In the present study, we attempted to determine a mechanism for how p21 expression is required for the regulation of resection-induced intestinal adaptation. Deletion of Rb results in a hyperplastic phenotype with no further adaptive changes following SBR. This seems to be specific to Rb as deletion of other members of its pocket protein family, p107 and p130, results in normal adaptive responses. We have identified that Rb protein levels are significantly elevated in the context of p21 deletion. Knocking down Rb expression in the context of p21 deficiency restores the adaptive capacity of p21-null mice. Taken together, these results suggest that in adapting enterocytes, p21 functions to regulate Rb protein levels, thereby providing a plausible mechanism for p21 as a required protein for resection-induced intestinal adaptation.
Rb deletion produces a unique hyperplastic intestinal phenotype not seen with deletion of other members of its pocket protein family. Rb has been previously shown to be required for normal epithelial cell homeostasis.14,15
The hyperplasia seen with Rb deletion is associated with defects in epithelial cell terminal differentiation as well as increased epithelial cell proliferation in mutant animals. At the molecular level, perturbation of the Rb pathway results in increased expression of transcription factors, such as Math1, Cdx1, and Cdx2, which regulate the proliferation and differentiation of the intestinal epithelium.14
Additionally, Rb, but not p107 or p130, has been demonstrated to be required for the maintenance of the post-mitotic epithelial cells in quiescence so that absorptive enterocytes can complete differentiation.15
It seems counterintuitive that the deletion of a cell cycle inhibitor (Rb) would result in no adaptive response to SBR. Because Rb deletion results in a hyperplastic mucosa that already resembles the adaptive growth seen after SBR, perhaps there is a plateau or a maximal level of enterocyte proliferation that the added proliferative stimulus of SBR cannot further enhance. Alternatively, because deletion of Rb results in a marked proliferative stimulation to enterocytes, the alteration of multiple counter-regulatory molecular pathways could mask the contribution of Rb to adaptation. However, deletion of other important transcription factors, such as p277
or the other pocket proteins p107 and p130, does not result in abrogated adaptation to intestinal resection.
The proliferative response to SBR is seen as early as 12–24 h after resection, although it is maximally increased at day 3.18
Because adaptation is maintained over time by increased rates of proliferation and Rb-null mice have baseline elevated proliferative rates, we evaluated adaptation in the Rb-null animals over a range of postoperative time intervals from early (3 days) to late (28 days). However, there was no difference in adaptation between the early vs. middle vs. late time points, and the higher levels of baseline proliferation did not translate into further increases in villus height or crypt depth following massive SBR.
Apoptosis is also a critical hallmark of the crypt enterocyte response to intestinal resection.20
After SBR, the intrinsic pathway of apoptosis is initiated via the p38α-dependent activation of Bax in intestinal epithelial cells.21
New data suggest that Rb is directly phosphorylated by p38α independent of the cell cycle, leading to the degradation of Rb, release of E2F1, and cell death.22
Although Rb-null mice have similar baseline rates of apoptosis as observed in WT mice, Rb deficiency prevented the usual increase in apoptosis following SBR. This observation suggests that in addition to Bax, resection-associated apoptosis may also require Rb, perhaps via a mechanism of p38α-induced Rb phosphorylation. Further, since Rb-nulls had similar rates of apoptosis at baseline, the contribution of apoptosis does not appear to represent a significant factor in the hyperplastic phenotype of these mice. Finally, in the Rb-null animals where the normal increase in apoptotic rate following SBR is prevented, exaggerated adaptation does not occur.
The function of p21 as a cell cycle inhibitor vs. cell cycle inducer seems to be situation and tissue dependent. While the predominant literature supports the concept of p21 involvement in cell cycle arrest, such as in the blood and brain,24, 25
increased expression of this CDKI has been reported in other models in association with induction of proliferation. Following partial hepatectomy, p21 expression was found to be elevated in the regenerating liver.23
Increased expression of p21 in the cytosol of vascular smooth muscle cells was associated with increased cell cycle progression.24
There is increasing evidence in the literature for the regulation of Rb by p21 and that their dual oncogenic/tumor suppressor functions are intimately associated. Through the inhibition of CDK2/4 complexes and other transcription factors, p21 affects cell survival, morphology, and gene expression.26
Phosphorylation by CDK2 and CDK4/6 inactivates Rb in proliferating cells; therefore, the induction of p21 results in Rb dephosphorylation, activation, and G1 arrest.16
However, p21 has also been reported to be associated with the proteasomal degradation of Rb.27
The observations that p21 can both activate Rb transcription through dephosphorylation and then deactivate it through degradation suggest a powerful negative feedback regulation between these two proteins.16
Our observation that Rb protein levels are elevated in the context of p21 deletion fits the concept of regulation of Rb by p21. When p21 is absent, Rb is not degraded and thus accumulates in the cell. This accumulation of Rb inhibits resection-induced proliferation and explains the lack of adaptation seen in the p21-null mouse following SBR. Additionally, reducing Rb levels to near-normal in the context of p21 deletion resulted in restoration of the proliferative response to SBR. This highlights not only the important regulatory relationship between p21 and Rb but also that the presence of Rb is critical for adaptation.