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, 27 (42), 5599-611

Activation of NF-kappaB Is Required for Mediating Proliferative and Antiapoptotic Effects of Progastrin on Proximal Colonic Crypts of Mice, in Vivo

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Activation of NF-kappaB Is Required for Mediating Proliferative and Antiapoptotic Effects of Progastrin on Proximal Colonic Crypts of Mice, in Vivo

S Umar et al. Oncogene.

Abstract

Mice overexpressing progastrin (PG) in intestinal mucosa (fatty acid-binding protein (Fabp)-PG mice) are at an increased risk of proximal colon carcinogenesis in response to azoxymethane. Here, we report a significant increase in the length of proximal colonic crypts in Fabp-PG mice, associated with potent antiapoptotic effects of PG, which likely contributed to the previously reported increase in colon carcinogenesis in Fabp-PG mice. Phosphorylation of kinase of IkappaBalpha (IKKalpha/beta), inhibitor of kappaB (IkappaB)alpha and p65NF-kappaB was significantly elevated in proximal colonic crypts of Fabp-PG versus wild-type mice, which was associated with degradation of IkappaBalpha and nuclear translocation/activation of p65. Surprisingly, distal colonic crypt cells were not as responsive to elevated levels of PG in Fabp-PG mice. Annexin II, recently described as a high-affinity receptor for PG, strongly co-localized with PG intracellularly and on basolateral membranes of proximal crypt cells, providing evidence that annexin-II binds PG in situ in colonic crypt cells. Proliferative and antiapoptotic effects of PG on proximal crypts of Fabp-PG mice were attenuated to wild-type levels, on treatment with NEMO peptide (an inhibitor of nuclear factor-kappaB (NF-kappaB) activation), demonstrating for the first time a critical role of NF-kappaB in mediating hyperproliferative affects of PG on colonic crypts of Fabp-PG mice, in vivo. Thus, downregulation of NF-kappaB may significantly reduce the increased risk of colon carcinogenesis in response to PG.

Figures

Figure 1
Figure 1
A. H&E stain of formalin-fixed cryosections (5μm) from distal (D) and proximal (P) colons of WT and Fabp-PG mice (magnification, ×4). (n = 10 mice/group; scale bar = 150 μm). B. Representative images of the intact distal and proximal colonic crypts from WT and Fabp-PG mice (n = 10 mice/group; scale bar = 50μm). C. The length of intact colonic crypts isolated from both WT and Fabp-PG mice were measured using Metamorph image analysis software as described in Methods and the results from 150 crypts/group of mice are shown in C. * = P <0.05 versus WT as measured by students' t-test.
Figure 2
Figure 2
A-B. Proliferative index. Representative sections from Fabp-PG versus WT, stained with proliferating cell nuclear antigen antibody (A). PI was calculated based on number of positive cells along the longitudinal crypt axis (B). Data in B were derived from 55-102 crypts/5-10 mice/group. C-F. Apoptotic Index. Representative H&E stained sections from proximal colons of AOM-treated WT and Fabp-PG mice are shown in C. Arrowheads identify apoptotic bodies, and data from 76-224 crypts/group of mice (5-10 mice/group) are shown in D as mean ± SEM. TUNEL staining of representative longitudinal sections of colonic crypts from proximal colon is shown in E; arrows point to positively stained (apoptotic) cells and data from 48-98 crypts/group of mice is presented in F as mean ± SEM. G-H. IHC for activated caspase 3. Representative longitudinal sections of proximal colons, immunostained for activated caspase 3, are shown in G at 40× and 200 × magnifications. Arrows point to positively stained cells. Number of apoptotic cells/75 fields of view from proximal colons of 5 mice in a group are shown as mean ± SEM in H. * = P<0.05 versus WT values in each case.
Figure 3
Figure 3. Immunolocalization of PG and ANX-II in distal and proximal colons
Frozen sections prepared from distal and proximal colons were stained with anti-PG-Abs and ANX-II-antibodies, which were detected by fluorescent microscopy using second antibodies tagged with either Texas Red (against PG-Abs) or FITC-green (against ANX-II-Abs). Representative sections from distal (D) and proximal (P) colons of WT mice and Fabp-PG (Tg) mice are shown (n = 3; 200×) ANX-II staining was localized predominantly in the apical-baso-lateral membranes of distal and proximal colons with minimal cytoplasmic staining in sections from WT mice. No staining for PG was detected in sections from WT mice. In the case of Fabp-PG (Tg) mice, in sections from distal colons, intense baso-lateral staining for ANX-II, which extended throughout the longitudinal crypts axis with minimal cytoplasmic staining was observed. However, crypts from proximal colons of Tg mice exhibited dramatic increase in cytoplasmic staining for ANX-II with a gradient of increasing crypt base: surface immunoreactivity, along with perinuclear and nuclear staining. Equivalent levels of hgastrin gene were expressed in distal and proximal colons of Tg mice (as measured by quantitative Real Time PCR). However, relatively low levels of PG were retained in distal colonic crypts of Tg mice, while significant levels were retained within the proximal colonic crypts of Tg mice. Merged images of PG and ANX-II staining, demonstrated co-localization of PG and ANX-II only at the baso-lateral and apical membranes in the distal colonic crypts of Tg mice, with no co-localization intracellularly, which essentially resembled the pattern of ANX-II staining observed in the distal colons of Tg mice. In few mice, higher levels of PG were retained in the distal colonic crypts (representative data are presented in Supplementary Fig 1A). In spite of higher retention of PG in distal crypts of 10-20% of PG mice, PG, bound to ANX-II, remained co-localized at the apical and lateral membranes of the distal crypts (Supplementary Fig 1A). The merged images of proximal colonic crypts from Tg mice, on the other hand, demonstrated marked co-localization of PG and ANX-II staining at both the apical/lateral poles and within the cytoplasm and even nucleus (Fig 3). Arrows = co-localization of ANX-II and PG at plasma membranes; arrowheads = co-localization of ANX-II and PG intracellularly.
Figure 4
Figure 4. Phosphorylation of IKKα/β and IκBα in colons of Fabp-PG and WT mice
A. IKKα/β. Western blots of cellular extracts from isolated crypts were densitometrically analyzed, and ratio of phospho IKKα/β (pIKKα/β) to total IKK for WT samples were arbitrarily assigned a 100% value. % change in ratios for Fabp-PG vs. WT samples are shown as bar graphs. Data in each bar graph represents Mean ± SEM of 3 blots from 3 mice. * = P<0.05 versus WT values. B. IκBα. Representative Western Blots for pIκBα and total IκBα in cellular extracts from proximal and distal colons of Fabp-PG and WT mice are shown; corresponding β-actin in the samples are presented as loading controls. +C = Hela cell extract used as positive control. In C, ratio of % changes in pIκBα versus total IκBα is shown as Mean ± SEM of 3 blots from 3 mice. D. IHC of Ser32/36-pIκBα revealed significant accumulation in proximal (P) but not distal (D) colons of Fabp-PG mice (lower panel) while the differences were less dramatic in proximal versus distal colonic crypts of WT mice (upper panel). (n = 3; bar = 50μm). Arrow heads = staining of p-IκBα in cytosol; Arrows = staining of phosphorylated IκBα in nuclei.
Figure 5
Figure 5. Phosphorylation and DNA binding of activated NF-κB p65
Representative Western Blots of cellular (A) and nuclear (B) extracts for p-p65276, p-p65536 and total p65 are presented from distal and proximal crypts of wild type (WT) and Fabp-PG (Tg) mice. C. DNA binding assay. Relative levels of activated p65NF-κB in nuclear extracts of proximal and distal colons of WT and Tg mice, measured in a DNA binding assay using the TransAm™ p65 NF-κB Chemi Transcription Factor Assay Kit from Active Motif, are shown. Each bar graph represents mean ± SEM of three measurements from three mice. *=p<0.05 vs all other values. Di. IHC of frozen sections with p-p65276 antibody. Cytoplasmic and nuclear staining in proximal (P) and distal (D) colons of Fabp-PG mice (lower panel) and in distal and proximal colons from WT mice (upper panel) are shown. (n = 3; bar = 50μm). Arrowheads = phosphorylated p65-Ser276 in the nuclei of colonic crypts. Arrows= p-p65276 in cytosol of crypts. Dii. Bar graph representing % cells/crypt positive for nuclear staining for p-p65276. The data demonstrate several fold increase in % cells positive for phosphorylated p65 in nuclei of proximal colons of Tg mice compared to that in distal colons (n = 5).
Figure 6
Figure 6. Effect of NEMO peptide on the levels of phosphorylated ERKs, IKKα/β, IκBα (A-C), p65NFκB (Di-iii), proliferation (Ei-ii) and apoptosis (F) in colonic crypts
Mice were divided into two groups and injected, once a day for four days with either NEMO or control peptide (as described in Methods). Two hours after last injection, colonic crypts were isolated and fractionated into cellular and nuclear extracts. Data obtained from proximal colonic crypts of Fabp-PG mice are presented. A-C: Representative Western blots from NEMO versus control peptide treated samples are shown from one of four mice/group. The data in the Western blots of the cellular extracts were densitometrically analyzed from all mouse samples, and the ratios of: phospho ERK1/2 (pp44/42; A), phospho IKKα/β (p-IKKα/β); B) and phospho IκBα (p-IκBα); C) to β-actin, were arbitrarily assigned a 100% value. % change in ratios for NEMO peptide versus control peptide values are shown as bar graphs. Data in each bar graph represents Mean ± SEM of 4 blots from 4 mice. Di. DNA binding assay. NEMO peptide significantly inhibited NFκB activation, measured in a DNA binding assay with nuclear extracts from proximal, but not distal, colonic crypts of Fabp-PG mice, compared to levels measured in control peptide samples (upper panel, Di); % change in the relative levels of activated NFκB in control peptide vs NEMO peptide treated samples are shown in lower panel of Di. Each bar represents mean ± SEM values from 3 measurements from 3 separate mice. *=p<0.05 vs corresponding control values. Dii. Phosphorylation status of p65. Representative Western blots of nuclear extracts were densitometrically analyzed, and the ratio of p-p65276 to LaminB, was arbitrarily assigned a 100% value. % change in ratios for NEMO peptide versus control peptide treated values are shown as bar graphs. For data in A-D, *=P<0.05 versus control peptide values. Diii. IHC of frozen sections with anti- p-p65276 antibody. Cytoplasmic and nuclear staining (arrows) in proximal colons of Fabp-PG mice, receiving either control peptide (left panel) or treated with NEMO (right panel), are shown (n = 3; bar = 50μm). Note significant reduction in staining for p-p65276 in sections prepared from proximal colon of NEMO-treated animals. E. Effect of NEMO peptide on PI of colonic crypts. Ei. Representative sections from Fabp-PG mice treated with either control peptide (left panel) or NEMO (right panel) were stained with BrdU antibody. Similar to Diii, NEMO peptide significantly reduced BrdU incorporation into S-phase cells, to basal levels. Arrows point to BrdU staining of nuclei. Eii. PI was calculated from 40 crypts/group of 4 mice each, based on number of positive cells along the longitudinal crypt axis. PI decreased significantly in NEMO-treated colonic crypts. Each bar = mean ± SEM of 40 crypts. *=p<0.05 vs control values F. Effect of NEMO peptide on Caspase 3 activation. Representative Western blot of cellular extracts from one of four mice/group is presented. Treatment of Fabp-PG mice with NEMO peptide resulted in a significant increase in the relative levels of activated (A) caspase 3, with a concomitant decrease in relative levels of pro-caspase 3, in proximal colonic crypts when compared to that in mice treated with the control peptide. β-actin was used as loading control. The relative levels of phosphorylated kinases, PI and activated caspase 3 in distal crypts of Fabp-PG mice was not significantly altered in NEMO versus control treated mice (data not shown), since these levels were not changed significantly in response to PG as presented in Figs 1, 2, 4, and 5. Similarly, the relative levels of phosphorylation of the indicated kinases, PI and activated caspase 3 were not altered significantly in NEMO versus control peptide treated WT mice (data not shown), suggesting that IKKα/β/NFκB pathway does not play a significant role in the basal growth of colonic crypts in the WT mice as suggested by data presented in Figs 1, 2, 4, and 5.

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