PKG inhibits TCF signaling in colon cancer cells by blocking β-catenin expression and activating FOXO4

Kwon, In-Kiu; Wang, Rui; Thangaraju, Muthusamy; He, Shuang; Liu, Kebin; Dashwood, Roderick H.; Dulin, Nickolai O.; Ganapathy, Vadivel; Browning, Darren D.

doi:10.1038/onc.2010.91

Cited by 62 publications

(57 citation statements)

References 56 publications

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“…The MUC2-luciferase reporter assay was described previously. 18,34 c-Jun-and ATF2-luciferase reporter assays were performed using PathDetect c-JUN transReporting System (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocols. Dominant-negative JNK was a generous gift from Dr. Shuang Huang (Georgia Regents University, Augusta, GA, USA).…”

Section: Discussionmentioning

confidence: 99%

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

et al. 2013

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Analysis of knockout animals indicates that 30 ,5 0 cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2 þ / þ and Prkg2 À / À mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2 þ / þ mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2 þ / þ but not Prkg2 À / À mice. Treatment of Prkg2 À / À mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.

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Section: Discussionmentioning

confidence: 99%

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

et al. 2013

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“…Gene-specific primers were designed using the National Center for Biotechnology Information Primer Blast Software as follows: human PKG2 (forward: 5=-AGCCCGCTTCAGGCCTCT CC-3=, reverse: 5=-AGTCGACCCTCT-GCCAGCAC-3=), human Sox9 (forward: 5=-GATCTGAAGAAGGA GAGCGAGGAGGACAAG-3=, reverse: 5=-CGTTGGGGGAGATGT-GCGTCTGCTC-3=), human MUC2 (forward: 5=-CCGCTGGAGCCG-TATCTGCG-3=, reverse: 5=-CGGGGCAGGGCAGGTCTTTG-3=), human CDX2 (forward: 5=-AGGCAGAAGAGCCGCGAGGA-3=, reverse: 5=-CCAGTCCTCCCGGAGTGGG G-3=), mouse PKG2 (forward: 5=-GTGGCGGAGGACGCCAAGAC-3=, reverse: 5=-AGGCCTG-CAGTGG GCTTCCT-3=), and common hypoxanthine phosphoribosyltransferase 1 (HPRT1) (forward: 5=-GCGTCGTGATTAGCGATGAT-GAAC-3=, reverse: 5=-CCTCCCATCTCCTTCATGACATCT-3=). Luciferase reporter assays were performed as described previously (19). The Flag-Sox9, MUC2-, and CDX2-luciferase reporter constructs were described previously (2).…”

Section: Methodsmentioning

confidence: 99%

“…In vitro studies have identified several pathways that potentially mediate the antiproliferative effects of cGMP, including calcium entry through cyclic nucleotide gated channels (16,35), inhibition of ERK (37), ␤-catenin/TCF (19), and more recently, AKT (24). However, there are significant gaps in knowledge of the immediate signaling events, and comparatively very little known about cGMP signaling mechanisms in vivo.…”

mentioning

confidence: 99%

Type 2 cGMP-dependent protein kinase regulates proliferation and differentiation in the colonic mucosa

Wang

Kwon

Thangaraju

et al. 2012

American Journal of Physiology-Gastrointestinal and Liver Physi

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Signaling through cGMP has emerged as an important regulator of tissue homeostasis in the gastrointestinal tract, but the mechanism is not known. Type 2 cGMP-dependent protein kinase (PKG2) is a major cGMP effector in the gut epithelium, and the present studies have tested its importance in the regulation of proliferation and differentiation in the mouse colon and in colon cancer cell lines. Tissue homeostasis was examined in the proximal colon of Prkg2(-/-) mice using histological markers of proliferation and differentiation. The effect of ectopic PKG2 on proliferation and differentiation was tested in vitro using inducible colon cancer cell lines. PCR and luciferase reporter assays were used to determine the importance of Sox9 downstream of PKG2. The colons of Prkg2(-/-) mice exhibited crypt hyperplasia, increased epithelial apoptosis, and reduced numbers of differentiated goblet and enteroendocrine cells. Ectopic PKG2 was able to inhibit proliferation and induce Muc2 and CDX2 expression in colon cancer cells, but did not significantly affect cell death. PKG2 reduced Sox9 levels and signaling, suggesting possible involvement of this pathway downstream of cGMP in the colon. The work presented here demonstrates a novel antiproliferative and prodifferentiation role for PKG2 in the colon. These homeostatic functions of PKG2 were reproducible in colon cancer cells lines where downregulation of Sox9 is a possible mechanism. The similarities in phenotype between PKG2 and GCC knockout mice positions PKG2 as a likely mediator of the homeostatic effects of cGMP signaling in the colon.

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“…35 Briefly, cells were cultured in 12-well plates, and at 80% confluence the assays were performed by transfecting triplicate wells with luciferase reporter plasmids using Lipofectamine …”

Section: Foxo Reporter Assaymentioning

confidence: 99%

cGMP Signaling Increases Antioxidant Gene Expression by Activating Forkhead Box O3A in the Colon Epithelium

Wang

Islam

Bridges

et al. 2017

The American Journal of Pathology

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Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMPdependent protein kinase (PKG2) to activate forkhead box O (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. FoxO3a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. FoxO3a levels were reduced in Prkg2 À/À animals, and FoxO target genes were unaffected by 8Br-cGMP challenge in vitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized FoxO3a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors. (Am J Pathol 2017, 187: 377e389; http://dx

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PKG inhibits TCF signaling in colon cancer cells by blocking β-catenin expression and activating FOXO4

Cited by 62 publications

References 56 publications

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

Type 2 cGMP-dependent protein kinase regulates proliferation and differentiation in the colonic mucosa

cGMP Signaling Increases Antioxidant Gene Expression by Activating Forkhead Box O3A in the Colon Epithelium

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