The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling

Members of the Hedgehog (Hh) family of intercellular signaling molecules play crucial roles in animal development. Aberrant regulation of Hh signaling in humans causes developmental defects, and leads to various genetic disorders and cancers. We have characterized a novel regulator of Hh signaling through the analysis of the zebrafish midline mutant iguana (igu). Mutations in igu lead to reduced expression of Hh target genes in the ventral neural tube, similar to the phenotype seen in zebrafish mutants known to affect Hh signaling. Contradictory at first sight, igu mutations lead to expanded Hh target gene expression in somites. Genetic and pharmacological analyses revealed that the expression of Hh target genes in igu mutants requires Gli activator function but does not depend on Smoothened function. Our results show that the ability of Gli proteins to activate Hh target gene expression in response to Hh signals is generally reduced in igu mutants both in the neural tube and in somites. Although this reduced Hh signaling activity leads to a loss of Hh target gene expression in the neural tube, the same low levels of Hh signaling appear to be sufficient to activate Hh target genes throughout somites because of different threshold responses to Hh signals. We also show that Hh target gene expression in igu mutants is resistant to increased protein kinase A activity that normally represses Hh signaling. Together, our data indicate that igu mutations impair both the full activation of Gli proteins in response to Hh signals, and the negative regulation of Hh signaling in tissues more distant from the source of Hh. Positional cloning revealed that the igu locus encodes Dzip1, a novel intracellular protein that contains a single zinc-finger protein-protein interaction domain. Overexpression of Igu/Dzip1 proteins suggested that Igu/Dzip1 functions in a permissive way in the Hh signaling pathway. Taken together, our studies show that Igu/Dzip1 functions as a permissive factor that is required for the proper regulation of Hh target genes in response to Hh signals.     

shRNA-mediated silencing of Gli2 gene inhibits proliferation and sensitizes human hepatocellular carcinoma cells towards TRAIL-induced apoptosis

Aberrant activation of the Hedgehog (Hh) signaling pathway has been reported in various cancer types including hepatocellular carcinoma (HCC). As a key effector of this signaling, Gli2 plays a crucial role in carcinogenesis, including the activation of genes encoding apoptosis inhibitors and cell-cycle regulators. In this study, we examined the role of Gli2 proliferation and survival of HCC cells. First, the expression levels of Hh pathway components were detected in a subset of HCC cell lines. To establish the role of Gli2 in maintaining the tumorigenic properties of HCC cells, we developed small hairpin RNA (shRNA) targeting Gli2 and transfected it into SMMC-7721 cell, which was selected with high level of Hh signaling expression. Next, effects of Gli2 gene silencing, on cell proliferation and on the expression of cell cycle-related proteins were evaluated, then, whether down-regulation of Gli2 renders HCC cell susceptible to TRAIL was examined in vitro. Knockdown of Gli2 inhibited cell proliferation and induced G1 phase arrest of cell cycle in SMMC-7721 cell through down-regulation of cyclin D1, cyclinE2, and up-regulation of p21-WAF1. Also, Gli2 gene siliencing sensitized SMMC-7721 cell to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by reducing the expression of the long and short isoform of c-FLIP and Bcl-2, and then augmented the activation of initiator caspases-8/-9 and effector caspases-3, which induces PARP cleavage. In conclusion, our data suggest that Gli2 plays a predominant role in the proliferation and apoptosis resistance of HCC cells, and that knockdown of Gli2 may be a novel anticancer strategy for the treatment of HCC.     

Functions of B56-containing PP2As in major developmental and cancer signaling pathways

Members of the B'/B56/PR61 family regulatory subunits of PP2A determine the subcellular localization, substrate specificity, and catalytic activity of PP2A in a wide range of biological processes. Here, we summarize the structure and intracellular localization of B56-containing PP2As and review functions of B56-containing PP2As in several major developmental/cancer signaling pathways.