Mechanism Of DGK In Regulating Hedgehog Signaling Pathway

The Hedgehog signaling pathway is important for the tissue morphogenesis and the cell differentiation and is closely related to the embryonic development, the regeneration of the organs and the developing of cancer in vertebrate.In the early period of the development, abnormal inhibition or activation of the Hedgehog signaling pathway caused by gene mutation will lead to different kinds of congenital malformations. And the abnormal activation of the Hedgehog signaling pathway contributes to the happening of many kinds of tumors. Recently, the clinical trial data of some antagonists of the Hedgehog signaling pathway shows promising efficacy in treating basal cell carcinoma and medulloblastoma.The FDA approved Erivedge (vismodegib capsules) for treating the metastatic basal cell carcinoma on the January 30,2012. But all these drugs are for the single target--Smo, if the mutation of the Smo gene happens, these drugs is no more useful due to the drug-resistant. Therefore, the study of the Hedgehog signaling pathway is not only an important issue in the field of the molecular cell biology and the developmental genetics, and is also important for the clinical treatment of the tumors caused by the Hedgehog signaling pathway.The Hedgehog signaling pathway is evolutionally conserved from Drosophila to vertebrate. In vertebrates, while there is no Hedgehog ligand, the twelve-transmembrane receptor Ptchl will inhibit the activation of the seven-transmembrane receptor Smo. Gli2 and Gli3, encoded by the gli gene, will be degradated into their repressor form by the proteasome, thereby inhibiting the activation of the Hedgehog signaling pathway. When the Hedgehog ligand exists, the Hedgehog ligand will interact with the Ptchl, so the Smo can be activated to transduce the signal to the Gli protein, thus these full-length activators can activate the Hedgehog signaling pathway.The transduction of the Hedgehog signaling pathway depends on the primary cilia in vertebrates. And the intraflagellar transport (IFT) is important for the formation and the maintenance of the primary cilia. The IFT can be divided into two kinds:the anterograde IFT (from the basal body to the tip of primary cilia) and the retrograde IFT (from the tip to the basal body of primary cilia). Kinesin-2, as a motor protein, is involved in anterograde IFT with IFT B complex. And another motor protein Dynein and IFT A complex are responsible for the retrograde IFT. Normally expressed Kinesin-2, Dynein and the IFT protein are necessary for the formation and maintenance of the primary cilia and the Hedgehog signaling pathway. However, how the IFT regulates the Hedgehog signaling pathway? Is there any unknown protein which is responsible for this regulation? These questions remain unknown.We use the antibody of an IFT protein-IFT88 to find protein which has interaction with IFT88 through Immunoprecipitation (IP) experiment, and we find out DGKδ, IFT81, Klra19. We are so interested in the DGKδ as it can physically interact with β-arrestin which can regulate the localization of Smo. In order to verify that IFT88 can specifically interact with DGKδ, we transfect the 293T cells with the plasmids which can express IFT88 protein and DGKδ protein respectively. And by co-IP experiment, we find out that IFT88 can specifically interact with DGKδ as well as six other DGKs:DGKα, DGKβ, DGKγ, DGKε, DGKζ and DGKi. As these DGKs share two common domains:the C1 domain and the catalytic domain, we guess that the interaction of IFT88 and DGK protein is related to the C1 domain or/and the catalytic domain.As DGKδ can specifically interact with IFT88 which contributes to the regulation of the Hedgehog signaling pathway, can DGKδ also regulate the Hedgehog signaling pathway? To answer this question, first, we designed siRNAs specifically knockdown DGKδ. After we knockdown the DGKδ with the siRNA, we detect the mRNA level and the protein level of Glil as it is the target gene of the Hedgehog signaling pathway. And we find that the knockdown of DGKδ can obviously reduce the mRNA level and the protein level of Gli1. To verify this conclusion, we adopt two DGK inhibitors:R59022 and R59949 which can inhibit the kinase activity of all the DGKs. First, we use ShhN and PURM to activate the Hedgehog signaling pathway separately. At the same time, we treat these cells with these two inhibitors of different concentration. And then we test the activity of the Hedgehog signalling pathway by Reporter Assay and the protein level of Glil. We find out that when Hedgehog signaling pathway is activated by ShhN ligand, the two DGK inhibitors can both reduce the activity of the Hedgehog signaling pathway. But when the Hedgehog signaling pathway is activated by the Smo agonist PURM, neither of the two DGK inhibitors can reduce the activity of the Hedgehog signaling pathway. These interesting results suggest that DGKS works on the upstream of Smo. In order to verify that the Inhibition to the Hedgehog signaling pathway by DGK inhibitors is not due to drug toxicity, we reduce the time of drug treatment and then test the Gli3 localization in primary cilia by immunofluorescence experiment. We find out that DGK inhibitors can inhibit Gli3 localization in the primary cilia. In summary, we can conclude that DGKδ can upregulate the Hedgehog signaling pathway.The purpose of this study is to explore how IFT regulates the Hedgehog signaling pathway. We find that IFT88 can specifically bind DGKδ and DGKδ can upregulate the Hedgehog signaling pathway. This work suggests that there is a connection between the lipid metabolism and the Hedgehog signaling pathway. This is important for us to understand the Hedgehog signaling pathway better.