Homeostatic control of the crypt-villus axis by the bacterial enterotoxin receptor guanylyl cyclase C restricts the proliferating compartment in intestine

Abstract

Guanylyl cyclase C (GC-C), the receptor for diarrheagenic enterotoxins and the paracrine ligands guanylin and uroguanylin, regulates intestinal secretion. Beyond volume homeostasis, its importance in modulating cancer cell proliferation and its uniform dysregulation early in colon carcinogenesis, reflecting loss of ligand expression, suggests a role for GC-C in organizing the crypt-villus axis. Here, eliminating GC-C expression in mice increased crypt length along a decreasing rostral-caudal gradient by disrupting component homeostatic processes. Crypt expansion reflected hyperplasia of the proliferating compartment with reciprocal increases in rapidly cycling progenitor cells and reductions in differentiated cells of the secretory lineage, including Paneth and goblet cells, but not enteroendocrine cells. GC-C signaling regulated proliferation by restricting the cell cycle at the G(1)/S transition. Moreover, crypt expansion in GC-C(-/-) mice was associated with adaptive increases in cell migration and apoptosis. Reciprocal alterations in proliferation and differentiation resulting in expansion associated with adaptive responses in migration and apoptosis suggest that GC-C coordinates component processes maintaining homeostasis of the crypt progenitor compartment. In the context of uniform loss of GC-C signaling during tumorigenesis, dysregulation of those homeostatic processes may contribute to mechanisms underlying colon cancer.

Figure 1

GC-C expression in NCM460 cells and GC-C^−/− mice. A: GC-C mRNA is present in normal NCM460 colonocytes and T84 colon carcinoma cells (positive control) as assessed by RT-PCR (1 μg RNA of sample/reaction). B: Dose response of ¹²⁵I-ST binding to NCM460 membranes (cold competitor, 1 μmol/L ST). kDa, ST binding affinity, Bmax, maximum number of ST binding sites. C: Total binding of ¹²⁵I-ST to NCM460 membranes in the presence of the vehicle (CTR, control) or the inactive ST analog TJU 1-103 (TJU, 1 μmol/L), ST (1 μmol/L), or guanylin (1 μmol/L) as cold competitors. In NCM460 cells, ST induces cGMP accumulation in a dose-dependent manner (D), an effect mimicked by guanylin (1 μmol/L) but not the vehicle (CTR, control) or the inactive analog TJU 1-103 (TJU, 1 μmol/L) (E; ST, 1 μmol/L). F: PCR on DNA extracts yielded the expected 195-bp (GC-C^+/+) and 146-bp (GC-C^−/−) amplicons. G: Specific ¹²⁵I-ST binding (cold competitor, 1 μmol/L ST) to intestinal mucosa membranes isolated from GC-C^+/+ (hatched columns) and GC-C^−/− (black columns) mice. H: H&E staining of representative GC-C^+/+ and GC-C^−/− mouse intestinal sections. Du, duodenum; Je/Ile, jejunum and ileum; PC, proximal colon; and DC, distal colon. *P < 0.05, two-tailed t-test. Original magnifications, ×100.

Figure 2

GC-C^−/− mice exhibit expanded proliferating compartments. A: Crypt depth was increased in GC-C^−/− intestinal segments (black columns) compared with their GC-C^+/+ counterparts (hatched columns). B: Ki-67 IHC staining of representative GC-C^+/+ and GC-C^−/− mouse intestinal sections. C: Ki-67⁺ cells per crypt section were increased in GC-C^−/− mice. D: Ki-67 labeling index (Ki-67⁺ cell number/total cell number/crypt section) in different intestinal sections. E: PCNA⁺ cells per crypt section were increased in GC-C^−/− mice. F: PCNA labeling index (PCNA⁺ cell number/total cell number/crypt section) in different intestinal sections. Du, duodenum; Je, jejunum; Ile, ileum; PC, proximal colon; and DC, distal colon. *P < 0.05, **P < 0.01, ***P < 0.0001 by linear mixed effects model. n = 9 mice/genotype. Original magnifications, ×400.

Figure 3

GC-C inhibits proliferation of normal human colonocytes. A: DNA synthesis in human colonic mucosa sheets treated with the vehicle (CTR, control), ST (1 μmol/L), or 8-br-cGMP (1 mmol/L). B: DNA synthesis in NCM460 cells treated with the vehicle (CTR, control), 1 μmol/L of either the inactive ST analog TJU 1-103 (TJU), ST or guanylin, or 8-br-cGMP (1 mmol/L). Experiments with control, ST, TJU 1-103 or guanylin were performed in the presence of 3-isobutyl-1-methylxanthine (1 mmol/L). C: Dose-dependent inhibition of DNA synthesis by 8-br-cGMP in NCM460 cells. Data in A–C reflect ³H-thymidine incorporation into nuclear DNA expressed as the percentage of parallel control incubations exposed to the appropriate vehicle. *P < 0.05, two-tailed t-test.

Figure 4

GC-C^−/− crypt cells exhibit shorter cell cycles. A: Representative BrdU IHC staining of GC-C^+/+ mouse duodenal slides, after cumulative intraperitoneal injections with BrdU. Proliferating cells are progressively recruited in the BrdU-labeled compartment as a function of their cell cycle time. B: BrdU-labeling curves in GC-C^−/− (dashed lines) and GC-C^+/+ (solid lines) mice were generated by calculating the percentage of BrdU-labeled cells per crypt at each time point relative to the average genotype number of PCNA⁺ cells per crypt in the corresponding intestinal segment. C: Cell cycle rates in GC-C^−/− (black columns) and GC-C^+/+ (hatched columns) intestinal segments. D: Representative immunoblots of cell cycle mediators cyclin D1 and phosphorylated Rb (p-Rb) in jejunal mucosa from GC-C^+/+ and GC-C^−/− mice. E: Mean densitometry of cyclin D1 and p-Rb immunoblots, normalized to GAPDH, from jejunal mucosa of five individual GC-C^+/+ or GC-C^−/− mice, respectively. Intestinal segments are labeled as in Figure 2. A–C: *P < 0.05, by linear mixed effects model. E: *P < 0.05, **P < 0.01, two-tailed t-test. Original magnifications, ×400.

Figure 5

GC-C induces a G₁/S transition delay in normal human colonocytes. Growth curves (A, B) and cell doubling times (C, D) of NCM460 cells treated with 1 μmol/L ST (A, C) or 1 mmol/L 8-br-cGMP (B, D). E: Time course of cell cycle distribution by flow cytometry of NCM460 cells treated with PBS (solid lines) or 1 mmol/L 8-br-cGMP (dashed lines). F: Representative immunoblots for cyclin D1 and phosphorylated Rb (p-Rb) in NCM460 cells. G: Mean densitometry of cyclin D1 and p-Rb immunoblots, normalized to GAPDH, from three independent experiments. Con, control (cells treated with PBS); 8br, 8-br-cGMP. *P < 0.05, two-tailed t-test.

Figure 6

Adaptive increases in migration and apoptosis along the GC-C^−/− crypt-villus axis. A: BrdU IHC staining of representative intestinal sections from mice receiving a single BrdU injection to quantify cell migration. B: Quantification of intestinal epithelial cell migration. C and D: Apoptosis was quantified by TUNEL staining (C) after scoring 100 to 800 crypts/intestinal segment in five GC-C^+/+ (hatched columns) and five GC-C^−/− (black columns) mice (D). E: Representative immunoblots of cleaved caspase 3 in jejunal mucosa from GC-C^+/+ and GC-C^−/− mice. F: Mean densitometry of cleaved caspase 3 immunoblots, normalized to GAPDH, from jejunal mucosa of five individual GC-C^+/+ or GC-C^−/− mice. Intestinal segments are labeled as in Figure 2. *P < 0.05; **P < 0.01, two-tailed t-test. Original magnifications: ×200 (A, jejunum); × 400 (A, proximal colon; C).

Figure 7

Lineage-specific cell commitment is altered in GC-C^−/− mice. A–D: Goblet cells were detected by Alcian blue staining (A) and enumerated in 3 to 15 villi per mouse (B). Expression of intestinal trefoil factor (ITF), a goblet cell marker, was detected by immunoblot analysis (C) and quantified by average densitometry, after normalization to GAPDH, in colons from five individual GC-C^+/+ and GC-C^−/− mice, respectively (D). E–G: Paneth cells (E, F) were detected by lysozyme staining and enumerated in ∼10 crypts per mouse (G). H–L: Enteroendocrine cells (H, I, crypts; J, villi) were quantified in ∼100 crypts per mouse (K) or ∼20 villi per mouse (L) by chromogranin A staining. Data in B, D, G, K, and L reflect analysis of jejunal (Je) sections from nine GC-C^+/+ (hatched columns) or nine GC-C^−/− (black columns) mice, respectively. *P < 0.05, two-tailed t-test. Original magnifications: ×200 (A, E, H); ×400 (F, I, J).

Figure 8

The transition from proliferating to differentiated absorptive cells is retained in GC-C^−/− mice. IHC staining of representative small (jejunum, Je) and large (proximal colon, PC) intestinal sections from GC-C^+/+ and GC-C^−/− mice. Mature enterocytes and proliferating crypt cells were visualized in adjacent sections after staining for villin and Ki-67, respectively. Double-headed arrows indicate the cell position of the transition from proliferation to enterocyte differentiation (first villin⁺/Ki-67⁻ cell from the crypt bottom). Original magnifications, ×400.