Retinoblastoma Protein (pRb), but Not p107 or p130, Is Required for Maintenance of Enterocyte Quiescence and Differentiation in Small Intestine

Jun Guo,Shannon Longshore,Rajalakshmi Nair,Brad W Warner

doi:10.1074/jbc.m806133200

Jun Guo, Shannon Longshore + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m806133200

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Abstract

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.

Highlights

As a gauge of enterocyte proliferation, the ratio of BrdUrdpositive cells to total crypt cells increased about 20% in VCϩRb(f/f) small intestine when compared with the wild type littermates
We have clearly demonstrated a critical role for pRb, but not the other pocket proteins, as being indispensable for normal exit from the cell cycle as crypt enterocytes emerge to the villus in small intestine
We reveal a unique pattern of phosphorylation of pRb in small intestine and cell cycle regulatory protein expression as a direct consequence of pRb deficiency

Summary

Introduction

The generated pRb intestine conditional nulls still expressed pRb, at a reduced level These mice did not demonstrate an abnormal small intestinal phenotype, but they did show ectopic cell cycle in colon enterocytes. Another group crossed collagen 1A1 Cre mice with Rb (flox/flox) mice to delete pRb in the small intestine [10]. This phenotype, could not be conclusively tied to conditionally deleting pRb in enterocytes, as the author acknowledged that the collagen 1A1 promoter is not enterocyte-specific Another group used Villin Cre transgenic mice that were crossed with Rb (flox/flox) mice to delete pRb in the intestine [11]. We found a unique and not previously described phenotype in the pRb ablation mice in which pRb expression was undetectable, and we discovered that pRb deficiency alone was sufficient to perturb the cell cycle and differentiation in villus cells

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