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 pathway s that control cell growth and differentiation. The G alpha i-2 gene 5 '-flanking region contains a putative binding site (5'-CGCCCCCGC-3') f or EGR-1 that may allow it to be a target gene for EGR-1 mitogenic sig naling. We now demonstrate in LLC-PK1 renal cells the temporal express ion of EGR-1 protein by immunoblotting and immunocytochemistry coincid ent with the maximal activation of the G alpha i-2 gene during cell gr owth. To determine whether G alpha i-2 or EGR-1 influence epithelial c ell growth, LLC-PK1 cells were transiently transfected with plasmids e ncoding 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 con taining either 10 or 0.1% fetal bovine serum. Only cells transfected w ith plasmids encoding G alpha i-2 and EGR-1 had growth rates greater t han that of serum replete cohorts. To assess whether EGR-1 was contrib uting to the transcriptional activation of the G alpha i-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luci ferase reporter genes fused to 5'-flanking areas of the G alpha i-2 ge ne 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 transcripti on was found only in LLC-PK1 cells following their transfection with p lasmids that contained both the EGR-1 binding site and overexpressed E GR-1 protein. Utilizing mobility shift assays, which compared nuclear extracts from cells before and after cell polarization, a probe contai ning the EGR-1 motif detected induced nuclear protein complexes during transcriptional activation of the G alpha i-2 gene. An anti-EGR-1 ant ibody specifically retarded the mobility of the induced nuclear comple xes, indicating that the EGR-1 protein was a component of these comple xes. These data provide direct evidence for a novel mitogenic signalin g 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.