Abstract
Mutations in the APC (adenomatous polyposis coli) tumor suppressor gene cause uncontrolled proliferation and impaired differentiation of intestinal epithelial cells. Recent studies indicate that human colon adenomas and carcinomas lack retinol dehydrogenases (RDHs) and that APC regulates the expression of human RDHL. These data suggest a model wherein APC controls enterocyte differentiation by controlling retinoic acid production. However, the importance of APC and retinoic acid in mediating control of normal enterocyte development and differentiation remains unclear. To examine the relationship between APC and retinoic acid biosynthesis in normal enterocytes, we have identified two novel zebrafish retinol dehydrogenases, termed zRDHA and zRDHB, that show strong expression within the gut of developing zebrafish embryos. Morpholino knockdown of either APC or zRDHB in zebrafish embryos resulted in defects in structures known to require retinoic acid. These defects included cardiac abnormalities, pericardial edema, failed jaw and pectoral fin development, and the absence of differentiated endocrine and exocrine pancreas. In addition, APC or zRDHB morphant fish developed intestines that lacked columnar epithelial cells and failed to express the differentiation marker intestinal fatty acid-binding protein. Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes. Downstream of retinoic acid production, we identified hoxc8 as a retinoic acid-induced gene that, when ectopically expressed, rescued phenotypes of APC- and zRDHB-deficient zebrafish. Our data establish a genetic link supporting a critical role for retinoic acid downstream of APC and confirm the importance of retinoic acid in enterocyte differentiation.
Highlights
Mutations in the tumor suppressor gene APC result in an inherited colon cancer predisposition known as familial adenomatous polyposis [1,2,3]
Recent studies indicate that human colon adenomas and carcinomas lack retinol dehydrogenases (RDHs) and that APC regulates the expression of human RDHL
We demonstrated previously that colon adenomas and carcinomas have elevated -catenin target genes, they showed a deficiency of retinoic acid biosynthetic enzymes [10]
Summary
Mutations in the tumor suppressor gene APC (adenomatous polyposis coli) result in an inherited colon cancer predisposition known as familial adenomatous polyposis [1,2,3]. The second step involves conversion of retinal into retinoic acid via aldehyde dehydrogenases [11] Given this required biosynthetic conversion, retinoic acid production is limited to cells harboring the necessary enzymes for conversion of vitamin A. In establishing a link between APC and control of retinoic acid biosynthesis, introduction of APC into an APC mutant colon carcinoma cell line increased retinoic acid biosynthesis in parallel with the transcriptional induction of retinol dehydrogenase L (RDHL) [10]. Despite these observations, we currently protein; RDH, retinol dehydrogenase; MO, morpholino; HPLC, high pressure liquid chromatography; hpf, hours postfertilization; i-FABP, intestinal fatty acid-binding protein; TTNPB, (4-(E)-2-(5,6,7,8-tetrahydro5,5,8,8-tetramethyl(-2-naphtalenyl-propenyl) benzoic acid. The phenotypes evoked by knockdown of either APC or zRDHB were rescued by the addition of exogenous retinoic acid or injection of hoxc mRNA
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