Gastroenterology
Volume 136, Issue 3 , Pages 763-766, March 2009

Bak to Basics of Colonocyte Renewal

  • Nathan J. Susnow

      Affiliations

    • Gastroenterology/Hepatology Division, Department of Medicine, University of Washington, Seattle, Washington
    • Corresponding Author InformationReprint requests Address requests for reprints to: Nathan J. Susnow, MD, Gastroenterology/Hepatology Division, Department of Medicine, University of Washington, Box 356424, Suite AA103, 1959 NE Pacific Street, Seattle, Washington 98195-6424. fax: (206) 764-2147
    • ,
  • David M. Hockenbery

      Affiliations

    • Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington

published online 23 January 2009.

Article Outline

 

See “Suppression of apoptosis, crypt hyperplasia and altered differentiation in the colonic epithelia of bak-null mice,” by Duckworth CA, Pritchard DM, on page 943.

Apoptosis is a common mode of cell death utilized for removal of excess cells during development, homeostatic turnover of cells in adult organisms, and unscheduled death of cells owing to extrinsic factors. The decision of a cell to undergo apoptosis is a complex process that involves cell-intrinsic proteins, some specialized for apoptotic pathways, and other components including entire organelles and lipid membranes. Although apoptosis may function to eliminate irreversibly damaged cells, its principal in vivo role seems to be the control of tissue cellularity, perhaps to match the availability of nutrients, growth factors, or substratum space.

Normal control over tissue cellularity must be inhibited in cells undergoing malignant transformation. Oncogenes that promote cell proliferation or growth often trigger apoptosis, unless the cell death machinery is disabled. A critical set of components in this machinery is the Bcl-2 family of proteins. In general terms, the balance between anti-apoptotic and pro-apoptotic Bcl-2 family members determines whether cells survive under a variety of stressful conditions.

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Cell kinetics in the colon 

The normal colon mucosa consists of a luminal surface of columnar epithelial cells arranged as millions of closely spaced crypts interspersed with table regions. Colonic crypt architecture is established by birth, with expansion of crypt number by crypt fission until maturity.1 Individual cell loss from crypts increases during development to a turnover time for mature crypts in steady state estimated at 2 to 3 days. Colonocytes undergo transitions from proliferating, pluripotent precursors to noncycling, terminally differentiated cells during their migration out of crypts. Cells are shed at the epithelial surface, accompanied by biochemical and, less reliably, morphologic signs of apoptosis. A small number of cells within the crypt are destroyed by apoptosis. The only cells with long-term residence in the colon crypt are a small cohort of stem cells located at the crypt base, estimated at 5 to 20 per crypt.2, 3

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In GASTROENTEROLOGY this month 

Expression patterns of Bcl-2–related genes in colon epithelium may provide clues as to how apoptosis of colonocytes is regulated.4 Bcl-2 is expressed in a few cells at the crypt base, whereas Bcl-xL is expressed in the upper crypt and surface epithelium.5, 6 Spontaneous apoptosis and apoptosis induced by 5-fluorouracil (5-FU) and γ-irradiation occurs at the base of colon crypts and is increased in Bcl-2−/− mice, at least at early time points, with a tendency toward reduced clonogenic crypt survival.4, 7, 8 Bcl-xL–null mice have an embryonic lethal phenotype, and a tissue-specific deletion would be required to test its role in colonic epithelium. Bcl-w−/− mice have increased spontaneous and induced apoptosis in a similar location as Bcl-2−/− mice.9 It is probable that Bcl-w expression is highest in the basal crypt, although the pattern of expression pattern in tissue sections has not been reported.

Bax is expressed at the luminal surface10 and x-irradiation up-regulates Bax in mouse colon.11 A study of Bax-null mice found only marginally significant reductions in 5-fluorouracil– and γ-irradiation–induced apoptosis using a modified median test.7 In summary, these results indicate that Bcl-2 and, to a lesser extent, Bcl-w, help to maintain survival of colon stem cells and/or proliferating early transit cells (Figure 1). Conspicuously missing, however, is a pro-apoptotic effector for crypt death, and any clues to the regulation of colonocyte turnover at the surface epithelium.

  • View full-size image.
  • Figure 1. 

    Distribution of primary regulators of apoptosis within colon crypts. As cells migrate outward from the crypt base, Bak expression is maximal at the crypt table and lower crypt, while Bcl-2 is confined to basal cell positions.

In this issue of Gastroenterology, Duckworth et al analyze intestinal morphology and epithelial cell kinetics in mice deficient in the pro-apoptotic Bak gene.12 Bak expression in the colon is more extensive than Bax, with little expression in mid-crypt, but increasing gradients of expression toward the surface epithelium and crypt base.13 Analyzing Bak−/− mice (previously reported as normal14), Duckworth et al demonstrate a remarkable increase in colonic crypt height, with a 50% increase in cell number per crypt compared with normal littermates. Several possible changes in colonocyte kinetics could account for this finding: reductions in shedding/apoptosis at the crypt table, increased production of committed daughter cells by crypt stem cells, or proliferation in the transit amplifying compartment. The data presented here are supportive of multiple mechanisms.

Apoptotic cells at the colon surface are significantly less evident in Bak-deficient mice compared with wild-type mice, suggesting that terminally differentiated Bak−/− colonocytes hang around longer. Cell proliferation indices in the lower half of the crypt are also increased in Bak-null mice, most notably as a shift toward higher cell positions from the base.

Do these results indicate that Bak has both anti-proliferative and pro-apoptotic functions? Studies in mouse embryonic fibroblasts suggest that Bax and Bak oppose cell cycle arrest in G0 by down-regulating p27, a cyclin-dependent kinase inhibitor, opposite from the reduced p27 levels demonstrated in crypt lysates of Bak-null mice.15 Cell-type–specific effects could be operating, as antisense-mediated suppression of Bak expression in intestinal IEC-18IEC-8 cells is associated with transformation with decreases in cell doubling time.16

Alternatively, a small number of apoptotic cells seen in normal healthy colon crypts are scattered in the proliferative zone. Fortuitous rescue of an early transit amplifying colonocyte (and its progeny) could give rise to 128 to 256 additional cycling cells, although Duckworth et al noted no differences in crypt apoptosis. Finally, as the positional shift in cycling cells is of similar magnitude to the increase in cell number per hemicrypt (∼10) in Bak−/− mice, local mesenchymal signals supporting colonocyte proliferation may have expanded along the crypt axis. More detailed studies of cell kinetics, including migration rate, using radiation crypt survival assays, and estimation of stem cell number using newer markers of stem cell identity (eg, Lgr5, Musashi-1, PTEN), will be necessary to tease apart the cellular basis of the expanded proliferation zone.

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Bak as tumor suppressor 

Genotoxic carcinogens induce apoptosis as an early event, although the correlation between levels of DNA damage and apoptosis across tissues is fairly weak. Rather, apoptosis functions as a tumor-suppressive mechanism by eliminating cells that otherwise would accumulate fixed mutations and other genetic lesions.17 Duckworth et al demonstrate that Bak−/− mice are resistant to azoxymethane-induced apoptosis. As predicted, these mice develop more aberrant crypt foci than wild-type mice soon after 5-fluorouracil treatment, consistent with a direct role for apoptosis in preventing tumor initiation. The expanded stem/transit amplifying cell zone in Bak−/− may also be a contributing factor. Further analysis of dysplastic foci and tumorigenesis is necessary to confirm that Bak functions as a tumor suppressor. It would also be interesting to examine the effect of Bak deletion in ApcMin mice.

Krajewska et al found reduced BAK expression in 90% of colorectal adenocarcinomas and 92% of adenomatous polyps,18 although the prognostic significance of BAK expression is not well established.19 In contrast with BAX mutations frequently identified as early events in microsatellite-unstable colon cancers, BAK missense mutations are rare, late events in gastrointestinal cancers.20 In mouse tumor models, germline loss of Bax has been shown to enhance tumorigenesis, although Bax expression stimulates tumorigenesis in p53-deficient T-cell lymphomas and seems to promote Myc-induced mammary tumors.21, 22 The recent demonstration that the sequence of acquired mutations affects tumor phenotype and prognosis is instructive,23 and suggests that the ability to recapitulate discrete steps in human cancer progression may be worth the extra investment in generating these complex mouse models.

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Bak as lineage determinant 

A curious result reported by Duckworth et al is the shift in secretory cell phenotype toward goblet cells and away from enteroendocrine cells in Bak−/− crypts. There is little evidence to suggest that intrinsic differences in cell turnover or migration exist among differentiated cell lineages in the large intestine (as opposed to Paneth cells in the small intestine).24 In the absence of other explanations, the possibility that Bak has direct effects on differentiation as another cell fate decision must be entertained. There are precedents for Bcl-2 proteins affecting cell lineage choice. For example, survival of a pluripotent hematopoietic cell line in growth factor-depleted media is accompanied by monocytic differentiation if Bcl-2 is expressed, or erythrocytic differentiation for Bcl-xL.25

As discussed, small intestinal morphology and appearance of spontaneous apoptosis are normal in both Bax and Bak knockout mice. It would be surprising if enterocyte turnover is normal in Bax−/−/Bak−/− double knockout mice, because these genes act redundantly in many in vitro and in vivo examinations of apoptosis26 and current models propose that Bak and/or Bax are required for mitochondrial pathways of apoptosis. Intestinal abnormalities are not reported in double knockout mice,14 although a detailed analysis may still uncover changes in enterocyte kinetics despite normal morphologies. A third multidomain pro-apoptotic family member, Bok, is expressed in intestinal crypts, and may provide an additional level of redundancy.6 Finally, death receptor signaling may trigger apoptosis independently of Bak/Bax-regulated mitochondrial pathways in certain cell types (type I cells). Kopitz et al have reported that expression of acyl-CoA synthetase 5 (ACSL5) sensitizes intestinal epithelial cells to the death receptor ligand TRAIL by increasing cell surface expression of its receptor and down-regulating FLIP, an inhibitor of death receptor signaling.27 Splicing of ACSL5 is regulated along the crypt–villus axis, favoring expression of the full-length ACSL5 isoform at the villus tip.

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Summary 

Duckworth et al seem to have found the missing pro-apoptotic link controlling apoptosis in the colon. The phenotypic changes in the large intestine, involving spontaneous and induced-apoptosis, colonocyte proliferation, and aberrant crypt formation, are more pronounced than in previous studies of apoptotic dysregulation in the intestine, and elevate Bak to a principal role in epithelial homeostasis. The observations of non-apoptotic functions for Bcl-2–related proteins, largely derived from in vitro studies, may explain the pleiomorphic effects of deleting Bak on colonic epithelium. If so, this seems to be the first in vivo demonstration of an expanded role for a Bcl-2 family member in tissue homeostasis, perhaps yielding new clues into the parallel integration of cues for cell division, differentiation, and apoptosis.

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References 

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 Conflicts of interest The authors disclose no conflicts.

PII: S0016-5085(09)00051-1

doi:10.1053/j.gastro.2009.01.021

Refers to article:

  • Editorial Accompanies this Article Suppression of Apoptosis, Crypt Hyperplasia, and Altered Differentiation in the Colonic Epithelia of Bak-Null Mice , 21 November 2008

    Carrie A. Duckworth, D. Mark Pritchard
    Gastroenterology March 2009 (Vol. 136, Issue 3, Pages 943-952)

Gastroenterology
Volume 136, Issue 3 , Pages 763-766, March 2009

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