Abstract
The thyroid hormone (triiodothyronine, T3) is essential for normal brain maturation. To determine the mechanisms by which T3 controls neuronal proliferation and differentiation, we have analyzed the effect of this hormone on the expression and activity of cell cycle-regulating molecules in neuroblastoma N2a-beta cells that overexpress the beta1 isoform of the T3 receptor. Our results show that incubation of N2a-beta cells with T3 leads to a rapid down-regulation of the c-myc gene and to a decrease of cyclin D1 levels. T3 also causes a strong and sustained increase of the levels of the cyclin kinase inhibitor p27(Kip1). This increase is secondary, to the augmented levels of p27(Kip1) transcripts as well as to stabilization of the p27(Kip1) protein. The increased levels of p27(Kip1) lead to a significant increase in the amount of p27(Kip1) associated with cyclin-dependent kinase 2 (CDK2), and to a marked inhibition of the kinase activity of the cyclin.CDK2 complexes. As a consequence, the retinoblastoma protein (pRb) and the retinoblastoma protein-related protein p130 are hypophosphorylated in T3-treated N2a-beta cells. This study shows for the first time that T3-mediated growth arrest and neuronal differentiation are associated with an increase in the levels of a cyclin kinase inhibitor, which does not allow the inactivation of retinoblastoma proteins required for progression through the restriction point in the cell cycle.
Highlights
The thyroid hormone is essential for the normal development of the central nervous system, the specific mechanisms by which these hormones control neuronal proliferation and differentiation are currently unknown
This study shows for the first time that T3-mediated growth arrest and neuronal differentiation are associated with an increase in the levels of a cyclin kinase inhibitor, which does not allow the inactivation of retinoblastoma proteins required for progression through the restriction point in the cell cycle
Our results show that incubation of N2a- cells with T3 leads to a rapid downregulation of the c-myc gene, to a decrease of cyclin D1 levels, and to a sustained induction of the cyclin kinase inhibitor (CKI)1 p27Kip1
Summary
Cell Cultures—The clonal cell line Neuro-2a stably transfected with the 1 isoform of the human thyroid hormone receptor (N2a- cells) was grown as described previously in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) T3-depleted serum [3]. Aliquots containing 50 g of lysate were boiled in 5ϫ Laemmli sample buffer, and the proteins were separated by SDSpolyacrylamide gel electrophoresis. Proteins were transferred onto nitrocellulose membranes (Schleicher & Schull), the membranes were blocked with 5% nonfat milk in Tris-buffered saline and 0.1% Tween 20 This was followed by incubation with the corresponding diluted antibody for 1 h at room temperature. Cells were exposed to the DNA precipitates for 16 h, washed, and incubated in DMEM containing 10% T3-depleted serum for 48 h. Cells were fixed with methanol/acetic acid/ water (90:5:5, v/v) for 30 min at room temperature, stained with a 1:500 dilution of -galactosidase antibody (Promega), and subsequently with a BrdUrd monoclonal antibody (Amersham Pharmacia Biotech) as suggested by the supplier. Cells were examined using fluorescence microscopy with the appropiate filters
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