Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein alpha i-2 protooncogene transcription

The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene G alpha i-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The G alpha i-2 gene 5'-flanking region contains a putative binding site (5'-CGCCCCCGC-3') for EGR-1 that may allow it to be a target gene for EGR-1 mitogenic signaling. We now demonstrate in LLC-PK1 renal cells the temporal expression of EGR-1 protein by immunoblotting and immunocytochemistry coincident with the maximal activation of the G alpha i-2 gene during cell growth. To determine whether G alpha i-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for G alpha i-2 (pRSV G alpha i-2) or EGR-1 (pRSV EGR-1) driven by a viral Rous sarcoma promoter enhancer to overexpress each protein. Following transfection, cell growth was examined in media containing either 10 or 0.1% fetal bovine serum. Only cells transfected with plasmids encoding G alpha i-2 and EGR-1 had growth rates greater than that of serum replete cohorts. To assess whether EGR-1 was contributing to the transcriptional activation of the G alpha i-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5'-flanking areas of the G alpha i-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (5'-AAAAACCGC-3'). A 320% enhancement of G alpha i-2 transcription was found only in LLC-PK1 cells following their transfection with plasmids that contained both the EGR-1 binding site and overexpressed EGR-1 protein. Utilizing mobility shift assays, which compared nuclear extracts from cells before and after cell polarization, a probe containing the EGR-1 motif detected induced nuclear protein complexes during transcriptional activation of the G alpha i-2 gene. An anti-EGR-1 antibody specifically retarded the mobility of the induced nuclear complexes, indicating that the EGR-1 protein was a component of these complexes. These data provide direct evidence for a novel mitogenic signaling pathway coupling proximal signaling events that activate EGR-1 gene expression to a target protooncogene G alpha i-2 that is participatory for growth and differentiation in renal cells.