Regulation of monocyte chemoattractant protein-1 by the oxidized lipid, 13-hydroperoxyoctadecadienoic acid, in vascular smooth muscle cells via nuclear factor-kappa B (NF-kappa B).
The leukocyte- type 12/15-Lipoxygenase (12/15-LO) enzyme and its oxidized lipid products play important roles in vascular smooth muscle cell (VSMC) growth, migration, and matrix responses associated with hypertension, atherosclerosis, and restenosis. However, much less is known about their inflammatory effects. In this study, we showed that the 12/15-LO product of linoleic acid, 13-hydroperoxyocta decadienoic acid (13-HPODE) can transcriptionally upregulate the expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in VSMC. We also observed reduced activation of the transcription factor, NF-kappa B and reduced expression of MCP-1/JE mRNA in VSMC from 12/15-LO knock-out mice relative to WT. To confirm the role of NF-kappa B in 13-HPODE-induced MCP-1 expression and to selectively block the induction of such inflammatory genes in VSMC, we designed novel molecular approaches to knockdown NF-kappa B with short interfering RNAs (siRNAs). We designed siRNAs to human NF-kappa B p65 transcriptionally active subunit by using a rapid PCR-based approach that generates sense and antisense siRNA separated by a hairpin loop downstream of the U6 promoter. siRNA PCR products targeting seven different sites on p65 cDNA could induce upto 92% reduction in HA-p65 protein levels. A six-fold decrease in NF-kappa B-dependent luciferase activity was also seen. Transfection of human VSMC with these siRNA PCR products resulted in 70% reduction in p65 protein levels. We cloned the PCR products into a pCR3.1 vector and these p65 siRNA expressing plasmids very effectively blocked 13-HPODE-induced expression of both MCP-1 and TNF-alpha genes. These results show for the first time that 13-HPODE can induce MCP-1 in the vasculature via activation of NF-kappa B.
Dwarakanath
Roopashree S
RS
Departments of Diabetes, Beckman Research Institute of City of Hope, Duarte, CA 91010, USA.
Sahar
Saurabh
S
Reddy
Marpadga A
MA
Castanotto
Daniela
D
Rossi
John J
JJ
Natarajan
Rama
R
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis* / Q000378:metabolism; D003001:Cloning, Molecular; D015536:Down-Regulation; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007249:Inflammation; D008041:Linoleic Acids / Q000494:pharmacology; D050356:Lipid Metabolism; D008054:Lipid Peroxides / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D016328:NF-kappa B / Q000378:metabolism*; D009841:Oligonucleotides / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D018932:Chemokine CCL2; D008041:Linoleic Acids; D008054:Lipid Peroxides; D016328:NF-kappa B; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D008156:Luciferases