No abstract

b-catenin is the major effector of the canonical Wnt signaling pathway. Mutations in components of the pathway that stabilize b-catenin result in augmented gene transcription and play a major role in many human cancers. We employed microarrays to identify transcriptional targets of deregulated b-catenin in a human epithelial cell line (293) engineered to produce mutant b-catenin and in ovarian endometrioid adenocarcinomas characterized with respect to mutations affecting the Wnt/b-catenin pathway. Two genes strongly induced in both systems-FGF20 and DKK1-were studied in detail. Elevated levels of FGF20 RNA were also observed in adenomas from mice carrying the Apc Min allele. Both XFGF20 and Xdkk-1 are expressed early in Xenopus embryogenesis under the control of the Wnt signaling pathway. Furthermore, FGF20 and DKK1 appear to be direct targets for b-catenin/TCF transcriptional regulation via LEF/TCF-binding sites. Finally, by using small inhibitory RNAs specific for FGF20, we show that continued expression of FGF20 is necessary for maintenance of the anchorage-independent growth state in RK3E cells transformed by b-catenin, implying that FGF-20 may be a critical element in oncogenesis induced by the Wnt signaling pathway.

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag‐pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site‐directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag‐pro shift site, dependent on the same structure at the junction of the two stems.

Objective It is not understood why cultured fibroblast‐like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) often display a persistently activated phenotype, despite removal from an inflammatory environment. Previously, we found that these FLS expressed high levels of both Wnt‐5A and Frizzled 5 (Fz5), a receptor–ligand pair implicated in both limb bud and bone marrow stem cell development. The objective of the present experiments was to determine whether Wnt‐5A/Fz5 signaling contributes to FLS activation. Methods Wnt‐5A expression in FLS was inhibited by transfection with both antisense and dominant negative (dn) vectors. Fz5 signaling was blocked with an antibody to the extracellular domain of the receptor. The effects of these treatments on the expression of the proinflammatory cytokines interleukin‐6 (IL‐6) and IL‐15 and on the expression of receptor activator of nuclear factor κB ligand (RANKL) were assessed by reverse transcriptase–polymerase chain reaction and immunoblotting. Results Both antisense Wnt‐5A and dnWnt‐5A vectors, but not empty vector, diminished IL‐6 and IL‐15 expression in RA FLS. Anti‐Fz5 antibody exerted similar effects and also reduced RANKL expression. Conclusion Wnt‐5A/Fz5 signaling may contribute to the activated state of FLS in RA. Receptor antagonists of Fz5 should be considered for the treatment of refractory synovitis.