Although disruption of neither p21 nor p27, nor both genes led to significant differences in differentiation or proliferation, disruption of p27 led to downregulation of Cyclin D2 and Cyclin D3 in the colon unveiling a critical role for p27 in regulating these Cyclins in the gastrointestinal tract. MATERIAL AND METHODS Animals Generation of p21?/? (Cdkn1atm1Tyj) 19, p27?/? (Cdkn1btm1Kiyo; 51) 24 and p21?/?; p27 ?/? double knockout mice 25 mice was previously described. These Cdk inhibitors are not needed in vivo for either assembly of Cdk/Cyclin complexes that drive active proliferation, or inhibition of Cdk/Cyclin complexes during cell cycle exit. However, expression of Cyclin D2 and to a lesser degree Cyclin D3 was reduced in p27?/? and p21/p27?/? mice, indicating a unique role for p27 in the regulation of these specific D-type Cyclins in vivo. In the absence of p27, reduced levels of Cyclin D2 and D3 may help to counteract increased proproliferative signals in the intestine. strong class=”kwd-title” Keywords: p21, p27, p57, Cip, Kip, Cyclin D2, Cyclin D3, duodenum, colon INTRODUCTION The intestinal epithelium represents a unique model system for studying cell proliferation and differentiation, because it undergoes rapid and continuous renewal throughout life (reviewed in 1). In the small intestine, proliferation is restricted to the crypts that contain anchored stem cells that give rise to rapidly proliferating progenitor cells that produce four different epithelial cell lineages. Three types of epithelial cells (absorptive enterocytes, mucus-producing goblet cells, and peptide hormone secreting enteroendocrine cells) differentiate during an upward migration from the crypt to an adjacent villus. The fourth epithelial lineage, Paneth cells, differentiate as they migrate downwards to Febuxostat (TEI-6720) the base of the crypt. Although the small intestine is frequently analyzed in mouse models of intestinal cancer, most human intestinal cancers arise in the colon. In contrast to the small intestine, the colon lacks Paneth cells and villi. Stem cells are located at the base of the crypts in the colon and give rise to colonocytes, goblet cells and endocrine cells that differentiate as they migrate upwards and out of the crypt 2. Rapid proliferation coupled with continuous differentiation makes the intestine an ideal model for addressing contributions of cell cycle regulators to differentiation. G1 progression Febuxostat (TEI-6720) is regulated by the D-type Cyclins (D1, D2, D3) that associate with either Cdk4 or Cdk6, and Cdk2 that associates with Cyclin E and A. The Cip/Kip family of Cyclin dependent kinase (Cdk) inhibitors, including p21 (Cip) and p27 (Kip), bind a variety of Cdk/Cyclin complexes with different outcomes 3. Although initially isolated as Cdk inhibitors, p21 and p27 were detected in active Cyclin D/Cdk4 complexes 4C7. p21 was shown to stabilize interactions between Cdk4 and Cyclin D and promote the formation of active complexes 7, 8. In contrast, p21 and p27 association with Cdk2 led to Cdk inhibition, although genetic evidence indicates that Cdk2 is usually dispensable for p21 and p27 mediated inhibition of cell cycle progression 9. In addition to inhibiting Cdk2, p27 also regulates Cdk1 activities 10. In the last few years novel functions for p21 and p27 in regulating cytoskeletal dynamics and migration have also been proposed 11. Both p21 and p27 have been implicated as regulators of intestinal epithelial cell differentiation. Expression of p21 is usually induced as epithelial cells exit Febuxostat (TEI-6720) the cell cycle and begin the process of terminal differentiation in the small and Rabbit polyclonal to BZW1 large intestine 12C15. Similar to p21, p27 expression has been localized to differentiated epithelial cells in the small intestine 14, 16, 17 and colon 18. Although initial reports did not support a role for p21 in regulating differentiation in the gut 19, subsequent studies showed that expression of p21 was induced following expression of dominant unfavorable TCF-4 in colon cancer cells, and that ectopic expression of p21 in the Ls174T colon cancer cell line promoted epithelial cell differentiation associated gene expression 15. A variety of studies, often using human colon cancer cell lines, have suggested that p27 plays a direct role in promoting intestinal epithelial cell differentiation 16, 20C23. We used wild type and knockout mice to examine contributions of p21 and p27 to intestinal epithelial cell differentiation in vivo. Febuxostat (TEI-6720) Although disruption of neither p21 nor p27, nor both genes led to significant differences in differentiation or proliferation, disruption of p27 led Febuxostat (TEI-6720) to downregulation of Cyclin D2 and Cyclin D3 in the colon unveiling a critical role for p27 in regulating these Cyclins in the gastrointestinal tract. MATERIAL AND METHODS Animals Generation of p21?/? (Cdkn1atm1Tyj) 19, p27?/? (Cdkn1btm1Kiyo; 51) 24 and p21?/?; p27 ?/? double knockout mice 25 mice was previously described. The genotypes of the mice were confirmed by genomic DNA isolation and PCR. Mice were fed a commercial diet and water ad libitum, and sacrificed at 8C10 weeks of age. When possible littermates were used in individual experiments and results.