cAMP regulates G-protein α(i-2) subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor

T. B. Kinane, C. Shang, Jonathan Finder, L. Ercolani

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (G(s)) and inhibitory pertussis toxin-sensitive G-proteins (G(i)). Two G(i) genes encoding the G(i) isoforms Gα(i-2) and Gα(i-3) are expressed in LLC-PK1 cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by Gα(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 (Holtzman, E. J., Kinane, T. B., West, K., Soper, B. W., Karga, H., Ausiello, D. A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975). We now demonstrate after stimulating polarized LLC-PK1 cells with forskolin, which raises intracellular cAMP levels 50-fold, Gα(i-2) but not Gα(i-3) protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with Gα(i-2) or Gα(i-3) gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased Gα(i-2) transcription 3-fold but inhibited Gα(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 Gα(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. Following systematic reduction and mutation of this DNA segment, a 'CCAAT' box motif was identified that bound the induced nuclear protein complex during forskolin-induced Gα(i-2) gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstrate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal promoter/firefly luciferase reporter gene. The transcriptional response of these plasmids in polarized forskolin-treated cells was similar in magnitude but retarded temporally to that observed in cells stably transfected with the Gα(i-2) firefly luciferase gene. These studies demonstrate that chronic cAMP elevation initiates a counter-regulatory response in fully polarized renal cells that includes increased transcription of the Gα(i-2) gene. In contrast to other cAMP-responsive genes, increased Gα(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.

Original languageEnglish (US)
Pages (from-to)24669-24676
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number33
StatePublished - Jan 1 1993
Externally publishedYes

Fingerprint

LLC-PK1 Cells
Transcription
GTP-Binding Proteins
Genes
Colforsin
Firefly Luciferases
Nuclear Proteins
Base Pairing
Protein Isoforms
Gene encoding
Cells
Reporter Genes
Transcriptional Activation
DNA
Plasmids
Avian Sarcoma
Chemical activation
Steroid hormones
Hormones
Kidney

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

cAMP regulates G-protein α(i-2) subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor. / Kinane, T. B.; Shang, C.; Finder, Jonathan; Ercolani, L.

In: Journal of Biological Chemistry, Vol. 268, No. 33, 01.01.1993, p. 24669-24676.

Research output: Contribution to journalArticle

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abstract = "Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (G(s)) and inhibitory pertussis toxin-sensitive G-proteins (G(i)). Two G(i) genes encoding the G(i) isoforms Gα(i-2) and Gα(i-3) are expressed in LLC-PK1 cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by Gα(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 (Holtzman, E. J., Kinane, T. B., West, K., Soper, B. W., Karga, H., Ausiello, D. A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975). We now demonstrate after stimulating polarized LLC-PK1 cells with forskolin, which raises intracellular cAMP levels 50-fold, Gα(i-2) but not Gα(i-3) protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with Gα(i-2) or Gα(i-3) gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased Gα(i-2) transcription 3-fold but inhibited Gα(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 Gα(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. Following systematic reduction and mutation of this DNA segment, a 'CCAAT' box motif was identified that bound the induced nuclear protein complex during forskolin-induced Gα(i-2) gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstrate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal promoter/firefly luciferase reporter gene. The transcriptional response of these plasmids in polarized forskolin-treated cells was similar in magnitude but retarded temporally to that observed in cells stably transfected with the Gα(i-2) firefly luciferase gene. These studies demonstrate that chronic cAMP elevation initiates a counter-regulatory response in fully polarized renal cells that includes increased transcription of the Gα(i-2) gene. In contrast to other cAMP-responsive genes, increased Gα(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.",
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T1 - cAMP regulates G-protein α(i-2) subunit gene transcription in polarized LLC-PK1 cells by induction of a CCAAT box nuclear binding factor

AU - Kinane, T. B.

AU - Shang, C.

AU - Finder, Jonathan

AU - Ercolani, L.

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N2 - Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (G(s)) and inhibitory pertussis toxin-sensitive G-proteins (G(i)). Two G(i) genes encoding the G(i) isoforms Gα(i-2) and Gα(i-3) are expressed in LLC-PK1 cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by Gα(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 (Holtzman, E. J., Kinane, T. B., West, K., Soper, B. W., Karga, H., Ausiello, D. A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975). We now demonstrate after stimulating polarized LLC-PK1 cells with forskolin, which raises intracellular cAMP levels 50-fold, Gα(i-2) but not Gα(i-3) protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with Gα(i-2) or Gα(i-3) gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased Gα(i-2) transcription 3-fold but inhibited Gα(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 Gα(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. Following systematic reduction and mutation of this DNA segment, a 'CCAAT' box motif was identified that bound the induced nuclear protein complex during forskolin-induced Gα(i-2) gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstrate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal promoter/firefly luciferase reporter gene. The transcriptional response of these plasmids in polarized forskolin-treated cells was similar in magnitude but retarded temporally to that observed in cells stably transfected with the Gα(i-2) firefly luciferase gene. These studies demonstrate that chronic cAMP elevation initiates a counter-regulatory response in fully polarized renal cells that includes increased transcription of the Gα(i-2) gene. In contrast to other cAMP-responsive genes, increased Gα(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.

AB - Heterotrimeric G-proteins function as signal transducers for a variety of hormone-coupled enzyme systems in eukaryotic cells. In LLC-PK1 renal cells, vasopressin-stimulated adenylylcyclase activity is regulated in part, by the counterbalancing activity of stimulatory G-proteins (G(s)) and inhibitory pertussis toxin-sensitive G-proteins (G(i)). Two G(i) genes encoding the G(i) isoforms Gα(i-2) and Gα(i-3) are expressed in LLC-PK1 cells. In polarized cells, these isoforms are topographically segregated to different membranes, which allows for the selective inhibition of adenylylcyclase by Gα(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 (Holtzman, E. J., Kinane, T. B., West, K., Soper, B. W., Karga, H., Ausiello, D. A., and Ercolani, L. (1993) J. Biol. Chem. 268, 3964-3975). We now demonstrate after stimulating polarized LLC-PK1 cells with forskolin, which raises intracellular cAMP levels 50-fold, Gα(i-2) but not Gα(i-3) protein is increased 3-fold at 12 h and remains elevated above control values by 24 h. In cells stably transfected with Gα(i-2) or Gα(i-3) gene 5'-flanking sequences fused to firefly luciferase cDNA reporter gene, forskolin treatment increased Gα(i-2) transcription 3-fold but inhibited Gα(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 Gα(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. Following systematic reduction and mutation of this DNA segment, a 'CCAAT' box motif was identified that bound the induced nuclear protein complex during forskolin-induced Gα(i-2) gene transcriptional activation. Induction of this nuclear protein complex was prevented in forskolin-treated cells by cycloheximide. To demonstrate functional activity of the CCAAT box motif, cells were transiently transfected with plasmids encoding either the minimal 135-bp segment or a multimerized CCAAT box segment fused to a Rous sarcoma minimal promoter/firefly luciferase reporter gene. The transcriptional response of these plasmids in polarized forskolin-treated cells was similar in magnitude but retarded temporally to that observed in cells stably transfected with the Gα(i-2) firefly luciferase gene. These studies demonstrate that chronic cAMP elevation initiates a counter-regulatory response in fully polarized renal cells that includes increased transcription of the Gα(i-2) gene. In contrast to other cAMP-responsive genes, increased Gα(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.

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