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

T. Bernard Kinane, Jonathan Finder, Akira Kawashima, Dennis Brown, Mauro Abbate, Cheng Shang, William J. Fredericks, Frank J. Rauscher, Vikas P. Sukhatme, Louis Ercolani

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene Gαi-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The Gαi-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 Gαi-2 gene during cell growth. To determine whether Gαi-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for Gαi-2 (pRSV Gαi-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 Gαi-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 Gαi-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5'-flanking areas of the Gαi-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (5'-AAAAACCGC-3'). A 320% enhancement of Gαi-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 Gαi-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 Gαi-2 that is participatory for growth and differentiation in renal cells.

Original languageEnglish (US)
Pages (from-to)27503-27509
Number of pages7
JournalJournal of Biological Chemistry
Volume269
Issue number44
StatePublished - Nov 4 1994
Externally publishedYes

Fingerprint

LLC-PK1 Cells
Transcription
GTP-Binding Proteins
Up-Regulation
Genes
Kidney
Cell growth
Growth
Plasmids
Binding Sites
Chemical activation
Transcriptional Activation
Early Growth Response Transcription Factors
Transfection
Proteins
Avian Sarcoma
Firefly Luciferases
Guanine Nucleotides
5' Flanking Region
Zinc Fingers

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Kinane, T. B., Finder, J., Kawashima, A., Brown, D., Abbate, M., Shang, C., ... Ercolani, L. (1994). Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein αi-2 protooncogene transcription. Journal of Biological Chemistry, 269(44), 27503-27509.

Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein αi-2 protooncogene transcription. / Kinane, T. Bernard; Finder, Jonathan; Kawashima, Akira; Brown, Dennis; Abbate, Mauro; Shang, Cheng; Fredericks, William J.; Rauscher, Frank J.; Sukhatme, Vikas P.; Ercolani, Louis.

In: Journal of Biological Chemistry, Vol. 269, No. 44, 04.11.1994, p. 27503-27509.

Research output: Contribution to journalArticle

Kinane, TB, Finder, J, Kawashima, A, Brown, D, Abbate, M, Shang, C, Fredericks, WJ, Rauscher, FJ, Sukhatme, VP & Ercolani, L 1994, 'Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein αi-2 protooncogene transcription', Journal of Biological Chemistry, vol. 269, no. 44, pp. 27503-27509.
Kinane, T. Bernard ; Finder, Jonathan ; Kawashima, Akira ; Brown, Dennis ; Abbate, Mauro ; Shang, Cheng ; Fredericks, William J. ; Rauscher, Frank J. ; Sukhatme, Vikas P. ; Ercolani, Louis. / Growth of LLC-PK1 renal cells is mediated by EGR-1 up-regulation of G protein αi-2 protooncogene transcription. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 44. pp. 27503-27509.
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abstract = "The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene Gαi-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The Gαi-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 Gαi-2 gene during cell growth. To determine whether Gαi-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for Gαi-2 (pRSV Gαi-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 Gαi-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 Gαi-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5'-flanking areas of the Gαi-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (5'-AAAAACCGC-3'). A 320{\%} enhancement of Gαi-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 Gαi-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 Gαi-2 that is participatory for growth and differentiation in renal cells.",
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AU - Brown, Dennis

AU - Abbate, Mauro

AU - Shang, Cheng

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N2 - The early growth response zinc finger transcription factor (EGR-1) and the heterotrimeric guanine nucleotide binding protein encoded by the protooncogene Gαi-2 each play pivotal roles in signaling pathways that control cell growth and differentiation. The Gαi-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 Gαi-2 gene during cell growth. To determine whether Gαi-2 or EGR-1 influence epithelial cell growth, LLC-PK1 cells were transiently transfected with plasmids encoding cDNAs for Gαi-2 (pRSV Gαi-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 Gαi-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 Gαi-2 gene, cells were cotransfected with pRSV EGR-1 and plasmids encoding firefly luciferase reporter genes fused to 5'-flanking areas of the Gαi-2 gene containing either the EGR-1 binding site or a mutated EGR-1 binding site (5'-AAAAACCGC-3'). A 320% enhancement of Gαi-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 Gαi-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 Gαi-2 that is participatory for growth and differentiation in renal cells.

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