GDNF/RET Signaling Regulates Hedgehog Signaling And Its Significance On The Biological Behavior Of Carcinoma

A large amount of researches showed that the aberrant expression of proto-oncogene RET or Hedgehog (Hh) signaling pathway was closely associated with many diseases in organogenesis and tumorigenesis. But the regulation of Hh signaling by RET was seldom reported. To figure out whether RET could regulate Hh signaling, Gli-1(an important downstream target of Hh signaling) dual luciferase assay was used to detect the activity of Hh signaling. First, the RET agonist Glial cell-line derived neurotrophic factor (GDNF) increased the Gli-1luciferase activity in SH-SY5Y cell line, a neuroblastoma cell line expressing RET endogenously. Then, by transfecting RET overexpressing plasmid and Gli-1luciferase reporter plasmid into293T cells and SH-SY5Y cells, overexpression of RET activated Gli-1luciferase activity, indicating that RET regulated Hh signaling. Overexpression the constitutively activated form of Akt, a downstream molecular of RET, also led to enhancement of Gli-1luciferase activity, while specific inhibition of Akt by MK-2206or lentiviral-mediated knockdown of Akt inhibited the RET-induced Hh signaling, confirming that RET regulated Hh signaling via Akt. Furthermore, in order to ascertain which molecule in Hh signaling was affected by RET, we used Cyclopamine to specifically inhibit Smoothened (SMO), a key molecule of Hh signaling, finding that activated RET could still increase Gli-1 mRNA levels, suggesting that RET regulate Hh signaling in SMO downstream pathway. By detecting the phosphorylation level of SMO downstream related molecules, including PKA, GSK3β and CK1, we found that activated RET or Akt inhibited the phosphorylation of GSK3β.Since GSK3β regulates Hh signaling negatively, its inhibition activates Hh signaling. And this effect was not blocked by MK-2206or RET knockdown, indicating RET/Akt activated Hh signaling by inhibiting GSK3β.Finally, with the aid of cell proliferation assay, we found that RET knockdown, MK-2206or Cyclopamine treatment inhibited the proliferation of SH-SY5Y cells. In conclusion, this study demonstrated that RET could regulate Hh signaling and cell proliferation by Akt activation, and the inhibition of GSK3β phosphorylation.