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Modulation of heparan sulfate/chemokine interactions using RNA interference
Author(s) -
Arzoumanian Hrant,
Bennet Terry,
Watson Zara,
Carter Noel
Publication year - 2004
Publication title -
international journal of experimental pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.671
H-Index - 72
eISSN - 1365-2613
pISSN - 0959-9673
DOI - 10.1111/j.0959-9673.2004.0390m.x
Subject(s) - sulfation , chemokine , heparan sulfate , microbiology and biotechnology , chemistry , glycosaminoglycan , rna interference , biology , biochemistry , rna , receptor , gene
The glycosaminoglycan heparan sulfate (HS) on the luminal surface of the vascular endothelium plays a vital role in promoting leucocyte migration during inflammation via specific interaction chemokines. HS is modified by sulfation and this appears to regulate chemokine binding. We previously observed that under pro‐inflammatory conditions [tumour necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ)], the endothelial cell line HMEC‐1 showed an increased sulfation HS. This promoted a significant increase binding of the chemokine RANTES and transendothelial migration of leucocytes. TNF‐α and IFN‐γ clearly modulated HS biosynthesis, in part by up‐regulating the NDST 1 and 2 genes responsible for N ‐sulfation of the glycan, resulting in structural changes that favour HS/chemokine interactions (Carter et al . 2003). We have attempted to down‐regulate the expression of the gene NDST1 by RNA interference in order to investigate the structural and functional effects of reduced HS N ‐sulfation. Materials and methods A double‐stranded interference RNA was designed and synthesized in vitro to target the sulfotransferase NDST‐1 and used to tranfect the HMEC‐1 cell line. A number of assays measured its effect on HS structure and function. Cell surface N ‐sulfation was measured by flow cytometry using the antibody F58 10E4 (Seikagaku, Japan). Cell surface binding of the chemokine MCP‐1 was visualized by immunohistochemistry. The functional effect of MCP‐1 binding was measured using a trans ‐endothelial migration assay. Results Surprisingly, the transfected cells appeared to show a small but marked increase in cell surface N ‐sulfation. This was consistent with previous results transfecting a full‐length NDST‐1 antisense construct into the cell line, A549 (unpublished). The increase in N ‐sulfation observed was supported functional date. Namely, transfected cells show an increased binding of the chemokine MCP‐1 and this promoted a statistically significant increase in transendothelial migration of leucocytes. Discussion The results observed were contrary to expectations. It would appear that the interference RNA molecule is in some way interfering in the regulation of the NDST‐1 expression leading to an increase not decrease in gene expression. This has yet to be verified but we are beginning real time expression studies to validate this. We have hypothesized two possible reasons for this observation; endothelial cells may be able to sense a reduction in NDST activity and up‐regulate gene expression. Or the presence of the interference RNA itself promotes NDST‐1 expression. The latter is supported by growing evidence that short double‐stranded RNAs are thought to up‐regulate the pro‐inflammatory STAT transcription factor family (Shuey et al . 2002).