocytes
to populate damaged villi. This plasticity allows for complete recovery
from the injury. An understanding of the control mechanisms for
enterocyte proliferation and maturation is in its infancy. One reason
for this is the inability to maintain normal enterocytes in culture for
research study.
What clues are there from cell models that will enhance our
understanding of maturation mechanisms of the enterocyte? In the current article in focus (Ref. 2; see p. C1193 in this
issue), Ding and colleagues address the timing of cell cycle checkpoint activation and differentiation of Caco-2 intestinal cells. Caco-2 cells
spontaneously differentiate to an enterocyte phenotype when grown to
confluency and have been previously shown to accumulate at the
G1 cell cycle checkpoint by 3 days
after reaching confluency. In a series of experiments comparing
"preconfluent," confluent, and postconfluent cells after 3, 6, and 12 days, the authors discovered that accumulation of cells at the
G1 checkpoint at
day 3 postconfluency preceded the ascertainment of sucrase and alkaline
phosphatase activity, measurements of differentiation, which occurred
by day 6 postconfluency. The events that lead
to cells accumulating at the G1
checkpoint on day
3 appear to be partially the result
of increased expression of the universal cyclin-dependent
kinase (CDK) inhibitor,
p21Waf1/Cip1, binding to CDK2, a
critical kinase that allows transition through G1 in association with cyclin E. Immunodepletion experiments suggest that another CDK inhibitor is
present, but this inhibition is not due to
p27Kip1/Pic2 and
p57Kip2. Continued
accumulation at the G1 phase on
days
6 and
12 postconfluency was due to decreased
levels of cyclin E. Interestingly, CDK4 kinase levels also decrease on
day 3 postconfluency. This reduction does not appear to be due to inhibitors
but is more likely due to decreased levels of the partners of CDK4
kinase, the D cyclins, and is sustained through
days
6 and
12 postconfluency.
For immature enterocytes to differentiate, it appears that they must be
in the G1 phase of the cell cycle.
Logically, a cell cannot devote energy to proliferation while
simultaneously performing complex physiological processes. This is the
opposite of neoplastic cells, in which proliferation is selected for,
and cells often regress from a terminally differentiated state. During
the G1 phase of the cell cycle, a
trigger occurs that activates gene transcription in the enterocyte for
terminally differentiated proteins. Whether differentiation is
specifically triggered by the increased expression of
p21Waf1/Cip1 or other inhibitory
proteins or by a decrease in cyclin expression is not known. Potential
candidates for activation of differentiation include homeobox genes,
such as Cdx2 (4). One potential area for study would be to assess Cdx2
expression in the Caco-2 cell model. Indeed, by conditional expression
of Cdx2, IEC-6 cells, another undifferentiated intestinal cell line,
arrested cellular proliferation for several days, followed by a period
of growth and differentiation (4). Fetal intestinal cells transformed by simian virus 40 (SV40) large T antigen displayed an irreversible growth arrest mediated by
p21Waf1/Cip1 and 1-2 days
later acquired increases in brush-border enzymes (3). Transgenic mice
for wild-type and mutant SV40 T antigen linked to an intestinal fatty
acid-binding protein gene promoter suggests that downregulation of CDK2
and/or cyclin D1 expression is important for control of the
proliferation status of the enterocyte and commencement of terminal
differentiation (1). These other reports are consistent with the
findings of Ding et al. (2) but they utilize transforming proteins or
overexpress genes in their models. At this time, the Caco-2 model of
confluency may be the best model that simulates the neighboring contact
and interaction between enterocytes. Although it now appears that
we know the cell cycle state and timing of differentiation in this
model, we still do not know the trigger. A better understanding of
interaction between G1 phase
regulators and transcription activators of intestinal differentiation
might be fruitful in the future.
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REFERENCES |
1.
Chandrasekaran, C.,
C. M. Coopersmith,
and
J. I. Gordon.
Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators.
J. Biol. Chem.
271:
28414-28421,
1996[Abstract/Free Full Text].
2.
Ding, Q.-M.,
T. C. Ko,
and
B. M. Evers.
Caco-2 intestinal cell differentiation is associated with G1 arrest and suppression of CDK2 and CDK4 activities.
Am. J. Physiol.
275 (Cell Physiol. 44):
C1193-C1200,
1998[Abstract/Free Full Text].
3.
Quaroni, A.,
and
J. F. Beaulieu.
Cell dynamics and differentiation of conditionally immortalized human intestinal epithelial cells.
Gastroenterology
113:
1198-1213,
1997[Medline].
4.
Suh, E.,
and
P. G. Traber.
An intestine-specific homeobox gene regulates proliferation and differentiation.
Mol. Cell. Biol.
16:
619-625,
1996[Abstract].
Am J Physiol Cell Physiol 275(5):C1191-C1192
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society