cAMP regulates G-protein alpha sub(i-2) subunit gene transcription in polarized LLC-PK sub(1) cells by induction of a CCAAT box nuclear binding factor

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK sub(1) renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (G sub(s)) and inhibitory pertussis toxin-sensitive G-proteins (G sub(i)). Two G sub(i) genes encoding the G sub(i) isoforms G alpha sub(i-2) and G alpha sub(i-3) are expressed in LLC-PK sub(1) cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by G alpha sub(i-2). The genes encoding these isoforms are similarly regulated in these cells during growth and differentiation but differ in response to steroid hormone signals. We now demonstrate after stimulating polarized LLC-PK sub(1) cells with forskolin, which raises intracellular cAMP levels 50-fold, G alpha sub(i-2) but not G alpha sub(i-3) protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with G alpha sub(i-2) or G alpha sub(i-3) gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased G alpha sub(i-2) transcription 3-fold but inhibited G alpha sub(i-3) transcription by 50% at 12 h. In vivo footprinting of forskolin-treated cells was performed to examine the molecular basis for activation of the G alpha sub(i-2) gene. Protected guanosines were identified in a 135-base pair (bp) area previously associated with enhancer activity of this gene in non-polarized cells. This DNA segment did not contain the classical cAMP response element 5'-TGACGTCA-3'. Utilizing the 135-bp DNA segment as a probe in mobility shift assays, which compared nuclear extracts from cells before and after forskolin treatment, an induced nuclear protein complex was identified. The studies demonstrate that chronic cAMP elevation initiates a counter-regulatory response in fully polarized renal cells that includes increased transcription of the G alpha sub(i-2) gene. In contrast to other cAMP-responsive genes, increased G alpha sub(i-2) gene transcription appears to be mediated by a cAMP pathway that requires induction of a member of the CCAAT box family of DNA-binding proteins.