The guanylate cyclase-C signaling pathway is down-regulated in inflammatory bowel disease

Øystein Brenna, Torunn Bruland, Marianne W Furnes, Atle van Beelen Granlund, Ignat Drozdov, Johanna Emgård, Gunnar Brønstad, Mark Kidd, Arne K Sandvik, Björn I Gustafsson, Øystein Brenna, Torunn Bruland, Marianne W Furnes, Atle van Beelen Granlund, Ignat Drozdov, Johanna Emgård, Gunnar Brønstad, Mark Kidd, Arne K Sandvik, Björn I Gustafsson

Abstract

Objective: Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn's disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined.

Material and methods: Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH).

Results: GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3',5'-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C.

Conclusions: GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Trial registration: ClinicalTrials.gov NCT00516776.

Keywords: cyclic GMP; guanylate cyclase-C; guanylin; inflammatory bowel disease; uroguanylin.

Figures

Figure 1.
Figure 1.
Individual log2 expression for (A) GUCA2A, (B) GUCA2B and (C) GUCY2C for IBD Right colon, IBD Left colon, IBD-U and Healthy. Log2 expressions are significantly different for all genes between both IBD Right colon and Healthy and IBD Left colon and Healthy. There are no significant differences in log2 expressions neither between IBD-U and Healthy nor between IBD Right colon and IBD Left colon. Filled circles represent UC (n = 37) or UC-U (n = 42) samples and open circles represent CD (n = 7) or CD-U (n = 19) samples in A-C. (D) and (E) show individual log2 expression values for TNFA and GUCA2A and TNFA and GUCY2C, respectively. In IBD (both UC and CD) there is negative correlation between both TNFA and GUCA2A (r2 = 0.27, p = 0.0003) and TNFA and GUCY2C (r2 = 0.31, p <0.0001). (F) In IBD samples (both UC and CD) log2 expression of CDX2 positively correlates with log2 expression of GUCY2C (r2 = 0.43, p <0.0001). Figures in D-F are shown with regression lines; open circles represent individual log2 expression in IBD (both UC and CD) samples, while filled circles represent individual log2 expression in Healthy.
Figure 2.
Figure 2.
(A) Representative endoscopic pictures from one rat at Days 3, 7 and 12 after induction of colitis with TNBS. (B) Average MEICS score is portrayed with corresponding fold change (FC) of Guca2a, Gucy2c, Il1a and Il1b relative to baseline (two days before colitis induction). FC of Guca2a and Gucy2c is inversely related to both MEICS score and FC of Il1a and Il1b.
Figure 3.
Figure 3.
Detection of peptides/proteins (IHC) and mRNA (ISH). (A) Immunoreactivity score (IRS) for GN and GC-C in healthy mucosa, un-inflamed IBD patients (IBD-U) and inflamed IBD. Healthy (n = 6) is indicated with target circle, UC (n = 5) and UC-U (n = 5) with filled circles and CD (n = 3) and CD-U (n = 5) with open circles. Mean IRS for GN was 11.33, 7.70 and 3.38 for Healthy, IBD-U and IBD. Mean rank difference for GN was 6.50 for Healthy vs. IBD-U (p = 0.14) and 13.63 for Healthy vs. IBD (p = 0.0006). Mean IRS for GC-C was 8.67, 6.55 and 4.56 for Healthy, IBD-U and IBD. Mean rank difference for GC-C was 5.05 for Healthy vs. IBD-U (p = 0.32) and 9.44 for Healthy vs. IBD (p = 0.02). (B) Representative GN and GC-C stained tissue specimens from Healthy, IBD-U and IBD (Magnification, 200×). (C) Mucosal expression of GUCA2A/Guca2a, GUCA2B/Guca2b and GUCY2C/Gucy2c in endoscopic biopsies from healthy human colon and from endoscopic biopsies from healthy and inflamed rat colon after induction of TNBS colitis. There is marked expression of GUCA2A/Guca2a and quite significant expression of GUCY2C/Gucy2c in healthy colonic mucosa. After induction of TNBS colitis only a few cells are positive for Guca2a (arrows with open arrowheads) and Gucy2c (dashed arrow). The pattern of expression is similar in healthy human and rat colon. GUCA2A/Guca2a and GUCY2C/Gucy2c are expressed in both goblet and columnar cells. Guca2b has no noticeable expression in rat samples neither before nor after colitis induction, whereas in human colonic mucosa GUCA2B is strongly expressed in solitary epithelial cells (arrows with solid arrowheads) and weaker in other epithelial cells (Magnification, 200×).
Figure 4.
Figure 4.
Proposed GC-C signaling in colitis. Direction of filled arrows in frames indicates significantly down- or up-regulated expression of genes encoding transcription factors, GC-C, GC-C ligands (GN and UGN) or cGMP downstream mediators in IBD (blue) and TNBS colitis (red). Down-regulation of transcription factors leads to reduced synthesis of GN (and UGN), which both act in a paracrine, luminocrine and autocrine manner, and reduced synthesis of GC-C, with subsequent decreased intracellular cGMP generation. Reduced cGMP and down-regulation of cGMP-dependent protein kinase II (PKGII) reduce phosphorylation of CFTR and diminish chloride efflux. In addition bicarbonate efflux through an unknown channel decreases. The ion channels CFTR and sodium-hydrogen exchanger (NHE) are also down-regulated. These processes have implications for fluid and ion homeostasis. Both lower cGMP levels and down-regulation of cyclic nucleotide-gated channels (CNG) lead to diminished calcium influx and calcium-sensing receptor (CaR) signaling, with reduced anti-proliferative effects as a consequence [38]. cGMP-dependent phosphodiesterase (PDE5) which facilitates conversion of cGMP to GMP is down-regulated in IBD and up-regulated in TNBS colitis. cGMP is transported through the basolateral cell membrane, possibly by multidrug-resistance transporter (MRP) 5. Reduced availability of cGMP in the lamina propria and submucosa could hamper inhibition of nociceptors. The figure was adapted and modified from Basu et al. and Fiskerstrand et al. [37,39].

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