siRNA knocks down Hsp27 and increases angiotensin II-induced phosphorylated NF-kappaB p65 levels in aortic smooth muscle cells.
OBJECTIVE
Angiotensin II (Ang II) induces the proinflammatory nuclear factor kappaB (NF-kappaB) in the vasculature. Heat shock and elevated levels of heat shock proteins (Hsps) decrease Ang II-induced NF-kappaB transcriptional activity and inflammation, but little is known about the role of specific Hsps. Here we used small interfering RNA (siRNA) technology to examine the role of Hsp27 in the Ang II-induced NF-kappaB signaling pathway.


METHODS
Hsp27 siRNA was transfected into rat aortic vascular smooth muscle (A10) and 48 hrs later, the cells were stressed with 100 nM of Ang II for up to 24 hrs. Hsp27 levels were determined by immunofluorescence microscopy and Western analysis and inhibitor kappaB-alpha (I kappaB-alpha), the p65 subunit of NF-kappaB, and I kappaB kinase (IKK) levels were determined by Western analysis.


RESULTS
When Hsp27 was specifically knocked down with Hsp27 siRNA in A10 cells there was a trend toward an increase in Ang II-induced phosphorylated p65. I kappaB-alpha and IKK-beta levels were not changed by the knockdown of Hsp27.


CONCLUSIONS
Hsp27 may regulate the phosphorylation of the p65 subunit of NF-kappaB in the Ang II-induced signaling pathway of NF-kappaB in A10 cells. The proinflammatory effects of Ang II on NF-kappaB in vascular smooth muscle cells may be through a non-canonical pathway and be dependent on p65 phosphorylation.
Voegeli
Tracy S
TS
Department of Anatomy & Neurobiology, Faculty of Medicine, Dalhousie University, Halifax, NS B3H 1X5, Canada.
Currie
R William
RW
D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics*; D051381:Rats; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000378:metabolism*
D055551:HSP27 Heat-Shock Proteins; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D000804:Angiotensin II