Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Feb;140(2):583-595.e4.
doi: 10.1053/j.gastro.2010.08.054. Epub 2010 Sep 6.

Annexin A2 mediates up-regulation of NF-κB, β-catenin, and stem cell in response to progastrin in mice and HEK-293 cells

Shubhashish Sarkar  1 , Rafal Swiercz, Carla Kantara, Katherine A Hajjar, Pomila Singh
Affiliations

Annexin A2 mediates up-regulation of NF-κB, β-catenin, and stem cell in response to progastrin in mice and HEK-293 cells

Shubhashish Sarkar et al. Gastroenterology. 2011 Feb.

Abstract

Background & aims: Prograstrin induces proliferation in colon crypts by activating p65nuclear factor-κB (NF-κB) (p65) and β-catenin. We investigated whether Annexin A2 (AnxA2), a progastrin receptor, activates NF-κB and β-catenin in vivo.

Methods: ANXA2-null (ANXA2(-/-)) and wild-type (ANXA2(+/+)) mice were studied, along with clones of progastrin-responsive HEK-293 cells that stably expressed full-length progastrin (HEK-mGAS) or an empty vector (HEK-C). Small interfering RNA was used to down-regulate AnxA2, p65NF-κB, and β-catenin in cells.

Results: Proliferation and activation of p65 and β-catenin increased significantly in HEK-mGAS compared with HEK-C clones. HEK-mGAS cells had a 2- to 4-fold increase in relative levels of c-Myc, cyclooxygenase (COX)-2, CyclinD1, double cortin CAM kinase-like 1 (DCAMKL+1), and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and β-catenin, as well as levels of DCAMKL+1. Surprisingly, down-regulation of β-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of β-catenin in HEK-mGAS clones. Loss of either p65 or β-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of progastrin. Lengths of colon crypts and levels of p65, β-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2(+/+) mice compared with either ANXA2(-/-) mice given progastrin or ANXA2(+/+) and ANXA2(-/-) mice given saline.

Conclusions: AnxA2 expression is required for the biologic effects of progastrin in vivo and in vitro and mediates the stimulatory effect of progastrin on p65NF-κ, β-catenin, and the putative stem cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and cocarcinogenic effects of progastrin.

PubMed Disclaimer

Conflict of interest statement

DISCLOSURE: None of the authors have any conflict of interest with any entity related to the data presented in here.

Figures

Fig1
Fig1
A–C – Proliferative potential of HEK-mGAS and HEK-C cells. (A) Expression of full length 9-kDa progastrin peptide by HEK-mGAS clones. (B) Growth response of an equal number of HEK-C and HEK-mGAS cells to increasing concentrations of FCS, measured in an MTT assay. Absorbance values are plotted against FCS concentrations. Each value represents data from 6 separate wells/experiment, from a representative of 2 similar experiments. (C) Relative levels of PCNA in the cellular lysates of sub-confluent clones, growing in 5% FCS, measured by immunoblot analysis. A representative blot from a total of 6 blots from 3 experiments is presented. Basal intensity of bands, determined densitometrically, is plotted as percent change in the ratio of PCNA: β-actin (ratio for HEK-C cells was arbitrarily designated 100%); data from 6 blots are presented as mean±SEM, in bar graphs. *=p<0.05 versus HEK-C values. Fig1D. Co-localization and internalization of progastrin/AnxA2 complexes, in response to exogenous progastrin (PG). wtHEK-293 cells on glass cover slips were incubated with progastrin for indicated time periods and processed for detection of progastrin/AnxA2 by confocal microscopy. Red-fluorescence=progastrin-staining, green fluorescence=AnxA2-staining. Co-localization of progastrin/AnxA2 appears bright yellow in merged images at 60x magnification. Insets presented for merged images are computer enhanced. Co-localized progastrin/AnxA2 on membranes (short arrows) and intracellularly (long arrows) are shown.
Fig2
Fig2
A, B. Activation of p65NF-κB/β-catenin in response to autocrine-progastrin in HEK-mGAS clones. Relative levels of pp65Ser276/COX-2 (A), and β-catenin/c-Myc/CyclinD1/β-actin (B) in cellular lysates of HEK-mGAS versus HEK-C cells, measured by immunoblot analysis, are presented. In A, B, data from a representative blot of 4 blots/2 experiments are presented. In A, B, percent change in ratio of the indicated proteins to β-actin, are presented as mean±SEM of data from 4 blots, as described in Fig1 legend. In each case *=p<0.05 versus HEK-C values. Fig2C. Down-regulation of AnxA2 in HEK-mGAS cells results in loss of relative-levels of pp65NF-κB and β-catenin. HEK-C/HEK-mGAS clones were treated with either control-siRNA or AnxA2-siRNA, and cellular-lysates processed for immunoblot analysis. β-actin was analyzed as an internal control. Representative blots from a total of 4 blots/2 experiments are presented in the left panel. In the right panel, data from 4 blots are presented as percent change in the ratio of indicated proteins: β-actin as mean±SEM. *=p<0.05 versus HEK-C values. Figs2D–E. Down-regulation of AnxA2 in HEK-mGAS cells results in loss of activation of NF-κB and β-catenin. D. HEK-C/HEK-mGAS cells, were transfected with either control- or AnxA2-specific-siRNA for 48h, followed by transfection with either FOPFlash or TOPFlash plasmids. 24h after transfection, promoter-activity was measured in terms of luciferase-units, and data from 6 separate samples/2 experiments are presented. E. Cells were transfected with either control- or AnxA2-specific-siRNA for 48h, followed by preparation of nuclear-extracts, and processed for measuring binding of activated-NF-κB in a DNA-binding-assay; data from 6 separate samples/2 experiments are presented as mean±SEM.
Fig3
Fig3
A–C. Autocrine progastrin up-regulates DCAMKL+1/CD44 in HEK-mGAS versus HEK-C cells in an AnxA2-dependent manner. A. HEK-C/HEK-mGAS cells growing on cover-slips were stained for the indicated stem cell marker, as described in legend of Fig1D. 3B–C. HEK-C/HEK-mGAS cells were transfected with either control or specific AnxA2-siRNA, followed by preparation of cellular-extracts after 48h. Lysates were processed for immunoblot analysis, and data from a representative blot from a total of 4 blots/2 experiments are shown in B. Data from 4 blots are presented as percent change in the ratio of indicated proteins: β-actin as mean±SEM in C. *=p<0.05 versus HEK-C values. Figs3D, E. Effect of down-regulating β-catenin/p65NF-κB on growth of HEK-C/HEK-mGAS cells. HEK-C/HEK-mGAS cells in culture were transfected with either control or β-catenin/p65 specific-siRNA. After 72h, cells were either processed for immunoblot analysis (FigD) or counted (E). Cell-numbers in 6 separate dishes/experiment were measured and presented as mean±SEM in Cii. Data presented are representative of 3 similar experiments. *=p<0.05 versus corresponding HEK-C values; †=p<0.05 versus corresponding control-siRNA values.
Fig4
Fig4
A, B. Down-regulation of p65NF-κB attenuates β-catenin activation in HEK-mGAS cells. Cells were transfected with either control-siRNA or p65NF-κB-specific-siRNA, and processed for immunoblot analysis. Representative blots from a total of 4 blots/2 experiments, are presented in A. β-actin and histone levels were used as internal-controls for cellular/nuclear lysates, respectively. Ratio of indicated proteins to either β-actin (total-p65/p65276/total-β-catenin/COX-2) or histone (nuclear-β-catenin) are presented as mean±SEM from all 4 blots in B, left panel: control-siRNA and Right panel: p65-specific-siRNA. *=p<0.05 versus corresponding HEK-C values. Fig4B. Activation of p65NF-κB is independent of β-catenin activation in HEK-mGAS cells. Cells were transfected with either control-siRNA or β-catenin-specific-siRNA and processed for immunoblot analysis of cellular-lysates. β-actin levels were measured as an internal-control. Representative blots of a total of 4 blots/2 experiments are presented in C. Ratio of indicated proteins: β-actin, determined from densitometric-analysis of immunoblot bands, is presented as mean±SEM of data from all 4 blots in D (as described above for B). *=p<0.05 versus HEK-C values. β-cat = β-catenin; CycDi = Cyclin D1.
Fig5
Fig5. ANXA2 expression is required for the growth/signaling effects of progastrin in vivo
A. Representative immunoblot data demonstrating absence of AnxA2 expression in colonic crypts of ANXA2− /− mice. B. Representative tissue sections from mid-colons of ANXA2+/+/ ANXA2− /− mice, treated with either saline (onM) or 10nM-rhPG. Dashed lines represent average length of colonic crypts in the indicated mice. C. To obtain accurate measurements of colonic-crypt lengths, colons were processed for preparation of isolated colonic-crypts (Supplementary Fig4), and lengths measured as previously described (12,13). Each bargraph=mean±SEM of 30-50 isolated-crypt lengths from 3–5 mice. *=p<0.05 versus control (saline-treated) mice:†=p<0.05 versus respective ANXA2+/+ levels. D. Isolated colonic crypts from mid-colons of the indicated genotypes were processed for immunoblot analysis. Representative blots of 6–8 blots from 3–4 mice are shown in left panel. Percent change in the ratio of p65Ser276:total p65 and β-catenin: β-actin, is shown in right panel; each group represents mean±SEM of data from 3–5 separate mice. *=p<0.05 versus corresponding-control (0nM PG) group.
Fig6
Fig6. AnxA2 expression is required for stimulatory effect of progastrin on CD44/DCAMKL+1 expression in colonic crypts
Colonic crypts were isolated from the mice and processed for immunoblot analysis as described in Fig5. Immunoblots from a representative mouse, of a total of 3-5 mouse blots, are shown in A. Immunoblot data from all the mice are presented in B, as percent change in the ratio of indicated proteins: β-actin. *=p<0.05 versus corresponding control (0nM PG) values. C. Mouse colon sections from the indicated 4 groups of mice were processed for immunofluorescence staining with either anti-DCAMKL+1-IgG (green) or anti-CD44-IgG (red) (Supplementary Fig5). Representative enlarged images from stained colonic crypts of indicated mice are presented in C. Relative staining/cell for both DCAMKL+1/CD44 appears to be increased in PG-treated ANXA2+/+ colonic crypts, but no significant differences were observed in PG-treated ANXA2− /− mice compared to corresponding controls (Figs6C, D, Supplementary Fig5). The percent cells (within a viewing field at 20X magnification) positive for either DCAMKL+1 or CD44 were counted in ten sections from 3-5 mice. Data are presented as mean±SEM in D. *=p<0.05 versus corresponding control mouse sections; †=p<0.05 versus corresponding ANXA2+/+ levels.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Rengifo-Cam W, Singh P. Role of progastrins and gastrins and their receptors in GI and pancreatic cancers: targets for treatment. Curr Pharm Des. 2004;10:2345–2358. - PubMed
    1. Grabowska AM, Watson SA. Role of gastrin peptides in carcinogenesis. Cancer Lett. 2007;257:1–15. - PubMed
    1. Baldwin GS, Hollande F, Yang Z, et al. Biologically active recombinant human progastrin contains a tightly bound calcium ion. J Biol Chem. 2001;276:7791–7796. - PubMed
    1. Singh P, Lu X, Cobb S, et al. Progastrin1–80 stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites. Am J Physiol. 2003;284:G328–G339. - PubMed
    1. Wang TC, Koh TJ, Varro A, et al. Processing and proliferative effects of human progastrin in transgenic mice. J Clin Invest. 1996;98:1918–1929. - PMC - PubMed

Publication types

MeSH terms