Abstract
Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation. Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene. In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene. Ca(2+) signals are necessary for the induction of MKP-1 in response to TRH but not to EGF. Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF. By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block. Ca(2+) signals are important to release the MKP-1 elongation block in a manner similar to the c-fos oncogene. These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells.
Highlights
Long term cellular processes such as proliferation, differentiation, and neuronal plasticity are controlled by extracellular stimuli and require the synthesis of new gene products
We show a rapid and massive increase in MAP kinase phosphatase 1 (MKP-1) mRNA triggered by either thyrotropin-releasing hormone (TRH) or epidermal growth factor (EGF) in GH4C1 neuroendocrine cells
Rapid Induction of MKP-1 mRNA by TRH and EGF in Pituitary GH4C1 Cells Depends on Transcriptional Activation—
Summary
Materials—The thyrotropin-releasing hormone TRH (Roche Molecular Biochemicals) and the epidermal growth factor EGF (Sigma) were diluted in H2O at 27.6 mM and stored in aliquots at Ϫ80 °C. Autoradiographic signals detected in Northern and RNase protection assays for MKP-1 were quantified in arbitrary units using a PhosphorImager (Molecular Dynamics, Inc.) and normalized to the corresponding GAPDH values to correct variations in RNA loading. TaqMan RT-PCR—Quantification of the MKP-1 mRNA was performed by TaqMan RT-PCR (PerkinElmer Life Sciences) from the total RNAs extracted from 10 series of GH4C1 cells cultured in nonstimulated or stimulated conditions. The standard curve was prepared from the total RNA of a TRH-treated sample with a high level of expression of MKP-1, diluted from 100 ng/l to 1 pg/l. The primers and probe for the quantification of MKP-1 were designed using Primer Express 1.0 software from PerkinElmer Life Sciences These primers amplified an 87-bp fragment of the rat’s MKP-1 gene comprising the exon 1–2 boundary, to avoid amplification of genomic DNA. Blots were developed by autoradiography and quantified with a Molecular Dynamics PhosphorImager
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