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

Abstract

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.

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

“…Cells with inducible PKG2 expression have been described previously. 26 The doxycycline and 8Br-cGMP were from Calbiochem (San Diego, CA). NP-40, Tween-20, and puromycin were from Sigma (St. Louis, MO).…”

“…The type 2 PKG knockout (Prkg2 À/À ) mouse was provided by Dr. Franz Hofmann (Technical University of Munich, Munich, Germany), with 129S1/Sv  129X1/SvJ genetic background, 25 and was bred and genotyped as previously described. 26,27 All mouse procedures were approved by the Augusta University (Augusta, GA) Institutional Animal Care and Use Committee.…”

“…26 Quantitation of Western blots was performed on low-exposure scans using ImageJ software version 1.48 (NIH, Bethesda, MD; http:// imagej.nih.gov/ij). 37 Data from different experiments were normalized to control (untreated) lanes.…”

“…37 Data from different experiments were normalized to control (untreated) lanes. Animal tissues were processed for histological analysis, as described previously, 26 and probed using antibodies to FoxO isoforms using the same antibodies as for immunoblotting. Histological interpretation and analysis was done independently by two individuals who were blinded to treatments (A.B.…”

“…24,25 PKG2 knockout mice also exhibit crypt hyperplasia and increased apoptosis of the luminal epithelium, indicating that PKG2 mediates the homeostatic effects of cGMP in the colon. 26 We recently demonstrated that PKG2 suppresses apoptosis in the colon epithelium, in part by increasing dual specificity protein phosphatase 10 expression, which suppresses c-Jun N-terminal kinase (JNK) activity. 27 It has also been reported that cGMP can attenuate AKT signaling in the gut mucosa, but the role of PKG2 in that pathway is not known.…”