The Myb-p300-CREB axis modulates intestine homeostasis, radiosensitivity and tumorigenesis

Abstract

The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300-Myb-CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeostasis.

Figure 1

p300 hypomorphic mutant (plt6/6) mouse intestinal defects phenocopy Myb mutant mice. (a) Like Myb hypomorphs, plt6/6 mutant mice have shorter crypts than WT mice (P=0.03). (b) This reduced crypt length corresponds to reduced proliferation, as determined by PCNA staining at crypts positions from the base (P<0.05–0.01). (c) Expression of ISC gene Myb is significantly reduced in colonic crypts isolated from plt6/6 mutants compared with WT as is Myb target and ISC gene, lgr5, P<0.01. (d) p300 and Myb can be viewed as being part of a larger complex of interacting TFs, including β-catenin, and most particularly, closely related CREB family members (CREB, CREM and ATF1) and more distantly related, ATF2. Linear protein lengths are shown in amino acid (AA) residues, and key interaction and function domains (DNA-binding domain (BD), transactivation domain (T/A), negative regulation domain (NRD) with leucine zipper-like domain (LZ), C=conserved homology domains (Ch), kinase interaction domain (KIX) and key conserved phosphorylation site (P). Means±S.E.M. one-tailed t-test

Figure 2

Complete intestinal epithelial deletion of CREB. creb^fl/fl mice (a and c) and creb^fl/fl x villinCre^ERT2 mice (b and d) were proved chow with Tamoxifen ad libitum for 4 weeks before cull, and preparation of colon and SI sections. These were subjected to IHC with an antibody specific to CREB to show complete loss of antigen in epithelial cells associated with crypts, but not lamina propria (LP), Peyer's patches (PP) or muscle (Mu). (Insert panel) Metaphase (Me) figures within the epithelial compartments were CREB−ve (arrow)

Figure 3

Modestly reduced proliferation in CREB−ve intestinal crypts despite uniform epithelial KO. (a and b) PCNA IHC was performed and determined at each crypt position from the base, showing a modest reduction in proliferation in the transit-amplifying region of the colonic crypt (d and e) and throughout the SI crypt. (e) Overall, the total PCNA staining in the colon was not significantly different (f), whereas a statistically reduced number of PCNA+ve nuclei were found in the SI. (g) Relative levels of mRNA for CREB target gene pcna was found to be statistically reduced following KO. Means±S.E.M., two-tailed t-test. *P=0.01; ***P=0.001

Figure 4

No apparent compensation for CREB loss by family members, CREM and ATF1, including following irradiation. (a) WT and KO sections were subjected to IHC with phospho-CREB antibodies to show that under steady state KO, SI epithelial cell showed no signal. (b)This antibody also detects closely related phospho-CREM and phospho-ATF1. (c and d) When mice were exposed to whole-body irradiation and intestines processed, 5 days later it was apparent that the small but significant difference in proliferation observed in unirradiated mice in the SI was not exacerbated with radiation treatment. (e) It was notable that the extent of phospho-CREB/CREM/ATF1 was more restricted to the base of crypts in WT mice (SI) and that all three CREB members were not present in the KO crypts (colon) with or without radiation, indicating that these were not induced by radiation damage at this time point

Figure 5

In vitro culture of SI organoids reveals a defect in colony-forming ability in KO mice. (a) Organoid cultures were initiated from creb^fl/fl x villinCre^ERT2 mice and these were exposed to 4OHT to show reduced colony-forming efficiency in the KO cultures; Means±S.E.M. two-tailed t-test; **P<0.01. (b) Representative images are shown, (mag= × 10)

Figure 6

Proliferation and phospho-CREB in intestinal adenomas and colon adenocarcinoma are concordant. (a and b) Apc^min/+ mice develop intestinal adenomas, which contain a large fraction of cells that are highly proliferative as shown by PCNA staining. (c and d) The pattern of PCNA staining mirrors that for phosphor-CREB. (e and f) MC38 colon adenocarcinoma cells form large tumors in syngeneic mice (C57Bl/6), which show high levels of PCNA+ve and pCREB+ve nuclei (green arrows indicate a focus to align nuclei in each micrograph)

Figure 7

Messenger RNA expression changes in selected genes identified by RNAseq studies validated by qRT-PCR. (a) Significant changes in mRNA levels for phosphatases, Pmm1b, pTen, multidrug resistance gene Abcc/Mrp2 and tyrosine kinase gene, Yes1. Four genes that encode proteins engaged in chromosome cohesion, Stag1, Stag 2, Smc2 and Smc6, are similarly lower in the crebKO small intestine crypts. Cartoons depicting the structural roles and general theme of interactions of the cohesion molecules are shown (b). Models of the structural convergence of several putative CREB target gene products, Stag1, Stag 2, Smc2 and Smc6, all of which are involved in cohesion functions, are shown

Figure 8

Multidrug transporter MRP2 is underexpressed in creb KO intestine. (a) Small intestine, and (b) colon sections were stained with αMRP2 antibodies (yellow arrow) to reveal reduced expression in creb^fl/fl/villinCre^ERT2crypts compared with WT, following 4-weeks exposure to Tamoxifen. (c and d) Organoid cultures were established in the presence of 4OHT for 3 days, and subsequently subjected to treatment with Oxaliplatin after cultures were allowed to proceed for another 7 days. These results show that as expected, the growth (MTT) and organoid-forming ability was impeded by crebKO alone, but these measures were significantly reduced compared with WT in the presence of Oxaliplatin. Means±S.E.M., two-tailed t-test. *P=0.01; ***P=0.001