OR14-04 Musashi-1 Is A Cell-type Specific Regulator Of Type 2 Deiodinase-mediated Thyroid Hormone Activation

Abstract

Abstract Disclosure: P. Mohacsik: None. E. Halmos: None. Y. Ruska: None. C. Fekete: None. B. Gereben: None. Thyroid hormone (TH) exerts a profound effect on the regulation of energy homeostasis, metabolism, brain development and the determination of cell fate in various tissues. Type 2 deiodinase enzyme (D2) is the crucial regulator of TH activation by converting the prohormone thyroxine to T3 that can bind the TH receptor. This represents a fundamental step of cell-type specific TH action. D2 regulation is tightly controlled at various molecular levels to allow quick adaptation of TH action to specific challenges. Within the brain, D2 is expressed in hypothalamic tanycytes that play a crucial role in the regulation of the hypothalamo-pituitary-thyroid (HPT) axis and in astrocytes of extrahypothalamic brain regions. Under changing TH economy, D2 activity undergoes homeostatic alteration in astrocytes while remains largely unaffected in hypothalamic tanycytes. Our aim was to identify regulators that could underlie this phenomenon as a cell-type specific regulator of D2-dependent TH action. Musashi (Msi)-1 is a highly conserved RNA-binding protein that regulates cell cycle and play a crucial role during brain development. We found, that Msi-1 was co-expressed with D2 both in hypothalamic tanycytes and cortical astrocytes in mice and T3 treatment (24h T3 1µg/bwg, i.p.) increased Msi-1 protein level in both cell types indicating its TH-dependent regulation. Then we identified multiple potential Msi-1 binding sites in the extremely long 3’UTR both of the human, mouse, rat and chicken D2 mRNA and used RNA electromobility shift assay that revealed direct binding of Msi-1 to the 3’UTR of the D2 mRNA. Studies on the impact of Msi-1 level on D2 activity showed, that Msi-1 overexpression decreased D2 activity in HEK293 cells while this effect was abolished by the deletion of a 3 kb region of the 3’UTR of D2 containing the Msi-1 binding sites. Furthermore, Msi-1 knock down by shRNA in H4 glioma cells increased endogenous D2 activity. A mouse model, lacking Msi-1 protein exclusively in the brain and pituitary, showed increased D2 activity in the cortex accompanied with a slightly decreased serum fT4 and decreased liver dio1 expression. Increased cortical D2 activity was not a consequence of the mildly hypothyroid periphery since cortical expression of dio3, the T3-regulated T3-degrading enzyme remained unchanged along with enpp2, another T3-regulated gene. In contrast, D2 activity remained unchanged in hypothalamic tanycytes. Our results identify Msi-1 as a TH-dependent cell-type specific regulator of D2 activity in the mouse brain that acts via direct binding to the 3’UTR of the D2 mRNA. In summary, Msi-1 regulates D2 activity cell type specifically and may contribute to the homeostatic regulation of cortical D2 while at the same time allows TH-resistant D2 function in tanycytes that is required by proper HPT function. Presentation: Friday, June 16, 2023