Differentiation of a Catecholaminergic CNS Cell Line Modifies Tyrosine Hydroxylase Transcriptional Regulation

Abstract

Recently, a tyrosine hydroxylase (TH)-expressing CNS-derived cell line, CAD, was obtained that is capable of undergoing reversible morphological differentiation. The isolation of the CAD line allowed us to ask whether different DNA regulatory elements direct TH transcription when cells are growing and undifferentiated versus postmitotic and differentiated. To this end, we compared expression of a transiently transfected bacterial chloramphenicol acetyltransferase reporter gene under the transcriptional control of TH 5' flanking DNA in CAD cells grown in the presence and absence of serum. Mutational analysis indicates that CAD cells differently regulate TH transcription depending on their state of differentiation. In both states, the cyclic AMP response element and APi site each activate transcription. However, in undifferentiated cells, the dyad/E-box element represses expression by~2.7-fold, whereas it modestly activates transcription in differentiated cells. The role of the dyad/ E-box as a repressor correlates well with the two-to threefold lower amount of endogenous TH protein present in the undifferentiated CAD cells. This study demonstrates the differential use of TH DNA regulatory elements in proliferating, undifferentiated and nonproliferating, differentiated immortalized neuronal cells.

Epigenetic, transcriptional and posttranscriptional regulation of the tyrosine hydroxylase gene

“…The regulatory function of the DSE/E‐box depends on the degree of cell line differentiation. In undifferentiated CAD cells, the DSE/E‐box sequence acts as a silencer, but once they have reached the catecholaminergic phenotype, plays a role as a TH gene enhancer (Lazaroff et al, 1998). DSE of the bovine TH gene silences both the basic and angiotensin II stimulated levels of TH transcription in primary cultures of bovine chromaffin cells (Kim et al, 1996).…”

Studies of the central nervous system‐derived CAD cell line, a suitable model for intraneuronal transport studies?

“…Molecular mechanisms of neurite outgrowth and cell differentiation have been studied in the CAD cell line by adding promoting compounds/ proteins to the culture medium (Donai et al, 2000;Satoh et al, 2000). DNA regulation of tyrosine hydroxylase transcription, investigated in CAD cell cultures transfected with calcium phosphate/DNA precipitates, was demonstrated to differ between undifferentiated and differentiated CAD cells (Lazaroff et al, 1998). The CAD cell line has also been used for the study of cAMP signaling pathways by transfecting the cells with different agents (Johnston et al, 2002(Johnston et al, , 2004Chen et al, 2005).…”

Interference of alpha-synuclein with cAMP/PKA-dependent CREB signaling for tyrosine hydroxylase gene expression in SK-N-BE(2)C cells

“…TH activity is tightly regulated during development and differentiation (Lazaroff et al, 1998) and by a variety of physiological factors, such as trans-synaptic signals, hormones, growth factors, and stressors, in adulthood (Wong and Tank, 2007). Long-term and short-term mechanisms are involved in the regulation of TH activity; longterm regulation of TH includes transcriptional regulation, alternative RNA splicing, RNA stabilization, and translational regulation, and short-term modulation occurs through phosphorylation/dephosphorylation at four different serine residues (Ser8, Ser19, Ser31, and Ser40) in the N-terminal regulatory region of TH (Lindgren et al, 2000;Dunkley et al, 2004).…”