THYROID HORMONE-INDUCED SONIC HEDGEHOG SIGNAL UP-REGULATES ITS OWN PATHWAY IN A PARACRINE MANNER IN THE XENOPUS LAEVIS INTESTINE DURING METAMORPHOSIS. We have shown previously that during Xenopus laevis metamorphosis, Sonic hedgehog (Shh) is directly induced by thyroid hormone (TH) at the transcription level as one of the earliest events in intestinal remodeling. However, the regulation of other components of this signaling pathway remains to be analyzed. We have now analyzed the spatiotemporal expression of Shh receptors Patched (Ptc)-1 and Smoothened (Smo), and its downstream transcription factors Gli1, Gli2 and Gli3 during natural and TH-induced intestinal remodeling. We showed that all of the genes examined were transiently up-regulated in the mesenchymal tissues during intestinal metamorphosis. Interestingly, in the presence of protein synthesis inhibitors, Gli2 but not the others was induced by TH, suggesting that Gli2 is a direct TH response gene, while the others are likely indirect ones. Furthermore, we demonstrate by the organ culture experiment that overexpression of Shh enhances the expression of Ptc-1, Smo and Glis even in the absence of TH, indicating that Shh regulates its own pathway components during intestinal remodeling. These findings indicate that Shh expressed in the epithelium regulates mesenchymal tissue development, which in turn facilitate the development of the adult intestinal stem cells during metamorphosis. THYROID HORMONE ACTIVATES PROTEIN ARGININE METHYLTRANSFERASE 1 EXPRESSION BY DIRECTLY INDUCING C-MYC TRANSCRIPTION DURING XENOPUS INTESTINAL STEM CELL DEVELOPMENT. We have previously shown that protein arginine methyltransferase 1 (PRMT1) is upregulated in and required for adult intestinal stem cells during metamorphosis and that this role is evolutionally conserved in vertebrates. PRMT1 upregulation is the earliest known molecular event for the developing stem cells and is also conserved during zebrafish and mouse adult intestinal stem cell development. To analyze how PRMT1 is specifically upregulated during the formation of the adult intestinal stem cells, we cloned Xenopus PRMT1 promoter and characterized it in CaCo-2 cells, a human cell line with intestinal stem cell characteristics. Through a series deletion and mutational analyses, we showed that the stem cell associated transcription factor c-Myc could bind to an evolutionally conserved site in the first intron to activate the promoter. Furthermore, we demonstrated that during metamorphosis, both c-Myc and PRMT1 were highly upregulated specifically in the remodeling intestine but not the resorbing tail and that c-Myc was induced by TH prior to PRMT1 upregulation. In addition, we showed that TH directly activated c-Myc gene during metamorphosis in the intestine via binding of TH receptor to the c-Myc promoter. These results suggest that TH induces c-Myc transcription directly in the intestine and c-Myc in turn activates PRMT1 expression and that this is an important gene regulation cascade controlling intestinal stem cell development.
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