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.
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
T B
TB
Kinane
J D
JD
Finder
A
A
Kawashima
D
D
Brown
M
M
Abbate
C
C
Shang
W J
WJ
Fredericks
F J
FJ
Rauscher
V P
VP
Sukhatme
L
L
Ercolani
D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D016764:Cell Polarity; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000235:genetics; D013329:Structure-Activity Relationship; D013552:Swine; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D019204:GTP-Binding Proteins