Abstract
We investigated the intracellular events involved in the 3,3',5-triiodo-L-thyronine (T3)-induced accumulation in acetylcholinesterase (AChE) activity in neuroblastoma cells (neuro-2a) that overexpress the human thyroid receptor beta 1 (hTR beta 1). Treatment of these cells with T3 increased AChE activity and its mRNAs after a lag period of 24-48 h, and these levels increased through stabilization of the transcripts by T3. T3 had no effect on the transcriptional rate or processing of AChE transcripts. The protein kinase inhibitor H7 inhibited T3-induced accumulation in AChE activity and its mRNAs, whereas okadaic acid (a potent inhibitor of phosphatases 1 and 2A) potentiated the effect of T3. Okadaic acid and H7 have no effect on the binding of hTR beta 1 to T3 or the transcriptional rate of the AChE gene. Finally, treatment of cells with T3 stimulated cytosolic serine/threonine, but not tyrosine kinase, activities. The time course analysis reveals that the increase in serine/threonine activity precedes the effect of T3 on AChE mRNAs. These results suggest that activation of a serine/threonine protein kinase pathway might be a link between nuclear thyroid hormone receptor activation and stabilization of AChE mRNA.
Highlights
Acetylcholinesterase (AChE; EC 3.1.1.7)1 is responsible for the hydrolysis of acetylcholine at peripheral and central cholinergic synapses
Our results suggest that activation of a serine/threonine protein kinase pathway may be the link between thyroid receptor activation and the stabilization of AChE mRNA induced by T3
AChE Activity and mRNA Levels Are Induced by T3—Neuroblastoma cells that overexpress the thyroid receptor 1 were used to analyze the effect of T3 on AChE gene expression
Summary
Tissue Culture—Neuro-2a cells that overexpress hTR1 were cultured in Dulbecco’s modified Eagle’s medium and F-12 in a 1:1 (v/v) ratio, supplemented with 10% fetal calf serum as described previously [11]. The serum was depleted of thyroid hormones according to the procedure of Samuels et al [32]. Cells were grown at 37 °C in a humidified atmosphere of 5% CO2, 95% air. RNA Preparation and Northern Blot Hybridization—Total RNA was prepared by the guanidinium thiocyanate procedure [33], and poly(A)ϩ mRNA was prepared as described by Hartmann et al [34]. For Northern blot, 6 g of poly(A)ϩ mRNA or 15 g of total RNA was separated on 1.5% agarose gels containing 0.66 M formaldehyde and transferred to nitrocellulose membranes as described [11]
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