Interleukin-6 silences y gene expression via the jak/stat signal transduction pathway

Heather A. Foley, Stuart Critz, Betty Sue Pace

Research output: Contribution to journalArticle

Abstract

We have previously shown that bipotential K562 cells treated with interleukin-6 (IL6) exhibit a concentration dependant decrease in y globin mRNA levels with a concurrent increase in the platelet specific marker glycoprotein lib (J. Biol. Chem. 272:230, 1997). This suggests that IL-6 may be involved in erythroid versus megakaryocytic lineage commitment. We subsequently tested the effects of two known megakaryocyte inducers, thrombopoietin and 1L-11 in K562 cells. Neither agent had an effect on y mRNA levels, suggesting a unique role for IL-6 in y gene silencing. IL-6 is known to phosphorylate the transcription factors Jak2, Stall and StatS to activate gene expression. To gain further insight into the mechanism for y gene silencing by IL-6 we examined the Jak/Stat signal transduction pathway. K562 cells were grown in serum-free media for 24 hours, then treated for 5 minutes with genistein (10nM), a general tyrosine kinase inhibitor, followed by the addition of 1L6 (100ng/ml). y mRNA levels were calculated as a ratio of GAPD mRNA and compared to untreated cells. K562 cells treated with either genistein alone or followed by IL-6, showed a 4.3-fold increase in y mRNA levels, demonstrating the ability of genistein to abrogate the inhibitory effect of IL-6 on y gene expression. These results suggest that Stat proteins may bind in the y promoter to alter y gene expression. Gel mobility shift assay (GMS A) experiments were completed to identify potential Stall and/or Stat3 binding sites. A sequence located in the y gene from+1 to +25 (AyStatS) contained a potential Stat3 site that was tested further. We observed decreased binding for a major DNA-protein complex with IL-6 (100ng/ml) treated K562 nuclear extract at 2 and 4 hours, however pre-treatment with AG-490 (25|iM), a Jak2 specific inhibitor, for 16 hours prevented the decreased binding observed with IL6 treatment alone. Supershift experiments with monoclonal IgG antibodies to Stall and Stal3 revealed lhal Ihe DNA-prolein complex established with the AyStat3 probe contained both Stall and Stat3 proteins. These experiments suggest that Stat3 binding in the y gene promoter may mediate the inhibitory effect of IL-6 on y gene expression. In other systems, an inhibitory splice variant, Stat3β has been identified. Experiments will be completed to test whether the Stat3β variant mediates y gene silencing by IL-6.

Original languageEnglish (US)
JournalBlood
Volume96
Issue number11 PART II
StatePublished - Dec 1 2000

Fingerprint

Signal transduction
Gene expression
Interleukin-6
Signal Transduction
Gene Expression
K562 Cells
Genes
Genistein
Gene Silencing
Messenger RNA
Experiments
Thrombopoietin
Glyceraldehyde-3-Phosphate Dehydrogenases
Proteins
Monoclonal antibodies
Megakaryocytes
Globins
DNA
Serum-Free Culture Media
Electrophoretic Mobility Shift Assay

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

Cite this

Interleukin-6 silences y gene expression via the jak/stat signal transduction pathway. / Foley, Heather A.; Critz, Stuart; Pace, Betty Sue.

In: Blood, Vol. 96, No. 11 PART II, 01.12.2000.

Research output: Contribution to journalArticle

Foley, Heather A. ; Critz, Stuart ; Pace, Betty Sue. / Interleukin-6 silences y gene expression via the jak/stat signal transduction pathway. In: Blood. 2000 ; Vol. 96, No. 11 PART II.
@article{d76320a67c364f26882ea4a586e5c0ca,
title = "Interleukin-6 silences y gene expression via the jak/stat signal transduction pathway",
abstract = "We have previously shown that bipotential K562 cells treated with interleukin-6 (IL6) exhibit a concentration dependant decrease in y globin mRNA levels with a concurrent increase in the platelet specific marker glycoprotein lib (J. Biol. Chem. 272:230, 1997). This suggests that IL-6 may be involved in erythroid versus megakaryocytic lineage commitment. We subsequently tested the effects of two known megakaryocyte inducers, thrombopoietin and 1L-11 in K562 cells. Neither agent had an effect on y mRNA levels, suggesting a unique role for IL-6 in y gene silencing. IL-6 is known to phosphorylate the transcription factors Jak2, Stall and StatS to activate gene expression. To gain further insight into the mechanism for y gene silencing by IL-6 we examined the Jak/Stat signal transduction pathway. K562 cells were grown in serum-free media for 24 hours, then treated for 5 minutes with genistein (10nM), a general tyrosine kinase inhibitor, followed by the addition of 1L6 (100ng/ml). y mRNA levels were calculated as a ratio of GAPD mRNA and compared to untreated cells. K562 cells treated with either genistein alone or followed by IL-6, showed a 4.3-fold increase in y mRNA levels, demonstrating the ability of genistein to abrogate the inhibitory effect of IL-6 on y gene expression. These results suggest that Stat proteins may bind in the y promoter to alter y gene expression. Gel mobility shift assay (GMS A) experiments were completed to identify potential Stall and/or Stat3 binding sites. A sequence located in the y gene from+1 to +25 (AyStatS) contained a potential Stat3 site that was tested further. We observed decreased binding for a major DNA-protein complex with IL-6 (100ng/ml) treated K562 nuclear extract at 2 and 4 hours, however pre-treatment with AG-490 (25|iM), a Jak2 specific inhibitor, for 16 hours prevented the decreased binding observed with IL6 treatment alone. Supershift experiments with monoclonal IgG antibodies to Stall and Stal3 revealed lhal Ihe DNA-prolein complex established with the AyStat3 probe contained both Stall and Stat3 proteins. These experiments suggest that Stat3 binding in the y gene promoter may mediate the inhibitory effect of IL-6 on y gene expression. In other systems, an inhibitory splice variant, Stat3β has been identified. Experiments will be completed to test whether the Stat3β variant mediates y gene silencing by IL-6.",
author = "Foley, {Heather A.} and Stuart Critz and Pace, {Betty Sue}",
year = "2000",
month = "12",
day = "1",
language = "English (US)",
volume = "96",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "11 PART II",

}

TY - JOUR

T1 - Interleukin-6 silences y gene expression via the jak/stat signal transduction pathway

AU - Foley, Heather A.

AU - Critz, Stuart

AU - Pace, Betty Sue

PY - 2000/12/1

Y1 - 2000/12/1

N2 - We have previously shown that bipotential K562 cells treated with interleukin-6 (IL6) exhibit a concentration dependant decrease in y globin mRNA levels with a concurrent increase in the platelet specific marker glycoprotein lib (J. Biol. Chem. 272:230, 1997). This suggests that IL-6 may be involved in erythroid versus megakaryocytic lineage commitment. We subsequently tested the effects of two known megakaryocyte inducers, thrombopoietin and 1L-11 in K562 cells. Neither agent had an effect on y mRNA levels, suggesting a unique role for IL-6 in y gene silencing. IL-6 is known to phosphorylate the transcription factors Jak2, Stall and StatS to activate gene expression. To gain further insight into the mechanism for y gene silencing by IL-6 we examined the Jak/Stat signal transduction pathway. K562 cells were grown in serum-free media for 24 hours, then treated for 5 minutes with genistein (10nM), a general tyrosine kinase inhibitor, followed by the addition of 1L6 (100ng/ml). y mRNA levels were calculated as a ratio of GAPD mRNA and compared to untreated cells. K562 cells treated with either genistein alone or followed by IL-6, showed a 4.3-fold increase in y mRNA levels, demonstrating the ability of genistein to abrogate the inhibitory effect of IL-6 on y gene expression. These results suggest that Stat proteins may bind in the y promoter to alter y gene expression. Gel mobility shift assay (GMS A) experiments were completed to identify potential Stall and/or Stat3 binding sites. A sequence located in the y gene from+1 to +25 (AyStatS) contained a potential Stat3 site that was tested further. We observed decreased binding for a major DNA-protein complex with IL-6 (100ng/ml) treated K562 nuclear extract at 2 and 4 hours, however pre-treatment with AG-490 (25|iM), a Jak2 specific inhibitor, for 16 hours prevented the decreased binding observed with IL6 treatment alone. Supershift experiments with monoclonal IgG antibodies to Stall and Stal3 revealed lhal Ihe DNA-prolein complex established with the AyStat3 probe contained both Stall and Stat3 proteins. These experiments suggest that Stat3 binding in the y gene promoter may mediate the inhibitory effect of IL-6 on y gene expression. In other systems, an inhibitory splice variant, Stat3β has been identified. Experiments will be completed to test whether the Stat3β variant mediates y gene silencing by IL-6.

AB - We have previously shown that bipotential K562 cells treated with interleukin-6 (IL6) exhibit a concentration dependant decrease in y globin mRNA levels with a concurrent increase in the platelet specific marker glycoprotein lib (J. Biol. Chem. 272:230, 1997). This suggests that IL-6 may be involved in erythroid versus megakaryocytic lineage commitment. We subsequently tested the effects of two known megakaryocyte inducers, thrombopoietin and 1L-11 in K562 cells. Neither agent had an effect on y mRNA levels, suggesting a unique role for IL-6 in y gene silencing. IL-6 is known to phosphorylate the transcription factors Jak2, Stall and StatS to activate gene expression. To gain further insight into the mechanism for y gene silencing by IL-6 we examined the Jak/Stat signal transduction pathway. K562 cells were grown in serum-free media for 24 hours, then treated for 5 minutes with genistein (10nM), a general tyrosine kinase inhibitor, followed by the addition of 1L6 (100ng/ml). y mRNA levels were calculated as a ratio of GAPD mRNA and compared to untreated cells. K562 cells treated with either genistein alone or followed by IL-6, showed a 4.3-fold increase in y mRNA levels, demonstrating the ability of genistein to abrogate the inhibitory effect of IL-6 on y gene expression. These results suggest that Stat proteins may bind in the y promoter to alter y gene expression. Gel mobility shift assay (GMS A) experiments were completed to identify potential Stall and/or Stat3 binding sites. A sequence located in the y gene from+1 to +25 (AyStatS) contained a potential Stat3 site that was tested further. We observed decreased binding for a major DNA-protein complex with IL-6 (100ng/ml) treated K562 nuclear extract at 2 and 4 hours, however pre-treatment with AG-490 (25|iM), a Jak2 specific inhibitor, for 16 hours prevented the decreased binding observed with IL6 treatment alone. Supershift experiments with monoclonal IgG antibodies to Stall and Stal3 revealed lhal Ihe DNA-prolein complex established with the AyStat3 probe contained both Stall and Stat3 proteins. These experiments suggest that Stat3 binding in the y gene promoter may mediate the inhibitory effect of IL-6 on y gene expression. In other systems, an inhibitory splice variant, Stat3β has been identified. Experiments will be completed to test whether the Stat3β variant mediates y gene silencing by IL-6.

UR - http://www.scopus.com/inward/record.url?scp=33748540726&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33748540726&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:33748540726

VL - 96

JO - Blood

JF - Blood

SN - 0006-4971

IS - 11 PART II

ER -