RhoA mediates angiotensin II-induced phospho-Ser536 nuclear factor κB/RelA subunit exchange on the interleukin-6 promoter in VSMCs

Ruwen Cui, Brian Tieu, Adrian Recinos, Ronald Tilton, Allan R. Brasier

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor κB (NF-κB). The classical NF-κB activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-κB activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces ≈1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA-mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II-inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II-induced NF-κB activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.

Original languageEnglish (US)
Pages (from-to)723-730
Number of pages8
JournalCirculation Research
Volume99
Issue number7
DOIs
StatePublished - Oct 2006

Fingerprint

Vascular Smooth Muscle
Angiotensin II
Smooth Muscle Myocytes
Interleukin-6
Blood Vessels
Phosphorylation
Inflammation
Exotoxins
Chromatin Immunoprecipitation
Vasoconstrictor Agents
Serine
Small Interfering RNA
Aorta
Monocytes
Atherosclerosis
Transcription Factors
Cytokines
Gene Expression

Keywords

  • Atherosclerosis
  • Nuclear factor κB
  • RhoA
  • Vascular inflammation

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

RhoA mediates angiotensin II-induced phospho-Ser536 nuclear factor κB/RelA subunit exchange on the interleukin-6 promoter in VSMCs. / Cui, Ruwen; Tieu, Brian; Recinos, Adrian; Tilton, Ronald; Brasier, Allan R.

In: Circulation Research, Vol. 99, No. 7, 10.2006, p. 723-730.

Research output: Contribution to journalArticle

Cui, Ruwen ; Tieu, Brian ; Recinos, Adrian ; Tilton, Ronald ; Brasier, Allan R. / RhoA mediates angiotensin II-induced phospho-Ser536 nuclear factor κB/RelA subunit exchange on the interleukin-6 promoter in VSMCs. In: Circulation Research. 2006 ; Vol. 99, No. 7. pp. 723-730.
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AU - Tieu, Brian

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AU - Tilton, Ronald

AU - Brasier, Allan R.

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N2 - The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor κB (NF-κB). The classical NF-κB activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-κB activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces ≈1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA-mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II-inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II-induced NF-κB activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.

AB - The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor κB (NF-κB). The classical NF-κB activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-κB activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces ≈1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA-mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II-inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II-induced NF-κB activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.

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