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

Angiotensin II (ANG II) plays important roles in cardiac extracellular matrix remodeling via its type 1A (AT(1A)) receptor. The cytokines tumor necrosis factor-alpha and interleukin-1 beta (IL-1 beta) were shown previously to upregulate AT1A receptor mRNA and protein, thereby increasing the profibrotic response to ANG II in cardiac fibroblasts. The present experiments implicate increased nuclear factor-kappa B (NF kappa 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-kappa B was previously deemed necessary for cytokine-induced AT(1A) receptor mRNA upregulation. Computer analysis of upstream DNA sequences revealed putative NF-kappa B elements at -365 and -2540 bp. Both isolated elements were shown to bind NF-kappa 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 beta treat-ment. 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 AT(1) protein levels by altering both transcription and splicing.