In the present study, we sought to determine a mechanism for why p21waf1/cip1-null mice fail to demonstrate an intestinal adaptation response to massive SBR. We specifically tested the hypothesis that deficient expression of this protein would result in an attenuated population of stem cells within the intestinal crypt. We found no evidence that the intestinal stem cell pool was affected by either massive SBR or p21waf1/cip1 deficiency. These results suggest that expansion of stem cells does not occur after SBR and the lack of resection-induced adaptation in p21waf1/cip1-null mice is due to another mechanism.
Identification of specific stem cells within the intestinal crypt has been difficult, primarily due to the lack of specific, universally agreed-upon stem cell markers. Indeed, the stem cell genes that were screened in our array kit contained gene constructs associated with stem cells in other locations and tissues. As such, one limitation of this study is that we could have overlooked expression differences in intestine-specific stem cell genes that are either presently unknown, or not present on the array. This latter consideration is the rationale for performing a separate analysis of Musashi-1 and Lgr5. These are two markers that have been most directly associated with stem cells of the intestine. An additional possibility is to consider that we have overlooked alterations in genes that might have occurred at earlier or later time points. The 3rd
postoperative day time point was chosen as we have already demonstrated measurable changes in proliferation by 3 days under normal conditions 12
In addition to expression differences, stem cells have been classically considered to reside in roughly the fourth cell position from the crypt base 16
. Another cell nested between Paneth cells in the crypt termed crypt-based columnar cells (CBC) has more recently been targeted as the crypt stem cell 17,18
. By virtue of relative ease of identification on histologic sections, we were able to determine that there were no differences in either the CBC number or expression of the CBC-associated protein Lgr5 as a consequence of p21waf1/cip1
deficiency or SBR.
Our findings of lack of changes in stem cell populations (as determined by CBC numbers and expression of various stem cell markers) is at variance with the study by Dekaney, et al in which an expansion of the intestinal stem cell population was observed after ileocecal resection in mice 19
. In that report, increased crypt fission and Musashi-1 immunohistochemistry staining was observed after 7 days. It is possible that changes in stem cell numbers and/or expression of various markers might have occurred in our model had we extended our observations to later postoperative time points. Another reason for the disparate findings between this study and ours is that they analyzed the jejunum following a distal ileal and cecal resection while we evaluated the ileum following a proximal jejunal resection. It is therefore possible that intestinal stem cell responses to SBR may be dependent upon the intestinal segment removed. Finally, the expression of Musashi-1 in their study was gauged by immunohistochemistry versus direct Western blotting in the present study. We would argue that the method of explicit enterocyte isolation, protein, quantification, and Western blotting would be more objective and accurate when compared with simple immunohistochemistry.
The involvement of p21waf1/cip1
on stem cells has been mostly studied in bone marrow-derived hematopoietic stem cells. Some investigators have concluded that p21waf1/cip1
is solely a cell cycle inhibitor 20
, while others believe it is involved in proliferation 21,22
, and yet others like ourselves have recognized p21waf1/cip1
to have a dual role in the cell cycle: necessary for proliferation at low levels, but inhibitory at high levels 23,24
In neural stem cells, loss of p21waf1/cip1
leads to long term loss of stem cells, thought to be a consequence of hyper-proliferation and subsequent exhaustion of the stem cell population 25
. Hematopoietic stem cells underwent normal proliferation in p21waf1/cip1
deficient mice under normal conditions, but when perturbed, their stem cell population was depleted resulting in death 26,27
. This protein has also been found to be necessary for maintenance of umbilical cord blood stem cells 28
. This group concluded that p21waf1/cip1
was necessary for the maintenance and self-renewal of the stem cell population and is the rationale for the experiments in this report.
Mantel’s group found p21waf1/cip1
to be necessary for proliferation of the hematopoietic stem cell population 29
. They found absolute numbers of bone marrow progenitors to be significantly decreased in p21waf1/cip1
deficient mice. This same group also described an antiapoptotic role for p21waf1/cip1
in the hematopoietic progenitor cell pathway 30
, further supporting the importance of p21waf1/cip1
in the kinetics of cell turnover.
A dual role for p21waf1/cip1
has been described by several groups. An inverse relationship was found between p21waf1/cip1
levels in megakaryocytes and the cycling state of the cells 31
was necessary for megakaryocyte differentiation, but arrested the cell cycle at high levels 32
. More recently another role for p21waf1/cip1
was suggested in that p21waf1/cip1
can activate retinoblastoma protein (Rb) via dephosphorylation and yet inactivate Rb via degradation, suggesting it can regulate cell cycle progression and/or arrest 33
. Work from our laboratory has revealed p21waf1/cip1
was required for stabilization of the Cyclin D/Cdk 4 complexes and therefore necessary for intestinal cell proliferation 34
Our data would suggest p21waf1/cip1 deficiency does not decrease or deplete the intestinal stem cell population after massive small bowel resection. Additional mechanisms for the requirement of p21waf1/cip1 in the adaptation story must be explored. A more thorough understanding of p21waf1/cip1 role in intestinal proliferation and adaptation will undoubtedly contribute to optimization of future therapies for patients suffering from the short bowel syndrome.