Selective inhibition of intestinal guanosine 3,5-cyclic monophosphate signaling by small-molecule protein kinase inhibitors

The guanosine 3,5-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the C-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII

WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum.

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called W(ZsGreen). In the gastrointestinal tract of heterozygous W(ZsGreen)/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel W(ZsGreen)/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Downregulation of two novel genes in Sl/Sld and W(LacZ)/Wv mouse jejunum.

Interstitial cells of Cajal (ICC) are the so-called pacemaker cells of the gut. W(LacZ)/Wv and Sl/Sld mice lack ICC surrounding the myenteric plexus (MP) in the jejunum. We compared the gene expression profile of wild type (WT) and W(LacZ)/Wv and Sl/Sld mice using suppression subtractive hybridization (SSH), generating a cDNA library of 1303 clones from which 48 unique sequences were differentially expressed with Southern blot. Among them, we identified heme oxygenase2, TROY, and phospholamban in ICC using immunohistochemistry. Using RT-qPCR, c-Kit and two new transcripts Dithp and prenylcysteine oxidase1 were significantly lower expressed in Sl/Sld and W(LacZ)/Wv versus WT. Prenylcysteine oxidase1 appeared cytotoxic for COS-7 cells and was highly expressed in liver while Dithp was mainly expressed in small intestine. The combination of SSH, Southern blot, RT-qPCR, and immunohistochemistry turned out to be a useful approach to identify rarely expressed genes and genes with small differences in expression.Journal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cancer Cells Differentially Activate and Thrive on <i>De Novo</i> Lipid Synthesis Pathways in a Low-Lipid Environment

<div><p>Increased lipogenesis is a hallmark of a wide variety of cancers and is under intense investigation as potential antineoplastic target. Although brisk lipogenesis is observed in the presence of exogenous lipids, evidence is mounting that these lipids may adversely affect the efficacy of inhibitors of lipogenic pathways. Therefore, to fully exploit the therapeutic potential of lipid synthesis inhibitors, a better understanding of the interrelationship between <i>de novo</i> lipid synthesis and exogenous lipids and their respective role in cancer cell proliferation and therapeutic response to lipogenesis inhibitors is of critical importance. Here, we show that the proliferation of various cancer cell lines (PC3M, HepG2, HOP62 and T24) is attenuated when cultured in lipid-reduced conditions in a cell line-dependent manner, with PC3M being the least affected. Interestingly, all cell lines - lipogenic (PC3M, HepG2, HOP62) as well as non-lipogenic (T24) - raised their lipogenic activity in these conditions, albeit to a different degree. Cells that attained the highest lipogenic activity under these conditions were best able to cope with lipid reduction in term of proliferative capacity. Supplementation of the medium with very low density lipoproteins, free fatty acids and cholesterol reversed this activation, indicating that the mere lack of lipids is sufficient to activate <i>de novo</i> lipogenesis in cancer cells. Consequently, cancer cells grown in lipid-reduced conditions became more dependent on <i>de novo</i> lipid synthesis pathways and were more sensitive to inhibitors of lipogenic pathways, like Soraphen A and Simvastatin. Collectively, these data indicate that limitation of access to exogenous lipids, as may occur in intact tumors, activates <i>de novo</i> lipogenesis is cancer cells, helps them to thrive under these conditions and makes them more vulnerable to lipogenesis inhibitors. These observations have important implications for the design of new antineoplastic strategies targeting the cancer cell's lipid metabolism.</p></div

Addition of very-low density lipoproteins (VLDL), fatty acids and cholesterol to lipid-reduced (LR) growth conditions reverses the increased activation of the lipogenic pathway.

<p>Gene expression of FASN, ACLY, HMGCR and ACSS2 was analyzed by qPCR in T24 cells were cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL <b>(a)</b>, different fatty acid mixtures <b>(b)</b> and different concentrations cholesterol <b>(c)</b>. VLDL was added at a concentration of 607 µg triglycerides/ml serum (corresponding to the concentration triglycerides in normal FBS). Fatty acid (FA) mixtures were as follows, FA Mix 1: 20 µM linoleic (18∶2), 20 µM α-linolenic (18∶3), 5 µM arachidonic (20∶4), 5 µM docosahexaenoic acid (22∶6), FA Mix 2: 10 µM 18∶2, 15 µM 18∶3, 10 µM 20∶4, 15 µM 22∶6 and FA Mix 3: 20 µM 18∶2, 20 µM 18∶3, 5 µM 20∶4, 5 µM 22∶6, 30 µM oleic acid, 30 µM palmitic acid. Different cholesterol (Ch) concentrations are as indicated in the figures (25 µM, 50 µM or 100 µM). Data are normalized to 18S and represented as mean ± S.D. (triplicate per experiment and n = 3). Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001) from normal medium control. ^#Significantly different (^#p≤0,05; ^##p≤0,01; ^###p≤0,001;^####p≤0,0001 ) from LR control. <b>(d, e, f)</b>¹⁴C-incorporation into cellular lipids was determined in T24 cells, cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL <b>(d)</b>, different fatty acid mixtures <b>(e)</b> and different concentrations cholesterol <b>(f)</b> as mentioned in (a, b and c). During the last 4 hours ¹⁴C-acetate was added and the incorporation of radioactivity in cellular lipids was normalized to sample DNA content. Representative experiment is shown, experiment was repeated two times. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001) from normal medium control. ^#Significantly different (^#p≤0,05; ^##p≤0,01; ^###p≤0,001; ^####p≤0,0001) from LR control.</p

Lipid-reduced (LR) growth conditions differentially increase expression of FASN, HMGCR, ACLY and ACSS2 in different cancer cell lines.

<p>Gene expression of FASN, ACLY, ACSS2 and HMGCR was analyzed by qPCR analysis in <b>(a)</b> HOP62 <b>(b)</b> HepG2 <b>(c)</b> PC3M <b>(d)</b> T24 cells. Cells were cultivated in normal or LR medium for 48 hours. Data are expressed as mean ± S.D of triplicate samples, normalized to TFRC for HOP62, HepG2 and PC3M or to 18S for T24. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001), n.s. not significant (p>0,05). <b>(e)</b> FASN and ACLY expression at protein level was analyzed by Western blot analysis in HOP62, HepG2, PC3M and T24 cells cultivated under normal (N) or LR medium for 72 hours. Beta-actin was used as a loading control.</p