Contributions made by CDC25 phosphatases to proliferation of intestinal epithelial stem and progenitor cells - PubMed (original) (raw)

Contributions made by CDC25 phosphatases to proliferation of intestinal epithelial stem and progenitor cells

Gwanghee Lee et al. PLoS One. 2011.

Abstract

The CDC25 protein phosphatases drive cell cycle advancement by activating cyclin-dependent protein kinases (CDKs). Humans and mice encode three family members denoted CDC25A, -B and -C and genes encoding these family members can be disrupted individually with minimal phenotypic consequences in adult mice. However, adult mice globally deleted for all three phosphatases die within one week after Cdc25 disruption. A severe loss of absorptive villi due to a failure of crypt epithelial cells to proliferate was observed in the small intestines of these mice. Because the Cdc25s were globally deleted, the small intestinal phenotype and loss of animal viability could not be solely attributed to an intrinsic defect in the inability of small intestinal stem and progenitor cells to divide. Here, we report the consequences of deleting different combinations of Cdc25s specifically in intestinal epithelial cells. The phenotypes arising in these mice were then compared with those arising in mice globally deleted for the Cdc25s and in mice treated with irinotecan, a chemotherapeutic agent commonly used to treat colorectal cancer. We report that the phenotypes arising in mice globally deleted for the Cdc25s are due to the failure of small intestinal stem and progenitor cells to proliferate and that blocking cell division by inhibiting the cell cycle engine (through Cdc25 loss) versus by inducing DNA damage (via irinotecan) provokes a markedly different response of small intestinal epithelial cells. Finally, we demonstrate that CDC25A and CDC25B but not CDC25C compensate for each other to maintain the proliferative capacity of intestinal epithelial stem and progenitor cells.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Targeted disruption of Cdc25B in mice.

(A) Structure of targeting vector and chromosomal organization of Cdc25B locus before and after Cre-mediated excision. The genomic organization of the mouse Cdc25B gene was disrupted by inserting into intron 1 the neomycin phosphotransferase cDNA driven by the phosphoglycerine kinase promoter (pGK-neo) as a selectable marker. Exons are represented by black boxes. The location of Hind III (H), Bam HI (B) and Kpn I (K) site is indicated and loxP sites are represented by yellow triangles. Sizes of upstream (3.3 kb) and downstream (4.5 kb) homologous arms are indicated. Position of probes used for Southern blotting are shown. Red triangles depict the locations of PCR primers used for genotyping. Abbreviations: +, wild type allele; R, recombinant allele; F, floxed allele; WT, wild type. (B–C) Southern blot analysis demonstrating homologous recombination in the Cdc25B locus. ES cell genomic DNA was digested with Hind III (B) and Bam HI (C), and Southern blotting was performed using the 5′ and 3′ probes shown in panel A. The genotype of each ES cell line is indicated. The location of size markers is shown on the left. (D) Southern blot analysis demonstrating Cre-mediated recombination in the Cdc25B locus. ES cell clones containing the recombinant allele were expanded and transiently transfected with a plasmid encoding Cre recombinase. Genomic DNA was digested with Kpn I (K), and Southern blotting was performed using the internal probe shown in panel A. The genotype of each ES cell line is indicated. Location of size markers is shown on left. (E) PCR analysis of mouse tail DNA. Mouse tail DNA was amplified with PCR primers depicted as red triangles in panel A. The wild type (+) allele produced a 383 bp PCR product and floxed allele (F) produced a 433 bp PCR product. The genotype of each mouse is indicated. The location of size markers is shown on the right.

Figure 2

Figure 2. Specific deletion of Cdc25A in intestinal epithelial cells.

(A, B) vR26R;Af/− and v_Af/−_ mice were injected with tamoxifen for 5 consecutive days and then sacrificed 3 days after the final injection. Frozen sections of small intestines were prepared and stained with X-gal to visualize Cre-mediated deletion frequencies. Scale bar: 200 µm. (C) Genomic DNA isolated from the small and large intestines of vAf/− mice 3 days after the final tamoxifen-injection was digested with Bst XI followed by Southern blotting. Deletion frequencies are shown below the each lane. (D) Mice were weighed prior to injection and 3 days after the final tamoxifen injection. Data is presented as mean +/− standard error of the mean (SEM). Asterisks indicate significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. The actual P-values are 0.86 (vil-Cre-ERT2), 0.15 (vAKO), 0.87 (vBKO), 0.00007 (vABKO), 0.11(vACKO) and 0.0004 (vTKO).

Figure 3

Figure 3. Loss of homeostasis in small intestines of _vABKO a_nd vTKO mice.

(A) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Small intestines were isolated and length determinations were made. Small intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. The actual P-values are 0.38 (WT), 0.76 (vil-Cre-ERT2), 0.003 (vAKO), 0.31 (vBKO), 0.002 (vABKO), 0.46 (vACKO) and 0.004 (vTKO). The small intestinal lengths of vAKO, vABKO and vTKO mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.40 (vil-Cre-ERT2), 0.03 (vAKO), 0.10 (vBKO), 0.00006 (vABKO), 0.36 (vACKO) and 0.0006 (vTKO). (B) Duodenums isolated from mice treated as described in A were photographed under a dissection microscope. Scale bar: 0.5 mm. (C) Significant shortening of villi in small intestines of vABKO and vTKO mice. Length of individual villi shown in panel B were measured (30 villi per mouse). Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.00008 (vABKO) and 0.007 (vTKO). Villi lengths of vil-Cre-ERT2, vABKO and vTKO mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.001 (vil-Cre-ERT2), 0.27 (vAKO), 0.45 (vBKO), 0.0005 (vABKO), 0.25 (ACKO) and 0.03 (vTKO).

Figure 4

Figure 4. Tamoxifen injection induced efficient recombination within Cdc25A and Cdc25B loci.

(A) Genomic DNA isolated from the small and large intestines of tamoxifen-treated mice were assessed for Cre-mediated excision by Southern blotting. Deletion frequencies are shown below each lane and were determined by measuring band intensities using a Molecular Dynamics Storm imager. (B) Total RNA isolated from the small intestine (jejunum) of tamoxifen-treated WT and vBf/− mice was reverse-transcribed into cDNA. qRT-PCR was used to determine relative amounts of Cdc25B mRNA. The data is presented as mean +/− SEM. Asterisk (*) indicates significantly different from WT, P = 0.012 by Student's t-test. Note that vBf/− mice are generated by crossing Cdc25B null mice with Cdc25B conditional mice. The PCR primers detect transcript arising from the null allele but not the deleted floxed allele. Thus, a 50% decrease in relative expression indicates complete loss of Cdc25B expression.

Figure 5

Figure 5. Significant shortening of large intestine of vABKO and vTKO mice.

Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. Large intestines were isolated and length determinations were made. Large intestine lengths were normalized to body weights, which were determined prior to the first tamoxifen-injection. Data is presented as mean +/− SEM. Asterisks indicate significantly different after tamoxifen injection as determined by a Student's t-test. P-values are 0.059 (WT), 0.32 (vil-Cre-ERT2), 0.020 (vAKO), 0.27 (vBKO), 0.0009 (vABKO), 0.12 (vACKO) and 0.003 (vTKO). Large intestinal lengths of vABKO, vACKO and vTKO mice were significantly different from WT mice injected with tamoxifen as determined by a Student's t-test. P-values are 0.94 (vil-Cre-ERT2), 0.82 (vAKO), 0.30 (vBKO), 0.00034 (vABKO), 0.041 (vACKO) and 0.0077 (vTKO).

Figure 6

Figure 6. Response of crypt epithelial cells to loss of CDC25s.

(A) Mice were treated with tamoxifen for five consecutive days and sacrificed 3 days after the final injection. Small intestines were isolated and fixed. 5 µm tissue sections were prepared and stained with Hematoxylin and Eosin. Arrow heads indicate Paneth cells. Scale bar: 0.5 mm. (B) Crypts within the proximal portion of the small intestine of tamoxifen treated mice were examined for epithelial cells and Paneth cells. Areas containing Brunner's gland were excluded from analysis. Twenty crypts were counted per mouse and three mice of each genotype were evaluated. Similar patterns were observed in mid and distal portions of the small intestine (data not shown). Data is presented as mean +/− SEM. Asterisk (*) indicates significantly different from WT mice as determined by a Student's t-test (total epithelial cells). *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.41 (vil-Cre-ERT2), 0.21 (vAKO), 0.23 (vBKO), 0.002 (vABKO), 0.15 (ACKO) and 0.0007 (vTKO). vBKO mice have slightly more mature Paneth cells per crypt compared to WT mice as determined by a Student's t-test. P-values are 0.52 (vil-Cre-ERT2), 0.77 (vAKO), 0.044 (vBKO), 0.28 (vABKO), 0.076 (ACKO) and 0.098 (vTKO).

Figure 7

Figure 7. Proliferation and apoptosis in crypts of _Cdc25_-disrupted mice.

(A) Mice were injected with tamoxifen for five consecutive days and then sacrificed 3 days after the final injection. One hour prior to sacrifice, mice were injected with BrdU. Intestines were isolated, and sections were stained with BrdU antibody (brown) and counterstained with hematoxylin (blue). Scale bar: 20 mm. (B) Mice were injected with tamoxifen for five consecutive days and were sacrificed 3 days after last tamoxifen injection. Intestines were isolated and sections were stained for cleaved caspase-3. 3, 3′-diaminobenzidine (DAB, brown) was used as a substrate, and sections were counter-stained with hematoxylin. Arrows indicate cells at the tip of villi, which stain positive for cleaved caspase-3. Scale bar: 0.1 mm. (C–D) Crypts within the proximal portion of the small intestine of tamoxifen treated mice were examined for mitotic cells (presence of mitotic figures) (C) and apoptotic cells (presence of fragmented nuclei) (D). Areas containing Brunner's gland were excluded from analysis. Twenty crypts were counted per mouse and three mice of each genotype were evaluated. Similar patterns were observed in mid and distal portions of the small intestine (data not shown). Data is presented as mean +/− SEM. Asterisks in panel C indicate significantly different from WT mice as determined by a Student's t-test (M-phase cells). *, P<0.05; **, P<0.01; ***, P<0.001. Actual P-values are 0.22 (vil-Cre-ERT2), 0.045 (vAKO), 0.16 (vBKO), 0.00056 (vABKO), 0.17 (ACKO) and 0.00055 (vTKO). Asterisks in panel D indicate significantly different number of apoptotic cells from WT mice as determined by a Student's t-test. P-values are 0.48 (vAKO), 0.33 (vBKO), 0.0011 (vABKO), 0.064 (ACKO) and 0.0010 (vTKO). (E) Mice were injected with tamoxifen as described in A and small intestines were isolated, sectioned and stained for inactive Cdk1 (phosphorylated on Tyr-15, brown) and counterstained with hematoxylin (blue). Arrows indicate epithelial cells positively stained with the phospho-Tyr 15 CDK1 antibody. Scale bar: 20 µm.

Figure 8

Figure 8. Enhanced Wnt-signaling and differentiation of CBC cells in vABKO and vTKO mice.

(A) Tissue sections of small intestines prepared from Vil-Cre-ERT2 (left panel), vABKO (middle panel) and vTKO (right panel) mice were stained with antibodies specific for beta-catenin (green). Nuclei were stained with hematoxylin (blue). Green hatched lines outline nuclei. Scale bar: 20 µm. (B) Quantitation of nuclear beta-catenin staining is shown in A. Data is presented as mean +/− SEM. Asterisks in panel B indicate significantly different from vil-Cre-ERT2 mice as determined by a Student's t-test. (C) Intestinal sections were stained with Periodic Acid Schiff/alcian blue to label Paneth cells and goblet cells from Vil-Cre-ERT2 (left panel), vABKO (middle panel) and vTKO (right panel) mice. Insets are magnifications of boxed regions shown in middle and right panel. Asterisks in left panel indicate CBC cells. Asterisks in middle and right panels indicate immature Paneth cells. Arrows in middle and right panel indicate small granules of immature Paneth cells. Scale bar: 10 µm.

Figure 9

Figure 9. Differentiation of CBC cells into immature Paneth Cells in _Cdc25_-disrupted mice.

Small intestines were dissected and processed for Transmission Electron Microscopy. EM sections of crypts from vil-Cre-ERT2 (A), vABKO (B, C) and vTKO (D, E) mice. Insets in panels B and D are shown at higher magnification in panels C and E, respectively. The red-hatched lines in panel A demark two CBC cells separated by a mature Paneth cell. Blue-hatched lines in panels B-D outline borders of immature Paneth cells arising from premature differentiation of CBC cells. Note that differentiating CBC cells also undergo hypertrophy. Scale bar: 10 µm (A, B, D), 5 µm (C, E).

Figure 10

Figure 10. Response of small intestinal epithelial cells to irinotecan.

(A) Significant decrease in the number of BrdU postive cells in the small intestinal crypts of irinotecan-treated mice. WT mice were injected with PBS (cont) or irinotecan (CPT) for 6 consecutive days and sacrificed on day 7. One hour prior to sacrifice, mice were injected with BrdU. Intestines were isolated and sections were stained with an antibody specific for BrdU. Dotted lines demark crypt margins. Red: BrdU, Blue: nuclei (DAPI). Scale bar: 10 µm. (B, C) Significant loss of crypts in the small intestines of irinotecan-treated mice. Intestines were isolated from irinotecan-treated mice (B) and TKO mice (C), and sections were stained with H & E. Arrows in (B) indicate remaining crypts. Scale bar: 50 µm. (D–F) Irinotecan induces significant apoptosis in the crypts of treated mice at early time points after irinotecan-treatment. (D) Intestinal sections were prepared after two irinotecan administrations and stained with H & E. Arrows depict apoptotic cells. Scale bar: 10 µm. (E) Intestinal sections from WT mice treated with PBS (cont)) and irinotecan (CPT) were stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Green: TUNEL, Blue: nuclei (DAPI). Scale bar: 100 µm. (F) Intestinal sections from WT mice treated with PBS (left) and irinotecan (right) were stained for cleaved caspase-3. DAB (3, 3′-diaminobenzidine) was used as a substrate (brown) and sections were counter-stained with hematoxylin (blue). Scale bar: 50 µm. (G) Differentiation along the enterocyte lineage is not affected in irinotecan-treated mice. Intestines isolated from PBS- or irinotecan-treated WT mice were stained with the enterocyte marker, L-Fabp. Dotted lines demark crypt margins. Green: L-Fabp, Blue: nuclei (DAPI). Scale bar: 10 µm. (H) Loss of goblet cells in irinotecan-treated mice. Intestinal sections were stained with Periodic Acid Schiff/alcian blue to label goblet cells (indicated with arrow). Sections from WT (left), TKO (center) and irinotecan-treated WT mouse (right) are shown. Scale bar: 20 µm. (I) Significant infiltration of neutrophils in crypts of irinotecan-treated mice. The numbers of neutrophils were counted in 10 randomly chosen microscopic fields at 40X magnification in untreated WT, TKO and irinotecan-treated WT mice (CPT). Asterisks indicate significantly different from WT as determined by Student's t-test. WT and TKO: not significant, P = 0.33; WT and CPT: significant, P = 0.0006; TKO and CPT: significant, P = 0.02.

References

    1. Lee MS, Ogg S, Xu M, Parker LL, Donoghue DJ, et al. cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2. Mol Biol Cell. 1992;3:73–84. - PMC - PubMed
    1. Dunphy WG, Kumagai A. The cdc25 protein contains an intrinsic phosphatase activity. Cell. 1991;67:189–196. - PubMed
    1. Gautier J, Solomon MJ, Booher RN, Bazan JF, Kirschner MW. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell. 1991;67:197–211. - PubMed
    1. Sebastian B, Kakizuka A, Hunter T. Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15. Proc Natl Acad Sci USA. 1993;90:3521–3524. - PMC - PubMed
    1. Strausfeld U, Labbe JC, Fesquet D, Cavadore JC, Picard A, et al. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human cdc25 protein. Nature. 1991;351:242–245. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources