A Novel Role for Flotillin-Containing Lipid Rafts in Negative-Feedback Regulation of Thyroid-Specific Gene Expression by Thyroglobulin.
Thyroglobulin (Tg) stored in thyroid follicles regulates follicular function in thyroid hormone (TH) synthesis by suppressing thyroid-specific gene expression in a concentration-dependent manner. Thus, Tg is an intrinsic negative-feedback regulator that can restrain the effect of thyrotropin (TSH) in the follicle. However, the underlying mechanisms by which Tg exerts its prominent autoregulatory effect following recognition by thyrocytes remains unclear.



In order to identify potential proteins that recognize and interact with Tg, mass spectrometry was used to analyze immunoprecipitated Tg-bound proteins derived from Tg-treated rat thyroid FRTL-5 cells.



Flotillin 1 and flotillin 2, two homologs that are integral membrane proteins in lipid rafts, were identified as novel Tg-binding proteins with high confidence. Further studies revealed that flotillins physically interact with endocytosed Tg, and together these proteins redistribute from the cell membrane to cytoplasmic vesicles. Treatment with the lipid raft disrupter methyl-β-cyclodextrin abolished both the endocytosis and the negative-feedback effect of Tg on thyroid-specific gene expression. Meanwhile, siRNA-mediated knockdown of flotillin 1 or flotillin 2 also significantly inhibited Tg effects on gene expression.



Together these results indicate that flotillin-containing lipid rafts are essential for follicular Tg to be recognized by thyrocytes and exert its negative-feedback effects in the thyroid.
1 Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University , Tokyo, Japan .|2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .|3 Department of Pathology, Faculty of Medicine, University of Yamanashi , Yamanashi, Japan .
Yuqian
Y
Luo
2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .
Takeshi
T
Akama
4 Advanced Medical Research Center, Yokohama City University , Yokohama, Japan .
Akiko
A
Okayama
1 Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University , Tokyo, Japan .|2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .|5 Department of Education Planning and Development, Faculty of Medicine, Toho University , Tokyo, Japan .
Aya
A
Yoshihara
2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .|6 Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Toho University , Tokyo, Japan .
Mariko
M
Sue
1 Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University , Tokyo, Japan .|2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .|6 Division of Diabetes, Metabolism and Endocrinology, Department of Internal Medicine, Toho University , Tokyo, Japan .
Kenzaburo
K
Oda
1 Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University , Tokyo, Japan .|2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .
Moyuru
M
Hayashi
1 Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University , Tokyo, Japan .|2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .
Yuko
Y
Ishido
3 Department of Pathology, Faculty of Medicine, University of Yamanashi , Yamanashi, Japan .
Hisashi
H
Hirano
5 Department of Education Planning and Development, Faculty of Medicine, Toho University , Tokyo, Japan .
Naoki
N
Hiroi
3 Department of Pathology, Faculty of Medicine, University of Yamanashi , Yamanashi, Japan .
Ryohei
R
Katoh
1 Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University , Tokyo, Japan .|2 Laboratory of Molecular Diagnostics, Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases , Tokyo, Japan .
Koichi
K
Suzuki
D000818:Animals; D002417:Cattle; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D025461:Feedback, Physiological / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D020249:Hormone Replacement Therapy; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering; D051381:Rats; D013954:Thyroglobulin / Q000737:chemistry / Q000378:metabolism*; D000072637:Thyroid Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
D008565:Membrane Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; D013954:Thyroglobulin
flotillin; lipid rafts; negative-feedback effect; thyroglobulin; thyroid cell biology