Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene

Aug 24, 2006American journal of physiology. Endocrinology and metabolism

A key DNA element is required for exendin-4 to activate the rat EGR-1 gene

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Abstract

A critical region in the EGR-1 gene promoter between -73 and -46 is essential for its activation by exendin-4.

Exendin-4 activates early growth response factor-1 (EGR-1), which is necessary for the transcriptional activation of cyclin D1 (Ccnd1).
Mutation of the proximal cAMP response element (CRE) at -69 led to a significant decrease in EGR-1 transcription.
The distal CRE at -139 did not affect EGR-1 transcription when mutated.
Binding of phosphorylated cAMP response element-binding protein (CREB) to the proximal CRE increased following exendin-4 treatment.
Inhibition of CREB binding through specific mutations resulted in a significant decrease in exendin-4-induced EGR-1 transcription.
The protein kinase A (PKA) signaling pathway appears to play a more critical role in EGR-1 induction compared to the MEK/ERK pathway.

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Glucagon-like peptide-1 and its potent agonist exendin-4 induce several immediate early response genes (IEGs) that code for transcription factors implicated in cell proliferation, differentiation, and apoptosis. We recently observed that early growth response factor-1 (EGR-1), an IEG product, was required for transcriptional activation of Ccnd1 (cyclin D1) gene by exendin-4. Herein, the regulatory mechanism whereby exendin-4 activates the transcription of EGR-1 gene was investigated in the pancreatic beta-cell line INS-1. Deletion analysis of rat EGR-1 promoter identified a critical region between -73 and -46 for the activation of EGR-1 in response to exendin-4. Mutation of the proximal putative cAMP response element (CRE, 5'-GTACGTCA-3') located at -69 resulted in a significant decrease in the EGR-1 transcription, whereas the mutation of the distal putative CRE at -139 was without such an effect. In immune supershift assays using exendin-4-treated cells, binding of cAMP response element-binding protein (CREB) phosphorylated on Ser(133) to the proximal CRE was increased. Employment of a CREB mutant containing Ala substitution at Ser(133) or a dominant negative CREB mutant that inhibits the binding of endogenous CREB to DNA significantly decreased the exendin-4-induced EGR-1 transcription. In experiments using specific protein kinase inhibitors, the effect of H-89 was more prominent than PD-98059, indicating the predominance of the PKA signaling over the MEK/ERK in induction of EGR-1. Therefore, it appears that the proximal CRE site is critical and the binding with CREB phosphorylated on Ser(133) is necessary for induction of the EGR-1 transcription by exendin-4.

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Proximal cyclic AMP response element is essential for exendin-4 induction of rat EGR-1 gene

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