Gastroenterology

Gastroenterology

Volume 133, Issue 1, July 2007, Pages 232-243
Gastroenterology

Basic–alimentary tract
p27kip1 Regulates cdk2 Activity in the Proliferating Zone of the Mouse Intestinal Epithelium: Potential Role in Neoplasia

https://doi.org/10.1053/j.gastro.2007.04.043Get rights and content

Background & Aims: Reduced p27kip1 expression is a marker of poor prognosis in colorectal neoplasia, and inactivation of p27 in mice (p27Δ51/Δ51) causes increased intestinal epithelial cell proliferation and small and large intestinal neoplasia in a diet-dependent manner. Here, we addressed the role of p27 in untransformed intestinal epithelial cells in vivo and the consequence of its targeted inactivation. Methods: A sequential fractionation procedure was used to isolate murine intestinal epithelial cells relative to their position along the crypt-villus axis, and the levels of cyclins, cyclin-dependent kinases (cdks), and cdk inhibitors and of the complexes formed among them was determined by immunoprecipitation-immunoblotting and kinase assays. Results: As cells exited the proliferative crypt compartment, expression and activity of both cdk2 and cdk4 decreased, in parallel with reduced expression of cyclin A and proliferating cell nuclear antigen (PCNA); expression of cyclin D1, D2, and cyclin E showed little change. As expected, expression of the cdk inhibitors p21, p57, and p16 was highest in differentiated villus cells. Unexpectedly, p27 protein expression was highest in cells of the proliferative crypt compartment where it bound both cdk2 and cdk4. Cdk2 activity was increased in crypt cells from p27Δ51/Δ51 mice, although cyclin D-associated kinase activity was unchanged (indeed, cyclin D1/2-cdk4 complex levels were reduced). Importantly, cdk2 activity was unchanged in crypt cells from p21−/− mice, which do not develop intestinal tumors. Conclusions: We propose that p27 contributes to intestinal epithelial homeostasis by regulating cdk2 activity in proliferating cells, thus gating cell cycle progression and suppressing intestinal neoplasia.

Section snippets

Mice

Mice with functional inactivation of both p27 alleles (p27Δ51/Δ51) because of disruption of the N-terminal cdk inhibitory domain (giving an approximately 20-kilodalton truncated p27 protein, Δ51, unable to bind to cdk2 and cdk4) have been described previously.18 p21−/− mice have been described previously.24 Mice were maintained on AIN-76 diet from Harlan-Teklad (Madison, WI) from weaning at approximately 3 weeks after birth until death at 13 to 14 weeks of age (prior to onset of tumor-related

Results

We first used a biochemical approach to determine expression and activity of G1/S-regulatory proteins along the normal murine small intestinal crypt-villus axis. Cells were fractionated according to their position along the crypt-villus axis using a method we have extensively validated,23 and the fractionation procedure was monitored in each experiment by determining expression of PCNA (a marker of proliferative crypt epithelial cells) and villin (a marker of differentiated villus cells) (

Discussion

Although cells undergo rapid proliferation in the small intestinal crypt, it is likely that, in the normal crypt, this is regulated. Here, we showed that the cdk inhibitor p27 is uniquely localized to the proliferative crypt compartment. Moreover, using a mouse with a targeted inactivation of p27, we showed that p27 significantly limits the extent of cdk2 activity in the crypt, and we propose that this regulates cell cycle progression within the proliferative compartment. Unlike p21, p57, and

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    The authors have no conflict of interest to disclose.

    Supported in part by NCI grants RO1 CA114265 and U54 CA100926.

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