DIFFERENTIAL REGULATION OF TWO HISTIDINE AMMONIA-LYASE GENES DURING XENOPUS DEVELOPMENT IMPLICATES DISTINCT FUNCTIONS DURING THYROID HORMONE-INDUCED FORMATION OF ADULT STEM CELLS. We have shown earlier that the de novo formation of adult intestinal stem cells that requires TH signaling in both the larval epithelium (Ep) and non-epithelial tissues (non-Ep). To understand the underlying molecular mechanisms, it is critical to determine the genes and signaling pathways involved in this process. Thus, we have profiled the gene expression programs in the epithelial and non-epithelial tissues in X. laevis intestine to systematically determine changes underlying the cell autonomous and cell-cell interaction-dependent processes that are required for stem cell formation (1). One of the genes thus identified is the histidine ammonia-lyase (HAL) gene, which encodes an enzyme known as histidase or histidinase (2). We showed that there are two duplicated HAL genes, HAL1 and HAL2, in both Xenopus laevis and Xenopus tropicalis, a highly related but diploid species. Interestingly, only HAL2 is highly upregulated by TH and appears to be specifically expressed in the adult intestinal progenitor/stem cells while HAL1 is not expressed in the intestine during metamorphosis. Furthermore, when analyzed in whole animals, HAL1 appears to be expressed only during embryogenesis but not metamorphosis while the opposite appears to be true for HAL2. Our results suggest that the duplicated HAL genes have distinct functions with HAL2 likely involved in the formation and/or proliferation of the adult stem cells during metamorphosis. DIRECT ACTIVATION OF SOX3 GENE TRANSCRIPTION BY THYROID HORMONE IN THE DEVELOPING ADULT INTESTINAL STEM CELL DURING XENOPUS METAMORPHOSIS. In addition, we also discovered from our microarray studies that the transcription factor Sox3, well known for its involvement in neural development, was upregulated in the intestinal epithelium during metamorphosis. Here, we have now shown that Sox3 is highly and specifically expressed in the developing adult intestinal progenitor/stem cells. We further demonstrated that Sox3 is a direct target gene of TH as its induction by TH is independent of new protein synthesis, suggesting that TH activate the promoter directly via TH receptor. Thus, TH activates Sox3 as one of the earliest changes in the epithelium and that Sox3 in turn may facilitate the dedifferentiation of the larval epithelial cells into adult stem cells. THE CATALYTIC SUBUNIT OF THE SYSTEM L1 AMINO ACID/THYROID HORMONE TRANSPORTER (LAT1 OR SLC7A5) FACILITATES NUTRIENT SIGNALING IN MOUSE SKELETAL MUSCLE. To regulate cellular processes, TH has to be actively transported into cells and this process is mediated by several different types of transporters. One of our previously identified TH-response genes in the intestine, LAT1, encodes the light chain of a heterodimeric system L type of TH transporter, which also transports several amino acids. Interestingly, LAT1 is highly upregulated at the climax of metamorphosis in the tadpole intestine, coinciding with the formation and rapid proliferation of the adult intestinal stem cells. We recently found out that LAT1 was also highly expressed in the mouse intestine during the neonatal period when the mouse intestine matured into the adult form, a process that appears also involves TH-dependent formation and/proliferation of the adult intestinal stem cells. Through a collaborative study, we generated a mouse line with the LAT1 gene floxed, which allows conditional knockout of the LAT1 upon expression of the Cre recombinase (3). When we crossed floxed LAT1 mice with mice expressing Cre driven by a global promoter, we obtained global LAT1 heterozygous knockout (Slc7a5+/-) animals, although failure to produce homozygous Slc7a5 animals indicate that the global knockout of Slc7a5 is embryonically lethal. Slc7a5 mRNA expression and functional System L1-type transport activity were reduced in the Slc7a5+/- animals, although no growth phenotype was detected. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout mice were established and used to study the role of SLC7A5 in the intracellular delivery large neutral amino acids (LNAA) such as leucine, which are required for full activation of the mTOR-S6K signaling pathway promoting protein synthesis and cell growth. Activation of muscle mTOR-S6K (Thr389 phosphorylation) by intraperitoneal leucine injection was blunted in homozygous MCK Cre-Slc7a5-Flox mice relative to wild-type animals. Leu and Ile concentrations in gastrocnemius muscle were reduced by 40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in Slc7a5-knockout muscle, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MCK Cre-Slc7a5-Flox mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signaling in mice, although it appears non-essential for maintenance of normal muscle mass. In addition, we had earlier shown through another collaboration that LAT1 is critical for antigen receptor-mediated the metabolic re-programming (4), a process essential for T cell differentiation, suggesting LAT1 plays diverse roles in multiple tissues/organs.
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