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Background Rearranged during transfection ( RET ) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB). Methods We have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches. Results The key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3β, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts. Conclusion GDNF/RET/AKT/GSK3β signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth. General significance Targeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.

Background Epithelial to mesenchymal transition (EMT) is critical for human placental development, trophoblastic differentiation, and pregnancy-associated diseases. Here, we investigated the effects of hedgehog (HH) signaling on EMT in human trophoblasts, and further explored the underlying mechanism. Methods Human primary cytotrophoblasts and trophoblast-like JEG-3 cells were used as in vitro models. Quantitative real-time RT-PCR and Western blot analysis were performed to examine mRNA and protein levels, respectively. Lentiviruses expressing short hairpin RNA were used to knock down the target genes. Reporter assays and chromatin immunoprecipitation were performed to determine the transactivity. Cell migration, invasion and colony formation were accessed by wound healing, Matrigel-coated transwell, and colony formation assays, respectively. Results Activation of HH signaling induced the transdifferentiation of cytotrophoblasts and trophoblast-like JEG-3 cells from epithelial to mesenchymal phenotypes, exhibiting the decreases in E-Cadherin expression as well as the increases in vimentin expression, invasion, migration and colony formation. Knockdown of GLI1 and GLI2 but not GLI3 attenuated HH-induced transdifferentiation, whereas GLI1 was responsible for the expression of HH-induced key EMT regulators including Snail1, Slug, and Twist, and both GLI1 and GLI2 acted directly as transcriptional repressor of CDH1 gene encoding E-Cadherin. Conclusion HH through GLI1 and GLI2 acts as critical signals in supporting the physiological function of mature placenta. General significance HH signaling through GLI1 and GLI2 could be required for the maintenance of human pregnancy.

Osteoclasts dissolve mineralized bone matrix at bone resorption sites and release large amounts of calcium (Ca 2+ ) and phosphate (PO 4 3− ) ions into the extracellular fluid. However, the exact nature of Ca 2+ and PO 4 3− on osteoblasts remains unclear. We proposed that Ca 2+ and PO 4 3− ions are required for the expression of sodium-dependent vitamin C transporter (SVCT) 2 and a differentiation marker, osteopontin (OPN), in osteoblasts as a response to the osteoclastic degradation. Results from Northern blotting indicated that a deficiency of Ca 2+ or PO 4 3− inhibited both SVCT2 and OPN expression in a time-dependent manner, whereas elevated Ca 2+ (1 to 4 mM) or PO 4 3− (1 to 4 mM) dose-dependently induced SVCT2, OPN expression and OPN promoter activity. In addition, the L-type calcium channel blocker, nifedipine (5 to 20 μM) and the phosphate transporter inhibitor, foscarnet (0.15 to 0.6 mM), dose-dependently abolished Ca 2+ - and PO 4 3− -induced SVCT2, OPN expression and OPN promoter activity. Furthermore, the results from l -ascorbic acid uptake assay and Western blotting indicated that the stimulatory effect of Ca 2+ and PO 4 3− on functional SVCT2 protein expression. These findings suggested that Ca 2+ and PO 4 3− regulate osteoblastic phenotype by entering into cells to stimulate SVCT2 and OPN expression.