Abstract
The I-κB kinase (IKK) subunit NEMO/IKKγ (NEMO) is an adapter molecule that is critical for canonical activation of NF-κB, a pleiotropic transcription factor controlling immunity, differentiation, cell growth, tumorigenesis, and apoptosis. To explore the functional role of canonical NF-κB signaling in thyroid gland differentiation and function, we have generated a murine strain bearing a genetic deletion of the NEMO locus in thyroid. Here we show that thyrocyte-specific NEMO knock-out mice gradually develop hypothyroidism after birth, which leads to reduced body weight and shortened life span. Histological and molecular analysis indicate that absence of NEMO in thyrocytes results in a dramatic loss of the thyroid gland cellularity, associated with down-regulation of thyroid differentiation markers and ongoing apoptosis. Thus, NEMO-dependent signaling is essential for normal thyroid physiology.
Highlights
The nuclear factor-B (NF-B) signaling pathway controls a variety of important biological functions, including immune and inflammatory responses, differentiation, cell growth, tumorigenesis, and apoptosis [1]
Gene-targeting experiments have shown that mice lacking p65/RELA, IKK2/IKK, or NF-B essential modulator (NEMO) die during embryonic development due to liver apoptosis [7,8,9,10,11,12]
Previous studies have clearly established a role for NF-B in thyroid tumor progression (30 –32), this is the first work that examines the role of canonical NF-B signaling in normal thyroid development and physiology
Summary
Ethics—Procedures involving animals were conducted as indicated in the Italian National Guidelines (D.L. 100/2006 and D.L. 116/1992) and in the pertinent European Directives (EEC Council Directive 86/609, 1.12.1987), adhering to the Guide for the Care and Use of Laboratory Animals (United States National Research Council). Primary antibodies were incubated overnight at 4 °C, and sections were incubated with secondary anti-rabbit HRPconjugated antibody (Dako). Thyroid sections were incubated overnight at 4 °C with anti-NIS antibody Extracted proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences). Anti-NEMO, anti-IKK␣/, anti-p65, anti-p50, anti-phospho-CREB, antiCREB, anti-caspase-3, and anti--actin antibodies were obtained from Santa Cruz Biotechnology, Inc. All immunoblots were done at least three times using different biological material as sources. Thyroid lobes were dissected aseptically and placed on a microscope slide containing a drop of Eagle’s minimum essential medium. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide Assay—A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric assay for cell viability was performed in a 96-well flat-bottomed tissue culture plates as described previously [22].
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