EDITORIAL FOCUS
Intestinal Cell Differentiation: Cellular
Mechanisms and the Search for the Perfect
Model Focus on "Involvement of p21(WAF1/Cip1) and
p27(Kip1) in intestinal epithelial cell
differentiation"
B. Mark
Evers
Department of Surgery, The University of Texas Medical Branch,
Galveston, Texas 77555-0533
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ARTICLE |
THE MAMMALIAN INTESTINAL MUCOSA, a remarkable example
of high efficiency and intricate complexity, is in a constant state of
self-renewal, with complete mucosal turnover occurring every 3-5
days. Pluripotent stem cells, localized near the base of the crypt, are
characterized by rapid proliferation and ascension along the
crypt-villus axis, with cessation of proliferation and subsequent
differentiation into one of the four primary cell types: absorptive
enterocytes, goblet cells, Paneth cells, and enteroendocrine cells (1,
11). The cellular mechanisms regulating this highly regimented process
of cell cycle arrest and differentiation have not been clearly defined.
The elucidation of these mechanisms would be useful not only in
delineating normal cell processes leading to the differentiated
phenotype but also in providing information regarding abnormal
processes, such as neoplasia formation, that can occur when these
mechanisms go awry. Although an area of active research
interest, this field of investigation has been hampered by the lack of
cellular models that accurately reflect what is occurring in vivo.
For the most part, investigators have relied on human colon cancer cell
lines that possess the property of differentiating to an
enterocyte-like phenotype to investigate mechanisms of normal cellular
differentiation (16). For example, two well-characterized cell lines
include the Caco-2 and HT-29 colon cancer cell lines that differentiate
either spontaneously or in response to changes in media or
differentiating agents, such as sodium butyrate. In the current article
in focus by Tian and Quaroni (Ref. 14, see p. C1245 in this
issue), a novel approach has been utilized to generate a
human fetal intestinal cell line (tsFHI), immortalized with a
temperature-sensitive SV40 T-antigen (SV40 T-Ag) (12). At the
permissive temperature (i.e., 32°C), tsFHI cells actively proliferate and display crypt cell markers. However, with a shift to
the nonpermissive temperature (i.e., 39°C), the cells undergo irreversible growth arrest and shift to a differentiated phenotype, as
noted by expression of differentiated brush-border membrane proteins,
including aminopeptidase N, dipeptidyl-peptidase IV, and
sucrase-isomaltase. Whereas other temperature-sensitive cell lines have
been derived from the rat and mouse (10, 15), the tsFHI cell line was
derived from human intestinal cells and obviously is quite attractive
in this regard. This cell line recapitulates, in a number of respects,
the well-coordinated in vivo process of active proliferation with
subsequent growth arrest and a shift to a differentiated phenotype.
Although this cellular model is appealing in its approach, the results
obtained must be interpreted with some caution, since the functional
SV40 T-Ag may interfere with certain cellular signaling pathways. That
being said, this novel cell line should still prove to be quite
beneficial and valuable in further delineating the pathways that
trigger intestinal cells to differentiate.
Cellular factors that determine whether cells continue to proliferate
or cease dividing and differentiate appear to function during the first
gap phase (G1) of the cell
cycle. Progression through the cell cycle is regulated by a highly
conserved family of serine/threonine protein kinases that are composed
of a regulatory subunit (the cyclins) and a catalytic subunit
[the cyclin-dependent kinases (Cdks)] (9, 13). Progression
through the G1 phase requires
association of D-type cyclins with Cdk4 and Cdk6, whereas cyclin E
binding to Cdk2 is required for the
G1/S transition. The activities of
the Cdks can be inhibited by the binding of Cdk-inhibitory proteins, of
which two families have been identified (4). The first family consists
of universal inhibitors of the cyclin/Cdk complexes and includes
p21(WAF1/Cip1) and p27(Kip1/Pic2). Another family appears
to selectively bind and inhibit Cdk4 and Cdk6 and includes p15(Ink4b),
p16(Ink4a), p18(Ink4c), and p19(Ink4d). In addition, important targets
of Cdk4 and Cdk2 include the retinoblastoma protein (pRb) and the
pRb-related proteins, p107 and p130 (7). Previous studies have shown
that differentiation of the Caco-2 cell line is associated with a
suppression of Cdk2 and Cdk4 activities, which precede the
G1/S cell cycle block and most
likely contribute to this process (3). In addition, induction of the
Cdk inhibitor protein p21(WAF1/Cip1) contributes to this initial cell
cycle block (3, 5, 8). The maintenance of the differentiated phenotype
appears to then involve downregulation of cyclin and Cdk protein
expression and inhibition of cyclin/Cdk complex formation. Findings in
this study by Tian and Quaroni (14) using the tsFHI cell line
corroborate many of the findings noted with Caco-2 cell differentiation
and, moreover, greatly expand our current knowledge regarding the
complicated interactions that occur with intestinal cell
differentiation. The authors further emphasize that the increase of
p21(WAF1/Cip1) is rapid and transient and may be involved in the early
stages of differentiation; however, involvement of p21(WAF1/Cip1) in
the maintenance of the differentiated phenotype is probably not
required. These results are consistent with findings in the differentiated Caco-2 cell line as well as in transgenic mice that lack
the p21 gene (2). A working hypothesis is proposed regarding the
involvement of these cell cycle-related proteins in intestinal cell
differentiation. Thus multiple (and potentially redundant) mechanisms
are likely responsible for the initial cell cycle arrest and the
maintenance of differentiated phenotype.
In summary, the report by Tian and Quaroni (14) is important for the
assessment of intestinal cell differentiation from several different
standpoints. First, it represents the use of a novel human intestinal
cell line that should be useful in further delineating signaling
pathways that are important for the switch from active proliferation to
differentiation. While not perfect, the results using this cell line
will be highly illuminating. In addition, the authors further emphasize
that multiple and potentially redundant mechanisms are at work to
achieve and maintain a differentiated phenotype. Correlation of
findings using this cell line with transgenic models of intestinal
differentiation (6) should prove immensely useful in unraveling the
complex series of events that lead to the remarkably efficient and
orderly process of intestinal cell proliferation and differentiation.
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FOOTNOTES |
Address for reprint requests and other correspondence: B. M. Evers,
Department of Surgery, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0533 (E-mail:
mevers{at}utmb.edu).
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Am J Physiol Cell Physiol 276(6):C1243-C1244
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