Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)(Met30) ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response. We show here that SMER3 inhibits SCF(Met30) in vivo and in vitro, but not the closely related SCF(Cdc4). Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

Induction of fibroblast growth factor 10 (FGF10) in the ileal crypt epithelium after massive small bowel resection suggests a role for FGF10 in gut adaptation.

We have previously reported that fibroblast growth factor 10 (FGF10) is crucial for the survival and proliferation of progenitor cells during embryonic gastrointestinal development. We sought to characterize the potential role of FGF10 signaling in the adaptive response following small bowel resection. Adult wild-type and Fgf10(LacZ) mice underwent 50% small bowel resection (SBR) or sham operation. Tissues were harvested 24 or 48 hr after surgery for histology, immunohistochemistry, and in situ hybridization. After SBR, Fgf10 expression was demonstrated in the epithelium at the base of the crypts. Moreover, there was a statistically significant increase in proliferating cells and goblet cells after SBR. In vitro studies using rat intestinal epithelial crypt (IEC-6) cells exposed to medium with or without recombinant FGF10 showed increased proliferation and phosphorylation of Raf and AKT with the addition of FGF10. Our results suggest that FGF10 may play a therapeutic role in diseases involving intestinal failure.

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development.

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in alpha-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.

The fibroblast growth factor pathway serves a regulatory role in proliferation and apoptosis in the pathogenesis of intestinal atresia.

BACKGROUND/PURPOSE: Intestinal atresia occurs in 1:5000 live births and is a neonatal challenge. Fibroblast growth factor receptor 2b (Fgfr2b) is a critical developmental regulator of proliferation and apoptosis in multiple organ systems including the gastrointestinal tract (GIT). Fgfr2b invalidation results in an autosomal recessive intestinal atresia phenotype. This study evaluates the role of Fgfr2b signaling in regulating proliferation and apoptosis in the pathogenesis of intestinal atresia. METHODS: Wild-type and Fgfr2b-/- embryos were harvested from timed pregnant mice. The GIT was harvested using standard techniques. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) was used to evaluate apoptosis and bromodeoxyuridine to assess proliferation by standard protocols. Photomicrographs were compared (Institutional Animal Care and Use Committee-approved protocol 32-02). RESULTS: Wild-type and mutant GIT demonstrate that deletion of the Fgfr2b gene results in inhibition of epithelial proliferation and increased apoptosis. Inhibited proliferation and increased apoptosis are specific to those tissues of normal Fgfr2b expression, corresponding to the site of intestinal atresia. CONCLUSIONS: The absence of embryonic GIT Fgfr2b expression results in decreased proliferation and increased apoptosis resulting in GIT atresia. The regulation of proliferation and apoptosis in intestinal cells as a genetically based cause of intestinal atresia represents a novel consideration in the pathogenesis of intestinal atresia.

Fibroblast growth factor-10 serves a regulatory role in duodenal development.

PURPOSE: Duodenal obstruction occurs in 1 of 6000 live births and requires urgent surgical intervention. Duodenal atresia previously has been ascribed to a developmental failure of luminal recanalization; however, the cause of duodenal atresia remains incompletely understood. Although familial intestinal atresias have been described and syndromic associations are known, no specific genetic link has been established. Fibroblast growth factor-10 (Fgf10) is a known regulatory molecule relevant to mesenchymal-epithelial interactions, and mice deficient in Fgf10 demonstrate congenital anomalies in several organ systems including the gastrointestinal tract. The authors hypothesized that Fgf10 could serve a regulatory role in establishing normal duodenal development. METHODS: Wild-type mice with beta-galactosidase under the control of the Fgf10 promoter were harvested from timed-pregnancy mothers. The expression of Fgf10 in the duodenum during development was evaluated by developing the embryos in X-Gal solution. Wild-type and mutant Fgf10(-/-) embryos were harvested from timed-pregnancy mothers at 18.5 days postconception (near term) and were analyzed for duodenal morphology (Institutional Animal Care and Use Committee-approved protocol 32-02). Photomicrographs were reviewed. RESULTS: Fibroblast growth factor-10 is active in the duodenum at a late stage of development. The Fgf10(-/-) mutants demonstrate duodenal atresia with a variable phenotype similar to clinical findings. The duodenum fails to develop luminal continuity and has proximal dilation. The phenotype occurs in an autosomal recessive pattern with incomplete penetrance (38%). CONCLUSIONS: Fibroblast growth factor-10 serves as a regulator in normal duodenal growth and development. Its deletion leads to duodenal atresia and challenges traditionally accepted theories of pathogenesis. This novel, genetically mediated duodenal malformation reflects an animal model that will allow further evaluation of the pathogenesis of this surgically correctable disease. By studying the mechanism of Fgf10 function in foregut development, the authors hope to better understand these anomalies and to explore possible therapeutic alternatives.

Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway.

BACKGROUND/PURPOSE: Colonic atresia occurs in 1:20,000 live births, offering a neonatal surgical challenge. Prenatal expression of fibroblast growth factor 10 (Fgf10), acting through fibroblast growth factor receptor 2b (Fgfr2b), is critical to the normal development of the colon. Invalidation of the Fgf10 pathway results in colonic atresia, inherited in an autosomal recessive pattern. Classically, disturbance of the mesenteric vasculature has been thought to cause many forms of intestinal atresia. The purpose of this study was to evaluate the role of vascular occlusion in the pathogenesis of colonic atresia. METHODS: Wild type (Wt), Fgf10(-/-), and Fgfr2b(-/-) mutant mouse embryos were harvested from timed pregnant mothers. Immediately following harvest, filtered India ink was infused via intracardiac microinjection. The gastrointestinal tract was dissected, and photomicrographs of the mesenteric arterial anatomy were taken at key developmental time points. RESULTS: Photomicrographs after India ink microinjections demonstrate normal, patent mesenteric cascades to the atretic colon at the time points corresponding to the failure of colonic development in the Fgf10(-/-) and Fgfr2b(-/-) mutants. The mesenteric arterial anatomy of the colon demonstrates no difference between the Wt and mutant colonic atresia. CONCLUSIONS: The absence of embryonic expression of Fgf10 or its receptor Fgfr2b results in colonic atresia in mice. India ink microinjection is a direct measure of mesenteric arterial patency. Colonic atresia in the Fgf10(-/-) and Fgfr2b(-/-) mutants occurs despite normal mesenteric vascular development. Thus the atresia is not the result of a mesenteric vascular occlusion. The patent colonic mesentery of the Fgf10(-/-) and Fgfr2b(-/-) mutants challenges an accepted pathogenesis of intestinal atresia. Although colonic atresia can occur as a result of vascular occlusion, new evidence exists to suggest that a genetic mechanism may play a role in the pathogenesis of this disease.

Fibroblast growth factor receptor 2 IIIb invalidation--a potential cause of familial duodenal atresia.

BACKGROUND/PURPOSE: Duodenal atresia (DA) occurs in 1 in every 6,000 live births and represents a significant surgically correctable cause of intestinal obstruction in the neonate. Familial or congenital DA has been reported, implying that at least some cases of DA are the result of genetic, heritable abnormalities. The genes controlling duodenal development are incompletely understood. Fibroblast growth factor receptor 2IIIb (Fgfr2b) is known to play a critical role in the development of multiple organ systems including other gastrointestinal tract (GIT) structures. This study shows the key role of Fgfr2b in normal duodenal development and the pathogenesis of DA. METHODS: Wild type (Wt) and Fgfr2b-/- embryos were harvested from timed pregnant mothers at stage E18.5 and were analyzed for duodenal phenotype. RESULTS: Inactivation of Fgfr2b results in DA. DA is present in the Fgf2b-/- mutants with a 35% penetrance. The duodenal phenotype of the Fgf2b-/- mutants ranges from normal to a mucosal web, type I, and type III atresia. CONCLUSIONS: Fgfr2b is a critical regulatory gene in the development of the duodenum. Fgfr2b invalidation (Fgfr2b-/- mutant) results in a reproducible, autosomal recessive duodenal atresia phenotype with incomplete penetrance and a variable phenotype.