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Effects of cytokine treatment on angiotensin II type 1A receptor transcription and splicing in rat cardiac fibroblasts

Department of Medicine, Divisions of ¹Cardiology and ²Endocrinology, University of California, San Diego, California

Submitted 28 January 2005 ; accepted in final form 4 May 2005

Angiotensin II (ANG II) plays important roles in cardiac extracellular matrix remodeling via its type 1A (AT_1A) receptor. The cytokines tumor necrosis factor- and interleukin-1 (IL-1) were shown previously to upregulate AT_1A receptor mRNA and protein, thereby increasing the profibrotic response to ANG II in cardiac fibroblasts. The present experiments implicate increased nuclear factor-B (NF-B)-dependent transcription and also, to a lesser extent, altered mRNA splicing in the mechanism of receptor upregulation. Cytokine stimulation was found to increase AT_1A heterogeneous nuclear RNA levels, which strongly suggests that mRNA upregulation occurs transcriptionally. The transcription factor NF-B was previously deemed necessary for cytokine-induced AT_1A receptor mRNA upregulation. Computer analysis of upstream DNA sequences revealed putative NF-B elements at –365 and –2540 bp. Both isolated elements were shown to bind NF-B (using gel-shift assays) and to transactivate a minimal promoter (using reporter assays), although the element at –365 bp appeared stronger. Three splice variants of AT_1A receptor mRNA that have different 5' untranslated regions were detected in rat tissues, namely, exons 1-2-3 (predominant), 1-2-3+6, and 1-3. Cytokine treatment of fibroblasts upregulated all splice variants, but exon 1-3 increased more than the others. This differential upregulation, albeit of modest magnitude, was statistically significant with IL-1 treatment. Exon 2 contains an inhibitory minicistron and a predicted inhibitory hairpin structure. Luciferase reporter assays indicated that each splice variant translates at a different efficiency, with exon 1-2-3+6 (both minicistron and hairpin) < exon 1-2-3 (minicistron only) < exon 1-3 (neither minicistron or hairpin). These results provide evidence that cytokines increase AT₁ protein levels by altering both transcription and splicing.

messenger RNA; heart; nuclear factor-B; tumor necrosis factor-; interleukin-1

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